MEANS FOR REMOVING MICROORGANISMS FROM TISSUE

Abstract: Means for removing microorganisms from tissue, such as mucous membranes, skin and wound tissue, which means comprises at least one protein or fragment(s) or residue(s) thereof which can be bound to the microorganisms, and optionally a carrier to which the protein(s) or the fragment(s) or residue(s) thereof is bound. The invention further concerns use of protein(s) or fragment(s) thereof for removing of microorganisms from tissue.

Description: MEANS FOR REMOVING MICROORGANISMS FROM TISSUE The present invention concerns means for removing micrα- orgartisms from tissue, such as mucous membranes, skin and wound tissue. /* On skin and mucous membranes there is an indigenous flora of nαrf-pathogenic as well as potentially pathogenic microorga¬ nisms. The presence of the indigenous flora serves certain use¬ ful functions and can prevent colonisation of pathogenic mic¬ roorganisms. Someone who in this way carries pathogenic micro¬ organisms is a potential carrier of pathogens as the patho¬ genic microorganisms from such a person can be transferred to a tissue wound . With the expression microorganisms is meant Gram-positive bacteria, such as different species of staphylαcocci , strepto¬ cocci and pneumococci , Sram-positive rods, such as mycobac- teria, e.g. Mycobacterium leprae, Gram-negative bacteria, such as different species of the family Enterobacteriaceae, e.g. Escherichia coli, Klebsiella, Proteus, Enterobacter or Vibriα- nacae, or Pseudomonas, as well as Gram-negative cocci, such as gonococci . This expression also includes anaerobic bacteria, such as Bacteroides, fusobacteria, anaerobic cocci, e.g. pep- tαcocci and peptostreptococci , as well as spirillae and spirα- chetes. Further included are fungi, such as Candida, e.g. Can¬ dida albicans, and der atophytes, as well as pathogenic amoebae, such as Entamσeba histolytica, and Giardia Lamblia and Leishmania. The ways of removing microorganisms from tissue vary due to type of tissue and microorganism, respectively. For removing pathogenic microorganisms from mucous membranes and skin tissue a disinfectant or antibiotic is often used. A drawback with this is that the indigenous flora is removed as well, and thus the functions it serves. Further drawbacks are that dis¬ infectants are cell toxic and that today many pathogenic strains have developed resistence against a large number of antibiotics. The removal of pathogenic microorganisms from wαund tissue is carried out in several ways. One way is a direct attack on the microorganisms by treatment with dis¬ infectants or antibiotics, but this is unefficient and results in the above mentioned drawbacks the indigenous flora. Another way is mechanical cleaning αf wound tissue by means of moist saline dressings.The drawbacks with this are i.a. that the tissue healing is inhibited and that such dressings must be changed very.often. The latter is an example of a more in¬ direct way of removing pathogenic microorganisms from wound tissue. The secretions from the wound tissue are excellent substrate for microorganisms, and by reducing the amount thereof the growth of the microorganisms will also be de¬ creased. Other methods wherein this indirect way is used are treatment of wounds by means of suitable polymers, -e.g. in particle form , which polymers absorb the secretions from the wound tissue. Some of these secretion-absorbing polymers con¬ tain complexly bound iodine (so called iodαphαres), and with these a secretion—absorption and a disinfection are simul¬ taneously achieved. The present invention concerns means for removing microorga¬ nisms from tissue without influencing the indigenous flora in any greater extent. A predominant cause of primary wound infections and postsur- gical infections are certain Gram-pαsitive cocci, such as Staphylαcoccus aurεus and so called $-hemolysing streptococci from group A, C and G. Pathogenic staphylococci exist every¬ where in the environment, also that of a hospital where up to 80 % of the personnel can be carriers αf pathogens. Yellow staphylococci (S.aureus) colonize especially in certain parts αf the skin, such as the axilla, the bottom of the pelvis and on the hand as well as in the nose. Even in dry eczema there is a frequent existence of white as well as yellow staphylo¬ cocci. The commαn tonsillitis bacteria Streptococcus pyogenes Cgrαup A streptococcus) exists in a high frequency on nursing personnel as well as patients. Nosocomial infections are often transferred exogenic (from e.g. personnel tα a patient) or endogenic (patients having these microorganisms on skin or mucous membranes infect their own wounds) and this has became an increasing problem. In order to decrease the frequency of these infections classical preventive steps have so far been taken, such as careful pre-surgical cleaning and bandaging of infected eczema in separate rooms. The first step in most infection processes is the binding of the microorganism to tissue. The microorganisms bind speci¬ fically, either direct to a component on the surface of the epithelium cell or to extracellular molecules in blood clot or in connective tissue being exposed to the microorganisms in an epithelium wound. The present invention is based upon a completely new principle for