JP2002541924A - Methods for making autograft fibrin sealants and allogeneic - Google Patents

Abstract

SUMMARY OF THE INVENTION In general, the present invention is directed to an autograft bioadhesive sealant wherein all blood components for the bioadhesive sealant are derived from a patient to whom the bioadhesive sealant will be applied. It relates to a two-phase method for forming a composition or fibrin glue. First, platelet-rich plasma and platelet-poor plasma are formed by centrifuging the amount of anticoagulated whole blood drawn from a patient earlier. The platelet-rich and platelet-poor plasma is then divided into two parts. To the first part used for the first phase, a compound that reverses the effect of the anticoagulant is added to form a clot. The clot is then crushed and the resulting serum containing autologous thrombin is recovered. The serum obtained from the first phase is then mixed with the second portion of the platelet-rich or platelet-poor plasma used in the second phase to form the bioadhesive sealant of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

This application is a continuation-in-part of US patent application Ser. No. 08 / 640,278, filed Apr. 30, 1996, which is a method for making an autografted fibrin sealant. is there.

TECHNICAL FIELD [0002] The present invention relates to autograft bioadhesive sealant compositions, and more particularly to the convenient and substantial preparation of bioadhesive sealants from blood components, particularly from patients to whom the bioadhesive sealant will be applied. A two-phase method.

BACKGROUND OF THE INVENTION When the lining of a blood vessel is damaged, a complex sequence of events occurs, preventing loss of the blood vessel and ultimately restoring the integrity of the blood vessel. Physical factors such as short-term vasoconstriction and pressure of the ejected blood on the vascular wall may play a role in homeostasis, but the major factors in the homeostasis mechanism are the platelets and the blood coagulation system.

[0004] Blood clotting is the result of a complex interaction of several blood clotting factor proteins through a cascade, see FIG. Generally, damage to the vascular endothelium exposes subendothelial structures that attract platelets and induce them to reversibly aggregate. The protein thrombin formed during the activation of the coagulation pathway produces insoluble cross-linked fibrils of the protein fibrin, causing reversible assembly of platelets. The resulting platelet-fibrin clot is an effective protection against blood loss from the vasculature and serves as a scaffold for subsequent repair of the lining of blood vessels.

[0005] Bioadhesive sealants or fibrin glues are a relatively new technological advance that repeats the biological processes of the final stages of blood coagulation. Clinical reports have demonstrated the utility of fibrin glue in various surgical fields, such as, for example, cardiovascular and thoracic transplants, head and neck, oral, gastrointestinal, orthopedic, neurological and plastic surgery. At the time of surgery, two initial components, fibrinogen and thrombin, containing fibrin glue, are mixed to form a clot. Blood clots need tissues, bones,
Or adheres to nerves but is slowly reabsorbed by the body in approximately 10 days due to fibrinolysis. Important features of fibrin glue include: (1) achieving homeostasis in vascular anastomotic connections, especially in areas where the suturing approach is difficult or where the place of suturing poses undue risk; (2) suturing alone (3) its ability to obtain homeostasis in heparinized patients or patients with coagulation disorders, which inhibits bleeding from incapable needle holes or arterial lacerations. Borst, HG,
et al., J. Thorac.Cardiovasc. Surg., 84: 548-553 (1982); Walterbusch, G.
J., et al., Thorac.Cardiovasc. Surg., 30: 234-235 (1982); Wolner, FJ,
See, et al., Thorac. Cardiovasc. Surg., 30: 236-237 (1982).

[0006] Despite the usefulness and successful use of fibrin glue by European healthcare professionals, neither fibrin glue nor its essential components, fibrinogen and thrombin, are widely used in the United States. This was due to the 1978 US Food and Drug Administration's ban on the sale of commercially prepared fibrinogen concentrates made from stored donors, and in most cases, viral infections, especially HBV and HCV (non-A (Also known as non-B hepatitis virus) due to the risk of transmission of viral infections that cause hepatitis. In addition, more recently, the emergence of AIDS-related HIV, and also other lipid foreskin viruses such as cytomegalovirus (CMV), Epstein-Barr virus and herpes simplex virus in fibrinogen preparations, for the foreseeable future. No change was possible. For similar reasons, human thrombin is also currently not licensed for human use in the United States. Bovine thrombin, which is licensed for human use in the U.S., may have other bovine pathogens, such as bovine spongiform encephalopathy,
Obtained from bovine sources that do not appear to have a significant risk for HIV and hepatitis.

Various methods have evolved for the preparation of fibrin adhesives. For example, Rose et al. (A) screened one or more blood borne diseases, for example, syphilis, hepatitis or acquired immune disease syndrome, at about 80 ° C. for at least 6 hours, preferably at least about 12 hours. Freezing fresh frozen plasma from a single donor, such as a human or other animal such as a cow, sheep or pig, (b) supernatant and cryoprecipitate suspension containing fibrinogen and factor XIII Raising the temperature of the frozen plasma to, for example, between about 0 ° C. and room temperature to form a (c) recovering the cryoprecipitated suspension, which is useful as a precursor in the preparation of a fibrin glue. A method for preparing a cryoprecipitate suspension containing fibrinogen and factor XIII is disclosed. And applying the fixed volume of the cold precipitation suspension described above to a location where the fibrin glue is to be converted into fibrin glue which solidifies in gel form there in the suspension, and It is prepared by applying a composition containing a sufficient amount of thrombin, for example, human, bovine, ovine or porcine thrombin. See U.S. Patent No. 4,627,879.

A second technique for preparing a fibrin glue is disclosed by Marx in his US Pat. No. 5,607,694. Cryoprecipitates, essentially as previously described, serve as a source of fibrinogen components, to which Marx adds thrombin and liposomes. Title “Immunization with bovine thrombin used with fibrin glue during cardiovascular surgery” (J. Thorac. Cardiovasc. Surg., 105 (5
): 892-897 (1992)), the third method discussed by Beruyer, M. et al., Only with bovine thrombin and human coagulation proteins such as fibrinogen, fibronectin, factor XIII and plasminogen. Rather, it reveals a fibrin glue prepared by also mixing with urea protinin and calcium chloride.

The above-mentioned patents by Rose et al. And Marx and the technical articles by Berruyer, et al. Each disclose methods for the preparation of fibrin sealants, but both of these methods involve activators. As a consequence of using bovine thrombin as A serious and life-threatening importance associated with the use of fibrin glue containing bovine thrombin was that patients were reported to have a hemorrhagic constitution after receiving topical bovine thrombin.
This complication occurs when the patient develops antibodies to bovine factor V in a relatively impure bovine thrombin preparation. These antibodies cross-react with human factor V,
It causes a factor V deficiency that will be severe enough to induce bleeding and even death. Rapaport, SI, et al., Am. J. Clin.Pathol, 97: 84-91 (1992)
; Berruyer, M., et al., J. Thorac. Cardiovasc. Surg., 105: 892-897 (1993).
; Zehnder, J., et al., Blood, 76 (10): 2011-2016 (1990); Munteen, W., et.
al., Acta Paediatr., 83: 84-7 (1994); Christine, RJ, et al., Surgery, 1
27: 708-710 (1997).

[0010] A further disadvantage associated with the method disclosed by Marx and Rose, et al. Is that cryoprecipitation preparation requires a lot of time and money to prepare. In addition, much care must be taken to ensure that there is no contamination of any virus.

A final disadvantage associated with the previously disclosed method is that while it is planned to use human thrombin as an active agent, human thrombin is not available for clinical use and the patient has antigenicity against human thrombin. There is no evidence that there is no response. By analogy, it was shown that human recombinant factor VIII elicits an antigenic response in hemophilia patients. Biasi, R. de., Thrombosis and Hoemostasis,
71 (5): 544-547 (1994). Therefore, merely assuming that the use of recombinant human thrombin will obviate the antigenicity problems associated with bovine thrombin until further clinical studies are performed on the effects of human recombinant thrombin. Can not. A second difficulty with thrombin is that it is autocatalytic, that is, it tends to self-destruct, where handling and permanent storage are problematic.

[0012] Thus, a simple and substantial process for the preparation of a bioadhesive sealant composition such that the resulting bioadhesive sealant results in zero risk of disease transmission and no risk of causing adverse physiological reactions. There is still a need for a new method.

DISCLOSURE OF THE INVENTION It is therefore an object of the present invention to provide a complete autograft bioadhesive sealant composition.

[0014] Another object of the present invention is to provide an autograft bioadhesive sealant composition that eliminates the risks associated with the use of bovine and recombinant human thrombin.

