Note: Descriptions are shown in the official language in which they were submitted.
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I
UROGEN~TAL AND INTLSTINAL COMPOSITIONS
TECHNICAL FIELD
This application relates to compositions useful in preventing and/or treating
urogenital and ;~le~ disorders.
BACKGROUND OF THE INVENTION
1~ Complex, microscopic ecosystems pervade the urogenital and intestin~l tracts of
warm blooded ~nim~lc. Trillions of microo~ , comprising hundreds of species,occupy the urogenital and intçstin~l tracts of m~mm~lc, infl~enc:in~ and ,~ .g
digestive and urologic functions. MicroorD~ c occupying these regions range frompotentially pathogenic strains, such as Esche.ricl~ia coli, enterococci, c~n~ gardnerella,
15 klebsie 11~ and clostridia, to the relatively nonpathogenic, such as lactobacillus and
bifidobacterium. Deviations from this delicate flora balance have been etiologically linked
to a number of urogenital and/or gastrointestin~l tract disorders; such imh~l~nces usually
r~sulting in the proliferation and predo.,.h~allce of pathogenic species~ Establishing and/or
preserving such a delicate flora balance is;, therefore, eSsçnti~l to ~ g optimal
20 health.
One way of establishing or 1..~ ;"i,~ the body's flora is by ~riminictering
to~cii~lc ~he use of lactobacillus to treat urogenital and intestin~l disorders has been
proposed, for in~t~ncP, in C~n~ n Patent 1,298,556, issued April 4, 1992, to Bruce et al.,
PCT Application Serial Number WO 93/09793, published May 27, 1993, to Reid et al. and
U.S. Patent 5,176,911, issued Januarv 5, 1995, to Tosi et al. Researchers hypothesize that
tolo~c.illl~s provide host protection via adherence to intestin~l or vaginal epithelial cells.
N~lw;l~ tling such proposals, several factors have been identified, casting doubt
on the use of lactobacillus as single agent therapy for urogenital and/or gastroint.ostin~l
infection. 1) Variation exists among strains of lactobacillus concerning the degree to
which individual strains of lactobacillus can adhere to epithelial surfaces. Reid, G. et al.,
,.,.;nO~ion of strains of Lactobacillus for properties that may inflllençe bacterial
illlel~el~ce in the urinary tract. J. Urol., 138:330-335;}987. This variation may also
extend to individual microorg~i-;s...s. 2) Pathogenic microo- ~ are most reliably
~Yr.lll~7ed where lactobacillus colonies are established prior to pathogenic invasion.
35 E~awthorn, L.A. et al., Exclusion of uropatho~en adhesion to polyrner surfaces by
Lactobacillus acidophilus~ Jour. Biomed. Mat. Res., 24;39-46; 1990; a nonexistent
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situation, however, barring in vitro design. 3) Viability issues exist regarding orally
ingested live bacterial cultures. Clinical research reveals that not all live bacteria survive
the digestive fluids of the stomach and upper intestin~l tract. Those which do survive may
emerge too weak or too few in number to effectively colonize the lower intestine.
5 Moreover, recognizing the need for sound clinical studies in the area, some researchers
express further concern as to the efficacy of certain lactobacillus species. Therefore,
despite the disclosures relating to lactob~ s products, there still remains a need for
improved urogenital and gastrointestin~l tract compositions incorporating lactobacil1us.
The present inventors have found that compositions incorporating plants or
10 extracts of the Ericaceae family with microencapsulated Lactobacillus andlor
Bifidobacterium provide improved compositions for treating and/or preventing urogenital
and gastrointestin~l disorders by modifying the interaction of pathogens with cellular
tissue. Recent studies also suggest the value of Ericaceae extracts in treating urinary tract
infections. -Researchers have observed that various species of vaccin~ (e.g. cranberry
~5 and blueberry) contain a polymeric compound which inhibits the adhesion of common
urinary pathogens (e.g., E. coli) to infection sites within the urinary tract. Ofek I et al.,
Anti-Escherichia coli Adhesin Activity of Cranberry and Blueberry Juices. N Engl ~ Med
1991;324;1599. Su~ ingly, the compositions ofthe present invention provide improved
environments more conducive to the colonization of lactic acid bacteria. Accordingly, it is
20 an object of the present invention to provide compositions for dietary supplPrnent~tion
Another obiect of the present invention ;s to provide improved compositions
comp~ ing a viable colony of microencapsulated lactobacillus and/or bifidobacteria.
A further object of the present invention is to provide compositions effective in
preventing andlor treating urogenital and intestin~l disorders.
2~ A still further object of the present invention is to provide topical compositions for
vaginal use.
An even further object of the present invention is to provide methods for
preventing and/or treating urogenital and intestin~l disorders.
These and other objects will become readily a,oparel,l from the disclosure whichfollows.
SUMMARY OF THE INVENTION
The present invention relates to compositions for the l.eaLI,,e.lL or prevention of
urog~nit~l and tntestin~l disorders, comprising:
a.) an ~nfi lh~sive amount of at least one plant species of the Ericaceae
family or its extract; and
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b.) a viable culture of at least one species of microencapsulated bacteria
selected from the group eonsisting of lactobacillus, bifidobacterium and
mixtures thereof.
The present invention also relates to the above compositions filrther comprising a
S growth factor for fz.cilitz~ting the growth of lactic acid bacteria.
The phrase "urogenital and intestin~l compositions," as used herein, means a
product which in the ordinaly course of usage may be retained in the oral cavity,
swallowed or applied topically to provide urogenital and/or intPstinZll activity.
