Patent Description:
In the United States there are currently no regulations regarding the sterilization requirements of topical antiseptic solutions. Therefore, antiseptic solutions currently sold in the United States generally do not undergo a sterilization process. In other jurisdictions, however, such as European Union (EU) countries, some degree of sterilization is required. A known antiseptic solution containing <NUM>% w/v chlorhexidine gluconate in <NUM>% v/v isopropanol in water (i.e., an alcoholic solution), manufactured by CareFusion Corp. , is sterilized for EU countries using a known sterilization method.

It is the industry belief that high temperature sterilization is not suitable due to the expected degradation. See, for example, <NPL> and <NPL>.

A known method of sterilization involves heat treating glass ampoules containing the chlorhexidine gluconate alcoholic solution in a convection oven at <NUM>-<NUM> for <NUM>-<NUM> hours. It was believed that relatively low temperature and relatively long processing time is necessary to sufficiently sterilize the antiseptic alcoholic solution without overly degrading the antimicrobial molecules, thereby avoiding reducing the concentration and purity of the chlorhexidine gluconate contained therein as an antiseptic. Applicant's copending <CIT> describes an alternative method for sterilizing an alcoholic solution.

However, there is no known method of sterilizing an aqueous antiseptic solution that affords a sterile solution without overly degrading the antimicrobial molecules. Thus, there is an unmet need in the art for a method of sterilizing aqueous antiseptic solutions.

Aspects of the present invention overcome the above identified problems, as well as others, by providing systems, methods, and devices for efficiently sterilizing antimicrobial solutions while maintaining antimicrobial efficacy as an aqueous antiseptic and purity of the active drug moiety to comply with regulatory requirements.

The invention as defined in claim <NUM> is a method for sterilizing an aqueous antiseptic solution, the method comprising providing a container containing the aqueous antiseptic solution, the aqueous antiseptic solution comprising about <NUM>% w/v chlorhexidine gluconate and a solvent containing <NUM>%v/v water, heating the aqueous antiseptic solution to a predetermined temperature, maintaining the aqueous antiseptic solution at the predetermined temperature for a predetermined time, and terminating the heating of the aqueous antiseptic solution when the predetermined time expires; wherein the predetermined temperature and the predetermined time are selected such that after terminating the heating, the aqueous antiseptic solution is sterile; characterized in that wherein the sterilization temperature and the sterilization time are selected such that after terminating the heating, the antiseptic solution has a post-sterilization purity of at least about <NUM>% and the percentage point change in purity from the initial purity to the post-sterilization purity is at most about <NUM>%, the predetermined temperature is between about <NUM> and <NUM>,
the sterilization time is:.

In another example aspect, the selected sterilization temperature and the selected sterilization time are chosen such that after terminating the heating, the antiseptic solution has a post-sterilization purity of at least about <NUM>% and the percentage point change in purity from the initial purity to the post-sterilization purity is at most about <NUM>%.

Additional advantages and novel features relating to aspects of the present invention will be set forth in part in the description that follows, and in part will become more apparent to those skilled in the art upon examination of the following or upon learning by practice thereof.

Aspects of the present invention overcome the above identified problems, as well as others, by providing systems, methods, and devices for sterilizing an aqueous antiseptic solution while maintaining antimicrobial efficacy and while complying with regulatory requirements.

Various aspects of an antiseptic applicator may be illustrated with reference to one or more exemplary embodiments. As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other embodiments of sterilization methods disclosed herein.

The term "about" as used herein means ±<NUM>% and preferably ±<NUM>% of the provided value.

Aspects of the present invention include a method of sterilizing aqueous antiseptic solution contained in a container. The method may include heating aqueous antiseptic solution contained within a container or ampoule to a certain predetermined temperature and maintaining the temperature for a certain amount of predetermined time sufficient to sterilize the solution while maintaining sufficient purity of the antiseptic solution to comply with regulatory requirements. The antimicrobial efficacy directly relates to the purity of the antiseptic solution. Generally, when the purity of the antiseptic molecules is too low, the solution is not as effective in serving the function of an antimicrobial solution. Furthermore, higher levels of impurities within an antiseptic solution can have a deleterious impact on patient health.

