U.S. patent application number 10/644787 was filed with the patent office on 2004-12-23 for strains of bacteria and pharmaceutical composition containing one or more of such strains and use of same for preventing and treating diseases asociated with or caused by altered metabolism of bile acids.
Invention is credited to Cavaliere Vesely, Renata Maria Anna, De Simone, Claudio, Vesely, Marco.
Application Number | 20040259227 10/644787 |
Document ID | / |
Family ID | 11373572 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259227 |
Kind Code |
A1 |
Cavaliere Vesely, Renata Maria Anna
; et al. |
December 23, 2004 |
Strains of bacteria and pharmaceutical composition containing one
or more of such strains and use of same for preventing and treating
diseases asociated with or caused by altered metabolism of bile
acids
Abstract
Strains of bacteria characterized by exhibiting: (a) a
7.alpha.-dehydroxylase activity of less than 50%, and (b) a bile
acid deconjugation activity of less than 50%, and descendants,
mutants and derivatives thereof preserving activities (a) and (b);
and a pharmaceutical composition using one or more of such strains
and use of same for preventing and treating diseases associated
with or caused by an altered metabolism of bile acids.
Inventors: |
Cavaliere Vesely, Renata Maria
Anna; (US) ; De Simone, Claudio; (Ardea,
IT) ; Vesely, Marco; (Milano, IT) |
Correspondence
Address: |
KENYON & KENYON
1500 K STREET, N.W., SUITE 700
WASHINGTON
DC
20005
US
|
Family ID: |
11373572 |
Appl. No.: |
10/644787 |
Filed: |
August 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10644787 |
Aug 21, 2003 |
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09796432 |
Mar 2, 2001 |
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09796432 |
Mar 2, 2001 |
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08813776 |
Mar 7, 1997 |
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6225104 |
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Current U.S.
Class: |
435/252.3 |
Current CPC
Class: |
A61K 31/7016 20130101;
A61P 1/16 20180101; A61P 1/00 20180101; C12R 2001/46 20210501; C12R
2001/225 20210501; A61K 35/747 20130101; A61K 31/575 20130101; A61K
35/744 20130101; C12N 1/205 20210501; A61K 35/744 20130101; A61K
2300/00 20130101; A61K 31/7016 20130101; A61K 2300/00 20130101;
A61K 35/747 20130101; A61K 2300/00 20130101; A61K 31/575 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
435/252.3 |
International
Class: |
C12N 001/12; C12N
001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 1996 |
IT |
MI96A000468 |
Claims
1-11. cancel
12. A pharmaceutical composition for preventing and/or treating
diseases associated with or caused by an altered metabolism of bile
acids, comprising an effective amount capable of producing a
normalizing effect on such an altered metabolism in a patient
suffering therefrom, of (1) at least one bacteria strain provided
with: (a) a 7 .alpha.-dehydroxylase activity of less than 50%, and
(b) a bile acid deconjugation activity of less than 50%, and
descendants, mutants and derivatives thereof preserving activities
(a) and (b), and (2) a pharmaceutically acceptable carrier.
13. The pharmaceutical composition of claim 12, wherein said at
least one bacteria strain is a gram-positive bacteria strain.
14. The composition of claim 12, wherein said at least one bacteria
strain belongs to a species selected from the group consisting of
Streptococcus thermophilus, Streptococcus faecium, and
Lactobacillus bulgaricus.
15. The composition of claim 14, wherein the bacteria strain is
Streptococcus thermophilus YS 52 deposited with the CNCM,
Collection Nationale de Cultures de Microorganismes, Institut
Pasteur, under the accession number I-1670.
16. The composition of claim 14, wherein the bacteria strain is
Streptococcus thermophilus YS 46 deposited with the CNCM,
Collection Nationale de Cultures de Microorganismes, Institut
Pasteur, under the accession number I-1668.
17. The composition of claim 14, wherein the bacteria strain is
Streptococcus thermophilus YS 48 deposited with the CNCM,
Collection Nationale de Cultures de Microorganismes, Institut
Pasteur, under the accession number I-1669.
18. The composition of claim 14, wherein the bacteria strain is
Streptococcus faecium SF 3 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1671.
