WO2004058324A2

WO2004058324A2 - Antithrombotic venous access devices and methods

Info

Publication number: WO2004058324A2
Application number: PCT/US2003/040888
Authority: WO
Inventors: Mcdonald K. Horne, Iii
Original assignee: The Government Of The United State Of America As Represent By The Secretary Of The Department Of Health And Human Services
Priority date: 2002-12-23
Filing date: 2003-12-22
Publication date: 2004-07-15
Also published as: AU2003299802A8; AU2003299802A1; WO2004058324A3

Abstract

Catheters are disclosed which are treated with recombinant hirudin, such as lepirudin, to reduce thrombosis in patients. Also disclosed are medical devices, such as venous access devices that are treated with various recombinant hirudins to reduce their thrombogenesis properties in patients. Related inventions for methods of treating medical devices with recombinant hirudin to reduce their thrombogenesis properties in patients are further disclosed. Methods for reducing the risk of thrombosis caused by venous access devices, and kits for treating the exterior of venous access devices for reducing their thrombogenesis properties are also disclosed.

Description

ANTITHROMBOTIC VENOUS ACCESS DEVICES AND METHODS

FIELD OF THE INVENTION

[0001] The present invention relates to the field of venous access medical devices. More particularly, the invention relates to devices and methods to reduce the risk of thrombosis associated with venous access devices.

BACKGROUND OF THE INVENTION

[0002] Chronically ill patients, particularly those with cancer, often have catheters inserted in the large veins of their upper chest in order to receive medications and blood products. These "venous access devices" (VADs) remain in place for months at a time and may cause clotting (thrombosis) of the veins, which results in swelling and pain and leaves the veins permanently damaged. In an attempt to prevent VAD-induced thrombosis, catheters have been coated with the anticoagulant heparin using various chemical techniques (e.g., Huang et al., U.S. Patent Application Publication No. US 2002/0068183 Al), but so far none of these coated catheters has demonstrated sufficient efficacy to be introduced in the clinic. Attempts have also been made to coat medical devices with other anticoagulants, such as disodium ethylenediamine tetraaceticacid (EDTA), a combination of citrate and heparin, enoxaparin sodium, anisindione, protamine sulfate, streptokinase, urokinase, anti-thrombin HI, and native hirudin (Welle et. al, U.S. Patent No. 6,187,768).

[0003] Hirudin is a potent thrombin inhibitor derived from the salivary glands of the medicinal leech, Hirudo medicinalis (J.H. Sohn et al., Appl. Microbiol. Biotechnol (2001) 57:606-613). Hirudin has been intensively researched for its therapeutic purposes, and its investigation has contributed to the understanding of the mode of action of thrombin and the clotting system. Natural hirudin is a protein comprising three isoforms of 65 or 66 amino acids, and each of the isoforms has a sulfated tyrosine in position 63 or 64. Its structure basically comprises a compact, hydrophobic core comprising the N-terminal half of the molecule and an extended, hydrophilic C-terminal region. Id. The core region has three disulfide bridges that restrict the configuration of the core. Hirudin generates its antithrombotic action by binding directly to thrombin at multiple sites: the N-terminal globular domain binds near the active site of thrombin, while the C-terminal segment, which is abundant in acidic residues and includes the sulfated tyrosine, makes both ionic and hydrophobic interactions with the fibrinogen recognition exosite (FRE) of thrombin. It appears that the sulfated tyrosine enhances the thrombin exosite binding of hirudin. Id.

[0004] In view of its role as a potent thrombin inhibitor in preventing the coagulation of blood, researchers have attempted to coat in-dwelling medical devices, such as catheters, with natural hirudin to reduce thrombosis. To prevent desorption of the hirudin researchers have developed methods to chemically or physically bind hirudin molecules to medical device surfaces. For example, in U.S. Patent 5,053,453, the disclosure of which is incorporated herein by reference thereto in its entirety, Ku discloses thromboresistant materials comprising hirudin or hirudin derivatives covalently linked to support materials such that the resultant composition reportedly has substantially the same biological activity as hirudin.

[0005] Fearnot, et. al., WO 95/06487, the disclosure of which is incorporated herein by reference thereto in its entirety, reports the inclusion of various antithrombogenic agents, e.g., hirudin, in formulations for the base material and or a coating material of intravascular medical devices to inhibit the formation of thrombus on the surface of the medical devices.

[0006] Ito, et al., WO 92/05748, the disclosure of which is incorporated herein by reference thereto in its entirety, reports a soluble, biocompatible, pharmacological agent intended for inhibiting thrombin generation and thrombus formation, and methods for producing the same. The pharmacological agent or conjugate includes a soluble, biocompatible carrier and a thrombogenesis inhibitor immobilized thereto via a component of the carrier which binds the inhibitor, hirudin, or an active analog or active fragment thereof. The thrombogenesis inhibitor may be bound to the component of the carrier via a bifunctional cross-linking reagent.

[0007] Raad et al., U.S. Patent 5,688,516, the disclosure of which is incorporated herein by reference thereto in its entirety, reports compositions and methods of employing compositions in flushing and coating medical devices. The compositions include selected combinations of a chelating agent, anticoagulant, or antithrombotic agent, with a non- glycopeptide antimicrobial agent, such as a tetracycline antibiotic. Particular combinations of the claimed combinations include minocycline or other non-glycopeptide antimicrobial agent together with EDTA, EGTA, DTP A, TTH, heparin and/or hirudin in a pharmaceutically acceptable diluent.

[0008] Chirm et al., U.S. Patent 6,416,549, the disclosure of which is incorporated herein by reference thereto in its entirety, reports antithrombogenic annuloplasty rings, and methods for making the same. At least some portion of the annuloplasty ring has incorporated into or onto its structure one or more antithrombogenic agents, e.g., hirudin, or materials in a manner which reportedly reduces the likelihood of thrombosis following implantation.

[0009] Welle et al., U.S. Patent No. 6,187,768, the disclosure of which is incorporated herein by reference thereto in its entirety, reports a kit and a method for flushing a medical device. The kit includes a container containing a mixed solution a unit dose of a pharmacologically effective amount of an antimicrobial agent and a second agent. The second agent is an anticoagulant, an antithrombotic agent, e.g., hirudin, or a chelating agent. The kit and method are reported to be useful for maintaining the patency of indwelling medical devices such as catheters and for preventing infections caused by bacterial growth in catheters.

