WO2001097826A2

WO2001097826A2 - Hemostatic compositions, devices and methods

Info

Publication number: WO2001097826A2
Application number: PCT/US2001/019145
Authority: WO
Inventors: Charles R. Spillert
Original assignee: University Of Medicine And Dentistry Of New Jersey
Priority date: 2000-06-16
Filing date: 2001-06-15
Publication date: 2001-12-27
Also published as: WO2001097826A3; AU2001266926A1

Abstract

A hemostatic composition which comprises at least one procoagulant metal ion, such as silver (I) or mercury (II), and at least one procoagulant biopolymer, such as collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, Factor VIIa, Factor VIII, Factor IXa, Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulant venom, a plasminogen activator inhibitor, glycoprotein IIb-IIIa, a protease, or plasma. The composition in the form of a paste, dough, glue, liquid, lyophilized powder or foam, may be provided, for application to a wound. A hemostatic device is also described which comprises a hemostatic composition as described above. The device may be in the form of, for example, a plug, bandage, gauze, cloth, tampon, membrane or sponge. Methods are also provided for prophylaxis or treatment of bleeding at a site by application to the site of the composition or device as described.

Description

HEMOSTATIC COMPOSITIONS, DEVICES AND METHODS

BACKGROUND OF THE INVENTION Uncontrolled hemorrhage from any type of wound, whether accidental or iatrogenic, may result in dire consequences. In particular, bleeding attendant with arterial damage is particularly troublesome, especially in an individual with a coagulopathy, congenital, the result of disease, or induced by administration of anticoagulants. Moreover, recovery from surgical and other procedures involving transarterial cannulation, particularly in anticoagulated individuals, presents a challenge to the medical profession as such individuals often present prolonged bleeding times from such wounds, necessitating close monitoring and increased length of hospital or in-patient stays. In such patients, manual pressure, pressure bandages, sandbags, and other means have been used to hasten hemostasis at the wound site. Not only is the excessive bleeding potentially dangerous to the patient, but the burden ofthe excess attention to the wound is dangerous to the already overburdened health care system.

The foregoing discussion is also pertinent to arterial and other major hemorrhagic wounds suffered from traumatic injury such as those inflicted on the battlefield, as a result of vehicular accidents, and from knife and gunshot wounds. Means to stop potentially fatal hemorrhage outside ofthe hospital environment is desirable.

Numerous hemostatic agents, compositions, and devices are known. Many such agents and compositions employ naturally procoagulant biopolymers, such as clotting factors and connective tissue proteins that have known hemostatic roles in vivo. Devices may include an inflatable member for blocking an opening in an artery, at which site a hemostatic agent may be introduced; other devices comprise agents or compositions in a particular form for application to a hemorrhaging site or plugging a puncture-type wound. For example, U.S. Patent 5,951,583 describes lyophilized thrombin, reconstituted with buffer and mixed with collagen, to form a viscous mixture. U.S. Patent 4,891,359 describes a hemostatic collagen paste composition, which optionally includes thrombin. U.S. Patent 5,595,735 described a hemostatic composition comprising thrombin in a polyethylene glycol base. U.S. Patent 5,948,425 described a hemostatic plug comprising collagen for sealing an incision which comprises a bleeding blood vessel. These and all other citations herein are incorporated herein by reference in their entireties.

Certain metal ions have known hemostatic properties. The use of silver nitrate as a topically applied cautery agent is known. As described in copending application Serial No. 09/022,449, filed February 12, 1998, now U.S. patent 6,245,573, metal ions such as mercuric ion, silver ion, ion, cadmium ion, copper ion, barium ion, tin ion, selenate ion and tungstate ion are useful to modulate the clotting rate of blood in order to diagnose coagulopathies. Silver metal, in the form of colloidal silver, has been used as an antimicrobial agent for many years, predating the use of antibiotics, but still used today particularly in the management of burn patients.

It is toward the development of improved hemostatic compositions and devices that the present invention is directed.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention is directed to a hemostatic composition which comprises at least one procoagulant metal ion and at least one procoagulant biopolymer. The metal ion may be, by way of non-limiting example, silver (I) or mercury (II). Preferably, silver (I) ion is used. Such ions, particularly silver, may be used at very low concentrations, for example, as low as the level of free silver ion available from an insoluble silver salt such as silver chloride. The procoagulant biopolymer may be, for example, one or more ofthe following: collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, Factor Vila, Factor VIII, Factor IXa, Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulant venom, a plasminogen activator inhibitor, glycoprotein Ilb-IIIa, proteases, or plasma. The hemostatic composition ofthe invention may also include a carrier, such as, but not limited to, polyethylene glycol, hyaluronic acid, methyl cellulose, or albumin. In a preferred embodiment, the concentration ofthe metal ion present in the biopolymer may be reduced below its effective hemostatic concentration in the absence ofthe biopolymer, as the instant inventors have found surprisingly a synergy between the procoagulant biopolymer and the procoagulant metal ion. In another embodiment, the level ofthe procoagulant biopolymer may be reduced well below its effective hemostatic concentration in the absence ofthe metal ion. In a further embodiment, the hemostatic composition comprises a procoagulant polymer at a sub-coagulant level in the absence of a metal ion, in combination with a metal ion at a sub-coagulant level in the absence of a procoagulant polymer.