19. The composition of claim 14, wherein the bacteria strain is
Lactobacillus bulgaricus LB 1 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1664.
20. The composition of claim 14, wherein the bacteria strain is
Lactobacillus bulgaricus LB 3 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1665.
21. The composition of claim 14, wherein the bacteria strain is
Lactobacillus bulgaricus LB 7 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1666.
22. The composition of claim 14, wherein the bacteria strain is
Lactobacillus bulgaricus LB 77 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1667.
23. The composition of claim 12, comprising 10.sup.3 to 10.sup.13
cells of the bacteria strain per gram of composition.
24. The composition of claim 12, further comprising lactulose.
25. The composition of claim 12, further comprising bile acid-based
preparations, such as ursodeoxycholic acid and tauroursodeoxycholic
acid.
26. A method for preventing and treating diseases caused by or
associated with an altered metabolism of bile acids, said method
comprising administering at least one bacteria strain characterized
by exhibiting: (a) a 7 .alpha.-dehydroxylase activity of less than
50%, and (b) a bile acid deconjugation activity of less than 50%,
and descendants, or a mutant or derivative thereof preserving
activities (a) and (b).
27. method of claim 26, wherein the at least one bacteria strain is
a gram-positive bacteria strain.
28. The method of claim 26, wherein the bacteria strain belongs to
a species selected from the group consisting of Streptococus
thermophilus, Streptococcus faecium, and Lactobacillus
bulgaricus.
29. The method of claim 28, wherein the bacteria strain is
Streptococcus thermophilus YS 52 deposited with the CNCM,
Collection Nationale de Culturess de Microorganismes, Institut
Pateur, under the accession number I-1670.
30. The method of claim 28, wherein the bacteria strain is
Streptococus thermophilus YS 46 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1668.
31. The method of claim 28, wherein the bacteria strain is
Streptococcus thermophilus YS 48 deposited with the CNCM,
Collection Nationale de Cultures de Microorganismes, Institut
Pasteur, under the accession number I-1669.
32. The method of claim 28, wherein the bacteria strain is
Streptococcus faecium SF 3 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes,Institut Pasteur, under
the accession number I-1671.
33. The method of claim 28, wherein the bacteria strain is
Lactobacillus bulgaricus LB 1 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1664.
34. The method of claim 28, wherein the bacteria strain is
Lactobacillus bulgaricus LB 3 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1665.
35. The method of claim 28, wherein the bacteria strain is
Lactobacillus bulgaricus LB 7 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1666.
36. The method of claim 28, wherein the bacteria strain is
Lactobacillus bulgaricus LB 77 deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, under
the accession number I-1667.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to strains of bacteria and
pharmaceutical compositions containing one or more of such strains
and the use of same for preventing and treating diseases associated
with or caused by an altered metabolism of bile acids.
[0003] 2. Discussion of the Background
[0004] Hepatic bile is a pigmented isotonic fluid with an
electrolyte composition resembling blood plasma. Major components
of bile include water (82 percent), bile acids (12 percent),
lecithin and other phospholipids (4 percent), and unesterified
cholesterol (0.7 percent). Other constituents include conjugated
bilirubin, proteins, electrolytes, mucus and the final products of
hepatic transformation of drugs, hormones, etc. The liver
production of bile, in basal conditions, is approximately 500-1000
ml/day.
[0005] The primary bile acids, cholic acid (CA) and
chenodeoxycholic acid (CDCA), are synthesized from cholesterol in
the liver, conjugated with glycine or taurine, and excreted into
the bile. Secondary bile acids, including deoxycholic acid (DCA)
and lithocholic acid (LA), are formed in the colon as bacterial
metabolites of the primary bile acids. Other bile acids, called
tertiary bile acids (e.g.: ursodeoxycholic acid--UDCA), are formed
in the gut following the enzymatic epimerization of --OH groups on
sterol rings by the intestinal flora.
