Source: http://www.google.com/patents/US8128589?dq=5,889,522
Timestamp: 2014-10-02 04:32:33
Document Index: 528179203

Matched Legal Cases: ['Application No. 05703144', 'Application No. 176972', 'Application No. 176972', 'Application No. 160090', 'Application No. 05703144', 'Application No. 176972', 'Application No. 176972', 'Application No. 2006', 'Application No. 2006', 'Application No. 2006', 'Application No. 2006']

Patent US8128589 - Apparatus and methods for enzymatic debridement of skin lesions - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsAn apparatus for debridement of devitalized tissue in skin lesions, that includes a plurality of height- and angle-adjustable inlet tubes and at least one outlet tube and a member that forms an occlusive seal around a skin lesion. The plurality of inlet tubes is adapted for directing a continuous stream...http://www.google.com/patents/US8128589?utm_source=gb-gplus-sharePatent US8128589 - Apparatus and methods for enzymatic debridement of skin lesionsAdvanced Patent SearchPublication numberUS8128589 B2Publication typeGrantApplication numberUS 11/493,381Publication dateMar 6, 2012Filing dateJul 25, 2006Priority dateJan 27, 2004Also published asCA2554330A1, CN1929883A, EP1720586A1, EP1720586B1, EP1720586B8, US7364565, US20040186421, US20070041960, US20120004627, WO2005070480A1Publication number11493381, 493381, US 8128589 B2, US 8128589B2, US-B2-8128589, US8128589 B2, US8128589B2InventorsAmihay Freeman, Eran Hirszowicz, Michal Be'eri-LippermanOriginal AssigneeRamot At Tel-Aviv University Ltd., Enzysurge Ltd.Export CitationBiBTeX, EndNote, RefManPatent Citations (96), Non-Patent Citations (21), Referenced by (2), Classifications (52), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetApparatus and methods for enzymatic debridement of skin lesionsUS 8128589 B2Abstract An apparatus for debridement of devitalized tissue in skin lesions, that includes a plurality of height- and angle-adjustable inlet tubes and at least one outlet tube and a member that forms an occlusive seal around a skin lesion. The plurality of inlet tubes is adapted for directing a continuous stream of enzymatic solution to the surface and into the entire volume of the wound bed of the lesion and the at least one outlet is adapted for removing the enzymatic solution, fluids draining from the lesion and tissue debris from the occluded skin lesion.
What is claimed is: 1. An applicator for treating a skin lesion, comprising:
19. The method according to claim 18, further comprising: (a) providing an apparatus comprising an applicator, said applicator comprising a housing unit having at least one aperture formed therein, and means for affixing the applicator, wherein said housing unit comprising : (i) a plurality of inlet tubes, each of said plurality of inlet tubes having a first longitudinal axis and configured to be adjustable along its longitudinal axis through said at least one aperture; and (ii) at least one outlet tube having a second longitudinal axis; (b) providing a spacer for occluding the skin lesion, the spacer having a lower plane facing the skin, an upper plane facing the housing of said applicator, wherein the lower plane comprises adhesive means for affixing the spacer to the skin at the circumference of said skin lesion; (c) affixing the lower plane of the spacer to the skin at the circumference of said skin lesion; (d) affixing the applicator to the upper plane of the spacer, thereby obtaining an occluded lesion; (e) connecting the plurality of inlet tubes to at least one reservoir by a fluid communication, wherein the at least one reservoir encompassing a debriding solution comprising at least one catalytically active protease; (f) initiating a flow of the debriding solution from the at least one reservoir through at least one inlet tube of the plurality of inlet tubes to the occluded lesion; and (g) draining said solution from said occluded lesion through the at least one outlet tube.
20. The method according to claim 18, wherein step (c) further comprises dispersing a sealing medium at the edge of the spacer that contacts the circumference of the occluded lesion.
21. The method according to claim 18, further comprising adjusting the position and angle of each of said plurality of inlets with respect to said housing unit.
22. The method according to claim 18, wherein connecting the plurality of inlet tubes to at least one reservoir by a fluid communication, provides a liquid-impermeable, vacuum-proof seal around the occluded lesion.
23. The method according to claim 18, further comprising providing control means being in fluid communication with at least one inlet tube of said plurality of inlet tubes and with the at least one reservoir, thereby initiating a controlled flow of fluids from the at least one reservoir through the at least one inlet tube, wherein the flow has a rate within the range of 1 ml/hour to 10 ml/hour.
24. The method according to claim 18, further comprising positioning the at least one reservoir on a higher level than the lesion, thereby initiating flow of debriding solution from the at least one reservoir through the plurality of inlet tubes to the lesion by gravitation.
25. The method according to claim 18, further comprising providing a plurality of control means, each control means being in fluid communication with a corresponding inlet tube of the plurality of inlet tubes and with the at least one reservoir, thereby controlling the flow rate within each inlet tube independently by a separate control means, wherein the flow rate within each inlet tube is within the range of 1 ml/hour to 10 ml/hour.
