OPTHALMIC NON-IRRITATING IODINE MEDICAMENT AND METHOD

Abstract: The present invention relates to an enzymatically generated iodine microbicide for the inactivation of pathogenic organisms that are contaminants in or on sensitive biological materials such as the human eye. The biocidal agent from the enzymatic reaction is free molecular iodine which is generated at a minimum level of 5 ppm and comprises at least 25 % of the total iodine species present on a molar basis.

Description: OPTHALMIC NON-IRRTTAΉNG IODINE MEDICAMENT AND METHOD Field of the Invention The present invention is a continuation in part of Serial No. 08/551,478 filed 1/01/95 which is a continuation in part of Serial No. 08/324391 filed 10/17/94 and relates generally to the enzymatic formation of a non-toxic and non-irritating germicidal iodine based microbicide which will inactive pathogenic organisms that are contaminants in and on mammalian ocular tissue. Background Of Invention Free molecular iodine is generated in situ in accordance with the present invention as a biocidal agent by the peroxidase catalyzed reaction between iodide and a peroxide for the purpose of inactivating pathogenic organisms that are contaminants in and on mammalian ocular tissue. Frequent contaminants include bacteria, viruses and fungi. Antibiotics are currently used to treat infected eyes. Antibiotics are not broad spectrum agents. No antibiotic has efficacy against bacteria, viruses and fungi. Widespread use of antibiotics has also led to antibiotic resistant pathogens. A preferred ocular anti-infective would be broad spectrum and not capable of generating resistant pathogens. A large variety of chemical germicides have been used in the medical profession and biomedical industry to inactivate unwanted bacteria, viruses and fungi. The majority of these chemical germicides are either toxic or destructive to organic material and, as a result, these disinfectants are not suitable for use with sensitive biological materials such as the human eye. U.S. Patent No. 5,185,371 describes a method to disinfect blood products that relies upon diluting a germicide in one of the following compositions: 0.9% aqueous sodium chloride, 5 % aqueous dextrose, sugar solutions or other diluents that ameliorate the chemical incompatibilities of the liquid germicides with blood and blood products. F.A. Highsmith et al. (Journal of Infectious Diseases, volume 167, page 1027,1993) describe methods that utilize a chemically modified polyvinylpyrrolidone- iodine complex. The compositions and methods described in Highsmith have been used to disinfect blood products. The compositions of Highsmith contain a large number of iodine species including free molecular iodine. All of the prior art related to decontamination of sensitive biological materials with iodine utilize iodophors. Iodophors are compositions that utilize povidone-iodine or another chemical complex or contains iodine bound to a high molecular weight polymer. The use of an iodophor increases the absolute concentration of iodine species that are required to inactivate pathogens as compared to a composition of free molecular iodine that does not contain high molecular weight polymers which bind iodine. Iodophors are not the preferred form of iodine for disinfection of sensitive biological material since all forms of iodine can be toxic. A conflict exists between biocidal activity and toxicity with respect to sensitive living cells such as those that comprise the human eye. In this regard the prior art does not directly address the critical parameters required to effectively utilize iodine for the disinfection of sensitive biological materials. Although iodophors have been used to disinfect the human eye the ratio of free molecular iodine relative to other iodine species is less than 1/100. In accordance with the present invention the level of free molecular iodine is controlled such that when a plurality of iodine species is simultaneously present free molecular iodine will be the principal iodine species relative to the other species of iodine with a preferred concentration of above at least 25% free molecular iodine on a molar basis. The free molecular iodine concentration is preferably between 5 and 330 ppm and may be formed in situ by enzymatically reacting peroxidase, iodide and hydrogen peroxide in an aqueous medium. The molar ratio of hydrogen peroxide to iodide should preferably be no greater than four and the maximum concentration level of hydrogen peroxide should preferably not be greater than 0.015% before applying the microbicide to the human eye so that the treatment will be effective to inactivate all unwanted pathogens in the human eye. In addition, it is highly preferred to substantially maintain the level of free molecular iodine under the conditions of pH and ionic strength that are not destructive or irritating to the eye. In order for free molecular iodine to be effective, the level of this biocidal agent must be substantially maintained for a period of time that is sufficient to inactivate all of the unwanted pathogens. However, the chemical activity of free molecular iodine must also be controlled in order to substantially maintain the integrity of the biological material of interest. Because of the conflicting attributes of biocidal activity and biological compatibility, it is important to eliminate or minimize other iodine species that contribute toxicity and chemical incompatibilities but do not necessarily contribute biocidal efficacy. In addition, the concentration of other non-biocidal cytotoxic chemicals should be minimized since they serve as a potential source of irritation and incompatibility. The pH that is most compatible with the overwhelming majority of biological materials contemplated under this application is pH 6.8 to 7.1. While