[0006] In normal bile, the ratio of glycine to taurine conjugates
is about 2:1, while in patients with cholestasis, increased
concentrations of sulfate and glucuronide conjugate of bile acids
are often found. The intestinal microflora transforms the bile
acids into different metabolites. These biotransformations include
the hydrolysis of the bond between the bile acid and taurine or
glycine, with formation of unconjugated or free bile acids and
taurine or glycine. The unconjugated bile acids are therefore made
available for the oxidation of the hydroxylic groups in positions
C3, C7, and C12 and for the dehydroxylation in positions 7.alpha.
and 7.beta.. This latter transformation leads to the formation of
the secondary bile acids DCA and LA. The primary bile acids,
deconjugated bile not transformed, and the secondary biliary acids
are reabsorbed from the gut lumen and enter the portal bloodstream,
then are taken up by hepatocytes, conjugated with glycine or
taurine and resecreted into the bile (enterohepatic
circulation).
[0007] Normally, the bile acid pool circulates approximately 5 to
10 times daily. Intestinal absorption of the pool is about 95%
efficient, so fecal loss of bile acids is in the range of 0.3 to
0.6 g/day. The fecal loss is compensated by an equal daily
synthesis.
[0008] For this reason the composition of the pool of biliary acids
present in the bile is the result of complex interactions occurring
between the liver and the microflora enzymes.
[0009] Deconjugation activity is a characteristic shared by many
bacteria, aerobes and anaerobes, but is particularly common among
the obligate anaerobic bacteria, i.e. Bacteroides, Eubacteria,
Clostridia, Bifidobacteria, etc. The majority of the bacteria is
active against both glycine and taurine conjugates; however, some
of them have a certain degree of specificity, depending on the
bound amino acid and the number of hydroxides bound to the steroid
nucleus. The free biliary acids obtained following the action of
the bacterial hydrolases can undergo the oxidation of the hydroxide
groups present at the C3, C7, and C12 positions by the
hydroxysteroidodehydrogenase.
[0010] The interest in the metabolic disorders of biliary acids
comes from the hypothesis that biliary acids and/or metabolites
thereof are involved in the pathogenesis of some hepato-biliary and
gastroenterologic diseases: biliary dyspepsia, cholelithiasis,
acute and chronic hepatopathies, inflammatory diseases of the
colon, etc.
[0011] Very often in literature the hydrophobicity of the bile acid
is correlated with detergency; the secondary bile acids are more
hydrophobic than the primary bile acids, the deoxycholic acid (DCA)
being actually more detergent than the cholic acid (CA). Therefore
an increased concentration of DCA in the bile may involve: a) an
augmentation of the secretion of cholesterol, with increased
saturation index; b) a cytotoxic effect on the liver cells.
[0012] For this reason a qualitative modification of the bile acids
pattern could be a decisive factor, especially in treating the
above-mentioned pathologies.
[0013] Thus, there remains a need for effective bacterial strains
or compositions that, by reducing the 7.alpha.-dehydroxylase
activity and at the same time deconjugation, can be used for
treating and/or preventing diseases associated with metabolic
disorders of the biliary acids.
[0014] No bacteria strains have been found that are capable of
qualitatively modifying the bile acid pattern in such a way.
SUMMARY OF THE INVENTION
[0015] Accordingly, it is one object of the present invention to
provide novel strains of bacteria, in particular gram-positive
bacteria, which are useful for treating and/or preventing diseases
associated with or caused by a metabolic disorder of biliary
acids.
[0016] It is another object of the present invention to provide
pharmaceutical compositions which contain one or more strains of
such bacteria and are useful for treating and/or preventing
diseases associated with or caused by a metabolic disorder of
biliary acids.
[0017] It is another object of the present invention to provide a
novel method for treating and/or preventing diseases associated
with or caused by a metabolic disorder of biliary acids.
[0018] The foregoing and other objects, which will become more
apparent during the following detailed description, have been
achieved by the inventors, who have found bacteria strains having a
reduced or zero 7.alpha.-dehydroxylase activity and a reduced or
zero ability to deconjugate bile acids. This is in contrast with
the previous known art. Accordingly, the present invention provides
the use of such strains to modify the bile acid metabolism in a
useful manner to prevent or treat diseases caused by or associated
with metabolic disorders of biliary acids.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] Thus, in a first embodiment, the present invention provides
novel strains of bacteria which have a 7.alpha.-dehydroxylase
activity of less than 50%, preferably less than 25%, and a
conjugated bile acid deconjugation activity of less than 50%,
preferably less than 25%.
