Micro-encapsulated lactobacilli for medical applications

Micro-encapsulated lactobacilli bacteria are orally administered to mammals, including humans, to treat or prevent antibiotic associated or other chronic or acute diarrhea. Micro-encapsulated lactobacilli bacteria are topically administered to the skin to treat or prevent recurrent skin infections, and are administered intra-vaginally to treat or prevent vaginal yeast infections.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is directed to a pharmaceutical composition in the 
form of capsules, tablets, creams, foams, ointments, powders, 
suppositories or the like containing micro-encapsulated lactobacilli for 
oral, topical and intra-vaginal administration for the treatment or 
prevention of antibiotic associated diarrhea, skin and vaginal infections. 
2. Brief Description of the Prior Art 
Antibiotic associated diarrhea is a common side effect of broad spectrum 
anti-bacterial therapy, and is believed to be due to a shift in intestinal 
flora as a result of therapy with antibiotics, and particularly due to 
loss of lactobacilli in the intestinal flora. This condition can become 
life-threatening to the patient and is very difficult to treat when it is 
caused by a Clostridium difficile bacterical infection. 
It has been attempted in the prior art to treat antibiotic associated 
diarrhea with oral administration of lactobacilli. This type of therapy, 
however, has been proven to be of only limited success, for two primary 
reasons. First, orally administered lactobacilli, in accordance with the 
prior art, are exposed to the destructive action of gastric acid. 
Therefore only an inadequately small number of viable lactobacilli tend to 
reach the lower intestinal tract where they would be needed to reestablish 
a healthy lactobacilli flora. Second, transient lactose intolerance is 
frequently found in persons suffering from antibiotic associated diarrhea. 
(The temporary lactose intolerance is attributed by those skilled in the 
art to the loss of the brush border of the intestinal villa, caused by the 
antibiotic.) Moreover, this type of transient lactose intolerance 
frequently occurs even in persons who, otherwise under normal conditions, 
have no prior history of dairy product or lactose intolerance. 
Lactobacilli preparations in accordance with the prior art usually contain 
traces or remnants of the media in which the lactobacilli bacteria were 
grown and therefore contain lactose, lactulose and other disacharides. 
These in turn, upon reaching the lower intestines of persons suffering 
from antibiotic associated diarrhea, tend to make worse the transient 
lactose intolerance condition. In light of the foregoing, there is room 
for improvement in the prior art. More particularly, there is room in the 
prior art for improvement of pharmaceutical compositions and methods for 
the treatment of antibiotic associated diarrhea, and also for treatment or 
prevention of certain skin and vaginal infections. The present inventions 
provides such improvements. 
SUMMARY OF THE INVENTION 
In accordance with one aspect of the present invention more than 
approximately 10.sup.3 viable micro-encapsulated lactobacilli in each unit 
dose, are orally administered in a pharmaceutical composition or delivery 
vehicle, to persons suffering from antibiotic associated diarrhea. The 
lactobacilli are administered for the treatment or prevention of the 
diarrhea and this therapy is preferably continued during and shortly after 
treatment with the antibiotic. Administration of the micro-encapsulated 
lactobacilli may also be coupled with administration of metronidazole or 
other antibiotic which kill Clostridium difficile antibiotics. The 
micro-encapsulated lactobacilli survive in sufficient numbers the exposure 
to gastric acid while traveling through the stomach and reach the lower 
intestines where they help establish or reestablish a healthy bacterial 
flora, and eliminate or alleviate the symptoms of antibiotic associated 
diarrhea. 
In another aspect of the present invention a pharmaceutical composition 
containing micro-encapsulated lactobacilli is applied topically to the 
skin to prevent or treat skin infections, or a vaginal cream, foam, 
ointment or suppository is used to prevent or treat vaginal infections. 
A large variety of delivery vehicles, such as gelatin capsules, tablets or 
liquid dosage forms may be used for oral administration of the 
micro-encapsulated lactobacilli. Similarly, a large variety of delivery 
vehicles, such as creams, foams, ointments, suppositories, or admixtures 
with inert powders such as starch or talcum powder may be used for topical 
and/or vaginal administration, as applicable, of the micro-encapsulated 
lactobacilli. 