The container is preferably a self-contained structure, formed of a material suitable for containing the antiseptic solution. In an aspect, the container may be made of a frangible material such that upon application of sufficient force the container fractures. For example, the material may comprise plastic or glass. The terms "container" and "ampoule" are used interchangeably herein. The wall of the container may have a thickness sufficient to withstand the sterilization process, transport, and storage. When the container is frangible, the material and thickness may also be sufficient to allow the container to be fractured upon the application of localized pressure. The thickness range may vary depending on the container size. Example thicknesses for glass or plastic containers include from about <NUM> to about <NUM>. In another example aspect, the container may comprise a non-frangible material, such as a metal (steel, aluminum, etc.) or such as a pouch comprising or consisting of a polymeric and/or foil material capable of withstanding the sterilization process. For example, the container may be a retort-like foil pouch having a composite material of polymeric and foil. An example thickness of the pouch may be about <NUM> inches to <NUM> inches (<NUM> to <NUM>).

While antiseptic solutions are of particular focus herein, the container may alternatively contain medicaments, chemical compositions, cleansing agents, cosmetics, or the like. For example, the container may be filled with antiseptic compositions (e.g., compositions comprising one or more antiseptic molecules), preferably an antimicrobial liquid or gel composition. For example, the antiseptic solution may contain non-active ingredients/agents with functions that include moisturizing, skin smoothing, visualization, solubility, stability, viscosity, wetting, etc..

In an aspect of the present invention, the antiseptic solution is aqueous. That is, the solvent of the solution is primarily water. It is disclosed that aqueous means at least about <NUM>% v/v water, more preferably at least about <NUM>% v/v water, more preferably at least about <NUM>% v/v water, more preferably at least about <NUM>% v/v water, more preferably at least about <NUM>% v/v water, more preferably at least about <NUM>% v/v, up to <NUM>% v/v water. As used herein, aqueous means <NUM>% v/v water. It is disclosed that, when the solution is less than <NUM>% v/v water, the remaining volume may include one or more additional solvents, for example, alcoholic solvents. Example alcoholic solvents include ethanol, isopropanol, and n-propanol. For example, the solution may contain less than about <NUM>% v/v, more preferably less than about <NUM>% v/v, more preferably less than about <NUM>% v/v, more preferably less than about <NUM>% v/v, more preferably less than about <NUM>% v/v, down to <NUM>% v/v alcohol. A preferred alcohol may be isopropanol.

The container may contain antiseptic solution of a sufficient amount, sufficient concentration, and sufficient purity to be applied to a desired surface and have an antimicrobial effect on the desired surface. In one aspect, the desired surface is a patient's skin. It will be appreciated that the amount of antiseptic solution may vary. In one aspect the amount of antiseptic solution may be <NUM>-<NUM> of antiseptic. More preferably, the amount of antiseptic solution needed may be about <NUM>-<NUM> and still preferably may be about <NUM>-<NUM>. Examples include <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> of antiseptic. In a situation where a larger amount of solution is desired, e.g., <NUM>, multiple smaller containers may be implemented in a single applicator (e.g., two <NUM> containers).

Aspects of the disclosure are suitable antiseptic molecules which include bis-(dihydropyridinyl)-decane derivatives (e.g. octenidine salts) and/or biguanides (e.g., chlorhexidine salts) according to the invention. As used herein, the term "derivative" refers to a) a chemical substance that is related structurally to a first chemical substance and derivable from it; b) a compound that is formed from a similar first compound or a compound that can be imagined to arise from another first compound, if one atom of the first compound is replaced with another atom or group of atoms; c) a compound derived or obtained from a parent compound and containing essential elements of the parent compound; or d) a chemical compound that may be produced from first compound of similar structure in one or more steps. Aspects of the disclosure are examples of biguanides/biguanide derivatives other than chlorhexidine/chlorhexidine salts including alexidine, alexidine salts, polyhexamide, polyhexamide salts, polyaminopropyl biguanide, polyaminopropyl biguanide salts, and other alkyl biguanides. Aspects of the disclosure are antiseptic agents including octenidine salts, such as octenidine dihydrochloride (a bis-(dihydropyridinyl)-decane derivative and a cationic surfactant), and chlorhexidine salts. According to the invention, the antiseptic molecule is chlorhexidine gluconate (a cationic biguanide). It is disclosed that the concentration of the antiseptic may vary depending on the specific antiseptic species used or the desired antimicrobial effect that is desired. For example, when using octenidine or an octenidine salt the concentration may vary from about <NUM> % w/v to about <NUM>% w/v, more preferably from about <NUM>% w/v to about <NUM>% w/v, and still more preferably from about <NUM>% w/v to about <NUM>% w/v. When chlorhexidine or a chlorhexidine salt is used, the concentration may be from about <NUM>% w/v to about <NUM>% w/v, more preferably from about <NUM>% w/v to about <NUM>% w/v, and still more preferably about <NUM>% w/v to about <NUM>% w/v.

