WO2008030964A2

WO2008030964A2 - Use of noncalcium zeolites with added calcium salt in hemostatic devices and products

Info

Publication number: WO2008030964A2
Application number: PCT/US2007/077763
Authority: WO
Inventors: Robert L. Bedard
Original assignee: Honeywell International, Inc.
Priority date: 2006-09-08
Filing date: 2007-09-06
Publication date: 2008-03-13
Also published as: CN101677848A; EP2059187A2; WO2008030964A3

Abstract

It is known that calcium exchanged zeolites are effective in hemostasis. The reason that Ca-exchanged zeolites are required has not been explained in the prior art, except that the presence of Ca2+ ions are important in the clotting mechanism. It has now been found that the addition of Ca2+ ions alone actually slow clotting of blood. The use of Ca-zeolites appear to be effective in hemostasis simply because they don't remove the critical Ca2+ from the blood by ion exchange. A NaA zeolite with an effective amount of added Ca2+ salt is nearly equally active in coagulation as the Ca-exchanged form of CaA zeolite.

Description

USE OF NONCALCIUM ZEOLITES WITH ADDED CALCIUM SALT IN HEMOSTATIC DEVICES AND PRODUCTS

FIELD OF THE INVENTION

[0001] The present invention relates to blood clotting agents/medical devices and methods of controlling bleeding in animals and humans. More particularly, the present invention relates to the effectiveness of zeolites that have not been ion exchanged with calcium in promoting blood clotting provided that calcium ions are separately added along with the zeolites to the wound.

BACKGROUND OF THE INVENTION

[0002] Blood is a liquid tissue that includes red cells, white cells, corpuscles, and platelets dispersed in a liquid phase. The liquid phase is plasma, which includes acids, lipids, solubilized electrolytes, and proteins. The proteins are suspended in the liquid phase and can be separated out of the liquid phase by any of a variety of methods such as filtration, centrifugation, electrophoresis, and immunochemical techniques. One particular protein suspended in the liquid phase is fibrinogen. When bleeding occurs, the fibrinogen reacts with water and thrombin (an enzyme) to form fibrin, which is insoluble in blood and polymerizes to form clots.

[0003] In a wide variety of circumstances, animals, including humans, can be wounded. Often bleeding is associated with such wounds. In some instances, the wound and the bleeding are minor, and normal blood clotting functions without significant outside aid in stopping the bleeding. Unfortunately, in other circumstances, substantial bleeding can occur. These situations usually require specialized equipment and materials as well as personnel trained to administer appropriate aid. If such aid is not readily available, excessive blood loss can occur. When bleeding is severe, sometimes the immediate availability of equipment and trained personnel is still insufficient to stanch the flow of blood in a timely manner.

Moreover, severe wounds can be inflicted in very remote areas or in situations, such as on a battlefield, where adequate medical assistance is not immediately available. In these instances, it is important to stop bleeding, even in less severe wounds, long enough to allow the injured person or animal to receive medical attention. [0004] In an effort to address the above-described problems, materials have been developed for controlling excessive bleeding in situations where conventional aid is unavailable or less than optimally effective. Although these materials have been shown to be somewhat successful, they are not effective enough for traumatic wounds and tend to be expensive. Furthermore, these materials are sometimes ineffective in all situations and can be difficult to apply as well as remove from a wound. Additionally, or alternatively, they can produce undesirable side effects.

[0005] Compositions for promoting the formation of clots in blood have also been developed. Such compositions generally comprise zeolites and binders. [0006] The use of activated zeolites (a dehydrated material) was disclosed by Hursey et al. in US 4,822,349. It was recognized that the use of these activated zeolites in the clotting of blood generated heat and Hursey et al. stated that the heat was important in achieving a cauterization effect as well as increasing coagulation of the blood. In US 2005/0074505 Al, there is described the use of a zeolite that is exchanged with calcium ions to a very high level. The application states that "[T]he zeolite used for a blood clotting composition of the present invention includes an adjusted calcium content such that the calcium content is up to 83 wt-% calcium and preferably 75 wt-% to 83 wt-% calcium." An individual skilled in the art would interpret this patent to mean that the zeolites used were 75% and 83% exchanged with calcium (with reference to 100% of the cation exchange sites) rather than having the weight percentages of calcium indicated, since the bulk of the weight of the zeolites are silicon and aluminum by definition. It would be impossible for the weight percentage of the zeolite to be at the levels disclosed in the patent.