[0010] Although the above-mentioned researchers report various ways of reducing thrombosis of indwelling medical devices, such as catheters, there is a continuing need to provide antithrombotic medical devices which do not require additional steps of binding an antithrombotic agent, such as hirudin, to the device. Moreover, there is a continuing need to develop medical devices having antithrombotic properties which do not require the use of scarce naturally-derived proteins, such as hirudin. Because natural hirudin derived from leeches is only produced in small amounts, about 20 micrograms per leech head, recombinant DNA technologies have been developed to provide recombinant hirudin analogues in quantities sufficient for human therapeutic uses.

[0011] Two recombinant hirudins have gained FDA approval for marketing. One of them is lepirudin (Refludan), which is a biotechnologically manufactured desulfatohirudin from S. cerevisiae and has a N-terminal of Leu-Thr. The other FDA-approved recombinant hirudin, desirudin (Revasc) differs from lepirudin only on the first two N-terminal amino acids (Nal-Nal). Although successfully produced, recombinant hirudin analogues expressed in several microorganisms lack the sulfate group on the tyrosine in position 63 or 64, and thus have a te«- fold reduced affinity for thrombin. (Sohn et al., Id.) [0012] Thus, there remains an important need in the art for effecting the use of more abundant forms of recombinant hirudin, such as desirudin and lepirudin, to provide antithrombotic medical devices, preferably without requiring a coating to bind the recombinant hirudins to the medical device's substrate material.

SUMMARY OF THE INVENTION

[0013] The present inventor has discovered that recombinant forms of hirudin can be effectively used to impart antithrombotic properties to venous access devices, such as catheters, without requiring a binding coating or requiring a covalently binding cross-linking reagent. In a first aspect of the present invention, there are provided venous access devices, which include a substrate material, and recombinant hirudin adsorbed to at least a portion of the surface of the substrate material. Surprisingly, within this aspect of the invention, recombinant hirudins are found to adhere sufficiently to a variety of substrates without requiring a binding coating or cross-linking reagents.

[0014] Within additional aspects of the invention, medical devices are provided that include a substrate material, and a recombinant hirudin adsorbed on the surface of the substrate material. In these aspects of the present invention, medical devices having sufficient antithrombotic properties are provided by treatment with a variety of recombinant hirudins, without requiring the use of a binding coating or cross-linking reagent.

[0015] In a related aspect of the present invention, there are provided methods of treating medical devices with a recombinant hirudin, which include contacting the substrate material of medical devices with a composition including the recombinant hirudin, and removing loosely bound recombinant hirudin from the substrate material. In this aspect of the present invention, medical devices having sufficient antithrombotic properties are provided using a variety of recombinant hirudins without requiring a binding coating or a cross-linking reagent.

[0016] In another aspect of the invention, there are provided methods for reducing the risk of thrombosis caused by a venous access device in vivo, which include: a) contacting at least a portion of a venous access device with a composition including recombinant hirudin, whereby the recombinant hirudin is adsorbed to at least a portion of the exterior substrate material of the venous access device; b) removing unbound or loosely bound recombinant hirudin from the exterior substrate material to give rise to an at least partially treated venous access device; and c) inserting at least a portion of the treated venous access device into a vein or artery of an animal or human. [0017] Related aspects of the present invention are also directed to kits for treating the exterior of a venous access device, the kit including a venous access device and an antithrombotically effective amount of recombinant hirudin adsorbed to at least a portion of the exterior substrate material of the venous access device. These and other aspects of the present invention are more fully described in the drawings, the detailed description, and the examples which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The invention may be understood by reference to the following description taken in conjunction with the accompanying drawings, in which:

[0019] FIG. 1 shows a schematic diagram illustrating a proposed mechanism as to why desulfato lepirudin remains adsorbed to hydrophobic VAD surfaces, whereas sulfated hirudin is washed away.

[0020] FIG. 2 shows a schematic diagram of one embodiment of a kit for treating a venous access device with an antithrombotically effective amount of recombinant hirudin.

[0021] FIG. 3 shows a schematic diagram of one embodiment of a kit for treating a venous access device with recombinant hirudin.

[0022] FIG. 4 shows the effect of treating catheter segments with whole blood alone compared to treating them with lepirudin followed by 30 minute soaking in whole blood.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0023] The instant invention is based in part on an unexpected finding that particular recombinant forms of hirudin, exemplified by lepirudin, can be effectively used to impart antithrombotic properties to venous access devices. Also surprising is the discovery detailed herein that these devices can be produced efficiently to yield excellent antithrombotic results, without requiring a binding coating or requiring a covalently binding cross-linking reagent. These novel features of the invention are advantageously used in various methods, devices and kits.

[0024] Without wishing to be bound by a particular theory or mechanism of operation, it is believed that the hydrophobic N-terminal portion of lepirudin adsorbs to hydrophobic surfaces, while the hydrophilic C-terminal portion does not adsorb, but remains "floating and active" above the substrate surface. It is further proposed that the desulfato tyrosine in the 63 position avoids interacting strongly with flowing blood components that would otherwise strip away the lepirudin molecule from the substrate surface.

[0025] Referring to FIG. 1, it is believed that the hydrophobic N-terminal portion 12 of a recombinant hirudin readily adsorbs to the hydrophobic surface 32 of a substrate material of a VAD 30, while the desulfato hydrophilic C-terminal portion 14 remains "floating and active" above the substrate surface 32. In this regard, the absence of sulfate groups on the tyrosine amino acids in position 63 or 64 helps to balance the thermodynamics of adsorption of recombinant hirudin molecules to hydrophobic surfaces with the binding energetics of blood components (e.g., thrombin) to adsorbed hirudin molecules. In other words, it is believed that recombinant hirudin works as well as it does at rendering VADs antithrombotic in the present invention for at least two reasons: first, the lack of sulfate groups in the C-terminal end reduces overall polarity, which favors adsorption to hydrophobic surfaces; second, the lack of sulfate groups helps to reduce binding interactions between the bound recombinant hirudin molecules and the blood components in the flow stream. This is further illustrated in Figure 1 , where the indicated blood components 20 are shown to have an affinity for the hydrophilic C-terminal groups 14. However, the blood components can desorb from the C-terminal groups 14, as illustrated by reference numeral 22, and then continue moving in the flow stream as illustrated by reference numeral 24. In contrast, sulfation of hirudin analogues in the C-terminal end increases overall polarity of the lepirudin molecule, which thereby reduces the strength of adsorption of the lepirudin molecule to the hydrophobic substrate surface.