[0015] Further objects, advantages and novel features of the invention will become apparent to those skilled in the art from examination of the portions of the description which follows, and the specification set forth below, or will be learned by the practice of the invention. Shown in parts. The objects and advantages of the invention will be better understood and attained by means of the instrumentalities, combinations, compositions and methods particularly pointed out in the appended claims.

[0016] In accordance with the intent of the present invention, and as embodied and broadly described herein, to achieve the foregoing and other objects, the method of the present invention involves the enrichment of platelets rich in anticoagulants. Includes the formation of an autograft bioadhesive sealant that includes forming a plasma or platelet-poor plasma. The platelet-rich or platelet-poor plasma is then split into two parts and the first part is allowed to clot and thus to recover to form a clot. The clot is then crushed and the resulting serum is collected. A sufficient volume of serum to become fibrinogen in a second portion of platelet-rich or platelet-poor plasma to convert the bioadhesive sealant composition to fibrin, which solidifies in gel form, is then added to the platelet. Is prepared by mixing a second portion of a fixed amount of rich or platelet-poor plasma.

BEST MODE FOR CARRYING OUT THE INVENTION Generally, the present invention relates to a method in which all blood components for a bioadhesive sealant are from the patient to whom the bioadhesive sealant is to be applied. , A self-implanted bioadhesive sealant composition or fibrin adhesive for the two-phase method shown in FIGS. First, platelet-rich and platelet-poor plasma is formed by centrifuging a large amount of anticoagulated whole blood previously drawn from a patient. The platelet-rich and platelet-poor plasma is then divided into two parts. To the first part used in the first phase, a compound that reverses the effect of the anticoagulant is added to form a clot. The clot is then crushed and the resulting autologous thrombin-containing serum is collected. The serum obtained from the first phase is then mixed with the second portion of the platelet-rich or platelet-poor plasma used in the second phase to form the bioadhesive sealant of the present invention.

The method of the present invention for preparing an autograft bioadhesive sealant composition, described in further detail below, is represented by the flow diagrams depicted in FIGS. The method of the present invention begins with the formation of anticoagulated whole blood 90 by collecting the patient's whole blood 80 in a culture containing an anticoagulant such as sodium citrate (citrate) or heparin. The act of drawing blood initiates the clotting reaction and a blood clot will form unless the process is stopped in any way. Clot formation is a multi-step process, and some of these steps require the presence of calcium ions. Removing the calcium ions present in whole blood, such as the effect when blood is collected in citrate, can prevent blood from clotting. Calcium is added back into the whole blood to reinitiate the clot formation process (recalcification). Calcium chelators are chemicals that react with calcium and are present in the blood in such a way that calcium can no longer function in blood clotting. The most common chelating agent is the salt of citric acid (citrate) because it has the fewest side effects on coagulation-based compositions
It is. By collecting blood into a culture solution containing a calcium chelating agent such as citric acid, sample collection and further preparation of a citric acid sample can be performed over a period of up to several hours.

Preferably, whole blood 80 is collected and a 3.8% solution of sodium citrate (referred to herein as a “citrate collection medium”), especially citrate recovery of blood Mix at a ratio of 9: 1 to culture. A 3.8% sodium citrate solution is prepared by adding 3.8 grams of sodium citrate per 100 ml of water. While 3.8% sodium citrate recovery cultures are commonly used to collect and store blood, those skilled in the art will appreciate that the ratio of sodium citrate to whole blood at a final concentration of about 10.9-12.9. It will be appreciated that it may be in the range of% mM / L.

The anticoagulated whole blood 90 is then subjected to approximately 20-50 r.cf (10-40 minutes).
Relative centrifugal force), centrifugation at 25 r.cf for 20 minutes,
As a result, two liquid phases are formed. The upper phase is platelet-rich plasma (PRP) 100
The lower layer is anticoagulated whole blood minus platelet-rich plasma.
0. Then, the platelet-rich plasma (PRP) 100 is slowly extracted and stored in a container. The anticoagulated whole blood 190 from which the remaining platelet-rich plasma has been removed is centrifuged for a further 15-30 minutes at a speed of approximately 3000-4500 r.cf, preferably refrigerated at 3850 r.cf for 20 minutes. Centrifugation at this higher speed results in the removal of red blood cells, white blood cells, and platelets from anticoagulated whole blood 190 that has been depleted of the platelet-rich plasma phase, and thus the sediment containing cellular constituents that will be removed ( (Not shown). The resulting platelet-poor plasma (PPP) 200 was decanted from the precipitate and stored in a container. Container for storing platelet-rich plasma 100 and platelet-poor plasma 200 (not shown)
Has either a wettable surface (such as silica, permeable soils, coaline, etc.) or a non-wettable surface (such as plastic, siliconized glass, etc.). Since the surface plays a role in activating blood coagulation, the surface of the container selected to store either platelet-rich plasma 100 or platelet-poor plasma 200 should be clot-forming immediately or desirably. Depends on what is desired. Chemically active agents such as coaline can also be used to speed up the clotting time, but they need to be removed later. In a preferred embodiment, glass tubes are the preferred containers used to collect platelet rich plasma 100 and platelet poor plasma 200.

In a preferred embodiment according to route 101, the platelet-rich plasma 100 is divided into two parts. The first part is approximately 1/4 to 1/2 of the total amount of platelet rich plasma 100 and is used in the first phase to prepare thrombin,
A second portion of the platelet-rich plasma 100 is used for the second phase. Once platelet-rich plasma 100 and platelet-poor plasma 200 are obtained, a preferred method for obtaining the bioadhesive sealant composition in an enhanced manner, ie, within 2 minutes, is shown in FIGS. 3 and 4, respectively. And is detailed in more detail in path 101 or 201 as discussed in detail below. However, if a longer clotting time, i.e., in the range of 2 to 8 minutes, is desired, the method of obtaining the bioadhesive sealant composition of the present invention is also schematically detailed in FIGS. May be proceeded along paths 105 and 205, discussed in detail below.

The first phase according to the preferred embodiment begins with restoring the clot formation process. To accomplish this, a reagent (regeneration reagent) that can reverse the effect of the anticoagulant is added back to the first portion of the platelet-rich plasma 100. In a currently preferred embodiment of the invention, the reversal of the effect of the anticoagulant is achieved by using calcium chloride. However, any substrate known or known to be functionally equivalent to calcium chloride, such as calcium gluconate, in regenerating the clotting activity of citrated blood may be used in the practice of the present invention. . Thus, although calcium chloride is the currently preferred calcium salt for use in the present invention, any calcium salt that functions in a manner similar to calcium chloride may be used in the present invention. Similarly, many blood clotting reactions are now believed to require calcium ions as cofactors, but are known or essentially known to be functionally equivalent to calcium by promoting these clotting reactions Any of the substrates mentioned may be used individually or in combination with calcium in the practice of the present invention. If the anticoagulant used was heparin, heparinase would be used to reverse the effect of the anticoagulant. The concentration of regeneration reagent used to reverse anticoagulation will depend on the concentration of anticoagulant in the platelet-rich plasma 100 and the stoichiometry of the chelation and coagulation reactions. However, the concentration of regeneration reagent used to reverse anticoagulation must be sufficient to achieve clot formation.

In the recovery of the platelet-rich plasma 100 as shown in FIG.
Will form naturally. The resulting clot 110 is then crushed by high speed centrifugation or crushed through a mesh and thus thrombin-containing serum 12
Emit 0. In a preferred embodiment, a second platelet-rich plasma (PRP) 100 is used to form the serum 120 within 2 minutes to form the bioadhesive sealant composition 130 of the present invention in an amount sufficient for clinical use. Mix with parts.

In another embodiment, the serum 120 is combined with the platelet-poor plasma 20 of the second phase.
0, thus forming the autograft bioadhesive sealant composition 140 of the present invention within 2 minutes.

In a third embodiment of the present invention, the pathway 105 shown in FIG. 3 is used to pass the original amount of platelets from whole blood 90 anticoagulated into a container having a wettable surface, such as glass. Plan to collect 100 rich plasma (PRP). The platelet-rich plasma 100 is then recalcified to form the bioadhesive sealant composition 150. The preferred bioadhesive sealant composition 150 will require approximately 2 to 8 minutes to form, for within 2 minutes as described in the preferred embodiment.

