EP0147428A1

EP0147428A1 - Vaginal capsules

Info

Publication number: EP0147428A1
Application number: EP84902197A
Authority: EP
Inventors: Ann Marie Doucette; Henrik Boisen
Original assignee: Chr Hansen Biosystems AS
Current assignee: Chr Hansen AS
Priority date: 1983-05-27
Filing date: 1984-05-22
Publication date: 1985-07-10
Also published as: JPS60501160A; WO1984004675A1; AU3018684A; DK242083D0

Abstract

Des capsules de gélatine solubles pour effectuer la régulation d'infections vulvo-vaginales contiennent un concentré viable, séché, stable, tel qu'un concentré lyophilisé, de bactéries d'acide lactique, en particulier le Lactobacillus acidophilus, dispersées dans un porteur fluide pharmaceutiquement actif possédant une viscosité d'au moins 2000 cps, de préférence 3000 cps, et en particulier 4000 cps. Le nombre de bactéries par capsule est d'au moins 1 x 106, de préférence au moins 1 x 107. L'activité de dilution (aw) du concentré bactérien et du porteur fluide se situe entre 0,00 et 0,2. Le porteur fluide est de préférence une huile non-hygroscopique contenant un agent d'accroissement de la viscosité comme l'amidon et/ou la vaseline. On a découvert que les capsules résultantes possédaient une activité d'inhibition envers Candida albicans in vitro et in vivo.Soluble gelatin capsules for regulating vulvovaginal infections contain a viable, dried, stable concentrate, such as a lyophilized concentrate, of lactic acid bacteria, in particular Lactobacillus acidophilus, dispersed in a pharmaceutically fluid carrier active with a viscosity of at least 2000 cps, preferably 3000 cps, and in particular 4000 cps. The number of bacteria per capsule is at least 1 x 10 6, preferably at least 1 x 10 7. The dilution activity (aw) of the bacterial concentrate and of the fluid carrier is between 0.00 and 0.2. The fluid carrier is preferably a non-hygroscopic oil containing a viscosity-increasing agent such as starch and / or petrolatum. The resulting capsules were found to have inhibitory activity against Candida albicans in vitro and in vivo.

Description

VAG I NAL CAPSULES

The present invention relates to a method and a capsule for control¬ ling vulvo-vaginal infections .

It has been known for a long time to use antibiotics in the treatment of vulvo-vaginal infections, but antibiotic treatment has proved to be disadvantageous as antibiotics tend to kill the desi red microflora in the vagina as well, whereby the natu ral healthy microbial balance of the vagina is disturbed . This in turn often results in the condition becoming re-occurring as the pathogenic microorganisms often re-in¬ vade the vaginal environment without other microorganisms to check their growth .

A more preferred way of controlling vulvo-vaginal infections is to employ lactic acid bacteria so as to simulate the normal vaginal envi- ronment. There is some disagreement as to the causes of vulvo-vagi¬ nal infections . It has been thought that infections occu r when the pH of the vagina is too high such as at menstruation, or when the hor¬ monal balance is disturbed because of pregnancy or when taking oral contraceptives, thus promoting the growth of potentially pathogenic microorganisms such as Candida albicans, Trichomonσs vaginalis, Staphyfococcus aureus, Gardnerella, - Streptococci and various anaer¬ obic microorganisms which, conversely, were not assumed to th rive under the normal, slightly acidic conditions in the vagina . Thus it was believed that, by introducing lactic acid bacteria in the vagina, the environment would attain its normal acidity so that the propa¬ gation of the pathogenic microorganisms would once more be inhibited.

However, more recent studies (cf . Annals of internal Medicine vol . 96 ( Part 2) , 1982, pp. 931 -34, J. din . Microbiol . , May 1980, pp . 479-84, Br. J. Veπer. Dis . vol . 56, 1980, pp . 107-10) indicate that it is not the acidity of the vagina as such which prevents the excessive growth of undesirable microorganisms as both Candida and Trichomo- nas grow under acidic conditions, but rather the ecological balance normally prevailing in the vagina, or rather on the vaginal mucosa, and that infections are more likely to occu r when , for some reason ,

OM the population of the various Lactobacillus species has become dimi¬ nished . The studies cited agree that there is a correlation between the abundance of Lactobacillus colonies and vaginal pH, in that the pH decreases with the number of Lactobaciili present, but that it is the actual presence of large numbers of Lactobaciili which prevents vulvo-vaginal infections.

In the known use of lactic acid bacteria, either a fermented milk product containing Lactobaciili is introduced manually, or a slurry of dried Lactobacillus acidophilus (e.g . the product known as Floranorm, marketed by Danapharm) is introduced by means of a suitable applica¬ tor such as a disposable syringe.

It has also been suggested to introduce lactic acid bacteria incorpora¬ ted in a capsule, vide US Patent No. 3,639,566 which discloses a hard gelatin capsule which contains estriol and living Doederlein bacteria (the type of Lactobaciili naturally occurring in the vagina) adsorbed to starch . This product has a moisture content of about 40%.

The non-encapsuled products, however, suffer from the disadvantage that their application is uneven, i . e. the distribution of the lactic acid bacteria in the vagina is not homogeneous . The application me- thod may also be found to be inconvenient. Also, as the products are rather fluid, they tend to run out of the vagina so that they do not have the desired effect. A disadvantage of the product known from US Patent No. 3.639.566 is that the bacteria incorporated in the capsules are likely to have a limited stability, due to the content of moisture absorbed by the starch incorporated in the capsule so that the capsules may only be stored for limited periods of time. Further¬ more, when applied in the vagina, the capsule wilt take up moisture for which reason the capsule content tends to get lumpy; this may result in an uneven distribution over the vaginal area and probably a reduced effect.