The phrase "~nti~-lhesive amount," as used herein, means an amount effective to
1 û reduce the number of pathogenic microorg~micmc on the epithelial and/or mucosal lining of
the urogenital and/or intestjnz I ~ract.
The term "urogenital," as used herein, means that system of organs concerned with
the production and excretion of urine and reproduction.
The terrn "intectinz I " as used herein, means of or relating to the intestin~c
The term "nonpathogenic," as used herein, means s~ a~Lially lacking the ability to
cause disease or abnormality in healthy m2mms~1c The term "healthy," as used herein,
means free of underlying disease and/or immunosuppression.
All percentages and ratios used herein are by weight unless otherwise specified.Also, all measurements referred to herein are made at 25~C unless otherwise specified.
DETAILED DESCRIPTION OF THE INVENTION
The e~.cç~ as well as optional components of the compositions of the present
invention are described in the following paragraphs.
ESSENTIAL COMPONENTS
Plants or extracts of the Family Ericaceae
The Ericaceae (heath) fannily, consisting of about 110 genera and 4,000 species, is
by far the most i."~o- I~IIL family of the Ericales order, enComraccing a wide variety of fruit
producing shrubbery and evergreen plants. Genera falling under Ericaceae family include
V~crinil~m, Arctostaphylos, ~Ja~-lt~-~ria, and Gaylnsc~ci~ The Arctostaphylos genus
inr~ 1e.s such species as the checkerberry and bearl,clly (Uva ursi). Other edible fruits
3 0 such as the creeping snowberry or moxie plum fall under the genus Gaultheria.
kieberries are a well known species of the genus Gayll~cs~ei~ The Vacrinium genus,
best known for its fruits, contain some of the most cornrnon of berries, inrlut1ing the
blueberry (e.g., V. australe), cranberry ~e.g., Y. macrocarpon) and bilberry (e.g., Y.
myrt~llus). The term "berry ~ies)," as used herein, means berries, drupes, plums and the
lilce.
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E. coli adherence results primarily from ar1hesin.c on the raised hair like fimbriae (or
pili) of the microol~ani~lll. These ~tlhe~inc are (lçcign~ted MS (mannose-sensitive) and
MR (mannose-resistent). Like most firuit, Ericacease fruit species contain fructose, an
inhibitor of MS ~h~cin~. However, it has been recently suggested that the plants or
extracts of Ericaceae species further contain an llnic~elltified, non-dialyzable polymeric
compound which inhibits the MR adhesins associated with pyelonephritogenic strains of E
Cofi. The llni~ntified polymeric compound, as studied in Vaccinium species, was found
to inhibit the adhesion of both urinary and fecal isolates of E. Coli, the urinary isolates
being inhibited to a greater extent. Ofek I et al., Anti-Escherichia coli Adhesin Activity of
Cranberry and Blueberry Juices. N Engl J Med 1991;324;1599. It has also been reported
that the ingestion of large quantities of cranberry.~uice increased the hippuric acid content
of urine by several grams a day. This increase in hippuric acid excretion was accompanied
by small decreases in urine pH. In vivo tests have established that hippuric acid was
bacteriostatic at pH 5.0 for common pathogens of the urinary tract, but this action was
1~ considerably decreased as the urine pH was raised. Papas, N.P., et al., Cranberry Juice In
the Treatment of Urinary Tract Infections. Southwestern Medicine, 47:No. I (Jan. 1966).
That the Ericaceae species of the present invention are effective against other pathogenic
bacteria ~e.g., Pseudomonas aeruginosa) is disclosed in U.S. Patent 5,474,774, issued
December 12, 1995, to Walker et al., herein incorporated by reference in its entirety; no
20 effect was observed with respect to adhesion of lactobacillus strains to cells. Without
being lim-ted by theory, it is believed that the pathogenic inhibition caused by the
Ericaceae plants or extracts results in decreased pathogenic interaction, providing a more
favorable, less antagonistic em,i,~,-"l-G--l for lactobacillus to initially adhere and m~int~in
adherence. The phrase "anti-adhesive activity," as used herein, means an amount effective
25 to inhibit the adhesion of pathogenic microoly,~ni~ to the epithelial and/or mucosal
lining ofthe urogenital and/or intçstin~l tract.
Plants or extracts useful in the compositions of the present invention come from a
wide range of Ericaceae genera incltl~in~, but not limited to, V~ccinillm Arctostaphylos,
~lllt~eria, and Gayl~ c~ c~.l~,d species ;nclude, V. ausfrale~ V. corymbosum, V.30 occidenfale, V. ovatum, V. myrffllus, V. parvifolium, V. uliginosum, V. macrocarpon, V.
o~ycoccus, ~ erythrocarpum, V. vitis-idaea Y. ausfrale~ V. macrocarpon. V~cr.inium
species most I)lefc-lcd for use in the present invention include V. ausfrale~ Y.macrocarpon, and V. myrtillus. Mixtures of Ericaceae plants and/or extracts may also be
used.
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The plants or extracts of the present invention are preferably concentrated, having a
ratio of at least about 4 pounds of plant concentrate or extracts per pound of concentrate,
more preferably from about 4 pounds of plant concentrates or extracts per pound of
~ concentrate to about ~0 pounds of plant concentrate or extracts per pound of concentrate.