26. The method according to claim 18, further comprising providing at least one element being in fluid communication with the at least one reservoir and at least one inlet tube of the plurality of inlet tubes, the element is selected from the group consisting of: a control means adapted for controlling the rate of flow from the at least one reservoir to the at least one inlet tube; a connector adapted for opening and closing the fluid communication between the at least one reservoir and the at least one inlet tube; a filter for filtering a solution flowing within the at least one inlet tube; a mixing means for mixing the solution within the at least one reservoir; a thermo-regulating means for affecting the temperature of the solution within the at least one reservoir; a thermo-regulating means for affecting the temperature of the solution flowing within the at least one inlet tube; a separator being in fluid communication with at least one inlet tube of said plurality of inlet tubes and with the at least one reservoir, thereby to reversibly disconnect and reconnect said at least one inlet tube from said at least one reservoir; a vacuum source being in fluid communication with the at least one outlet tube, adapted for generating a negative pressure at the occluded skin lesion; and a collecting reservoir being in fluid communication with the at least one outlet tube configured to collect the fluids drained from the proximal end of said at least one outlet tube.
CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International application PCT/IL2005/000101 filed Jan. 27, 2005, which is a continuation-in-part of application Ser. No. 10/768,749 filed Jan. 27, 2004 now U.S. Pat. No. 7,364,565. The entire content of each prior application is expressly incorporated herein by reference thereto.
FIELD OF THE INVENTION The present invention relates to apparatus and methods for debridement of devitalized tissue in skin lesions, the apparatus comprising a plurality of height-and angle-adjustable inlet tubes, at least one outlet tube and means to form an occlusive seal around a skin lesion.
BACKGROUND OF THE INVENTION Acute and chronic diseases, such as diabetes and psoriasis, or acute injuries result in a severe damage to the skin. This damage may involve the entire thickness of the skin and may often include deeper tissues wherein the depth of the damage varies over the entire damaged zone. The damaged skin loses the anatomic organization of a healthy skin as the stratum corneum is at least partially destroyed and consequently the inner layers of the skin are no longer protected from the external environment. Moreover, the damaged skin typically contains dead eschar, diseased and/or abnormal cells that must be removed in order to enable healing. Leaving the dead eschar in place extends and deepens the damage into the neighboring, undamaged tissues. This dead eschar also serves as a medium for bacteria growth, and a source of infection, contamination and sepsis which may be life threatening.
Removal of the dead eschar, diseased and/or abnormal cells, also known as �debridement�, is executed either by surgical procedures or by using enzymatic means. Surgery is one of the most common procedures of debridement wherein small necrotic areas are excised of the entire damaged skin. This method is limited to small non-tangential surfaces. It also involves the removal of large fractions of healthy tissue which, if preserved, could serve as a source for the natural healing processes. Surgical procedures are also long, expensive and require complicated medical resources.
U.S. Pat. No. 3,910,266 describes a method and apparatus that provide a jet of pressurized fluids which is used for penetrating the skin and inserting cosmetic or therapeutic agents into the skin. U.S. Pat. No. 5,697,920 describes means for mechanic debriding using a jet of pressurized fluids and a brush. U.S. Pat. Nos. 5,941,859; 5,989,211 and 6,264,666 describe medical instruments for supplying to and removing rinsing fluids from the skin. A hand-held surgical apparatus adapted to be used substantially as a sharp surgical tool for removal of diseased tissue by utilizing pressurized fluid jets, is described in U.S. Pat. Nos. 5,037,431. 5,358,494 describes an irrigation dressing comprising a conduit for supplying the irrigation fluids and pad attached at the tip of the conduit wherein the pad is adapted to fill the wound cavity, thereby supporting the walls of the wound. However, the methods and apparatus described in the above patents are not adapted for providing a sealed system that occludes a defined treatment zone. Furthermore, these methods and apparatus cannot provide a sealed environment that encompasses the wound and that is resistant to pressure accumulated therein.
Enzymatic debridement is advantageous over mechanical and surgical debridement mainly since it is less painful and does not involve the loss of a great deal of blood. The application of proteolytic enzymes for debridement is well known in the art (G. Rodeheaver, 1975, Am. J. Surg. 129(5):537-544). These enzymes include those generally found in to plant sources, such as papaya (papain), fig (ficin), and pineapple (bromelain). Hydrolytic enzymes derived from the pineapple plant that are useful for digestion, dissection and separation of non-viable, especially eschar tissue, from viable tissue in a mammalian host are described in U.S. Pat. Nos. 4,197,291; 4,226,854; 4,307,081; 4,329,430 and 5,830,739 among others. U.S. Pat. No. 6,017,531 describes a proteolytic composition which includes an extracellular neutral protease produced by Vibrio proteolyticus. The degree of therapeutic activity obtained from topical application of proteolytic enzymes is governed, inter alia, by the intrinsic catalytic characteristics of those enzymes. The major problems associated with topical use of compositions comprising proteolytic enzymes are that the catalytic activity of the enzymes rapidly attenuates due to the typical low pH at the lesion area, adsorption of enzyme molecules to the surface of the wound bed and/or the surface of the dressing thus preventing their accessibility to other regions at the wound bed and inhibition of enzymatic activity by moieties within the wound exudates. Accordingly, obtaining stable enzymatic formulations is often complicated.