[0020] The essential features of the strains according to the
present invention are defined in claim 1; specific strains having
said features are defined in the dependent claims 2 to 11.
[0021] The present invention also provides a pharmaceutical
composition for treating and/or preventing diseases associated with
or caused by an altered metabolism of biliary acids, said
composition comprising at least one bacteria strain according to
the present invention. The essential features of the composition
according to the present invention are defined in claim 12;
specific embodiments of said composition are defined in the
dependent claims 13 to 25.
[0022] In another embodiment, the present invention provides a
method for treating and/or preventing diseases caused by or
associated with an altered metabolism of biliary acids by
administering to a patient in need thereof one or more strains of
bacteria which have a 7.alpha.-dehydroxylase activity of less than
50%, preferably less than 25%, and a conjugated bile acid
deconjugation activity of less than 50%, preferably less than 25%,
or a pharmaceutical composition containing one or more such strains
of bacteria.
[0023] The essential features of said method are defined in claim
26; specific embodiments are defined in the dependent claims 27 to
36.
[0024] In the context of the present invention, the diseases
associated with or caused by a metabolic disorder of the biliary
acids include liver diseases and diseases of the digestive
apparatus, such as blind loop syndrome, biliary gallstones,
cirrhosis, chronic hepatopathies, acute hepatopathies, cystic
fibrosis, intrahepatic cholestasis, intestinal inflammatory
diseases, colonpathies, malabsorption. The present pharmaceutical
compositions may also be used to prevent the onset of biliary
gallstones in women during pregnancy or subsequent periods and in
subjects undergoing weight-loss programs or diets.
[0025] The 7.alpha.-dehydroxylase activity of the bacteria strain
should be less then 50%, preferably less than 25%. The
7.alpha.-dehydroxylase activity values are those measured by the
method described in Example 1 below. Specifically, the 10.sup.7
cells of the strain in question are incubated at 37.degree. C. for
48 hours, in 15 ml of the specific culture medium with the addition
of 2 mg/ml of glycocholic acid (GCA) or 2 mg/ml of taurocholic acid
(TCA), and then the amount of 7.alpha.-dehydroxylated product is
measured. The percentage value for the 7.alpha.-dehydroxylase
activity is calculated by the following formula: 1 7 -
dehydroxylase activity = mass of 7 - dehydroxylated product after
48 hours of incubation mass of GCA or TCA at the start of
incubation .times. 100
[0026] The 7.alpha.-dehydroxylase activity for any given strain is
the higher of the numbers measured for GCA and TCA.
[0027] Based on the above, the bacteria strain to be administered
should in addition have a conjugated bile acid deconjugation
activity of less than 50%, preferably less than 25%. The ability to
deconjugate bile acid is determined by using the same incubation
procedure described for measuring the 7.alpha.-dehydroxylase
activity followed by measuring the amount of deconjugated product
formed. The deconjugation activity is calculated using the
following formula: 2 Deconjugation activity = mass of deconjugated
GCA or TCA after 48 hours of incubation mass of GCA or TCA at the
start of incubation .times. 100
[0028] The deconjugation activity for any given strain is the
higher of the numbers measured for GCA and TCA.
[0029] The bacteria strains of the present invention may be
administered enterically. Preferably, the bacteria strains of the
present invention are administered orally.
[0030] Although a single bacteria strain may be administered, it is
also possible to administer a mixture of two or more bacteria
according to the present invention.
[0031] Although the exact dosage of bacteria to be administered
will vary with the condition and size of the patient, the exact
disease being treated, and the identity of the strains being
administered, good results have been achieved by administering
10.sup.3 to 10.sup.13 cells of the bacteria/g, preferably 10.sup.8
to 10.sup.12 of the bacteria strain/g. To achieve the good effects
of the present invention, it is preferred that the strain be
administered in an amount and a concentration sufficient to result
in the intestines of the patient being populated with an important
amount thereof. Thus, it is preferred that the strain be
administered in a composition which contains 10.sup.3 to 10.sup.13
cells of the strain/g, preferably 10.sup.8 to 10.sup.12 cells of
the strain/g and that the composition be administered in such a
regimen so that the patient receives 100 mg to 100 g of the
strain/day, preferably 1 g to 20 g of the strain/day, for a period
of 1 to 365 days, preferably 3 to 60 days in case of therapy, or
according to periodical cycles in case of prophylaxis. The bacteria
strain may be administered in any form suitable for enteral
administration, such as capsules, tablets, or liquids for oral
administration or liquids for enteral administration.