The following is a detailed description of the invention, and a summary of 
certain tests and results which demonstrate the effectiveness of the 
invention.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention Lactobacillus acidophilus, 
Lactobacillus rhamnosus or other lactobacillus species, such as L. 
bulgaricus, L. thermophilus, etc. bacteria are micro-encapsulated and used 
for oral, topical or vaginal application, for the prevention or treatment 
of diseases and conditions described below. The various species of 
lactobacilli are hereinafter collectively referred to under that name, 
unless a specific species is described which is then referred to by its 
more definite name. It was found in accordance with the present invention 
that micro-encapsulation of the bacteria is of great advantage in terms of 
therapeutic utility when compared to treatment or attempted treatment of 
the same conditions with lactobacilli which are not micro-encapsulated. 
First, the micro-encapsulation significantly increases the shelf-life of 
the bacteria. Even more importantly, however, after application to the 
human or other mammalian body, orally, topically or intra-vaginally, the 
encapsulating film slowly disintegrates and gradually releases the 
lactobacilli bacteria into its environment, namely into the lower 
intestines, on the skin, or in the vagina, as applicable. When the 
micro-encapsulated lactobacilli are used for oral administration, the 
micro-encapsulation provides protection to the lactobacilli bacteria 
against gastric juices, and allows viable lactobacilli to reach the lower 
intestines where they are able to have beneficial therapeutic effect. 
Whereas several forms of delivery may be used to deliver the 
micro-encapsulated lactobacilli bacteria to its intended place of action 
(orally to the lower intestines, topically to the skin, or 
intra-vaginally) it is generally speaking necessary to have at least 
approximately 10.sup.3 viable lactobacilli bacteria in each unit dosage 
form, whether it be a gelatin capsule, cream, ointments, powder applied to 
a skin area or vaginal suppository. This means that generally speaking 
there is at least 10.sup.3 viable micro-encapsulated lactobacilli in each 
gram or milliliter of the pharmaceutical composition of the invention. 
More preferably, the concentration of the lactobacilli bacteria in the 
delivery vehicle or pharmaceutical composition is in the range of 
approximately 10.sup.3 to 10.sup.12 viable bacteria per gram, or per 
milliliter of the pharmaceutical composition, with the range of 10.sup.5 
to 10.sup.12 being even more preferred. In this regard it is noted that 
10.sup.12 bacteria per gram or per milliliter essentially represents the 
highest number of lactobacilli bacteria which can be given in a gram or 
milliliter of material. The presently most preferred concentration of 
lactobacilli for topical or oral application is approximately 10.sup.6 to 
10.sup.7 viable micro-encapsulated bacteria per milliliter or gram of 
composition. 
In the preferred embodiments of the invention, the bacteria are of the 
Lactobacillus rhamnosus species. Although some bacteriologists may 
consider Lactobacillus rhamnosus as a separate but related species to 
Lactobacillus acidophilus, rhamnosus is more properly considered a simple 
variant of the acidophilus species. As is known in the art, both 
Lactobacillus acidophilus and Lactobacillus rhamnosus and other 
lactobacilli species are "friendly" bacteria, and form a healthy 
intra-vaginal bacterial flora and are also part of the healthy lower 
intestinal flora. Both of these bacteria are known to produce certain 
bactericidins and hydrogen peroxide, which helps to suppress pathogenic 
bacteria. The advantage of Lactobacillus rhamnosus over the acidophilus 
variant in the present invention is in the facts that the rhamnosus 
variant is more prolific (about 8 to 10 times), and is capable of 
fermenting more carbohydrates (23 compared to 12 of the acidophilus 
variant) and that the rhamnosus variant produces L.sup.+ lactic acid 
instead of a racemic mixture of lactic acid produced by the acidophilus 
variant. The foregoing are advantageous because the rhamnosus variant's 
ability to ferment more types of carbohydrates makes it a more sturdy, 
survival-prone bacteria. Production of L.sup.+ lactic acid is 
advantageous because it is the L.sup.+ enantiomer which has substantial 
antifungal action. 