The Ericaceae extracts are preferably present at a level of at least lOmg, more preferably
from about IOOmg to about 18g, most pler~i~bly from about 250mg to about 4g per unit
dose. The amount of extract contained in each dose of product can be adjusted for the
dosage form. For example, the amount of extract in powdered form used in a drink mix
can range up to 18g per dose while the amount used in swallowable capsules might range
10 to about 4g. Plef~,led levels of the Ericaceae plants or extracts provide urinary and/or
intestin~l tract fluid concenllalions of the above mentioned unid~ntified, non-dialyzable
polymeric compound of from about 12 to about 25 micrograms per m~ ter Also, the
plants or extracts of the present invention pl erc:. ~bly retain greater than 2. 5% of their total
acid content and greater than about 0.1% of their benzoic acid content. The level is
15 selected to provide the desired level of a~nti-adhesin activity and can be modified as
desired. Cranberries and cranberry extracts are useful in the tre~tme-lt and/or prophylaxis
of urinary tract infections and are also useful as vaginal deodorants.
Species of Lactobacillus or Bifidobacterium
Another ~Sspnti~l component of the present invention is a viable colony of
20 microencapsulated Lactobacillus or Bifidobacterium. Bacteria of the Lactobacillus genus
are characterized as rod-shaped, gram-positive and non-spore-forrning bacteria. Of the
family Lactob~cill~e~e~ Lactobacillus inhabit the urogenital and gastrointestin~l tracts of
animals and h~ n~ and are important members of lactic acid producing group of bacteria.
Various species of Lactobacillus are used c;ommercially in the production of sour milks,
25 cheeses and yogurt. Lactobacilli also sha.re an important role in the m~n~lf~ct~lre of
fermented vegetables (e.g., pickles and sauerkraut), beverages ~e.g., beer, wine and
juices), sourdough breads, and some s~lls~çs.
T ~ctob~rillus species suitable for use in the present invention are those which 1.)
readily adhere to the epi~ l cells of either the urogenital or ~a~ oi~-~estin~l tracts of
30 ~Uhl~ ; 2.) produce hydrogen peroxide; 3.) ~.ol--ole low pH; and produce bacteriocins.
By "bacteriocins," as used herein, mea,ns p~ e;..~ceious, bacteriocidal substances
syr~t~e~i~P~d by b..cLe,ia, which usually ha~e a narrow spectrum of activity, inhibiting
strains of the same or closely related species. Bacteriocins appear to be capable of
~1i5p~ ~ or supplesslng the growth of other bacteria, and as such may provide an35 advantage to microorg~ni~m~ in -fellllell~ g the female genital tract ecosystem. Pl~r~llcd
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species of Lactobacillus include L. aci~ophilus, L. cate~ta~orme, L. brel~is, L. bulgaricus,
L. Iac~is, L. reu~erii, L. gasseri, L. helveticus, ~. casei, L. plantar1~m, L. delbru~chi, L.
t*ermop*ilis, L. Jensenii, L Crispatl~S, L. rogosae and r. fermerltum Species ofLactobacillus most prefe--ed for use in the compositions of the present invention include
L acidophilus, L. casei, L. crispatl~s, L. fermentum, and L. plantarum. Preferably7 the
Lactobacillus species of the present invention are hydrogen peroxide producing such as ~.
ac~dophilus, L. catenaJrorme, L. casei, L. crispatus, L. delbrueckii, L. jensenii, L rogosae.
r.. fermentum, L. gasseri and L. plantarum are also plerelled for use herein in view of
their adhesive properties.
Also inhabiting the urogenital and gastrointestin~l tracts of m~mm~lc and useful to
the compositions of the present invention are species of the genus Bifidobacterium (family
Actinomycet~ce~e). Bifidobacterium species are non-acid-fast, nonmotile gram negative
rods. Lactic and acetic acid producing Bifidobacteria are also considered important
regulators of the urogenital and inte5tin~1 flora of m~rnm~lc Species suitable for use in the
1~ present compositions in~lnd.q, but are not limited to, B. longllm B. breve Lactobacillus
B~f d~s and L~ctobacillus bif dus s2(bsp pennsylvanicus. Preferred for use in the present
compositions is B. Bif dum, most p-ere-led B. Bif dt~m subsp. Pennsylvanicus.
Mixtures of the Lactobacillus and/or Bifidobacterium species may also be used.
Any of the above species may be obtained either commercially or through laboratory
20 cultures.
The Lactobacillus andlor Bifidobacterium species are present as core and/or
coating con-ponents at levels of at least about 103 cells per unit dose7 pl~re-ably at levels
of ~om about 104 to about 1012 cells per unit dose and most preferably at levels of from
about 106 to about IOlQ cells per unit dose. The phrase "unit dose," as used herein,
25 means physically discrete units suitable as unitary dosages for ~tlminictration to m~mm~lc,
each such unit co..~ g a predetermined quantity of an active ingredient calculated to
produce the desired Lll~ pcutic effect in association with pharrnaceutically acceptable
carriers. The level is sçlectecl to provide the desired level of urogenital and gastrointestin~l
activity ~nd can be modified as desired. Lactobacillus may loose 4-6 fold of its viability at
30 room ttn~pc-~lure and during m~mlf~r,tllring, so depending on the m~n-lf~ctllring
conditions, an excess of Lactobacillus is added to IllAinlAi.~ an adequate number of viable
org~nicmC per final unit dose form. Alternatively, a patient can be a-lnninict~red the
equivalent of these conc~ ions of org~ni~m.c where the values are expressed by some
other measurement such as, for example, total protein concentration.