removing microorganisms from tissue, which principle is' based upon inhibition αf the binding of the microorganisms to the tissue proteins on mucous membranes, skin and in wounds. Pathogenic staphylococci have on their cell surface a protein (protein A) that specifically binds to the complement binding site (Fc-site) of i muπoglobulins of different classes in a great number of animal species. This binding specificity is used in immunological tests and for removal of antibodies in extracorpαral circulation by means of protein A bound to a solid phase. It is also known that staphylococci are agglutinated by normal serum from different animal species. This reaction is due to a specific binding between the surface of the bacteria ,aπd i- brin/fibrinαgen. It has now been found that receptors like those being present in staphylococci also exist in other microorganisms. Frequence studies on e.g. strains of staphylococci and streptococci isolated from wound infections and other types of infections show that the ability of binding e.g. fibronectin is contribu¬ ting to the virulence of the microorganisms. In infections caused e.g. by Staphylococcus aureus and σαagulase negative staphylococci this binding ability contributes to the ^ (JE.E OMPI ~ virulence by making an initial tissue colonisation passible. Surprisingly it has now been found that proteins or fragments or residues thereof, optionally bαund tα a carrier, are sui¬ table means for removing microorganisms from tissue. The present invention thus concerns means for removing av micro¬ organisms from tissue, such as mucous membranes, skin and wound tissue, which means comprises at least one protein or a fragment αr residue thereof which can be bound to the micro¬ organisms, and optionally a carrier to which the protein or fragment or residue thereof is bound. Suitable proteins to be used in accordance with the invention are fibrinαgen, fibrαnectin, an immunoglobulin, albumin, haptαglobin, β-2-microglobulin, collagen, laminin, eπtactin αr chαndronectin. Further, fragments or residues of these proteins are also suitable. These fragments or residues can be peptides having a molecular weight of 5x10 2 r 105 , preferably 5xl0 2 - SxlQ 4 . Suitable carriers to be used in accordance with the invention are polymer materials, such as agarαse αr a derivative there¬ of, starch or a derivative thereof, cellulose or a derivative thereof, dextran or a derivative thereof, or an alginate. Further, a fiber material being a part αf absorption products, such as a dressing αr a tampon, is also suitable. Proteins or fragment(s) or residue(s) thereof can be bound to the carrier in a manner known- per se, and that through a cova- lent bond via the carbohydrate part, the primary amino groups, the sulfhydryl or the carboxy. groups in the protein, e.g. by means of divinyl sulfon, epichlorohydrin, carbodiimide, by means αf transa ination or via cyanobrαmide bridges, or by biospecific interactions known per se, such as the binding of fibrαnectin to gelatin, the binding of glycαprαteins to lectin or the binding of antigens to antibodies. The proteins or fragments or residues thereof can also be absorbed directly on a carrier. The present invention is further illustrated in the following exarηples; -_____m____e_l ! Skin areas on human loin was washed with a physio- 7 q logical saline solution. Then about 10 and 10 Staphylococcus aureus (strain Newman) were applied on several skin areas. A gel of Sepharose ® 4B, to which fibronectin had been bound, was applied to the skin areas on which strain Newman had been applied, and that in 2 - 3 mm thick layers. The fibronectin had been bound to a cyanobromide activated gel of Sepharose ® 4B by incubating 10 mg of fibronectin per ml cyanobromide activated gel for 2 h at room temperature and under a gentle stirring. The reaction was terminated by adding 1M glyσine solution to the reaction mixture, whereafter the incubation was continued for further 30 minutes. Before use the gel was thoroughly washed. Pure Sepharose 48 was used as control gel and applied in the same manner on the other skin areas on which strain Newman had been applied. All skin areas were dried with a fan (cold air). After 20-30 minutes test gel as well as control gel were removed by a sterile scalpel. Ther- after fresh material of both gels were applied and the pro¬ cedure was repeated until totally 3 treatments had been carried out, and that during a period of time of about 2 h. After the completed procedure cultivation shows that strain Newman had been removed almost completely (only single coloni¬ es of bacteria were observed) whereas cultivations from skin areas where control gel had been applied only showed a small decrease in bacteria number. Examgl_e_2: Example 1 was repeated, but instead of strain Newman Staphylococcus aureus (strain V 8) was used. The same type of control gel was also used. The cultivation check after the completed treatment showed a growth of strain V 8 that was considerably larger than that for strain Newman of example 1. Examgl_e_3: Example 1 was repeated, but instead of fibronectin fibrinogen was used. The binding of fibrinogen to the gel o ® Sepharose 4B was established in the same manner as for fibro¬ nectin in example 1. A gel of pure Sepharose ® 4B was used as a