US 2809149 A
Description (OCR text may contain errors)
United States Patent G ANTIBIOTIC-Ih/IPREGNATED GAUZE PADS AND NIETHOD OF MAKING SAME Frank Ramo Cusumano, Baltimore, Md.
N Drawing. Application December 23, 1954, Serial No. 477,425
6 Claims. (Cl. 167-84) My invention relates to an improvement in the prophylactic properties of gauze pads which are utilized for the protection of both minor and major wounds.
The value of the synergistic combination of polymyxin B, bacitracin and neomycin was described for the suppression of intestinal flora in man in May 1952 in Gastroenteral, vol. 21, pages 139147.
It has now been discovered that the same combination of antibioticshas synergism in topical applications.
It is well known that the function of gauze pads is to protect the wounds they cover from contamination with dirt, to prevent the clothing of the patient from coming into contact with the wound surfaces, to absorb serous exudates and pus from infected wounds, and to absorb serious fluids from uninfected wounds. Gauze pads are used extensively also in hospitals as sponges during operations. Except for the physical protection from contamination with the outside dirt and debris, the common gauze pad ofiers no protection against the development/and multiplication of bacteria in and around the.
woundsurfaces. .Practically all wounds, be they major or minor, are contaminated with bacteria at the time they occur. In many of these Wounds there are suflicient body defenses to protect the patient from invasion by these contaminating bacteria. As a matter of fact, many minor Wounds rarely become frankly infected because of the hosts natural resistance to the contaminating bacteria. The relatively few bacteria contaminating wounds are usually phagocytised, by the leukocytes (engulfedand destroyed). These white blood cells are one of the agents of the bodys resistance. However, there are a number of wounds which become so heavily infected at the time they are obtained that the large number of bacteria present overcome the resistance of the individual and serious and sometimes. fatal infections ensue. This is particularly true in the war wounded where large wounds are incurred under most difficult conditions. Such wounds become badly contaminated with mud, dirt, and debris which are carried into the wound by the impact of shrapnel, bullets, or metallic fragments and, because of the distances back to base hospitals, the bacteria have time to develop and multiply. Even in minor wounds, bacteria will multiply if the hosts resistance is not sufficient to overcome the bacterial invasion.
It is well known that the various antibiotics are both bacteriostatic and bactericidal; that is, they not only inhibit the growth of some bacteria but certain antibiotics actually kill gacteria on contact. For example, penicillin is considered to be a bactericidal agent (kills bacteria) while the other antibiotics such as aureomycin, chlorarnphenicol, and terramycin are considered mainly to be bacteriostatic (inhibit growth of bacteria). In addition to the fact that some antibiotics are bacteriostatic and others bactericidal, it is 'well known that all antibiotics are selective in their action against microor ganisms. For example, penicillin is active against the so-called Gram-positive bacteria such as streptococci and staphylococci whilean antibioticsuch'as streptomycin Patented Oct. 8,, 1957 or neomycin is more active against the so-called Gramnegative bacteria such as those of the colonaerogenes group of organisms. Furthermore, antiobiotics such as aureomycin have activity against some Gram-positive and Gram-negative bacteria but are active also against certain of the large viruses and rickettsia. It is well known by those in the antibiotic field that no one antibiotic is capable of antagonistic action against all Gram-positive and Gram-negative bacteria. A proper combination of two or more antiobiotics would be most valuable in the prophylaxis of wounds if the patient could be protected against the large variety of bacteria likely to infect such wounds.
It is an object of my invention, therefore, to produce a gauze pad impregnated with antibiotics that will be effective against all or practically all bacteria which may be expected to infect and develop in a wound.
It is known that most antibiotics are not stable in solution and that they must be dried to a very low moisture content if they are to retain their effectiveness. It is therefore another object of my invention to impregnate gauze bandages with antibiotics so that the antibiotics will remain stable and effective when put to practice.