The present invention provides capsules which are superior with respect to efficient introduction and distribution of an effective a- mount of lactic acid bacteria into the vagina, and correspondingly provides an efficient method for controlling vulvo-vaginal infections . The capsules of the invention are improved over the known art by containing stabilized cells of lactic acid bacteria, which means that they may be stored at room temperature for several months while maintaining a high percentage of viability.

Thus, one aspect of the invention relates to a soluble capsule for controlling vulvo-vaginal infections which contains a stable, dried, viable concentrate of lactic acid bacteria dispersed in a pharmaceuti¬ cally acceptable fluid carrier.

I n the present context, the term "dried" indicates that the concen¬ trate has a water activity (a ) of not more than 0.2, calculated w according to the formula described by e. g . D . Demeyer, Fleischwirt- schaft 59(7) , 1979, p. 940.

The capsule is preferably a gelatin capsule, in particular a soft gelatin capsule. The capsule may, however, also be a hard, but soluble gelatin capsule. The capsule may be of any suitable shape such as a spherical or oblong shape, but for easy application it is preferred that the capsule be oval or egg-shaped . The volume of the capsule is in the range of about 0.2-5.0, preferably about 1 .2 ml . The capsule volume is in fact rather critical as the physical proper^¬ ties of the paste vide below) set limits to the amount of paste mate¬ rial and thus the amount of bacteria per capsule which may adhere or be adsorbed to the vaginal mucosa without causing any significant discharge problems . The capsules preferred for use according to the invention are those produced substantially according to the disclosu re of US Patents Nos . 1 ,970,396, 2, 152, 101 , 2,234,479 and 2, 288,327.

It is well known to prepare concentrates of lactic acid bacteria . Methods for preparing such concentrates are described, e. g . , in US Patent No. 4, 115, 199, utilizing a polyphosphate salt such as sodium hexametaphosphate or sodium tripolyphosphate as an additive to the cultu re medium prior to concentration by centrifugation . The wet bacterial concentrate is then dried, preferably freeze-dried, under stabilizing conditions in the presence of stabilizing additives as de¬ scribed in US Patent No. 3,897,307.

OMPI By using a freeze-dried concentrate, it becomes possible to obtain a high count of stable, viable bacteria in a capsule for intravaginal administration . According to the invention, the count of viable lactic acid bacteria is at least 1 x 10^s , preferably at least 1 10⁷, more - preferably at least 1 x 10^s and most preferably in the range between about 1 10⁹ and 1 10¹¹, such as about 1 x 10⁹ and 1 10^{l α} per capsule.

The dried, stabilized bacterial concentrate, when incorporated in the capsules of the invention, is not totally moisture-free, but contains some internal moisture. I n order to obtain a stable, dried concentrate with such a high cell count of viable bacteria, it is, however, desi¬ rable that the water activity of the concentrate is in the range of 0.00-0.2, preferably in the range of 0.00-0.1 . A suitable instrument for determining water activity is available from Novasima AG, Zurich, Switzerland .

I n order to prevent an excessive uptake of water and oxidation of the concentrate, resulting in a substantial loss of viability, which is likely to occur if the dried concentrate was to be incorporated in the gelatin capsules as such, the concentrate is mixed with a fluid car- rier. According to the invention , it has been found that particular types of fluid carrier are extremely well suited both for an even distribution in the vagina and for maintaining a high stability (i . e. long-term viability) of the concentrate.

The carrier is thus primarily so selected that it will protect the freeze-dried bacterial culture from physical and/or chemical reactions which have a negative effect on cell stability (expressed as a loss of viability) . It has been found that the most important single factor for maintaining the stability of the concentrate is the low water activity of the culture/carrier mixture for the reasons stated above. The carrier should also have a water activity in the range of 0.00-0.2, preferably 0.00-0.1 . The carrier is preferably a non-hygroscopic carrier to substantially prevent water uptake th rough the capsule wall which would decrease bacterial stability.

OMPI S A IO The fluid carrier should also be one which secures a homogeneous distribution of the bacteria in the vagina and an optimal contact with the vaginal mucosa upon the release of the carrier from the partly- dissolved capsule but which, on the other hand, does not cause the carrier material to run from the vagina immediately after application . From a manufacturing point of view, though , it is normally advanta¬ geous that the carrier is relatively low in viscosity during processing conditions and during the filling of the capsules . Therefore, it is desirable that a carrier containing a bacterial concentrate has a certain thixotropicity, i . e. increase in viscosity, once it has been filled into the capsules so as to prevent sedimentation of the bacteria in the carrier and so as to prevent excessive movement of the carrier in the capsule in order to cause as little of the bacterial concentrate as possible to come into contact with the relatively wet capsule wall during the manufactu ring process . This relative immobility minimizes the amount of moisture which might possibly be taken up by the dry bacterial concentrate which might in tu rn cause a decrease of bacte¬ rial stability. Also, when such capsules are stored at normal storage temperatures, it is believed to be an advantage that the viscosity of the carrier within the capsule is sufficiently high to ensu re that there is little movement of the carrier during handling or transportation of the capsules, thereby avoiding any movement of the dried concentrate which might increase the exposu re of the concentrate to the inner capsule wall and thus increase the possibility that the cultu re might receive moistu re from the su rroundings . This emphasis on a certain viscosity of the fluid carrier distinguishes the capsule of the inven¬ tion from capsules intended for oral administration , which contain lactic acid bacteria in a fluid carrier (e. g . known under the trade name Lactoflora™, marketed by Camette ApS, Esbjerg, Denmark, as the fluid carrier in the known capsules does not have a sufficient viscosity to prevent sedimentation of the bacteria and possible uptake of moisture so that cell stability is likely to be impai red .