contcαl gel . Cultivation after the completed procedure shows that strain V 8 had been almost completely removed ( only single colonies were observed) while cultivations from skin areas being treat¬ ed with control gel only showed a small decrease in bacteria number. These examples show that strain Newman has a fibronectin receptor whereas strain V 8 is bound very weakly to fibrα¬ nectin, but has a fibrinogen receptor. It is further evident that a strain having a Fn receptor is removed specifically. Examp_I.es_4_-_l_9: Example 1 was repeated using Staphylococcus aureus, strain SA 113 and strain Cowan 1, and group A strepto¬ cocci, strain H 15757 and strain 11270. The proteins used were albumin, immunσglobulin G, fibrinogen and fibronectin, respectively, and these were bound to a gel of Sepharose 4B in the same way as was fibronectin in example 11.. IInn aallll tthhe examples gel αf pure Sepharose ® 4B was used as a control gel . The procedure described in example 1 was carried out and subs¬ equent cultivation showed the hereinafter given results for the different organisms and proteins, respectively. When fibronectin was used Staphylococcus aureus (strain SA 113) was cultivation negative (only single colonies of bac¬ teria were observed) , whereas positive cultivation results of this strain were obtained when albumin, immunoglobulin G and fibrinogen, respectively, were used. Staphylococcus aureus (strain Cowan 1) was cultivation negati¬ ve when immunoglobulin G, fibrinogen and fibrαnectin, respect¬ ively, was used, whereas positive cultivation results of this strain were obtained when albumin was used. Group A streptococcus (strain H 15757) was cultivation negati¬ ve wjnen albumin and fibronectin, respectively, were used, whereas positive cultivation results of this strain were ob¬ tained when immunoglobulin G and fibrinogen, respectively, were used. Group A streptococcus (strain 11270) was cultivation negative when fibrinogen was used, whereas positive cultivation results αf this strain were obtained when albumin, immunoglobulin G and fibronectin, respectively, were used. Cultures from skin areas where control gel had been applied gave in all cases positive cultivation results. E__a.mj3l.e_2_. Hospital personnel wi'th a recognized colonisation of antibiotic resistent Staphylococcus aureus were divided into two groups , each of the same size (test and control group, respectively). Samples from all persons in the two groups exhibited repeated positive cultivation results. The test group was treated with cotton swabs, that had been dipped in a fibronectin solution, which swabs were plugged in the nostrils and kept there for a couple of hours, whereafter they were exchanged with fresh swabs, also being dipped in the fibronectin solution. The procedure was repeated so that the treatment was carried out totally 3 times. The control group was treated in the same way, but unprepared swabs were used. Samples from the persons in the control group after the com¬ pletion procedure were cultivation positive, whereas samples from the persons in the test group were completely negative. Ex.amgl.e_21.: Mice (20 - 30 g , CBA strain) were shaved and subjected to a second or third degree burn according to an international standard on an area αf lxl cm (ethanαl flame, 2 minutes). Staphylαcoccus aureus (strain Cowan 1) was used as micrαorgaism in this example. Suspensions αf 10 microorgan¬ isms per ml saline solution (phosphate buffered) was prepared and all mice were painted with 0.5 ml of the suspension on the burnt area . The mice were then divided into a test and a control group. The mice of the test group were treated with ® fibr_anectin bounded to a gel of Sepharose 4B whereas the mice ® of the control group were treated with pure Sepharose 4B. The treatment was carried out so that the gel was applied to the burnt area in a confluent layer being about 2-3 mm thick. After removing of one layer by scraping with a scalpel a fresh layer was applied, and the treatment was repeated until totally three layers αf gel had been applied and removed. Then samples were taken from the burnt area, which samples were cultivate . Samples from mice in the test group were cultivation negative (i.e. 1-3 colonies) after 1 day. Samples from mice in the control group were cultivation positive (more than 3 colonies up ta confluent growth) still after 3 days. __________: The same type αf mice as those of example 21 was used. Instead of S. aureus Staphylococcus capitis, K 499 was used, but besides that the procedure of example 21 was repeated . Samples from mice in the test group were cultivation positive on day 2, whereas samples from mice in the control group were cultivation positive still on day 4. E_.am_t_e_23. The same type of mice as those of example 21 was used. Instead αf S. auerues Staphylococcus haemolyticus was used, but besides that the procedure of example 21 was repeated. Samples from mice in the test as well as control group were cultivation positive after day 3. Example 24: The same procedure and strain as in examples 21 were used and also the same type of mice, but the mice in the test group were treated with fibrinogen bound to a gel of ® Sepharose 4B. Samples from mice in the test group were cultivation negative on day 2, whereas samples