[0026] Moreover, hirudin generates its antithrombotic action by binding directly to thrombin at multiple sites; the N-terminal globular domain binds near the active site of thrombin, while the extended C-terminal segment makes ionic interactions with the fibrinogen recognition exosite (FRE) of thrombin. (Sohn et al., Id.) Because the sulfate group in the C-terminal segment increases the affinity of hirudin for thrombin (apparently through the FRE), a stable complex is formed that may be stripped from the surface by the hydrodynamic velocity gradient

(i.e., the fluid shear field near a surface, as indicated by 50), leaving behind a potentially thrombogenic surface. Furthermore, once it is tightly complexed with thrombin, even hirudin that remains adsorbed has no potential for further antithrombotic activity. However, the corresponding lack of sulfation in recombinant hirudins, e.g., lepirudin and desirudin, results in reduced affinity for thrombin. This promotes reversible binding between the recombinant hirudins and thrombin. Therefore, the hydrodynamic velocity gradient is more likely to remove thrombin from the adsorbed recombinant hirudin than to remove the intact complex of recombinant hirudin and thrombin from the surface. Free thrombin that is swept away will encounter natural inhibitors in the blood and become neutralized, whereas the recombinant hirudin that is left adsorbed is available to bind other thrombin molecules close to the surface. Therefore, the lack of sulfation of recombinant hirudins allows them to serve as a renewable source of antithrombotic activity at the surface. Thus, the overall effect of lack of sulfation of recombinant hirudins is to increase their overall adsorption energy to hydrophobic surfaces while sufficiently decreasing their affinity for thrombin to make them a more persistent source of antithrombotic activity.

[0027] Suitable recombinant hirudins are obtainable through recombinant DNA technologies. In certain embodiments, the recombinant hirudin contains a compact hydrophobic core containing the N-terminal half of the molecule and an extended, very hydrophilic C- terminal region. The core-region typically has three disulfide bridges that restrict the configuration of the core. Suitable recombinant hirudins comprise an amino acid sequence (54)- Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-65 (SEQ ID NO. 1) in the hydrophilic C- terminal portion of the protein molecule, which is typical for the HV-1 and HV-2 isoforms of hirudin. Other isoforms of recombinant hirudin can be used in various embodiments of the present invention. For example, several suitable isoforms of recombinant hirudin vary in the amino acid terminal group, which may be leucine or isoleucine. Often, the recombinant hirudins lack a sulfate group on the tyrosine in the C-terminal portion, which is typically at position 63 or 64. (Sohn et al., Id.) Other suitable hirudin isoforms lack a sulfate group on the tyrosine in position 63 and have a leucine at the amino terminal. Several recombinant hirudin analogues have been developed in quantities sufficient for human therapeutic uses. One recombinant hirudin, lepirudin (Refludan (TM), Hoechst Marion Roussel, Frankfurt am Main, Germany), is a desulfatohirudin having an N-terminal of Leu-Thr. Lepirudin is biotechno logically manufactured from S. cerevisiae, is FDA approved, and is useful in various embodiments of the invention. Another suitable recombinant hirudin, desirudin (Revasc™, Rhone-Poulenc-Rorer), is also FDA-approved, and differs from lepirudin only on the first two N-terminal amino acids (Val-Val).

[0028] In one embodiment of the invention there are provided venous access devices that include a substrate material and recombinant hirudin adsorbed to at least a portion of the exterior surface of the substrate material. In this aspect of the invention, the hirudin is found to adhere sufficiently to a variety of substrates without requiring a binding coating or cross-linking reagents.

[0029] Suitable venous access devices (VADs), such as peripheral intravenous catheters for short-term use and central venous catheters for long-term use. can be made antithrombotic by treatment with recombinant hirudin. These venous access devices typically do not require a binding coating or cross-linking reagents to ensure suitable adherence of recombinant hirudin to a variety of substrates. Instead, recombinant hirudin is found to adhere sufficiently to various types of substrate materials that comprise VADs.

[0030] In one embodiment of the present invention, a suitable VAD substrate material includes a hydrophobic material to sufficiently bind recombinant hirudin. Suitable hydrophobic materials typically include a polymeric material such as polysiloxanes, polyurethanes, polyvinyl chlorides, polyolefins, fluoropolymers such polytetrafiuoroethylene, and any combinations thereof. In carrying out certain preferred aspects of the present invention, it is desirable that the hydrophobic polysiloxane substrate materials include a silicone rubbery material. Preferably, suitable hydrophobic materials are flexible. As used herein, the term "flexible" typically means that a VAD of the present invention is capable of deforming by flexion to accommodate manual or remote insertion into a vein or artery.

[0031] The venous access devices in certain embodiments of the present invention include recombinant hirudin molecules, typically lepirudin, adsorbed onto their substrate surfaces. Typical amounts of surface-adsorbed lepirudin effective for reducing thrombosis range from about 1 ng /cm² to about 1000 ng /cm², and in more detailed embodiments from about 10 ng/cm² to about 500 ng /cm², often from about 50 ng /cm² to about 300 ng /cm² , and in certain embodiments from about 100 ng /cm to about 200 ng /cm . In preferred embodiments of the invention, the amount of adsorbed lepirudin is enough to form about at least one monolayer of lepirudin. Although certain embodiments may include surfaces having less than a monolayer of adsorbed lepirudin, it is preferred that the VADs will have at least about one monolayer of adsorbed lepirudin. In certain embodiments, higher amounts of lepirudin, i.e., greater than a monolayer, may be advantageous for forming thicker layers of lepirudin on the VAD substrate. Thicker lepirudin layers may be useful for temporarily increasing the antithrombotic activity of a newly inserted VAD.