The hemostatic composition ofthe invention may be in the form of, for example, a paste, dough, glue, liquid, lyophilized powder or foam, for application to a wound.

In another aspect, the invention is directed to a hemostatic device which comprises a hemostatic composition as described above. The device may be in the form of, for example, a plug, bandage, gauze, cloth, tampon, membrane or sponge.

A method for the prophylaxis or treatment of bleeding at a site is provided herein by applying to the site a hemostatic composition as described above, or a hemostatic device as described above. In one embodiment, the methods and compositions ofthe invention are useful in treating hemorrhaging, hypothermic patients.

The present invention is further directed to a method for the prophylaxis or treatment of bleeding at a site by applying to the site a composition comprising at least one procoagulant metal ion and at least one procoagulant biopolymer. The procoagulant metal ions and biopolymers are as described hereinabove. The composition may include a carrier, as described above. The hemostatic composition ofthe invention may be in the form of, for example, a paste, dough, glue, liquid, lyophilized powder or foam. Other forms ofthe composition are embraced herein.

In another aspect, the invention is directed to a method for the prophylaxis or treatment of bleeding at a site by applying to the site a device comprising a composition comprising at least one procoagulant metal ion and at least one procoagulant biopolymer. The procoagulant metal ions and procoagulant biopolymers are as described hereinabove. As noted above, the device may be in the form of a plug, bandage, gauze, cloth, tampon, membrane or sponge. Other forms ofthe device are embraced herein.

These and other aspects ofthe present invention will be better appreciated by reference to the following drawings and Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of various concentrations of silver (I) ion on clotting of whole blood and plasma.

Figure 2 shows the effect of collagen on clotting of whole blood.

Figure 3 shows the effect of silver (I) ion and collagen on the clotting of heparinized whole blood.

Figure 4 shows the effect of silver (I) ion and collagen on the clotting of heparinized plasma.

Figure 5 shows further the effect of silver (I) ion and collagen on the clotting of heparinized plasma.

Figure 6 shows further the effect of silver (I) ion and collagen on clotting of whole blood.

Figure 7 shows the effect of silver (I) and collagen on clotting time of whole blood with low molecular weight heparin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is broadly directed to hemostatic compositions and devices comprising at least one procoagulant metal ion and at least one procoagulant biopolymer, optionally with a carrier. Although silver (I) ion has been used as a cautery agent to stop bleeding by direct application of a soluble silver salt such as silver nitrate to a wound, it was found herein by surprise that an improved hemostatic composition was preparable using the combination of at least one procoagulant metal ion, such as silver (I) ion, and at least one procoagulant biopolymer, such as collagen. Furthermore, the inventor has found that the combination of a low concentrations of a procoagulant metal ion and a procoagulant biopolymer provides an effective hemostatic composition at concentrations wherein the individual components alone are less or not at all effective. For example, the concentration of free silver ion available in a composition ofthe invention comprising what is generally regarded as the water-insoluble silver salt silver

-A- chloride is adequate to promote hemostasis in combination with a procoagulant polymer. Thus, a composition ofthe present invention achieves the desired hemostatic activity with low concentrations of components, offering less potential toxicity, more rapid biodegradation, lower cost of raw materials, and superior activity in wound dressings and arterial plugs. A further advantage is that the clot formed in the presence ofthe instant compositions are stronger than clots formed in the absence ofthe compositions.

The inventor has found that the present procoagulant compositions are useful for inducing clotting in both whole blood and in plasma. As will be seen below, the procoagulant properties ofthe present compositions are also useful in the presence of anticoagulant factors such as heparin. As patients undergoing anticoagulant therapy are prone to prolonged and thus dangerous bleeding, the present compositions are particularly useful for use in surgery or trauma care in such patients.

The invention has been described in co-pending provisional application serial no. 60/212,229, filed June 16, 2000, and is incorporated herein by reference in its entirety. Moreover, priority of the present application is claimed to the aforementioned provisional application under 35 U.S.C. § 119(e). As shown therein, various metal ions at low concentrations in combination with a procoagulant biopolymer were found to act synergistically to promote coagulation or clotting of whole blood.