[0032] Typically, the administration of the bacteria strain
according to the present invention can be prescribed after the
diagnosis of metabolic disorders of the biliary acids. However, in
the case of the prophylaxis of biliary gallstones, the strain may
be administered when the subject is determined to belong to an
at-risk population, such as becoming pregnant or beginning a
weight-loss program or diet. In addition, the present strain of
bacteria may be administered after a patient has had their
gallbladder removed.
[0033] In a preferred embodiment, the coadministration of lactulose
is provided when the disease being treated is cirrhosis. Suitably,
the lactulose is administered in an amount of 100 mg to 100 g/day,
preferably 1 g to 20 g/day.
[0034] In another preferred embodiment, the coadministration of
bile acid-based preparations, such as ursodeoxycholic acid or
tauroursodeoxycholic acid, is provided. Suitably, the
ursodeoxycholic or tauroursodeoxycholic acid is administered in an
amount of 10 to 3,000 mg/day, preferably 50 to 800 mg/day.
[0035] The present invention finally provides novel pharmaceutical
compositions for treating and/or preventing the metabolic disorders
of the biliary acids which comprise (a) one or more strains of
bacteria having a 7.alpha.-dehydroxylase activity of less than 50%,
preferably less than 25%, and a bile acid deconjugation activity of
less than 50%, preferably less than 25%, and (b) a pharmaceutically
acceptable carrier. Preferably, the present pharmaceutical
compositions contain the strain(s) of bacteria in a concentration
of 10.sup.3 to 10.sup.13 cells/g, preferably 10.sup.8 to 10.sup.12
cells/g. The pharmaceutically acceptable carrier may be any which
is suitable for enteral administration and is compatible with the
strain of bacteria, such as dextrose, calcium carbonate together
with different additional substances such as starch, gelatin,
vitamins, antioxidants, stains or taste-improving substances.
[0036] As an optional component, the compositions of the invention
may possibly contain a drug compatible with the bacteria employed
and capable of potentiating the activity of the active ingredients
present. Anticholinergic drugs, antihistamines, adrenergic,
antiulcer, antiacid, antidiarroic, and anti-inflammatory drugs,
sedatives, antipyretis, choleretics antirheumatic agents, analgesic
drugs, diuretics, antiseptic agents, antilipemic hepatoprotective
drugs and drugs active on the gastrointestinal motility (e.g.,
trimebutine) may be herein mentioned.
[0037] When treating cirrhosis, it is preferred that the
pharmaceutical composition further comprise lactulose. Suitably,
the composition will contain sufficient lactulose to result in the
administration of 100 mg to 100 g/day, preferably 1 g to 20 g/day
of lactulose. When treating biliary cirrhosis and chronic
hepatitis, it is preferred that the pharmaceutical composition
comprise bile acid-based preparations, such as ursodeoxycholic acid
or tauroursodeoxycholic acid. Suitably, the composition will
contain sufficient bile acid preparation to result in the
administration of 10 to 3,000 mg/day of such bile acid
preparations, preferably 50 to 800 mg/day of ursodeoxycholic acid
or tauroursodeoxycholic acid.
[0038] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLES
Example 1
[0039] Strains of the following species have been tested:
Streptococcus thermophilus, Streptococcus faecium, Lactobacillus
acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum,
Bifidobacterium infantis. Each strain (10.sup.7 CFU) was cultivated
in duplicate in specific nutrient broths (15 ml); "CFU" means
"colony forming units".