In an aspect, when a biguanide, e.g., the chlorhexidine salt, is used, the purity of the solution, when applied to the skin (e.g., after the sterilization method described herein), may be at least about <NUM>% pure, more preferably at least about <NUM>% pure, still more preferably at least about <NUM>% pure. As used herein, purity means the percent concentration of antiseptic molecules in solution relative to the total concentration of antiseptic molecules plus concentration of substances that are derived from or related to the antiseptic molecule. For example, a <NUM>% pure antiseptic solution means that if there are <NUM> molecules that are either antiseptic molecules or molecules derived from or related to the antiseptic molecule, <NUM> of the molecules are the antiseptic molecule and <NUM> of those molecules are derived from or related to the antiseptic molecule. These molecules derived from or relating to the antiseptic molecule have reduced or no antimicrobial activity. Thus, a lower purity solution will have lower antimicrobial efficacy as fewer of the target antiseptic molecules are delivered to the patient's skin. Further, a lower purity solution will not comply with regulatory requirements. By measuring the concentration of antiseptic molecules in solution as compared to concentration of antiseptic molecules and molecules derived from or related to the antiseptic molecule, one can determine the purity of the solution and whether the purity is sufficient to comply with regulatory requirements.

The antiseptic solution provided in the container comprises, consists essentially of, or consists of water as the only solvent and about <NUM>% w/v antiseptic molecule which is chlorhexidine gluconate.

It has been found that when the aqueous antiseptic solution within the container is brought to a particular temperature and maintained at that temperature for a particular amount of time, the solution is sufficiently sterilized while maintaining sufficient antimicrobial efficacy as an antiseptic and while satisfying regulatory requirements. The antiseptic solution is brought to a temperature (also referred to herein as the "sterilization temperature") from about <NUM> to about <NUM>.

As used herein, the term "predetermined sterilization time" means the length of time at which the solution is at the sterilization temperature. That is, the "sterilization time" does not include the time it takes for a solution to reach the sterilization temperature (i.e., does not include "ramp up" time) and also does not include the time it takes for the solution to return to the temperature the solution was at prior to the heating (i.e., does not include "cool down" time). The time it takes for the temperature of the solution to reach the sterilization temperature is referred herein as the "ramp up" time and the time to return to the starting temperature is referred herein as the "cool down" time. As used herein, the term "predetermined sterilization temperature" means the temperature or temperature range that the solution reaches and maintains during the sterilization time, independent of the starting temperature of the solution. For purposes of illustration only, a sterilization time of <NUM> minutes and a sterilization temperature of <NUM> for a solution starting at <NUM> would mean that the period of time starting from the moment the solution reaches <NUM> and ending the moment the solution falls below <NUM> during the beginning of the cool down process is <NUM> minutes. Thus, the time it takes from the solution to rise from <NUM> to <NUM> (i.e., ramp-up time) and the time it takes for the solution to return to <NUM> (i.e., cool-down time) is not included in the sterilization time.

The predetermined sterilization time and sterilization temperature provided herein generally assume the thermal exposure during the ramp-up and the cool-down does not contribute to the sterilization of the drug product as on a small scale these processes can be considered instantaneous. However, on a commercial scale, the time spent heating the product up will contribute to the overall lethality of the sterilization process, allowing the steady-state sterilization time to be decreased. When the ramp-up and cool-down contributions to the cycle are applied, the sterilization of the drug product can be described by the F-value calculated for each predetermined sterilization time and sterilization temperature using the following equation (see "<NPL>): <MAT> where:.