Both the Lactobacillus acidophilus and Lactobacillus rhamnosus variant 
bacteria used in the preferred embodiments of the present invention can be 
purchased from commercial sources, or can be obtained from laboratory 
strains. The Lactobacillus rhamnosus variant used in the below described 
preferred embodiments is obtained from the Institute Rosell Montreal, 
Quebec Canada. The Lactobacillus variant is micro encapsulated in 
accordance with the present invention, and is formulated into a 
pharmaceutical composition suitable for oral, topical or vaginal 
application. Several methods or procedures for micro encapsulating these 
bacteria are described below. The purpose of micro encapsulating the 
bacteria is to increase their shelf-life, assure that sufficient number of 
viable lactobacilli bacteria survive to reach the lower intestines in case 
of oral administration, and to provide a slow and gradual release in case 
of topical and intra-vaginal administration. The material or coating which 
encapsulates the bacteria is selected in such a manner in accordance with 
the present invention that the material loses its structural integrity as 
a film in the intestinal or vaginal environment, or upon application to 
the skin (primarily due to moisture) and releases the lactobacilli 
bacteria. 
Several methods or procedures for micro-encapsulating the lactobacilli 
bacteria are described below. 
ENCAPSULATION METHODS 
Viable, lyophilized lactobacilli bacteria that have been lyophilized after 
the removal of the media are used for encapsulation. The bacteria can be 
obtained from commercial sources, or can be obtained from laboratory 
strains. In the currently preferred embodiments Lactobacillus rhamnosus 
bacteria are purchased from Institute Rosell Montreal, Quebec, Canada. The 
organisms are grown to log phase in nutrient media. Suitable media include 
Thayer-Martin media, Trypticase Soy, Brain-Heart Infusion Broth, or any 
other enriched media suitable for the cultivation of these organisms, as 
no particular media is critical. The only important factors are the 
viability and quantity of the micro-organisms that are always determined 
by standard clinical laboratory dilution methods, such as plating the 
quantified dilution of bacteria on to blood agar plates or other enriched 
media, incubating at 37 degrees C. for 24-48 hours in a 5-10% carbon 
dioxide atmosphere, and then performing a colony count. The removal of the 
nutrient media is done by centrifugation at 14,000.times.g at 
0.degree.-4.degree. C., and then washing with sterile, balanced salts and 
5% glucose solution at least three times after the initial centrifugation. 
The bacteria are then "snap frozen" with liquid nitrogen and then 
lyophilized under high vacuum. 
Encapsulation Method A 
The freshly obtained, washed and lyophilized bacteria obtained as described 
above are suspended in 10 ml of 5% glucose saline solution in such volume 
so as to obtain a heavy suspension of bacteria which contains between one 
and ten billion organisms per ml, at 0-4 degrees C. All of these 
procedures are performed in the 0-4 degrees C. temperature range unless 
otherwise noted, in order to maintain viability of the lactobacilli 
bacteria which at room temperature lose viability. The suspension of 
bacteria is rapidly, but gently, stirred while 0.2-0.4 ml of sodium 
alginate solution (1.5% weight by volume) is added. The above mixture is 
then transferred into a 4 liter round bottom flask by using a nitrogen 
stream through a sheathed 14 gauge needle. The 4 liter round bottom flask 
was previously washed with a 5% albumin solution, and thereafter heated 
for at least 10 hours at 65 degrees C., and the needle and the tubing used 
in the process have also been treated this way. 
Thereafter the above mixture is forced through a 30 gauge multi-beveled 
needle under pressure using a large syringe and nitrogen stream. Very 
small droplets are generated at the end of the needle which are dried by 
the nitrogen and air stream around the 30 gauge needle, and the droplets 
are collected in an aqueous solution of 1.3-2% calcium chloride where they 
gel. Thereafter, they are washed at least three times with 0.08-0.13% 
2-(N-cyclohexyl-amino) ethanesulfonic acid (CHES) solution and 1.0-1.5% 
calcium chloride solution. 
The gelled droplets or little spheres are further washed with at least a 
five fold excess of the 0.1% CHES 1.1% calcium chloride, and normal saline 
solution. The resultant spheres are then "snap frozen" in liquid nitrogen 
and then lyophilized. After these steps, the encapsulated organisms can be 
used in the formulations of the present invention. 