Further studies herein have confirmed and expanded the scope ofthe invention. Measurements ofthe procoagulant effects ofthe compositions ofthe invention have been carried out using a SONOCLOT miniviscometer to detect early clot formation. In such studies, anti-coagulated, citrated whole blood is recalcified to initiate the clotting process in a device which sensitively monitors the formation ofthe clot. Studies have been expanded from whole blood to include plasma.

Various metal ions known to have procoagulant properties are useful in the compositions and devices ofthe present invention. The present invention embraces all such procoagulant metal ions. These ions are set forth herein as the element followed by the number of positive charges ofthe ion in parentheses, such as silver (I) or mercury (II) [also known as mercuric]. Any salts of these ions are suitable for use intended herein that provide a effective procoagulant level of free metal ion. Using silver (I) as an example, the nitrate salt provides adequate free silver (I) ion, whereas the chloride salt provides less, being less soluble and with a low dissociation constant. However, as mentioned above, certain metal salts which are considered insoluble have a large- enough dissociation constant to permit an effective procoagulant level ofthe metal ion to be present in the compositions herein which comprise the insoluble metal salt. The skilled artisan will be able to readily determine the suitable salt form ofthe metal ion that provides the procoagulant properties in combination with the procoagulant biopolymer. Furthermore, the skilled artisan will be aware ofthe compatibility ofthe salt forms ofthe metal(s) and other components ofthe composition to maintain adequate levels ofthe metal ion(s) in solution to provide procoagulant activity. Of course, considerations must be given to the toxicity ofthe metal salt, whether soluble or not, at the concentrations needed for an effective hemostatic composition.

As noted herein, the composition may comprise one or more procoagulant metal ions, such as silver (I) or mercury (II). For the example of silver (I) ion, effective levels may be achieved by using water-soluble silver salts such as the nitrate, selenate, nitrite, or perchlorate salts, or organic acids such as lactate. In the case of mercury (II), nitrate, acetate and chloride salts are examples. Mercury (II) ion in the form of MERCUROCHROME(R) (2',7'-dibromo-5- [hydroxymercuricjfluorescein) may also be used. As noted above, when such salts are used in combination, one must be cognizant ofthe compatibilities ofthe salts to avoid reducing the solubility ofthe procoagulant ion(s) below their effective concentration(s). Furthermore, certain components may provide oxidative or reductive properties which in contact with the metal ion(s), may alter their oxidation state and effectiveness. Such considerations are well within the realm ofthe skilled artisan in preparing an effective procoagulant composition in accordance with the disclosure herein.

As noted above, a composition ofthe present invention may have more than one metal salt, such as the combination of silver (I) and mercury (II).

With regard to the procoagulant biopolymer ofthe instant composition, this component embraces naturally occurring, recombinant, isolated, and any and all other forms of procoagulant proteins and other biologically compatible polymers with procoagulant activity. By way of non-limiting example, procoagulant proteins embraced herein include but are not limited to collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, Factor Vila, Factor VITJ, Factor IXa, Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulant venom, a plasminogen activator inhibitor, glycoprotein Ilb-IIIa, proteases, or plasma. These may be used singly or in combination. Such proteins include those which have a net effect of procoagulant activity, such as clot dissolution inhibitors including plasminogen activator inhibitor. Included herein is plasma, which comprises numerous procoagulant proteins, and proteases, which are procoagulant. An example of a procoagulant venom is Russells' viper venom. Examples of proteases are described in the catalog of Sigma Chemical Company, from numerous sources.

The procoagulant proteins ofthe invention may be prepared by any of numerous methods or obtained from commercial sources. Many such proteins are available in purified form, for example, from Sigma Chemical Co., American Diagnostica, and Critichem Inc., to name only a few examples.

A preferred procoagulant polymer is collagen, and more preferred, type I collagen. It is available from any of a number of sources in various forms, including fibrillar, amorphous, and others. Particular preparations used for certain ofthe studies herein are sponge collagens, woven collagens and fibrillar collagens from the Collagen Products Division of Datascope Corp., Mahwah, New Jersey, U.S.A.