[0007] Calcium-exchanged zeolites do not function as hemostatic materials by release of Ca²⁺ into the blood. They appear to function by surface activation of the intrinsic coagulation pathway. The calcium form of a zeolite can be effective because it does not remove Ca²⁺ by ion exchange from the blood during the activation process. Surprisingly, it has now been found that hemostatic products can be made from zeolites without a Ca ⁺ exchange step; provided that an effective amount of water soluble calcium salt is added to the zeolite as it is formulated into the hemostatic product. Much faster clotting has been found than is present with either the calcium salt or the noncalcium exchanged zeolite when used independently. In fact, the use of Na- zeolites alone have an anticoagulant effect on blood because they remove enough Ca ⁺ to stop the progression of the clotting pathway in spite of the high probability that the Na-zeolite surface is efficiently activating the initial phase of intrinsic hemostasis. This initial activation cannot progress further in the case of Na-zeolite alone because of a concurrent Ca²⁺ ion exchange process in which the zeolite is removing Ca²⁺ from the blood, therefore making Ca²⁺ unavailable for further progression of the intrinsic pathway.

SUMMARY OF THE INVENTION

[0008] Currently, calcium-exchanged zeolite A is being sold in an activated form as a hemostatic treatment for hemorrhages. The market at this time is primarily military, with substantial business being generated by the wars in Afghanistan and Iraq. Previously, it had been considered necessary for the zeolites to be calcium exchanged since calcium has been found to be an important element in promoting the functioning of some of the factors involved in blood clotting. It has now been found that it is not necessary for zeolites to undergo a cation exchange step in order to be effective in accelerating hemostasis. Noncalcium-exchanged zeolites have been found to accelerate blood clotting substantially as effectively as partially or fully dehydrated forms of calcium-exchanged zeolites when they are applied together with a calcium salt. These noncalcium exchanged zeolites can include sodium zeolites, potassium zeolites, lithium zeolites, magnesium zeolites and combinations thereof. The calcium salt is of low toxicity with noncoordinating anions and with solubility greater than 2 grams per 100 cc in 20⁰C water. Useful calcium salts that can be used include calcium nitrate, calcium acetate, calcium bromide, calcium iodide, calcium nitrate and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0009] It has been believed in the art that the use of Ca-exchanged zeolites in hemostasis actually accelerates blood clotting because the material releases Ca²⁺ to some extent, and because of the critical role of calcium in the activity of many of the blood clotting enzymes and cofactors. However, thromboelastographic data shows that the addition of Ca²⁺ solutions to blood actually slow clotting. The following protocol was used to test the blood samples. [0010] The apparatus that was used was a TEG® analyzer from Haemoscope Corp. of Morton Grove, Illinois. This apparatus measures the time until initial fibrin formation, the kinetics of the initial fibrin clot to reach maximum strength and the ultimate strength and stability of the fibrin clot and therefore its ability to do the work of hemostasis — to mechanically impede hemorrhage without permitting inappropriate thrombosis. On unactivated samples: i. Pipet 360 uL from red topped tube into cup, start TEG test On activated samples: i. First, obtain the zeolite or other powder sample to be tested from lab. They should be weighed, bottled, oven activated (if needed), and capped prior to the start of the experiment. Zeolite samples are bottled in twice the amount that need to be tested. For example, if channel two is to test 5 mg of zeolite A and blood, the amount weighed out in the bottle for channel two will be 10 mg. For 10 mg samples, 20 mg is weighed out, etc. See note below for reason. ii. For one activated run, 3 zeolite samples were tested at a time. An unactivated blood sample with no additive is run in the first channel. Channels 2, 3 and 4 are blood samples contacted with zeolite. iii. Once ready to test, set one pipet to 720 uL and other pipet to 360 uL. Prepare three red capped tubes (plain polypropylene-lined tubes without added chemicals) to draw blood and prepare three red additional capped tubes to pour zeolite sample into, iv. Draw blood from volunteer and bring back to TEG analyzer. Discard the first tube collected to minimize tissue factor contamination of blood samples. Blood samples were contacted with zeolite material and running in TEG machine prior to an elapsed time of 4-5 minutes from donor collection. v. Open bottle 1 and pour zeolite into red capped tube, vi. Immediately add 720 uL of blood to zeolite in tube, vii. Invert 5 times. viii. Pipet 360 uL of blood and zeolite mixture into cup. ix. Start TEG test.