In my Patent No. 2,680,701 I disclosed a process of impregnating gauze bandages with a synergistic combination of streptomycin, bacitracin and polymyxin which comprises dissolving the combination in absolute methanol, impregnating the pad with the resultant solution and removing the methanol from the pad by evaporation. That process is inoperative with the composition of the present invention because neomycin is not soluble in methanol. It has now been found that a solvent comprising from to methanol and from 5% to 20% water can be used to dissolve a mixture of neomycin, polymyxin and bacitracin, that a gauze pad can be impregnated with the resultant solution, and that the solvent can be removed from the pad by evaporation to leave the pad impregnated with the antibiotic combination which is stable and effective and has not been damaged by the impregnation process. For best results the solvent should be 95% methanol, and 5% water. As the water content of the solvent mixture rises and the methanol content falls, there is an increasing tendency to injure the activity of polymyxin and bacitracin. Neomycin will not dissolve in solutions having more than 97% methanol and the ease with which it is dissolved increases rapidly as the water content is raised from 3% to 5%. Other objects and uses of my invention will become apparent from the following description.
Ibelieve, however, that the use of the three antibiotics neomycin, bacitracin and polymyxin, in combination in the manner in which I am about to disclose, is distinctly new. These three antibiotics I choose to utilize for impregnation of gauze pads because of their wide antibacterial spectrum and because of the synergistic action obtained when these three antibiotics act together. By synergism I means that the combined eifect of the three drugs is greater than the sum of the effects of the individual drugs. A combination of these three antibiotics in the proper proportions, properly impregnated in the pad to prevent deterioration of them, I have discovered will act against both Gram-positive and Gram-negative bacteria. It is not my intention that these impregnated gauze pads be utilized alone for the treatment of infected wounds but rather as a prophylactic preparation to prevent the development and spread of bacteria which normally contaminate most wounds. There are other and more effective means of treatment for such wounds and it is my opinion that the antibiotic-impregnated gauze pads would not be suflicient in themselves for such treatment once/spread of infection has occured. These padscouldbe used, however, as a supplement to other;
forms of treatment such as parenteral and oral therapy with antibiotics or other chemotherapeutic agents.
In accordance with this invention, I have discovered that by combiningneomycin, polymyxin, and. bacitracin in solution and impregnating them in gauze pads, a preparation is obtained which approachesv the ideal for prophylactic treatment of wounds. This preparation has an important advantageover the use of 'a. single antibiotic for this purpose in that it provides antibacterial activity against both the Gram-positive and Gram-negative bacteria but in addition a synergistic effect is obtained. It is made readily available at the site of infection and in suflicient concentrations to inhibit growth or destroy the bacteria present in the wound. During and following operations even under the best of aseptic conditions in a hospital, minor contaminations occur from air borne bacteria, such pads used as sponges and as a covering for the operative Wound are of great value in protecting the patient against bacterial invasion.
The antibiotics terramycin, aureomycin and chloramphenicol, are active to a degree against some Gram-positive and Gram-negative bacteria. Because of their selectivity and similarity of action however, they would not be efiicient when used alone or together in controlling all organisms that might possibly infect wounds.
I choose to use a combination of neomycin, bacitracin, and polymyxin for the following reasons. All three of the drugs, aureomycin, terramycin, and chloramphenicol, not only act similarly on Gram-positive and Gram-negative bacteria but in addition it is known that organisms that become resistant to one of these drugs are resistant to the other two as well. This is not true in the case of the combination neomycin-bacitracin-polymyxin, since organisms resistant to neomycin are not resistant to other antibiotics. I choose to use bacitracin in place of penicillin, both of which are active against similar organisms, because bacitracin does not cause allergic (sensitivity) manifestations when used locally in wounds as does penicillin. Another advantage in the neomycin-bacitracin-polymyxin combination is that the latter drug, namely, polymyxin, is active against organisms in the Pseudomonas aeruginosa group and this is not true to the same degree for any of the other antibiotic drugs mentioned. Organisms in the Pseudomonas aeruginosa group are those that cause development of so-called green pus and these organisms are commonly found in infected wounds.