The concentrations ofthe procoagulant metal ion(s) and procoagulant biopolymer(s) in the composition of the invention may be prepared in accordance with the desired properties of the composition, the form ofthe device in which the composition is delivered or applied to the wound, and other factors that one or ordinary skill in the art would take into account in preparing the composition or device ofthe invention. Of course, the composition must have low toxicity, and if left in place, preferably needs to biodegrade after it has achieved its desired function. As noted above, it was found that the concentration ofthe procoagulant metal ion(s) in the instant composition may be reduced to a level below which it would not be effective as the sole hemostatic agent. For example, for a particular sample of patient blood, a composition comprising silver (I) and collagen may comprise 1% collagen and 0.1% silver nitrate, wherein 0.1% silver nitrate alone would not impart significant hemostasis in a wound. Therefore, the hemostatic compositions ofthe present invention may, in one embodiment, include either the procoagulant biopolymer or the procoagulant metal ion, or both, at a concentration lower than would be hemostatically effective alone. Examples of suitable compositions ofthe invention comprising one procoagulant biopolymer include, but are not limited to, silver (I) and collagen, silver (I) and thrombin, silver (I) and prothrombin, silver (I) and fibrin, silver (I) and fibrinogen, silver (I) and heparinase, silver (I) and Factor Vila, silver (I) and Factor VIII, silver (I) and Factor IXa, silver (I) and Factor Xa, silver (I) and Factor XII, silver (I) and von Willebrand Factor, silver (I) and a selectin, silver (I) and a procoagulant venom, silver (I) and a plasminogen activator inhibitor, silver (I) and glycoprotein Ilb-IIIa, silver (I) and a protease, silver (I) and plasma; mercury (II) and collagen, mercury (II) and thrombin, mercury (II) and prothrombin, mercury (II) and fibrin, mercury (II) and fibrinogen, mercury (II) and heparinase, mercury (II) and Factor Vila, mercury (II) and Factor VIII, mercury (II) and Factor IXa, mercury (II) and Factor Xa, mercury (II) and Factor XII, mercury (II) and von Willebrand Factor, mercury (II) and a selectin, mercury (II) and a procoagulant venom, mercury (II) and a plasminogen activator inhibitor, mercury (II) and glycoprotein Ilb-IIIa, mercury (II) and a protease, mercury (II) and plasma.

Preferred compositions include silver (I) and collagen, and mercury (II) and collagen. A preferred collagen is type I collagen. The collagen may be derived from any source, preferably mammalian and most preferably bovine in origin, but is it not so limiting. Human collagen, and in particular recombinantly-prepared human collagen, is another preferred procoagulant biopolymer.

Of course, the composition may contain a second or additional procoagulant metal ion, or additional procoagulant biopolymer(s). The invention embraces any combination of additional procoagulant metal ions or procoagulant polymers, as well as other excipients, carriers, stabilizers, preservatives and other additives to a hemostatic product normally needed to ensure stability, shelf life, and other commercial consideration. The known bacteriostatic activity of metal ions may provide an endogenous level of preservation in the compositions ofthe invention; moreover, the bacteriostatic properties are an additional benefit ofthe present compositions in use in wound care.

Thus, other hemostatic compositions ofthe invention include, but are not limited to, silver (I) and mercury (II) and collagen, silver (I) and mercury (II) and thrombin, silver (I) and mercury (II) and prothrombin, silver (I) and mercury (II) and fibrin, silver (I) and mercury (II) and fibrinogen, silver (I) and mercury (II) and heparinase, silver (I) and mercury (II) and Factor Vila, silver (I) and mercury (II) and Factor VIII, silver (I) and mercury (II) and Factor IXa, silver (I) and mercury (II) and Factor Xa, silver (I) and mercury (II) and Factor XII, silver (I) and mercury (II) and von Willebrand Factor, silver (I) and mercury (II) and a selectin, silver (I) and mercury (II) and a procoagulant venom, silver (I) and mercury (II) and a plasminogen activator inhibitor, silver (I) and mercury (II) and glycoprotein Ilb-IIIa, silver (I) and mercury (II) and a protease, silver (I) and mercury (II) and plasma.

The hemostatic composition ofthe invention may also include a carrier, such as, but not limited to, polyethylene glycol, hyaluronic acid, cellulose, oxidized cellulose, methyl cellulose, or albumin. These may be used to provide a matrix, a suitable viscosity, deliverability, adherence, or other properties desired to be imparted to the compositions herein for easy in application to a wound. Numerous other carrier which impart these characteristics are embraced herein.

The form ofthe hemostatic composition ofthe invention may be prepared in any form suitable for use in the intended application. Various carriers and other materials may be provided in the composition to achieve the desired form, in addition to the at least one procoagulant metal ion and at least one procoagulant biopolymer. For example, the composition in the form of a paste may be prepared for application to a surgical site; a thicker paste, or dough, may be molded by the surgeon for application at a site ofthe removal of a transarterial cannula. A glue may be prepared with hemostatic properties; other forms include a liquid, a lyophilized powder or foam, for application to or in a wound. As will be described in more detail below in reference to devices comprising the hemostatic composition ofthe invention, the composition may be dried or lyophilized for form a particular shape, such as a plug, that is useful for particular applications, such as placement to stop the bleeding of a removed transarterial cannula. A bandage comprising a composition ofthe invention may also be provided. All of these various forms and other are embraced by the present invention.