Encapsulation Method B 
As an improvement over Encapsulation Method A, the following further steps 
are performed to render the bacteria more resistant to the cationic 
antimicrobials. The steps are performed at 0-4 degrees C. Thus, after the 
washings described in Encapsulation Method n the materials are reacted 
with poly L-lysine (Sigma) solution (0.05% w/v) spheres for ten minutes. 
The spheres are then washed with normal saline buffered to pH 4.5 with 
lactic acid. The resultant spheres are then "snap frozen" in liquid 
nitrogen and then lyophilized. After these steps, the encapsulated 
organisms can be used in the formulations of the present invention. 
Encapsulation Method C 
At 0-4 degrees C., the freshly obtained, washed, lyophilized bacteria are 
mixed with hydroxypropylmethylcellulose to achieve a weight to weight 
ratio of bacteria to the hydroxypropylmethylcellulose of 10/90, although 
the range can vary from 1/99 to 99/1, respectively. This will effect the 
final mass and viability of encapsulated organisms. It should be 
understood that higher ratios of cellulose tend to "protect" the bacteria 
in the encapsulation process. The mixture of lyophilized bacteria and 
hydroxypropyl methylcellulose is encapsulated by "pan" coating. This is 
done by using a stainless steel round bottom flask which first had been 
"coated" with about 1% magnesium stearate, suspension in water. A 
combination of a freely water permeable acrylic methacrylic acid ester 
copolymer and a partially water permeable acrylic methacrylic acid ester 
copolymer, (EUDRAGIT RL198 and EUDRAGIT RS198, respectively (obtained from 
Rohm Parm. Ltd., Germany) is suspended at 5-10% concentration in 
acetone-isopropanol, 1:1, containing a 1% w/v of castor oil. The ratio of 
the two copolymers can vary from 1:1 to 1:10, with a preferred ratio of 
1:2. The suspension is contained in the stainless steel round bottom 
flask. As the suspension of the copolymer kills bacteria rapidly, the 
process has to be performed rapidly with a high ratio of the 
hydroxypropylmethyl cellulose to bacteria. Thus, the mixtures of bacteria 
and cellulose are added to the stainless steel flask in small amounts, 
agitating vigorously for 3-10 minutes while the material is being dried 
over a nitrogen stream. 
Encapsulation Method D 
The freshly obtained, washed, lyophilized bacteria are added using rapid, 
but gentle stirring at 0-4 degrees C. to a thick suspension of 
polyvinylpyrrolidone (commercially available BASF, Germany) which may or 
may not be crosslinked, for 2 to 12 hours with a one percent (1%) solution 
of divinylbenzene (Biorad) in a 5% glucose balanced salts solution at a pH 
of 5.0 (range 4.5-8.0). The lactobacilli become encapsulated by stirring 
in this mixture for 1-12 hours. The material is then "snap frozen" and 
lyophylized. 
Encapsulation Method E 
The freshly obtained, washed, lyophilized bacteria are added using rapid, 
but gentle stirring to a suspension of polyvinylpovidone 
(Crospovidone.TM.). Specifically, ten grams of lyophilized bacteria are 
added to a suspension of 50 gm of polyvinylpovidone (Crospovidone.TM.) at 
0-4 degrees C. The encapsulation occurs by stirring for 30-60 minutes, 
although longer times can be used. Moisture is then removed from the 
mixture with a vacuum in a desiccator, or the material is "snap frozen" 
and lyophylized. 
Oral Administration of Micro-Encapsulated Lactobacilli 
Micro-encapsulated lactobacilli (in accordance with the presently preferred 
embodiment micro-encapsulated Lactobacillus rhamnosus variant) are 
administered orally to persons who suffer from antibiotic induced diarrhea 
or other form of acute or chronic diarrhea and its resultant 
complications. It was discovered in accordance with the present invention 
that such treatment with micro-encapsulated lactobacilli significantly 
reduces or eliminates the symptoms of antibiotic induced diarrhea. 