In a fourth embodiment, schematically depicted by the path 201 in FIG. 4, as already discussed in the preferred embodiment, the platelet-poor plasma 200 rather than the platelet-rich plasma 100 is divided into two parts. Separate. About 1/4 to 1 of the original amount
The first part used for the first phase, / 2, is stored in a container with a wettable surface, and the regeneration reagent, preferably calcium chloride, is added directly to the platelet-poor plasma 200. Surface activation of the stored platelet-poor plasma 200 results in a clot forming on the surface of the container. The resulting clot was crushed as described above and serum 220 was collected. The serum 220 is then mixed with the second phase platelet rich plasma 100 to form the autograft bioadhesive sealant composition 230.

In a fifth embodiment, bioadhesive sealant composition 240 is formed within two minutes by mixing serum 220 with second phase platelet poor plasma 200.

In a sixth embodiment, the original amount of platelet-poor plasma 200 from anticoagulated whole blood 190, excluding platelet-rich plasma, is removed by pathway 205 shown in FIG. Collect in a container with a wettable surface. The platelet-poor plasma 200 is then recalcified to form a bioadhesive sealant composition 250.

A seventh example contemplates mixing human recombinant thromboplastin directly with platelet rich plasma to form a bioadhesive sealant composition (not shown). Alternatively, human recombinant thromboplastin is used to produce thrombin in small amounts of plasma, and the resulting thrombin is combined with platelet-rich plasma to form a bioadhesive sealant. Thromboplastin may be later removed by centrifugation.

[0030] The elongation strength of the bioadhesive sealant composition of the present invention can be affected by the addition of calcium ions. Thus, using the methods described and disclosed above in pathways 101 and 102 in FIGS. 3 and 4, respectively, if a stronger bioadhesive sealant composition is desired, more calcium ions will be transferred to the platelet-rich It may be added when introduced into plasma 100 or platelet poor plasma 200. Alternatively, if the method of preparation of the bioadhesive sealant composition follows routes 105 and 205 depicted in FIGS. 3 and 4, respectively, calcium ions can be introduced directly into platelet rich plasma 100 or platelet poor plasma 200. Well, bioadhesive sealant compositions 150 and 250, respectively, will form.

As described in further detail below, the time interval required to form the bioadhesive sealant composition of the present invention depends on the amount of serum added. A ratio of serum to platelet rich or platelet poor plasma of 1: 4, 1: 2 and 3: 4 results in the formation of a bioadhesive gel composition for approximately 90, 55 and 30 seconds, respectively. Was. Furthermore, due to the fact that thrombin is autocatalytic, it is important that the serum be used within 5 hours of preparation, preferably within 2 hours, ideally immediately. Alternatively, the serum can be refrigerated or frozen indefinitely.

Once the bioadhesive sealant composition of the present invention has gelled, the wound is sealed by applying an appropriate amount of platelet rich or platelet poor plasma to the wound. May be used for Furthermore, the probability of introducing a new blood-borne disease to a patient is zero due to the fact that the bioadhesive sealant composition of the present invention is simply prepared from blood components from the patient who is to receive the bioadhesive sealant. The method of the present invention relates to the formation of a bioadhesive sealant composition as a homeostasis reagent, but also as an additive to wound healing, and a matrix for delivery of drugs and other biologically active proteins. It may be further modified to function as For example, it is well known that fibrin glue has a high affinity for binding to bone fragments that is useful in bone remodeling, such as in plastic surgery, or in healing large fractures. Thus, by maintaining the autograft properties of the bioadhesive sealant composition of the present invention, the autograft bone from the patient can be ground into a powder or the like, and the second phase of the method of the present invention. Can be mixed into the platelet-rich plasma obtained in the above. The serum containing thrombin is then mixed with the platelet-rich plasma and bone fragments in an amount sufficient to apply the resulting gel to the desired location where it freezes.

In the case where the desired bioadhesive sealant composition of the present invention further functions as a mediator of drugs and other biologically active proteins, the method of the present invention may be modified as follows. The serum containing thrombin obtained in the first phase was
A wide variety of drugs and other biologically active proteins may be added to the phase 2 platelet-rich plasma prior to addition to the phase platelet-rich plasma. Examples of drugs added to the platelet-rich plasma prior to the addition of serum include analgesic compounds, antibacterial compounds including bactericidal and bacteriostatic compounds, antibiotics (eg, adriamycin, erythromycin, gentamicin, penicillin, tobramycin, etc.) ), Antifungal compounds, antiinflammatory agents, anthelmintic compounds, antiviral compounds, enzymes, enzyme inhibitors, glycoproteins, growth factors (eg, lymphokines, cytokines, etc.), hormones, steroids, glucocorticosteroids , Immunomodulators, immunoglobulins, minerals, neuroleptics, proteins, peptides, lipoproteins, tumoricidal compounds,
Non-limiting examples include oncostatic compounds, toxins and vitamins such as vitamin A, vitamin E, vitamin B, vitamin C, vitamin D or derivatives thereof. It is envisioned that some or all of the above selected fragments, portions, derivatives or analogs may also be used.

Several different medical devices and test methods exist for measuring and determining blood coagulation and coagulation-related activities. These devices and methods provide the actives necessary to form the bioadhesive sealant compositions of the present invention, namely, thrombin,
It can be used to help determine the best formula for calcium and plasma. Some of the more successful techniques for assessing blood clumping and coagulation are described in the Co, all of which are incorporated herein by reference and all are referred to the designated agent of the present invention.
U.S. Patent No. 4,599,219 to Jackson et al.
No. 4,752,449 to U.S. Pat. No. 5,752,449 and to U.S. Pat. No. 5,174,961 to Smith.

Automated devices that use plunger technology to measure and detect coagulation and coagulation-related activity are generally plunger sensor cartridges or cartridges and a microprocessor controller with the cartridge inserted therein. including. The device works on a cartridge and places a blood sample there to induce and detect coagulation-related events. The cartridge contains a number of test cells, each of which is located by a plunger assembly and is determined by a tube-like member having an upper reaction chamber for performing analytical tests, a reagent chamber containing reagents or reagents. For example, for an activated clotting time (ATC) test, the reagents include active agents that activate blood clotting. A plug member seals the bottom of the reagent chamber. As the test begins, the contents of the reagent chamber are extruded into the reaction chamber to mix with a sample of fluid, typically human blood or a component thereof. An actuator, which is part of the device, lifts the plunger assembly and lowers it to exchange the plunger assembly through a pool of fluid in the reaction chamber. The plunger assembly lowers due to gravity and resists due to the nature of the fluid in the reaction chamber, such as stickiness. If the nature of the sample changes in a predetermined manner as a result of the onset or development of coagulation-related activity, the rate of descent of the plunger assembly will change. By a sufficient change in the descent rate, coagulation-related activity is detected and indicated by the device.

Using the methods discussed above, the cartridge is loaded into the reagent chamber with a platelet lip.
Serum and CaCl obtained from either plasma or platelet poor plasma _Two Collected in. The clotting time test was performed using the blood dispersed in the reaction chamber of the cartridge.
Either platelet-rich plasma (PRP) or platelet-poor plasma (PPP)
Was performed by an automated process. As shown in FIG. 5, in the first experiment,
The amount, the type of plasma from which the serum is derived, and the type of
It was examined to determine the fixed time. Serum, platelet-rich plasma or blood examined
Platelet-to-poor plasma ratios include 1: 4, 1: 2 and 3: 4. FIG.
In a second set of experiments with the results shown in and 7 the bioadhesive sealant composition of the invention
Add serum from the relationship between the actual gel time for
The clotting times were compared with cartridges with a 30 or 60 minute delay. 8 and
A third set of experiments, the results of which are shown in FIGS.
The effect of calcium addition on time was investigated. Final experiment of the results shown in FIG.
In group No. 3, the effect of the addition of calcium on the coagulation time was examined.

Although clotting times vary from donor to donor, comparison of clotting times for individual donors shows a clear effect of serum to plasma ratio and calcium concentration. For all donors, the shortest clotting time occurs at a ratio of 3: 4, 47% shorter than the clotting time of a 1: 4 ratio. The difference in clotting time for the 3: 4 ratio and the 1: 2 ratio was not statistically significant, but the clotting time using the 3: 4 ratio was consistently shorter for all donors. These results indicate that clotting times may be reduced by increasing the ratio of serum to platelet-rich plasma. Similarly, clotting time is clearly influenced by the amount of calcium added, with the shortest clotting time being obtained when no calcium is added, which is the reason that serum recalcifies citrated platelet-rich plasma. To contain sufficient calcium levels. Preliminary results from the scale-up experiment indicate that the experimental clotting time in the cartridge correlates with the actual gel time.

