JPS6349510B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to blood coagulants or hemostatic agents.
More particularly, it relates to prepackaged bandages or absorbent pads saturated with blood clotting agents that can be effective in hemostasis at the site of trauma in a reliable and effective manner.

Treated bandages and patches have been devised in the past to help stop the flow of blood from trauma, cuts, scratches, and the like. A commonly employed method was to formulate a composition containing a hemostatic agent, with or without bactericidal or antiseptic agents, and to saturate a patch or piece of gauze over a bandage with the solution. . For example, Curtis U.S. Pat. No. 2,579,367 discloses that an aqueous solution or paste of proteins is applied to the area to be treated, and that following application of a protein blood clotting agent it forms an insoluble, semi-permeable scab. Discloses a bandage formulation that will change. The composition of the protein blood coagulant suggested in that patent consists of water-soluble salts of divalent zinc, manganese and cobalt and selected metal salts including in particular zinc acetate, cobalt sulfate and manganese sulfate. The composition is applied to surgical gauze and allowed to dry, and the gauze is then applied to the protein glue coating over the wound so that the glue reacts with the metal salts of the bandage. to form metal caseinates. Others have suggested the use of variously shaped bandages or patches treated with hemostatic agents. For example, Beardsley, US Patent No. 2,442,111;
No. 3,731,683; Edeinbaum No. 3,342,183; Shattner No. 3,317,376; and Anderson No. 3,328,259. however,
In the past, an effective method for preparing a prepackaged blood coagulant composition which, upon proper storage, can be used as a wound medicinal material and effectively works to convert fibrinogen in the blood to fibrin fragments has been described. No one has invented it.

The importance of acting directly on fibrinogen can best be appreciated by consideration of blood coagulation mechanisms. Generally speaking, unchanged clotting factors known as proenzymes are converted to active enzymes, which in turn can convert thousands of substrate molecules into active enzymes. This initiates a chain reaction by which activation of one proenzyme molecule will lead to activation of the entire blood coagulation mechanism. On the other hand, the body's fibrinolytic system works against the blood coagulation mechanism to maintain a balance between clot formation and clot dissolution. In the fibrinolytic system, a series of proenzymes are converted into enzymes that can dissolve blood clots when activated.
The lytic or degrading enzyme is called plasmin. However, many factors can occur that disrupt the equilibrium between clot formation and clot dissolution. In discussing the background of the present invention, it is not the purpose of discussing these factors that may interrupt the chain reaction or chain in blood coagulation. However, it is most desirable that the blood clotting activity be at some low point in the chain or chain reaction, so that the clotting mechanism will not be interrupted by other perturbing factors in the chain. And it is important to recognize that this should occur particularly at the site of the wound. This method will minimize the risk of blood clotting agents entering the blood and will result in the most rapid development of clotting and especially at the site of the wound.

Thrombin is a sterile protein that is prepared from prothrombin through interaction with thromboplastin, usually added in the presence of calcium. In concentrated form, it has a strong clotting effect on the blood and converts the same into fibrin clots, especially in clotting plasma fibrinogen. Traditionally, thrombin is mixed with trace amounts of other ingredients, such as sodium chloride, calcium chloride, glycine, and benzethonium chloride, which helps somewhat as a preservative when thrombin is stored in dry or powdered form. It is available in powdered form. It was once used as an absorbable gelatin sponge that was applied directly to the cut or injury in the form of a solution or after closure of a surgical incision and as a sponge that was gradually absorbed over a long period of time. It has been used in combination with other hemostatic agents. However, no method or formulation of thrombin has been satisfactorily devised by which it can be prepackaged, sealed, and stored in solution form for subsequent application to a wound. Seegers U.S. Patent No. 2,433,299
The purpose is to preserve thrombin by storage in a sucrose solution that can be sprayed or applied to the bleeding tissue along with physiological saline. However, even though this patent highlights some of the problems associated with thrombin storage, sucrose does not make it practically available as a prepackaged material for blood coagulation.

A significant difficulty that must be evaluated is providing a commercially portable product. The product must have a satisfactory shelf life under the temperature conditions to which the prepackaged plasma coagulation composition would be expected to be subjected.