For the purposes for illustration only, a sterilization temperature of <NUM> with a predetermined sterilization time of <NUM> minutes (i.e. ramp-up and cool-down do not contribute to the sterilization of the drug product) corresponds to a minimum F-value of <NUM> minutes at <NUM> (F<NUM>) in order to sterilize the drug product. This minimum required F-value can be used to quantify a process in which the ramp-up and cool-down do contribute to the sterilization of the drug product. In such a process, the contribution for the ramp-up and cool-down on the minimum required F-value can be calculated. If during a sterilization cycle defined by an F<NUM> = <NUM> minutes a temperature of <NUM> is not reached, the cycle parameters could still be met per the calculation of F<NUM> as a summation of thermal input during the actual cycle.

It has been found that combinations of sterilization temperature and sterilization time can be selected to provide a sterilized aqueous antiseptic solution having sufficient purity to comply with regulatory requirements when used as an antiseptic. For a sterilization temperature of about <NUM>, the sterilization time is from about <NUM> minutes to about <NUM> hours. For a sterilization temperature of about <NUM>, the sterilization time is at least about <NUM> minutes and up to about <NUM> hours. For a sterilization temperature of about <NUM>, the sterilization time is at least about <NUM> minutes to about <NUM> hours. For a sterilization temperature of about <NUM>, the sterilization time is at least about <NUM> minute and up to about <NUM> hours. The above sterilization temperatures and sterilization times are applied to an antiseptic solution comprising about <NUM>% v/v water and about <NUM>% w/v chlorhexidine gluconate.

It has been found that heating the antiseptic solution contained in the container to the above sterilization temperatures and maintaining the temperature for the above sterilization times, sufficiently sterilizes the solution, while maintaining sufficient purity to comply with regulatory requirements. The amount of degradation of the antiseptic molecule can be quantified by measuring the initial purity of antiseptic solution prior to the ramp up time (i.e., prior to the process of bringing the solution up to the sterilization temperature) and measuring the post-sterilized purity of antiseptic solution after the cool down time (i.e., after the antiseptic solution returns to the temperature the solution was at prior to the process of bringing the solution up to the sterilization temperature). Thus, as used herein, the "initial purity" is the purity prior to ramp up and "post-sterilization purity" is the purity of the solution after cool down. In an aspect of the present invention, the initial purity of the antiseptic solution, chlorhexidine gluconate, may be at least about <NUM>%, preferably at least about <NUM>%, and more preferably at least about <NUM>%. The meaning of purity is provided above. The resulting post-sterilized solution is found to have sufficient purity to provide the desired antimicrobial efficacy as an antiseptic and to comply with regulatory requirements.

In an example aspect, it has been found that chlorhexidine gluconate molecules degrade into one or more the following molecules when heat treated: N-[[<NUM>-[[[(<NUM>-chlorophenyl)carbamimidoyl]carbamimidoyl]-amino]hexyl]carbamimidoyljurea, N-(<NUM>-chlorophenyl)guanidine, N-(<NUM>-chlorophenyl)urea, <NUM>-(<NUM>-aminohexyl)-<NUM>-(<NUM>-chlorophenyl) biguanide, N-(<NUM>-chlorophenyl)-N'-[[<NUM>-[[[(<NUM>-chlorophenyl)carbamimidoyl]carbamimidoyl] amino]hexyl]carbamimidoyljurea, <NUM>-(<NUM>-chlorophenyl)-<NUM>-[<NUM>-[[(phenylcarbamimidoyl) carbamimidoyl]lamino]hexyllbiguanide, <NUM>-[<NUM>-(carbamimidoylamino)hexyl]-<NUM>-(<NUM>-chlorophenyl)-biguanide, and <NUM>-chloroaniline. Thus, in an example aspect, the purity of the solution can be determined by comparing the amount of chlorhexidine gluconate to all of the above-listed chlorhexidine gluconate related substances. However, it should be noted that the above list is not exhaustive. One having ordinary skill in the art would be able to determine which molecules are degradants of the antiseptic molecule after the sterilization process.