it is not necessary to decontaminate ocular material at a pH of 7.0 it is highly preferred to maintain a pH that is as close to 7.0 as possible in order to minimize the potential for irritation. A neutral pH introduces an additional factor that complicates the use of free molecular iodine in disinfection. Free molecular iodine is not stable at a neutral pH and, as a result, the duration of treatment is a critical parameter. One of the disinfectants identified in U.S. Patent No. 5,185,371 is 10% povidone- iodine which is frequently represented as PVP-I2. PVP-I2 is a complex of iodine with povidone. It contains not less than 9.0% by weight, and not more than 12% by weight of thiosulfate titratable iodine. In addition, PVP-I2 typically contains not more than 6.6% by weight of iodide ions. Additionally, iodate and other inorganic species are typically added to PVP-I2. PVP-I2 is available commercially from many different manufacturers and the pH of these products will vary by manufacturer. However, PVP-I2 is strongly buffered at acidic conditions that typically range between pH 3.5 and 4.5. The distribution of iodine species in PVP-I2 does not favor its use for sensitive biological materials. PVP-I2 is not a preferred degerming agent for biological materials since less than 0.2% of the iodine contained in such compositions is in the form of free molecular iodine. In addition, the pH of these compositions are toxic to most of the cells and tissues contemplated under this application. PVP-I2 was designed to be applied directly to intact skin. The epidermis of mammalians consists of dead cells that are significantly less sensitive to harsh chemicals than living cells. U.S. Patent No. 5,185,371 describes the treatment of blood and blood products with PVP-I2 at concentrations that range from 0.01 % to 0.5% by weight for up to 5 minutes. This application prescribes a concentration range for PVP-I2 that corresponds to a 1/200 to a 1/10,000 dilution of PVP-I2. When PVP-I 2 is diluted in normal saline at 1/200 the pH of the resulting solution is 4.6 and the level of free molecular iodine is 10 ppm. When PVP-I2 is diluted in normal saline at 1/10,000 the pH of the resulting solution is — 5.4 and the level of free molecular iodine is —0-0.5 ppm. The combination of free molecular iodine, pH and contact time described in U.S. Patent No. 5,185,371 is not adequate to effectively inactivate most of the bacteria and viruses of interest in the presence of a competing bioburden. It is known that a significant amount of iodine and/or extended contact times are required to completely inactivate non-lipid-enveloped viruses (Highsmith F.A. et al., Transfusion, volume 34, page 322, 1994). Accordingly, there presently is a need to provide an iodine based method to disinfect sensitive biological materials such as mammalian ocular tissue. Iodine based compositions have been formulated to disinfect sensitive biological materials but these compositions have several drawbacks. Iodine is an effective biocide and unlike other biocides, iodine is required for proper human nutrition and thyroid function. The difficulty with previous iodine based compositions is that they have unfavorable characteristics of pH and ionic strength and that active biocidal iodine (i.e., free molecular iodine) is (a) combined with other chemical species that are not biocidal and (b) not the predominant iodine species. Summary of the Invention The objectives of the present invention are accomplished by generating an iodine based disinfectant under conditions such that the free molecular iodine so generated in a concentration range of between 5 and 330 ppm. Such an iodine composition can be formed in an aqueous environment that is maximally compatible with ocular tissue. Surprisingly, such a solution will not only disinfect biological products but will do so without damage to the material of interest. It is presently believed that the disinfectant compositions of the present invention will render sensitive biological materials like the human eye safe, i.e., inactivate harmful bacteria, viruses and virus-like agents that may be present. Iodine based germicides are well known in the art. The method used to generate the iodine based germicide of this application is to use a peroxidase in combination with iodide and hydrogen peroxide. It is known from Kessler (U.S. Pat. No. 4,227,161, U.S. Pat. No. 5,169,455, U.S. Pat. No. 4,996,146 and U.S. Pat. No. 4,937,072), Orndoff (U.S. Pat. No. 4,370,199) and Montgomery (U.S. Pat. No. 4,576,817) that a combination of a peroxidase, a peroxide and iodide anions will form a bactericide in an aqueous environment. The bactericidal efficacy of this combination results from the enzymatic reaction that occurs when peroxidase, hydrogen peroxide and iodide react in solution. Peroxidase is known to effect the transfer of electrons from iodide to hydrogen peroxide. Hydrogen peroxide is converted into water by this reaction. Several possible reaction products have been postulated for the iodide anion including: 1) iodine free radicals (Nunez and Pommier, European Journal of Biochemistry, volume 7, pages 286-293, 1969); 2) hypoiodite ion (Morrison and Schonbaum, Annual Review of Biochemistry, volume 45, pages 861-888, 1976); and 3) iodinium ion (Ohtaki, Nakagawa, Kimura and Yamakazi, Journal of Biochemistr , volume 256, pages 805-810, 1981). It has been observed that under certain conditions this reaction will generate significant levels of free molecular iodine wherein said free molecular iodine comprises a significant percentage of all of the iodine species present. The phrase "free molecular iodine" is a term of art that refers to elemental iodine, the chemical species that is represented as 12 which is capable of being titrated with sodium thiosulfate. It has been observed that