Title:  Vaginal lactobacillus medicant

United States Patent:  6,468,526

Issued:  October 22, 2002

Inventors:  Chrisope; Gerald L. (Boulder, CO)

Assignee:  GyneLogix, Inc. (Louisville, CO)

Appl. No.:  027472

Filed:  December 21, 2001

Disclosed are novel isolated strains of bacteria of the genus Lactobacillus which are useful in a vaginal medicant. Also disclosed are medicants containing such Lactobacilli, a novel preservation matrix for microorganisms, a method for preserving microbial cells within a medicant, and methods for preventing and treating vaginal and gastrointestinal infections.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to human strains of bacteria of the genus Lactobacillus which have desirable characteristics suitable for oral use or intravaginal use as a medicant for prophylaxis of vaginal infections. More particularly, the desirable characteristics include the ability to adhere to and colonize vaginal epithelial cells, production of hydrogen peroxide, specific potency, shelf-life stability, inhibition of vaginal infectious agents, production of lactic acid and size of individual microbial cells. Further, this invention relates to a vaginal medicant having a substantially pure culture of preserved microbial cells, and to a method for preserving the microbial cells within the medicant so as to maintain the purity, the genetic stability and the desirable characteristics listed above throughout a storage period of at least about 12 months.

Currently available commercial Lactobacillus products are often contaminated or do not contain appropriate vaginal strains of H₂ O₂ -producing Lactobacilli in sufficient quantities. Furthermore, currently available Lactobacillus strains are often not able to colonize vaginal epithelial cells. In addition to adequate potency and strain selection, existing products may lack efficacy due to the method of preservation employed during their commercial production. In many products, the Lactobacillus strain often loses its ability to adhere to exfoliated vaginal epithelial cells in vitro, and to colonize vaginal epithelial cells in vivo. In addition, the shelf-life of preserved strains is often short, with viable cell counts decreasing rapidly over 2-3 months. Some of the currently used preservation methods were developed for use in the preservation of foods but are perhaps not optimal for preservation of microorganisms. This is critical as the preservation method must protect and suspend the growth of the cells while allowing them to adhere to the vaginal wall in the metabolically inactive state (i.e., the preserved state).

The present invention provides several advantages over the dietary and vaginal supplements that are currently available. Use of modern DNA based technology has suggested that the most common Lactobacillus in the vagina is not Lactobacillus acidophilus, which is the species upon which many current dietary supplements and vaginal treatments are based. The present inventors have identified and isolated novel strains of Lactobacillus crispatus and Lactobacillus jensenii which are superior to any currently available strain for use in a vaginal medicant. Moreover, the present inventors have determined the optimal conditions for producing the strains on both a small and large scale without losing the desirable characteristics during propagation, harvest, preservation, placement in a vaginal delivery system and storage. The present inventors also provide a novel preservation matrix and method for preserving a microbe in a format which enables the preservation of the desirable characteristics of the microbe. More particularly, the preservation matrix of the present invention allows the Lactobacillus strains to adhere to vaginal epithelial cells in vivo in a metabolically inactive state and retain placement while returning to an active state capable of producing functional inhibitory by-products. Such a capability of a microbe/preservation matrix combination has not been described prior to the present invention. The preservation matrix of the present invention also is capable of preserving microbes at room temperature or refrigerated temperatures for long periods of time in storage, and provides the flexibility to allow various drying methods for the production of a commercial medicant. This is the first demonstration of a preservation matrix with such capabilities. The present inventors are unaware of any currently known Lactobacillus vaginal medicant which exhibits efficacy equal to or superior to the efficacy demonstrated by the present medicant both in vitro and in vivo.

According to the present invention, a "vaginal medicant" is a medicant (i.e., medicament or medicine) which is used to prevent or treat infections, diseases, or other disorders directly or indirectly related to the vagina, including infections and diseases which can gain entry to the body through the vagina. Although a vaginal medicant of the present invention is primarily described herein for its use related to vaginal infections, it is to be understood that such a vaginal medicant can be used to treat infections and conditions which are not necessarily related to vaginal infections, such as gastrointestinal infections, in which case a medicant of the present invention can be referred to as a gastrointestinal medicant.

One embodiment of the present invention relates to a vaginal medicant which includes a substantially pure bacterial culture of an isolated strain of the genus Lactobacillus having identifying characteristics which include (i) a percent vaginal epithelial cell (VEC) cohesion value (as defined below) of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H₂ O₂. The vaginal medicant also includes a preservation matrix, which contains and preserves the bacterial culture. Such a matrix includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. The matrix is capable of maintaining at least about 10⁶ viable, genetically stable cells for a period of at least about 12 months in vitro. In one embodiment, the vaginal medicant can comprise an inert carrier including, maltodextrin beads or a gelatin capsule.

According to the present invention, an isolated strain of a microbe is a strain that has been removed from its natural milieu. As such, the term "isolated" does not necessarily reflect the extent to which the microbe has been purified. In contrast, a "substantially pure culture" of the strain of microbe refers to a culture which contains substantially no other microbes than the desired strain or strains (i.e., the "suppository strain" or "medicant strain") of microbe. In other words, a substantially pure culture of a strain of microbe is substantially free of other contaminants, which can include microbial contaminants as well as undesirable chemical contaminants.