As noted above, the purity of the antiseptic solution after the heating has been terminated and when the solution has returned to the temperature the solution was at prior to the process of bringing the solution up to the sterilization temperature (for example ambient temperature) is referred herein as the post-sterilization purity. As noted above, the post-sterilization purity is preferably measured when the antiseptic solution has cooled because degradation may occur during cooling. In an aspect of the present invention, by selecting an appropriate combination of sterilization temperature and sterilization time, the post-sterilization purity may be maintained relatively close to the initial purity, while still being sterile. In particular, the combination of sterilization temperature and sterilization time are chosen such that the percentage point change in purity from the initial purity to the post-sterilization purity is at most about <NUM>%, more preferably at most about <NUM>%, more preferably at most about <NUM>%, and most preferably at most about <NUM>%. It should be understood that the percentage point change refers to the absolute percentage point difference between the initial purity and the post-sterilization purity. For example, a change in initial purity of <NUM>% to a post-sterilization purity of <NUM>% is a percentage point change of <NUM>%.

In addition to maintaining a sufficient purity, it has been found that the proper combination of sterilization temperature and sterilization time can be selected such that the solution is sterile. As used herein, sterile means "<NUM> day sterility" as tested following the procedures described in <NPL>. Sterile also means completely free of microbes, immediately following sterilization. In an aspect, Bacillus subtilis may be used as a test microbe. Thus, in an aspect, a sterile solution would have no growth of Bacillus subtilis shown by the '<NUM> day sterility' testing described above. In another aspect, a solution inoculated with Bacillus subtilis would be completely free of viable Bacillus subtilis immediately following the sterilization method.

In another aspect of the present invention, it was found that the inventive method has a sterility assurance level (SAL) of at least about <NUM>-<NUM> under particular combination of sterilization temperature and sterilization time. SAL is a measurement of probability of a microorganism occurring on an item following a sterilization procedure. A SAL of <NUM>-<NUM> means there is a <NUM> in <NUM>,<NUM>,<NUM> chance of a viable microorganism occurring in a sterilized product. Thus, the SAL measures the probability of a sterilization method resulting in a non-sterilized product. The calculation to determine SAL is described in more detail in the below examples. For example, it has been found that a method of exposing the aqueous antiseptic solution to a temperature of <NUM> for about <NUM> minutes, a temperature of <NUM> for about <NUM> minutes, or <NUM> for about <NUM> minutes would each have a SAL of at least <NUM>-<NUM> (i.e., a <NUM>/<NUM>,<NUM>,<NUM> chance that a viable microbe will be present in a sterilized solution).

As noted above, after the sterilization time ends, the solution may be cooled. For example, it may take about <NUM> to about <NUM> minutes to cool the antiseptic solution following the sterilization time. The time can be shortened using a cooling device. This additional time correlates with the particular sterilization temperature. For examples, a higher sterilization temperature (e.g., <NUM>) as compared to a lower sterilization (e.g., <NUM>) would take longer to return to room temperature after sterilization. Thus, the overall processing time, including cool down, may include an additional about <NUM> to about <NUM> minutes longer than the sterilization time.

It is within the scope of the invention that any machine capable of heating the antiseptic solution to the sterilization temperature and maintaining the solution at the sterilization temperature for the sterilization time may be used, while preferably limiting the ramp up time. Example equipment may include a water bath, oil bath, autoclave, convection oven, cascading water sterilizer, and the like. When using the cascading water sterilizer the ramp up time may be about <NUM> minutes, while the cool down time may be about <NUM> minutes. The cascading water sterilizer provides a constant stream of water which heats the solution to the sterilization temperature, maintains the sterilization temperature over the entirety of the sterilization time, and finally cools the solution.

The sterile aqueous solution, after undertaking the above-described sterilization process, may be implemented in a variety of situations. For example, the original container in which the solution was sterilized may be placed into an antiseptic applicator. For example, the sterile solution in the original container may be placed within a multi-product kit, or a subcomponent of another final product.

A sample of aqueous antiseptic solution of <NUM>% v/v water and <NUM>% w/v chlorhexidine gluconate contained in a glass ampoule was tested in each of the following examples. The below experiments were performed by heating the indicated type of bath (e.g., water or oil) to the temperature indicated (e.g., <NUM> - <NUM>). The solution had the initial purity indicated and the purity was tested at the times indicated in the tables. The purity percent values listed in the tables are the absolute purity of the chlorhexidine gluconate after heat treatment and cooling to ambient temperature. Each temperature was tested in duplicate. The Δpurity percent values are the percentage point change relative to the baseline purity. For example, in Table <NUM>, a water bath was heated to <NUM> and a solution having <NUM>% purity was tested. At <NUM> hours the purity of chlorhexidine gluconate solution was <NUM>%, which is a <NUM>% percentage point change from the initial purity of <NUM>%.