it is possible to generate defined levels of free molecular iodine across a wide pH range using compositions comprised of a peroxidase, peroxide and iodide. The exact level of available iodine in these compositions is a function of the concentration levels of peroxide, iodide, peroxidase, buffering agents, pH and other additives. It is understood that if materials like a human eye are to be disinfected, the disinfectant composition must be substantially isotonic in order to minimize toxicity. The disinfectant concentration and the time required to effectively inactivate any pathogenic organisms are dependent upon the level of free molecular iodine in the final environment. Suitable concentrations of free molecular iodine are a function of the disease state and treatment regime. In general, the concentration of free molecular iodine that is suitable ranges from 5 to 330 ppm. The amount of time that is required to inactivate the pathogenic organisms of interest is a direct function of the level of free molecular iodine, the pathogens of interest and the level of biological material in the composition. In order to practice this invention to disinfect the eye of mammals there are several important considerations. It is highly preferred to make the composition isotonic to human lachrymal fluid. Alternately, one can control the ionic strength of the composition such that it lies between 280 and 300 milliosmols. It is well understood by one skilled in the art that this can be accomplished by adding agents such as sodium chloride to the composition. Polymers such as hydroxyethyl cellulose, methylcellulose and polyvinylalcohol are frequently used in ocular compositions as are many other polymers that are familiar to one skilled in the art. These materials are suitable for the compositions disclosed in this application provided that they are combined in a fashion that does not substantially increase the overall concentration of iodine necessary to achieve the desired biocidal activity. The ocular compositions contemplated under this application include conditions caused by viruses, bacteria, fungi and acanthameba. The free molecular iodine is allowed to dissipate via hydrolysis in the composition after the free molecular iodine contacts the sensitive biological material of interest. Alternatively the iodine in the composition can be separated by a plurality of means that are familiar to one skilled in the art including adding materials, like cyclodextrin, that bind iodine or passing the composition through columns that contain iodine binding materials or by adding iodine reducing agents to the composition such as lactose which act to reduce iodine slowly. Detailed Description of the Invention Inactivation of unwanted pathogenic organisms in the human eye is based upon the unexpected discovery that elevated concentrations of free molecular iodine are compatible with mammalian eyes. In accordance with this invention free molecular iodine is generated in situ and contacted with the ocular tissue of interest. The level of free molecular iodine that is required to be in contact with ocular tissue is between 5 and 330 ppm which corresponds to an input level of iodide anion that is between 25 ppm and 1660 ppm. The concentration of free molecular iodine should represent at least 25% of the thiosulfate titratable iodine present. It is understood by one skilled in the art that the ratio of free molecular iodine to triiodide and the ratio of free molecular iodine to thiosulfate titratable iodine can be varied. It is also recognized by one skilled in the art that different concentration constraints, either minima or maxima, can exist for both free molecular iodine and triiodide depending upon the specific application. While 25% represents a minimum ratio of free molecular iodine to total iodine, it is also useful to define a further limitation on the formulations that comprise this application with respect to the maximum concentration of thiosulfate titratable iodine that can be present. This limitation is useful with respect to maintaining an acceptable toxicological profile for the compositions in this application. The concentration of thiosulfate titratable iodine should not be higher than 650 ppm even if the free molecular iodine comprises over 40% of the total iodine present. The pH of the aqueous environment must be controlled to be between pH 5.0 and 7.1. The preferred pH range in which to treat the biological material of interest is between pH 6.0 and 7.0. The frequency of administration for the free molecular iodine can be varied widely and will depend upon the level of organic material in the composition and the disease state. The frequency of administration for treatment of any particular disease state must be derived empirically. Free molecular iodine is introduced by using a peroxidase, iodide, and a source of peroxide. It is obvious that the concentration of free molecular iodine used must be adequate to inactivate the contaminating pathogens; however, what is less obvious and equally critical is to generate an iodine based germicide whose principal component is free molecular iodine. By contacting the material of interest with an iodine composition that is principally comprised of free molecular iodine one minimizes the potential for chemical incompatibilities and toxicity. Free molecular iodine should comprise at least 25% on a molar basis of all of the iodine species present after the iodine based germicide has been activated in an aqueous environment. The reactants that comprise the peroxidase catalyzed oxidation of iodide must be controlled to yield a suitable result. The reaction should be established so as to efficiently convert iodide ions into free molecular iodine. Additionally, it is preferable to establish reaction conditions that convert at