Consequently, the capsule of the invention should contain a fluid car¬ rier with a viscosity of at least 2000 cps at a temperatu re of 20°C, as this viscosity is sufficient to secu re a satisfactory cell stability, while not being too high to prevent a "melting down" in the vagina to ob- tain a sufficiently low viscosity at the vaginal temperature for the above-mentioned homogeneous distribution to take place. It has been found that when the carrier has a viscosity of at least 3000 cps, in particular at least 4000 cps at 20°C, it will still have a behaviou r with respect to consistency which is very well suited for effective distribu¬ tion of the bacterial culture under the conditions prevailing in the vagina . A capsule containing such a carrier with a concentrate of lactic acid bacteria may suitably be administered when the patient goes to bed. A few minutes after administration , the capsule will disintegrate to such an extent that it releases its carrier content which is then distributed in the vagina, causing an even distribution of the concentrate of viable lactic acid bacteria which adhere to the vaginal wall and, in the conditions prevailing in the vagina, such as temperature and moisture, will become biologically active and multiply. When the patient stands, any remainder of the carrier will tend to leave the vagina, but will not give rise to noteworthy discomfort.

In accordance with the present invention, the fluid carrier is a sub¬ stantially anhydrous paste preferably comprising a substantially non- hygroscopic oil which is either inherently of a suitable viscosity, e.g . comprises a mixture of oil and fat, such as cocoa butter, or which may include a particulate or dissolved or polymeric viscosity-increasing agent to obtain a suitable viscosity.

The viscosity-increasing agent may be selected from solid or semi- solid hydrocarbons capable of forming a homogeneous system with the oil, such as vaseline or polyethylene, particulate inorganic substances such as fumed silica, talc, zeolite or bentonite, and carbohydrates or carbohydrate derivatives, preferably high molecular weight carbo¬ hydrates such as starch and starch derivatives . It is, however, believed to be necessary that the viscosity-increasing agents employed should have as low a water activity as possible; preferably the a does not exceed 0.1 . This means that the viscosity-increasing agent should have a certain , but limited hygroscopicity . Thus, sugars such as dextrose or maltodextrine have been attempted as viscosity-in¬ creasing agents, but have proved unsuitable due to their poor ad- sorption thermes, i . e. the amount of water bound to the adsorbent per defined increase in water activity. According to the invention , it has been found that a combination of an oil and a carbohydrate derivative such as a starch , in particular corn starch , is especially advantageous for maintaining a high stabili¬ ty of the dried lactic acid bacteria concentrate dispersed therein .

Furthermore, an oil and corn starch mixtu re is the preferred carrier because it has been found that there is a synergism between a sub¬ stantially anhydrous oil and corn starch with respect to preserving the viability of lactic acid bacteria . Although the present invention is not to be limited to any theory, it is believed that the synergism is a combined effect of 1 ) the fact that the corn starch (which is prefe¬ rably in a freeze-dried or dehydrated form prior to its incorporation in the oil so as to have a water activity of almost 0: 00) has a balan¬ ced water activity which tends to attract water from the oil in the final system, thus competing with the dried bacterial cultu re (which is very hygroscopic in the freeze-dried state) which will also have a tendency to absorb any small amount of free water present in the oil, and 2) the starch such as corn sta rch in particular may have an inherent stabilizing effect on lactic acid bacteria . However, in order to avoid any substantial dessication of the capsules which may be caused by the incorporation of freeze-dried or dehydrated starch , the starch may advantageously be admixed with vaseline in an amount of about 10-50% by weight of the starch . Thus, a combination of a sub- stantially anhydrous oil and the starch such as corn starch or, especially, the starch/vaseline mixtu re, e. g . a weight ratio in the range from about 2: 5 to about 5: 2, preferably about 1 : 1 , has been found to be a most suitable carrier for efficiently intravaginal ly administering lactic acid bacteria .

As examples of useful oils may be mentioned mineral or vegetable oils such as paraffin or sunflower oil .

The lactic acid bacteria concentrate incorporated in the capsules according to the invention may be comprised of any type of bacteria which produce lactic acid, such as bacteria belonging to the genus Streptococcus or Lactobacillus . The species of Lactobacillus employed according to the invention are principally L . acidophilus, L . bulgari- cus, L . lactls, L . hefveticus, L . bifid us, L . casei , L. plantarum, L. delbrueckii , L . thermophilus or L . fermentum. Preferred among Streptococcus species are S. lactis, S. cremoris, S. diacetylactis, S. thermophilus or S. faecium. The lactic acid bacteria may also be incorporated in the form of a mixture of two or more of these species . Lactic acid bacteria of the species L . acidophilus have proved particu¬ larly advantageous . The strain of L . acidophilus which has proved to be particularly advantageous has been deposited in the Northern Regional Research Center, Peoria, USA under the accession number NRRL No. B-15260 and is publicly available.

The conditions treated by administering the capsule according to the invention are vulvo-vaginal infections caused by, i . a . , microorganisms such as Candida alb leans, Trichomonas vaginal is and Staphylococcus aureus as well as various anaerobic microorganisms . The capsule of the invention has been found to be particularly advantageous in the treatment of recurrent vulvo-vaginal infections which has hitherto been difficult to cu re with conventional preparations such as anti¬ biotics. As mentioned above, a particularly advantageous species of lactic acid bacteria is L . acidophilus, as described in Example 5. The superior qualities of L . acidophilus are most likely due to the fact that this species not only produces lactic acid (a decrease of pH is often not enough to control or reduce the growth of pathogenic micro¬ organisms) , but has also been found to produce one or more antimi¬ crobial metabolites described as acidophilin ^rvide e. g. Shahani, K. M. et al : "Natural Antibiotic Activity of Lactobacillus acidophilus and bulgaricus. 2. Isolation of Acidophilin from L, acidophilus" , Cult. Dairy Prod. J. 12, 1977, p. 8. ) , acidolin Cvide e. g . "Lactobacillus acidophilus I I . Antimicrobial agents. Cult. Dairy Prod. J. 10, 1975, p. 18) , and lactosidin (vide e. g . "Antibacterial activity associated with Lactobacillus acidophilus" , J. Bacteriol . 78, 1959, p . 477) . These antimicrobial agents have been shown to possess an inhibitory effect on a variety of microorganisms .