from mice in the control group were cultivation positive on day 4. ______e_25 : The same procedure and strain as in example 22 were used and also the same type of mice, but the mice of the test group were treated with fibrinogen bound to a gel αf Sepharose ®4B. Samples from mice in the test as well as the control group were cultivation positive after 4 days. Exam____2_: The same procedure and strain as in example 23 were used and also the same type of mice, but the mice in the test group were treated with fibrinogen bound to a gel of Sepharose ®4B. Samples from mice in the test as well as the control group were cultivation positive after 4 days. From the examples is evident that different microorganisms bind to different proteins. The bindings shown between the microorganism and protein, respectively, have been confirmed by in vitro experiments. These have been carried out by incub¬ ating respectively microorganism with respectively protein, the latter being labelled with radioactive iodine. After comp¬ leted incubation the amount of radioactively labelled proteins bound to respective microorganism was measured. In those cases where the microorganisms have been removed by the treatment with a protein (cultivation negative samples), in vitro experiments have proved that respective microorganism has been bound to the radioactively labelled protein, whereas in those cases where the microorganisms have not been removed (cultiva¬ tion positive samples) after the treatment with a protein, in vitro experiments have proved that there is nα bond between the corresponding microorganism and radioactively labelled protein. OMPI The proteins can also be bound to fiber materials being a part of an absorption product, such as dressings or tampons in ordeαr to remove microorganisms from wound or other tissue. Dressings, tα which the proteins are bound are suitable for the treatment of wounds. Tampons to which a protein has been bound can e.g. be used in surgery for absorbing from surgical wounds, or as catamenial tampans, where they can be used for removing the microorganisms being the cause of the so called tampon disease. The means of the present invention can also be used for remov¬ ing microorganisms in wound hollows, who e.g. are the result of an opening of abscesses, such as anal abscesses, in which case the means preferably is bound to a dressing having the shape of the wound hollow in order to fill this up and to inhibit the closing of the abscess. Such a dressing material can e.g. be in fiber or granular form in an absorption material enclosed in a hydrαfαbic material. The protein can further be in the form of a rinsing solution for removing of microorganisms in local infections.

Claims: C L A I M S 1. Means for removing microorganisms from tissue, such as mucous membranes, skin and wound tissue, characterized in that it comprises at least one protein or fragment(s) or residue(s) thereof which can be bound to the microorganisms, and optio¬ nally a carrier to which the protein(s) αr the fragment(s) or residue(s) thereof is bound. 2. The means of claim 1, characterized in that the protein or fragment(s) or residue(s) thereof is fibrinogen, fibronec¬ tin, an immunoglobulin, albumin, haptoglobin, 6 -2-micrαg lα- bulin, collagen, laminin, entactin or chondronectin. 3. The means of claim 1, characterized in that the frag¬ ment^) or residue(s) of the protein(s) is derived from fibrinogen, fibronectin, an immunoglobulin, albumin, hapto¬ globin, β-2-microglobulin, collagen, laminin, entactin or chαdrαnectin. 4. The means of claim 3, characterized in that the fragment ment(s) or residue(s) is a peptide having a molecular weight of 5xl0 2 -10 5 . 5. The means of any of the claims 1-4, characterized in that the carrier is a polymer material, such as agarose or a derivative thereof, starch or a derivative thereof, cellulose αr a derivative thereof, dextrane or a derivative thereof or an alginate. 6. The means of any of the claims 1-4, characterized in that the carrier consists αf a fibrous material being a part αf absorption products, such as a dressing or a tampon. 7. The means of any of the claims 1-6, characterized in that the protein(s) or the fragment(s) or residue(s) thereof is linked/bound to the carrier by means αf divinyl sulfαn, epichlorohydrin, carbodiimid b means of transa ination, via cyanobromide bridges αr by biospecific interaction. 8. Jhe means of any of the claims 1-6, characterized in that the protein(s) αr the fragmentt ' s) or residue(s) thereof is absorbed on the carrier. -? . _ Use of proteins, such as fibrinogen, fibronectin, an immu¬ noglobulin, albumin, haptoglαbin, β-2-microglαbuli , colla¬ gen, laminin, entactin or chondroπectin, or fragments or resi¬ dues thereof of for removing αf icroorganisms from tissue, such as mucous membranes, skin and wound tissue. 10, The use of proteins or fragment(s) at- residue(s) thereof according to claim 9, characterized in that the protein(s ) or fragment's ') ac residue(s" 1 thereof is bound to or absorbed on a carrier.

Inventors: HOEOEK MAGNUS, WADSTROEM TORKEL, HOEOEK, MAGNUS, WADSTROEM, TORKEL
Applicants: HOEOEK MAGNUS, WADSTROEM TORKEL, HOEOEK, MAGNUS, WADSTROEM, TORKEL
Application year: 1983
Main classification: A61L15
IPC codes: A61F13, A61K9, A61K38, A61L2, A61L15, A61L26