Although the antibiotics neomycin, bacitracin, and polymyxin may be incorporated in gauze bandages of a variety of sizes, for demonstration and simplicity I choose to use gauze pads approximately 3 inches by 3 inches containing approximately of a yard of material. Furthermore, although a number of concentrations of antibiotics per square inch of pad are successful in the prophylactic treatment of wounds, I have found that 1,000 micrograms of neomycin per square inch, 1,000 micrograms of polymyxin per square inch (the polymyxin utilized is 50% to 70% of purity), and 50 units of bacitracin per square inch are adequate. In the preparation of antibiotic-impregnated gauze 'pads, it is essential that at least a 10% coverage be utilized to account for losses which occur during manufacture.
All the antibiotics presently in use, and these include the major ones, penicillin, streptomycin, neomycin, dihydrostreptomycin, aureomycin, chloramphenicol, terramycin, bacitracin, and polymyxin, are not stable to any great degree in solution. Thus in the process of impregnation of the gauze pads with antibiotics, it is essential that precautions be taken to prevent deterioration of the antibiotics during the processing. If the antibiotics in solution are heated above room temperature C.) during the process of manufacture, the antibiotics are even more unstable; and when sufficient heat is applied the antibiotics may be completely destroyed. In this process of degradation orbreakdown of antibiotics, two
factors are involve'dtemperature and time. For example, when penicillin is placed in aqueous solution and refrigerated, it will maintain its potency for at least 48 to 72 hours without significant loss of activity. However, the same penicillin solutions will lose their potency in a matter of a few hours if held at incubator temperature (37. C.). Thus it becomes important in the impregnation of gauze with antibiotics that the time and temperature elements be carefully controlled. If the antibiotics are placed in aqueous solutions and then impregnated into gauze pads, the time of drying to eliminate the water may be inordinately long so that destruction or partial loss of potency of the antibiotics results. This loss of potency just described can be overcome to a considerable extent if the gauze pads containing the water solutions of antibiotics are frozen and then dried under vacuum. However, this is a time consuming and expensive operation.
I have discovered that the time element which affects stability, and the temperature element which similarly affects stability can be obviated for all practical purposes by the use of a solvent mixture containing about 5% water and about methanol. capable of dissolving readily all three antibiotics, namely, neomycin (neomycin A which is actually neomycin sulfate), bacitracin, and polymyxin (polymyxin B). Other neomycin salts have been tested and their solubility characteristics appear to parallel those of neomycin sulfate. Methanol is extremely volatile and therefore it facilitates quick removal of the solvent mixture from the gauze pads by evaporation. Evaporation is hastened by the application of a blast of warm air.
The importance of the proper solvent mixture is illustrated in Table A below, which includes a tabulation of the results of a series of experiments in which neomycin, bacitracin and polymyxin were individually and collectively placed in various solvent mixtures and the solubility characteristics were noted along with the potency of the individual antibiotics after impregnation into gauze pads in accordance with the first example listed below:
In the above tests the assay methods used for determining the concentration of antibiotics in neomycinbacitracin-polymyxin gauze and for determining stability of antibiotics during the impregnation studies were as follows:
Use twelve representative samples of the gauze and soak in 0.1 molar phosphate buffer at pH 7.8-8.0 for. one hour with frequent agitation. Assay by the following methods:
Neomycin sulfate Using 0.10 molar buffer furtherdilute to an estimated-- This solvent mixture is 10 micrograms of activity per ml. Assay against known neomycin working standard on agar plates seeded with Micrococcus pyogenes var.'aureus 209-P (American Type Culture Collection #6538P may also be used) using cylinder method and determine the neomycin content by comparison with a standard curve prepared by determining points on the working standard.