Less severe cases of vulvo-vaginal infections may suitably be treated by administering 1 -2 capsules a day for 3-6 days, or a similar dosage may be administered prophylactically for a few days after each men-

OMPI struation . A suitable dosage in more severe cases may be 1 capsule a day until the first menstrual period, and for a 7-day period after each menstruation . The capsule may be inserted with the fingers or by means of a suitable applicator.

The capsules may be produced by homogeneously dispersing a dried, viable, stable culture of lactic acid bacteria in a substantially an¬ hydrous fluid carrier, filling the resulting dispersion into soft gelatin capsules and drying the capsules after sealing.

More specifically, in order to make the mixing of the ingredients re- latively easy, especially if the paste is to include a viscosity-in¬ creasing agent, it is preferred to add the culture to the paste ingre¬ dient which has the lowest viscosity followed by adding the viscosi¬ ty-increasing agent in the form of other dry matter or optionally a more highly viscous ingredient. This procedu re saves time which is vital with respect to the amount of moisture taken up by the cultu re from the air. In order to avoid moisture uptake, an inert and dry gas may be exposed to the mixing su rface.

The admixture of the cultu re and optionally other dry matter and the oil may, for instance, be performed by means of a slowly operating mixer. I n this way, the uptake of air in the mixtu re is minimized, thus minimizing the risk of uptake of moistu re and oxygen which are detrimental to bacterial stability . I n order to fu rther reduce the moisture content of the final product, the ingredients are incorpo¬ rated in the oil in a dry state. Thus, the freeze-dried bacterial concentrate has a water activity not exceeding 0.2 and preferably a far lower water activity, and when the viscosity-increasing agent employed is a starch such as corn starch , it is preferably subjected to freeze-drying prior to use, substantially to a water activity of 0.00.

However, a finely dispersed and homogeneous product is not obtained by merely mixing the ingredients as described above, and is provided e. g . by dispersing the cultu re by means of a roller mill operating at the pressu re and friction by which a fi ne division of the bacterial

OMPI culture is secured without, on the other hand, heating the paste or killing the bacteria . The space between the rollers will normally be between 150 and 300 μm. The resulting particle size will be about 100 urn. After this grinding process, the paste is gently stirred in order to impart homogeneity to the paste which is important to obtain in order to make it possible to dose accurate amounts of bacteria into each capsule.

During the mixing and grinding processes, admixture of a certain amount of air in the paste is unavoidable. As stated above, this may have a detrimental effect on bacterial stability as well as influence the accuracy with which the capsules are filled. Production of the paste should therefore also include removal of air from the paste which is performed in a vacuum chamber at a pressu re of 12 mm Hg in which the paste is atomized by means of a fast- rotating, horizontal plate. During the entire process, the paste is subjected to a total tempera¬ ture increase of 3-5°C relative to room temperature.

The paste is then incorporated in gelatin capsules, preferably soft gelatin capsules, by a process described in US Patents Nos . 1 ,970,396, 2, 152, 101 , 2,234,479 and 2,288,327. After shaping, filling and sealing, the capsules may be washed with an agent which prevents intercapsular adhesion, such as perchloroethylene, to which a lubricant such as lecithin has optionally been added whereby intercapsular adhesion and deformation of the capsules during the subsequent drying process are avoided . The drying itself may be performed in a two-stage process . Firstly, the still wet capsules may be subjected to a strong air-flow, normally with ordinary atmospheric air with a relative humidity of about 20-60%. When the capsules have become sufficiently hard, the drying is continued on trays with ventilation for up to several days until the desired hardness and loss of humidity have been obtained . For improved storage, the final product is preferably stored at a temperature below 20°C, such as at a refrigeration temperature of about 3-5°C.

The invention is further illustrated by the following examples . EXAMPLE 1

Preparation of the Bacterial Culture

A dry, stable, viable concentrate of L. acidophilus was prepared according to the procedures described in US Patents Nos. 4,115,199 and 3,897,307. The strain of L. acidophilus used is deposited with the Northern Regional Research Center and is publicly available under the accession number NRRL No. B-15260. The bacterial concentrate was dried by freeze-drying after adjusting the pH to 6.0-6.2, and the addition of 16 g L-ascorbic acid, 10 g inositol and 10 g monoso- dium glutamate per 100 g of the dry concentrate. The resulting stabilized, dry concentrate contains about 1.00 x 10¹¹ CFU (colony- forming units) per gramme.

EXAMPLE 2

Production of Paste

The compositions stated below were prepared according to the method described above with the exception of composition C.

The amount of paste material varies according to the desired number of cells in the finished capsules and thus the amount of bacterial con¬ centrate incorporated, in order to obtain the desired viscosity.

Composition A:

Paraffin oil of low viscosity¹ 5.5 kg Freeze-dried corn starch CPC 3401 4.5 kg Freeze-dried L. acidophilus culture (1.05 x 10^1X CFU/g) 1.0 kg

^l as described in Deutsches Arzneibuch, vol. 7/8

VflPO This composition is presently preferred .