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The Lactobacillus and/or Bifidobacterium species of the present invention must
also be microencapsulated. Viable I,actobacillus and/or Bifidobacterium bacteria that have
been Iyophilized after the removal of the growth media can be used for encapsulation. The
bacteria can be obtained from commercial sources, or can be obtained from laboratory
S strains. Suitable media include MRS, Thayer-Martin media, Trypticase Soy, Brain-Heart
Infusion Broth, or any other enriched rnedia suitable for the cultivation of these or~ni~m.c
as no particular med;a is critical to the success of this suppository. The only important
factors are the viability and quantity of the micro-o,~lis-"s that are always determined by
standard clinical laboratory dilution methotis, such as plating the quantified dilution of
bacteria on to blood agar plates or other enriched media, incubating at 37~C for 24-48
hours in a 5-10% carbon dioxide atmosphere, and then perforrning a colony count. The
removal ofthe nutrient media is done by centrifugation at 14,000 x g at 0~-4~C., and then
was~ing 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
1~ lyophilized under high vacuum. The bact.eria are then microencapsuled according to
conventional micro~nc~rsul~tion technolo~y. Suitable methods of encapsulation are
disclosed in U.S. Patent 5.466.463. issued November 14, 1995, to Ford and U.S. Patent
.407.609. issued April 18, 1995, to Tice, both of which are herein incorporated by
le~cl~ce in their entirety.
OPTION~L CO~DPO~nENTS
Lactobacillus Growth Factor
Also useful to the compositions of the present invention is a growth factor for
f~cilit~ting the growth of lactic acid bacteria. The phrase "a growth factor for f~r.ilit~tin~
the growth of lactic acid bacteria," as used herein is meant a nutrient source or media
25 which supplies a necessary source of food and/or energy for f~çilit~ting the growth of
lactic acid producing bacteria. The growth factor is preferably selective for establishing
and ..~ g the growth of lactic acid bacteria, preferably Lactobacillus and/or
Bifi~obacterium, without f~ it~tin~ extreme growth of pathogenic bacteria The various
nutritional requi.t;nle"Ls essential for bactelial and/or colony growth are normally met
30 when the growth factor contain fermentable carbohydrate, peptone, meat and yeast
extract. Suppiç~ l;ons with tomato juice, m~n~nç~, acetate and oleic acid esters,
especially Tween 80, are stim~ tory or even eS.~enti~l for most species and are, therefore,
in~ .lded in most M~S mefli-lm Lactic acid bacteria adapted to very particular substrates
rnay require special growth factors.
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Examples of suitable growth factors include, but are not limited to, yeast extracts;
gangliosides; salicin; mono-, di- and polysaccharide sugars such as glycogen, glucose,
fructose, rhamnose, lactulose, methyl-a-D-mannoside, p-nitrophenol-a-D-mannoside,
maltose, maltodextrin, dextrin, dextran, levan, sialic acid and acetylglucosamine as well as
5 oligosaccharides such as, but not limited to, fructooligosaccharides,
galactooligosaccharides and soybean oligos~cch~rides. Fiber or fermentable substrates
such as psyllium may be used in the present compositions as may gums such as guar gum
and x~nthum gum. Similarly, proteinacious materials such as, peptone, keratin; vegetable;
soy and unsaturated fatty acids such as lauric acid and teichoic acids such as lipoteichoic
10 acid and esters such as glycerophosphates or ~-glycerophosphates are also useful as
growth factors. The growth factor is preferably selective for establishing and m~int~ining
the growth of lactic acid bacteria, most preferably Lactobacillus and/or Bifidobacterium
species. Growth factors preferable for use in the compositions of the present invention
include lactose, lactulose, rhamnose, oligosaccharides and glycogen. Mixtures of these
15 nutrients may also be used.
More preferably the growth factor of the present invention is an oligosaccharidesuch as, but not limited to, galactooligosaccharides, soybean oligosaccharides and
fructooligosaccharides. Oligosaccharides possess bioadhesive properties which help fix
the location of these growth factors for easier access by lactic acid bacteria. Most
20 p, ~l, ed for use herein are fructooligos~cch~rides. Lactic acid bacteria, such as
l,actobacillus and Bifidobacterium, partially utilize fructooligosaccharides as an energy
source ~y converting it, via rw.,lenlalion, to lactic acid or a mixture of lactic acid, acetic
acid, and CO2. The lactic acid and other fatty acids produced by this carbohydrate
fermentation contribute to the ~n~int~n~nce of low pH which is an important control
25 mPr.h~nicm for preventing colonization of pathogens.
Ch~mic~lly~ oligofructose is the oligosaccharide fraction of inulin. It is composed
of the GFn and Fn type [G = glucose; F = fructose; n = number of frutose moieties linked
by ,~ (2,1) linkages in a ratio of about 2:1, with n = 2-6, and an average degree of
pol~meli~Lion of 4. Inulin is p~t;paled by hot water extraction of chicory roots and is
30 composed of molecllles of the GFn type, n ranging as high as 60 with an average degree of
polymerization of 10. Fructooligosaccharides suitable for use herein may or may not have
non-fructosyl units in place of fructosyl end units. The same is true for other
olis~os~cr.h~rides with respect to their osyl end units. Non-fructosyl units may include, but
are not limited to, polyalcohols such as xylitol, m~nnitol, and sorbitol.
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Fructooligos~cçh~rides most preferred for use in the present compositions are inulin or
oligofructose. Mixtures of these nutrients may also be used.
Without being limited by theory, it is believed that, upon ingestion, growth factors
increase the number of Lactobacillus and/or Bifidobacterium species available to displace
5 pathogenic microorganisms from epithelial surfaces. The increase in the number of
Lactobacillus and/or Bifidobacterium species con,peLiLively exclude the pathogens causing
them to be displaced and excreted; the resLlt is an overall reduction of host pathogens.
Moreover, as vaginal infections are generally believed to result from pathogenic migrating
from the rectum to the vagina, the number of pathogens invading the vagina are also
10 reduced as the number of migratory pathogens decreases.
Growth factors are preferably incorporated into the compositions of the present
invention at from about 5% to about 75%, more preferably from about 20% to about70%, and most pl~;re-~bly from about 30% to about 65% per unit dose.