As mentioned above, the invention is also directed to a hemostatic device which comprises a hemostatic composition as described above. The device may be provided in any number of formats useful for the control of bleeding. For example, the composition ofthe invention may be provided in the foπn of a plug, for placement in a wound; or provided on a bandage, gauze, cloth, tampon, membrane or sponge, for application to or placement in a wound. Certain materials may be biodegradable and suitable for placement in a closed surgical wound. Other materials may be provided for later removal. Devices for emergency application to hemorrhaging areas or severed arteries may be useful for emergency use. In another embodiment ofthe invention, the hemostatic device comprising the composition may be prepared in an asymmetric fashion, of contain components asymmetrically applied, such that blood is restricted from flowing completely through a device. For example, a blood flow altering agent may be provided at the distal part ofthe device from that to which is applied to the wound, such that blood will not penetrate beyond the device. A bandage may comprise the instant composition on one side, and a blood- impermeant membrane on the other.

The present invention also embraces various methods for the prophylaxis or treatment of bleeding at a site in the body. This method is carried out by the application or placement of a hemostatic composition or a hemostatic device comprising the hemostatic composition ofthe invention to or in the site. The present invention embraced various methods and means used for the delivery or placement ofthe device in the site of desired hemostatic activity. The various forms ofthe composition and ofthe device are embraced within this method.

The present invention may be better understood by reference to the following non-limiting

Examples, which are provided as exemplary ofthe invention. The following examples are presented in order to more fully illustrate the preferred embodiments ofthe invention. They should in no way be construed, however, as limiting the broad scope ofthe invention.

Example I

Effect of silver (I) ion and heparinase on clotting time of heparinized blood

The hemostatic compositions ofthe invention were evaluated in two different systems. In one system, clotting time of recalcified, citrated whole blood in the presence ofthe hemostatic composition was determined using a SONOCLOT Coagulation Analyzer (Sienco Inc., Wheat Ridge CO). Other instrumentation for providing clotting data are equally useful. Results were expressed as clotting time in seconds. In a second system, the hemostatic composition was dried on a cellulose matrix (filter paper), and 7μl aliquot of fresh blood or recalcified citrated whole blood was placed in the cellulose matrix; resulting in the spreading (perfusion) ofthe applied blood as an enlarging disk. Upon clotting or reduced perfusion, the further spread ofthe blood ceases. The surface area ofthe spread blood in mm² is used as an indication ofthe clotting rate ofthe blood. Results are expressed as mean ± standard deviation. The number of samples run is indicated at the top of each data column. Silver (I) ion was provided as silver nitrate, 5% stock solution. It was diluted 100 fold to 0.05%, or in some cases to 0.01% in the clotting studies. In perfusion studies, a lOμl aliquot ofthe solution was applied to the filter paper then dried.

Collagen was from Sigma Chemical Co. In the clotting time studies, the final concentration of collagen was 0.1-0.2%. Heparinase was from Haemoscope Corp. and used at a final concentration of 1 U/mL.

Citrated blood anticoagulated with heparin (2 U/mL) was used as a model ofthe utility ofthe compositions ofthe present invention to induce hemostasis in an individual treated with anticoagulants (including fibrinolytics and antiplatelet therapies) as may occur in patients with myocardial infarction, stroke, unstable angina, thrombophlebitis, peripheral vascular disease, traumatic injury, post-surgical patients and those undergoing cancer chemotherapy. Recalcification time was measured. Treatment Clotting time (sec) n^

Control 287 ± 21

Control + silver(I) 94 ± 35

Control + heparin 1930 ± 83

Control + heparin + heparinase 360 ± 22 Control + silver (I) + heparinase 872 ± 353

Control + silver (I) + heparin + heparinase 157 ± 30

These data show that silver (I) ion is capable of returning the recalcification time of blood towards normal; moreover, in combination with heparinase, is capable of reducing the clotting time to below normal values.

Example II Effect of silver (I) ion and collagen on blood perfusion

Recalcified, citrated whole blood was applied to filter paper impregnated with 5% AgNO₃, 2% collagen, or the combination of 2.5% silver nitrate and 1% collagen. The size ofthe blood disk on the paper was measured. Study I Treatment Perfusion (mm) (n=13

5% AgNO₃ 18 ± 4

2% collagen 35 ± 23 2.5%AgNO₃ + 1% collagen 7 ± 4

Study II

Treatment Perfusion (mm) ^=5)

5% AgNO₃ 17 ± 2 l% AgNO₃ 42 ± 18

2% collagen 28 ± 10

2.5% AgNO₃ + 1 % collagen 7 ± 0.4

0.5% AgNO₃ + 1 % collagen 15 ± 5

This study shows the effectiveness ofthe combination of AgNO₃ and collagen, and furthermore, that the combination of a lower concentration of silver (I) ion, 0.5%, with collagen (1%) can achieve the hemostatic effect provided by 5% AgNO₃ alone.