Composition B :

Paraffin oil of low viscosity¹ 7.0 kg yellow vaseline² 6.0 kg L . acidophilus culture

(1 .05 x 10^{1 1} CFU/g) 1 .2 kg

¹ as described in Deutsches Arzneibuch , vol . 7/8

² as described in Deutsches Arzneibuch, vol . 7

Composition C:

Plastibase¹ 9.4 kg

"Alkatheπe" 23 powder² 2.5% Liquid paraffin³ 97.5% L . acidophilus culture (1 .05 x 10¹¹ CFU/g) 1 .0 kg

^x prepared according to Farmaceutisk Tidende, No. 10, vol . 67, 1957, pp. 113-15 ² a polyethylene powder (023.030) marketed by 1 CI

EXAMPLE 3

Comparison and Stability Tests

As described above, it is a requirement that the carrier for the bacterial cultu re has as low an uptake of water as possible, and pre¬ ferably no uptake of water at all .

In order to determine the protective properties of various carrier materials, especially with respect to uptake of water from the sur- roundings, hygroscopicity analyses were made of the following mate¬ rials and mixtures of materials as shown in Table 1 . The materials were spread on petri dishes with a diameter of 9 cm, and a thickness of the material of 7-8 mm, the materials having a substantially smooth surface.

The materials analyzed were:

Paraffin oil Paraffinum liquidum tenui as described in

Deutsches Arzneibuch, vol. 8, purchased from

Mecobenzon.

Sunflower oil Commercial edible oil purchased from Irma. Corn starch Globe® 03401 purchased from CPC, freeze-dried to an A of 0.00. w

Culture Freeze-dried concentrate of L. acidophilus NRRL No. B-15260 (1 10¹¹ CFU/g), produced by Christian Hansen's Laboratories.

"Plastibase" As described in Farmaceutisk Tidende No. 10, vol. 67, 1957, pp. 113-15, 2.5% polyethylene in liquid paraffin.

Vaseline Vaselinum Ph. Nord. 63 purchased from Meco¬ benzon.

Table 1

Analysis of Hygroscopicity (% weight increase)

Composition 2 weeks 3 weeks (% by weight)

Paraffin oil 0.1 -

Paraffin oil * culture (10%) 6.9 17.0²

Paraffin oil ⁺ corn starch (1:1) ⁺ culture (10%) 6.8 10.0

Sunflower oil 0.2 0.3

Sunflower oil ⁺ culture (10%) 6.3 9.7 Sunflower oil ⁺ corn starch (2:1) ⁺ culture (10%) 7.6 10.4 Sunflower oil ⁺ corn starch (1 : 1 ) ⁺ culture (10%) 14.9 18.4

Vaseline 0.0 0.0

"Plastibase" (2 1/2%) ⁺ culture (10%) 2.9 5.4

"Plastibase" (2 1/2%) 0.01 0.08 - Corn starch 20.4 -

Culture (100%) 115.6 149.7

¹ defined and analysed as water uptake: % weight increase during 2-3 weeks by standing at 100% RH and 20°C (± 2°C) . ² complete sedimentation of the culture from the oil.

It appears from the results that the water uptake of the oils, the vaseline and polyethylene is minimal , whereas the culture itself is extremely hygroscopic. When the cultu re is mixed with one or more of the carriers, the suitability of the selected carriers with respect to protection against water uptake may be seen . Addition of a visco¬ sity-increasing agent such as corn starch which in comparison to the culture is only slightly hygroscopic does not appear to change the water uptake properties of the total mixture to any significant extent. However, the water uptake in the sunflower oil mixture is directly proportional to the amount of corn starch added . A mixture such as "Plastibase" is demonstrated to afford the best protection of the culture against water uptake.

With respect to protection against sedimentation , i . e. the viscosity of the paste, the following paste compositions were made:

1) Vaseline 100 g 5) Sunflower oil 100 g

Paraffin oil 100 g Cultu re 10 g

Cultu re 20 g

2) Paraffin oil 100 g 6) Sunflower oil 100 g

Culture 10 g Corn starch 50 g Culture 10 g 1 w

3) "Plastibase" 2 1/2% 200 g 7) Sunflower oil 50 g

Culture 10 g Corn starch 50 g Culture 10 g

4) Paraffin oil 50 g 8) ^x Corn starch 100 g

Corn starch 50 g Cultu re 10 g

Cultu re 10 g

¹ not prepared in a roller mill - the ingredients were mixed in a plastic bag

An unsatisfactory sedimentation of the culture on standing, i . e. too low a viscosity of the mixtu re, was only observed in mixtu res 2) and 5) .

Stability tests

I . The stability of the bacterial cultures when incorporated in various types of pastes or other carriers as described above was tested by means of an accelerated storage test (AST) in which 10 samples of each mixture of cultu re and carrier were subjected to increasing temperatures (from 30-75°C) in a water bath for 3 days, one sample of each mixture being removed from the water bath every 8th hour and analysed . The results are shown in Figs . 1 -3.

I n Figs . 1 -3, the "white" star indicates the L . acidophilus culture alone, the square with the filled-in ci rcle indicates oil and viscosity- increasing agent (paraffin oil and corn starch in Fig . 1 , sunflower oil and corn starch in Fig . 2, paraffin oil and vaseline in Fig . 3) , the square indicates the viscosity-increasing agent alone (corn starch in Figs . 1 and 2, vaseline in Fig . 3) , the filled-in ci rcle indicates the oil (paraffin oil in Figs . 1 and 3) , and the filled-in star indicates "Plastibase" (as defined above) ; "log CFU" indicates the logarithm of colony-forming units per gramme of carrier.

It should be noted that the cou rses of the cu rves cannot be directly interpreted as stability measurements (percentage of loss of viability)

ΠΌ in a conventional storage test. However, the curves may be compared among themselves, especially with respect to the slope of the first, straight line and "breakpoint" (45°) , i. e. the temperature at which the curve changes its course (usually at 59-60°C for cultures with a satisfactory stability; indicated by the dotted line in Figs . 1 -3) .