[0040] List of the Employed Culture Media Depending on the
Different Species
1 Bifidobacterium infantis: MRS + 0.5% glucose (added after
sterilization by diluting a 20% sterile solution) Streptococcus
thermophilus: M17 All the remaining strains MRS
[0041] Composition of the MRS Broth:
2 g/liter universal peptone 10.0 g meat extract 5.0 g yeast extract
5.0 g D(+)-glucose 20.0 g potassium hydrogen phosphate 2.0 g Tween
80 1.0 g dibasic ammonium citrate 2.0 g sodium acetate 5.0 g
magnesium sulfate 0.1 g manganous sulfate 0.05 g
[0042] Preparation: dissolve 50 g/l in distilled water, sterilized
at 121.degree. C. for 15 minutes--pH 6.5.+-.0.1 at 25.degree.
C.
[0043] Composition M17 broth (Merck):
3 g/liter soybean flour peptone 5.0 g meat peptone 2.5 g casein
peptone 2.5 g yeast extract 2.5 g meat extract 5.0 g D(+)-lactose
5.0 g ascorbic acid 0.5 g sodium-.beta.-glycerophosphate 19.0 g
magnesium phosphate 0.25 g
[0044] Preparation: dissolve 42.5 g/l in distilled water,
sterilized at 121.degree. C. for 15 minutes--pH 7.2.+-.0.1 at
25.degree.
[0045] Bifidobacterium infantis was cultivated under anaerobic
conditions since it is known that it is an anaerobic bacterium.
After 24 hours of incubation at 37.degree. C. to each tube was
added an amount of bile salt equivalent to 30 mg in order to obtain
a final concentration of 2 mg/ml. The bile acids employed are:
glycocholic acid (GCA) and taurocholic acid (TCA), obtained from
Sigma Chemicals. Each bile acid was added separately to each series
of bacterial cultures.
[0046] After 48 hours of incubation, isopropanol, 3 ml, was added
for 2 minutes. Then it was centrifuged at 400 rpm for 15 minutes
and the supernatant was collected (5 ml). The supernatant was kept
refrigerated at -30.degree. C. until it was analyzed. The
percentage of conjugated bile salt present was determined by HPLC
(high performance liquid chromatography) utilizing a Gilson
apparatus equipped with a detector Diode array mod 1000 and a
Spherisorb 5 .mu.m ODS 2 C18 reverse phase column, a mobile phase
composed by methanol/buffered phosphate (20 mMol), pH 2.5 in
water/acetonitrile/water (150:60:20:20 by volume), a fluid speed of
0.85 ml/min, at a wavelength of 205 nm; 100 .mu.l of the sample to
be tested, dried under nitrogen, were extracted with 100 .mu.l of
the mobile phase containing as an internal standard
7.alpha.-OH-12.alpha.-OH-- dihydroxy-58-cholanic acid (Calbiochem
U.S.A.) at a concentration of 2 mg/ml.
[0047] The recovery percentage of the bile acid incubated with the
bacterial cultures was calculated by the ratio of the area of the
bile acid to be detected (GCA or TCA) to the area of the internal
standard. When the quantity of the conjugated bile acid found in
the bacterial cultures after 48 hours of incubation was less than
50%, thin layer chromatography (TLC) was performed on silica 60 gel
plates to detect the presence of CA and DCA, using a mobile phase
of cyclohexane/isopropanol/a- cetic acid (30:10:1 by volume). On
every plate, 20 .mu.l of the alcoholic extract of the sample, 20
.mu.l of a solution of CA and DCA, and 20 .mu.l of CA, 20 .mu.l of
DCA, were spotted. The plates after development at room
temperature, were treated with sulfuric acid and warmed at
145.degree. C. until the appearance of the colored spots.
[0048] The results of the deconjugation experiments (Table I) show
that 5 out of the 16 strains tested with GCA were able to
completely deconjugate the bile acid added to the culture, as
previously reported in the literature and widely known to all
researchers. Surprisingly, ten strains were able to deconjugate GCA
but not completely, ranging from 9 to 90 percent (Table I). There
was no difference among aerobic and anaerobic bacteria. Two
strains, Streptococcus thermophilus YS 52 and Bifidobacterium
infantis Bi 6 do not have any deconjugating activity for GCA. The
strain YS 52 in addition does not attack the bile acid--taurine
bond.