least 50% of the the initial concentration of hydrogen peroxide into water and the concentration of hydrogen peroxide that is left in contact with ocular tissue after initiation of the enzymatic reaction should not be above a level of 0.015%; this prevents ocular irritation. In order to optimally convert iodide into free molecular iodine the molar ratio of hydrogen peroxide to iodide should be initially established in a 1/2 molar proportion. It is highly preferred to limit this ratio to no greater than 4. The preferred oxidant of this invention is hydrogen peroxide. Any material that acts as a source of hydrogen peroxide when admixed in an aqueous environment is suitable for the present invention. The term "source of peroxide" for purposes of the present invention and as used herein shall mean any material alone or in combination which can serve as precursors for hydrogen peroxide including metal peroxides, percarbonates, persulphates, perphosphates, peroxyesters, urea peroxide, peroxyacids, alkylperoxides, acylperoxides and perborates. Alternately methyl, ethyl and other higher molecular wieght peroxides can be used as a source of hydrogen peroxide but these are not preferred. Mixtures of two or more of these substances can also be used. The preferred concentration for hydrogen peroxide is between 0.001 and 0.01 % in the final composition prior to initiation of the oxidation of iodide. The donor molecule of this invention is iodide anion. Suitable dry sources of iodide anion for this invention include sodium iodide and potassium iodide as well as other salts of iodide. Any compound which yields iodide anion upon dissolution in an aqueous environment is suitable for this application. The simple salts of iodide are preferred and have the advantage of being less costly. Additionally, they have a long shelf life in solid and liquid form. Iodide anion can be provided to the system in a liquid form if it is kept stable prior to use. Specifically, it is preferred not to contact the iodide anion with hydrogen peroxide. The concentration iodide that will yield a suitable level of iodine varies with the pH of the contemplated formulation. In addition, the required iodide level will vary dramatically depending upon the ratio of peroxide to iodide. When the preferred ratio of hydrogen peroxide to iodide of 1/2 is used, the preferred range for iodide anions is between 0.0015 and 0.10 grams per liter in the final reconstituted formulation prior to initiation of the enzymatic reaction. However, for certain disease states of general prophylaxis of the eye, it is possible to convert iodide into free molecular iodine less efficiently and therefore the potentially useful range is for iodide anion is between 0.025 and 0.30 grams per liter in the final reconstituted formulation prior to initiation of the enzymatic reaction. These ranges of iodide anion in conjunction with pH and the concentration of the other additives are anticipated to yield a concentration of free molecular iodine within a range of 5 to 330 ppm. The peroxidase enzyme of this invention is identified by the International Union of Biochemistry and the International Union of Pure and Applied Chemistry by the Enzyme Commission identification No. E.C. 1.11.1.7 although certain members of E.C. 1.11.1.8 can also be used. These classes of peroxidase can be obtained from a wide variety of sources including milk (lactoperoxidase), soy bean, and human leukocytes (myerloperoxidase). Within these two E.C. classes the further requirement for a suitable peroxidase as defined in this application is that it is capable of oxidizing iodide to iodine within the pH range of 4 to 7.1. The least expensive peroxidases suitable for this application is horseradish peroxidase and soybean peroxidase. It is anticipated that peroxidase that has been cloned from either horseradish, milk or human leukocytes or other sources will be suitable as a source of peroxidase for this application. Additionally, it has been observed that chemically modified peroxidase is suitable for use in this application. Modifications to the amino, carboxyl or carbohydrate moieties yield a suitable catalytic agent for inclusion in this application. The chemical modifications to peroxidase include cross-linking of enzyme molecules to each other, to solid surfaces or to other proteins. The chemical agents used for crosslinking include glutaraldehyde, maleimides, succinimides, carbodiimides, dicarboxylates, activated glycols, imidoesters, photoreactive azides and other agents known to one skilled in the art. The aforementioned forms of peroxidase can be provided in a dry form such as the lyophilized peroxidase offered commercially or in a largely aqueous environment. If the peroxidase is supplied in an aqueous environment it typically will be incorporated into a medium that provides increased stability such as glycerol, dextrans, or other polyols or sugars in a suitable buffering medium that optimally contains calcium. The peroxidase of this application can be combined with many additives whether it is supplied dry or in an aqueous environment. The concentration range that peroxidase can be used over is between 0.00005 and 0.005 mg/mL in the final composition. The preferred range is between 0.0005 and 0.01 mg/mL in the final composition. Suitable buffering agents for inclusion in the compositions contemplated in this application include water and hydroalcoholic mixtures buffered with glycine-glycine'HCl, potassium hydrogen phthalate-phthalic acid, citric acid-Na2HPO4, citric acid-KH2PO4- H3Bθ3-diethylbarbituric acid-NaOH, citric acid-sodium citrate, dimethylglutaric acid- sodium dimethylglutarate, acetic acid-sodium acetate, succinic acid-sodium succinate, potassium hydrogen phthalate-dipotassium phthalate, sodium cacodylate-cacodylic acid, sodium hydrogen