It appears from the Figures that the best results in terms of a slope approaching zero and a breakpoint at a temperature of about 59°C is obtained with the mixtu res of oils (paraffin and sunflower oil) and corn starch even though each of the component parts of the mixtures shows a steeper course of the curves with breakpoints before or at 59°C. It further appears that the course of the curves is improved in comparison with the culture itself. Thus, In these mixtures, an im¬ proved protection has been obtained for the cultures, whereby the bacteria obtained a very high stability.

I I . Capsules containing freeze-dried L . acidophilus bacteria dispersed in a mixture of sunflower oil and corn starch were also subjected to a conventional storage stability test in which the capsules were stored at 5°C and 20°C, respectively, and tested for their content of colo¬ ny-forming units (CFU) after 4, 8, 16 and 32 weeks at the respective temperatures . The initial count of CFU's was 150 x 10V2 ^' capsules . The results are shown in Table 2.

Table 2

4 weeks 8 weeks 16 weeks 32 weeks %Survival %Survival %Su rvival %Survival

5°C 91 92 75 43

20°C 87 67 49 25

It appears from the table that the capsules have a half life of about 4 months at 20°C at which time the capsules still contain an effective amount of bacteria . Due to the improved storability at 5°C, it is, however, preferred that the capsules be stored at about 5°C or less . EXAMPLE 4

Encapsulation and Drying

Soft gelatin capsules were prepared according to US Patents Nos . 1 ,970,396, 2, 152, 101 , 2,234,479 and 2,288,327. The capsule material may vary in composition within certain limits . The composition pre¬ ferred for the present purpose is shown in Table 3.

Table 3

Composition of Soft Gelatin Capsules

Gelatin ¹ 189.040 - 221 .916 mg

Glycerol² 62.411 - 73.265 mg

Anid isorb 85/70 48. 609 - 57.063 mg Dyes³

E 171/77891 3.288 - 3.860 mg E 172/77492 0.252 - 0.296 mg Total weight of capsule mate¬ rial per capsule 330 mg ± 8%

¹ according to USP ² 85% Ph . Eur. ³ white, opaque, EWG Nos . and Col . I nd . 1956 Nos . listed

After shaping the capsules and filling them with the dispersion of bacterial cultu re incorporated in the paste and sealing, the capsules were washed in perchloroethylene to which had been added a limited amount of lecithin in order to avoid intercapsular adhesion and defor¬ mation of the capsules during drying .

The capsules were dried for 2 hours in a rotary drier with 6-8 com¬ partments by a strong ai r-flow with ordinary atmospheric air with a relative humidity of about 20%. I n this manner, the capsules were dried to an extent corresponding to a loss of weight of about 40-45%. The capsules had then become sufficiently hard for continued drying

OMPI sy wipo to be performed on trays in a closed, ventilated cupboard at about 20°C (relative humidity 20%) for up to 4-6 days, until a suitable hardness and dessication had been obtained.

EXAMPLE 5

Inhibition of Candida albicans in vitro

To demonstrate the inhibitory effect of L . acidophilus on the growth of the yeast Candida albicans which is one of the most common causes of vulvo-vaginal infections in women, the following in vitro experiment was made.

A liquid growth medium. (MRS-Oxoid CM 359 - 5.2%) was inoculated with the following lactic acid bacteria :

Lactobacillus acidophilus NRRL No. B -15260

Lactobacillus bulgaricus¹ CH-1

Streptococcus thermophilus¹ CH-1

¹ Available from Chr. Hansen's Laboratories .

Each of the strains was inoculated in growth flasks with 100 ml MRS- broth to a cell count of 1 -5 x 10^s CFU/ml . To some of these flasks as well as to some flasks which had not previously been inoculated were simultaneously added one of two cultu res of Candida albicans which were freshly grown from women with yeast infections (vulvo-vaginal infections) . Furthermore, MRS-broth was inoculated with L . acidophi¬ lus in an amount of 5 x 10^s CFU/ml and incubated at 37°C for 24 hours after which the living cells were centrifugated off and the supernatant were finally filtered under sterile conditions . The final pH was 4.18. This broth was inoculated with one of the Candida cultures . All flasks were incubated at 37°C, and the broths were plated for the specific microorganism after 0 hours, 24 hours and 48 hours .

" JC OMPI The results are shown in Figs. 4-7.

In Fig. 4 a and b, the dotted lines represent the growth of L. acido¬ philus (expressed as cell count), the filled-in star indicating the growth of L. acidophilus when grown alone, and the square indicating the growth of L. acidophilus when grown together with C. albicans (two different strains in 4 a and 4 b). It appears that the growth of L. acidophilus. is not inhibited by the presence of Candida. The full lines represent the growth of C. albicans, the "white" star indicating the growth of Candida when grown alone, and the circle indicating the growth of Candida when grown together with L. acidophilus. It appears that both Candida cultures are significantly inhibited (ap- prox. 4 log,,, units) when inoculated together with the L. acidophilus strain used.

Figs. 5 and 6 are analogous with Fig. 4 with the exception that Fig. 5 shows the use of L. bulgaricus and Fig. 6 the use of S. thermo¬ philus instead of L. acidophilus. From the graphs, it appears that neither L. bulgaricus nor S. thermophilus (the dotted lines) are inhibited by the growth of C. albicans. It appears from Fig. 5 that there is a slight inhibition of C. albicans when grown together with L. bulgaricus. From Fig. 6 it appears that S. thermophilus does not exert any inhibitory effect against C. albicans.

The above tests indicate that the chosen strain of L. acidophilus shows the most efficient inhibition on C. albicans compared with L. bulgaricus whereas no effect was shown from S. thermophilus .

