Patent Application: US-201414325750-A

Abstract:
a wound dressing includes a backing material , and thrombin , applied to the backing material . preferably , the backing material includes biodegradable polyester , such as electrospun polycaprolactone . the thrombin preferably includes salmon thrombin adsorbed onto the backing material . a process of fabricating a wound dressing includes applying thrombin to the backing material . preferably , the thrombin is prepared from the plasma of a salmonid . the thrombin is applied to the backing material by adsorbing the thrombin onto the backing material , preferably after applying a detergent solution to the backing material . the dressing can be lyophilized and vacuum - sealed within an air - tight wrapping .

Description:
the process preferably begins with the consistent and reproducible conditions under which donor fish are reared . all fish used as plasma sources preferably are progeny of domesticated broodstock , inspected for fish disease according to the american fisheries society “ blue book ” standards , sexually immature , in the log - phase of growth , larger than two kilograms , reared by standard husbandry methods , and fed a commercially pelleted food appropriate to the species . water temperature at the time of bleeding is preferably 4 ° c . to 15 ° c . the fish are preferably starved for five days before bleeding to reduce proteolytic enzymes and non - protein nitrogen . each fish is stunned by a blow to the head , or by immersion in ice - water , or in water containing co 2 or other fish anesthetic , in order to stun the fish to a level of loss of reflex activity ( unconsciousness ). whole blood is then drawn from the caudal artery or vein with a sterile needle and syringe or vacuum tube containing an anticoagulant such as acd ( acid citrate dextrose ), trisodium citrate , or other anticoagulant commonly used in human blood - banking . whole blood is held , preferably for no more than four hours at 2 - 4 ° c ., and then centrifuged at 2 - 4 ° c . thrombin then can be prepared by the method of michaud et al . 2002 . briefly , prothrombin is extracted from plasma , for example , with a series of barium chloride and ammonium sulfate precipitations . the prothrombin is activated by echis carinatus venom or equivalent proteolytic enzymes that cleave the site required to activate prothrombin , and the thrombin is passed over a heparin column or other binding matrix such as sp - sepharose . thrombin is eluted from the column with a high salt buffer and frozen at & lt ;− 60 ° c . the thrombin may then be lyophilized . alternately , plasma fractionation or recombinant techniques can be used . these techniques are illustrative of those currently in use , but other techniques for preparation of thrombin or its precursor prothrombin may be equally effective . for example , frozen salmon thrombin was thawed at 4 - 6 ° c ., analyzed for activity , and held on ice for less than one hour . electrospun polycaprolactone ( pcl ), purchased from sns nanofiber technology , was cut into 2 cm squares , and 200 μl of thrombin was applied to each square . of course , pcl or other biodegradable polyester obtained from another source can be used . pcl is known to be hydrophobic , so it would not absorb the liquid thrombin . the pcl squares were therefore soaked in a detergent solution ( 0 . 5 mg / ml tween 80 ) and the result was that the thrombin penetrated between the fibers and was adsorbed on the pcl , other detergent solutions , such as triton x - 100 , may be used in place of the tween 80 . also , instead of soaking , the detergent solution can be applied by pipette , spraying , or any other method that allows it to soak into the backing material . alternatively , the detergent solution can be mixed with the thrombin and applied to the backing material with the thrombin . the pcl squares ( dressings ) with the adsorbed thrombin were then frozen at − 80 ° c ., lyophilized , vacuum packed in foil , and stored at refrigerator or room temperature ( fig1 ). after 90 and 180 days , the dressings were rehydrated with 200 μl of distilled water and held at room temperature for less than 5 minutes . the eluate containing thrombin was then pressed from the dressing and analyzed for thrombin activity . fig2 shows that more than 90 % of the original activity was retained for at least 180 days , demonstrating the stability of the dressing . although mammalian thrombins are contraindicated for treatment of pain , we have found that salmon - derived thrombin applied to an injury site is highly effective for reducing pain originating from injury to nerves and other tissue . this effectiveness is independent of any healing or structural relief provided at the injury site ; that is , the thrombin provides analgesic relief . the beneficial effects of salmon thrombin are likely related to differing cellular receptors . in contrast to the very similar reactivity of salmon and human thrombin to fibrinogen - based substrates ( michaud et al . 2002 ), we have found substantial differences in their ability to activate protease activated receptors ( par ). at 37 ° c . salmon thrombin cleaves par1 approximately three times more slowly than human thrombin ( p = 0 . 0013 ). these findings illustrate some of the functional differences in mammalian and salmon thrombin . salmon thrombin is an effective hemostat ( rothwell et al ., 2005 ), and the pain of serious injury is frequently accompanied by bleeding . therefore , a dressing that would treat both pain and bleeding is desirable . we have demonstrated the anti - inflammatory properties of salmon thrombin by experiments in a rat injury mode as shown in the following example . this anti - inflammatory effect adds a new and important dimension to the hemostatic / analgesic dressing . as an example of inflammation after injury , we chose a rat incisional model of post - surgical swelling and inflammation . the rat plantar - incision model is a well - accepted and predictive model of human swelling and inflammation . it allows investigation of inflammation by comparing the effects of test substances during and after surgery ( whiteside et al . 2004 ; brennan et al . 1996 ). rats were anesthetized with isoflurane ( 2 - 3 %) vaporized in a nose - cone . the plantar left hind paw was prepared in a sterile manner with an iodine solution and 70 % ethanol . a 1 cm - long incision starting 0 . 5 cm from the heel and extending toward the toes was made with a number 10 blade , through the skin and fascia of the plantar aspect of the paw including the underlying muscle ( brennan et al . 1996 ). the plantaris muscle was then elevated and longitudinally incised , leaving the muscle origin and insertion intact . the wound was blotted with a gauze pad and 50 μl salmon thrombin or a control ( distilled water ) was applied directly to the wound cavity . the skin was then closed with two mattress sutures of 5 - 0 nylon . at the end of surgery , anesthesia was stopped , and rats were allowed to recover before being returned to their cages . before surgery and 12 , 24 , and 48 hours after surgery , groups of rats ( n =− 4 ) were evaluated for inflammation by measuring paw volume using an ugo - basile paw plethysometer . the rat paw is placed initially in a calibrated cylinder filled with a mixture of distilled water , salt and a wetting agent . baseline and post - surgery paw volumes were obtained by measurement of the solution displacement , with the value being an average of two or three separate determinations . rats receiving the salmon thrombin treatment had significantly reduced swelling and inflammation at 12 , 24 , and 48 hours post - surgery compared to the animals treated with distilled water ( fig3 .) all rats gained weight , and the treatment was well tolerated as assessed by behavioral observations . thus , the present invention provides pain relief , hemostasis , and anti - inflammation in a single stable product ( dressing ) suitable for field , pre - hospital , or hospital use . a one - time application of the dressing is safe , effective , and long - lasting , without the addictive properties or need for multiple applications of other therapeutic treatments . the present invention has been described by way of example and in terms of preferred embodiments . however , it is to be understood that the present invention is not strictly limited to the particularly disclosed embodiments . to the contrary , various modifications , as well as similar arrangements , are included within the spirit and scope of the present invention . the scope of the appended claims , therefore , should be accorded the broadest possible interpretation so as to encompass all such modifications and similar arrangements . brennan t j , vandermeulen e p , gebhart g f . 1996 . characterization of a rat model of incisional pain . pain 64 : 493 - 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