KR20040050169A - Method of making fibrinogen particles suited for intravenous injections - Google Patents

Abstract

PURPOSE: A method of making fibrinogen particles suited for intravenous injections is provided. The fibrinogen particles are useful in significantly reducing the amount of blood consumed in surgery and drug therapy. CONSTITUTION: The method of making fibrinogen particles suited for intravenous injections comprises the steps of: (a) dissolving protein powder in a buffer solution with a suitable mole osmotic pressure concentration; (b) adding a surfactant selected from tetradecyl sodium sulfate, Tween-80 and Triton X-151 into the protein solution; (c) adding a crosslinking agent solution containing covalently bound micromolecules such as glutaraldehyde into the mixture of protein and surfactant; and (d) adding alcohol into the mixture of protein, surfactant and crosslinking agent, wherein the protein solution is human serum albumin solution; the surfactant is detergent; the buffer has pH 4 to 8; the mole osmotic pressure concentration of the buffer solution is 14 to 680 mOsm; and the alcohol is ethanol.

Description

Method of making fibrinogen particles suited for intravenous injections

FIELD OF THE INVENTION The present invention relates to a method for producing synthetic protein particles that are used to significantly reduce the blood consumption of a patient, especially during surgical and drug therapy, and to help fast recovery with such surgical and drug therapy, and especially fibrinogen suitable for intravenous injection. It relates to a method for producing the particles.

It is known that many surgical and drug measures, such as cancer surgery, often result in a large amount of blood consumption that can put the patient at risk of death, resulting in a lack of platelets in the patient's blood. One solution to blood loss is to use blood transfusions. However, blood transfusions can introduce fatal diseases such as HIV virus or hepatitis virus. Therefore, in the field of clinical medicine, there is a strong demand for providing a substance useful for blood aggregation or functioning substantially as a platelet of human blood. Such materials must meet the following requirements. First, the particle size of these materials should be as small as possible so that they can enter the human capillary network. The maximum diameter of these materials cannot, on average, exceed 7 μm, the diameter of human capillaries. Second, these substances should not themselves aggregate or initiate blood aggregation of blood cells in human blood, but should promote blood aggregation at the wound site. In particular, such materials should have the ability to engage in or promote clotting at the wound site.

Microspheres made from proteins are disclosed, which are synthesized by emulsifying aqueous protein solutions in oil, followed by heat treatment or extensive polymerization. [K.J. Widder et al. in "Magnetically Responsive Microspheres and Other Carriers for the Biophysical Targeting of Antitumor Agents", Advances in Pharmacology and Chemotherapy, vol. 6, pp. 213-271]. However, the lifespan of the microspheres prepared according to the method of Wither is short because proteins cause undesirable denaturation reactions in the preparation of such microspheres. In practice, the synthesized platelet-shaped particles should be preserved in human blood vessels for at least one day.

A method for preparing particles comprising a cross-linked matrix of naturally occurring polymer particles is disclosed [Oppenheim et al., In US Pat. No. 4,107,288 entitled "Injectable compositions, nanoparticles useful therein, and process of manufacturing same "]. In this method, the protein solution was brought to the desolvation limit by adjusting the concentration of the desolvator with a contrasting resolubilization agent. This process starts in an unstable state and continues with a wide range of polymerization reactions, which are not controlled for the degree of aggregation and form spheres. Aggregates larger than 7 microns in diameter can clot blood vessels and are therefore dangerous for intravascular applications. Thus, particles that do not aggregate and are considerably smaller than 7 microns in diameter are needed.

It is a primary object of the present invention to provide a method of making fibrinogen particles suitable for intravenous injection.

According to the invention, the microspheres formed in the presence of a crosslinker have an average diameter of less than 1 micron.

According to the present invention, fibrinogen can be used as a target molecule and can bring protein particles to the wound site, thus reducing blood consumption.

According to the invention, the synthesized material acts as a platelet of human blood. The synthesized materials of the invention are also called fibrinoplates or platelet aggregation substitutes.

According to the invention, the composition of the material is as follows (parts by weight):

Human albumin: 90-100 parts

Fibrinogen: 0.02 to 6 parts

Materials of the invention are prepared according to the following procedure:

1. Preparation of protein particle suspension is:

a. Dissolving the protein powder in a buffer solution with an appropriate molar osmolarity;

b. A surfactant is added to the preparation solution of step a;

c. Crosslinker is added to the mixed solution of step b;

d. Adding the alcoholic solution to the mixed solution of step c.