It is therefore an object of the present invention to provide new and improved blood coagulant compositions and said compositions in prepackaged form for application to cuts or injuries, either internal or external to the body. The purpose of this invention is to provide a method for manufacturing the same.

Another object of the present invention is that when applied to a wound, it is capable of coagulating blood while providing protection against the entry of foreign particles and facilitating its removal from the skin without reopening the wound. An object of the present invention is to provide a new and improved bandage containing a blood coagulant that can be used.

A further object of the invention is that it is suitable for use in conjunction with absorbent pads or dressings, wound dressings, bandages and the like, and for future use in the treatment of injuries or cuts in a safe and reliable manner.
The object of the present invention is to provide a prepackaged thrombin solution that can be stored and stored for a long period of time.

For the purpose of both rapidly and effectively coagulating blood, prepackaged blood coagulant compositions and methods of making the same are devised in accordance with the present invention. Trauma dressings, bandages, absorbent patches and the like impregnated with blood coagulant solutions for use in producing superficial blood coagulation where needed, e.g. at the site of an injury or cut. Especially suitable. The compositions of the invention are comprised of a low but effective proportion of thrombin dissolved in saline with or without added preservatives.

The main preservatives are linear 3-6 fully saturated polyhydric alcohols: glycerol, mannitol, sorbitol. Desirably one or more sulfur-free essential amino acids are present as additional preservatives, and optionally polyethylene glycol, preferably having a molecular weight in the range of 2500 to 6000.

The sodium chloride content of the blood coagulant solution is nearly isotonic and prevents the rupture of red blood cells when applied to a lesion.

The bandage or gauze dressing is saturated with the resulting solution and, after being sealed, such as in a sealed container or sealable package, is stored at a cool temperature, preferably under refrigerated conditions, for maximum shelf life. In other words, it is stored at 2-8°C. The bandage, dressing, dressing, or the like can then be removed from its container or packaging and applied directly to the injury or cut without any special preparation or activation of the blood clotting agent composition.

In one preferred embodiment of the invention, thrombin is dissolved in a saline solution along with lysine, glycerol and polyethylene glycol. The conditioned dressings are saturated with the resulting mixture and individually sealed and stored for future use. In this embodiment, the shelf life and effectiveness of thrombin are significantly extended due to the combined preservative effects of glycerol, polyethylene glycol, and lysine.

In one modified embodiment, thrombin is combined with lysine and glycerol and introduced into a saline solution, thoroughly mixed or dissolved, and then immediately applied to the bandage or patch until completely saturated with the same. . The dressing is sealed by placing it in an overwrap or other sealable container and stored at a cool temperature to minimize evaporation of the solution and preserve the active state of the thrombin.

Hereinafter, specific embodiments of the present invention will be described with reference to the accompanying drawings.

According to the invention, a blood coagulant solution of 50 to 2000 units per ml, preferably 10 to 100 units per ml of thrombin is combined with a small amount of sulfur-free essential amino acids, polyhydroxy compounds, and optionally polyethylene glycol. A blood coagulant solution is prepared by blending together.

The saline blood coagulant solution will have a final concentration of sodium chloride on the order of 0.50% to 0.95% by weight.

As already noted, the dilute saline blood coagulant solution is isotonic or near isotonic to avoid unnecessary rupture of red blood cells at the site of injury.

The amino acids, which will preferably be introduced in solid form, are in a molar ratio of 0.01 to 0.25. For example, L-lysine would be employed at a concentration of 0.05 molar, or 0.91325 grams per 100 ml of blood coagulant solution.

One consideration regarding the practice of this invention is that some of the lesions to which the compositions of this invention may be applied are inside body cavities, ie, the oral cavity. BACKGROUND OF THE INVENTION Trauma dressings and dressings saturated with blood coagulant solutions may be well employed to control bleeding after tooth extractions or other oral surgery. Some of the blood coagulant solution from the wound dressing may be swallowed. Therefore, it is appropriate to employ preservatives that are generally recognized as safe in food, including in particular the use of only essential amino acids (those without sulfur moieties) in blood coagulant solutions. . As already pointed out, the amino acids are present in solution in a concentration of 0.01-0.025 molar.