Example IV Effect of silver (I) ion and collagen on clotting time

The final concentrations ofthe composition in blood is provided below for a similar experiment. Treatment Clotting time (sec) [n=4]

Control 273 ± 46

Control + 1 % collagen 264 ± 37 Control + 0.6 mM AgNO₃ 220 ± 50

Control + 0.3 mM AgNO₃ + 0.5% collagen 178 ± 42

This study demonstrates the combined effect of silver (I) ion and collagen on hemostatic measured by clotting time of recalcified, citrated whole blood.

Example V Effect of silver (I) ion on clotting time of whole blood, platelet-rich blood, and platelet-poor blood This study was undertaken using platelet-rich and platelet-poor plasma to simulate an individual with normal and reduced platelet levels.

Treatment Clotting time (sec) [n=8]

Normal Blood 262 ± 26

Normal blood + 2.9 mM AgNO₃ 109 ± 20

Platelet Rich Plasma (PRP) 314 ± 70

PRP + 2.9 mM AgNO₃ 83 ± 29

Platelet Poor Plasma (PPP) 353 ± 62

PPP + 2.9 mM AgNO₃ 128 ± 32

These studies show the effectiveness of silver (I) ion as a hemostatic agent in blood with low platelet numbers.

Example VI Clot strength after silver (I) ion induced clotting of heparinized blood

Using the SONOCLOT, tracings of heparinized whole blood clotting in the presence of silver (I) ion were obtained. While the clotting time of heparinized blood is shortened from 1930 seconds to 872 seconds in the presence of silver (I) ion, the physical characteristics ofthe silver-induced clot as shown by the analyzer demonstrates a stronger clot. In non-heparinized blood, the clotting time is shortened from 217 seconds to 81 seconds in the presence of silver; furthermore, the clot is stronger. In a second study, silver (I) ion reduces the clotting time from 588 to 168 seconds, also with a stronger clot.

Example VI Silver (I) ion and thrombin potentiates clotting time compared to either alone

Thrombin was used at a final concentration of 0.025 U/mL. Silver ion was present at a final concentration of 2.9 mM. Treatment Clotting time (sec) [n=8]

Control 236 ± 38

Control + thrombin 156 ± 42

Control + silver 91 ± 14

Control + silver + thrombin 71 ± 7

Example VII Effect of various concentrations of silver (I) ion on clotting of whole blood and plasma

Figure 1 depicts the clotting time of citrated whole blood (A-D) and plasma (E-H) in the presence of 0.05% silver (I) ion (B, F), 0.005% silver (I) ion (C, G), 0.0005% silver (I) ion (E, H), or no silver ion (A, E).

Example VIII Effect of collagen preparations and silver (I) ion on clotting of whole blood

Figure 2 depicts the clotting times of whole blood alone (A) or in the presence of 1 mg/ml sponge collagen (B), 1 mg/ml fibrillar collagen (C), 0.05% silver (I) ion (D), 0.05% silver (I) ion and 1 mg/ml sponge collagen (E), and 0.05% silver (I) ion and 1 mg/ml fibrillar collagen (F).

Example IX Effect of collagen and silver ion on clotting of heparinized whole blood

Figure 3 shows the clotting times of heparinized whole blood (1 U/ml) alone (A) or in combination with 0.05% silver (I) ion (B), with 1 mg/ml fibrillar collagen (C), and in the presence ofthe combination of 0.05% silver (I) ion and 1 mg/ml sponge collagen (D). Bar E shows the clotting time ofthe heparinized whole blood with 1 mg/ml sponge collagen; and in combination with 0.05% silver (I) ion in (F).

Figure 4 similarly shows the effect on heparinized whole blood clotting time in the presence of

0.05% silver (I) ion, collagen and 0.25 U/ml heparin (A), and under similar conditions with 0.3% silver (I) ion (B). However, in the absence of collagen at either silver (I) levels, a prolonged clotting time is seen C and D, respectively. Figure 5 shows the clotting time of heparinized whole blood (1 U/ml) with various concentrations of silver ion or mercuric ion. Heparinized whole blood alone (A), with sponge collagen (B), with sponge collagen and 0.05% silver (I) ion (C), with sponge collagen and 0.3%> silver (I) ion (D), or with sponge collagen and 0.1% mercuric (II) ion (E). The same series with fibrillar collagen are shown in F-I, respectively.

Figure 6 shows the clotting time of heparinized whole blood (2U/ml): whole blood (A), heparinized whole blood (B), activated clotting time of heparinized whole blood (C), clotting time of heparinized whole blood plus fibrous collagen (D), or sponge collagen (E); the clotting time of heparinized whole blood with fibrous collagen and 0.05% silver (I) ion (F), the same with 0.5% silver (I) ion (G), or sponge collagen collagen with 0.05% silver (I) ion (H) or 0.5% silver (I) ion (I). Heparinized whole blood with 0.05% silver (I) ion is shown in J, and with 0.5% silver (I) ion in K.