SUMMARY OF THE INVENTION The present invention relates to apparatus and methods for debriding the devitalized tissue of skin lesions. The apparatus of the present invention overcomes the drawbacks of the background art by providing a continuous flow of therapeutic solutions into the wound bed of the skin lesion, through a plurality of adjustable inlet tubes. In an embodiment of the present invention, the therapeutic solutions may include catalytically active proteolytic enzymes.
It is to be understood that the terms �skin lesion� and �lesion� as used herein are to be construed according to their broadest meaning, to describe damaged skin comprising devitalized tissue, including but not limited to, chronic cutaneous ulcers (e.g. diabetic ulcers and decubitus ulcers) and burns. The skin lesion may extend through all or through part of the skin layers and may further extend through the underlying muscles and tissues.
(a) a housing unit having at least one aperture formed therein; and (b) means for affixing the applicator to the skin around the circumference of skin lesion; wherein said housing unit comprising:
(i) a plurality of inlet tubes, each of said plurality of inlet tubes having a first longitudinal axis and configured to be adjustable along its longitudinal axis through said at least one aperture; and (ii) at least one outlet tube having a second longitudinal axis. According to one embodiment, the housing unit comprises a plurality of apertures, wherein each of said plurality of inlet tubes and the at least one outlet tube extend through a corresponding one of each of said plurality of apertures.
(a) a spacer for occluding an area comprising the skin lesion, the spacer having a lower plane facing the skin, an upper plane facing the housing, wherein the lower plane comprises adhesive means for affixing the spacer to the skin at the circumference of said skin lesion; and (b) an applicator comprising a housing unit, said housing unit comprising:
(i) a plurality of inlet tubes, each of said plurality of inlet tubes having a first longitudinal axis and configured to be adjustable along its longitudinal axis through said at least one aperture; (ii) at least one outlet tube having a second longitudinal axis; and (iii) means for affixing the applicator to the upper plane of the spacer. According to one embodiment, the spacer comprises an elastomer. According to another embodiment, the elastomer is a foam-like material. According to yet another embodiment, the spacer comprises a material selected from the group consisting of: silicone, silicone foam, polyurethane, natural rubber, neoprene and ethyl vinyl acetate foam. According to yet another embodiment, the adhesive means for affixing the spacer to the skin comprising a material selected from the group consisting of: thermoplastic resin, pressure sensitive adhesive, hydrocolloid adhesive and rubber. According to another embodiment, the lower plane of the spacer is covered with a protective detachable film
(i) a plurality of inlet tubes, each of said plurality of inlet tubes having a first longitudinal axis and configured to be adjustable along its longitudinal axis through said at least one aperture; and (ii) at least one outlet tube having a second longitudinal axis; (b) placing against the skin at the circumference of said skin lesion said means, thereby affixing the apparatus to the skin at the circumference of said skin lesion to obtain an occluded lesion; (c) connecting the plurality of inlet tubes to at least one reservoir by a fluid communication, wherein the at least one reservoir encompassing a debriding solution comprising at least one catalytically active protease; (d) initiating a flow of the debriding solution from the at least one reservoir through at least one inlet tube of the plurality of inlet tubes to the occluded lesion; and (e) draining said solution from said occluded lesion through the at least one outlet tube. According to one embodiment, the method further comprises adjusting the position and angle of each of said plurality of inlet tubes with respect to said housing.
(i) a plurality of inlet tubes, each of said plurality of inlet tubes having a first longitudinal axis and configured to be adjustable along its longitudinal axis through said at least one aperture; and (ii) at least one outlet tube having a second longitudinal axis; (b) providing a spacer for occluding the skin lesion, the spacer having a lower plane facing the skin, an upper plane facing the housing of said applicator, wherein the lower plane comprises adhesive means for affixing the spacer to the skin at the circumference of said skin lesion; (c) affixing the lower plane of the spacer to the skin at the circumference of said skin lesion; (f) affixing the applicator to the upper plane of the spacer, thereby obtaining an occluded lesion; (g) connecting the plurality of inlet tubes to at least one reservoir by a fluid communication, wherein the at least one reservoir encompassing a debriding solution comprising at least one catalytically active protease; (h) initiating a flow of the debriding solution from the at least one reservoir through at least one inlet tube of the plurality of inlet tubes to the occluded lesion; and (i) draining said solution from said occluded lesion through the at least one outlet tube. According to yet another embodiment step (c) further comprises dispersing a sealing medium at the periphery of the occluded lesion which contacts the edge of the spacer but is not covered thereby.
BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B are schematic isometric and side views of the apparatus, constructed and operative in accordance with an embodiment of the invention;
The terms �skin lesion� and �lesion� are interchangeable used herein to describe damaged skin comprising devitalized tissue, including but not limited to, chronic cutaneous ulcers (e.g. diabetic ulcers and decubitus ulcers) and burns. The skin lesion may extend through all or through part of the skin layers and may further extend through the underlying muscles and tissues.