A limited number of humectant compounds for the blood coagulant compositions of the present invention, namely linear _C3 - _C6 fully saturated polyhydric alcohols and hexacarbon sugar alcohols, particularly and preferably including glycerol. is planned. Mannitol, sorbitol, and mixtures thereof belong to the preferred polyhydric alcohols. Its polyhydric alcohol content is from 10 to 50% by weight of the blood coagulant solution. Glycerol, a desirable polyhydric alcohol, appears to impart anti-coagulant adhesion properties to the bandage, which allows for easier removal of the bandage from clots at the site of injury. When employing glycerol, its concentration may range from 10 to 50% by volume, with a preferred concentration being about 30% by volume of the blood coagulant solution.

The presence of polyethylene glycol is optional, but its addition is desirable. When added, polyethylene glycol is
It is 0.01 to 4.0%. Polyethylene glycols with molecular weights within the range of 2500-6000 are preferred.

Thus, for example, for the embodiment of a desirable blood coagulant solution, lysine up to a concentration of 0.05 molar,
and polyethylene glycol with a molecular weight of about 6000 is introduced at a level of 0.2 grams or 0.2% per 100 ml of physiological saline solution of the blood coagulant, along with 6500 international units of thrombin per 100 ml of the blood coagulant. Glycerol is added at a rate of about 30 parts by volume of the solution. In the resulting solution described above, lysine, glycerol, and polyethylene glycol each act as protein preservatives, and their combined use greatly preserves thrombin activity when applied to patches and other absorbent materials. It is becoming clear that the amount of electricity is increasing rapidly.

Thrombin in saline solution is stable over long periods of time if care is taken to maintain the solution between 2°C and 8°C. However, as a practical matter, it is extremely difficult to avoid the possibility that any batch of thrombin solution would be exposed to room temperature or higher temperatures. Inevitably, some shipments of blood coagulant products from manufacturing sites may be left in unrefrigerated trucks over holidays or weekends, or left in the sun for hours at a loading dock. . If the presence of heat-inactivated thrombin is not confirmed prior to use, failure of the dressing or wound dressing to induce blood clotting will be attributable to the manufacturer. The gist of the invention is to provide a thrombin solution that can withstand inactivation due to temperature conditions to which certain shipments are likely to be exposed.
In other words, one of the important objectives realized here is the preparation of blood coagulant compositions that remain active at ambient temperature for extended periods of time, ie, for several months.

Experimental results have shown that the primary stabilizing or preservative agent is a polyhydric alcohol wetting agent.
The amino acids stabilize thrombin to a lesser extent, but cumulatively. Although it is not clear whether polyethylene glycol contributes to thermal stability, the presence of polyethylene glycol is nevertheless considered desirable and preferred.

In any event, the compositions of the present invention, particularly in their preferred embodiments, have shown a shelf life of over 100 days at 37°C. At 37°C, thrombin solutions in saline become inactivated within a few days. The compositions of the present invention are suitable for distribution from the point of manufacture through normal commercial channels in the processed form of the present invention, such as absorbent dressings, wound dressings, bandages, and the like. Sufficiently (temperature-wise) stable.

To produce a blood coagulant composition, sodium chloride is dissolved at the desired concentration in sterile water (deionized or distilled) at room temperature, and then some of the saline solution is added to thrombin (usually in powdered form). It is used to pre-dissolve the The bulk of the saline solution and the entire amount of polyhydric alcohol used in the batch are mixed, followed by the thrombin solution. All of the amino acid (in powder form), ie lysine, and, if used, all of the polyethylene glycol (in solid form) are then added by weight relative to the (final) volume of solution and mixed until dissolved. Correction to the final volume of the coagulant composition is then made by adding further saline solution.

As illustrated in FIG. 1, the solution is then applied to bandage 10 so that it is sufficiently saturated. The dressing can, for example, consist of one or more layers of gauze strips numbered 12 enclosed within a porous plastic film 13, according to routine practice. It is. The bandage or patch consists of a foil lining 15 and a polyethylene film layer 1.
It is enclosed in a paper envelope 14 having a diameter of 6. Inner film layers 16 have heat-sealed borders 17 along their outlined edges, which are applied together to seal the bandage in place. Once packaged as described above, the dressing is stored at a cool temperature, preferably on the order of 2°C to 8°C. The steps involved in preparing the solution itself and as previously described are shown in FIG.