[0032] The recombinant hirudin typically completely covers the exterior surface of the

VAD, although various embodiments include incomplete coverage. By "exterior surface" is meant the exposed surfaces of the VAD, which may be the outside and/or the lumenal surface of a vascular graft, or any surface, e.g., of a valve implant exposed to the blood. For example, it is desirable for certain embodiments of the present invention to ensure that recombinant hirudin molecules are substantially absent from the portion of the VAD that traverses a venipuncture site. The portion of the VAD that traverses the venipuncture site typically includes the end of the VAD the passes through the vein wall and out of the patient. Keeping the recombinant hirudin substantially absent from this portion of the VAD helps to reduce bleeding arising from the VAD passing through the vein wall.

[0033] The present invention is not limited to only recombinant hirudin-treated venous access devices, but also includes other medical devices that reside within the body cavity or are exposed to blood and can be a source of thrombosis. In these embodiments of the present invention, there are provided medical devices which include a substrate material, and a recombinant hirudin adsorbed on the surface of the substrate material. Examples of suitable medical devices typically include indwelling hemodialysis shunts and tubing in hemodialysis instruments and other instruments for extracorporeal circulation, such as heart-lung bypass machines. Examples of other suitable medical devices which can be treated with recombinant hirudin also include just about any type of device or surgical material that can be implanted in a body and be in contact with blood, such as artificial cardiac valves, patches, implantable devices like drug pumps, pacemakers, bone repair devices/implants, sutures, and pins.

[0034] The medical devices of the present invention are typically constructed from substrate materials that are hydrophobic. In certain embodiments, the hydrophobic surfaces of the substrate materials provide for attractive hydrophobic-hydrophobic interactions with the N- terminal end of recombinant hirudin molecules, while repelling the hydrophilic C-terminal end. Suitable hydrophobic materials can be provided by a variety of polymeric materials, examples of which include polysiloxane, polyurethane, polyvinyl chloride, and polytetrafluoroethylene, as well as any combinations thereof. In more detailed aspects, of the invention, it is desirable for the medical devices to include rubbery materials, such as a silicone rubber. Rubbery materials are particularly desirable where flexibility, a resilient surface, or a combination of both resiliency and flexibility are desired. In certain embodiments, the medical devices of the invention include a layer of recombinant hirudin adsorbed to the substrate surface of the device. Although this recombinant hirudin layer may only partially cover the substrate surface, it may completely cover the substrate surface. Examples include venous access devices, indwelling hemodialysis shunts, and tubing in hemodialysis instruments and other instruments for extracorporeal circulation, such as heart-lung bypass machines.

[0035] The medical devices in certain embodiments of the invention typically include recombinant hirudin molecules adsorbed onto a substrate surface of the device. Typical amounts of surface-adsorbed recombinant hirudin effective for reducing thrombosis of in-dwelling medical devices range from about 1 ng/cm² to about 1000 ng/cm², while various detailed embodiments feature alternative amounts of surface-adsorbed recombinant hirudin as detailed above. In preferred embodiments of the invention, the amount of adsorbed recombinant hirudin is sufficient to form about at least one monolayer on the substrate surface. Although certain embodiments may include surfaces having less than a monolayer of adsorbed recombinant hirudin, it is more preferred that the medical devices will have at least about one monolayer of adsorbed recombinant hirudin. In certain embodiments, higher amounts of recombinant hirudin, i.e., greater than a monolayer, may be advantageous for forming thicker recombinant hirudin layers. Thicker layers may be useful for temporarily increasing the antithrombotic activity of a newly inserted VAD.

[0036] The recombinant hirudin typically completely covers the exterior surface of the medical device, while various embodiments may include devices having incomplete coverage. For example, it is desirable for certain embodiments of the present invention to ensure that recombinant hirudin molecules are substantially absent from a portion of the medical device that may cause bleeding. Portions of medical devices that are prone to cause bleeding include, for example, sections that traverse a venipuncture site. The portion of the medical device that traverses the venipuncture site typically includes the end of the medical device the passes through the vein wall and out of the patient. Keeping the recombinant hirudin substantially absent from this portion of the VAD helps to reduce bleeding arising from the VAD passing through the vein wall.

[0037] The medical devices of the present invention can be prepared using various methods in which any of the recombinant hirudin analogues are contacted to a substrate surface of a medical device. The methods of treating a medical device with a recombinant hirudin typically include contacting the substrate surface(s) of medical devices with a composition comprising a recombinant hirudin to effect adsorption for the recombinant hirudin molecules to the substrate surfaces, and removing unbound or loosely bound recombinant hirudin. As used herein, the term "loosely bound" is meant that recombinant hirudin is readily removed from the substrate surface by submersion in, or a single rinsing with, a suitable rinsing solution, such as saline. Loosely bound recombinant hirudin is typically not bound covalently to the substrate surface. Loosely bound recombinant hirudin is typically not absorbed into the substrate material or an absorbent material residing on the substrate. While all of the accessible substrate surfaces of the medical devices may be contacted with recombinant hirudin, it is typically sufficient to contact those substrate surfaces that are exposed to blood flow when placed in vivo.

[0038] In one embodiment of the present invention, medical devices having an external substrate surface made from a hydrophobic material are coated with a solution including a recombinant hirudin. While these solutions may contain almost any type of liquid, solvent or non-solvent, the solutions are typically water-based, i.e., containing water. Typical formulations of recombinant hirudin solutions that are useful in carrying out these methods contain sodium chloride, glucose, organic and inorganic buffers.

[0039] The coating step can include any of a variety of coating processes known in the coating art, examples of which include but are not limited to soaking, flow coating, dip coating, spray coating, roller coating, and channel coating. The present invention typically does not require a precoating or treatment step, such as with a binding coating or a cross-linking reagent, to ensure the efficacy of the adhesion strength of the adsorbed recombinant hirudin to the substrate surface.

[0040] In one embodiment of the present invention, a medical device is dipped into an aqueous solution of a recombinant hirudin and dried. The coated medical device is removed from the aqueous solution and excess solution is allowed to drip off prior to drying. Drying can be optionally carried out in a sterile and clean environment. In this embodiment, the amount of adsorbed recombinant hirudin can be controlled by the concentration and viscosity of the recombinant hirudin solution. Viscosity modifiers know in the art can be formulated into the aqueous solution for controlling the coating thickness of liquid coatings.