�Stage X� is commonly used to classify skin lesions. Lesion types are classified in stages according to the severity of the lesion. The staging system applies to burn wounds, decubitus ulcers and several other types of ulcers and lesions. STAGE I is a superficial lesion characterized by a surface reddening of the skin. The skin is unbroken. This lesion may be, inter alia, a beginning decubitus ulcer and tends to heal spontaneously when pressure is relieved on the area. STAGE II is characterized by a blister either broken or unbroken wherein at least a partial layer of the skin is injured. Stage II decubitus ulcer or pressure wound may develop into Stage III decubitus ulcer or pressure wound. STAGE III lesion extends through all of the layers of the skin and may involve a serious infection. STAGE IV lesion extends through the skin and involves underlying muscle, tendons and bone. This type of lesion can produce a life threatening infection if not aggressively treated. STAGE V is an older classification of a lesion that is extremely deep, having gone through the muscle layers and involving underlying organs and bone. Amputation may be necessary is some situations.
The term �devitalized tissue� as used herein refers to necrotic tissue or eschar, from cutaneous ulcers or burns which consists of a complex mixture of dried blood, purulent exudates, and denatured proteins normally found in the epidermal and dermal skin layers. The denatured proteins are primarily collagen, elastin, fibrin, hemoglobin, and other coagulated proteins. Collagen comprises about 75% of the skin's dry weight and is the main constituent of the necrotic debris and of eschar. Strands of semi-viable, compromised collagen, whose protective mucopolysaccharide sheath has been damaged or destroyed, anchor the necrotic tissue to the wound surface. These strands must be fully eliminated in order for the necrotic material to be separated from its base. This complete debridement then permits development of granulation tissue during the healing process.
The term �debridement� as used herein refers to the process of removing the non-viable tissue from a lesion to prevent infection and to facilitate healing as healing of lesion is a complex process which is often further complicated by the presence of non-viable, necrotic tissue in the wound bed.
The term �wound bed preparation� as used herein is to be construed in its most general sense and refers to the global management of the wound to accelerate endogenous healing or to facilitate the effectiveness of therapeutic modalities. Wound bed preparation of acute wounds includes debridement and removal of necrotic tissue and bacteria. In chronic wounds, wound bed preparation is more complicated as most of the necrotic matter cannot not be easily accessed, and since the preparation further includes removal of exudates.
Preferably, each inlet and outlet tube should be fitted with a suitable sealing unit, such as an �O� ring, for example, to prevent the passage of fluids between the external diameter of the tubes and its corresponding aperture.
As used herein the phrase �in fluid communication� refers mainly to the capability of selective or non-selective transfer of fluid and/or semi-fluid substances between the specified elements. Such transfer may be accomplished by, for example, channels, tubes, membranes, conduits, pores and/or capillaries.
In yet a further embodiment of the present invention, first reservoir 10 is in fluid communication with a filter 16 which serves for sterilization of the protease solution prior to its application. Filter 16 is preferably a sealed (except for inlet and outlet ports), sterilized housing containing a filtering member excluding particles greater than, for example, 0.25 microns, eliminating common bacterial contamination. One such commercially available filter is distributed under the name Complete Sterifil System (Sigma Chemical Company, Inc.). In a further embodiment of the present invention, first reservoir 10 is in fluid communication with a pump 18 which serves for streaming the protease solution from first reservoir 10 to an applicator 24 (illustratively described in detail hereinbelow) under positive pressure. Thus, the protease solution is delivered to the site of treatment with sufficient force to effect a mechanical, �stripping� action in addition to the enzymatic digestion of matrix proteins. The novel combination of a directional, mechanical force and enzymatic disruption of the lesion tissue provided by the present invention enables the removal of cells and tissue from the treated surfaces.
The term �height� as used herein refers to the length of the inlet tube that extends beyond the opening 108 towards the lesion. This length may also correspond to the distance between opening 108 and the tip of the inlet tube which faces the lesion.
One embodiment of applicator 24 is illustrated in FIGS. 10-12. Inlet port 20 and outlet port 22 provide directional fluid motion for the stream of protease solution, enabling a mechanical �stripping� effect enhancing the enzymatic disruption of the intracellular matrix and removal of cells from the treated lesion surface. Applicator 24 may be engaged with the skin surface by skin-ward pressure applied by attendant operators or treated subject, weight, adhesive connection to adjacent skin surfaces or other means, suitable for the body part bearing the lesion to be treated. In one preferred embodiment applicator 24 comprises an engaging mechanism 26, which comprises two or more flexible elements adjustably connected to allow encirclement of a cylindrical body part (such as a limb or torso) and application of skin-ward pressure through tension, such as a strap and buckle or toothed belt fastener.
Applicator 24 may be constructed of durable, non-porous material including, but not limited to, glass, metal, plastic or rubber, and may be reusable or preferably disposable. Applicator 24 is preferably capable of sterilization by gas, chemicals, moist or dry heat, or radiation, and is supplied sealed and sterilized for use. In one alternative embodiment, applicator 24 is a �push-pull� cannula typically employed in tissue perfusion techniques, for example, as described by Arancibia, S., in �Push-pull Perfusion Technique In Neuroendocrinology�, Ann. Endocrinol. (Paris) 48, 410-18 (1987), which comprises an inflow tube recessed within a wider, outflow tube, creating localized flow of protease solution confined to the outer diameter of the wider, outflow tube.