The presence of the suppository strain or strains and the absence of contaminating strains in a culture can be determined by any method, including by analyzing the microorganisms in a culture for (1) DNA homology using labeled DNA probes, (2) DNA fingerprints and/or (3) cell wall fatty acid profile. For example, strains within a culture can be analyzed for DNA homology to the desired, suppository strains by determining whether DNA from the culture hybridizes under stringent hybridization conditions to DNA from the suppository strain. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify molecules having similar nucleic acid sequences. Stringent hybridization conditions typically permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used as a probe in the hybridization reaction. Such standard conditions are disclosed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press. The reference Sambrook et al., ibid., is incorporated by reference herein in its entirety. Examples of such conditions include, but are not limited to, the following: Oligonucleotide probes of about 18-25 nucleotides in length with T_m 's ranging from about 50^oC. to about 65^oC. (e.g., DNA from the suppository strain), for example, can be hybridized to nucleic acid molecules (e.g., DNA from the culture to be tested) typically immobilized on a filter (e.g., nitrocellulose filter) in a solution containing 5.times. SSPE, 1% Sarkosyl, 5.times. Denhardts and 0.1 mg/ml denatured salmon sperm DNA at 37^oC. for about 2 to 12 hours. The filters are then washed 3 times in a wash solution containing 5.times. SSPE, 1% Sarkosyl at 37^oC. for 15 minutes each. The filters can be further washed in a wash solution containing 2.times. SSPE, 1% Sarkosyl at 37^oC. for 15 minutes per wash. Randomly primed DNA probes can be hybridized, for example, to nucleic acid molecules typically immobilized on a filter (e.g., nitrocellulose filter) in a solution containing 5.times. SSPE, 1% Sarkosyl, 0.5% Blotto (dried milk in water), and 0.1 mg/ml denatured salmon sperm DNA at 42^oC. for about 2 to 12 hours. The filters are then washed 2 times in a wash solution containing 5.times. SSPE, 1% Sarkosyl at 42^oC. for 15 minutes each, followed by 2 washes in a wash solution containing 2.times. SSPE, 1% Sarkosyl at 42^oC. for 15 minutes each.

Methods to identify the suppository strain or strains using DNA fingerprinting by Repetitive Sequence Polymerase Chain Reaction (Rep PCR) or using cell wall fatty acid analysis are described in detail below in the Examples section.

A Lactobacillus strain suitable for use in a medicant of the present invention (i.e., a suppository strain) can be any Lactobacillus strain which has the above-described identifying characteristics. Lactobacillus strains can be detected and isolated from natural sources through the use of appropriate screening techniques which are known in the art. The identifying characteristics of Lactobacillus strains suitable for use in the present invention and methods to screen for these characteristics are discussed in detail below. Preferred species of Lactobacillus include Lactobacillus acidophilus, Lactobacillus jensenii and Lactobacillus crispatus, with Lactobacillus crispatus being particularly preferred. Preferably, a suitable strain of Lactobacillus is isolated from the vagina of a human. A particularly preferred strain of Lactobacillus is a strain having all of the identifying characteristics of the Lactobacillus crispatus CTV-05 strain, with Lactobacillus crispatus CTV-05 being the most preferred.

It is within the scope of the present invention that, in addition to known species and strains of Lactobacillus, newly identified species and strains from nature and mutant strains derived from known or newly identified strains can be used in a medicant of the present invention. Naturally occurring mutants of a parental strain of Lactobacillus that have the identifying characteristics of a Lactobacillus suitable for use in a medicant of the present invention can be isolated by, for example, subjecting a parental strain to at least one round of chemical and/or radiation mutagenesis, in order to increase the rate of mutagenesis, thereby increasing the probability of obtaining a microorganism having improved desired characteristics. It will be obvious to one of skill in the art that mutant microorganisms of the present invention also include microorganisms that can be obtained by genetically engineering microorganisms to, for example, have increased percent VEC cohesion values (defined below). As used herein, a "mutated microorganism" is a mutated parental microorganism in which the nucleotide composition of such microorganism has been modified by mutation(s) that occur naturally, that are the result of exposure to a mutagen, or that are the result of genetic engineering.

One identifying characteristic of a Lactobacillus that is suitable for use in a vaginal medicant of the present invention is that the Lactobacillus strain has a percent vaginal epithelial cell (VEC) cohesion value of at least about 50%, and more preferably at least about 65%, and even more preferably at least about 80%, and most preferably at least about 95%. According to the present invention, the terms "cohesion" and "adherence" can be used interchangeably. Adherence of microbial cells to vaginal epithelial cells is critical for colonization and biological effect. As used herein, colonization refers to the establishment of a site of microbial reproduction on a cell or material which does not necessarily result in tissue invasion or damage. Successful adherence of a Lactobacillus cell of the present invention to a vaginal epithelial cell will result in successful colonization of the vaginal epithelial cell. According to the present invention, "percent VEC cohesion value" is defined as the percentage of VECs to which at least one Lactobacillus cell is adhered in the total number of VECs in an identified group. This is a different measure of adherence than has typically been used in the past. Heretofore, in vitro adherence efficacy has been determined by counting the number of adhered microbial cells on the first pre-defined number (e.g., 50) of vaginal epithelial cells (VECs) observed in a stained preparation and calculating the average, or mean value, of adhered microbial cells per VEC. This mean value has previously served as an indicator of efficacy of a particular strain of microbe. Without being bound by theory, the present inventors believe that determination of "percent VEC cohesion", as described above, is a better measurement of efficacy. The present inventors believe that, with an overall emphasis on the practical perspective, in vitro adherence means nothing if it does not correlate with in vivo adherence and colonization in human subjects. Therefore, rather than using the conventional mean value of adhered cells per VEC in a total count of VECs as a measure of in vitro and in vivo efficacy, the present inventors instead calculate the percentage of VEC cells that have at least one adhered microbial cell in a total count of VECs (i.e., percent VEC cohesion value). The present inventors believe that this value is of greater significance since it is a good predictor of whether a significant number of VECs will accept microbial cells in vitro and in vivo. In contrast, the use of conventional mean values for adherence may be skewed and lead to erroneous interpretations of efficacy. For example, in the following three scenarios in which a technician counts the number of microbial cells adhered to a population of 50 VECs, the two methods of determining adherence (i.e., average adherence versus percent VEC cohesion value) provide surprisingly different evaluations of in vitro adherence efficacy:

A. The technician counts 1500 microbial cells adhered to 10 VECs in a total count of 50 VECs, leaving 40 VECs (80%) with no adhered microbial cells (in this scenario, there are 150 microbial cells on each of the 10 VECs). By conventional mean value calculation, the average adherence value is 30 microbes/cell and the percent VEC cohesion value is 20%.

B. The technician counts 1500 microbial cells adhered to 50 VECs in a total count of 50 VECs, leaving zero VECs (0%) with no adhered microbial cells (in this scenario, there are 30 microbial cells on each of the 50 VECs). By conventional mean value calculation, the average adherence value is again 30 microbes/cell, but the percent VEC cohesion value is 100%.