[0041] In another embodiment a catheter is plugged at one or both ends and is dipped into an aqueous solution of a recombinant hirudin. Alternatively, an aqueous solution of recombinant hirudin is coated onto the catheter. In these embodiments, the aqueous solution typically comprises a concentration of recombinant hirudin in the range of from about 0.25 mg/ml to about 200 mg/ml, more typically from about 0.5 mg/ml to about 100 mg/ml, even more typically from about 1 mg/ml to about 50 mg/ml, further typically from about 2.5 mg/ml to about 25 mg/ml, and preferably in the range of from about 5 mg/ml to about 10 mg/ml. Such aqueous solutions are typically sodium chloride in water.

[0042] In one embodiment of the present invention, an untreated silicone rubber catheter is initially soaked in recombinant hirudin for about one to about five minutes and is then washed extensively with saline to remove loosely bound lepirudin. In this embodiment, a thin layer of adsorbed lepirudin remains adsorbed to the silicone rubber substrate surface.

[0043] In another aspect of the present invention, there are provided methods for reducing the risk of thrombosis caused by a venous access device in vivo. Methods to reduce the risk of thrombosis typically include first contacting at least a portion of a venous access device with a composition comprising recombinant hirudin. In these methods, this contacting step typically gives rise to recombinant hirudin molecules being adsorbed to at least a portion of the exterior substrate material of the venous access device. Contacting can be carried out by flushing, flowing, coating, dipping, spraying, soaking or any combination thereof. After this contacting step, loosely bound recombinant hirudin molecules are removed from the exterior substrate material to give rise to an at least partially treated venous access device. Here, it is desirable to remove excess recombinant hirudin that may otherwise contribute to excessive bleeding at the venipuncture site. Subsequently, the treated venous access device is inserted into a vein or artery of an animal or human. Although the entire length of the treated venous access device may be inserted into a vein, typically a portion of the venous access device that is exterior to the body does not require such treatment.

[0044] In certain embodiments to reduce the risk of thrombosis by venous access devices, such as in an operating environment, the wet treated venous access is simply wiped with a sterile wiper to remove excess recombinant hirudin solution clinging to the substrate surface prior to insertion. Air drying is not required in such embodiments as the aqueous component can be readily absorbed by the body.

[0045] In other embodiments of the present invention, the venous access device having wet recombinant hirudin solution clinging to it can be rinsed with a suitable rinsing solution, such as saline, to remove the excess recombinant hirudin solution. The rinsed venous access devices will have recombinant hirudin molecules clinging to the substrate surface. The rinsed venous access devices may be subsequently wiped with a sterile wiper, air dried, or both wiped and died, prior to insertion. Alternatively, the catheter may be inserted wet.

[0046] In one embodiment, the risk of thrombosis by a catheter can be reduced by initially soaking a catheter in recombinant hirudin and then washing it extensively with saline to remove loosely bound recombinant hirudin. While the soaking time needs to be sufficient to permit adsorbtion of lepirudin to the catheter surface, a soaking time of about one to five minutes is typical. Removing the loosely bound lepirudin helps to prevent local bleeding, as loosely bound lepirudin may be stripped off from the catheter as it is inserted through the skin or tunneled through subcutaneous tissue. In addition to reducing thrombosis, local bleeding is reduced at the point where the catheter traverses the vein wall.

[0047] In one embodiment, reducing the risk of thrombosis by adsorbing a catheter with recombinant hirudin is performed by an operator, such as a surgeon, nurse, radiologist, or other medical professional, who implants the catheter. This allows the operator to treat only the portion of the catheter that will be intravenous, while avoiding the portion that will traverse the vein wall and pass extravascularly. Avoiding the coating of recombinant hirudin on the portions of the catheter that traverse the vein wall, or pass extravascularly, or both, typically will help to reduce bleeding. Here, it is desirable that the untreated portion of the inserted venous access device contacts the wall of the vein or artery. Typically, such an untreated portion in contact with the wall of a vein or artery is at least about one centimeter long. The catheter may be treated any time before insertion, but is typically treated within about 30 minutes before insertion, more typically within about 10 minutes of insertion, and even more typically immediately before insertion.

[0048] In certain embodiments of the invention, it is desirable for an operator to reduce the risk of deep vein thrombosis when using uncoated VADs. As used herein, "uncoated venous access device" means that the venous access device does not include a coating that increases the binding strength of recombinant hirudin to the substrate surface. Suitable uncoated venous access devices include devices that have a bare hydrophobic substrate material present at a surface. For example, uncoated VADs comprising a bare hydrophobic polymer have hydrophobic polymer molecules present at the surface of the bare hydrophobic substrate material. Thus, uncoated VADs prepared from fluorinated polymeric materials have fluorinated polymers present at the surface of the bare substrate material. Likewise, uncoated VADs prepared from rubbery silicone materials have silicone polymers present at the surface of the bare substrate material. In these embodiments, recombinant hirudin is typically adsorbed to the exterior substrate material of an uncoated VAD through a hydrophobic interaction arising from the hydrophobic core of the recombinant hirudin and the substrate surface.

[0049] In other embodiments of the invention, it is desirable for an operator to reduce the risk of deep vein thrombosis when using long catheter lengths that require shortening prior to insertion. In these methods, long untreated catheters can be trimmed prior to insertion into the vein or artery. Trimming VADs is necessary in order adapt their length to the body size of different patients. Trimming can be performed before or after treating the catheter with recombinant hirudin solution. In this embodiment, trimming is preferably performed before this treatment step.

[0050] Kits for treating venous access devices with an antithrombotically effective amount of recombinant hirudin are also provided by the present invention. The recombinant hirudin used in the kits may have any physical form, but it is typically in the form of a solution, a dispersion, or a dry mass, e.g., a powder. Typically, the recombinant hirudin is separately packaged in the kit, such as in a vial, an ampule, or a syringe.

[0051] In one embodiment, the recombinant hirudin in the kit is in the form of a lyophilized powder. Lyophilization can be carried out by freezing an aqueous solution of a recombinant hirudin to about -80 degrees centigrade, and then subliming away the water under vacuum. This process is typically very gentle on the recombinant hirudin proteins. Lyophilized desirudin is commercially available from Berlex Laboratories, Montville, New Jersey, and lyophilized lepirudin is commercially available from Rhone-Poulenc Rorer, Collegeville, Pennsylvania.