It will be noted that the fluid outflow from cell collector 30 contains largely still active protease solution, devoid of the cellular and tissue debris fractions removed by filter 32 and/or centrifuge 40 which may be recycled for reuse. Thus, in one preferred embodiment the fluid outflow of cell collector 30 is reintroduced to the stream of at least one protease solution �upstream� of applicator 24 and pump 18. Fluid communication between the cell collector outflow and the stream of protease solution may be effected by a one-way valve connection, ensuring uni-directional streaming of fluid towards applicator 24. Thus, significant economy of operation is achieved by reuse of the cell collector 30 outflow, effectively reducing the volume of protease solution required per treatment.
FIGS. 7-9 depict enzymatic surgery apparatus 80 designed to receive prepared reservoirs or ampoules of protease, protease solution and/or protease activating solution. In one embodiment, illustrated in FIG. 7, a receptacle 42 is designed to receive modular reservoir or ampoule 44, containing catalytically active protease solution, effecting fluid communication with applicator 24, cell collector 30 and additional �downstream� elements of apparatus 80. Thus, apparatus 80 may be operated with standardized, pre-prepared, stored protease solution(s), increasing simplicity of use and accuracy of protease activity delivered, and decreasing risk of contamination of treated skin surfaces.
As used herein in the specification and in the claims section below, the terms �reservoir� and �ampoule� interchangeably refer to a separate, enclosed container capable of establishing fluid communication with other containers, receptacles or devices. Such reservoirs or ampoules typically contain fluids or fluid-like substances, and may be designed to be accurately engaged by a complementary receptacle or housing. Sealed reservoirs or ampoules provide convenient, standardized means of preparation and storage of active solutions and reagents for the operation of, for example, enzymatic surgery apparatus 80.
The ability of proteases to gently disrupt the integrity of dermal tissue has led to the therapeutic use of proteolytic enzymes as an adjunct, or alternative to mechanical or laser surgical treatment of skin lesions. In order for such enzymatic treatment to overcome the abovementioned disadvantages of surgical, electrosurgical, cryosurgical and laser-surgical methods (pain, scarring, traumatic stress, hyperpigmentation and destruction of neighboring tissue), it is desirable for the proteolytic method to readily and thoroughly hydrolyze a wide variety of proteins found in skin lesions; function at physiological pH and temperature; be compatible with adjunct therapies (e.g., anesthetics, cleansing agents, topical antibiotics); and not interfere with normal wound heating or complicate skin grafting. In addition, it is important to provide means of retention and preservation of the viability of the isolated, removed cells for histological examination or cell culture; to allow for localized and confined application of the protease and provide for stability of the enzyme formulations from the effects of pH, temperature and autoproteolysis. These and other beneficial considerations are addressed, for the first time in an integrative approach, by the present invention. Thus, benefits provided by the present invention include gentle enzymatic tissue removal enhanced by mechanical �stripping� action of the locally directed protease stream, superior pain reduction and wound healing provided by inclusion of anesthetics, coagulants/anticoagulants and antibiotics in the protease solution and availability of removed skin cells for histological examination and/or cell culture from the treated lesions. In addition, control of temperature, ph and flow rate of the stream of protease solution, and provision for on-site activation of stabilized enzyme preparations ensure delivery of accurate, effective levels of catalytic activity, to the lesion surface.
As used herein, the term �protease� refers to any biologically active molecule, typically a polypeptide, possessing enzymatic peptide hydrolase activity, including endopeptidase and/or exopeptidase activity.
In one preferred embodiment of the present invention, the protease is, but not limited to, vibriolysin, krill protease, chymotrypsin, trypsin, collagenase, elastase, dipase, proteinase K, Clostridium multifunctional protease and Bacillus subtillis protease. These represent proteases commonly employed in therapeutic methods, have demonstrated low incidence of undesirable side effects, and are commercially available in pure, purified or genetically engineered form, for example, Esperase, Subtilisin A, Savinase, and Durazyme, available from Novo Nordisk Bioindustry Japan K.K.; Protease N �Amano�, Protease S �Amano�, available from Amano Pharmaceutical K.K.; Bioprase, available from Nagase Seikagaku Kogyo K.K.; and Purified Collagenase, available from Advance Biofactures, Lynbrook, N.Y. Clostridium multifunctional protease and krill protease are easily prepared by one skilled in the art, for example, as disclosed in U.S. Pat. No. 6,416,626 to Markert et al., and U.S. Pat. No. 5,958,406 to de Faire et al., respectively.
As used herein, the phrase �local anesthetic� refers to any agent applied within a proscribed region (e.g., not systemically) effecting significant reduction or inhibition of activity of nonciceptive substances, receptors and/or neural pathways. Non-limiting examples of commonly used local anesthetic agents are cyclo-oxygenase inhibitors (e. g. ibuprofen, indomethacin and ketorolac), 5-hydroxytryptamine receptor antagonists (e.g. amytryptyline), bradykinine receptor antagonists and histamine receptor antagonists.
As used herein, the term �coagulant� is defined as any agent that promotes clotting, or coagulation of blood, which may be safety applied to a dermatological lesion. A non-limiting example of such a coagulant material comprising gelatin, thrombin and calcium is described in U.S. Pat. No. 6,045,570 to Epstein, et al. Likewise, the term �anti-coagulant� refers to any agent which retards, inhibits or prevents the clotting or coagulation of blood, which�may be safely applied to a dermatological lesion, such as heparins, coumarins or other agents possessing thrombolytic activity.