Examples of antibiotics, the diarrhea side effects of which are 
significantly diminished or eliminated by the treatment in accordance with 
the present invention, include clindamycin, ampicillin or tetracycline. 
Those skilled in the art will readily understand that the antibiotics are 
administered to the patient as a medical necessity for treatment of an 
underlying infection, and that the treatment with the micro-encapsulated 
lactobacilli in accordance with the present invention accompanies the 
antibiotic treatment to eliminate or reduce its side effects. In the 
event, a patient suffers from infection with Clostridium difficile, 
treatment with the drug metronidazole, vancomycin or other suitable drug 
is also recommended in accordance with the present invention, because 
these drugs kill the C. difficile bacteria. 
Many vehicles well known in the art which are normally suitable for oral 
delivery of drugs or pharmaceuticals can also be utilized for the oral 
administration of micro-encapsulated lactobacilli, in accordance with the 
present invention. Generally speaking the oral dose of micro-encapsulated 
lactobacilli for an adult patient suffering from antibiotic induced or 
other form of acute or chronic diarrhea is approximately 4.times.10.sup.3 
to 4.times.10.sup.12 viable bacteria per day, which is preferably provided 
in 4 substantially equal doses per day. Even more preferably, 
approximately 10.sup.5 to 10.sup.12 viable micro-encapsulated bacteria are 
administered orally, approximately 4 times a day. The oral doses of the 
bacteria may be admixed with pharmaceutical excipients which are otherwise 
well known in the art, to form tablets, powders, oral liquid doses, drops, 
or the micro-encapsulated bacteria may be contained and administered in 
capsules, such as gelatin capsules. Liquid doses and drops however, should 
be prepared freshly, otherwise the liquid breaks down the 
micro-encapsulation film and adversely affects the viability of the 
bacteria when exposed to gastric juices. The presently preferred mode of 
oral administration of micro-encapsulated lactobacilli is in capsules. In 
this regard it is noted that a capsule such as a gelatin capsule serving 
as a container for micro-encapsulated lactobacilli is considered for the 
purposes of the present description a "pharmaceutically acceptable 
excipient" because it serves to deliver oral doses of the "active 
ingredient", namely the micro-encapsulated lactobacilli. Other 
pharmaceutically acceptable excipients are materials well known in the art 
for tablet or powder formation, such as starch, microcrystalline 
cellulose, buffering and flavoring agents. Still more excipients are 
described below in connection with the descriptions of delivery vehicles 
for topical (skin) and intra-vaginal application. Although this is not the 
presently preferred embodiment, the micro-encapsulated lactobacilli can 
also be combined in a formulation for oral delivery with an antibiotic or 
other drug which is to be administered to the patient. 
Effectiveness of orally applied micro-encapsulated lactobacilli for 
significantly reducing or eliminating the diarrhea side effects of 
antibiotics is demonstrated by the following clinical test. 
Clinical Test for Oral Application 
For this study, five patients with a history of recurrent, antibiotic 
associated diarrhea were given oral encapsulated lactobacilli to prevent 
their antibiotic associated diarrhea. The encapsulated lactobacilli were 
given four times a day at the initiation of the antimicrobial therapy and 
were continued for three days after the antibiotic therapy was 
discontinued. All of these patients were given antibiotics for appropriate 
medical indications that were completely independent of this study. An 
oral consent was obtained and the potential risks and benefits of this 
study were explained in detail. The patients were treated with 10.sup.6 
encapsulated lactobacilli per dose that had been prepared according to 
Encapsulation Method B, which involved a poly-lysine base that was safe 
for oral administration. The preparation was given in a gelatin capsule 
filled with the encapsulated micro-organisms at the above dose 
(approximately 10.sup.6 viable bacteria per capsule). 
TABLE 1 
______________________________________ 
Patient Profiles with Antibiotic Associated Diarrhea 
Usual Duration 
of Antibiotic 
Associated 
Patient Diarrhea by Dose of 
Number Sex Age History Antibiotic 
antibiotic 
______________________________________ 
1 F 29 6-7 Days Clindamycin 
300 mg q.i.d. 
2 F 33 4-5 Days Clindamycin 
300 mg q.i.d. 