[0052] In another embodiment, kits including recombinant hirudin in a dry mass form can further include a carrier solution for reconstituting the powder. Typical carrier solutions include water or saline (0.9% sodium chloride in water). The carrier solution is typically packaged separately in the kit, such as in a vial, syringe, or ampule. When mixed, an antithrombotically effective amount of recombinant hirudin combined with the carrier solution gives rise to a concentration of recombinant hirudin in the range of from about 1 mg/ml to about 50 mg/ml. Typically, the carrier solution is included in a separate package in the kit, such as in a vial, syringe, or ampule.

[0053] In another embodiment of the present invention, the kit may further contain a venous access device, such as a catheter for short-term peripheral or central venous access or long-term central venous access. In using such kit, the kit is opened, and the venous access device is not rinsed prior to treating with an antithrombotically effective amount of recombinant hirudin solution that is supplied with, or prepared from, recombinant hirudin as described above.

[0054] In another embodiment of the present invention, the kit may further include an applicator for applying the antithrombotically effective amount of recombinant hirudin to the venous access device. In this embodiment, any type of applicator suitable for contacting a solution to a venous access device may be used.

[0055] In the various embodiments of the invention, it is also envisioned that the kits further include an instruction set to instruct an operator to treat a venous access device using the kit. As an example, an instruction set lists the steps to take to use the components of a kit to treat a venous access device. For example, the instruction set for a kit to treat a catheter would indicate to an operator the following: preparing a solution of recombinant hirudin (such as mixing a lyophilized recombinant hirudin powder and a diluent); preparing a catheter for treatment (such as by rinsing the catheter thoroughly with saline); applying the recombinant hirudin solution to the catheter; rinsing off excess recombinant hirudin solution from the catheter; and inserting the treated catheter into a body.

[0056] Referring to FIG. 2, one embodiment for a kit 100 includes a catheter 102 that is packaged inside a piece of tubing 104. Although the tubing 104 shown in this embodiment is straight, in other embodiments, particularly for long catheters, the tubing may be coiled. The external portion of the catheter 106 to the tubing passes through a water-tight seal 108 into the tubing 104. The end of the tubing opposite to the external portion of the catheter is equipped with a fitting 110 for a syringe 112. The recombinant hirudin solution is prepared by the operator by dissolving a lyophilized powder of a recombinant hirudin into a diluent in a suitable container. Both the recombinant hirudin lyophilized powder and the diluent can be separately packaged (e.g., in vials) in the kit (not shown). The recombinant hirudin solution is then drawn into the syringe 112. The operator measures the distance on the patient from the expected venipuncture site to the external end of the catheter (i.e., the portion of the catheter expected to remain outside of the vein plus the portion expected to traverse the venipuncture site) and pulls the external portion of the catheter 106 out of the tubing. The syringe 112 containing the recombinant hirudin solution 114 is then injected into the tubing 104. The portion of the catheter remaining in the tubing is then bathed in a solution of recombinant hirudin injected from the syringe. The catheter is typically left in the recombinant hirudin bath for at least five minutes. When ready to insert the catheter into a vein, the operator pulls the remainder of the catheter from the tubing. The catheter can be inserted into the vein with an introducer that protects the venipuncture site from the lepirudin coating on the catheter (not shown).

[0057] In another embodiment of the present invention, the kit may further include a rinsing fluid for removing loosely bound recombinant hirudin from the treated venous access device. Typical rinsing fluids include sterile water and saline (0.9% sodium chloride in water). The rinsing fluid can be packaged separately in the kit, such as in a vial, syringe, or ampule.

[0058] An alternate embodiment includes kits (FIG. 3) having break-seal packages 200 for holding dry-mass recombinant hirudin 208 (e.g., lyophilized powders) in compartment 206 and a carrier solution 204 in compartment 202. In this embodiment, a breakable seal 210 separating the dry-mass recombinant hirudin from the carrier solution in a single package can be broken by an operator, and the components mixed by shaking. The breakable seal can be any type of material (e.g., glass or plastic) that keeps the carrier solution separated from the dry mass recombinant hirudin prior to use. Mixing the contents of the two compartments reconstitutes the dry mass recombinant hirudin into a solution suitable for treating venous access devices.

Optionally, the break-seal package may include a porous surface 218 through which the recombinant hirudin solution can be applied to a catheter. In embodiments wherein the package is made of a suitably flexible material, such as plastic, the recombinant hirudin solution can be applied to a suitable VAD by squeezing the solution out of the package through the porous surface 218, which can be used as an applicator for applying the recombinant hirudin solution to the VAD or medical device. As shown in FIG. 3, the porous surface 218 is flat, however, it may also be shaped to conform to the outer surfaces of VADs or medical devices to function as a suitable applicator. Also, the breakable seal 210 is depicted flat, however, it may also be shaped, for example, to enhance rupture when used. Although the break-seal package 200 is shown as substantially cylindrical in shape, it should be apparent to those skilled in the art that other shaped packages are also envisioned to be within the scope of the present invention.

[0059] An alternate embodiment includes kits having a break-seal package for holding dry-mass recombinant hirudin, a carrier solution, and a rinsing fluid in separate chambers. In this embodiment, a seal separating the dry-mass recombinant hirudin from the carrier solution can be broken and these two components can be mixed by shaking the break-seal package. Mixing the two components reconstitutes the dry mass recombinant hirudin into a solution suitable for treating venous access devices. Excess recombinant hirudin solution is rinsed off the treated venous access device by aid of the rinsing solution, which is accessible by an opening in the package or a second break seal. Optionally, the break-seal package may include an integral applicator for applying the recombinant hirudin solution to a venous access device. The optional integral applicator may also aid in rinsing the excess recombinant hirudin solution from the treated venous access device.

[0060] These and other aspects of the present invention are more fully described in the examples which follow.

EXAMPLES

[0061] Sources of Materials Used:

[0062] Lepirudin, lyophilized, obtained from Hoechst Marion Roussel, Frankfurt am Main, Germany.