FIG. 3 is a graph showing the blood coagulation properties based on a stability test of a blood coagulant solution containing thrombin and various preservatives according to the present invention. The tests carried out showed that thrombin was present in the proportions described above, i.e. an amount of 50 to 2000 units of thrombin per milliliter of solution, and polyhydric alcohol at a level of 10 to 50 parts by weight of the coagulant solution;
This concerns the case of For example, if the proportion of glycerol in the blood coagulant solution exceeds 50 parts,
It tends to be very viscous or thick and appears to retard contact activation.
This means that thrombin becomes less accessible as a coagulant for fibrinogen. At amounts below 10 parts of glycerol in solution, glycerol loses its preservative effect.

As already pointed out, the amino acid range is 0.01
It is on the order of molar to 0.25 molar. Preferred amino acids are lysine, glycine, and isoleucine. Polyethylene glycol ranges from 0.01 to 4.0% by weight in its blood coagulant solution
It is.

In the following examples of alternative formulations of blood coagulant solutions, presented here to illustrate the practice of the invention and the results that can be obtained therefrom, relative proportions are expressed either in percentage concentrations or by weight by volume. It shows.

Experimental results obtained from blood coagulants prepared as described by Examples 3 to 3 are shown in FIG. The ratios and experimental conditions selected for the Examples were selected to present approximately idealized results of the illustrative results that can be obtained by practicing the present invention. Compositions of preferred embodiments are illustrated in the Examples.

EXAMPLE A blood coagulant solution containing the following ingredients was prepared. Thrombin 1500 units per 100 ml of ingredients Lysine 0.1 molar polyethylene glycol 0 Glycerol 30 parts by volume Physiological saline 0.95% NaCl concentration (equilibrium) Ingredients were thoroughly mixed as described and placed in a vial, accelerated stabilization It was stored at a temperature of 40° C. for the purpose of determining its stability by carrying out sexual experiments. From Figure 3, it can be seen that, based on accelerated stability experiments, after 6 days of storage at 40°C, thrombin in solution is stable with the ability to clot blood within 10 seconds. . The accelerated experiment is equivalent to a shelf life of 8 months at room temperature or 12 months at 4°C. The results of accelerated stability tests for the blood coagulant solutions of Examples are shown in FIG.

Example A blood coagulant solution was prepared by the following method. Thrombin 1500 units per 100 milliliters of ingredients Lysine 0.05 molar glycerol 30 parts (by volume) Polyethylene glycol 0 Physiological saline 0.95% NaCl concentration (equilibrium) Ingredients were thoroughly mixed as described and adsorbed in the solution. The agent patch was saturated and then packaged and sealed according to the method described for ease of use. In the absence of polyethylene glycol, lowering the amount of lysine appeared to decrease the stability and coagulability of the solution.

EXAMPLE A blood coagulant solution was prepared as follows. Thrombin 1500 units Lysine 0 Polyethylene glycol (molecular weight 6000) 0.2 g Glycerol 30 min (by volume) Physiological saline 0.95% NaCl concentration (equilibrium) Ingredients are thoroughly mixed as described and adsorbent A patch was saturated with the solution and then packaged and sealed for ease of use. In the absence of lysine, blood clotting time increased substantially, thereby showing a significant decrease in stability.

Example A blood coagulant solution was prepared by the following method. Thrombin 1500 units per 100 milliliters of ingredients Lysine 0.1 molar glycerol 30 parts (by volume) Polyethylene glycol 0.2 grams Saline 0.95% NaCl concentration (equilibrium) The ingredients are thoroughly mixed and absorbed as described. A patch of the agent was saturated with the solution and then packaged and sealed according to the method described for ease of use. Due to the presence of lysine and polyethylene glycol, stability was enhanced and shelf life was significantly extended.