3 F 42 4-5 Days Ampicillin 
500 mg q.i.d. 
4 F 45 3-4 Days Ampicillin 
500 mg q.i.d. 
5 M 40 4-5 Days Tetracycline 
500 mg q.i.d. 
______________________________________ 
"q. i. d." is 4 times daily 
Results of Oral Therapy with the Encapsulated Lactobacilli 
In all of these patients there were no problems with diarrhea or other 
changes in bowel habits or symptoms. In this group of patients, there were 
no untoward effects of any kind from the therapy. This successful 
therapeutic result in this population could only be attributed to the oral 
therapy with the encapsulated lactobacilli. Oral administration of 
micro-encapsulated lactobacilli in accordance with the present invention 
is suitable for treatment not only for humans, but other mammals as well. 
Clinical Test for Topical Application of Micro-encapsulated Lactobacilli to 
Prevent Skin Infections 
For this study, five patients with a history of recurrent skin infections 
were given topical encapsulated lactobacilli to prevent skin infections. 
These patients were selected because they all had conditions that 
predisposed these individuals to chronic, recurrent skin infections. In 
all of these patients an oral consent was obtained and the potential risks 
and benefits of this study were explained in detail. The topical 
micro-encapsulated lactobacilli were applied to the affected skin area 
three times a day for six months. Each administration of the lactobacilli 
was made with 10.sup.6 encapsulated organisms which had been prepared 
according to Encapsulation Method D. The encapsulated organisms were mixed 
with talc to give a homogeneous powder to apply to the application site. 
The concentration of viable bacteria in the formulation which was applied 
to the skin was approximately 10.sup.6 bacteria per gram, and depending on 
the area treated approximately 1/2 to 1 gram was used in application. 
TABLE 2 
______________________________________ 
The Topical Use of Encapsulated Lactobacilli to Retard 
Skin Infections 
Fungal 
Patient Pre-Disposing 
Sex and 
Site of Organisms 
Number Age Factor(s) Race Infection 
Isolated 
______________________________________ 
1 31 Anabolic White Groin T.r., T.v. 
Steroid Abuse 
2 33 Anabolic Black Groin T.r., T.v. 
Steroid Abuse 
3 27 Anabolic White Groin T.r., T.m. 
Steroid Abuse 
4 59 Morbid Obesity 
White Abdominal 
C.a. 
Diabetes Mellitus 
Female 
pannus 
5 39 Diabetes White Abdominal 
C.a. 
Mellitus Female 
pannus 
______________________________________ 
C.a. is Candida albicans, T.r. is Trichophyton rubrum, T.v. is 
Trichophyton verrucosum, and T.m. is Trichophyton mentagrophytes. 
Results of Topical Application of Encapsulated Lactobacilli 
In this study with patients using the topically applied, micro-encapsulated 
lactobacilli three times a day, there were no yeast infections observed in 
any of the patient for the entire study period. Also, there were no 
untoward reactions of any kind in any of these patients. It should be 
understood in this regard, that topical use of micro-encapsulated 
lactobacilli in accordance with the present invention is not limited to 
the skin areas which were treated in the 5 patients in the above-described 
test. Rather the pharmaceutical composition of the present invention is 
suitable for treating or preventing skin infections in substantially any 
area of the skin in humans, and in mammals. 
Clinical Test for Intra-Vaginal Application of Micro-encapsulated 
Lactobacilli to Decrease the Risk of Infections 
It is the consensus of current medical and scientific thinking and is 
reflected in a great volume of scientific literature that a "normal" 
vaginal flora must contain appropriate lactobacilli as the major species. 
Moreover, it is desirable that the lactobacilli of the flora should 
produce both hydrogen peroxide and microbiocidins. In order to demonstrate 
the utility of the topical vaginal application of micro-encapsulated 
lactobacilli to prevent vaginal infections, five patients with a history 
of more than six, recurrent vaginal infections per year and with 
predisposing factors to vaginal infections were selected for the topical, 
vaginal therapy with micro-encapsulated lactobacilli. In all of these 
patients an oral consent was obtained and the potential risks and benefits 
of this study were explained in detail. The topical, micro-encapsulated 
lactobacilli were applied once a day, nightly at bedtime for six months. 