[0063] Bovine serum albumin, lyophilized, obtained from ICN Biomedicals, Inc., Aurora, OH.

[0064] S2238™, obtained from Chromogenix, Milano, Italy.

[0065] Throughout the experiments lepirudin was dissolved in tris-buffered saline (TBS: 0.1 M NaCl, 0.04 M tris, pH 7.4) and maintained in polypropylene tubes. The silicone catheters and tubing were handled wearing latex gloves to prevent adsorption of skin proteins.

[0066] Sections of catheters (Leonard™ Dual-Lumen CV Catheter, Bard Access Systems, Salt Lake City, Utah) were cut with a sharp razor into 6.5 cm sections. The open ends of the sections were occluded with small plastic plugs. The sections were then formed into loops and submerged for 5 minutes in 1 mL volumes of TBS containing 1% bovine serum albumen (BSA) or 5 mg/mL lepirudin in 1.5 mL polypropylene tubes. Care was taken to keep the ends of the catheter loops above the solution. The submerged portion of each loop measured 5 cm. After removal from solution each catheter segment was gently wiped with Precision Wipes (Kimberly-Clark). Some segments were then immediately washed in a continuous stream of TBS at 1.5 mL/sec with a total volume of 200 mL. Others were submerged for 10, 30, or 60 minutes in 3 mL of freshly drawn whole blood anticoagulated with 11 mM sodium citrate. Other segments were not exposed to lepirudin but were soaked in blood for 30 minutes. After the exposure to blood each catheter segment was blotted with Precision Wipes and washed with 200 mL of TBS as described above.

[0067] Each segment was again blotted and placed lengthwise into a 1 mL polypropylene cylinder containing 0.6 mL of approximately 1 unit/mL thrombin. Care was taken to submerge the entire segment in the solution. After 10 minutes each segment was removed, and the solution was assayed for thrombin activity using a chromo genie substrate (S2238).

[0068] In preliminary experiments it was shown that catheter loops that had only been exposed to TBS inhibited trace amounts of thrombin, presumably by adsorption of the protein. The result was similar with the catheter loops were first soaked in TBS containing 1% BSA. In subsequent experiments TBS/BSA-treated catheter segments were used to control for this slight reduction in thrombin activity.

[0069] Figure 4 shows the effect of treating the catheter segments with whole blood alone compared with treating them with lepirudin followed by 30 minute soaking in whole blood. Although exposure to whole blood alone resulted in a degree of thrombin inhibition (9.5 ±6.0 mU), pretreatment with lepirudin increased this threefold (30.0 ± 17 mU) (p=0.007).

[0070] While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of examples, in the drawings, and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as embodied by the appended claims.

Claims

What is Claimed is:

1. A venous access device, comprising:

a) a substrate material; and

b) recombinant hirudin adsorbed to at least a portion of the exterior surface of said substrate material.

2. The venous access device according to claim 1, wherein said venous access device comprises a catheter.

3. The venous access device according to claim 1, wherein said substrate material is uncoated.

4. The venous access device according to claim 1, wherein said substrate material comprises a hydrophobic material.

5. The venous access device according to claim 4, wherein said hydrophobic material comprises a polymeric material.

6. The venous access device according to claim 5, wherein said polymeric material comprises at least one of a polysiloxane, a polyurethane, a polyolefin, a polyvinylchloride, a fluoropolymer, and any combinations thereof.

7. The venous access device according to claim 6, wherein said polysiloxane comprises a silicone rubber.

8. The venous access device according to claim 1, wherein said recombinant hirudin is physisorbed to said substrate material.

9. The venous access device according to claim 1, wherein said recombinant hirudin forms a layer on said substrate material.

10. The venous access device according to claim 9, wherein said layer is a monolayer.

11. The venous access device according to claim 10, wherein said monolayer completely covers said exterior surface.

12. The venous access device according to claim 1, wherein said recombinant hirudin comprises a desulfato hirudin.

13. The venous access device according to claim 1, wherein said desulfato hirudin comprises lepirudin, desirudin, or combinations thereof.

14. The venous access device according to claim 1, wherein said recombinant hirudin comprises SEQ ID NO. 1.

15. The venous access device according to claim 14, wherein said amino acid sequence lacks a sulfate group on the tyrosine in position 63.

16. The venous access device according to claim 1, wherein said recombinant hirudin has a leucine at the amino terminal.

17. The venous access device according to claim 1, wherein said recombinant hirudin lacks a sulfate group on the tyrosine in position 63 and has a leucine at the amino terminal.

18. The venous access device according to claim 1, wherein said recombinant hirudin comprises lepirudin.

19. A medical device, comprising:

a) a substrate material; and

b) a recombinant hirudin adsorbed to the surface of said substrate material.

20. The medical device according to claim 19, wherein said medical device is a venous access device.

21. The medical device according to claim 20, wherein said venous access device is a catheter.

22. The medical device according to claim 19, wherein said substrate material comprises a hydrophobic material.

23. The medical device according to claim 22, wherein said hydrophobic material comprises a polymeric material.

24. The medical device according to claim 23, wherein said polymeric material comprises at least one of a polysiloxane, a polyurethane, a polyolefin, a polyvinylchloride, a fluoropolymer, and any combinations thereof.

25. The medical device according to claim 24, wherein said polysiloxane comprises a silicone rubber.

26. The medical device according to claim 19, wherein said recombinant hirudin is physisorbed to said substrate material.

27. The medical device according to claim 19, wherein said recombinant hirudin forms a layer on said substrate material.

28. The medical device according to claim 27, wherein said layer is a monolayer.

29. The medical device according to claim 28, wherein said monolayer completely covers said exterior surface.

30. The medical device according to claim 19, wherein said recombinant hirudin comprises lepirudin or desirudin.

31. The medical device according to claim 19, wherein said recombinant hirudin comprises SEQ ID NO 1.

32. The medical device according to claim 31, wherein said amino acid sequence lacks a sulfate group on the tyrosine in position 63.

33. The medical device according to claim 31 , wherein said recombinant hirudin has a leucine at the amino terminal.

34. The medical device according to claim 31 , wherein said recombinant hirudin lacks a sulfate group on the tyrosine in position 63 and has a leucine at the amino terminal.