Fig. 7 shows that, when a broth is acidified (to a pH of 4.18 by means of the selected strain of L. acidophilus) and physically sterili¬ zed as described above, the C. albicans culture (indicated by the "white" circle) cannot tolerate the concentration of the antimicrobial metabolites, i.e. the cell counts drop during the first 24 hours peri- od. In comparison, the growth of C. albicans in normal broth is indicated by the filled-in circle. A chemical acidification of MRS-broth with lactic and phosphoric acid to a pH 4-4.2 has no influence, or only very little influence, on the growth of C. albicans. Thus, it is demonstrated that it is the pro¬ duction of antimicrobial agent by certain Tactic acid bacteria, prin- cipally L . acidophilus, which is responsible for the inhibition in the growth of yeast cells.

EXAMPLE 6

Inhibition of β- streptococci (Streptococcus G roup B) In vitro

I n the field of human pathology, ^-Streptococci are recognized as important infectious agents especially in the u rogenital area, and a significant correlation has been demonstrated between the presence of ^-streptococci and the diagnosis of vaginitis (cf. Ugeskrift for Laager 747/15, 1979, pp. 992-994; Scand . J. Infect. Dis. 11 , 1979, pp. 199- 202; Scand. J. Infect. Dis. 12, 1980, pp. 101 -104) . To demonstrate the inhibitory effect of L . acidophilus on the growth of these potential pathogens in vitro experiments were performed essentially as described in Example 5 using laboratory reference strains from Statens Veteri- πeere Seruminstitut, Ringsted, Denmark (Types I A, I B, I I and I I I ) . It appeared from these tests that the growth of ξ>- streptococci is inhibited when the streptococci are grown together with L . acidophilus (partly a question of substrate competition) . The growth is also largely inhibited in a substrate with a pH below about 5.00 (the op¬ timal pH of (5- streptococci is about 7.4) . It was also demonstrated that the growth is inhibited more than 2 logs in a neutralized MRS-broth in which L . acidophilus had been grown , compared to an MRS-sub- strate to which the equivalent amount of sodium lactate had been added.

Thus, it was demonstrated that L . acidophilus production of antimi¬ crobial agents combined with the decrease in pH is responsible for the inhibition of the growth of (3- streptococci . 21

EXAMPLE 7

Inhibition of vulvo-vaginal infections in vivo

Preliminary clinical studies of the inhibitory effect of the capsules of the invention on vulvo-vaginal infections were carried out at the dermatological clinics of several hospitals.

I. The study was performed at the dermatological clinic of Odense Sygehus, Denmark, involving 8 women of ages between 18 and 30 years (23 years on average) suffering from vulvo-vaginal infections, who had been referred to the clinic for treatment. The test comprised administering one capsule twice daily for 7 days so that four of the patients received the capsule of the invention and four of the patients received a placebo capsule used as control. After 1 week, a subjec¬ tive as well as objective evaluation of the condition of the patients was carried out, comparing it to their condition before treatment. The criteria of evaluation were: recovery (scored as 1), improvement (2), no change (3), and deterioration (4).

By a t-statistical analysis of the means of two samples, the following results were obtained.

Table 4

Evaluation Mean Standard deviation t-test prob. t>

Capsule Placebo Capsule Placebo of the of the invention invention

Subjective 1.75 • 3 0.5 0.0 0.0012

Objective 2.25 3 0.5 0.816 0.084

OMPI The subjective evaluation of the effect of the capsule of the invention compared to the placebo capsule showed a statistical significance of 99.88%, whereas the objective evaluation is almost significant at a level of 91 .6%.

I I . The study was carried out at the dermatological clinic of Rigs- hospitalet, Copenhagen, Denmark, involving 10 women of ages be¬ tween 21 and 43 years (average age: 30 years) who had suffered from chronic or recurrent vaginitis for 1/2-8 years (3 years on average) . The symptoms were constant in 2 patients, recurred once a month in 6 patients and ocurred 4-10 times a year in 2 patients .

The capsule according to the invention was administered once a day for 2-6 months (4 months on average) . Du ring the period of treat¬ ment, C. albicans infections recurred in 6 of the patients, and the presence of Cardnerelfa vaginalis was demonstrated in 3 cases . 2 Patients received antibiotics and 3 patients received antimycotics for brief periods during the period of treatment with the capsule of the invention .

After the test period one patient reported recovery and 3 patients reported their condition improved (abating symptoms at greater inter- vals) . In 4 patients no change had occurred after 2 months' treat¬ ment, and 2 patients reported a deterioration of their condition (in¬ creased irritation of the vaginal mucosa) after 3 months' treatment.

I n the summary of the test, it was concluded that the capsule of the invention is also useful in the treatment of women suffering from severe, ch ronic or recurrent vaginitis .

11 1 . I n a subjective study of the inhibition of vulvo-vaginal infecti¬ ons, a group of 27 women of ages between 17 and 38 years who suffered from recurrent vulvo-vaginal infections which had repeatedly been treated with the usual vaginal compositions, were treated by administering one capsule of the invention a day for 6 subsequent days . Results (subjective evaluation) :

5 patients reported that the capsules had no effect, 12 patients reported that the capsules had had a satisfactory effect, 8 patients reported that the capsules had had an excellent effect, and 2 patients did not report.

Claims

1 . A soluble capsule for controlling vulvo-vaginal infections which contains a stable, dried, viable concentrate of lactic acid bacteria dispersed in a pharmaceutically acceptable fluid carrier which has a viscosity of at least 2000 cps .

2. A capsule according to claim 1 in which the fluid carrier has a viscosity of at least 3000 cps, in particular at least 4000 cps .

3. A capsule according to claim 1 which is a gelatin capsule, in parti¬ cular a soft gelatin capsule.

4. A capsule according to any of claims 1 -3 in which the count of viable lactic acid bacteria in the capsule is at least 1x10^s, preferably at least l xlO⁷.