C. The technician counts 600 microbial cells adhered to a total of 40 VECs in a total count of 50 VECs, leaving 10 VECs (20%) with no adhered microbial cells (in this scenario, there are 15 microbial cells on each of the 40 VECs). By conventional mean value calculation, the average adherence value is 12 microbes/cell, and the percent VEC cohesion value is 80%.

Therefore, using the preferred adherence measurement of the present invention (percent VEC cohesion value), even though the mean number of microbes adhered per VEC is the same in both scenario A and scenario B, the microbes in scenario B would be selected over the microbes of scenario A for their adherence efficacy since more VECs have accepted the microbial cells in scenario B (100% versus 20%). Indeed, even the microbes tested in scenario C would be selected over the microbes of scenario A, because even though the microbes of scenario C have a conventional mean adherence value of 12 microbes/VEC compared to 30 microbes/VEC in scenario A, a greater number of VECs (80% versus 20%) have accepted microbial cells in scenario C. In view of the above scenarios, the percent VEC cohesion value is a more sensitive and relevant calculation of adherence efficacy.

The present inventors believe that a "percent VEC cohesion" value is more predictive of in vivo long-term colonization than the conventional average, or mean, adherence value, especially considering the self-regulation process exhibited by Lactobacilli in the vaginal ecosystem. It has been proposed that overgrowth of H₂ O₂ -producing Lactobacilli in the vagina is prevented by self-inhibition when the Lactobacilli population becomes "over crowded". This built-in safety factor minimizes the possibility of detrimental effects of excessive numbers of vaginal Lactobacilli. Considering this phenomenon, in the present invention, a suppository strain which will spread itself over a larger range of VECs (e.g., has a high percent VEC cohesion value) is preferred over a strain that would adhere in large numbers to only a few VECs. It is likely that the latter situation may lead to self-inhibition of H₂ O₂ -producing Lactobacilli on a few over-crowded VECs, thus decreasing the likelihood for long-term survival and colonization of the microbe. Long-term in vivo colonization is the ultimate objective of the products and methods of the present invention and it is believed that smaller numbers of Lactobacilli cells adhered to a larger number of VECs will better achieve this objective. In one embodiment, an isolated Lactobacillus strain for use in a medicant of the present invention is identified by its ability to sustain colonization of vaginal epithelial cells for at least about 1 month.

Another identifying characteristic of a Lactobacillus which is suitable for use in a medicant of the present invention is the ability to produce hydrogen peroxide (H₂ O₂). As discussed above, hydrogen peroxide has been shown to be directly responsible for the killing of other microorganisms by the Lactobacillus. Preferably, the Lactobacillus is able to produce greater than about 0.5 ppm of H₂ O₂ under normal growth conditions. More preferably, the Lactobacillus is able to produce at least about 10 ppm of H₂ O₂, and even more preferably, the Lactobacillus is able to produce at least about 20 ppm of H₂ O₂ under effective growth conditions, herein defined as any medium and conditions capable of promoting production of H₂ O₂. Effective growth conditions include both in vitro growth conditions (e.g., an effective culture medium and conditions) and in vivo growth conditions (e.g., successful colonization of a vaginal epithelial cell).

H₂ O₂ production by a Lactobacillus of the present invention can be quantitated by any means for measuring H₂ O₂ production. For example, H₂ O₂ production can be measured by quantitation of the intensity of a blue pigment formed when Lactobacillus is inoculated onto tetramethylbenzidine medium (TMB) and incubated under anaerobic conditions. H₂ O₂ production can also be measured using commercially available H₂ O₂ detection strips (e.g., available from EM Sciences).

In one embodiment, another identifying characteristic of a Lactobacillus suitable for use in a medicant of the present invention is the genetic stability of the Lactobacillus over time both in vivo and in vitro. According to the present invention, genetic stability refers to the ability of successive generations of a Lactobacillus strain to substantially maintain the identical genetic profile of the mother strain. In other words, successive generations of a genetically stable strain will not acquire substantial mutations in its DNA over a period of time. More importantly, successive generations of a genetically stable strain will not acquire substantial mutations in DNA related to one of the above described identifying characteristics over time. Most importantly, successive generations of a genetically stable strain will not acquire substantial mutations (e.g., mutations that significantly change the phenotype of the encoded protein) in DNA related to the identifying characteristics of vaginal epithelial cell cohesion value, hydrogen peroxide production, or the ability to adhere to vaginal epithelial cells in a metabolically inactive state as described herein. Preferably, a Lactobacillus strain of the present invention which has colonized vaginal epithelial cells in vivo will maintain genetic stability in vivo for at least about 12 months of vaginal colonization, and more preferably at least about 18 months, and even more preferably at least about 24 months of vaginal colonization. In vitro, the genetic stability of a microorganism can be affected by the culturing conditions of the microorganism and by the preparation and storage format of the vaginal medicant of the present invention. Such conditions are discussed in detail below. Due to the superior qualities of the preservation matrix of the present invention, a Lactobacillus strain of the present invention preserved in a preservation matrix of the present invention is preferably genetically stable for at least about 12 months in vitro, and more preferably, at least about 18 months in vitro and even more preferably at least about 24 months in vitro during storage at room temperature or at refrigeration temperature (2-8^oC.). Genetic stability can be evaluated by any method of evaluating mutations or identifying selectable genetic markers. For example, genetic marker profiles based on restriction endonuclease patterns can be performed to establish the stability of genetic profile of a particular culture compared to the mother strain. Repetitive Sequence Polymerase Chain Reaction (Rep PCR) has been used by the present inventors to distinguish as many as 40 different strains of Lactobacillus from each other, and to confirm genetic stability of a particular strain of Lactobacillus over time after either in vitro storage or in vivo colonization of vaginal epithelial cells.

In one embodiment, an identifying characteristic of a Lactobacillus suitable for use in a medicant of the present invention is the ability to produce lactic acid. Lactic acid has been shown to inhibit the growth of pathogens in vitro. Preferably, a Lactobacillus produces at least about 0.75 mg/100 ml lactic acid, and more preferably at least about 4 mg/100 ml lactic acid, and even more preferably at least about 8.8 mg/100 ml lactic acid under effective growth conditions.