As used herein in the specification and in the claims section below, the phrase �pharmaceutically acceptable carrier� refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid filter, diluent, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can perform its intended function. Each carrier must be �acceptable� in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar, buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; fruit acids, pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
As used herein, an �effective amount� of antibiotic is intended to include the amount of antibiotic sufficient to significantly prevent and inhibit at least 50%, preferably 75% and most preferably 100% of microbial growth within a dermatological lesion of the subject being treated, such effective amount determined by one skilled in the art.
It will be appreciated, in the context of the present invention, that autolysis and loss of functional enzyme concentration from catalytically active preparations of proteases constitutes a significant disadvantage of therapeutic administration of enzymes in topical, injected and/or other compositions. Active shelf life of the protease is limited, and precise control of enzyme activity at the site of administration is virtually unattainable, once injection or topical application is completed. A number of inventions have proposed the storage of biologically active substances, including enzymes, in contact with substances or under conditions limiting their native activity, effectively inactivation and stabilization, until contacted with substantially adequate amount of activating substance, or conditions sufficient to restore biological activity. For example, Edens, et al (U.S. Pat. No. 6,117,433) teach the stabilization of biologically active substances, such as vitamins, enzymes and antibiotics in high concentrations by preparation in water activity lowering agents such as salts, polyols, sequestering agents such as EDTA, phyate or gluconate, or antioxidants such as sulphites, glutathione, cysteine or ascorbic acid. Crystallized compositions of biologically active substances, typically more stable than aqueous preparations, are mixed with viscosifying agents to retard precipitation and ensure homogeneity of the biologically active composition. The disclosure further describes a dispensing system for such stabilized formulations, activating the biologically active substance by dilution with an aqueous composition. Nakagawa et al. in U.S. Pat. No. 5,409,546 describes the stabilization of serine protease derived from bacteria belonging to genus Bacillus for contact lens cleanser composition by addition of polyols, and the specification of a defined range of temperatures (room temperature to about 58� C.) within which the enzyme retains catalytic activity. Rowan et al. in U.S. Pat. No. 5,106,621 teaches the restoration of catalytic activity of a plant cysteine protease for treatment of burn wounds by addition of cysteine for regeneration of thiol groups. None of the aforementioned examples, however, relate to the administration of proteases for treatment of living cells, nor provide for ongoing, precise control of the activation of catalytic activity at the site of application.
Thus, in an embodiment of the present invention, there is provided a method for treating skin lesion protease is activated shortly prior to streaming the solution containing the effective amount of the at least one protease, over, and in contact with, the treated skin portion. The method wherein the protease is activated may be effected by: (a) keeping the protease at a first temperature in which the protease is substantially catalytically inactive and heating and/or cooling the at least one protease to a second temperature in which the at least one protease is catalytically active; and/or (b) providing the protease in a powder form and mixing the powder with a solution in which the protease is catalytically active; and/or (c) providing the protease in a first solution in which the protease is substantially catalytically inactive and mixing the first solution with a second solution so as to achieve a mixed solution in which the protease is catalytically active. The second solution may differ from the first solution with respect to pH, ion concentration, free metal concentration, hydrophilicity and hydrophobicity. For example, FIG. 3 depicts enzymatic surgery apparatus 80 in fluid communication with thermoregulator 14, enabling filling of first reservoir 10 with protease solution at sub-optimal, stabilizing temperatures, restoring catalytic activity by raising the temperature of the protease solution only shortly prior to application at the lesion site. Typically, enzymes are substantially inactivated at temperatures below 10� C., preferably 4� C. Activation of enzyme catalytic activity may be accomplished by heating and/or cooling the protease solution to optimal temperature, typically in the range of 30 to 40� C., preferably 37� C.
As used herein, the term �hydrophilicity� refers to the polar nature of a solution or compound, indicating its tendency to be attracted to other solutions or compounds exhibiting significant dipole moments. Likewise, the term �hydrophobicity� refers to the non-polar nature of a compound or solution, indicating its tendency to be repelled by and immiscible in other compound or solutions exhibiting significant dipole moments.
As used herein, the term �inactivation� refers to the reversible or irreversible suppression or loss of catalytic activity, for example, inactivation rendering proteolytic enzymes incapable of catalyzing hydrolysis of peptide bonds.
It will be appreciated, in the context of the present invention, that catalytic activity of enzymes may be modified by activators and inhibitors. One such mode of regulation of enzyme activity is reversible inhibition, effected by the interaction of substrate analogs or regulatory molecules which cause changes in substrate binding and/or enzyme kinetics, effectively reducing catalytic activity, for example, as described in �Enzymes�, chapter 3, in Molecular Cell Biology (1986): Darnell, J, Lodish, H and Baltimore, D, eds., Scientific American Books, Inc. Since such reversible inhibition of enzyme activity is concentration dependent, restoration of catalytic activity is achieved by contacting the inhibited enzyme preparation with appropriate volumes of diluent devoid of inhibitors. Thus, in a further embodiment of the present invention, stabilization of the protease solution is effected by the inclusion of an effective amount of reversible enzyme inhibitor(s). Activation of the stabilized protease preparation is effected by dilution with adequate volumes of activating solution devoid of inhibitor/and or inhibitor activity.