5. A capsule according to claim 4 in which the count of lactic acid bacteria in the capsule is at least 1 χl0⁸ , preferably in the range be- tween about 1 χl0⁹ and l xlO^{1 1}, such as about 1 χl0^{1 0} .

6. A capsule according to any of claims 1 -5 in which the concentrate is a freeze-dried concentrate.

7. A capsule according to claim 6 in which the freeze-dried concentra¬ te has a water activity in the range of 0.00-0.2, preferably 0.00-0.1 .

8. A capsule according to any of claims 1 -7 in which the fluid carrier is a substantially anhydrous paste.

9. A capsule according to claim 8 in which the paste has a water activity in the range of 0.00-0.2, preferably 0.00-0. 1 .

10. A capsule according to any of claims 7-9 in which the paste comprises a substantially non-hygroscopic oil .

11 . A capsule according to claim 10 in which the oil contains a visco¬ sity-increasing agent.

12. A capsule according to claim 11 in which the viscosity-increasing agent is a substance with a water activity of not more than 0.2, pre- ferably not more than 0. 1 .

13. A capsule according to claim 11 or 12 in which the viscosity-in¬ creasing ingredient is selected from solid or semi-solid hydrocarbons capable of forming a homogeneous system with the oil , such as vase¬ line or polyethylene, particulate inorganic substances such as fumed silica, talc, zeolite or bentonite, or carbohydrates or carbohydrate derivatives, preferably high molecular weight carbohydrates such as starch and starch derivatives , or a mixtu re thereof .

14. A capsule according to claim 13 in which the viscosity-increasing agent is a starch .

15. A capsule according to claim 14 in which the starch is corn starch .

16. A capsule according to claim 14 or 15 in which the starch is admixed with vaseline.

17. A capsule according to claim 16 in which the amount of vaseline is about 10-50% by weight of the starch .

18. A capsule according to any of claims 10-17 in which the oil is a mineral or vegetable oil such as paraffin or sunflower oil .

19. A capsule according to any of claims 15-18 in which the weight ratio between the oil and the starch or starch/vaseline mixtu re is in the range from about 2: 5 to- about 5: 2, preferably about 1 : 1 .

20. A capsule according to any of claims 1 -19 in which the lactic acid bacteria are of the genus Streptococcus or Lactobacillus.

OMPI

21 . A capsule according to claim 20 wherein the species of Lacto¬ bacillus are L . acidophilus, L . bulgaricus, L . lactis, L . helveticus, L . bifidus, L . casei, L . plantarum, L . delbrueckii, L . thermophilus, L . fermentum or a mixture thereof.

22. A capsule according to claim 21 wherein the strain of L . acido¬ philus is L . acidophilus NRRL No. B -15260.

23. A capsule according to claim 20 wherein the species of Strep¬ tococcus are S. lactis, S. cremorls, S. diacety lactis, S. thermophilus, S, faeciu or a mixtu re thereof.

24. A method of producing a soluble capsule containing a stable dried viable concentrate of lactic acid bacteria, comprising homogeneously dispersing a dried, viable, stable culture of lactic acid bacteria in a substantially anhydrous fluid carrier, filling the resulting dispersion into soft gelatin capsules and drying the capsules after sealing .

25. A method according to claim 24 in which the fluid carrier has a viscosity of at feast 2000 cps, preferably at least 3000 cps, in parti¬ cular at least 4000 cps, at 20°C.

26. A method according to claim 24 or 25 in which the fluid carrier comprises a pharmaceutϊally acceptable, substantially anhydrous, non-hygroscopic oil .

27. A method according to claim 26 in which the fluid carrier addi¬ tionally comprises a viscosity-increasing agent.

28. A method according to claim 27 in which the viscosity-increasing agent is a substance with a water activity of not more than 0.2, pre- ferably not more than 0.1 .

29. A method according to claim 28 in which the viscosity-increasing agent is selected from solid or semi-solid hydrocarbons capable of forming a homogeneous system with the oil, such as vaseline or poly¬ ethylene, particulate inorganic substances such as fumed silica, talc, zeolite or bentonite or carbohydrates or carbohydrate derivatives, preferably high molecular weight carbohydrates such as starch and starch derivatives .

^' 30. A method according to claim 29 in which the viscosity-increasing agent is a starch .

31 . A method according to claim 30 in which the starch is corn starch .

32. A method according to claim 30 or 31 in which the starch is admixed with vaseline.

33. A method according to claim 32 in which the amount of vaseline is about 10-50% by weight of the starch .

34. A method according to any of claims 26-33 in which the oil is a mineral or vegetable oil such as paraffin or sunflower oil .

35. A method as according to any of claims 30-34 in which the weight ratio between the oil and the starch or starch/vaseline mixture is in the range from about 2: 5 to about 5: 2, preferably about 1 : 1 .

36. A method according_, to any of claims 24-35 in wh ich the bacterial concentrate is dispersed in the fluid ca rrier in an amount which is sufficient to secu re a cell count in the final capsules of at least 1 x 10^s , after which the viscosity-increasi ng agent is added .

37. A method according to any of claims 24-36 in which the concen¬ trate is a freeze-dried concentrate.

38. A method according to claim 32 in which the freeze-dried concen¬ trate has a water activity in the range of 0.00-0.2, preferably 0.00-0. 1 .

39. A method of controlling vulvo-vaginal infections which comprises intravaginally administering a capsule containing a stable, dried, viable concentrate of lactic acid bacteria dispersed in a pharmaceuti¬ cally. acceptable fluid carrier.

40. A method according to claim 39 in which the count of viable lactic acid bacteria in the capsule is at least 1 x10^s , preferably at least 1^χ10⁷.