maleate-disodium maleate, Na2HPO4-NaH2PO4, sodium bicarbonate-5% CO2, imidazole-imidazole 'HC1, boric acid-sodium borate, and the following buffers known to one skilled in the art as Good buffers Tris, MES, BIS-TRIS, ADA, ACES and PIPES. The iodine composition can be rinsed from the eye after contact with mammalian ocular tissue. However, one advantage of the compositions described herein is that it is not neccessary to rinse since the concentration level of free molecular iodine is controlled relative to all other iodine species. The examples contained in this application are proof of this fact. In addition, the maximum iodine level and the level of hydrogen peroxide is also controlled thereby forming a non-irritating therapeutic. The non-irritating microbicides described in this application have at least 25% free molecular iodine on a molar basis with the free molecular iodine concentration between 5 and 330 ppm formed by enzymatically reacting peroxidase, iodide and peroxide in an aqueous medium in a molar ratio of hydrogen peroxide to iodide of no greater than four and a maximum concentration level of hydrogen peroxide of 0.015% before applying the microbicide to the human eye. The preferred pH for an ophthalmic mendicament is between 4.5 and 7.1 althoug there are existing ophthalmic mendicatments with a pH that range from 3.5 to 8.5.. All of the foregoing operational principles and advantages of the present invention will be more fully appreciated upon consideration of the following detailed examples. Examples Example 1 Iodine based compositions were prepared consisting principally of free molecular iodine. These compositions were prepared by incubating horseradish peroxidase, sodium iodide and hydrogen peroxide at a pH of 5.5, allowing these materials to incubate for a defined time period and then performing an analytical characterization of the resulting formulation. The concentration of the following three iodine species were measured: free molecular iodine, triiodide and iodide anion. Free molecular iodine was measured potentiometrically by the method of Gottardi (Gottardi W., Fresenius Z. Anal. Chem., volume 314, page 582, 1983). Two Corning model 345 pH meters were used to make the potentiometric measurements. The Corning electrodes that were used included a standard platinum electrode, a reference electrode and an iodide ion selective electrode. Triiodide was determined by performing a sodium thiosulfate titration to determine the level of total iodine and then subtracting the level of free molecular iodine from the total iodine level. The iodide ion concentration was determined directly by using a Corning iodide ion selective electrode. There were three conditions explored using a citrate buffer at a pH of 5.5. All conditions used a final concentration of 5 ug/mL of horseradish peroxidase. The first condition (Treatment No. 1) used a concentration of sodium iodide that was 60 mg per liter and an equal molarity of hydrogen peroxide that was established using sodium percarbonate (0.025 grams per liter) as the source of hydrogen peroxide. Conditions two and three (Treatment Nos. 2 & 3) used 200 and 450 mg per liter of sodium iodide and 0.0833 and 0.187 grams per liter of sodium percarbonate respectively. The solutions were allowed to incubate for 60 minutes and the analytical measurements were made. The results are shown below in Table I. The conclusion we draw from the data shown in Table I is that it is possible to generate peroxidase based iodine germicides that are principally comprised of free molecular iodine. The data in Table shows the concentration of the three predominant iodine species: free moleuclar iodine, iodide anion and triiodide. The concentration of hypoiodous acid and iodate under the conditions used are insignificant as compared to the concentration of the three iodine species listed in Table I. Example 2 Iodine based compositions were prepared at a pH of 7.0 that consisted principally of free molecular iodine. These compositions were prepared by incubating horseradish peroxidase, sodium iodide and hydrogen peroxide at a pH of 7.0. These components were allowed to incubate for a defined time period and then analytical determinations were performed on the resulting composition. The weights and volumes of the chemical components used were: 0.2 grams of anhydrous citric acid, 450 mg of sodium iodide, 1.3 mL of 3% hydrogen peroxide, 100 mg of sodium bicarbonate and 3 mg of horseradish peroxidase. The composition was allowed to incubate for 15 minutes prior to performing the analytical determinations described in experiment 1. The results are shown below: The conclusion we draw is that it is possible to generate iodine based germicides at a pH of 7.0 that are principally comprised of free molecular iodine. Example 3 The three formulations described in experiment 1 were tested to determine their ability to inactivatepathogens, such as bacteria, in the presence of a bioburden. Suspensions of Staphylococcus aureus were prepared that contained 10" red blood cells (RBC) per mL. The effect that these formulations had on the cellular morphology of the RBCs was determined by microscopic examination. A control experiment was performed using dilutions of 10% PVP-I2; the dilutions ranged from 1/20 to 1/10,000. Whole blood was processed by the Ficoll lymphocyte isolation procedure. The red blood cells (RBC) were recovered and washed once with phosphate buffered saline and held at a concentration of 3.5 x 10" RBC/mL in Alsevers solution at 5 C. Agar plates were inoculated by washing the growth from Staphylococcus aureus slants with 5 mL of 0.25 molar phosphate buffer at a pH of 7.0 (PBDW) into 100 mL of PBDW. One mL of this suspension was added to each of 7 nutrient agar plates which were incubated for 18-24 hr at 35-37 . Bacterial