In another embodiment of the present invention, a suitable Lactobacillus strain has a relatively large cell size. Ranges of typical Lactobacilli as provided in Bergey's Manual of Determinative Bacteriology are 0.8-1.6 .mu.m (width).times.2.3-11 .mu.m (length). A preferred Lactobacillus strain for use in the present invention has a cell size of from about 1 to about 2 microns in width and from about 2 to about 4 microns in length. Without being bound by theory, the present inventors believe that the large dimensions exhibited by cells of a Lactobacillus strain of the present invention may allow it to better serve as a protective agent in biocompetitive exclusion. Biocompetitive exclusion refers to the ability of the suppository strain or strains of the present invention to competitively inhibit the growth of undesired bacterial strains. Such exclusion is attributed to the occupation of available space on a vaginal epithelial cell by the beneficial Lactobacilli cells (e.g., the suppository strain), thus preventing attachment of pathogenic, or undesirable, microbial cells.

A vaginal medicant of the present invention also includes a preservation matrix. Microbial cells are suspended in the preservation matrix for preservation and storage in the delivery format. This matrix, as well as the microbial culture media, the methods of harvesting microbial cells and the preservation process, all have a profound effect on cell viability during storage and performance of the preserved cells after rehydration (e.g., rehydration in the vagina). The preservation matrix of the present invention maintains all of the previously described desirable characteristics of the vaginal strain.

The preservation matrix of the present invention is comprised of ingredients to minimize the damaging effects encountered during the preservation process and to provide functional properties. As will be discussed in detail below, when a Lactobacillus strain of the present invention is added to the preservation matrix for preservation, it is preferably converted from an actively growing metabolic state to a metabolically inactive state. One further identifying characteristic of a Lactobacillus strain used in a vaginal medicant of the present invention is that it is able to adhere to vaginal epithelial cells even when in a metabolically inactive state. The preservation matrix of the present invention is therefore also formulated for optimal microbial cell resilience, such that upon rehydration in vivo, the microbial cells are immediately free to adhere to vaginal epithelial cells and then return to full metabolic activity without delay.

The preservation matrix of the present invention includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a carbohydrate refers to one or more carbohydrates or at least one carbohydrate. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

According to the present invention, a biologically acceptable binding agent is binding agent, preferably a protein, which is acceptable for use in vivo (e.g., does not have any activity or toxic effect in vivo), which affixes the cell matrix to an inert carrier (described below) during the preservative process and which provides protective effects (i.e., maintains cell viability) throughout preservation and storage of the microbial cells. Preferred biologically acceptable binding agents for use in a preservation matrix of the present invention include, but are not limited to a water-soluble gum, carboxymethyl cellulose and/or gelatin. A biologically acceptable binding agent typically comprises from about 10% to about 20% by weight of the preservation matrix, and preferably comprises about 14% by weight of the preservation matrix. In one embodiment, a preservation matrix of the present invention comprises about 14% gelatin by weight of the preservation matrix.

Antioxidants included in a preservation matrix of the present invention are provided to retard oxidative damage to the microbial cells during the preservation and storage process. A particularly preferred antioxidant is sodium ascorbate. An antioxidant typically comprises from about 0.1% to about 1.0% by weight of the preservation matrix, and preferably comprises about 0.5% by weight of the preservation matrix. In one embodiment, a preservation matrix of the present invention comprises about 0.5% sodium ascorbate by weight of the preservation matrix.

Polyols (i.e., polyhydric alcohols) included in a preservation matrix of the present invention are provided to maintain the native, uncollapsed state of cellular proteins and membranes during the preservation and storage process. In particular, polyols interact with the cell membrane and provide support during the dehydration portion of the preservation process. Preferred polyols include, but are not limited to xylitol, adonitol, glycerol, dulcitol, inositol, mannitol, sorbitol and/or arabitol. A polyol typically comprises from about 1% to about 12% by weight of the preservation matrix, and preferably comprises about 6% by weight of the preservation matrix. In one embodiment, a preservation matrix of the present invention comprises about 6% xylitol by weight of the preservation matrix.

Carbohydrates included in a preservation matrix of the present invention are provided to maintain the native, uncollapsed state of cellular proteins and membranes during the preservation and storage process. In particular, carbohydrates provide cell wall integrity during the dehydration portion of the preservation process. Preferred carbohydrates include, but are not limited to dextrose, lactose, maltose, sucrose, fructose and/or any other monosaccharide, disaccharide or polysaccharide. A carbohydrate typically comprises from about 0.5% to about 5% by weight of the preservation matrix, and preferably comprises about 2.5% by weight of the preservation matrix. In one embodiment, a preservation matrix of the present invention comprises about 2.5% dextrose by weight of the preservation matrix.

A proteinaceous material included in a preservation matrix of the present invention provides further protection of the microbial cell during the dehydration portion of the preservation process. Preferred proteinaceous materials include, but are not limited to skim milk and albumin. A proteinaceous material typically comprises from about 0.5% to about 5% by weight of the preservation matrix, and preferably comprises about 1.5% by weight of the preservation matrix. In one embodiment, a preservation matrix of the present invention comprises about 1.5% skim milk by weight of the preservation matrix.

In one embodiment of the present invention, a preservation matrix includes a biologically active binding agent that is at least about 10% of the preservation matrix by weight, an antioxidant that is at least about 0.1% of the preservation matrix by weight, a polyol that is at least about 1% of the preservation matrix by weight, a carbohydrate that is at least about 0.5% of the preservation matrix by weight, and a proteinaceous material that is at least about 0.5% of the preservation matrix by weight.

A particularly preferred preservation matrix of the present invention comprises about 14% gelatin, about 0.5% sodium ascorbate, about 2.5% dextrose, about 1.5% skim milk and about 6% xylitol, by weight of the preservation matrix.