It will be appreciated that the combination of mechanical �stripping� and enzymatic action of a stream of protease solution on the skin surface is suitable for removal of skin cells and debris for esthetic purposes. Thus, in a further embodiment of the present invention, controlled streaming of a protease solution may be used to cosmetically treat esthetically undesirable portions of the skin surface.
In another, more preferred embodiment the device and method of the present invention are employed to provide protease irrigation, removal and/or sampling for biopsy of a tissue surface or surfaces via the abovementioned �push-pull� cannula in a closed, fiber optic-directed surgical procedure. Non-limiting examples of such procedures are arthroscopy, cystoseopy, endoscopy, cholecystoscopy, laparoscopy, colonoscopy, and myringoscopy.
As used herein, the term �treatment� includes the diminishment or alleviation of at least one symptom associated or caused by the disorder being treated. For example, treatment can be diminishment of several symptoms of a disorder or complete eradication of a disorder.
In the context of the present invention, it will be appreciated that confining the enzymatic activity to a stream of protease solution directed at the lesion surface, rather than topical application of creams or intradermal injection, provides the opportunity for retention of the cells removed from the treated lesion. Thus, the present invention provides a method of removing and collecting cells from a skin portion of a subject inflicted with a dermatological lesion, the method effected by streaming a solution containing an effective amount of at least one protease, over, and in contact with, the skin portion, thereby removing the cells from the skin portion of the subject; and collecting the cells. The products of protease digestion at the site of treatment are removed through the at least one outlet tube and are transferred to cell collector container, which is in fluid communication with the applicator. Separation of tie fluid and cellular components of the outflow of protease solution from applicator 24 may be accomplished by filtration, or, in another embodiment, by continuous flow centrifugation, as described above. Small volume continuous flow centrifuges, commonly used for separation of blood components (for example, the OrthoPAT� System, Haemonetics Corporation, Braintree, Mass.) are commercially available and are easily adapted to the device of the present invention through fluid communication, as illustrated in FIG. 4. Alternatively, cell collection may be effected by retention on a column capable of adsorbing cells through interaction with proteinaceous, poly- and/or oligo saccharide or other cell-surface components.
Known cell separations involve several techniques, some of which are based on specific affinities. Other cell separation techniques rely on more serendipitous mechanisms such as entrapment of target cells in supports of various origins and structures. See, for example, Wigzell and Anderson, J. Exp. Med. 129:23-36, 1969; Rutishauser et al. Proc. Natl. Acad. Sci. 70, 1973; Wysocki and Sato, Proc. Natl. Acad. Sci. 75:2844-2848, 1978; Antoine et al. Immunochem. 15, 1987. See also, U.S. Pat. No. 6,008,040 to Datar. The basic process of affinity separation entails creating contact between cell mixtures to be separated and a support matrix to enable the target cells to preferentially attach, bind, adsorb or become trapped to and within the support, and then washing away the undesired cells, or vice-versa Specific affinity techniques use monoclonal antibodies to recognize specific markers on the membranes of cells and to �attract� the target cells to bind to the monoclonal antibodies. Specific affinity �attractions� of target cells also may occur by hydrophobic or hydrophilic interactions, metal-affinities, ion exchangers, and the like. Thus, in a further embodiment of the present invention, cell collection is effected by passage of the outflow stream from applicator 24 through cell collector 30 and contacting with a device, e.g. a cell-binding column, capable of retention of the cells and their separation from the outflow stream.
EXAMPLES Example 1 Enzymatic Debridement Using the Appartus and Methods of the Invention Materials and Methods
Histology: Following the 3 hours treatment, the mice and rats were sacrificed with an overdose of chloral hydrate (Fluka chemicals, Switzerland) and rabbit was sacrificed with an overdose of thiopenton sodium. Full-thickness skin samples (4�15 mm) were removed for histological analysis from the margins of the confined area to allow comparison of treated and non-treated areas in same slide. Tissue samples were immediately fixed in 4% phosphate buffered formaldehyde solution for 48 hours, processed by routine histological procedures and embedded in paraffin. Serial sections perpendicular to the skin surface were cut at 8 μ thickness. The sections thus obtained were stained with hematoxylin and eosin for observations.