Example A blood coagulant solution was prepared by the following method. Thrombin 1500 units per 100 milliliters of ingredients Lysine 0.05 molar glycerol 30 parts (by volume) Polyethylene glycol 0.2 grams per milliliter saline 0.9% NaCl concentration (equilibrium) As described, the ingredients were thoroughly mixed and the absorbent patch is saturated with the solution;
It was then packaged and sealed according to the method described for ease of use. Despite the reduced amount of lysine, the stability of the solution based on accelerated stability testing was excellent.

EXAMPLE A blood coagulant solution was prepared as described from the following ingredients. Thrombin 1500 units per 100 ml of ingredients Lysine 0.05 molar Glycerol None Polyethylene glycol 0.2 g/ml Physiological saline 0.90% NaCl concentration (equilibrium) The results of the accelerated aging experiment are shown as a curve in Figure 3 for the example blood coagulant. It was virtually the same as from solution.

EXAMPLES A prepared bandage of the type described above and shown in FIG. 1 was soaked in a blood coagulant solution of the composition disclosed in the Examples until completely saturated. By way of example only, for a bandage consisting of a Telhua patch measuring 5.08 cm x 7.62 cm x 0.1 cm:
Each component was present in the padding in the following amounts per cubic centimeter of padding material:

Thrombin ≧25.83 units/cm ³ Polyethylene glycol ≧
516 micrograms/cm ³ Glycerin ≧77 microliters/cm ³ Lysine (monohydrochloride) ≧2.36 milligrams/cm ³ The concentration per cm ³ mentioned above is based on the assumption that the concentration per cm 3 is evenly distributed in the Tarfua patch. be.

EXAMPLES Preferred formulations for the practice of this invention are disclosed below. The formulation is then prepared as described in the Examples and impregnated into a bandage or the like as described in the Examples. Ingredient ratio Water 70 parts by volume Glycerin 30 parts by volume L-lysine (monohydrochloride) 0.913 grams per 100 ml solution (0.05 M) Sodium chloride 0.9 grams/100 ml solution Polyethylene glycol 6000 Mw 0.2% grams per 100 ml solution *Bovine thrombin ( Perc Davis) 6500 international units per 100 ml of solution
It is 100-125 international units.

EXAMPLES Proportions of examples with various substitutions of mannitol and sorbitol for glycerin and isoleucine and glycine for L-lysine, and with the exclusion of polyethylene glycol and with the exclusion of amino acids, except as otherwise indicated. A series of studies were carried out at 2-8°C and 37°C.

The control system, thrombin in saline,
When measured by the thrombin clotting time test,
It was inactivated within about 1 day at 37℃, but at 2-8℃
There was no significant decrease in activity even after 75 days. All tests at 2-8°C in solutions outlined below confirmed the stability of thrombin in solution at low temperatures.

Saline solutions of thrombin with 0.05 M glycine, or isoleucine, or L-lysine, but without polyethylene glycol (PEG), or wetting agents, revealed some increase in stability at 37°C, and all were stable for at least 10 days without loss of activity, but after that they did not rapidly lose activity, and all solutions were completely deactivated within 40 days. Isoleucine and glycine performed somewhat better than L-lysine in this controlled test.

15% mannitol, or 30% glycerol,
or a saline solution of thrombin (no PEG, no amino acids) with 30% sorbitol.
They continued to exhibit thrombin activity for 40 days (sorbitol) and in some tests for 75 days (glycerin and mannitol) at 37°C. 0.2%
A similar solution containing PEG and 0.05M lysine also showed stability at both room temperature and 37°C for at least 40 days.

thrombin, 0.2% PEG, 30% glycerin, and 0.05M L-lysine or isoleucine,
A set of saline solutions containing either glycine or glycine exhibited clotting activity after storage for at least 120 days at room temperature and 37°C.

The thrombin employed in the foregoing examples is a proteinaceous substance of bovine origin, such as the type formulated and sold by Park Davis and Company of Detroit, Michigan. Its commercial products include thrombin powder, which contains 10,000 units of thrombin, approximately 180 to 200 milligrams;
17.4 milligrams of sodium chloride, 15.6 milligrams of calcium chloride, 19 milligrams of glycine,
and 0.2 milligrams of benzethonium chloride.