The pH of the preservation matrix is important for optimal stability of the preserved microbial cells. The optimal pH of the preservation matrix can be determined by preparing suspensions of Lactobacilli cells in matrices adjusted to various pHs. A preservation matrix of the present invention is typically from about pH 5.0 to about pH 7.0, and preferably, about pH 7.0.

The preservation matrix for use in a vaginal medicant of the present invention, in addition to the particular qualities described above, maintains at least about 10⁶ viable, substantially pure and genetically stable cells in vitro for a period of at least about 12 months. As discussed above, the term "substantially pure" refers to a culture of microbial cells of the present invention which are substantially free of any other undesirable microorganisms (e.g., contaminants). The importance of genetic stability of such cells has also been discussed previously herein. In a more preferred embodiment, a preservation matrix of the present invention is capable of maintaining at least about 10⁷, and even more preferably, at least about 10⁸ viable, substantially pure and genetically stable Lactobacillus cells for a period of at least about 12 months.

In another embodiment of the present invention, a preservation matrix used in a medicant of the present invention is preferably capable of maintaining at least about 10⁶ viable, substantially pure and genetically stable Lactobacillus cells for a period of at least about 18 months, and even more preferably for a period of at least about 24 months.

Vaginal medicants of the present invention can be stored either at room temperature or at refrigerated temperature, which is typically from about 4^oC. to about 6^oC. In yet another embodiment, a preservation matrix used in a medicant of the present invention is preferably capable of maintaining at least about 10⁶ viable, substantially pure and genetically stable Lactobacillus cells for a period of at least about 12 months at room temperature. In another embodiment, a preservation matrix used in a medicant of the present invention is preferably capable of maintaining at least about 10⁶ viable, substantially pure and genetically stable Lactobacillus cells for a period of at least about 12 months at refrigerated temperature.

The ability of the matrix to preserve a minimum number of viable cells is critical to the efficacy of the medicant of the present invention and has been particularly problematic in vaginal treatments prior to the present invention. More specifically, the number of viable, substantially pure, genetically stable cells that are delivered in a medicant unit (e.g., a single suppository or tablet) is directly related to the critical issue of potency of the medicant. As used herein, the term "efficacy" refers to the ability of a suppository strain to exhibit a biological effect (e.g., provide a statistically significant level of protection against vaginal infection). "Potency" directly relates to the number of viable microbial cells delivered per medicant unit (i.e., per suppository or tablet). According to the present invention, viable cells have the ability to grow and reproduce. For a Lactobacillus medicant to be efficacious in vivo, both colonization of the vaginal epithelial cells by the microbial cells at a potency of at least about 10⁶ and biological effect (e.g., as evidenced by absence of an infected state such as bacterial vaginosis) are necessary. The present inventors have discovered that there is a difference between the potency of a medicant that allows colonization of the suppository strain and the potency of a medicant which exhibits a biological effect. The present inventors have found that the ability of the suppository strain to colonize vaginal epithelial cells combined with the specific potency requirements for a biological effect are critical for an efficacious Lactobacillus medicant. More specifically, a concentration of viable microbial cells that results in vaginal colonization of the suppository strain is necessary, but may not be sufficient, for a medicant to be efficacious. For example, good colonization of vaginal epithelial cells can be achieved at very low potencies (e.g., 10⁵ microbial cells) using the Lactobacillus strains and preservation format of the present invention. However, biological effect is not demonstrated at this potency. Therefore, colonization of vaginal epithelial cells is necessary for a biological effect, but colonization in the absence of insufficient potencies will not lead to the numerical superiority necessary to demonstrate biologic effect. The preservation matrix of the present invention demonstrates the heretofore unobserved ability to maintain the necessary potency of biologically effective Lactobacillus cells both in vitro over extended periods of time and in vivo upon delivery to vaginal epithelial cells.

Another embodiment of the present invention relates to a method to make a preservation matrix as previously described herein. Such a method includes the steps of (a) providing components which include: (i) a sterile biologically active binding agent, which can include water soluble gum, carboxymethyl cellulose or gelatin; (ii) a sterile proteinaceous material which can include skim milk or albumin; (iii)a sterile polyol which can include xylitol, adonitol, glycerol, dulcitol, inositol, mannitol, sorbitol or arabitol; (iv) a sterile antioxidant; (v) a sterile carbohydrate which can include dextrose, lactose, maltose, sucrose, fructose, and other monosaccharides, other disaccharides and other oligosaccharides; and(vi) water; and (b) and mixing said components together to form a solution. The biologically active binding agent is provided in a liquid form, which typically requires heating of the agent to about 37^oC., since such agents are likely to be in solid phase at room temperature. The various components of the preservation matrix and the preferred amounts of each are discussed in detail above. The components of the preservation matrix can be sterilized by any suitable method of sterilization. In a preferred embodiment, the biologically active binding agent and the proteinaceous material are sterilized by autoclave and the polyol, carbohydrate and antioxidant are sterilized by filtration. After the components are mixed to form a preservation matrix solution, such a solution can be used immediately, held at 37^oC. for short periods of time, or frozen at about -20^oC.

In a preferred embodiment, a vaginal medicant of the present invention includes an inert carrier. According to the present invention, an inert carrier can be any inert material which is suitable for use in vivo and which can be used to carry or support the cell suspension matrix (i.e., preservation matrix combined with microbial cells) of the present invention in such a manner that the cell suspension matrix can be stored in vitro and/or administered in vivo. Inert carriers include, but are not limited to, maltodextrin beads and gelatin capsules. Such carriers are discussed in more detail below.

One embodiment of the present invention relates to a vaginal medicant which includes (a) an isolated, substantially pure bacterial culture of Lactobacillus crispatus CTV-05; (b) a preservation matrix, which includes about 14% gelatin, about 0.5% sodium ascorbate, about 2.5% dextrose, about 1.5% skim milk and about 6% xylitol. The preservation matrix maintains at least about 10⁶ viable, genetically stable cells in vitro for a period of at least about 12 months. In addition, the preservation matrix preserves the desirable characteristics of the Lactobacillus crispatus CTV-05. Such characteristics include, for example, an ability to adhere to vaginal epithelial cells in a metabolically inactive state, an ability to produce greater than about 0.5 ppm of H₂ O₂ under effective culture conditions, or a percent vaginal epithelial cell (VEC) cohesion value of at least about 50%. These characteristics have been discussed in detail above.

Another embodiment of the present invention relates to a vaginal medicant which includes a substantially pure bacterial culture of at least two different isolated strains of the genus Lactobacillus having identifying characteristics which include (i) a percent vaginal epithelial cell (VEC) cohesion value of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H₂ O₂ under effective culture conditions. The vaginal medicant also includes a preservation matrix, which contains and preserves the bacterial culture. Such a matrix includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate, and a proteinaceous material. The matrix is capable of maintaining at least about 10⁶ viable, genetically stable cells for a period of at least about 12 months in vitro. In one embodiment, the vaginal medicant can also include an inert carrier.

In this embodiment of the present invention, each of the Lactobacillus strains is selected for its ability to prevent and/or treat a vaginal infection which is different from the vaginal infection prevented or treated by the other Lactobacillus strains included in the medicant. Such infections can include, but are not limited to, bacterial vaginosis, symptomatic yeast vaginitis, gonorrhea, chlamydia, trichomoniasis, human immunodeficiency virus infection, urinary tract infection and pelvic inflammatory disease. For example, in a preferred embodiment, a vaginal medicant includes a first Lactobacillus strain which is useful for preventing bacterial vaginosis, and a second Lactobacillus strain which is useful for preventing symptomatic yeast vaginitis. In a preferred embodiment, a first Lactobacillus strain is Lactobacillus crispatus CTV-05 and a second Lactobacillus strain is a strain of Lactobacillus jensenii.

Another embodiment of the present invention relates to an isolated bacterial strain of the genus Lactobacillus. Such a strain has identifying characteristics which include (a) a percent vaginal epithelial cell (VEC) cohesion value of at least about 50% and (b) an ability to produce greater than about 0.5 ppm of H₂ O₂. In another embodiment, such an isolated strain preferably has a percent VEC cohesion value of at least about 65%, and more preferably of at least about 80% and even more preferably of at least about 95%. In further embodiments, such a strain preferably produces at least about 10 ppm of H₂ O₂, and even more preferably at least about 20 ppm of H₂ O₂.

An isolated bacterial strain of the genus Lactobacillus of the present invention can have one or more other desirable identifying characteristics. Such characteristics have been previously described herein. In particular, in one embodiment, an isolated bacterial strain of the genus Lactobacillus is able to sustain colonization of vaginal epithelial cells for at least about 1 month. In another embodiment, an isolated bacterial strain of the genus Lactobacillus maintains genetic stability over at least about 24 months of vaginal colonization. In yet another embodiment, such a strain adheres to vaginal epithelial cells when the strain is in a metabolically inactive state (i.e., when in a preserved state). A further identifying characteristic of such a strain is the ability of the strain to produce at least about 0.75 mg/100 ml of lactic acid under effective growth conditions. In yet another embodiment, such a strain is from about 1 micron to about 2 microns in width and from about 2 microns to about 4 microns in length.

In a preferred embodiment, such an isolated bacterial strain is isolated from the human vagina. In another preferred embodiment, the strain is of the species Lactobacillus crispatus or Lactobacillus jensenii. A particularly preferred strain has all of the identifying characteristics of Lactobacillus crispatus CTV-05, with Lactobacillus crispatus CTV-05 being the most preferred strain.

Yet another embodiment of the present invention relates to a bacterial strain of Lactobacillus which has substantially all of the identifying characteristics of Lactobacillus crispatus CTV-05. Such a strain is particularly useful for protecting a female from a vaginal infection.

One embodiment of the present invention relates to a method to preserve microbial cells within a preservation matrix to form a vaginal medicant. This method includes the steps of (a) suspending a culture of at least about 10⁶ microbial cells in a preservation matrix which includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material, to form a cell matrix suspension; (b) adding the cell matrix suspension to an inert carrier to form a delivery composition; and removing water from the delivery composition.

In a further embodiment of this method, the delivery compositions can be placed into a package to protect against moisture and oxygen during transport and storage. The package can be any suitable material for such protection such as Mylar or metallic film pouches. In one embodiment, the delivery compositions can be packaged in individual packages. Another embodiment may include packaging with multiple cavities, perhaps aligned with dosage.

Conventional methods of preserving microbial cells commonly employ air drying, spray drying or freeze drying. Air drying requires long periods of time, sometimes with somewhat elevated temperatures. Spray drying exposes the cells to hot air, turbulence and excessive levels of oxygen. Freeze drying requires dramatic fluctuations in temperature and the inherent risk of ice crystal formation. An advantage of the preservation matrix of the present invention is that the matrix allows removal of water from the cells by a variety of conventional drying methods with minimal damage to the microbial cells. Preferably, the method of producing the vaginal medicant of the present invention encompasses processing steps which are most likely to reduce stress to the cells during harvest, dispensing and preservation so as to maximize the likelihood of a final product with long shelf-life and capability of delivering viable cells of the suppository Lactobacillus strain having the desirable characteristics described above. Stresses to avoid include an excessive number of processing steps, fluctuations of temperature, use of vacuum, exposure to moisture and long processing times. The process preferably also limits the introduction of contaminating microorganisms, a common problem in existing commercial preparations of Lactobacilli. Particularly preferred methods of preserving microbial cells in a preservation matrix are discussed in detail below.

All methods of microorganism preservation require drying or removal of water. Water in microorganisms exists in both free and bound states. Removal of sufficient amounts of both states is necessary for preservation of the microorganism, but excessive removal of the bound water may be problematic. Other problematic factors in the preservation process include the amount of time employed to remove the cellular moisture and the temperature of the process. Generally, it is preferred to remove moisture quickly with careful attention to temperature control, particularly if higher temperatures are used.

The present invention provides an optimal method for preservation of microorganisms which is designed to (1) be able to accept the microbial cells in a protective matrix wherein the cells are added to the matrix immediately after they are harvested from the culture medium; (2) avoid exposure of cells to temperatures above 40^oC. at any time during the preservation process; (3) avoid exposure of cells to ice crystal formation; (4) avoid exposure of cells to high or low extremes in temperature fluctuation; (5) have a short duration of the preservation process, optimally less than 4 hours; (6) avoid exposure of cells to vacuum or compression; and (7) avoid exposure of cells to air that may carry contaminating microorganisms. The goal of the preservation method is to have the final medicant product rehydrated directly within the vaginal milieu.

In one embodiment, the method of preserving microbial cells within a preservation matrix includes coating the cell matrix suspension onto an inert carrier which preferably is a maltodextrin bead. The coated beads are then dried, preferably by a fluid bed drying method. Fluid bed drying methods are well known in the art. For example, maltodextrin beads are placed into a fluid bed dryer are dried at 33^oC. The air pressure is set to 14 psi, the cell suspension matrix is sprayed onto the beads and the heat is increased to 38^oC. The coated beads are then allowed to dry for an additional period of time. The coated maltodextrin beads can be stored as a powder, placed into gelatin capsules, or pressed into tablets.

In another embodiment of the present invention, a convenient suppository format for delivering viable Lactobacillus cells of the present invention in an exogenous fashion to the vaginal milieu is that of a hard gelatin capsule. Gelatin capsules are commercially available and are well known in the art. In this embodiment, the above preservation method further comprises dispensing the cell suspension matrix to a gelatin capsule, chilling the gelatin capsule until the cell suspension matrix forms a non-fluid matrix and to affix the gel to the interior wall of the gelatin capsule, and desiccating the gelatin capsule in a desiccation chamber. The step of dispensing can be accomplished by any means known in the art, and includes manual, semi-automated and automated mechanisms. The chilling step is performed at from about 4^oC. to about 6^oC. The step of desiccating the gelatin capsule can include the steps of (i) providing dry air to the desiccation chamber containing less than about 25% moisture, at a temperature from about 24^oC. to about 32^oC.; and (ii) removing humidified air from the desiccation chamber.

In this embodiment, the desiccation process can proceed for about 1 to about 6 hours. The desiccation chamber can include a compressor, at least one hydrocarbon scrubbing filter and a chilled air compressor with or without a desiccant silica gel (or any other suitable desiccant material) column, in series. It is a preferred embodiment that the air entering the chamber (dry air) should contain less than about 25% moisture, and more preferably less than about 15% moisture, and even more preferably less than about 5% moisture, down to as little as zero moisture. The dry air should preferably have a temperature from about 24^oC. to about 32^oC. The preferred rate of air flow is 2 air exchanges per minute. This method allows preservation of microbial cells in a controlled environment with room temperature air in a short period of time. Also, the microbial cells may be dispensed directly into the suppository delivery device and preserved in an in situ fashion in the same device, thus increasing the likelihood of maintaining desirable identifying characteristics of the microbial cell.

One step of the method for preserving microbial cells in a matrix includes suspending a culture of microbial cells in a preservation matrix. According to the present invention, microbial cells are harvested from the culture medium and immediately suspended in the preservation matrix at optimal cell matrix ratio. Optimally but not necessarily, the harvest and suspension process is accomplished within a Class 100 environment.

Lactobacillus cells of the present invention can be grown in any medium which provides effective growth of the microbe without contamination, loss of genetic stability, or loss of any other desirable identifying and functional characteristics of a Lactobacillus strain of the present invention (as previously described herein). More particularly, Lactobacillus strains of the present invention are grown in a culture medium which includes a source of assimilable organic carbon, a source of assimilable nitrogen and appropriate salts and trace metals. A preferred medium for culturing Lactobacillus strains of the present invention is MRS medium. MRS medium is described in detail in the Examples section.

The Lactobacillus microorganisms of the present invention can be cultured in conventional culture conditions, which include, but are not limited to agar surface culture or broth fermentation. Both agar surface culture and broth fermentation methods are well known in the art. The Lactobacillus are preferably cultured anaerobically or microaerophilically.

The temperature of the culture medium can be any temperature suitable for growth of Lactobacillus. For example, prior to inoculation of the culture medium with an inoculum, the culture medium can be brought to and maintained at a temperature in the range of from about 20^oC. to about 35^oC., and more preferably in the range of from about 25^oC. to about 35^oC.

The culture medium is inoculated with an actively growing culture of a Lactobacillus strain of the present invention in an amount sufficient to produce, after a reasonable growth period, a suitable cell density for transfer to the preservation matrix. Typical inoculation cell densities are within the range of from about 10⁶ CFUs/ml to about 10⁹ CFUs/ml, and more preferably from about 10⁸ CFUs/ml to about 10⁹ CFUs/ml, based on the dry weight of the cells. The cells are then grown to a cell density in the range of from about 10⁷ CFUs/ml to about 10⁹ CFUs/ml, and more preferably to about 10⁸ CFUs/ml. At this stage, the cells are harvested for preservation in the preservation matrix.

In the first step of this embodiment of the present invention, after reaching the desired cell density, the microbial cells are harvested, preferably by a method such as centrifugation. At least about 10⁷ microbial cells, and more preferably at least about 10⁸ microbial cells and even more preferably at least about 10⁹ microbial cells are suspended in a preservation matrix. Prior to addition of the cells to the matrix, the cells may be washed in a saline buffer. The preservation matrix and microbial cell mixture is referred to herein as the cell suspension matrix. The cell suspension matrix is typically maintained at 30-40^oC. with continuous mixing during the subsequent steps of adding the matrix to an inert carrier. It is to be understood that one of ordinary skill in the art will appreciate variations to the basic culturing, harvesting and suspending steps disclosed herein and as such, the present invention incorporates such variations.

Yet another embodiment of the present invention relates to a method to protect a female against vaginal infections. This method includes administering to a female a vaginal medicant which includes (a) a substantially pure bacterial culture of at least about 10⁶ isolated strain of the genus Lactobacillus having identifying characteristics which include (i) a percent VEC cohesion value of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H₂ O₂. The medicant also includes (b) a preservation matrix which includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. In a further embodiment, the vaginal medicant can include an inert carrier as described previously herein. The preservation matrix maintains at least about 10⁶ viable, substantially pure and genetically stable cells for a period of at least about 12 months in vitro.