Baeitracin Polymyxin B sulfate Dilute the sample to an estimated concentration of 100 units per ml. using 1% phosphate buffer at pH 6.0. Assay against known polymyxin B sulfate Working standard by the cup plate method using ATCC #4617 Brucella bronchiseptica.
In the preparation of antibiotic-impregnated gauze pads, it is essential (to avoid contamination of the pad with bacteria) that aseptic technique be used throughout the process. Thus the gauze pads utilized are heat sterilized prior to impregnation and all processing thereafter is carefully controlled to avoid contamination with bacteria. The sterilization may be done by steam sterilization at 120 C. for 20 minutes or by the application of dry heat at a temperature of 110 C. for from 8 to 12 hours.
The impregnation of the sterile gauze pads may be accomplished in the following manner.
(1) Prepare stock solution as follows in 95% methyl alcohol (methanol) and 5% water.
Neemyein A mcg./rnl. 6,000 Baeitraein units/ml. 300 Polymyxin B meg./ml. 6,000
(2) Prepare the working solution by mixing equal parts of the above stock solutions. Thus, the working solution will contain 2,000 meg/ml. of neomycin, 100 units/ml. of baeitracin, and 2,000 meg/ml. of polymyxin B.
The standard sterile 3 inch by 3 inch folded gauze pad containing approximately ,6 of a square yard of gauze is dipped into the working solution and, after it is thoroughly saturated, removed and the excess solution extruded by applying pressure. The pressure applied should be such that approximately 5 ml. of the working solution remains in the pad. The pressure necessary to extrude the excess solution can be predetermined by weighing a pad before immersion in the solution, weighing it after immersion, and then trial squeezing the pad to remove the excess solution to allow approximately 5 ml. to remain in the pad. The pad is dried in a blast of warm air until all the solvent has been evaporated and the pad is dry (this takes approximately five minutes). All operations are performed under aseptic conditions.
The preferred procedure described above was repeated 7 with the following stock solutions:
96% methyl alcohol plus 4% water 95% methyl alcohol plus water 90% methyl alcohol plus 10% water 85% methyl alcohol plus water 80% methyl alcohol plus water described above. The end of the gauge is allowed to pass under a second roller which is fixed in the pan containing the solution so that the gauze, as it passes under the roller, is made to pass through the solution. As the end of the gauze is pulled forward through the solution and passes the pan containing the solution, a blast of warm air drives out the excess solvent present, thus drying the gauze. The dry gauze is attached to a third roller and may be rewound either by hand or by power. All operations including the folding of the gauze to form the pads are carried out under aspetic conditions.
With the stock solutions described above, the impregnated gauze pads (folded) contain per square inch approximately 1,000 mcg. of neomycin, 1,000 meg. of polymyxin, and 50 units of baeitracin.
After preparation, the gauze pads described are placed in sterile envelopes. For assay purposes, i. e., to determine the amount of each antibiotic impregnated into the pads, several square inch portions of the pads are cut and extracted with water and the water assayed for its antibiotic content. The assay methods utilized for neomycin, bacitracin, and polymyxin are those described in sections 141.101, 141.401, and 141.39 of section 507 of the Food, Drug, and Cosmetic Act of 1938. On several occasions twelve individual square inch portion of antibiotics-impregnated gauze pads prepared by the preferred procedure as described above have been assayed. It was found that to 100% of the three antibiotics impregnated into the pads can be recovered by the assay procedures described above. These assay results are well within the experimental error of these methods.