[0049] Only one out of the 16 strains tested was able to totally
deconjugate the TCA: the Bifidobacterium infantis Bi 6.
[0050] The results of the dehydroxylation experiments (Table II)
show that only one (Bi 4) out of the 16 strains is able to
completely dehydroxylate GCA. Six strains did not dehydroxylate at
all: YS 52; SF 2; SF 4; LA 3; LA 10; and Bi 6. The other strains
were able to dehydroxylate GCA but not completely, ranging from 9%
to 90%. As to TCA, seven strains do not dehydroxylate it at all: YS
52; SF 3; LA 3; LA 10; LB 1; LB 7; and LB 77. One strain, Bi 6,
dehydroxylated TCA completely; the other strains dehydroxylated TCA
according to varying percentages.
4TABLE I Percentage of deconjugation of GCA and TCA by bacterial
cultures after 48 hours of incubation ACCESSION BACTERIUM NO. GCA %
TCA % Streptococcus thermophilus YS 46 I-1668 9 9 Streptococcus
thermophilus YS 48 I-1669 17 11 Streptococcus thermophilus YS 52
I-1670 0 0 Streptococcus faecium SF 2 100 3 Streptococcus faecium
SF 3 I-1671 27 0 Streptococcus faecium SF 4 100 12 Lactobacillus
acidophilus LA 3 100 80 Lactobacillus acidophilus LA 10 100 95
Lactobacillus bulgaricus LB 1 I-1664 9 0 Lactobacillus bulgaricus
LB 3 I-1665 20 12 Lactobacillus bulgaricus LB 7 I-1666 14 0
Lactobacillus bulgaricus LB 77 I-1667 20 0 Bifidobacterium infantis
Bi 2 80 15 Bifidobacterium infantis Bi 3 90 10 Bifidobacterium
infantis Bi 4 100 26 Bifidobacterium infantis Bi 6 0 100
[0051]
5TABLE II Percentage of dehydroxylation of GCA and TCA by bacterial
cultures after 48 hours of incubation ACCESSION BACTERIUM NO. GCA %
TCA % Streptococcus thermophilus YS 46 I-1668 9 9 Streptococcus
thermophilus YS 48 I-1669 17 11 Streptococcus thermophilus YS 52
I-1670 0 0 Streptococcus faecium SF 2 0 3 Streptococcus faecium SF
3 I-1671 27 0 Streptococcus faecium SF 4 0 12 Lactobacillus
acidophilus LA 3 0 0 Lactobacillus acidophilus LA 10 0 0
Lactobacillus bulgaricus LB 1 I-1664 9 0 Lactobacillus bulgaricus
LB 3 I-1665 20 12 Lactobacillus bulgaricus LB 7 I-1666 14 0
Lactobacillus bulgaricus LB 77 I-1667 20 0 Bifidobacterium infantis
Bi 2 80 15 Bifidobacterium infantis Bi 3 90 10 Bifidobacterium
infantis Bi 4 100 26 Bifidobacterium infantis Bi 6 0 100
[0052] These strains have been deposited with the CNCM, Collection
Nationale de Cultures de Microorganismes, Institut Pasteur, 28 rue
du Dr Roux, 75724 Paris Cdex 15, France, under the following
accession numbers:
6 Streptococcus thermophilus YS 46: I-1668 Streptococcus
thermophilus YS 48: I-1669 Streptococcus thermophilus YS 52: I-1670
Streptococcus faecium SF 3: I-1671 Lactobacillus bulgaricus LB 1:
I-1664 Lactobacillus bulgaricus LB 3: I-1665 Lactobacillus
bulgaricus LB 7: I-1666 Lactobacillus bulgaricus LB 77: I-1667
[0053] The following strains are on the contrary kept at the Centro
Ricerche Sitia-Yomo S.p.A.,--strada per mercino 3-ZELO BUON PERSICO
(MILAN)--ITALY, distinguished by the below-reported
identifiers:
7 Streptococcus faecium SF 2: SF 2 Streptococcus faecium SF 4: SF 4
Lactobacillus acidophilus LA 3: LA 3 Lactobacillus acidophilus LA
10: LA 10 Bifidobacterium infantis Bi 2: Bi 2 Bifidobacterium
infantis Bi 3: Bi 3 Bifidobacterium infantis Bi 4: Bi 4
Bifidobacterium infantis Bi 6: Bi 6
[0054] These results demonstrate that the majority of the strains
tested by us have a low capability to deconjugate the bile acids
and that there are strains that do not deconjugate at all. This
observation is surprising in that it has not been known that the
lactic acid bacteria deconjugated the biliary salts. Furthermore,
it is evident that the enzymes of the strains are selective for the
specific bile acid bound on the side chain. In this study, the
clearest example is offered by the Bifidobacterium infantis Bi 6.