The above data was then used to prepare an Arrhenius equation using the standard method in the art. The use of an Arrhenius equation is a well-known and accepted method of modeling temperature dependence on reaction rate. Using the Arrhenius equation, the following predicted values for purity were obtained.

The measured impact of various sterilization temperatures and sterilization times on the characteristics of the antiseptic are shown below. Table <NUM> summarizes the change in % purity for the chlorhexidine gluconate after exposure to various sterilization temperatures and sterilization times. The percent change in purity is made by comparing the purity of solution prior to the ramp up time (i.e., prior to the process of bringing the solution up to the sterilization temperature) with the purity of solution after the cool down time (i.e., after the solution returns ambient temperature). 'The 'X', 'Y' and 'Z' indicate that the sterilization temperature and sterilization time would result in a change of purity of not more than <NUM>%, <NUM>% and <NUM>%, respectively.

The same can be done for other threshold values (e.g., changes in purity below or higher than <NUM>%, such as <NUM>%, <NUM>%, and <NUM>%).

In addition to above testing, further testing was conducted to determine at what time the Sterility Assurance Level (SAL) of <NUM>-<NUM> can be reached at a certain temperature. The USP <NUM> "Biological Indicators - Resistance Performance Tests" procedures were followed to determine the SAL. Greater than or equal to <NUM>,<NUM>,<NUM> test spores of Bacillus subtilis, but less than <NUM>,<NUM>,<NUM>, were inserted into a <NUM> sample of an aqueous solution comprising <NUM>% w/v chlorhexidine gluconate in <NUM>% v/v water. The samples were tested at <NUM>, <NUM>, and <NUM> for various times. The results were as follows:.

The above data was then used to calculate the "D-values," in accordance with USP <NUM> procedures. The term D-value has the normal meaning as used in microbiology. Specifically, it refers to decimal reduction time and is the time required at a certain temperature to kill <NUM>% of the organisms being studied. Thus after a colony is reduced by <NUM> D, only <NUM>% of the original organisms remain, i.e., the population number has been reduced by one decimal place in the counting scheme. D-values can be calculated using the Survivor Curve Method, which is a data analysis known in the art (based on methods described in ISO <NUM>-<NUM>:<NUM>). Applying the Survivor Curve Method to the above Table <NUM>-<NUM> data, the resulting D-values were calculated along with upper and lower confidence limits:.

The D-values can be used to calculate a sterility assurance Level (SAL) (see USP <NUM> procedures). SAL is a term used in microbiology to describe the probability of a single unit being non-sterile after it has been subjected to a sterilization process. A <NUM>-<NUM> SAL means there is a <NUM>/<NUM>,<NUM>,<NUM> chance that a single viable microbe will remain in sterilized items. The D-values were used to calculate the following time to achieve <NUM>-<NUM> SAL:.

Thus, as indicated in Table <NUM>, exposing the antiseptic solution to a temperature of <NUM> for about <NUM> minutes, a temperature of <NUM> for about <NUM> minutes, or <NUM> for about <NUM> minutes could each have a SAL of <NUM>-<NUM> (i.e., a <NUM>/<NUM>,<NUM>,<NUM> chance that a viable microbe will be present following the sterilization process).

Using standard mathematical modeling for the data presented in tables <NUM>-<NUM>, an exponential predictive function having the following formula: <MAT> where y is time in minutes and x is temperature in degrees Celsius. Thus, Formula (I) indicates at a given temperature the minimum time for achieving at least a <NUM>-<NUM> SAL. Using Formula (I), the following predictive data points were generated:.

<FIG> illustrates the sterilization times and temperatures fit to functions which capture the parameter space (time and temperature) to maintain a specific change in purity following the sterilization process (area between curves). The data points in <FIG> include data points from Table <NUM> and Table <NUM> above. The black squares represent the data points from <NUM> to <NUM> where the corresponding times were sterile. The following formula was fitted to the square data points from <NUM> to <NUM>: <MAT> where T is the temperature in degrees Celsius and t is the time in minutes.