The present invention is further illustrated by the following non-limiting examples. All scientific and technical terms have the meanings as understood by those skilled in the art. The specific examples that follow show that the bioadhesive sealant composition of the present invention was prepared in a clinical setting,
Figure 4 illustrates a method that is not to be construed as limiting the invention in scope or area. The method may be modified to yield a composition included in the present invention, but is not specifically disclosed. Furthermore, variations in the methods for producing similar compositions in any of the different ways will be apparent to those skilled in the art.

EXAMPLES The examples in this specification are intended to illustrate various aspects of practicing the present invention, and are not intended to limit the invention in any way.

Example 1 Preparation of Bioadhesive Sealant Composition Using Platelet-Rich Plasma and Serum 10 cc of platelet-rich plasma is added to a sterile glass tube containing 33 cc of 10% calcium chloride. A stopper was placed on the tube, the contents were mixed slowly, and the tube was set aside. Gelling of the contents will occur in 2 to 8 minutes. The gel is passed through a dry sterile cup that squeezes out 4-6 cc of serum, from which 1 cc of air is drawn along with 1 cc of serum into a syringe containing 4 cc of platelet-rich plasma. The bioadhesive sealant composition will gel inside the syringe within approximately 2 minutes. Gel time may be reduced by increasing the amount of serum added to platelet rich plasma.

Example 2 10 cc of whole blood is drawn from a patient, placed in a sterilized glass tube, and allowed to clot naturally. The clot may be subsequently squeezed or centrifuged to release approximately 4-6 cc of serum.

Then, an equal volume of 10% calcium chloride is mixed with the serum. 1 cc of this calciumated serum was then mixed with 7-8 cc of platelet rich plasma and 2 cc of air. The resulting bioadhesive sealant will gel in approximately 1-2 minutes.

Example 3 10c into a sterile glass syringe containing 33cc of 10% calcium chloride
Add platelet-rich plasma in c. The mixture is allowed to stand for 2 to 8 minutes. Once the gel is properly sticky, apply it where desired.

The techniques described in Examples 1 and 2 may be simultaneously prepared (a) for an additional source of serum and (b) to establish successful coagulation.

The foregoing description is considered as illustrative only of the principles of the present invention. It is not preferred to limit the invention to the constructs and treatments themselves set forth above, as many more modifications and changes will readily occur to those skilled in the art. Accordingly, all suitable modifications and equivalents may be resorted to as falling within the scope of the invention as defined by the appended claims.

[Brief description of the drawings]

The accompanying drawings and description, which are incorporated in and constitute a part of the description of the preferred embodiment of the invention, serve to explain the principles of the invention.

FIG. 1 is a schematic representation of the blood coagulation cascade.

FIG. 2 is a flow chart showing the first part of the method of the present invention used to obtain platelet rich and platelet poor plasma.

FIG. 3 is a flowchart showing the last part of a method for preparing a bioadhesive sealant composition of the present invention using platelet-rich plasma as a starting material.

FIG. 4 is a flowchart showing the last part of a method for preparing a bioadhesive sealant composition of the present invention using platelet poor plasma as a starting material.

FIG. 5 is a chart of the effect of serum-plasma ratio on clotting time.

FIG. 6 is a chart of the relationship between coagulation time using blood drawn from a donor and actual gelation time.

FIG. 7 is a chart of the relationship between coagulation time using blood drawn from a donor and actual gelation time.

FIG. 8 is a chart showing the effect of calcium addition on coagulation time and gelation time using blood drawn from a donor.

FIG. 9 is a chart showing the effect of calcium addition on coagulation time and gelation time using blood drawn from a donor.

FIG. 10 is a chart showing the effect of calcium addition on the clotting time of platelet rich and platelet poor plasma.

[Procedure amendment]

[Submission date] February 22, 2001 (2001.2.22)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Contents of correction]

Patent application title: Method of Making Autograft Fibrin Sealant and Allogeneic

[Claims]

DETAILED DESCRIPTION OF THE INVENTION

This application is a continuation-in-part of US patent application Ser. No. 08 / 640,278, filed Apr. 30, 1996, which is a method for making an autografted fibrin sealant. is there.

TECHNICAL FIELD [0002] The present invention relates to autograft bioadhesive sealant compositions, and more particularly to a convenient method for preparing a bioadhesive sealant from blood components, particularly from patients to whom the bioadhesive sealant will be applied. And a practical two-phase method.

BACKGROUND OF THE INVENTION When the lining of a blood vessel is damaged, a complex sequence of events occurs, preventing blood loss and ultimately returning the blood vessel to its original state . Physical factors such as short-term vasoconstriction and the pressure of the ejected blood on the vascular wall may play a role in hemostasis , but the major factors in the hemostatic mechanism are the platelets and the blood coagulation system.

[0004] Blood coagulation is Ru results der composite interaction of some of the blood coagulation factor protein through the cascade as shown in Figure 1. Generally, damage to the vascular endothelium exposes subendothelial structures that attract platelets and induce them to reversibly aggregate. The protein thrombin formed during the activation of the coagulation pathway produces insoluble cross-linked fibrils of the protein fibrin, causing reversible assembly of platelets. The resulting platelet-fibrin clot is an effective protection against blood loss from the vasculature and serves as a scaffold for subsequent repair of the lining of blood vessels.

[0005] Bioadhesive sealants or fibrin glues are a relatively new technological advance that repeats the biological processes of the final stages of blood coagulation. Clinical reports have demonstrated the utility of fibrin glue in various surgical fields, such as, for example, cardiovascular and thoracic transplants, head and neck, oral, gastrointestinal, orthopedic, neurological and plastic surgery. When surgery, a fibrin glue is including two of the first component, the fibrinogen and thrombin to form clot was mixed-. Clot tissue needed within a few seconds, but adhere to the bone or nerves, and is re-absorbed approximately 10 days tapping Kkuri and body by fibrinolysis. An important feature of the fibrin glue, (1) in particular approach is not difficult area in the suture or suture to place ability to achieve hemostasis in vascular anastomotic coupling in areas such as bear undue risk, (2 ) ability alone sutures to inhibit bleeding from needle holes or arterial tears which can not be suppressed, and (3) ability to achieve hemostasis in patients with patient or clotting disorder heparinized treatment is. Borst, HG, e
t al., J. Thorac.Cardiovasc. Surg., 84: 548-553 (1982); Walterbusch, GJ
., et al., Thorac. Cardiovasc. Surg., 30: 234-235 (1982); Wolner, FJ, e.
See al., Thorac. Cardiovasc. Surg., 30: 236-237 (1982).

[0006] Despite the effect the successful record of use fibrin glue by the European health care workers, fibrin glue also, fibrinogen and thrombin of its essential components also, have not been widely used in the United States. This is due to ban US Food and Drug Administration in 1978 for the sale of fibrinogen concentrate prepared commercially made from a donor that is savings in most viral infections, especially HBV and HCV (Non a type, and the reason the risk of transmission of viral infections such cause hepatitis as also known) as non-B hepatitis virus. In addition, more recently, HIV associated with AIDS, and also cytomegalovirus (CMV)
), The appearance of Epstein-Barr virus and other lipid foreskin viruses such as herpes simplex virus in the preparation of fibrinogen, for the time being, no change in this policy is possible. For similar reasons, human thrombin is also currently not licensed for human use in the United States. Bovine thrombin, which has been licensed for human use in the United States, is a bovine source that may not be as dangerous for HIV and hepatitis, although other bovine pathogens such as bovine spongiform encephalopathy may exist. Obtain from source.

Preparation of Fibrin AdhesiveforVarious methodsDevelopedHave been. For example,
Roseetc(A) for example, one or more bloods of syphilis, hepatitis or acquired immune disease syndrome.
For liquid communicable diseases, at about 80 ° C, at leastabout6 hours, preferably at least about
A human or other animal, such as a cow, sheep or pig, screened for 12 hours.
Fresh from a single donorBloodFreezing the serum, (b)The temperature of the frozen plasmaAn example
For example, raise it to between about 0 ° C and room temperatureContaining fibrinogen and factor XIII
To form a supernatant and a cryoprecipitate suspension(C) cold precipitationobjectSuspensionBlood collectionYou
Preparation of a fibrin adhesive comprising:AtFibri useful as a precursor
Noogen and Factor XIIIMDisclosed is a method of preparing a cryoprecipitate suspension
I have. Then the fibrin glue and the fibrinogen in the suspension are
Place the above decision in a location that will convert it to a fibrin glue that solidifies in shape.
Full volume of cold precipitationobjectApply the suspension and also for example human, cow, sheep or
Like pig thrombinNatoLongbinWith enoughApplying the composition containing Therefore Prepare. See U.S. Patent No. 4,627,879.