35. The medical device according to claim 34, wherein said recombinant hirudin comprises lepirudin.

36. A method of treating a medical device with a recombinant hirudin, comprising:

a) contacting a substrate material of said medical device with a composition comprising said recombinant hirudin; and

b) removing loosely bound recombinant hirudin from said substrate material.

37. The method according to claim 36, wherein said medical device is a venous access device.

38. The method according to claim 0, wherein said venous access device is a catheter.

39. The method according to claim 36, wherein said composition comprises an aqueous solution of recombinant hirudin.

40. The method according to claim 39, wherein said aqueous solution comprises a concentration of recombinant hirudin in the range of from about 1 mg/ml to about 100 mg/ml.

41. The method according to claim 36, wherein said recombinant hirudin comprises lepirudin or desirudin.

42. The method according to claim 36, wherein said recombinant hirudin comprises a desulfato tyrosine group at position 63.

43. The method according to claim 36, wherein said recombinant hirudin comprises a leucine group at the amino terminal.

44. The method according to claim 36, wherein said recombinant hirudin comprises a desulfato tyrosine group at position 63 and a leucine group at the amino terminal.

45. The method according to claim 44, wherein said recombinant hirudin comprises lepirudin.

46. A method for reducing the risk of thrombosis caused by a venous access device, comprising:

a) contacting at least a portion of a venous access device with a composition comprising recombinant hirudin, said contacting to yield recombinant hirudin adsorbed to at least a portion of an exterior substrate material of said venous access device;

b) removing loosely bound recombinant hirudin from said exterior substrate material to give rise to an at least partially treated venous access device; and

c) inserting at least a portion of said at least partially treated venous access device into a vein or artery of an animal or human.

47. The method according to claim 46, wherein said composition comprises an aqueous solution of recombinant hirudin.

48. The method according to claim 47, wherein said aqueous solution comprises a concentration of recombinant hirudin in the range of from about 0.25 mg/ml to about 200 mg/ml.

49. The method according to claim 46, wherein said recombinant hirudin comprises lepirudin or desirudin.

50. The method according to claim 46, wherein said recombinant hirudin comprises an desulfato tyrosine group at position 63.

51. The method according to claim 46, wherein said recombinant hirudin comprises a leucine group at the amino terminal.

52. The method according to claim 46, wherein said recombinant hirudin comprises an desulfato tyrosine group at position 63 and a leucine group at the amino terminal.

53. The method according to claim 52, wherein said recombinant hirudin is lepirudin.

54. The method according to claim 46, wherein said substrate material is uncoated.

55. The method according to claim 46, wherein said substrate material comprises a silicone rubber.

56. The method according to claim 46, wherein said contacting of said venous access device with said recombinant hirudin is carried out by flushing, flowing, coating, dipping, spraying, or any combination thereof.

57. The method according to claim 46, wherein said venous access device is completely contacted with said recombinant hirudin, to yield a completely treated venous access device.

58. The method according to claim 46, wherein the untreated portion of the inserted venous access device contacts the wall of said vein or artery.

59. The method according to claim 58, wherein said wall is located where said venous access device is inserted into said vein or artery.

60. The method according to claim 58, wherein said untreated portion in contact with said wall of vein or artery is at least about one centimeter long.

61. The method according to claim 46, wherein said loosely bound recombinant hirudin is removed by rinsing with a fluid.

62. The method according to claim 61, wherein said fluid comprises saline.

63. The method according to claim 46, wherein said treated venous access device is prepared by an operator prior to insertion into said vein or artery.

64. The method according to claim 63, wherein said venous access device is prepared about 30 minutes prior to insertion into said vein or artery.

65. The method according to claim 64, wherein said venous access device is prepared about ten minutes prior to insertion into said vein or artery.

66. The method according to claim 46, wherein said adsorbed recombinant hirudin is physisorbed to said exterior substrate material through a hydrophobic interaction.

67. The method according to claim 46, further comprising trimming said venous access device prior to insertion into said vein or artery.

68. The method according to claim 67, wherein said venous access device is trimmed to shorten the length of said treated portion.

69. A kit for treating the exterior of a venous access device, comprising an antithrombotically effective amount of recombinant hirudin.

70. The kit according to claim 69, wherein said recombinant hirudin is in the form of a lyophilized powder.

71. The kit according to claim 70, further comprising a carrier solution for reconstituting said lyophilized powder.

72. The kit according to claim 71, wherein said antithrombotically effective amount of recombinant hirudin combined with said carrier solution gives rise to a concentration of recombinant hirudin in the range of from about 0.25 mg/ml to about 200 mg/ml.

73. The kit according to claim 69, wherein said recombinant hirudin comprises lepirudin or desirudin.

74. The kit according to claim 69, wherein said recombinant hirudin comprises SEQ ID NO. 1.

75. The kit according to claim 74, wherein said amino acid sequence comprises a desulfato tyrosine in position 63.

76. The kit according to claim 69, wherein said recombinant hirudin comprises leucine at the amino terminal.

77. The kit according to claim 69, wherein said recombinant hirudin comprises a desulfato tyrosine in position 63 and leucine at the amino terminal.

78. The kit according to claim 75, wherein said recombinant hirudin comprises lepirudin.

79. The kit according to claim 69, further comprising said venous access device.

80. The kit according to claim 79, wherein said venous access device is a catheter.

81. The kit according to claim 79, wherein said venous access device comprises a substrate material, said substrate material comprising a hydrophobic material.

82. The kit according to claim 81, wherein said hydrophobic material comprises a polymeric material.

83. The kit according to claim 82, wherein said polymeric material comprises at least one of a polysiloxane, a polyurethane, a polyolefin, a polyvinylchloride, a fluoropolymer, and any combinations thereof.

84. The kit according to claim 83, wherein said polysiloxane comprises a silicone rubber.

85. The kit according to claim 69, further comprising an applicator for applying said antithrombotically effective amount of recombinant hirudin to said venous access device.

86. The kit according to claim 85, wherein said applicator comprises a grooved channel, an orifice, an absorbent material, a syringe, or any combination thereof.

87. The kit according to claim 69, further comprising a rinsing fluid for removing loosely bound recombinant hirudin from the treated venous access device.

88. The kit according to claim 69, further comprising an instruction set.