Figure 7 shows the effect of low molecular weight (LMW) heparin and fibrous collagen on clotting time of whole blood. LMW heparinized whole blood (A), with 0.05% silver (I) ion (B), with 0.3%) silver (I) ion (C), LMW heparinized whole blood plus fibrous collagen (D), the same plus 0.05% silver (I) ion (E), or with 0.5% silver (I) ion (F).

The foregoing studies demonstrate the effect ofthe combination of a procoagulant polymer, sponge (cross-linked) or fibrous collagen, and silver (I) or mercury (II) ion, on promoting coagulation of plasma, whole blood, and heparinized plasma and whole blood.

The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope ofthe appended claims.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

^■ WHAT IS CLAIMED IS:

1. A hemostatic composition comprising at least one procoagulant biopolymer in combination with a procoagulant metal ion, said procoagulant metal ion present in said composition at a level below its effective hemostatic concentration in the absence of said procoagulant biopolymer.

2. The composition of claim 1 wherein said procoagulant metal ion is selected from the group consisting of silver (I) and mercury (II).

3. The composition of claim 2 wherein said procoagulant metal ion is silver (I).

4. The composition of claim 2 wherein said procoagulant metal ion is provided by an insoluble salt comprising said metal ion.

5. The composition of claim 4 wherein said procoagulant metal ion is silver (I).

6. The composition of claim 5 wherein said salt is silver chloride.

7. The composition of claim 1 wherein said at least one procoagulant biopolymer is selected from the group consisting of collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, Factor Vila, Factor VIII, Factor IXa, Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulant venom, a plasminogen activator inhibitor, glycoprotein Ilb-IIIa, a protease, and plasma.

8. The composition of claim 7 wherein said at least one procoagulant biopolymer is collagen.

9. The hemostatic composition of claim 1 selected from the group consisting of silver (I)

I and collagen, silver (I) and thrombin, silver (I) and prothrombin, silver (I) and fibrin, silver (I) and fibrinogen, silver (I) and heparinase, silver (I) and Factor Vila, silver (I) and Factor VIII, silver (I) and Factor IXa, silver (I) and Factor Xa, silver (I) and Factor XII, silver (I) and von Willebrand Factor, silver (I) and a selectin, silver (I) and a procoagulant venom, silver (I) and a plasminogen activator inhibitor, silver (I) and glycoprotein Ilb- Ilia, silver (I) and a protease, silver (I) and plasma, mercury (II) and collagen, mercury (II) and thrombin, mercury (II) and prothrombin, mercury (II) and fibrin, mercury (II) and fibrinogen, mercury (II) and heparinase, mercury (II) and Factor Vila, mercury (II) and Factor VIII, mercury (II) and Factor IXa, mercury (II) and Factor Xa, mercury (II) and Factor XII, mercury (II) and von Willebrand Factor, mercury (II) and a selectin, mercury

(II) and a procoagulant venom, mercury (II) and a plasminogen activator inhibitor, mercury (II) and glycoprotein Ilb-IIIa, mercury (IT) and a protease, and mercury (II) and plasma.

10. The composition of claim 1 further comprising a carrier.

11. The composition of claim 10 wherein said carrier is selected from the group consisting of polyethylene glycol, hyaluronic acid, cellulose, oxidized cellulose, methyl cellulose, and albumin.

12. The composition of claim 1 wherein said composition is in the form of a paste, glue, liquid, lyophilized powder or foam.

13. A hemostatic device comprising the hemostatic composition of claim 1.

14. The device of claim 13 comprising a plug, bandage, gauze, cloth, tampon, membrane or sponge.

15. A method for the prophylaxis or treatment of bleeding at a site comprising applying to said site the hemostatic composition of claim 1.

16. A method for the prophylaxis or treatment of bleeding at a site comprising applying to said site the device of claim 13.

17. A method for the prophylaxis or treatment of bleeding at a site comprising applying to said site a hemostatic composition comprising at least one procoagulant biopolymer in combination with a procoagulant metal ion, said procoagulant metal ion present in said composition at a level below its effective hemostatic concentration in the absence of said procoagulant biopolymer.