[0008] A second technique for preparing a fibrin adhesive is disclosed by Marx in his US Patent No. 5,607,694. In essence, cryoprecipitate as already described becomes a source of fibrinogen component, there Marx is Ru Tei added thrombin and liposomes. Title "Immunization with bovine thrombin used with fibrin glue during cardiovascular surgery" (J. Thorac. Cardiovasc. Surg., 105 (5): 89).
2-897 (1992)) in Berruyer, third methods discussed by M. etc., bovine thrombin, rather than fibrinogen, fibronectin, and human coagulation protein, such as factor XIII and plasminogen only, bovine aprotinin and discloses a fibrin adhesive prepared by mixing with calcium chloride.

The above-mentioned patents by Rose et al. And Marx and the technical articles by Berruyer et al . Each describe methods for the preparation of fibrin sealants, but each of these methods uses bovine thrombin as an activator. I am inconvenienced about what I do. A serious and life-threatening result associated with the use of a fibrin glue containing bovine thrombin has been that patients have been reported to become hemorrhagic after receiving local bovine thrombin. This complication occurs when the patient develops antibodies to bovine factor V in a relatively impure bovine thrombin preparation. These antibodies cross-reaction with human Factor V, you're it, bleeding and even death as to cause the Factor V deficiency Ru sufficiently serious der also results. Ra
paport, SI, et al., Am. J. Clin.Pathol, 97: 84-91 (1992); Berruyer, M.
, et al., J. Thorac.Cardiovasc. Surg., 105: 892-897 (1993); Zehnder, J.,
et al., Blood, 76 (10): 2011-2016 (1990); Muntean , W., et al., Acta Paed.
iatr., 83: 84-7 (1994); Christine, RJ, et al., Surgery, 127: 708-710 (19
See 97).

[0010] <br/> A further disadvantage associated with the method disclosed by Marx and Rose like is that it requires a lot of time and financial of hanging place for preparing the cryoprecipitate prepared. In addition, much care must be taken to ensure that there is no contamination with any viruses.

[0011] As a final disadvantage already associated with the disclosed method, while is envisaged that the use of human thrombin as the active agent, human thrombin is not available as a clinical use, the patient to human thrombin There is no evidence that there is no antigenic response. By analogy, it was shown that human recombinant factor VIII elicits an antigenic response in hemophilia patients. Biasi, R. de., Thrombosis and Hoem
See ostasis, 71 (5): 544-547 (1994). Therefore, until further clinical studies on the effect of human recombinant thrombin is carried out, the use of recombinant human thrombin will prevent antigenic associated with bovine thrombin problem, a single <br / > Cannot be estimated . The second difficulty with the thrombin, it is autocatalytic, i.e. is that the storage handling and long takes self-destruction is likely to be a problem.

Accordingly, the risk of diseases transmissible by bioadhesive sealant resulting is nil, and also such that none risk of causing adverse physiological responses, for the preparation of bioadhesive sealant compositions There is still a need for a simple and practical method.

Accordingly, it is an object of the present invention to provide a complete autograft bioadhesive sealant composition.

[0014] Another object of the present invention is to provide an autograft bioadhesive sealant composition that eliminates the risks associated with the use of bovine and recombinant human thrombin.

[0015] Further objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art from the following detailed description , or Will be confirmed by implementation . The objects and advantages of the invention will be better understood and attained by means of the instrumentalities, combinations, compositions and methods particularly pointed out in the appended claims.

[0016] In accordance with the intent of the present invention, in order to achieve the foregoing and other objects, embodied herein, as described extensively, how the present invention, it abundant platelet containing anticoagulant including the formation of autologous bioadhesive sealant comprising a step of forming a poor plasma plasma or platelets. The divided poor plasma rich plasma or platelets Platelet into two parts, that allowed <br/> of forming blood clots by clot to restore the first portion. The clot is then crushed and the resulting serum is collected. A sufficient volume of serum to become fibrinogen in a second portion of platelet- rich or platelet-poor plasma to convert the bioadhesive sealant composition to fibrin which solidifies in gel form, prepared by mixing a rich plasma or a second portion of the fixed amount of poor plasma of platelets.

BEST MODE FOR CARRYING OUT THE INVENTION Generally, the present invention relates to a method in which all blood components for a bioadhesive sealant are from the patient to whom the bioadhesive sealant is to be applied. , for the formation of autologous bioadhesive sealant compositions or fibrin glue, about 2 phase method shown in FIGS. 2, 3 and 4. First, the whole blood amounts of anticoagulant treatment in advance bled from a patient by centrifugation, to form formed poor plasma rich plasma and platelet platelet. The platelet- rich and platelet-poor plasma is then divided into two parts. To the first part used in the first phase, a compound that reverses the effect of the anticoagulant is added to form a clot. The clot is then crushed and the resulting autologous thrombin-containing serum is collected. The serum obtained from the first phase is then mixed with a second portion of the platelet- rich or platelet-poor plasma used in the second phase to form the bioadhesive sealant of the present invention .

[0018] are described in detail further below, the method of the present invention for the preparation of autologous bioadhesive sealant composition is represented by a flow chart which is shown in FIGS. 2, 3 and 4.
The method of the present invention, anticoagulated whole blood 90 performed by collecting blood whole blood 80 of the patient to the medium containing anticoagulant blood agents such as sodium citrate (citrate) or heparin
Begins with the creation of The act of drawing blood initiates the clotting reaction and a blood clot will form unless the process is stopped in any way. Clot formation is a multi-step process, and some of these steps require the presence of calcium ions. Blood like effect when bled into citrate can prevent blood from clotting by removing calcium ions present in the whole blood. Calcium is added back into the whole blood to reinitiate the clot formation process (recalcification). Calcium chelators are chemicals that react with calcium and are present in the blood in such a way that calcium can no longer function in blood clotting. The most common chelating agent is the salt of citric acid (citrate), since it has the fewest side effects on coagulation-based compositions. By bled blood culture medium containing calcium chelating agents such as citrate, sampling and preparation of further citric acid sample can be performed beyond the time up to several hours.

Preferably, it bled whole blood 80, 3.8% solution (herein, "citrate blood cultures (citrate collection medium)" hereinafter) of sodium citrate and, especially blood citrate blood Mix at a ratio of 9: 1 to culture. A 3.8% sodium citrate solution is prepared by adding 3.8 grams of sodium citrate per 100 ml of water. While 3.8% sodium citrate blood collection cultures are commonly used for collecting and storing blood , those skilled in the art will appreciate that the ratio of sodium citrate to whole blood at a final concentration of about 10.9-12.9. It will be appreciated that it may be in the range of% mM / L.

[0020]Anticoagulant90 whole blood processed,Next1 in0 to 40 minutes, approximately 20 to 50 r.c.f.
Centrifugation at the speed of relative centrifugal force)processingAnd centrifugation at 25 r.c.f., preferably for 20 minutes. processing As a result, two liquid phases are formed. Upper phase of plateletsAbundantPlasma (PRP)
100, lower layer is anti-Blood clotPlatelets from processed whole bloodAbundantMinus the fresh plasma
190. SoAfter,PlateletAbundantPlasma (PRP) 100be quietSampling,
Store in a container.

The anticoagulated whole blood 190 excluding the remaining platelet- rich plasma was further added to 15-30
Min, centrifuged at a speed of approximately 3000～4500R.Cf, preferably 20 minutes, cooled centrifuged at 3850R.Cf. As centrifugation results in the higher speed, red blood cells from whole blood 190 were anticoagulated excluding rich plasma phase of platelets, white blood cells and platelets were removed, it'll it will be removed later A precipitate ( not shown ) containing cell constituents is formed. The resulting platelet-poor plasma (PPP) 200 was decanted from the precipitate and stored in a container. Container to store poor plasma 200 rich plasma 100 and platelet platelet (not shown), (silica, diathermic soil, such as kaolin) wettable surface or (plastic, such as siliconized glass Do) may have either non-wettable surfaces. Since the surface has a role of activating blood clotting, The choice of container that have a surface for storing either poor plasma 200 rich plasma 100 or platelets in platelet, It depends on whether you want clots to form faster or slower . It is also chemically active agents such as kaolin can be used to hasten the inter clotting time <br/>, it that Do need to remove later them. In a preferred embodiment, glass tubes are the preferred containers used to collect platelet- rich plasma 100 and platelet-poor plasma 200.