To demonstrate the antibacterial activity of the gauze pads, square centimeter portions are cut and placed aseptieally on Petri dishes containing agar previously inoculated with the bacteria (a) Micrococcus flavus, and (b) Bacillus bronchz'septieus. The Petri dishes are then placed in an incubator at 37 C. for a period of 24 hours. At the end of this time immediately surrounding the square portions of the antibiotic-impregnated gauze pads and extending outward from the pad for one to two centimeters or more is found a clear zone of inhibition of growth of the bacteria with which the agar in the Petri dishes was inoculated. The antibiotics present in the pads are extraeted from the pads by the moisture present in the agar used as a medium for growth of the bacteria thus simulating the absorption of the antibiotics from the pads by the moisture or serous exudates of wounds. The antibiotics so withdrawn from the pads diffuse through the agar thus preventing the growth of, or killing the bacteria in the clear zone. Beyond the clear zone the bacteria grow profusely. The numbers of bacteria acted upon by these antibiotics in this demonstration exceed by several million times the numbers normally present in wounds, thus demonstrating inhibition of the bacteria which represent both the Gram-positive (Micrococcus flavus) and the Gram-negative (Bacillus bronchisepticus) groups of organisms.
Having thus described my antibiotic-impregnated gauze pad, its method of use, and its activity against both Grampositive and Gram-negative bacteria, I am aware that the exact portions of the three antibiotics may be varied and should be varied to suit different types of wounds and different size pads, but that such variation is well within the precepts of the invention and such as may be under stood to those experienced in such matters and that it does not depart from the spirit of my invention nor the scope of the impending claims.
1. The method of impregnating a gauze pad with a stable therapeutic, antibiotic composition having synergistic bactericidal action and a wide antibacterial spectrum which comprises dissolving neomycin, polymyxin and bacitracin in a solvent mixture containing from about 5% to 20% water and from about 80% to methanol, impregnating said pad with the resultant solution, and removing the solvent from said pad by evaporation, each of said antibiotic compounds being present in the dried impregnated gauze pad in a minimal amount which, if used alone, would produce a bacteriostatic effect.
2. The method of impregnating a gauze pad with a stable therapeutic, antibiotic composition having synergistic bactericidal action and a wide antibacterial spectrum which comprises dissolving neomycin, polymyxin and bacitracin in a solvent mixture containing about 95% methanol and about 5% water, impregnating said pad with the resultant solution, and removing the solvent from said pad by evaporation, each of said antibiotic compounds being present in the dried impregnated gauze pad in a minimal amount which, if used alone, would produce a bacteriostatic effect,
3. A gauze pad for the prophylactic treatment of wounds said pad being impregnated by the process as set forth in claim 1, with a therapeutic, antibiotic composition having synergistic bactericidal action and a Wide antibacterial spectrum, said composition comprising bacitracin, polymyxin and neomycin, each of said antibiotic compounds being present in a minimal amount which, if used alone, would produce a bacteriostatic effect.
4. The gauze pad set forth in claim 3 wherein said pad is impregnated per square inch with about 50 units bacitracin, about 1000 micrograms of polymyxin having a purity of 50 to 70 percent and about 1000 micrograms of neomycin.
5. The method of impregnating a gauze pad with a stable therapeutic antibiotic composition having synergistic bactericidal actionand a wide antibacterial spectrum which comprises forming stock solutions of neomycin, polymyxin and bacitracin using a solvent of 95 methanol and 5% water, mixing said stock solutions to form a working solution, saturating a dry gauze with said working solution, pressing out excess solution and evaporating the solvent with a blast of warm air, each of said antibiotic compounds being present in the dried impregnated gauze pad in the minimal amount, which if used alone would produce a bacterial static eifect.
6. An impregnated gauze pad produced by the method set forth in claim 5 and in which each square inch of the impregnated pad contains about 50 units bacitracin, about 1000 micrograms of polymyxin, having a purity of 50 to 70% and about 1000 micrograms of neomycin.
References Cited in the file of this patent UNITED STATES PATENTS 2,680,701 Cusumano June 8, 1954 OTHER REFERENCES Policitracina (neomycin,'bacitracin and polymyxin, in ointment form), Unlisted Drugs, November 1954, p. 118, dating back to July 5, 1954, Rome.
Gastroenterology, vol. 21, May 1952, pp. 139-447.