Thus, nebulous protein particle suspensions are prepared which are suitable for intravenous injection and which do not clot blood vessels.

According to the invention, the protein solution is a human albumin solution.

According to the invention, the surfactant is either a cleaning agent or a cleaning agent.

According to the invention, the surfactant may be selected from the group consisting of sodium tetradecyl sulfate, Tween-80 and Triton X-151.

According to the present invention, the crosslinker is a covalent small molecule such as glutaraldehyde.

According to the invention, the pH of the buffer solution is 4-8.

According to the present invention, the molar osmolarity is 14-680 mOsm.

2. After preparing the nebula protein particle suspension, the surface protein is added to the suspension. In so doing, the unreacted portion of the microspheres will convert surface proteins to fibrinogen by covalent bonds.

3. The human albumin sphere suspension containing the surface protein is then in powder form by dilution, filtration, dialysis, diafiltration, centrifugation, electrophoresis, column chromatography, vacuum freeze drying, and powder reconstitution. Is treated as. In some cases, as is known in the art, addition or change of buffer solution or addition of preservatives is required.

Accordingly, the present invention provides a method for preparing fibrinogen particles suitable for intravenous injection, wherein the composition of the protein particle suspension does not clot blood vessels but aggregates at the wound site, and the protein particles formed are human albumin crosslinks having an average diameter of less than 7 μm. It is made of fibrinogen.

The present invention relates to frozen protein particle compositions reconstituted by bio-soluble liquids and suitable for intravenous injection. The protein composition of the present invention does not clog blood vessels (except for wound sites). The resulting protein composition particles are formed of human albumin crosslinked fibrinogen with an average diameter of less than 7 μm.

The present invention relates to suspensions of protein particles suitable for intravenous injection. The protein composition of the present invention does not clog blood vessels (except for wound sites). The resulting protein composition is formed on the sphere surface from human albumin crosslinked fibrinogen by covalent bonds. The resulting protein particles have an average diameter of less than 7 μm.

The present invention relates to frozen protein particle compositions that are reconstituted with biosoluble liquids and suitable for intravenous injection. The protein composition of the present invention does not clog blood vessels (except for wound sites). The resulting protein particle composition is formed on the sphere surface from human albumin crosslinked fibrinogen by covalent bonds, and the fibrinogen encapsulates human albumin. The resulting protein particles have an average diameter of less than 7 μm.

According to another aspect of the invention, the invention provides a new utility of such protein particles into the human body via intravenous injection. Protein particles can migrate to new wounds and promote aggregation, thereby reducing blood consumption.

In addition, the protein particles of the present invention can be used in trauma patients at risk of high blood consumption.

Other objects, advantages and novel features of the invention will become more readily apparent from the following detailed description set forth in connection with the accompanying drawings.

The invention is illustrated by the following examples, but is not limited thereto. Although the following experiment uses human serum albumin (HSA) as the starting material, it can be seen that a number of common human proteins such as immunoglobulins, myoglobin, collagen, or gelatin can be used as components of the spheres.

1. Experiment 1: Formation of spheres containing no fibrinogen. Powdered human serum albumin (HSA) is dissolved in standard saline (NS, or 0.9% sodium chloride solution) and as a surfactant sodium tetradecyl sulfate or tween-80, or Triton X, in the concentration range of 0.05 mg / ml to 15 mg / ml A solution of 5% to 20% (weight per volume) containing -151 degrees is formed. For a series of tubes containing 2 ml of protein solution containing various concentrations of surfactant, 2 ml of glutaraldehyde diluted with standard saline in a concentration range of 0.01% to 3.0% is added to the protein solution. After 10 minutes, about 10 ml of ethanol (diluted to about 80% in water) is added. Observe turbidity immediately. After 10 minutes, the turbid suspension was diluted to 5 volumes with brine to see if it was redissolved, but did not dissolve. Thereafter, the particles are observed by x1000 retardation microscopy.

It was found that the average diameter of the spheres was less than 1 micron.

It was also found that 1 to 2.5 mg / ml Triton X-151 with an HSA concentration of 100 to 200 mg / ml gave the best formulation. It can be seen that sodium tetradecyl sulfate in the range of 1-12 mg / ml produces the most uniform formulation in terms of size distribution.