18. The method of claim 17 wherein said metal ion is selected from the group consisting of silver (I) or mercury (II).

19. The method of claim 17 wherein said metal ion is silver (I). 5

20. The method of claim 17 wherein said at least one procoagulant biopolymer is selected from the group consisting of collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, Factor Vila, Factor NIII, Factor IXa, Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulant venom, a plasminogen activator inhibitor, glycoprotein

10 Ilb-IIIa, a protease and plasma.

21. The method of claim 20 wherein said procoagulant biopolymer is collagen.

22. The method of claim 17 wherein said hemostatic composition is selected from the group L5 consisting of silver (I) and collagen, silver (I) and thrombin, silver (I) and prothrombin, silver (I) and fibrin, silver (I) and fibrinogen, silver (I) and heparinase, silver (I) and Factor Vila, silver (I) and Factor VIII, silver (I) and Factor IXa, silver (I) and Factor Xa, silver (I) and Factor XII, silver (I) and von Willebrand Factor, silver (I) and a selectin, silver (I) and a procoagulant venom, silver (I) and a plasminogen activator inhibitor,

JO silver (I) and glycoprotein Ilb-IIIa, silver (I) and a protease, silver (I) and plasma, mercury

(II) and collagen, mercury (II) and thrombin, mercury (II) and prothrombin, mercury (II) and fibrin, mercury (II) and fibrinogen, mercury (II) and heparinase, mercury (II) and Factor Vila, mercury (II) and Factor VIII, mercury (II) and Factor IXa, mercury (II) and Factor Xa, mercury (II) and Factor XII, mercury (II) and von Willebrand Factor, mercury

!5 (II) and a selectin, mercury (II) and a procoagulant venom, mercury (II) and a plasminogen activator inhibitor, mercury (II) and glycoprotein Ilb-IIIa, mercury (II) and a protease, and mercury (II) and plasma.

24. The method of claim 17 wherein said hemostatic composition further comprises a carrier.

⁽0

25. The method of claim 24 wherein said carrier is selected from the group consisting of polyethylene glycol, hyaluronic acid, cellulose, oxidized cellulose, methyl cellulose, and albumin.

26. The method of claim 17 wherein said composition is in the form of a paste, glue, liquid, lyophilized powder or foam.

27. A method for the prophylaxis or treatment of bleeding at a site comprising applying to said site a device comprising a hemostatic composition comprising at least one procoagulant biopolymer in combination with a procoagulant metal ion, said procoagulant metal ion present in said composition at a level below its effective hemostatic concentration in the absence of said procoagulant biopolymer.

28. The method of claim 27 wherein said metal ion is selected from the group consisting of silver (I) and mercury (II).

29. The method of claim 28 wherein said metal ion is provided by an insoluble salt comprising said metal ion.

30. The method of claim 29 wherein said metal ion is silver (I).

31. The method of claim 30 wherein said salt is silver chloride.

32. The method of claim 27 wherein said at least one procoagulant biopolymer is selected from the group consisting of collagen, thrombin, prothrombin, fibrin, fibrinogen, heparinase, Factor Vila, Factor VIII, Factor IXa, Factor Xa, Factor XII, von Willebrand Factor, a selectin, a procoagulant venom, a plasminogen activator inhibitor, glycoprotein Iib-HIa, a proteases and plasma.

33. The method of claim 32 wherein said procoagulant biopolymer is collagen.

34. The method of claim 27 wherein said hemostatic composition is selected from the group consisting of silver (I) and collagen, silver (I) and thrombin, silver (I) and prothrombin, silver (I) and fibrin, silver (I) and fibrinogen, silver (I) and heparinase, silver (I) and

Factor Vila, silver (I) and Factor VIII, silver (I) and Factor IXa, silver (I) and Factor Xa, silver (I) and Factor XII, silver (I) and von Willebrand Factor, silver (I) and a selectin, silver (I) and a procoagulant venom, silver (I) and a plasminogen activator inhibitor, silver (I) and glycoprotein Ilb-IIIa, silver (I) and a protease, silver (I) and plasma, mercury (II) and collagen, mercury (II) and thrombin, mercury (II) and prothrombin, mercury (II) and fibrin, mercury (II) and fibrinogen, mercury (II) and heparinase, mercury (II) and Factor Vila, mercury (II) and Factor VIII, mercury (II) and Factor IXa, mercury (II) and Factor Xa, mercury (II) and Factor XII, mercury (II) and von Willebrand Factor, mercury (II) and a selectin, mercury (II) and a procoagulant venom, mercury (II) and a plasminogen activator inhibitor, mercury (II) and glycoprotein Ilb-IIIa, mercury (II) and a protease, and mercury (II) and plasma.

35. A hemostatic composition comprising at least one procoagulant biopolymer in combination with a procoagulant metal ion, said procoagulant metal ion present in said composition at a level below its effective hemostatic concentration in the absence of said procoagulant biopolymer, and said procoagulant biopolymer present in said composition at a level below its effective hemostatic concentration in the absence of said procoagulant metal ion.

36. A method for the prophylaxis or treatment of bleeding at a site comprising applying to said site the hemostatic composition of claim 35.