In a preferred embodiment according to route 101, the platelet- rich plasma 100
Divided into two parts. The first part is approximately 1/100 of the total amount of platelet- rich plasma 100.
4 to 1/2, used in the first phase to prepare thrombin, while the second portion of platelet- rich plasma 100 is used in the second phase. Once platelet- rich plasma 100 and platelet-poor plasma 200 are obtained, a preferred method for obtaining the bioadhesive sealant composition quickly, ie, within 2 minutes, is shown in FIG. And 101 as shown in FIG. 4 and discussed in detail below.
01. However longer, that is, the longer the clotting time in the range from 2 minutes to 8 minutes is desired, obtaining a bioadhesive sealant compositions of the present invention is shown in details in the flowchart in FIGS. 3 and 4 be discussed in more detail below, it is proceeding me I along the path 105 and 205.

The first phase according to a preferred embodiment begins by restoring the clot-forming effect . To accomplish this, a reagent ( recovery agent ) that can reverse the effect of the anticoagulant is added back to the first portion of the platelet- rich plasma 100. In a presently preferred embodiment of the invention, the reversal of the effect of the anticoagulant is achieved by using calcium chloride. However, when restoring coagulation blood action of citrated blood, such as calcium gluconate, known der to be calcium chloride functionally equivalent Luke, any substance that has been confirmed, the present invention Can be used in practice. Thus , although calcium chloride is the currently preferred calcium salt for use in the present invention, any calcium salt that functions in a manner similar to calcium chloride can be used in the present invention. Similarly, many blood clotting reactions are now believed to require calcium ions as cofactors, but are known to be functionally equivalent to calcium by promoting these clotting reactions , or later Any of the identified materials can be used individually or in combination with calcium in the practice of the present invention. If the anticoagulant used is heparin, heparinase is used to reverse the effect of the anticoagulant . Concentration of recovery agent used to reverse the anticoagulant reaction will to some extent depend on the stoichiometry of the concentration and the chelation and clotting reaction anticoagulant rich plasma 100 platelets . However, the concentration of the recovery agent used to reverse the anticoagulant reaction must be sufficient to achieve clot formation.

[0024] In-rich recovery of plasma 100 platelet as shown in FIG. 3, clot 110 you naturally formed. And grinding the clot 110 the resulting high-speed centrifugation,
Or squashed through a mesh, thereby providing thrombin-containing serum 120
Release. In a preferred embodiment, the where serum 120, mixed with rich plasma (PRP) 100 second part of the platelet, within 2 minutes, bioadhesive sealant compositions of the present invention in an amount sufficient for clinical use An object 130 is formed .

In another embodiment, the serum 120 is combined with the phase 2 platelet poor plasma 20.
0, thereby producing the autograft bioadhesive sealant composition 140 of the present invention.
Is formed within 2 minutes.

[0026] a third embodiment of the present invention, the path 105 shown in FIG. 3, the initial amount of platelets obtained from whole blood 90 has been anticoagulated rich plasma (PRP) 100, a glass it is intended to collect it in a container that has a wettable surface, such as. The platelet- rich plasma 100 is then recalcified to form the bioadhesive sealant composition 150. This desired bioadhesive sealant composition 150 takes approximately two to three minutes to form, as described in the preferred embodiment, within two minutes.
I know that require 8 minutes.

[0027] In a fourth aspect which shows in flow diagram the path 201 in FIG. 4, as discussed previously preferred embodiment, divide poor plasma 200 of platelets rather than the rich plasma 100 of platelets in two parts . About 1/4 to 1 of the original amount
/ 2 is the first part to be used in the first phase and stored in a container having a wettable surface and the recovery agent, preferably adding calcium chloride, the poor plasma 200 directly platelets. Surface activation of the stored platelet-poor plasma 200 results in a clot forming on the surface of the container. The resulting clot was crushed as described above and serum 220 was collected. The autologous bioadhesive sealant composition 230 is then formed by mixing the serum 220 with the second phase platelet- rich plasma 100.

In a fifth embodiment, bioadhesive sealant composition 240 is formed within two minutes by mixing serum 220 with second phase platelet poor plasma 200.

In the sixth embodiment, platelets are collected by the route 205 shown in FIG.AbundantExcluding plasma Anticoagulant Processed whole blood 190Obtained fromOf platelet-poor plasma 200initialQuantity
Collect in a container with a wettable surface such as glass. And platelet-poor plasma 2
00 is recalcified to form a bioadhesive sealant composition 250.

The seventh embodiment is mixed with rich plasma directly platelets human recombinant thromboplastin, is shall be intended to form bioadhesive sealant composition (not shown) . Alternatively, it used to generate thrombin with a small amount of plasma human recombinant thromboplastin, and thrombin resulting, be coupled with rich plasma of blood platelets that form a bioadhesive sealant . Thromboplastin may be removed by centrifugation after.

The elongation strength of the bioadhesive sealant composition of the present invention depends on the addition of calcium ions.
Thus it can be affected. Therefore, paths 101 and 1 in FIGS. 3 and 4, respectively.
02, a stronger bioadhesive sealant composition using the method disclosed and described above in US Pat.
If more is desired, more calcium ions,AbundantPlasma
It may be added when introduced into 100 or platelet poor plasma 200. Or
If the method of preparation of the bioadhesive sealant composition is shown in FIGS.ToPortrayed
When following pathways 105 and 205, calcium ions are directly Abundant May be introduced into fresh plasma 100 or platelet-poor plasma 200, respectively.
The body adhesive sealant compositions 150 and 250 formYou.

As described in further detail below, the time interval required to form the bioadhesive sealant composition of the present invention depends on the amount of serum added. Serum, the ratio for poor plasma rich plasma or platelet Platelet 1: 4, 1: 2 and 3: 4, as a result, respectively about 90 seconds, bioadhesive gel composition at 55 seconds and 30 seconds It is formed. Furthermore, the fact that thrombin is autocatalytic reactive serum within 5 hours of preparation, preferably within 2 hours, important that ideally used immediately. Instead, serum wear <br/> by be kept refrigerated or frozen indefinitely.

Once the bioadhesive sealant composition of the present invention has gelled, the wound is sealed by applying an appropriate amount of platelet- rich or platelet-poor plasma to the wound. May be used for Furthermore, the probability of introducing a new blood-borne disease to a patient is zero due to the fact that the bioadhesive sealant composition of the present invention is simply prepared from blood components from the patient who is to receive the bioadhesive sealant.
The method of the present invention relates to a matrix for forming the bioadhesive sealant composition, not only as a hemostat , but also as an additive to wound healing, and for the delivery of drugs and other biologically active proteins. It may be further modified to function as For example, it is well known that fibrin glue has a high affinity for binding to bone fragments that is useful in bone remodeling, such as in plastic surgery, or in healing large fractures. Thus, by maintaining the autograft properties of the bioadhesive sealant composition of the present invention, the autograft bone from the patient can be ground to a powder or the like, and the second phase of the method of the present invention. Can be mixed into the platelet- rich plasma obtained in the above. The serum containing thrombin is then mixed with the platelet- rich plasma and bone fragments in an amount sufficient to apply the resulting gel to the desired location where it freezes.

The preferred bioadhesive sealant composition of the present invention further comprises a drug and other biology
Of the present invention
The method may be modified as follows. The serum containing thrombin obtained in the first phase was
Phase of plateletsAbundantWide variety of drugs and other biological activities before adding to the plasma
With the protein ofAbundantMay be added to fresh plasma. Before serum addition
Of plateletsAbundantExamples of drugs that can be added to plasma are analgesic compounds, bactericidal and bacteriostatic
Compounds, including antibiotic compounds, antibiotics (eg, adriamycin, erythroma
Isin, gentamicin, penicillin, tobramycin)AntiFungal compounds, Anti Inflammatory agents, anthelmintic compounds,AntiViral compounds, enzymes, enzyme inhibitors, glycoproteins
Quality, growth factors (eg, lymphokines, cytokines, etc.), hormones,
Teroids, glucocorticosteroids, immunomodulators, immunoglobulins, minerals,
Neuroleptic, protein, peptide, lipoprotein, tumoricidal compound, tumor stasis
Stop compounds, toxins and vitamins (eg vitamin A, vitamin E, vitamin B,
Vitamin C, vitamin D or derivatives thereof)
. Some or all of the above selected fragments, parts, derivatives or analogs may also be used.
I imagine that it may be used.