colonies were removed from the agar surface using 1 mL of PBDW and a sterile swab. The suspension was standardized to give about 10™ cfu /mL using a Milton-Roy Spec 20 spectrophotometer. One hundred mL of each test germicide was equilibrated at 25 C in a water bath. Two minutes prior to the start of the assay, RBCs were added at a level of 10" RBCs/mL. S. aureus was added at a level of 2.0 x 10^ cfu/mL. One mL samples were removed after 30, 60, 120, and 300 second exposures and neutralized in 10 mLs 0.5% thiosulfate. One mL and 0.1 mL of the neutralized samples were pour plated in quadruplicate using Plate Count Agar. This experiment determined the ability of the disinfectant to inactivate at least 10° cfu/m of Staphylococcus aureus. All of the disinfectants were effective against Staphylococcus aureus in some time frame with the exception of 10% PVP-I2 at a 1/1,000 dilution (i.e., 10 ppm total iodine). The peroxidase based iodine germicide exhibited a 6 log reduction of Staphylococcus aureus within 30 seconds at free molecular iodine levels of 63 and 114 ppm; at a free molecular iodine level of 13 ppm the peroxidase based iodine germicide required 5 minutes to effect a 6 log inactivation of Staphylococcus aureus. Microscopic examination of the RBCs treated with the peroxidase based iodine germicide RBCs showed no morphologic changes or reduction in counts of the cells. The RBCs treated with 10% PVP-I2 appeared discolored and shriveled with no reduction in count except for the 10% PVP-I2 sample that was diluted 1/1000. The level of free molecular iodine in the 1/1000 dilution of 10% PVP- ) was determined to be 6 ppm. This experiment demonstrates that a properly controlled formulation that consists principally of free molecular iodine can both serve as an effective biocide while remaining non-irritating to sensitive biological materials such as individual cells and tissues. Example 4 An enzyme based iodine germicide was tested to determine its ability to inactivatepathogens, such as viruses, in the presence of a bioburden. An enzyme based iodine germicide was generated and its ability to inactivate viruses in the presence of a high concentration of red blood cells in human serum was determined. The germicide was prepared by dissolving 450 mg of sodium iodide in one liter of 0.05 molar citrate buffer at pH 5.5 and then simultaneously dissolving 10 mg of soybean peroxidase. This solution was allowed to incubate for 10 minutes and then the virucidal efficacy of this formulation was determined. Whole blood was processed by the Ficoll lymphocyte isolation procedure. The red blood cells (RBC) were recovered and washed once with phosphate buffered saline and held at a concentration of 6.5 x 10^ RBC/mL in Alsevers solution at 5 C. The tissue culture infective dose 50 (TCID50) is the concentration of virus particles that causes 50% of a series of cultures to be infected. About 10^ units of TCID50 vesicular stomatitis virus were added to samples of pooled human serum that contained 10" RBCs per mL. This sample was incubated with the germicide for 5 minutes at 4°C and at 25°C. After five minutes the virus titier was determined as follows. In quadruplicate, the sample was serially diluted fivefold in minimal essential media (MEM) nonessential amino acids solution containing 10% fetal calf serum and 0.1 % sodium thiosulfate. The dilutions were plated onto 96- well plates containing Vero cells (ATCC CRL81) at approximately 80% confluency. The lowest dilution used in this assay was 1:5 times the original treated serum. The cultures were incubated for 3 days at 37°C in a 5% CO2 atmosphere and each well was examined for a cytopathic effect. At both 4 °C and 25°C no cytopathic effect was observed in mock infected and iodine- treated tissue culture wells. This indicates that the germicide was effective in inactivating a lipid enveloped virus in the presence of sensitive biological material. The effect that these formulations had on the cellular morphology of the red blood cells was determined by microscopic examination. Microscopic examination of the red blood cells indicated that the enzyme based iodine germicide did not cause a morphologic change or reduce the number of RBCs. None of the treated cells appeared discolored or shriveled. Example 5 Suspension cells (CEM Lymphoblastic leukemia cells ATCC CCL-21) were grown in RPMI 1640 with 20% fetal bovine serum to plateau phase which equals approximately 2 x 10^ cells per mL and then concentrated by centrifugation. These cells were then treated with free molecular iodine so that various ratios of free molecule iodine per cell were achieved (see Table 5a). Attachment cells (Vero) were grown to confluency in T-75 flasks. The cells were removed by trpypsinization and then concentrated by centrifugation. These cells were then treated with free molecular iodine so that various ratios of free molecule iodine per cell were achieved (see Table 5b). The percent viability of the cells appears to be inversely proportional to the level of free molecular iodine per cell. An upper limit to the ratio of free molecular iodine per cell is apparent. The data strongly suggests that it is feasible to expose sensitive biological materials to a concentration of free molecular iodine that is antibacterial but not cytotoxic. Example 6 An enzyme based iodine germicide was generated and its ability to inactivate bacteria in the eyes of three month old male and female New Zealand white rabbits was evaluated. The germicide was identical to the compositionn described as Treatment 1, In Example 1 enough sodium percarbonate was added to adjust the pH to 6.0 which lowered the level of free molecular iodine from 46 to 32 ppm. Prior to test instillation of the germicide, all test and control eyes were judged free of significant ocular irritation. To