2. Experiment 2: binding the target reagent, fibrinogen, to the protein spheres. Radiolabeled human serum albumin (using iodine-125) is used to prepare spheres using the concentration of Experiment 1. After centrifugation to precipitate the spheres, it was found that at least 95% of the protein was incorporated into the spheres, as measured by the radiation coefficient of the pellet to the supernatant. Increasing the molar osmolarity of the solution to 1.8% sodium chloride solution (twice the standard brine) or to 0.09% sodium chloride solution did not significantly affect the yield of the spheres. Reducing the pH of the buffer to less than 4 required alcohol above pH 8 to form spheres and the yield was slightly reduced at pH below 4.

The experiment was repeated to obtain optimal conditions, but human serum albumin labeled with fluorescein isothiocyanate was used. Using the optimal conditions as listed in Experiment 1, after the appearance of turbidity, fibrinogen (1 mg / ml) is added to the sphere suspension several times. It was found that the spheres were stable and fibrinogen was added within 15 minutes after the spheres were formed to obtain the best formulation.

To obtain a physiologically suitable suspension which can be administered immediately to a subject in need, dilution, filtration, dialysis, diafiltration, centrifugation, electrophoresis, column chromatography, addition or change of buffer or preservative, freeze drying, and Further treatment, such as reconstitution of the lyophilized powder with an aqueous fluid (water or standard brine or lactic acid-added Ringer's solution or sugar-containing solution), would be desirable for specific suspensions.

According to McGill et al., "Platelet membrane vesicles reduced microvascular bleeding times in thrombocytopenic rabbits, J. Lab Clin Med1987; 109: 127-33", rabbits are platelet reduced. Busulfan 70 mg / After kg treatment, the typical bleeding time of 10 rabbits was 597 s. Simultaneously with the injection of 1 mg / kg of fibrinogen-coated spheres (reconstituted from freeze dried powder to standard saline), the bleeding time was reduced to 115 s Standard deviation 43 sec) Fluorescence microscopic examination of the ear wound site revealed fluorescence particles in the clot, and a fluorescent sphere containing no fibrinogen or containing standard saline or fibrinogen solution (1 mg / ml). Infusion into platelet-reduced rabbits from which fibrinogen molecules act as an automatic inducer to bring the particles to the site of active clot formation, improving bleeding time and lowering blood consumption It can be seen that.

It can also be seen that these particles are effective for trauma or surgical patients that do not reduce platelets but can benefit from fast clotting times. It is found to be particularly useful when a large number of patients require treatment, such as in battlefield conditions or after nuclear war events, but platelet donors are not difficult to find.

Various changes and modifications will be made by those skilled in the art. Accordingly, the above description should be construed as being limited only within the limits and scope of the claims of the present invention.

Claims (19)

a. Dissolving the protein powder in a buffer of appropriate molar osmolarity; b. A surfactant selected from the group consisting of sodium tetradecyl sulfate, Tween-80 and Triton X-151 is added to the protein solution; c. Adding a crosslinker solution containing a covalent small molecule such as glutaraldehyde to a mixture of a protein and a surfactant; d. A process for producing protein particles suitable for intravenous injection, characterized in that alcohol is added to a mixture of protein, surfactant and crosslinker solution. The method of claim 1, wherein the protein solution is human serum albumin solution. The method of claim 1, wherein the surfactant is a detergent. The method of claim 1, wherein the pH of the buffer is 4 to 8. The method for preparing protein particles suitable for intravenous injection according to claim 1, wherein the solution has a molar osmolarity of 14 to 680 mOsm. 10. The method of claim 1, wherein the alcohol is ethanol. The method of claim 1, wherein the particles in suspension are given in powder form. 8. The method of claim 7, wherein the further treatment comprises filtration, drying and reconstitution. The method of claim 1, further comprising adding a surface protein to the sphere suspension. 10. The method of claim 9, wherein the surface protein is human fibrinogen. 10. The method of claim 9, wherein the protein solution is human serum albumin solution. 10. The method of claim 9, wherein the surfactant is a detergent. 13. The method of claim 12, wherein the surfactant is selected from the group consisting of sodium tetradecyl sulfate, Tween-80 and Triton X-151. 10. The method of claim 9, wherein the crosslinker molecule is a divalent reactive small molecule comprising glutaraldehyde. 10. The method of claim 9, wherein the alcohol is ethanol. 10. The method of claim 9, wherein the pH of the buffer solution is between 4 and 8. 10. The method of claim 9, wherein the solution has a molar osmolarity of 14 to 680 mOsm. 10. The method of claim 9, wherein the particles in suspension are given in powder form. 10. The method of claim 9, wherein the further treatment comprises filtration, drying and reconstitution.