This strain is not able to deconjugate the glycine-conjugated bile
acid but is able to totally deconjugate the taurine-conjugated bile
acid. Some other strains (LB 1, LB 7, LB 77, and SF 3) are unable
to deconjugate TCA but are able to deconjugate GCA to a certain
extent.
[0055] To conclude, strains have been discovered that have a weak
or zero capability to deconjugate and dehydroxylate.
Example 2
[0056] Three healthy volunteers were tested for their content of
bile acids following treatment with a lactobacilli preparation
containing 1.times.10.sup.11 cells of Streptococcus thermophilus YS
52 per gram for a daily total of 6 g for 28 days. Before beginning
the treatment and after 12 hours starvation, the subjects were
intubated and the gallbladder bile, following stimulation with
ceruletide, was collected and frozen at -80.degree. C. The
gallbladder contraction was assessed by echography and the position
of the tube, in the second portion of the duodenum was checked by
Rx (fluoroscopy).
[0057] After a 4 week treatment, the subjects underwent a second
intubation and collection of bile. The bile samples were then
tested for their content of some bile acids as previously
described. The results are shown in Table III.
8 TABLE III Patient #1 Patient #2 Patient #3 Bile Acid Before After
Before After Before After Glychenodeoxycholic 32 15 22 15 28 12
Glycodeoxycholic 6 5 9 2 4 3 Glycoursodeoxycholic 1 5 1 7 1 4
Taurocholic 9 26 15 25 12 21 Taurodeoxycholic 1 3 5 8 3 9 NOTE:
(the bile acids are listed following the hydrophilic capacity
order, that is in inverse relation to detergency) Taurocholic
Taurodeoxycholic Glycoursodeoxycholic Glycodeoxycholic
Glychenodeoxycholic
[0058] This experiment is a confirmation of what is shown in
Example No. 1, that is: a lower deconjugation in one of the primary
bile acids if bacteria being the object of the present invention
are administered. The achieved result is a longer maintenance of
the primary bile acids in the enterohepatic circulation.
[0059] The properties of the bile acids are reported in the note to
Table III. Thus, according to these results the administration of
selected strains of bacteria can reduce the detergency property and
therefore the cytolytic activity of the bile acids.
Example 3
[0060] Fourteen patients with chronic hepatitis were treated with a
bacterial preparation containing Streptococcus thermophilus YS 46
and YS 48 (two strains), and Lactobacillus bulgaricus LB 1, LB 7,
and LB 77 (three strains). Each strain had been brought to a
concentration of 150.times.10.sup.9 cells per gram before being
mixed with the others, to prepare a mixture containing the same
parts by weight of each strain. 6 grams per day of said mixture
were administered for 28 days. Liver enzymes were measured before
and after the treatment, and the results are shown in Table IV.
9TABLE IV Influence of the Treatment with the Bacterial Mixture on
Liver Enzymes Aspartate Transaminase (AST; SGOT) and alanine
transaminase (ALT; SGPT) AST (SGOT) ALT (SGPT) Patient Before After
Before After #1 92 59 102 46 #2 89 67 96 42 #3 174 86 97 39 #4 121
91 102 66 #5 116 81 111 55 #6 156 87 94 76 #7 163 66 69 37 #8 78 64
122 57 #9 109 39 87 86 #10 166 70 102 48 #11 56 24 118 62 #12 131
83 96 79 #13 137 86 94 74 #14 84 87 144 114 Mean 119 71 102 63
Standard deviation 36 19 17 21 Significance Student p < 0.001 p
< 0.001 t test for paired data
[0061] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *