KR20100079078A - Pharmaceutical compositions including lactobacillus fermentum no. 1969 for treatment of vaginal infections - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for treating bacterial vaginitis is provided to suppress antibiotics resistant staphylococcus and to ensure strong epithelial cell adhesion ability. CONSTITUTION: A pharmaceutical composition for intravaginal administration for treating bacterial vaginitis contains Lactobacillus fermentum No. 1969 strain(KCTC 11437BP) as an active ingredient. The Lactobacillus fermentum No. 1969 strain has abilities of generating hydrogen peroxide and adhering to vaginal epithelial cells. The Lactobacillus fermentum No. 1969 strain suppresses to staphylococcus. The Lactobacillus fermentum No. 1969 strain is contained in a freeze-dried powder form.

Description

Pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis, comprising Lactobacillus fermentum NFO. １６６ strain as an active ingredient. 1969 FOR TREATMENT OF VAGINAL INFECTIONS}

  The present invention relates to a pharmaceutical composition for treating bacterial vaginosis, and more particularly, to a pharmaceutical composition for intravaginal administration for bacterial vaginosis, comprising hydrogen peroxide-producing Lactobacillus fermentum as an active ingredient.

Vaginitis in women is caused by an imbalance in the normal environment of the vagina. That is, the bacterial vaginal symptoms (bacterial vaginosis) is the Lactobacillus bacteria (Lactobacillus) foil from vagina steroid (Bacteroides), pepto Streptococcus (peptostreptococcus), pepto Rhodococcus (Peptococcus), if the mobile Rune kusu. It is due to bacterial interaction because it is replaced by a number of anaerobic bacteria, including Mobiluncus G. vaginalis . Therefore, the role of normal flora in the vagina is very important to maintain the normal environment in the vagina.

Results of recent has haejugo support that the Lactobacillus bacteria (Lactobacillus) are, for example, maintain the normal bacterial balance in dynamic ecosystems, such as quality haejugo, it plays an important role in preventing the cause being the genital infection by the pathogenic microorganisms. Doderlein is the first acid-producing vaginal lactic acid bacterium It is said to inhibit the colony production of pathogens by lowering nitrate.

In addition, sulfonamides, antibiotics, and antiseptic drugs have been mainly used to treat bacterial vaginosis, vaginal dysfunction, vulvitis, and the like. In particular, in recent years when various antibiotics have been developed, antibiotic preparations have often been administered to patients. Such antibiotics can be fatal to Lactobacillus present in the vagina. Unfortunately, pathogenic bacteria (such as Staphylococcus ) have acquired resistance to antibiotics, making it difficult to administer antibiotics to these diseases.

In order to solve this problem, it has been proposed to use Lactobacillus in the gynecology for treating vaginal infections. For example, Korean Patent Application No. 10-1999-0015469 discloses a Lactobacillus combination for the preparation of a pharmaceutical composition for treating vaginal dysfunction and vaginitis, wherein the bacterial combination is Lactobacillus brevis. ( Lactobaccilus brevis ) and Lactobacillus salivarius subs. Lactobacillus salivarius subs. Salicinius species, and possibly Lactobacillus salivarius subs . Lactobacillus salivarius subs.salicinius , Lactobacillus jensenii , Lactobacillus catenaforme , Lactobacillus minutus and Lactobacillus minutus , and one gaseous gaseous strain from Lactobacillus. It can be combined with the above.

However, in the conventionally known gynecology, the technique of using Lactobacillus uses only various types of Lactobacillus combinations, and no technique using only one type of Lactobacillus strain has been developed.

That is, in order to use the Lactobacillus strain for the treatment of gynecological diseases, at least all of the following characteristics disclosed in Korean Patent Application No. 10-1999-0015469 must be satisfied.

(a) The selected Lactobacillus strain must have a high affinity to the vaginal epithelium, ie it is strongly attached to the vaginal epithelium, enabling bacterial interactions in both physiological and pathogenic conditions (ie at the epithelial receptor site). To be able to compete competitively with pathogenic microorganisms), to be able to recover microorganisms and optimum pH conditions;

(b) at least one of the selected Lactobacillus strains must have a good ability to produce hydrogen peroxide that acts as an inhibitory agent against pathogenic microorganisms;

(c) At least one of the selected Lactobacillus strains must have a good ability to interfere with Candida albicans in a competitive manner in adhesion to HeLa cells.

However, using various combinations of Lactobacillus strains as in the prior art including Korean Patent Application No. 10-1999-0015469 has the problem of culturing and combining several kinds of strains.

The present inventors have completed the present invention by developing a Lactobacillus strain-containing pharmaceutical composition that can treat the intravaginal bacterial infection using only one type of Lactobacillus strain as a result of research efforts to solve the above problems.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for intravaginal administration of bacterial vaginitis, which has an effect on treating vaginitis by using only one type of Lactobacillus strain.

Another object of the present invention is to provide a pharmaceutical composition for intravaginal administration of bacterial vaginitis, which is more effective than antibiotics for the treatment of bacterial vaginitis by having an inhibitory effect on staphylococcus, which is difficult to treat antibiotics.

It is another object of the present invention to provide a pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis having not only strong hydrogen peroxide production capacity but also strong epithelial cell adhesion compared to other types of Lactobacillus strains.

The objects of the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention to achieve the above object is Lactobacillus fermentum ( Lactobacillus fermentum ) No. Provided is a pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis comprising 1969 strains (Korean Collection for Type Cultures, Accession No .: KCTC 11437BP, Accession Date: December 3, 2008) as an active ingredient.

In a preferred embodiment, the Lactobacillus fermentum No. The 1969 strain had better hydrogen peroxide-producing ability and adhesion to vaginal epithelial cells than other strains belonging to Lactobacillus fermentum.

In a preferred embodiment, the Lactobacillus fermentum No. 1969 strain has an inhibitory activity against staphylococci.

In a preferred embodiment, the Lactobacillus fermentum No. 1969 strain has an inhibitory action against Staphylococcus aureus, which is a lactic acid bacterium isolated from saliva.

In a preferred embodiment, the Lactobacillus fermentum No. 1 per dosage of the pharmaceutical composition. 1969 strains were included at a concentration of 1.6 × 10 ⁸ CFU / g.

In a preferred embodiment, the Lactobacillus fermentum No. 1969 strains are included in lyophilized powder.

The present invention has the following excellent effects.

First, the pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis of the present invention has an effect on treating vaginitis by using only one type of Lactobacillus strain.

The pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis of the present invention is more effective than antibiotics for the treatment of bacterial vaginitis by having an inhibitory effect on staphylococci, which is difficult to treat antibiotics.

Intravaginal pharmaceutical composition for the treatment of bacterial vaginitis of the present invention has a strong epithelial cell adhesion as well as a strong hydrogen peroxide production capacity compared to other types of Lactobacillus strains.

The terms used in the present invention were selected as general terms as widely used as possible, but in some cases, the terms arbitrarily selected by the applicant are included. In this case, the meanings described or used in the detailed description of the present invention are considered, rather than simply the names of the terms. The meaning should be grasped.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals designate like elements throughout the specification.

Example

1. Lactobacillus fermentum No. 1969 Identification of Strains

In order to find the normal vaginal flora in the vaginal epithelial cells while producing strong hydrogen peroxide, a number of experiments were identified from the saliva samples of healthy children through the following process.

Saliva samples were diluted with phosphate buffer solution (PBS buffer) and inoculated in Rogosa agar medium and anaerobic incubated at 37 ° C. for 48 hours, and colonies of different forms were taken.

This was again inoculated, cultured and stored in MRS liquid medium, which was used for experiments. Among them, strain No. Lactobacillus fermentum No. 1969 was identified by 16S rRNA full sequencing analysis.

2. Preparation of pharmaceutical composition containing Lactobacillus fermentum No.1969 for administration to the vagina

In order to prepare a pharmaceutical composition containing Lactobacillus fermentum No.1969, bacteria were inoculated and cultured in a large amount of MRS liquid medium. After centrifugation (4000 rpm, 20 min, 4 ° C.), washing twice with sterile water, and freeze drying using a lyophilizer. Thereafter, 0.1 g (1.6 x 10 ⁸ ) was added to the 1.5 ml tube to prepare a pharmaceutical composition containing Lactobacillus fermentum No.1969, which was stored in a desiccator containing silica gel and charged with nitrogen. Pharmaceutical compositions containing Lactobacillus fermentum No.1969 can be used by intravaginal administration of one tube.

Particularly, after 1.6 x 10 ⁸ CFU / g of vaginal infection, 7 days later, vaginal peroxide distribution was significantly increased and vaginitis was treated.

Experimental Example 1

Lactobacillus fermentum No. In order to test the hydrogen peroxide (H ₂ O ₂ ) production capacity of 1969 strain, Lactobacillus fermentum No. Inoculated with 3µl of 1969 strain (left side of Figure 1) and commercial Lactobacillus fermentum (right side of Figure 1) by anaerobic incubation at 37 ℃ for 2 days and then exposed to air and then present in the culture medium It was observed whether the peroxidase reacts, H ₂ O ₂ was Once produced, the peroxidase present in the culture medium reacts with subsequent oxidation of TMB resulting in a change in color. In other words, the more hydrogen peroxide produced, the darker the color becomes.

As shown in Figure 1, Lactobacillus fermentum ( Lactobacillus fermentum ) No. Since the H ₂ O ₂ -colon produced by the 1969 strain was colored more blue than the commercial strain, Lactobacillus fermentum No. It can be seen that the hydrogen peroxide production ability of the 1969 strain is very excellent.

Experimental Example 2

Lactobacillus fermentum No. from the American Type Culture Collection (ATCC). The ability to attach to HeLa cells, a cell line originating from human carcinoma of the cervix of the 1969 strain, was tested as follows.

Lactobacillus fermentum No. HeLa cells of 1969 were maintained in a monolayer in a tissue culture bottle containing minimal essential medium (MEM) added with 10% (v / v) fetal bovine serum (FBS). Attachment reactions were performed using multi-well culture plates on tissue containing sterile cover slides (24 × 24 mm) in each well. Each well was inoculated with 1 ml of a HeLa cell suspension at a concentration of 1.0 x 10 ⁵ cell / ml and plates were incubated for 24 hours in a 5% CO ₂ incubator. Lactobacillus bacteria were incubated in MRS liquid medium, centrifuged (4,000 rpm, 20 min, 4 ° C), washed twice with PBS, and the bacteria were suspended in MEM. For adhesion test, HeLa cells in culture in wells were inoculated with the bacteria as multiplicity of infection (MOI) = 1: 250 (2.5 × 10 ⁷ bacteria / cell) and incubated for 30 minutes. After washing three times in PBS to remove the unattached microorganisms, the cells were fixed, Giemsa stained and examined under an optical microscope and the results are shown in Figure 2 and Table 1.

Lactobacillus fermentum No. for HeLa cells from Figure 2 and Table 1 It can be seen that the attachment capacity of 1969 is very good.

Experimental Example 3

The inhibitory effect of Lactobacillus fermentum No.1969 on Staphylococcus aureus was tested as follows, and the results are shown in FIG.

The number of colonies generated by inoculating BHI agar medium was measured by diluting for the determination of viable cell counts and the 1: 1 culture of Staphylococcus aureus and Lactobacillus fermentum , respectively.

L. fermentum 1969 suppressed S. aureus virulence on average by 1.6 x 10 ² compared to the single culture (see Figure 3), but the number of mixed cultures was more than 10 times compared to the single culture. It can be judged that the reduction effect of Lactobacillus fermentum No. 1969 against Staphylococcus aureus is excellent, because it can be judged that the reduction effect is excellent.

Experimental Example 4

In order to examine the effectiveness of Lactobacillus fermentum No.1969 pharmaceutical composition to treat vaginitis, a clinical study was conducted on 51 healthy women and 51 women with vaginitis.

In other words, the lyophilized Lactobacillus fermentum No.1969 strain containing the pharmaceutical composition was observed 7 days after the first dose of healthy women, 12 of the vaginitis patients 7 days after the first dose, 11 patients twice a day at intervals of 2 days After administration, samples were taken from the vagina, smeared on slide glass, stained with Gram, and scored using an optical microscope. The results are shown in Tables 2 and 3.

Score Large gram (+) rods (Lactobacilli) Small gram-variable rods
(Gardnerella) and small gram (-) rods (Bacteroides spp.) Curved gram-variable rods (Mobiluncus spp.) 0 4+ 0 0 One 3+ 1+ 1+ or 2+ 2 2+ 2+ 3+ or 4+ 3 1+ 3+ 4 0 4+

       0: No morphotypes 1+: less than 1 morphotype

2+: 1 ~ 4 morphotyes 3+: 5 ~ 30 morphotyes

4+: 30 or more morphotyes

Scoring system Spiegel criteria 0-3 Normal 4 to 6 Intermediate 7-10 Bacterial vaginosis

As a result, 18 of 23 patients with vaginitis showed significant improvement in symptomatic terms (the infection was cured), and the number of viable hydrogen peroxide-producing Lactobacillus bacteria was significantly increased. Could know.

As described above, the present invention, unlike the prior art pharmaceutical composition for treating vaginitis, contains a mixture of several kinds of Lactobacillus , containing one strain of Lactobacillus fermentum No. 1969 which actually has a particularly beneficial effect when administered to a patient. It is very effective in treating vaginitis by providing a pharmaceutical composition.

In other words, the Lactobacillus fermentum No.1969 strain-containing pharmaceutical composition of the present invention not only has a good effect on the treatment of vaginitis with a single bacterium but also a mixed bacterium, and is strongly adhered to vaginal epithelial cells, thereby mutually affecting bacteria in both physiological and pathogenic conditions. It has the ability to produce hydrogen peroxide, which enables the action, restores the microorganism and the optimum pH condition, and inhibits pathogenic microorganisms, and has the inhibitory action against Staphylococcus aureus , which is difficult to treat antibiotics. Therefore, it is more effective than antibiotic treatment for treating vaginitis.

As described above, the present invention has been illustrated and described with reference to preferred embodiments, but is not limited to the above-described embodiments, and is provided to those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

1 is Lactobacillus fermentum ( Lactobacillus fermentum ) No. Hydrogen peroxide (H ₂ O ₂₎ production capacity of the 1969 strain is a photograph of the experimental results,

Figure 2 is Lactobacillus fermentum No. for HeLa cells. A photograph of the results of the adhesion test of 1969,

3 is a graph showing the results of the inhibitory effect of Lactobacillus fermentum No.1969 on Staphylococcus aureus .

Claims (6)

Lactobacillus fermentum No. Pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis comprising 1969 strain (Deposit: Korean Collection for Type Cultures, Accession No .: KCTC 11437BP, Deposit date: December 3, 2008) as an active ingredient. The method of claim 1, The Lactobacillus fermentum No. 1969 strain is a pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis, characterized in that it has an ability to produce hydrogen peroxide and adhesion to vaginal epithelial cells superior to other strains belonging to Lactobacillus fermentum. The method of claim 1, The Lactobacillus fermentum No. 1969 strain is a pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginosis, characterized in that it has an inhibitory action against staphylococcus. The method of claim 1, The Lactobacillus fermentum No. 1969 strain is a pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis, characterized in that it has an inhibitory action against staphylococcus, characterized in that the lactic acid bacteria isolated from saliva. The method of claim 1, The Lactobacillus fermentum No. per dose of the pharmaceutical composition. Pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginosis, characterized in that 1969 strain is contained at a concentration of 1.6 × 10 ⁸ CFU / g. The method of claim 1, The Lactobacillus fermentum No. 1969 strain is a pharmaceutical composition for intravaginal administration for the treatment of bacterial vaginitis, characterized in that it is included in a lyophilized powder state.

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