For ethical reasons this study did not have a "placebo arm" and the 
patients' previous history was used as the "control". All of these 
patients in this test were treated with clotrimazole (trade name Mycelex-G 
500, one 500 mg suppository) at bedtime for one week prior to the 
initiation of this study. This therapy was necessary because all of these 
patients had symptomatic infections with the fungal organisms identified 
below in Table 3, which had to be eradicated prior to the initiation of 
this study. The patients were treated with 10.sup.6 encapsulated 
lactobacilli per dose that had been prepared according to Encapsulation 
Method C, which involved a cellulose base. The product of Encapsulation 
Method C can be formed in a mold into a solid or semi-solid 
suppository-shaped object which allows for the easy insertion into the 
vagina. Optionally, additional cellulose or lactose or other conventional 
ingredients can be added to the suppository. 
In this regard it is noted that, generally speaking, for topical 
application and for intra-vaginal use a great variety of delivery vehicles 
(formulations) otherwise known in the art can be used. For example, the 
micro-encapsulated lactobacilli can be mixed with corn-starch, talcum or 
other suitable powder material. Creams, foams and ointments and 
conventional suppositories can be prepared, where, in addition to the 
active ingredient micro-encapsulated lactobacilli, fillers such as 
micro-crystalline cellulose, hydroxypropylmethyl cellulose, magnesium 
stearate, silicon dioxide, and lactose may be used. Still further optional 
components or ingredients which may be included in the suppository, or 
cream, foam, ointment, powder or other formulation of the present 
invention are fragrances, menthol, eucalyptus oil, methyl salicylate or 
related salicylates as topical cooling agents; hydrocortizone or related 
antiinflammatory steroids (1 to 500 mg per suppository) as 
anti-inflammatory agents; EDTA as a wetting agent and mild antimicrobial; 
propylene glycol or other pharmaceutically acceptable glycols, methyl 
paraben or related paraben derivatives as wetting agents and for 
"texture"; para diisobutylphenoxy polyethoxyethanol, nonoxynol-9 or 
dodoecaethylene glycol monolaurate as spermicidal and mild antimicrobial 
agents; tritions and menfegol as spermicidal, mild antimicrobial and 
wetting agents. 
TABLE 3 
______________________________________ 
Intra-Vaginal Use of Encapsulated Lactobacilli to 
Retard Vaginal Infections 
Number of Dominate 
Dominate 
Vaginal Micro- Micro- 
Infections 
Organism(s) 
Organism(s) 
Patent Pre- Per Year Isolated 
Isolated 
Number Disposing Prior to Prior to 
After 
and Age 
Factor(s) Therapy Therapy Therapy 
______________________________________ 
1 Diabetes, 9 C.a. L.a., L.r. 
33 yrs. 
obesity 
2 Diabetes 10 C.a. L.a., L.r. 
45 yrs. 
obesity 
3 Obesity, 8 C.g. L.a., L.r. 
29 yrs. 
cytotoxic 
therapy 
4 Diabetes, 11 C.a., C.p. 
L.a., L.r. 
41 yrs. 
obesity, 
cytotoxic 
therapy, auto- 
immune 
disease 
5 Diabetes, 10 C.t. L.a., L.r. 
37 yrs. 
obesity, 
cytotoxic 
therapy 
______________________________________ 
C.a., is Candida albicans, C.g. is Candida glabrata, C.t. is Candida 
tropicalis, C.p. is Candida parapsilosis, L.a. is Lactobacillus 
acidophilus, and L.r. is Lactobacillus rhamnosus. 
Results of Vaginal Application of Encapsulated Lactobacilli 
In this study with patients using the topically, intra-vaginally applied, 
micro-encapsulated lactobacilli at bedtime, there were no yeast infections 
observed in any patient for the entire study period. Also, there were no 
untoward reactions of any kind in any of these patients, and as it can be 
seen after treatment the predominant organisms in the vaginal flora were 
the beneficial Lactobacillus acidophilus or Lactobacillus rhamnosus 
bacteria.