[0035] Several different medical devices and test methods exist for measuring and determining blood clotting and clotting-related activities. These devices and methods provide the actives necessary to form the bioadhesive sealant compositions of the present invention, namely, thrombin,
It can be used to help determine the best formula for calcium and plasma. Some of the more successful techniques for assessing blood clumping and coagulation are described in the Co, all of which are incorporated herein by reference and all are referred to the designated agent of the present invention.
U.S. Patent No. 4,599,219 issued to such oper, granted US patent issued to Jackson, etc. U.S. Patent No. 4,752,449 and Smith first 5,17
No. 4,961.

Automated devices that use plunger technology to measure and detect coagulation and coagulation-related activity are generally plunger sensor cartridges or cartridges and a microprocessor controller with the cartridge inserted therein. including. This device works on the cartridge, the blood sample is placed there in order to detect induced coagulation-related events. The cartridge contains a number of test cells, each of which contains a plunger assembly and an upper reaction chamber for performing analytical tests.
Determined by a tube-like member having a reagent chamber containing reagents or reagents. For example, for an activated clotting time (ATC) test, the reagents include an active agent that activates blood clotting. A plug member seals the bottom of the reagent chamber. As the test begins, the contents of the reagent chamber are extruded into the reaction chamber to mix with a sample of fluid, typically human blood or a component thereof. An actuator, which is part of the device, lifts the plunger assembly and lowers it to exchange the plunger assembly through a pool of fluid in the reaction chamber. The plunger assembly lowers due to gravity and resists due to the nature of the fluid in the reaction chamber, such as stickiness. If the nature of the sample changes in a predetermined manner as a result of the onset or development of coagulation-related activity, the rate of descent of the plunger assembly will change. By a sufficient change in the descent rate, coagulation-related activity is detected and indicated by the device.

Using the methods discussed above, cartridges were loaded with serum and CaCl 2 from either platelet- rich or platelet-poor plasma in a reagent chamber.
Collected in. Clotting time tests were performed by automated processing using either platelet rich plasma (PRP) or platelet poor plasma (PPP) dispersed in the reaction chamber of the cartridge. In The first experiment shows the results in Figure 5, the amount of serum,
Serum the underlying plasma type, and mixed types of plasma serum, were examined to determine the shortest coagulation time. The ratio of serum to platelet- rich or platelet-poor plasma examined includes 1: 4, 1: 2 and 3: 4. In a second set of experiments the results are shown in FIGS. 6 and 7, the relationship between time of actual gelling about bioadhesive sealant compositions of the present invention, during the time delay from the generation of the serum to adding serum , 0 minutes, 30 minutes or 60 minutes were compared with the clotting time in the cartridge. In a third experiment in which the results are shown in FIGS. 8 and 9, it was examined effects of calcium added in the coagulation time of the actual gel time versus cartridge. The experiment of the last representing the results in Figure 10, examined the effect of the addition of calcium in clotting time.

Although the clotting time varies between donors, the clotting time for individual donors
ComparisonBy, Obvious effect of serum to plasma ratio and calcium concentrationButShown
I have. For all donors, the shortest clotting time occurs at a ratio of 3: 4,
47% shorter than the clotting time of a 1: 4 ratio. Clotting time for 3: 4 ratio and 1: 2 ratio
Differences are not statistically significant, but use a 3: 4 ratio for all donors
Coagulation times were consistently short. These results indicate that the clotting time is
ofAbundantIncreasing the ratio to healthy plasmaByShow potential for shorterare doing.
Similarly, the clotting time is clearly influenced by the amount of calcium added, with the shortest clotting time
Was obtained when calcium was not added, and the serum was citrated platelets.
ofAbundantEnough to recalcify healthy plasmaOf levelCalciuTimeIncluding Suggestion are doing. Preliminary results from the scale-up experiments were
Make sure that the setting time correlates with the actual gel timeSuggest.

The present inventionToAnd the following non-limitingTypicalIllustrated by exampleYouYou. All scientific
And technical terms have the meanings as understood by those skilled in the art. Specific following
An example of the bioadhesive sealant composition of the present invention is prepared in a clinical setting.hand Is an example of how In a range or areaStayThe present inventionToRestrictionWhat to doInterpreted as Should not be .Of the present inventionThe way is,The composition contained in the present inventionManufactureTo do
Make changes toButNot specifically disclosed. Further, Somewhat differentSame in style
Like compositionManufactureYouWhoLawCan be modifiedIs,For those skilled in the artTakeWould be obvious
.

[0040] Example (Example) herein are intended to illustrate various aspects of carrying out the invention and are not intended to in any way to limit the present invention.

Example 1 Preparation of Bioadhesive Sealant Composition Using Platelet-Rich Plasma and Serum 10 cc of platelet- rich plasma was added to a sterile glass test tube containing 33 cc of 10% calcium chloride. Add to The test tube was stoppered , the contents were mixed slowly, and the test tube was set aside. Gelation of the contents is Ru Oko in 8 minutes from 2 minutes. 4 to 6c
in dry sterile cup Sutame out squeezed generates c serum, through a gel, drawing air of 1 cc, with serum 1cc into the syringe containing the platelet rich plasma of 4 cc.
Bioadhesive sealant composition you gelation inside the syringe within about 2 minutes. Therefore to increase the amount of serum was added to the platelet rich plasma can be reduced <br/> low gel time.

Example 2 10 cc of whole blood is collected from a patient, placed in a sterilized glass test tube , and allowed to clot naturally. Subsequently clot down Luke or by centrifuging, it is possible to release the serum of approximately 4～6Cc.

Next, an equal volume of 10% calcium chloride is mixed with the serum. Then, the calcium sera of 1 cc, was mixed with air rich plasma and 2cc platelet 7～8Cc. The resulting bioadhesive sealant you gelled in about 1-2 minutes.

Example 3 Add 10 cc of platelet- rich plasma to a sterile glass syringe containing 33 cc of 10% calcium chloride. The mixture is allowed to stand for 2 to 8 minutes. If Once the gel represents the appropriate sticky, Ru can be applied to the place he wanted.

The techniques described in Examples 1 and 2 may be prepared simultaneously (a) for an additional source of serum and (b) to establish successful coagulation.

The aforementioned serial placement is considered as illustrative only of the principles of the present invention. Further, since it will readily occur to those that modifications and many modifications to one of ordinary skill in the art, the present invention, Mashi Nozomu is to limit the above as indicated configuration and processing itself wards.
Accordingly, all suitable modifications and equivalents example is likely attributable to be included within the scope of the invention as defined by the appended claims.

Incorporated into BRIEF DESCRIPTION OF THE DRAWINGS application, the accompanying drawings, which form a part of the specification, which illustrates a preferred embodiment of the present invention, together with the description, serve to explain the principles of the present invention There is .

FIG. 1 is a schematic representation of the blood coagulation cascade.

FIG. 2 is a flow chart showing the first part of the method of the present invention used to obtain platelet rich and platelet poor plasma.

FIG. 3 is a flow chart showing the last part of a method for preparing a bioadhesive sealant composition of the present invention using platelet- rich plasma as a starting material.

FIG. 4 is a flow chart showing the last part of a method for preparing a bioadhesive sealant composition of the present invention using platelet-poor plasma as a starting material.

FIG. 5 is a chart of the effect of serum-plasma ratio on clotting time.

6 is a chart showing the relationship between the actual gelation time and clotting time using blood collected from donors.

7 is a chart showing the relationship between the actual gelation time and clotting time using blood collected from donors.

FIG. 8 is a chart showing the effect of calcium addition on coagulation time and gelation time using blood collected from a donor.

FIG. 9 is a chart showing the effect of calcium addition on coagulation time and gelation time using blood collected from a donor.