[0011] Note: The proportions are doubled for the initial mixing of blood and zeolite because some volume of blood is lost to the sides of the vials, and some samples absorb blood. Using double the volume ensures that there is at least 360 uL of blood to pipet into cup. The proportion of zeolite to blood that we are looking at is usually 5mg/360uL, 10mg/360uL, and 30mg/360uL [0012] The R(min) reported in the Tables below is the time from the start of the experiment to the initial formation of the blood clot as reported by the TEG analyzer. The TEG® analyzer has a sample cup that oscillates back and forth constantly at a set speed through an arc of 4°45'. Each rotation lasts ten seconds. A whole blood sample of 360 ul is placed into the cup, and a stationary pin attached to a torsion wire is immersed into the blood. When the first fibrin forms, it begins to bind the cup and pin, causing the pin to oscillate in phase with the clot. The acceleration of the movement of the pin is a function of the kinetics of clot development. The torque of the rotating cup is transmitted to the immersed pin only after fibrin-platelet bonding has linked the cup and pin together. The strength of these fibrin- platelet bonds affects the magnitude of the pin motion, such that strong clots move the pin directly in phase with the cup motion. Thus, the magnitude of the output is directly related to the strength of the formed clot. As the clot retracts or lyses, these bonds are broken and the transfer of cup motion is diminished. The rotation movement of the pin is converted by a mechanical-electrical transducer to an electrical signal which can be monitored by a computer.

[0013] The resulting hemostasis profile is a measure of the time it takes for the first fibrin strand to be formed, the kinetics of clot formation, the strength of the clot (in shear elasticity units of dyn/cm^) and dissolution of clot. [0014] The tables below shows clotting data from unadulterated human blood as well as from the same blood with 5 and 10 μL of 0.2M CaCl₂ and in another experiment using a second unadulterated blood sample and with 20, 30, and 40 μL of 0.2M CaCl₂ added (each time the total volume was 360 μL). It is clear that the clotting time [R(min)] increases with increasing quantities of the Ca²⁺ salt.

TABLE l Procedure Name R(min)

Run 5 Blood 18.2

Run 5 Blood with 5μL CaCl₂ 19.5

Run 5 Blood with lOμL CaCl₂ 21.2

Run 4 Blood 25.8

Run 4 Blood with 20μL CaCl₂ 33.4

Run 4 Blood with 30μL CaCl₂ 37.8

Run 4 Blood with 40μL CaCl₂ 47.7 [0015] If fully dehydrated Na zeolite A, 10 mg (~7xlO^~5 exchange equivalents) and an effective amount of CaCl₂ (35μL of 0.2 M; 1.4xlO^~5 equivalents) is added to the blood, the clotting time is significantly shortened over the unadulterated clotting times of runs 3 and 8 below. The standard experimental procedure was varied for results shown in Table 2. The quantities used were 760 microliters of blood; 10 mg hydrated NaA, plus 35 microliters of 0.2 M CaCl₂.

TABLE 2 Procedure Name R(min)

Run 8 CaCl₂ doped NaA zeolite 3.8

Run 8 CaCl₂ doped NaA zeolite 4.3

Run 3 CaCl₂ doped NaA zeolite 2.1

Run 3 CaCl₂ doped NaA zeolite 2.0

Run 8 Blood (control) 24.3

Run 3 B lood (control) 21.8

[0016] The clotting times for activated CaA zeolite as tested are given below. It is clear that the clotting times of the NaA zeolite doped with CaCl₂ are slightly longer than the Ca- exchanged A zeolite but they are still quite short. In these examples, the quantities used were 760 microliters of blood; 10 mg hydrated NaA, plus 35 microliters of 0.2 M CaCl₂.