The data points found in Table <NUM> above were also plotted in <FIG>. The black diamonds represent the data points from <NUM> to <NUM> where the corresponding times had at most a percent change in purity of <NUM>%. The black triangles represent the data points from <NUM> to <NUM> where the corresponding times had at most a percent change in purity of <NUM>%. The black circles represent data points from <NUM> to <NUM> where the corresponding times had at most a percent change in purity of <NUM>%. The following formula was fitted to the black diamond data points (i.e. the points having at most <NUM>% change in purity): <MAT>.

The following formula was fitted to the black triangle data points (i.e., the points having at most <NUM>% change in purity): <MAT> where T is the temperature in degrees Celsius and t is the time in minutes. The following formula was fitted to the black circle data points (i.e., the points having at most <NUM>% change in purity): <MAT> where T is the temperature in degrees Celsius and t is the time in minutes.

As can be seen in <FIG>, the area bounded by the functions defined in Formula (II) and Formula (III), for temperatures between <NUM> to <NUM>, represents temperature and time combinations that provide a sterile solution with at most a <NUM>% change in purity. This area can thus be presented by subtracting Formula (III) from Formula (II) <MAT> where T is temperature in degrees Celsius and t is time in minutes.

As can be seen in <FIG>, the area bounded by the functions defined in Formula (II) and Formula (IV), for temperatures between <NUM> to <NUM>, represents temperature and time combinations that provide a sterile solution with at most a <NUM>% change in purity. This area can thus be presented by the following relationship: <MAT> where T is temperature in degrees Celsius and t is time in minutes.

As can be seen in <FIG>, the area bounded by the functions defined in Formula (II) and Formula (V), for temperatures between <NUM> to <NUM>, represents temperature and time combinations that provide a sterile solution with at most a <NUM>% change in purity. This area can thus be presented by the following relationship: <MAT> where x is temperature in degrees Celsius and y is time in minutes.

The above results, specifically, that an aqueous solution of <NUM>% w/v chlorhexidine gluconate remains sufficiently pure after sterilization is surprising. Additional experiments were conducted where an aqueous solution having <NUM>% v/v water and <NUM>% w/v chlorhexidine gluconate was heated in sealed ampoules at <NUM> for various times. Comparison was made to an alcoholic solution of <NUM>% w/v chlorhexidine gluconate, <NUM>% v/v isopropyl alcohol, and the remainder water. The results indicated that the degradation rate of the aqueous <NUM>% w/v chlorhexidine gluconate was significantly slower (<NUM>-<NUM> fold) than the alcoholic <NUM>% w/v chlorhexidine gluconate at <NUM>. Previous studies have shown that the degradation rate of aqueous solution having <NUM>% v/v water and <NUM>% w/v chlorhexidine gluconate at <NUM> is nearly identical to the degradation rate of the alcoholic solution of <NUM>% w/v chlorhexidine gluconate. Because the aqueous <NUM>% w/v chlorhexidine gluconate had the same degradation rate as the alcoholic solution of <NUM>% w/v chlorhexidine gluconate, one would expect the aqueous <NUM>% w/v chlorhexidine gluconate to exhibit a similar degradation rate.

In order to predict if the differences in the extent of chlorhexidine gluconate degradation are inherent to the solutions and would exist at any time and temperature, an Arrhenius equation was established for aqueous <NUM>% w/v chlorhexidine gluconate and an aqueous solution having <NUM>% w/v chlorhexidine gluconate, alcoholic solution of <NUM>% w/v chlorhexidine gluconate by monitoring the increase in total related substances with respect to time and temperature. Two different manufacturers (referred herein as Manufacture A and Manufacture B) of the aqueous <NUM>% w/v chlorhexidine gluconate were tested. The two different supplied aqueous <NUM>% w/v chlorhexidine gluconate were diluted with water to <NUM>% w/v chlorhexidine gluconate for purposes of testing (i.e., remaining <NUM>% v/v water).