FIG. 10 is a chart showing the effect of calcium addition on the clotting time of platelet rich and platelet poor plasma.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Figure 2

[Correction method] Change

[Contents of correction]

FIG. 2

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] Figure 3

[Correction method] Change

[Contents of correction]

FIG. 3

[Procedure amendment 4]

[Document name to be amended] Drawing

[Correction target item name] Fig. 4

[Correction method] Change

[Contents of correction]

FIG. 4

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventors Bo and Robert F. East Windcrest Row 7926 (72) Parker, Colorado 80134, United States of America Inventor Lim, Lisa M. Aurora, E., 80013, Colorado, United States. Mansfield Place 20472 (72) Inventor Johnston, Julie S. Apartment Canyon Dee Highlands, Red Canyon Drive 6274 (72) United States, Colorado, United States 6274 (72) Inventor Rivera, John G. Aurora, E., 80015, Colorado, United States. Leighton Place 15191 F-term (reference) 4C077 AA12 BB04 EE01 KK01 NN02 NN03 PP29 4C081 AC04 BA11 BA12 BA14 BA15 CD18 CD23 CE03 CF21 DA15 DC12

Claims (54)

[Claims] 1. A method for producing an autograft bioadhesive sealant from a single whole blood sample, comprising forming an inactive platelet-rich plasma from the whole blood sample; Dividing the plasma into first and second portions, reacting the first portion of the inactive platelet-rich plasma to form a clot, and obtaining a serum containing autologous thrombin. A method comprising crushing said clot and mixing said serum with said second portion of said inert platelet rich plasma. 2. The method of claim 1, wherein said platelet-rich plasma comprises sodium citrate. 3. The method of claim 1, wherein said inactivated platelet-rich plasma comprises heparin. 4. The method according to claim 2, wherein said reaction takes place by the addition of calcium ions.
The method described in. 5. The method according to claim 3, wherein the reaction occurs by adding heparinase. 6. The platelet-rich plasma containing sodium citrate is combined with a 9: 1 ratio of whole blood to a 3.8% sodium citrate solution to form a citrated blood mixture. 3. The method of claim 2, wherein the blood mixture is formed by centrifuging the citrated blood mixture to collect whole blood and to form a platelet-rich plasma containing sodium citrate. 7. The method of claim 1, wherein the platelet-rich plasma is obtained from an individual to whom the autograft bioadhesive sealant will be applied. 8. A fibrin sealant comprising human recombinant thromboplastin and platelet-rich plasma. 9. The fibrin sealant of claim 8, wherein said platelet-rich plasma is isolated from an individual to whom the fibrin sealant is to be applied. 10. The platelet-rich plasma obtained from the individual to whom the fibrin sealant is to be applied, the platelet-rich plasma, and thrombin are converted to the portion of the platelet-rich plasma to form a clot. A fibrin sealant comprising the thrombin which is isolated from a portion of the platelet-rich plasma by reacting and crushing the clot to form the thrombin-containing serum. 11. The method of claim 1, wherein said grinding comprises squeezing said clot through a mesh to separate said clot from said serum. 12. The method of claim 1, wherein said milling comprises high speed centrifugation to separate said clot from said serum. 13. The method of claim 1, further comprising adding calcium ions during said mixing of said second portion of said serum and said platelet-rich plasma. 14. The method of claim 1, wherein the serum and the second portion of the platelet-rich plasma are mixed in a ratio of serum to plasma-rich plasma of 1: 4. 15. The method of claim 1, wherein the serum and the second portion of the platelet-rich plasma are mixed at a ratio of serum to plasma-rich plasma of 1: 2. 16. The method of claim 1, wherein said serum and said second portion of said platelet rich plasma are mixed in a ratio of serum plasma to rich plasma of 3: 4. 17. A method for producing an autograft bioadhesive sealant from a single whole blood sample, comprising forming inactive platelet-rich plasma and inactive platelet-poor plasma from said whole blood sample. Reacting the inactive platelet-rich plasma to form a clot; crushing the clot to obtain serum containing autologous thrombin; and mixing the serum with the platelet-poor plasma A method that includes: 18. The method of claim 17, wherein said platelet rich plasma and said platelet poor plasma each comprise sodium citrate. 19. The method of claim 18, wherein said reaction occurs with the addition of calcium ions. 20. The method of claim 17, wherein said platelet rich plasma and said platelet poor plasma each comprise heparin. 21. The method of claim 20, wherein said reaction occurs upon the addition of heparinase. 22. The platelet-rich and platelet-poor plasma comprising sodium citrate is combined with a 9: 1 ratio of whole blood to a 3.8% sodium citrate solution forming a citrated blood mixture. 20. The method according to claim 18, wherein the whole blood is collected by centrifugation and centrifuging the citrated blood mixture to form platelet-rich and platelet-poor plasma, each containing sodium citrate. The method described in 23. The method of claim 17, wherein said whole blood is obtained from an individual to whom said autologous bioadhesive sealant is to be applied. 24. The method of claim 17, wherein squeezing the clot through a mesh to separate the clot from the serum is included in the crushing. 25. The method of claim 17, wherein said milling comprises high speed centrifugation to separate said clot from said serum. 26. The method of claim 17, wherein the serum and the platelet-poor plasma are mixed at a ratio of serum to platelet-rich plasma of 1: 4. 27. The method of claim 17, wherein said serum and said platelet-poor plasma are mixed in a ratio of serum to platelet-rich plasma of 1: 2. 28. The method of claim 17, wherein the serum and the platelet-poor plasma are mixed in a ratio of serum to platelet-rich plasma of 3: 4. 29. A method for producing an autograft bioadhesive sealant from a single whole blood sample, comprising forming inactive platelet-poor plasma from the whole blood sample; Separating the first portion of the inactive platelet-poor plasma to form a clot; crushing the clot to obtain a serum containing autologous thrombin And mixing the serum with the second portion of the inert platelet-poor plasma. 30. The method of claim 29, wherein said platelet poor plasma comprises sodium citrate. 31. The method of claim 30, wherein said reaction occurs with the addition of calcium ions. 32. The method of claim 32, wherein said platelet poor plasma comprises heparin. 33. The method of claim 29, wherein said reaction occurs with the addition of heparinase. 34. The platelet-poor plasma containing sodium citrate is collected at a 9: 1 ratio of whole blood to a 3.8% sodium citrate solution forming a citrated blood mixture. 31. The method of claim 30, wherein the mixture is formed by centrifuging a citrated blood mixture to form platelet-poor plasma containing sodium citrate. 35. The method of claim 29, wherein the whole blood sample is obtained from an individual to whom the autograft bioadhesive sealant will be applied. 36. The method of claim 29, wherein squeezing the clot through a mesh to separate the clot from the serum is included in the crushing. 37. The method of claim 29, wherein said milling comprises high speed centrifugation to separate said clot from said serum. 38. The method of claim 29, wherein the serum and the second portion of the platelet-poor plasma are mixed in a ratio of serum to platelet-rich plasma of 1: 4. 39. The method of claim 29, wherein the serum and the second portion of the platelet-poor plasma are mixed in a 1: 2 ratio of serum to platelet-rich plasma. 40. The method of claim 29, wherein the serum and the second portion of the platelet-poor plasma are mixed in a ratio of serum to platelet-rich plasma of 3: 4. 41. A method for producing an autograft bioadhesive sealant from a single whole blood sample, comprising forming inert platelet-rich plasma and inert platelet-poor plasma from said whole blood sample. Reacting the inactive platelet-poor plasma to form a clot; crushing the clot to obtain serum containing autologous thrombin; and mixing the serum with the platelet-rich plasma A method that includes: 42. The method of claim 41, wherein said platelet rich plasma and said platelet poor plasma each comprise sodium citrate. 43. The method of claim 42, wherein said reaction occurs with the addition of calcium ions. 44. The method of claim 41, wherein said platelet rich plasma and said platelet poor plasma each include heparin. 45. The method of claim 44, wherein said reaction occurs with the addition of heparinase. 46. The platelet-rich and platelet-poor plasma containing sodium citrate is combined with a 9: 1 ratio of whole blood to a 3.8% sodium citrate solution forming a citrated blood mixture. 43. The method of claim 42, wherein the whole blood is collected by centrifugation, and formed by centrifuging a citrated blood mixture to form platelet-rich and platelet-poor plasma, each containing sodium citrate. The method described in 47. The method of claim 41, wherein said whole blood is obtained from an individual to whom said autograft bioadhesive sealant is to be applied. 48. The method of claim 41, wherein squeezing the clot through a mesh to separate the clot from the serum is included in the crushing. 49. The method of claim 41, wherein said milling comprises high speed centrifugation to separate said clot from said serum. 50. The method of claim 41, wherein the serum and the platelet-poor plasma are mixed at a ratio of serum to platelet-rich plasma of 1: 4. 51. The method of claim 41, wherein said serum and said platelet-poor plasma are mixed in a ratio of serum to platelet-rich plasma of 1: 2. 52. The method of claim 41, wherein the serum and the platelet-poor plasma are mixed in a ratio of serum to platelet-rich plasma of 3: 4. 53. A method of producing an autograft bioadhesive sealant from a single whole blood sample, comprising forming an inactive platelet rich plasma from said whole blood sample, wherein said inactive platelet rich. Dividing the plasma into first and second portions; adding human recombinant thromboplastin to the first platelet-rich plasma portion to produce thrombin; and converting the thrombin to the second inactive portion. Mixing with the rich plasma portion of healthy platelets. 54. The method of claim 53, wherein the whole blood sample is obtained from an individual to whom the autograft bioadhesive sealant will be applied.