TABLE 3

Procedure Name R(mir

Run 5 CaA zeolitelO mg 1.5

Run 5 CaA zeolite 10 mg 1.5

Run 5 CaA zeolite 25 mg 1.8

Run 6 CaA zeolite 5 mg 2.2

Run 6 CaA zeolite 10 mg 1.0

Run 6 CaA zeolite 30 mg 0.8

Run 6 CaA zeolite 5 mg 2.2

Run 6 CaA zeolite 10 mg 1.9

Run 6 CaA zeolite 30 mg 1.2

Run 3 Blood (control) 23.9

Run 3 Blood (control) 26.0

Run 5 Blood (control) 28.4

Run 6 Blood (control) 21.4

Run 6 Blood (control) 27.2

[0017] An experiment was also run to compare Hydrated ZB-100 (Na 4A) with 35uL of 0.2M CaCl₂: The runs testing the blood of two different individuals yielded quite similar results Individual # 1 : Blood (control) - R = 24.8

5mg 2.8

5mg 2.6

Individual #2: Blood (control)- R = 25.8

5mg 2.8 5mg 2.2

5mg 2.5

[0018] Zeolite powders that have not been ion exchanged with calcium have been found to be effective hemostats provided that a source of calcium is provided with the application of the zeolite. The clotting times were slightly higher than with calcium exchanged zeolites, but still provided a significant acceleration in the clotting process. In general these noncalcium exchanged zeolite promote blood clotting at a rate 2 to 12 times faster than in its absence. These noncalcium exchanged zeolites promote blood clotting in less than 10 minutes and preferably these noncalcium exchanged zeolite promotes blood clotting in less than 5 minutes.

[0019] These zeolite powders may be combined with a binder such as clay, alumina or silica. The zeolite powder that is functioning as a blood clot promoter may be contained within a porous carrier such as woven fibrous articles, non-woven fibrous articles, puffs, sponges and mixtures thereof. Fibers used to make such woven or non-woven fibrous articles may include aramids, acrylics, cellulose, polyester, chemically modified cellulose fibers and mixtures thereof. These zeolite powders can be used as free flowing powders or incorporated into a bandage, gauze or other formed product for treatment of wounds. [0020] Various materials may be mixed with, associated with, or incorporated into the zeolites to maintain an antiseptic environment at the wound site or to provide functions that are supplemental to the clotting functions of the zeolites. Exemplary materials that can be used include, but are not limited to, pharmaceutically-active compositions such as antibiotics, antifungal agents, antimicrobial agents, anti-inflammatory agents, analgesics (e.g., cimetidine, chloropheniramine maleate, diphenhydramine hydrochloride, and promethazine hydrochloride), bacteriostatics, compounds containing silver ions, and the like. Other materials that can be incorporated to provide additional hemostatic functions include ascorbic acid, tranexamic acid, rutin, and thrombin. Botanical agents having desirable effects on the wound site may also be added. [0021] Although this invention has been shown and described with respect to the detailed embodiments thereof, it will be understood by those of skill in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed in the above detailed description, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

CLAIMS:

1. A method for promoting blood clotting comprising contacting a blood clot promoter comprising a mixture of a noncalcium exchanged zeolite and a calcium salt with blood.

2. The method of claim 1 wherein said noncalcium exchanged zeolite is a sodium zeolite, potassium zeolite, lithium zeolite, magnesium zeolite or a combination thereof.

3. The method of claim 1 wherein said calcium salt is a salt with low toxicity, non- coordinating anions and with a solubility greater than 2 grams/100 cc in 20⁰C water.

4. The method of claim 1 wherein said calcium salt is selected from the group consisting of calcium chloride, calcium nitrate, calcium acetate, calcium bromide, calcium iodide, calcium nitrite, and combinations thereof.

5. The method of claim 1 wherein said blood clot promoter further comprises a binder.

6. The method of claim 1 wherein said blood clot promoter is contained within a porous carrier selected from the group consisting of woven fibrous articles, non-woven fibrous articles, puff, sponges and mixtures thereof.

7. The method of claim 1 wherein said noncalcium exchanged zeolite is in the form of a free flowing powder.

8. The method of claim 1 wherein said noncalcium exchanged zeolite promotes blood clotting at a rate 2 to 12 times faster than in its absence.

9. The method of claim 1 wherein said noncalcium exchanged zeolite promotes blood clotting in less than 10 minutes.

10. The method of claim 1 wherein said blood clot promoter further comprises antibiotics, antifungal agents, antimicrobial agents, anti-inflammatory agents, analgesics, bacteriostatics, compounds containing silver ions or mixtures thereof.