Many of the above embodiments have been described previously herein in detail. The vaginal medicant of the present invention can be used to prevent a variety of vaginal infections, including, but not limited to bacterial vaginosis, symptomatic yeast vaginitis, gonorrhea, chlamydia, trichomoniasis, human immunodeficiency virus infection, urinary tract infection and pelvic inflammatory disease.

According to the present invention, "to protect a female from a vaginal infection" refers to reducing the potential for a female to develop a vaginal infection. Preferably, the potential for a vaginal infection is reduced, optimally, to an extent that the female does not suffer discomfort and/or altered function from exposure to a vaginal infectious agent. For example, protecting a female from a vaginal infection can refer to the ability of a vaginal medicant of the present invention, when administered to the female, to prevent a vaginal infection from occurring or recurring.

Another embodiment of the present invention relates to a method to treat a vaginal infection by administering to a female having a vaginal infection a vaginal medicant of the present invention. As used herein, treating a female with a vaginal infection refers to the ability of a vaginal medicant of the present invention to cure or alleviate infection symptoms, signs or causes.

Preferably, a single vaginal medicant to be administered to a female to prevent or to treat a vaginal infection includes at least about 10⁶ and more preferably at least about 10⁷ and even more preferably, at least about 10⁸ viable, substantially pure and genetically stable Lactobacillus cells having the identifying characteristics described herein. A preferred administration protocol includes the dose and the frequency of administration of the medicant and can be readily determined by one of skill in the art. In one embodiment, a single medicant is administered at least about once a day for about two days, or in another embodiment, at least about once a day for about three days, or in another embodiment, at least about twice a day for about three days. Such dosage can be administered again, if needed, for example, on a monthly basis. A vaginal medicant of the present invention can be administered orally, vaginally or rectally, although any other modes of administration which can deliver the microorganisms to the desired site of action are encompassed herein. A preferred format for administration is a suppository (e.g., such as a tablet or a capsule).

A vaginal medicant of the present invention can be administered in conjunction with (e.g., simultaneously with, before, and/or after) any other therapy for the prevention or treatment of vaginal infections. For example, a vaginal medicant can be administered in conjunction with an antibiotic.

Another embodiment of the present invention relates to a method to reduce the risk of infection of a human by human immunodeficiency virus (HIV) by administering to a human a medicant of the present invention as described herein. Preferably, the medicant reduces the risk of HIV infection by the human by at least about 2-fold, and more preferably, at least about 4-fold, and even more preferably, by at least about 6-fold. The human to which such a medicant can be administered can be a male or a female. The medicant can be administered orally, vaginally or rectally to a female, and orally or rectally to a male. In a preferred embodiment, a single medicant is administered daily for two days.

Yet another embodiment of the present invention is a method to prevent symptomatic yeast vaginitis by administering to a human a medicant of the present invention as described herein. In a preferred embodiment, such a medicant includes Lactobacillus crispatus CTV-05.

Another embodiment of the present invention is a method to prevent preterm birth. As discussed previously herein, bacterial vaginosis is one of the most common genital infections in pregnancy. Women with bacterial vaginosis diagnosed during the second trimester of pregnancy are 40 percent more likely to give birth to a premature, low-birth--weight infant than women without bacterial vaginosis. A method to prevent preterm births according to the present invention includes the steps of administering to a pregnant female (a) antibiotics and (b) a vaginal medicant of the present invention as described herein. Preferred dosages are as described above for administration of a vaginal medicant to prevent a vaginal infection. Preferred antibiotics to be administered with a vaginal medicant of the present invention include any antibiotic useful in treating vaginal infections. Such antibiotics are known in the art.

Another embodiment of the present invention includes a method to assist metabolism of estrogen in the vagina and bowel. Hyperestrogenism is a condition afflicting women which can be treated using a medicant of the present invention. The Lactobacillus strains of the present invention appear to assist in proper metabolism of estrogen in the vagina and the bowel. This method includes the step of administering to a female a medicant of the present invention as described herein. Such a medicant can be administered vaginally, orally, or rectally. Dosage ranges are substantially similar to those provided herein for treatment of vaginal infections.

As mentioned above, although the above discussion of the medicant of the present invention has been primarily directed to the use of such a medicant to treat vaginal infections, a medicant of the present invention is not restricted for use in the treatment of infections of or related to the vagina. A medicant of the present invention can be used, for example, to treat a gastrointestinal disorder or infection. In this case, such a medicant is referred to as a gastrointestinal medicant. A gastrointestinal medicant of the present invention has all of the distinguishing features of a vaginal medicant of the present invention, including a substantially pure bacterial culture of an isolated strain of the genus Lactobacillus having identifying characteristics which include (i) a percent vaginal epithelial cell (VEC) cohesion value of at least about 50% and (ii) an ability to produce greater than about 0.5 ppm of H₂ O₂. In another embodiment, an initial strain useful in the treatment of gastrointestinal infection has VEC cohesion values and/or H₂ O_s production values less than those useful in the vagina while still retaining effective gastrointestinal medicant qualities. The gastrointestinal medicant also includes a preservation matrix, which contains and preserves the bacterial culture. Such a matrix includes a biologically active binding agent, an antioxidant, a polyol, a carbohydrate and a proteinaceous material. The matrix is capable of maintaining at least about 10⁶ viable, genetically stable cells for a period of at least about 12 months in vitro. The gastrointestinal medicant can comprise an inert carrier including, maltodextrin beads or a gelatin capsule. A medicant for use in the gastrointestinal tract is administered by any route by which the microorganisms of the medicant can reach and adhere to cells in the gastrointestinal tract. Such modes of delivery include, but are not limited to oral and rectal delivery.

Claim 1 of 23 Claims

What is claimed:

1. A vaginal medicant, comprising:

a bacterial culture of an isolated strain of the genus Lactobacillus having identifying characteristics comprising:

(i) a percent vaginal epithelial cell (VEC) cohesion value of greater than 50%; and

(ii) an ability to produce greater than 0.5 ppm of H₂ O₂ under effective culture conditions.
 

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