The Arrhenius equations for the two aqueous <NUM>% w/v chlorhexidine gluconate were compared to the Arrhenius equations for aqueous <NUM>% w/v chlorhexidine gluconate, and the alcoholic solution having <NUM>% w/v chlorhexidine gluconate. The results are shown in <FIG>. As shown in <FIG>, the degradation rate of both aqueous <NUM>% chlorhexidine gluconate solution from Manufacturer A and Manufacturer B is significantly slower than both the alcoholic <NUM>% w/v chlorhexidine gluconate and the aqueous <NUM>% w/v aqueous chlorhexidine gluconate. A prediction of the degradation rates at <NUM> for aqueous <NUM>% w/v chlorhexidine gluconate in comparison to aqueous <NUM>% w/v and alcoholic <NUM>% w/v chlorhexidine gluconate are shown in Table <NUM> below.

Further, as shown in <FIG> and Table <NUM>, it was determined that the degradation rate for the alcoholic <NUM>% w/v chlorhexidine gluconate is comparable to the aqueous <NUM>% chlorhexidine gluconate at all temperatures.

The above-described degradation results are particularly surprising in view of further testing showing that the difference in degradation rate cannot be directly correlated with only one of the following factors: alcohol content, the difference in pH / apparent pH, and the amount of dilution.

To investigate the effect of isopropyl alcohol on the degradation rate, formulations with increasing concentrations of isopropyl alcohol were prepared with either a non-buffered (water) or buffered system (<NUM> acetate buffer pH about <NUM>). A buffered system was used in addition to varying the isopropyl alcohol concentration in order to minimize the effect of differences in the solution's apparent pH when degrading the chlorhexidine gluconate formulations. The results, shown in <FIG>, indicate that as the isopropyl alcohol concentration increases, the chlorhexidine gluconate degradation rate (increase in formation of total related substances (TRS)) also increases.

To determine the effect of pH on the degradation rate of chlorhexidine gluconate, aqueous <NUM>% w/v chlorhexidine gluconate formulations were prepared at various pHs using an <NUM> acetate buffer (see <FIG>). The chlorhexidine gluconate degradation rate is independent of pH, when the aqueous solution pH in the range of approximately <NUM>-<NUM>. As the solution pH increases above pH <NUM>, the chlorhexidine gluconate degradation rate also increases.

Dilution of aqueous <NUM>% w/v chlorhexidine gluconate to achieve an aqueous <NUM>% w/v chlorhexidine gluconate solution does not significantly impact the solution pH (Δ pH about <NUM> pH units). Further, when the chlorhexidine gluconate is diluted, the pH of the aqueous <NUM>% w/v chlorhexidine gluconate solution (pH <NUM>) is higher than that of aqueous <NUM>% w/v chlorhexidine gluconate (pH <NUM>). Thus, the differences in the observed degradation rates between aqueous <NUM>% w/v chlorhexidine gluconate and aqueous <NUM>% w/v chlorhexidine gluconate isn't a pH phenomenon based on the results shown in <FIG>, where an increase in pH trends with an increase in degradation.

Claim 1:
A method for sterilizing an aqueous antiseptic solution, the method comprising:
providing a container containing the aqueous antiseptic solution, the aqueous antiseptic solution comprising about <NUM>% w/v chlorhexidine gluconate and a solvent containing <NUM>%v/v water;
heating the aqueous antiseptic solution to a predetermined temperature;
maintaining the aqueous antiseptic solution at the predetermined temperature for a predetermined time; and
terminating the heating of the aqueous antiseptic solution when the predetermined time expires,
wherein the predetermined temperature and the predetermined time are selected such that after terminating the heating, the aqueous antiseptic solution is sterile;
characterized in that
wherein the predetermined temperature and the predetermined time are selected such that the post-sterilization purity is at least about <NUM>% and a percentage point change in purity from the initial purity to the post-sterilization purity of at most about <NUM>%,
the predetermined temperature is between about <NUM> and <NUM>,
the sterilization time is:
<NUM>) from about <NUM> minutes to about <NUM> hours at <NUM>,
<NUM>) at least about <NUM> minutes and up to about <NUM> hours at <NUM>,
<NUM>) at least about <NUM> minutes and up to about <NUM> hours at <NUM>,
<NUM>) at least about <NUM> minute and up to about <NUM> hours at <NUM>; and
wherein the purity is defined as the percent concentration of chlorhexidine gluconate molecules in solution relative to the total concentration of chlorhexidine gluconate molecules plus concentration of substances that are derived from or related to the chlorhexidine gluconate molecules, and
"about" means ±<NUM>% of the provided value.