detect or confirm any pre-existing corneal injury, the eyes were treated with fluorescein stain and observed in a darken room with ultraviolet light. A small sample of the germicide (0.1 ml) was instilled into the lower conjunctival sac of the left eye of each rabbit and the lid held closed for one (1) second. The opposite eye of each rabbit remained untreated and served as the comparative control. The eyes were examined for ocular reactions at one hour after test article instillation and again: at day 1, 2, 3, 4, 7, 14 and 21. The flourescein staining procedure was conducted at each time interval. One drop of fluorescein was administered by eye dropper to assess tissue damage. The results of these tests for days 1, 14 and 21 are shown below in Table 6. No corneal opacity nor iritis was observed in any of the animals during the test period. Minimal transient conjunctivitis was observed in 2/6 animals within 24 hours after treatment. This condition was abolished by day 2. No accessory orbital growth nor inflammation to the surrounding ocular structures was observed in any of the animals. The conclusion that was drawn was that the germicide did not cause irritation to the eye. A second experiment was conducted wherein 0.1 ml of a saline solution containing 10" colony forming units of Staphylococcus aureus was instilled directly into the left eye of each of the rabbits used above. Five minutes was allowed to elapse and then the each eye was instilled with 0.1 ml of the germicide. The germicide was instilled every six hours for 24 hours and then the left eye from each rabbit was swabbed with cotton. The cotton was plated onto an agar plate and the agar plates were incubated at 37°C for five days. No colonies of Staphylococcus aureus were observed on any of the rabbit eyes. Example 7. This experiment demonstrates that high concentrations of free molecular iodine are not necessarily an ocular irritant if they are formulated within the specifications of this application. Thirty samples each of the three enzymatic iodine compositions described in Example 1 were prepared and two samples of each formulation type was reconstituted freshly daily for three days. The concentration of free molecular iodine was measured for each sample and the averageconcentration of free molecular iodine was determined to be: 53 ppm for Treatment 1; 162 for Treatment 2; and 324 for Treatment 3. These three different Treatment types (1, 2 and 3) were used to determine ocular irritancy of free molecular iodine in NZ albino rabbits using two different applications regimens over the course of three days. Rabbits were divided into 6 groups; each group consisted of four rabbits. Group I and π were dosed with the iodine composition Treatment I that contained 53 ppm free molecular iodine. Group III and IV were dosed with the iodine composition Treatment π that contained 162 ppm free molecular iodine. Group V and VI were dosed with the iodine composition Treatment in that contained 324 ppm free molecular iodine. Treatment Groups I, III and V were dosed with four drops every 2 hours for 3 days; the total number of doses per day for these animals was 4 i.e., 16 drops. Treatment Groups π, IV and VI were dosed with 8 drops every hour for 3 days; the total number of doses per day for these animals was 8 i.d., 32 drops. Eyes were examined using the scale for scoring ocular lesions defined in the Draize test (Draize J.H., G. Woodward, H.O. Calvery, J. Pharmacol. Exp. T er. Vol. 82, pp. 377-390, 1944) which includes conjunctival hyperemia and chemosis, ocular discharge, iritis and cornneal opacities. The overall ocular irritation scale ranged from 0 to 110 and contained the following quantiles: 0.0 - 0.5 (non-irritating), 0.6 - 2.5 (practically non- irritating), 2.6 - 15.0 (minimally-irritating), 15.1 - 25.0 (mildly irritating), 25.1 - 50.0 (moderately irritating), 50.1 - 80.0 (severely irritating), 80.1 - 100.0 (extremely irritating) and 100.1 - 110.0 (maximally irritating). The irritation results are shown below in Table 7a. The staining of eyes was also evaluated by comparison to known ocular staining from dilutions of Betadine. Undiluted Betadine and seven separate twofold serial dilutions of Betadine in water were used as the comparitive norms for staining in a scale that ranged from 0 to 4.0. The results of this study demonstrate that there was no staining of any cornea or conjunctiva for any type or treatment regimen tested on any day treated. The preparations of free molecular iodine tested in this study were minimally irritating to the NZ rabbit eye. These preparations did not cause any damage to the cornea of any rabbit regardless of the concentration of free molecular iodine or treatment regimen. The highest concentration of free molecular iodine was found to be the most irritating of the three Treatment types tested and even that degree of irritation was minimal. There was no overall difference in irritation between the treatment regimens (4 drops vs 8 drops for 3 days). Some of the minimal ocular irritation induced by these preparations may be due to the low pH of the different formulations. The pH's of these preparation's are in the acidic range (Treatment 1 - pH = 4.75, Treatment 2 - pH = 4.90, Treatment 3 - pH = 5.01). These defined compositions of free molecular iodine did not at any time stain the cornea or conjunctiva of any rabbit eye. All corneas remained clear and all conjunctivae white. Matching the color of the three Treatment formulstions to the colors of the dilution's of Betadine that were used for the iodine staining scale, the 324 ppm compositions of I 2 (Treatment HI) appeared to be equivalent to 0.625% Betadine, the 162 ppm I composition appeared to be equivalent to 0.3125% Betadine, and the 53 ppm I 2 composition appeared to be equivalent to 0.15625% Betadine.