1. Collagenase activity was assayed by the addition of 0.2 ml enzyme solution (1 mg/ml) into 3 ml of 0.25 mM Nα-Benzoyl-L-Arginine Ethyl Ester (B4500, Sigma) and 0.32 ml of 10 mM Dithioerythritol (D8255, Sigma) in 10 mM Tris buffer pH 7.5 containing 4 mM CaCl2 (102382, Merck) and measuring OD253 for 5 min. at room temperature. 2. Trypsin activity was assayed by the addition of 50 μl of enzyme solution [1 mg/ml] into one ml of substrate solution (5 mg of Nα-Benzoyl-DL-Arginine p-Nitroanilide (BAPNA, B4875, Sigma) dissolved in 0.5 ml of dimethylsulfoxide (DMSO, 102931, Merck) and added to 25 ml of Tris 10 mM pH 7.5. containing 4 mM CaCl2) and measuring OD405 for 5 minutes at room temperature. 3. Papain activity was assayed by the addition of 100 μl of enzyme solution (1 mg/ml) into 1 ml of BAPNA solution (prepared by dissolving 5 mg of BAPNA in 0.5 ml of DMSO and adding into 25 ml of 50 mM Phosphate buffer, pH 6.2 containing 5 mM Cysteine and 2 mM EDTA) and measuring OD405 for 5 minutes at room temperature. 4. Bromelain activity was assayed by the addition of 50 μl of the enzyme solution (1 mg/ml) into 5 ml of 1% Casein solution (44016, BDH) in 50 mM Tris pH 8.5 in test tubes, equilibrated to 37� C. Following incubation for 10 minutes at 37� C. and pH 8-8.5, five ml of 10% Trichloroacetic acid (TCA, 33731, Riedel-de Haen) were added and the mixture incubated for additional 5 minutes at 37� C. The mixture thus obtained was centrifuged at 7,000 rpm for 10 minutes and OD280 of the supernatant measured. 5. Thermolysin (Protease type X) activity was assayed as described above for Bromelain. 6. Pepsin activity assay: One ml of pepsin solution (0.01-0.05 mg/ml in 10 mM HCl) was added into 5 ml of 2% Hemoglobin solution (H2625, Sigma) in 10 mM HCl at 37� C. Following 10 minutes incubation, 10 ml of 5% TCA were added and the mixture incubated for additional 5 minutes at 37� C. The mixture thus obtained was centrifuged at 7,000 rpm for 10 minutes and OD280 of the supernatant measured.
Controlled streaming of enzymes could be readily and conveniently applied as series of consecutive treatments using a multichanel pump, as demonstrated in FIG. 13A for treatments of six anaesthetized rats or treatment of six different sites on a larger animal (FIG. 13B). Effective digestion of different skin layers was readily achieved by streaming diluted buffered enzyme solutions for 3 hrs. The controlled streaming of 2 mg/ml papain onto mice affected digestion and removal of the outer keratinized layer (FIGS. 3A with 3B). Detachment of the epidermis from the dermis was effected by trypsin (4 mg/ml) and bromelain (5 mg/ml) mixture (FIG. 14C). Controlled streaming of 8 mg/ml trypsin solution effected complete digestion of the epidermis layer (FIG. 14D). Streaming of 3 mg/ml pepsin resulted in deeper penetration and collagen fibers digestion (FIG. 14E). Streaming of a mixture of 3 mg/ml collagenase and 1.5 mg/ml thermolysin resulted in digestion similar to the shown in FIG. 14D. Similar results were obtained by streaming of same solutions on rat, rabbit and pig skin.
Example 2 Enzymatic Removal of Epidermis Using an apparatus including applicator 24 illustrated in FIG. 12, an enzyme solution containing Collagenase (1 mg/ml, Sigma Cat. No. C0130) and Thermolysin (0.5 mg/ml, Sigma type x, Cat No. P1512) in 0.1 M PBS buffer, pH 7.5, was applied onto a skin sample freshly removed from an adult female large-white pig (1 year old, 90 kg), mounted on a flat holder and pre-cleaned with 70% (v/v) aqueous ethanol, at a flow rate of 3-4 ml/hour for 3 hours at room temperature.
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USA, 70(12): 3894-3898 (1973).* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS8205352 *Jul 13, 2011Jun 26, 2012Applied Materials, Inc.Vapor dryer having hydrophilic end effectorUS20110266736 *Jul 13, 2011Nov 3, 2011Lewis John SVapor dryer having hydrophilic end effector* Cited by examinerClassifications U.S. Classification604/19International ClassificationA61K38/49, A61M27/00, A61N1/30, A61B17/34, A61M35/00, A61M3/02, A61K38/48, A61B17/00, A61B17/54, C12N5/071, A61B10/00, A61B10/02, A61B17/22Cooperative ClassificationA61K38/4886, A61M3/0287, A61B10/0035, A61M1/0088, A61B2017/00761, A61B2017/3411, A61B17/545, A61M3/0283, A61B10/02, C12M45/09, C12N2509/00, A61M3/0241, A61M27/00, A61K38/48, A61B2017/22082, A61M3/0258, A61B2017/00774, A61B2017/00769, C12N5/0629European ClassificationA61K38/48L, A61K38/49, A61K38/48K1, A61M1/00T6, A61K38/48N, A61K38/48K7, A61K38/48K36, A61M27/00, A61K38/48M, A61M3/02H4, A61M3/02D8B, A61K38/48, A61B10/02, A61B10/00E, C12N5/06B9K, C12M45/09, A61B17/54P, A61M3/02H2, A61M35/00Legal EventsDateCodeEventDescriptionNov 9, 2006ASAssignmentOwner name: ENZYSURGE LTD., ISRAELOwner name: RAMOT AT TEL-AVIV UNIVERSITY LTD., ISRAELFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FREEMAN, AMIHAY;HIRSZOWICZ, ERAN;BE ERI-LIPPERMAN, MICHAL;REEL/FRAME:018499/0897;SIGNING DATES FROM 20060920 TO 20061015Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FREEMAN, AMIHAY;HIRSZOWICZ, ERAN;BE ERI-LIPPERMAN, MICHAL;SIGNING DATES FROM 20060920 TO 20061015;REEL/FRAME:018499/0897RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google