Coating Material
The compositions of the present invention may further comprise a coating material.
Coating materials useful to the compositions of the present invention may be water soluble
as well as water insoluble. The coating material of the present invention is preferably dried
to a water activity (Aw) of less than about 0.6, more preferably less than about 0.45, most
preferably less than about 0.3. The term "wa.ter activity," as used herein, is well known in
20 the art as the measure of the ratio of the equilibrium vapor pressure of water above a
substance such as food ~solid or liquid) to the vapor pressure of pure water, both taken at
the same tel~ L-Ire. A more detailed description of water activity is found in U.K.
Patent 2,014,429.
Water soluble co~tin~S useful to the compositions of the present invention may
25 include sugar or organic coatings. Sugar cozltin~.~ useful to the present compositions may
be syrupy materials co..~ ;n~ monos~cch~rides or polymers of two or more saccharide
units. Organic coaLii)gs are also useful to the compositions of the present invention.
Useful organic polymers or copolymers used include those having a plurality of carboxylic
ac;d and ester groups. Such groups are largely responsible for physical or chem;e~l
30 il~lela-;Lions with an active ingredient for effective taste m~c~ing properties. The p~c;re~led
polymers or copolymers contain either a vinyl and acrylic acid and/or ester groups or
carboxylic acid and/or ester groups. The specific polymers/copolymers are readily
ascci.~a;lled by one of c,l-lh~a-y skill in the art. Generally, polymers/copolymers that are
pharm~ce~ltic~lly acceptable in terrns of safei:y and toxicity may be used. It is pl~rell~d
35 that such polymers/copolymers be soluble in a solvent or a mixture of solvents. Such
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polymers/copolymers include polymeric or resinous substances such as: co-polymers of
acrylic and substituted acrylic acids; cellulose esters; vinyl and substituted vinyl esters;
polysulfonic acids, their esters and amides. Specific examples include naturally occurring
materials such as shellac and zein and synthetic and semi-synthetic materials such as ethyl
S cellulose, cellulose ?Icet~tes, cellulose acetate phthallates, ethyl vinyl ~cet~tçs and/or
phth~l~tes, polyvinyl ~cet~tes and/or phth~l~te~, ethyl and/or methyl methacrylic acids,
esters and co-polymers, hydroxy alkyl cellulose ~cet~tes and/or phth~l~tPc Such
compounds include commercially available materials sold under trade names such as
Eudragit S (trademark of Rohm Pharma) and Phthalavin (trademark of Colorcon). The
10 coating material of the present invention is preferably a polymeric mixture of meth~crylic
acid and meth~crylate Mixtures of sugar and organic coating may also be used.
One particular embodiment of the present invention comprises a core cont~ining at
least one species o~the Ericaceae family and a coating material cont~ining a viable culture
of at least one species of the above described microencapsulated bacteria. Alternatively,
15 the micropnç~rs~ ted Lactobacillus can reside in the core coated with a coating material
co~ g the Ericaceae species. The latter may find use in providing an acidic
environment for the Lactobacillus to survive and grow. Additional coating layers may also
be added.
Additionally, Protein-like coating components may be included. Useful for
20 improvement of gastrointestin~l disorders, such components may contain branched amino
acid-modified proteins. Whey powders, for example, are treated with papain in the
presence of the amino acids ethyl L-leucine (16.1 parts), ethyl L-isoleucine (7.4 parts),
ethyl ~-valine (10.2 parts), cysteine hydrochloride (1.5 parts), and sodium carbonate (26
parts) in water at 40~C for 20 minlltes to m~n~-~ct--~e coated powders co~ 10%
25 free arnino acids and 43% branched arnino acids. The branched amino acid-modified
powders can be mixed with fats, dextrins, salts, vitamins, and the like to make tablets.
Other eA~mi)t-- of a tablet coating materials are zeolites and clays to make tablets
more p~l~t~le Zeolites have found use as bacterial feed coatings for domestic animals.
For example, in ehe domestic animal business, ti~mlllin fumarate is dissolved in methanol,
30 supported on mordenite-type zeolite or starch, dried and further premixed with the
supports to produce s~lst~ined-release, coated granules. Still other examples of tablet
cQ~tinge include co-",.l~ - carbohydrate and inclusion complexes.
The sugar and/or organic co~ting.~ of the present invention may be applied by
conventional means inclnding .,.ec~ ical methods such as pan coating, air suspension
35 techni~ues, multiorifice centrifugal techniques and spray drying techniques as well as
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.
physicochemical methods such as coacervation-phase separation. Coating procedures are
more fully discussed in Remington's Pharmz~ceutic~l Sciences (Alfonso Gennarol, editor),
1666-1675 (1990), herein incorporated by reference.
Bufferin~ A~ents
The compositions of the present invention may also contain a buffering agent~ For
oral compositions, buffering to an acidic pH to enhance flavor may be done. For products
used in the vaginal area, buffering agents suitable for use in the compositions of the
present invention are those capable of ~IIAi~ ill;llg a urogenital pH of about 3.0 to a~out
5.5. Any mild pharm~ce~tically acceptable acid, other than those found in the Ericaceae
species disclosed herein, can be used. Suitable acids include bonc acid, or organic acids
such as quinnic acid, proprionic acid, malic acid, pyruvic acid, hippuric acid, tartaric acid,
sorbic acid, benzoic acid, lactic acid, ascorbic acid, citric acid, or acetic acid, in
colllbhlalion with their respective sodium or other pharrn~ceutically acceptable salt ~to the
extent necess~ry to achieve the desired pH[). When buffered, the compositions of the
present invention are preferably buffered to a pH range of from about 3.5 to about 5.0,
preferably from about 3.7 to about 4.7, and plerel~bly using lactic acid with sodium lactate
or a collll~ lion lactic acid/sodium lactate and benzoic acid or lactic acid/sodium lactate
and proprionic acid.