Claims: WHAT IS CLAIMED IS : Claim 1- A method for therapeutically treating a mammalian eye to disinfect said eye of pathogenic organisms comprising the steps of; admixing in an aqueous medium a composition consisting essentially of an iodide-oxidizing peroxidase selected from the group consisting of horseradish peroxidase, lactoperoxidase, myleroperoxidase and soybeanperoxidase, a source of hydrogen peroxide and an iodide source for forming a non- irritating aqueous microbicide having a plurality of iodine species with the molar ratio of hydrogen peroxide to iodide maintained at between about 0.5 and 4.0 such that free molecular iodine is the principle iodine species present with said microbicide having a maximum concentration of hydrogen peroxide of 0.015 % and a maximum concentration of thiosulfate titratable iodine of 650 ppm. 2. A method as defined in claim 1 wherein said free molecular iodine constitutes at least 25% of said plurality of iodine species on a molar basis. 3. A method as defined in claim 2 wherein said composition further comprises a buffer agent to maintain the pH of said microbicide at between pH 4.5 and pH 7.1. 4. A method as defined in Claim 3 wherein said pH is maintained between 6.0 to 7.1. 5. A method as defined in Claim 3 wherein said buffering agent is selected from the group consisting of glycine, phthalate acid, citric acid, phosphate, borate, barbituric acid, glutaric acid, dimethylglutarate, acetic acid, sodium acetate, succinic acid-sodium succinate, cacodylic acid, sodium hydrogen maleate, sodium bicarbonate or other agents that yield carbonate anion is solution, imidazole and the following buffers known to one skilled in the art as Good buffers Tris (tris(hydroxymethyl)aminoethane), MES (2-[N- morpholinojethanesulfonic acid), BIS-TRIS (bis[2-hydroxyethyl]imino- tris[hydroxymethyl]methane; 2-bis[2-hydroxyethyl]amino-2-[hydroxyme yl]- 1 ,3- propanediol), ADA (N-[2-acetamido]-2-iminodiacetic acid; N- [carbamoylmethyl]iminodiacetic acid), ACES ((2-[(2-amino-2-oxoethyl)- amino]ethanesulfonic acid; N-[2-acetamido]-2-aminoethane-sulfonic acid) and PIPES (piperazine-N, N'-bis[2-ethanesulfonic acid] and 1,4-piperazinediethanesulfonic acid). 6. An opthalmic non-irritating iodine anti-infective medicament for treating the eye comprising an iodide-oxidizing peroxidase selected from the group consisting of horseradish peroxidase, lactoperoxidase, myleroperoxidase and soybean-peroxidase , a source of hydrogen peroxide and an iodide source in an admixure with water having a molar ratio of hydrogen peroxide to iodide of between about 0.5 and 4.0 and a maximum concentration of hydrogen peroxide of 0.015% such that free molecular iodine is the principle iodine species generated in the admixture with said admixture having a maximum concentration of hydrogen peroxide of 0.015% and a maximum concentration of thiosulfate titratable iodine of 650 ppm. 7. An opthalmic non-irritating iodine medicament as defined in claim 6 wherein a plurality of iodine species is formed in the microbicide with free molecular iodine representing at least 25% of the plurality of iodine species on a molar basis. 8. An opthalmic non-irritating iodine medicament as defined in claim 7 wherein said free molecular iodine is in a range of between 5 and 330 ppm. 9. An opthalmic non-irritating iodine medicament as defined in claim 8 wherein said composition further comprises a buffer agent to maintain the pH of said microbicide at between pH 4.5 and pH 7.1. 10. An opthalmic non-irritating iodine medicament as defined in Claim 9 wherein said pH is maintained between 6.0 to 7.1. 11. An opthalmic non-irritating iodine medicament as defined in Claim 10 wherein said buffering agent is selected from the group consisting of glycine, phthalate acid, citric acid, phosphate, borate, barbituric acid, glutaric acid, dimethylglutarate, acetic acid, sodium acetate, succinic acid-sodium succinate, cacodylic acid, sodium hydrogen maleate, sodium bicarbonate or other agents that yield carbonate anion is solution, imidazole and the following buffers known to one skilled in the art as Good buffers Tris (tris(hydroxymethyl)aminoethane), MES (2-[N-morpholino]ethanesulfonic acid), BIS-TRIS (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane; 2-bis[2-hydroxyethyl]amino-2- [hydroxymethyl]-l,3-propanediol), ADA (N-[2-acetamido]-2-iminodiacetic acid; N- [carbamoylmethyl]iminodiacetic acid), ACES ((2-[(2-amino-2-oxoethyl)- amino]ethanesulfonic acid; N-[2-acetamido]-2-aminoethane-sulfonic acid) and PIPES (piperazine-N, N'-bis[2-ethanesulfonic acid] and 1 ,4-piperazinediethanesulfonic acid).

Inventors: KESSLER JACK, KESSLER, JACK
Applicants: KESSLER JACK, SYMBOLLON CORP, KESSLER, JACK, SYMBOLLON CORPORATION
Application year: 1997
Main classification: A61K9
IPC codes: A61K9, A61K33, A61K38, A61K47, A61P27, A61P31