After manufacturing including sealing as described,
The resulting saturated patch can be shipped at room temperature, although it is advisable to store it under refrigerated conditions in order to extend the lifetime of the solution, and in particular to preserve the activity of thrombin as long as possible. In any case, the effectiveness of the blood coagulant solution can be determined upon opening the package. If the solution is dry, thrombin will not act effectively as a coagulant.

The diagram in FIG. 3 shows the examples, and the significant increase in thrombin shelf life when formulated in accordance with the examples. Accelerated testing at 40℃
The above formulation has a shelf life of at least 8 months at room temperature (+22°C) or at refrigeration temperature (2°C to 8°C).
It has been shown to give a stability of thrombin that is equivalent to a shelf life of at least 12 months at temperatures (°C). Accelerated testing at 40°C also demonstrated that the stability of the thrombin molecule was cumulatively enhanced by all three components: glycerol or glycerin, propylene glycol (PEG), and sulfur-free essential amino acids. was also shown. If the optional PEG is removed, the content of amino acids in the formulation should be at least 2 times that of that in the desired formulation to maintain adequate stability of thrombin in the absence of polyethylene glycol.
should be doubled (from 0.05 molar to 0.1 molar).

From the foregoing, it can be seen that the prepackaged proteinaceous blood coagulant solution as described is not only an extremely effective method and means of blood coagulation when applied to lesions, but also when it is necessary for individual use. This avoids the time consuming and costly incorporation and preparation of blood clotting agents each time. On the other hand, the shelf life of the blood coagulant solutions described here is sufficient to guarantee effectiveness after long storage periods. When applied to lesions, even when present in small amounts in dilute solutions, thrombin can act very low in the blood coagulation chain, as long as the patient has normal circulating fibrinogen.

Although a prepared bandage and a particular method of constructing it have been described, it will be appreciated that various modifications and changes may be made without departing from the spirit and scope of the invention.

[Brief explanation of the drawing]

FIG. 1 is a perspective view of a prepared bandage embodying the present invention in a sealable outer package; FIG. 2 is a perspective view of the preparation of a preferred form of prepackaged blood coagulant according to the present invention; is a flow diagram followed in;
and FIG. 3 is a graphical diagram illustrating the relative stability of coagulant solutions and coagulant times prepared in accordance with the present invention. (Explanation of symbols), 10: Bandage, 12: Gauze,
13: Plastic film, 14: Outer packaging, 1
5: Inner burr, 16: Inner layer, 17: Boundary.

Claims (1)

[Claims] 1. Sodium chloride in an approximately isotonic amount from 10 to
50−2000 per ml of aqueous solution containing 50% by weight long-chain 3 to 6 carbon fully saturated polyhydric alcohols
A blood clotting agent suitable for use as a surgical medicinal material for injuries, consisting of a solution of units of thrombin. 2. A blood coagulant according to item 1 above, which also contains in its solution a non-sulfur-containing amino acid in a molar concentration ratio of 0.01-0.25. 3. The blood coagulant composition according to item 2 above, wherein the amino acid is lysine. 4. The blood coagulant composition according to item 2 above, wherein the solution contains a mixture of glycerol and lysine. 5 In the above item 2, the solution is 2500-6000
A blood coagulant containing 0.1-4.0% by weight of polyethylene glycol with a molecular weight in the range of . 6. The blood coagulant composition according to item 5 above, wherein the solution contains glycerol, lysine, and polyethylene glycol. 7. The blood coagulant composition of paragraph 1 above, wherein glycerol is present as about 30 parts by volume of the solution. 8 Prepackaged blood coagulation devices applicable as wound dressings, surgical medical materials, etc.
a solution of thrombin containing sodium chloride in approximately isotonic proportions with 10-50% by weight of a linear, fully saturated polyhydric alcohol having 3 to 6 carbon atoms, and an absorbent substance saturated by said solution; A blood coagulation device comprising encapsulation means for hermetically encapsulating said saturated absorbent substance. 9. The blood coagulation device of clause 8, wherein the thrombin is present in a range of 25 to 250 units per cc of absorbent material. 10 In the above item 8, the blood is 0.01 to
A blood coagulation device containing essential amino acids in a molar concentration ratio of 0.25 that does not contain sulfur. 11. The blood coagulation device according to item 10 above, wherein the solution contains lysine.