Additional Plant Extracts
Additional therapeutic and/or medicinal plants or extracts may also be incorporated
into the compositions of the present invention. Such plants or extracts include echin~c~
allium, bucha, juniper gincerlg allicin, chlorella, algin and the like. Mixtures of these
additional plants or extracts may also be used.
Nutritional Additives
Nutritional additives may also be incorporated into the compositions of the present
invention. Such additives inchldç; but are not limited to, proteins and carbohydrates other
than those ,.~nLiol-ed herein as growth factors, vitamins, minerals, amino acids such as
glycine, phytochemicals and mixtures thereof. These additives may, alternatively, be first
incorporated into the Lactobacillus andlor Bifidobacterium of the present invention.
Pha~ ce~ltical Actives
The compositions of the present invention may also be used in combination with
pharrn~ce~ltic~l actives. The pharm~ce~ltical active is ,c~t;felably selected from at least one
of an ~n~lg~ic agent and/or a ga~LIui~ l agent. When incorporating pharm~ce~ltic~l
actives, appropliale measures should be taken to avoid contact with the microor~ni~m~ of
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lZ
the present invention. Such measures may include, but are not limited to, modifying the
microencapsulation process as suggested by U.S. Patent 5,466,463 to Ford.
Examples of analgesics ,~l efe~ ~ ~d for use in the present invention include
~cet~minophen, acetyl salicylic acid, indomethacin and optically active isomers or
5 r~cem~tee of ibuprofen, naproxen, flurbiprofen, carprofen, tiaprofenic acid, cicloprofen,
ketoprofen, ketorolac, etodolac, indometh~cin elllin~C7 fenoprofen, diclofenac,
piroxicam, benzydomine, nabumetone, their pharrn~centically acceptable salts and mixtures
thereof.
Examples of gastrointçstin~l agents preferred for use in the present invention
10 include anticholinergics including atropine, clirlin;~ln and dicyclomine; antacids including
aluminum hydroxide, bismuth subsalicylate, bismuth subcillate~ simethicone, calcium
carbonate and magaldrate; ~2-receptor antagonists including cimetidine, famotidine,
ni7~ti-1ine and ranitidine; laxatives including: docusate, phenolphthalein and c~e~nthrol;
gastroprotect~nts inclu-~ing sucralfate and sucralfate humid gel; gastrokinetic agents
15 inc~ ing metoclopramide and cisapride; proton pump inhibitors incl~ing omeprazole and
antidiarrheals inclll~ing f~iphenoxylate, kaolin pectin, attapulgite and loperamide.
Carrier Materials
The carriers into which the compositions of the present invention may be
incorporated are many and varied and depend largely upon the end use of the
20 compositions. These carriers are pharrn~celltically acceptable and include orally
acceptable as well as topical compositions. They may be completely inert or contain or
may be other active ingredients, yet the carriers must be co~ alible with the herein
disclosed compositions. The term "colllpalible,'' as used herein, means that the carrier
components are capable of being commin~le(l with the components of the present
25 invention, and with each other, in a manner such that there is no interaction which would
subst~nti~lly reduce the activity or viability of the compositions under ordinary use situa-
tions. Carrier materials must, of course, be of s~lfficiently high purity and sufficiently low
toxicity to render them suitable for arl~inietration to the human being treated. Preferably
the compo ,iLions of the present invention comprise from about 0.01% to about 99.99% of
30 one or more carrier materials.
Carriers suitable for topical ~minictration of the present compositions include
suppositories, vaginal tablets or capsules, ovules, creams, solutions for lavages, emulsions,
foams, gels, ~ c-~tc~ oils and ointmente, douches.
Creams, gels and other base formulations may be used in topical ~f~ Lion of
35 the present compositions to, for example, male or female ~nit~ (incl-l~ling the vulva
,
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13
and vagina~ and are prepared according to conventional methods for semi-solid
compositions using excipients like vaseline, paraffin, vaseline oil, vegetable oils, animal
oils, solid and liquid synthetic glycerides, waxes, lanolin, lanolin alcohols, sorbitan esters,
fatty alcohols, liquid/solid polyethylene glycols, propylene glycols, polyethylene, starch,
S acrylamides. meth~crylamides, derivatives of cellulose and carboxyvinylpolymers
Ovules, suppositories, vaginal capsules or tablets and effervescent tablets may also
be useful in topical application of the prevention. Ovules are similar to suppositories,
ovoidal shaped and the excipients mainly used are semi-synthetic glycerides and
polyethylene glycols and optionally also emulsifiers and surfactants.
The vaginal capsules are gelatinous envelopes or sachets within which is
subdivided the suspension which is generally anhydrous and contains liquid paraffin,
vaseline, vegetable oils and semi-synthetic oils and thickening agents. The tablets, shaped
suitably for vaginal use, contain as main excipients lactose, starch, polyvinylpyrrolidone,
cellulose derivatives, mAgnecium stearate, glycol. The effervescent tablets contain chemical
1~ components (i.e. sodium bicarbonate with citric acid or tartaric acid), which are nec~ss~ry
to develop carbon dioxide in order to produce effervescence.
The compositions of the present in~vention may also be incorporated into and
topically applied by woven or nonwoven fabric materials such as tissues, wipes, rell~illine
n~pkin.~, panty liners, l,llnpolls, diapers, incontinent care products and the like. Preferred
20 for use herein are nonwoven fabrics. Nonwoven fabrics suitable for incorporating the
present compositions are described in U.S. Patent 4,891,227 to Thaman et al., herein
incorporated by reference.
Oral dosage forms are also usefu1 as carriers for the present invention. These
dosage forms contain compatible solid or liquid filler diluents or encaps~llAting substances
25 which are suitable for oral ~ 1. ation to a human or lower animal.
Liquid dosage forms for oral ~rimini~tl-ation may comprise dissolving or suspending
the compositions of the present invention in a potable liquid, such as sterile or purified
water. Alternatively, liquid or dry oral ~ Lion forrns can comprise an enterically
coated capsule co..~ p the dosage forms. Suitable forms include emulsions,
30 suspensions, solutions, syrups, and elixirs co..lAil~ inert diluents commonly used in the
art, such as purified water, sugars, polysaccharides, silicate gels, gelatin, or an alcohol.
These inert tlih~nts do not actively participate in the therapeutic effect of the present
invention. However, such liquid forms may require special care where free water is
present with the Lactobacillus to prevent fel ll,c;~llalion or degradation of the Lactobacillus.
35 Besides the inert diluents, such colllposiLions; can also contain wetting agents, emulsifying
-
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14
agents, suspending agents, as well as additional therapeutic actives. For a more detaile
description of liquid and liquid-like dosage forms, see Remington's Pharmaceutical
Sciences, 17th ed., M[ack Publishing Company, Easton, Pa. (1990), pages lS19-1544,
herein incorporated by reference.
Tablets can be col,lplessed, molded, triturated, enteric-coated, sugar-coated, film-
coated or multiple colllpl essed, cont~inin~ suitable binders, lubricants, rliluents,
rli~inte~rating agents, and flow-inducing agents.
Also useful are soft or hard gelatin ç~ps-l~çs Preferably, the gelatin shell is
essentially Ll~llsl)alt;lll so as to enhance the aesthetic qualities of the capsule. Soft and
hard gelatin shells generally comprise gelatin, a plasticizer and water The starting gelatin
material generally used in the m~mif~l~t~re of these capsules is obtained by the partial
hydrolysis of collagenous material. Gelatin suitable for capsule m~nuf~ctllre iscommercially available from the Sigma Chemical Company, St. Louis, Mo.. One or more
plasticizers is incorporated to produce a gelatin shell. Useful plasticizers of the present
invention include glycerin, sorbitan, sorbitol, or similar low molecular weight polyols, and
mixtures thereof.
Techniques and compositions for making solid oral dosage forms are described in
Marshall, "Solid Oral Dosage Forms," Modern Pharrn~ce~ltics, Vol. 7, (Banker andRhodes, editors), 359-427 (1979), incorporated by reference herein. Techniques and
compositions for making tablets (COlll~l t;ssed and molded), capsules (hard and soft
gelatin), troches and pills are described in Remington's Pharm~ceutical Sciences (Arthur
Osol, editor), 1553-1593 (1980) and U.S. Patent 4,935,243, to ~orkan et al., issued ~une
1~, 1990; these two references being incorporated herein by reference in their entirety.
Specific examples of pharm~cel1ti~11y acceptable carriers and excipients that may be used
to forrnulate oral dosage forms, are described in U.S. Patent 3,903,297, Robert, issued
September 2, 1975, inco",ol~ed by reference herein.
Alternatively, the compositions of the present invention may be achieved by
incorporating the compositions of the present invention into freeze-dried or Iyophilized
tablets. Freeze-drying or Iyophilization f~ tAtes ~icintegration of the composition by
forming the dried composition into an open matrix network. In most cases, this results in
rapid perrmeation by the aqueous media, promoting timely delivery of the product.
Suitable metho-~s of freeze drying are well known in the art and eollllllonly employed.
Any suitable conventional method of freeze-drying may be l~tili7e(l A preferable method
of rree~ing and drying is to fast freeze the composition and then dry the composition to a
final moisture content of about 2% to about 5%. Suitable methods of freeze-drying and
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production are taught by U.S. Patent 4,642,903, Febru~ry 17, 1987, to Davies, U.S.
Patent 4,946,6847 August 7, 1990, to Blank et al., U.S. Patents 4,305,50~ and 4,371,516,
issued December 15, 1981 and February 1, 1983 respectively, to Gregory et al., and U.S.
- Patent 5,188,825, February 23,1993, to Iles et al.; which are all incorporated herein by
S reference.
Similarly, the compositions of the present invention may be vacuum dried.
Vacuum drying involves at least the partial drying of compositions at temperatures above
compositions' collapse temperature. Freeze drying, on the other hand, involves the drying
of compositions at temperatures below the compositions; collapse temperature. Any
10 suitable method of vacuum drying may be used. Suitable vacuum drying processes are
described in U.S. Patent 5,298,261, to Pebley et al., issued March 29, 1994, herein
incorporated by I efe, e.,ce.
One other form of tableting technology that may be applicable to the present
invention is a liquid/liquid extract developed by Janssen Pharrnaceutica Inc. and is
15 identified by the trade name QuicksolvTM. This technology is fully described in U.S.
Patent 5,215,756 herein incorporated by reference.
Other optional ingredients well known to the pharmacist's art may also be ir~luded
in amounts generally known for these ingredients, for example, natural or artificial
sweeteners, flavoring agents, colorants, perfilming agents, buffering agents and the like to
20 provide a palatable and pleasant looking final product, antioxidants, for example, butylated
hydroxy anisole or butylated hydroxy toluene, and preservatives, for example, methyl or
propyl p~,~e,n, potassium sorbate, or sodiurn benzoate, to prolong and çnh~nce shelf life.
A l)lere~,ed optional component is also c~ffeine
Those of ordinary skill in the art ~,-ill quickly reaiize other suitable ingredients,
25 rlilu~nts and dosage forms, or will be able to ascertain such, using routine c~ .h~nt~tion
Further, the ~ . alion of the various compositions can be carried out using standard
techniques common to those of ordinary slcill in the art.
Exarnples
The following examples further describe and demonstrate embodimente within the
30 scope of the present invention. These e~xamples are strictly given for illustration purposes
and are not to be construed as limitations of the present invention, as many variations are
possible without dep&- ling from the spirit and scope of the invention as set forth herein.
Example I
The T ~ctob~c~ e and/or Bifidobacterium species cultures can be freeze dried (or35 purchased freeze dried). To provide freeze dried cultures, an inoculum of Lactobacillus
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16
and/or Bifidobacterium is grown in a sterile nutrient media ~e.g. Trypticase Soy agar
broth). The media is removed by centrifugation. The bacteria isolates are washed using a
sterile b~l~nced salt and 5% glucose solution. The bacteria are then "snap frozen" with
liquid nitrogen and vacuum freeze dried. The freeze dried product is then checked for
5 bacterial plate count and then diluted so that the plate count per unit dose composition is
from about 103 to about 1012.
Example II
The freshly obtained, washed and Iyophilized bacteria obtained as described above
are suspended in 10 ml of 5% glucose saline solution in such volume so as to obtain a
10 heavy suspension of bacteria which contains between one to 101~ organisms per ml, at 0-
4~C. All of these procedures are performed in the 0-4~C temperature range unlessotherwise noted, in order to "~ 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
15 rnixture is then transferred into a 4 liter round bottom flask by using a nitrogen stream
through a ~he~thec~ 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 6~~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
20 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%
25 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 norrnal saline solution. The
rçslllt~nt spheres are then "snap frozen" in liquid nitrogen and then Iyophilized. After
these steps, the encapsulated org~nicm~ can be used in the forrnulations of the present
30 invention.
Example III
A tablet forrn of the present invention is made by combining the following
components using conventional mixing and tableting technology.
Ingredient % Weight
Concentrated Cranberry Extract 37 300%
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17
Lactobacillusl 29.900%
~, K2932 (5% ethanol soln)2 13.4.00%
Avicel (pH 101)3 13.400%
~, Explotab4 1.8%
Talc 3.600%
~ne~il-m stearate 0.6û0%
1 Microencapsulated ~.actobnr~ 2~ Caseii l~ar. rhamnosus (108 cfu/g).
2 Polyv~ py~ ollidone
3 TM for microcrystalline cellulose, a highl)~ purified particulate form of cellulose
10 4 Abrandofsodiumstarchglycolate
The cranberry extract is gr~n~ ted with half the Avicel and the K2932 in ethanol.
The granl~l~tin~n is then passed through a I2 mesh screen and dried at 120~F. The dried
granulation mixture is passed through a 20 mesh screen. To the sieved granulation is
added the lactobacill--~, re~inin~ Avicel, Explotab and talc and mixed until uniform.
15 ~gn~cillm stearate is then added to the uniforrn mixture with mixing. The resultant
granulation mixture is then con,pressed using conventional tableting processes.
Example IV
A oral capsule form of the present invention is made by combining the following
components using conventional mixing techr~ology.
Ingredient % Weight
Conce"Ll~led Cl~l.l,~.ly Extract 29.900%
Bifidobacteriuml 15.000%
fructooligosaccharide2 29.900%
Avicel3 22.400%
Ac Di Sol4 2.800%
Microencapsulated Lactobacillus bif dus subsp pennsylvanicus ( 108 cfu/g).
2 Available as NutraFlora FOS from Golden Technologies Company, Inc.
3 TM for microcrystalline cellulose, a highly purified particulate form of cellulose.
4 A brand of a cross-linked form of sodium carboxymethylcellulose.
Combine and mix the cranberry extract, fructooligosaccharide, Lactobacillus
culture, Avicel and Act Di Sol in a V-blender until uniro~ . Unit dose amounts of the
res~lt~nt mixture is then placed into suitably sized hard gelatin capsules.
Example V
A topical gel form of the present invention is made by colllbilfing the following
35 components using conventional mixing technolog,v.
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18
Ingredient % Weight
Concentrated (~ranberry Extract 0.50%
Lactobacillus 1 2.50%
fructooligosaccharide2 6.00%
Polyacrylamide and C 13-14
Isoparaffin and Laureth-73 4.00%
PPG-14 Butylether 8.00%
Water, Purified q.s.
Microencapsulated Lactobacillus Caseii var. rhamnost~s (108 cfu/g).
10 2 Available as NutraFlora FOS from Golden Technologies Company, Inc.
3 Available as Sepigel from Seppic Corporation.
Water is added to a suitable size container. While mixing at a moderate speed (300
rpm), the Polyacrylamide and C13 14 Isoparaffln and Laureth-7 is added to the water to
form a water phase. Separately, the PPG-14 Butyl ether is placed in a container and
15 covered. Using a r.i~htnin' Mixer with a 3 blade paddle prop, the cranberry extract and
fructooligos~cch~ride are added to the PPG-14 Butyl ether and mixed at a low speed (100
rpm) until the el ~nbe- l y extract and fructooligosaccharide are dissolved. Thelactobacillus culture is added to the water phase and mixed at low speed (100 rpm) until a
uniform solution results. The PPG-14 Butyl ether is slowly added to the water phase to
20 form a gel. The reslllting gel is mixed at moderate speed until uniform.
