U.S. patent application number 09/091588 was filed with the patent office on 2002-09-05 for cyclohexane carbocyclic ester derivative and cyclodextrin complex and composition for treatment of helicobacter pylori infections.
Invention is credited to KAMODA, OSAMU, MIZOGUCHI, JUN-ICHI, SATO, SEIJI, TAMAKI, EIJI, YANAGI, TOSHIHARU.
Application Number | 20020123508 09/091588 |
Document ID | / |
Family ID | 18439052 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020123508 |
Kind Code |
A1 |
KAMODA, OSAMU ; et
al. |
September 5, 2002 |
CYCLOHEXANE CARBOCYCLIC ESTER DERIVATIVE AND CYCLODEXTRIN COMPLEX
AND COMPOSITION FOR TREATMENT OF HELICOBACTER PYLORI INFECTIONS
Abstract
Abstract The present invention relates to a pharmaceutical
composition which is appropriate for eradication or extermination
of Helicobacter pylon wherein
[4-[4-(4-methylbenzyloxycarbonyl)phenyl]phenyl
trans-4-guanidino-methylcyclohexanecarboxylate or an acid addition
salt thereof are compounded and it also relates to a complex
consisting them.
Inventors: |
KAMODA, OSAMU; (HYOGO,
JP) ; YANAGI, TOSHIHARU; (HYOGO, JP) ; TAMAKI,
EIJI; (HYOGO, JP) ; SATO, SEIJI; (HYOGO,
JP) ; MIZOGUCHI, JUN-ICHI; (HYOGO, JP) |
Correspondence
Address: |
DIKE, BRONSTEIN, ROBERTS AND CUSHMAN
INTELLECTUAL PROPERTY PRACTICE GROUP
P.O. BOX 9169
EDWARDS AND ANGELL
BOSTON
MA
02209
US
|
Family ID: |
18439052 |
Appl. No.: |
09/091588 |
Filed: |
August 12, 1998 |
PCT Filed: |
December 20, 1996 |
PCT NO: |
PCT/JP96/03723 |
Current U.S.
Class: |
514/310 |
Current CPC
Class: |
A61K 31/24 20130101;
A61P 1/04 20180101; A61K 47/6951 20170801; A61P 31/12 20180101;
B82Y 5/00 20130101 |
Class at
Publication: |
514/310 |
International
Class: |
A61K 031/47; A61K
031/185 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 1995 |
JP |
7-354660/95 |
Claims
1. A pharmaceutical composition having an anti-Helicobacter pylori
action which contains 4-[4-(4-methylbenzyloxycarbonyl)phanyl]phenyl
trans-4-guanidinomethylcyclohexanecarboxylate or an acid addition
salt thereof and cyclodextrin.
2. A pharmaceutical composition according to claim 1 having an
anti-Helicobacter pylori action which contains
4-[4-(4-methylbenzyloxycar- bonyl)phenyl]phenyl
trans-4-guanidinomethylcyclohexanecarboxylate or an acid addition
salt thereof and cyclodextrin in a compounding ratio of 1:1-4
(molar ratio).
3. A pharmaceutical composition having an anti-Helicobacter pylori
action which contains an inclusion compound of
4-[4-(4-metbylbenzyloxycarbonyl)p- henyl]phenyl
trans-4-guanidinomethyloyclohexanecarboxylate or an acid addition
salt thereof with cyclodextrin.
4. A pharmaceutical composition according to any of claims 1 to 3
wherein cyclodexitrin is .beta.-cyclodextrin.
5. A complex of 4-[4-(4-methylbenzyloxycarbonyl) phenyl]phenyl
trans-4-guanidinomethyloyclohexanecarboxylate or an acid addition
salt thereof with .beta.-cyclodextrin.
Description
TECHNICAL FIELD
[0001] The present invention provides a pharmaceutical composition
which is effective for eradication Helicobacter pylori living in
human stomach and, accordingly, said invention is utilized in the
medical field.
[0002] The present invention also provides a complex of
4-[4-(4-methylbenzyloxycarbonyl) phenyl]phenyl
trans-4-guanidinomethylcyc- lohexanecarboxylate or an acid addition
salt thereof with.beta.-cyclodextrin.
BACKGROUND ART
[0003] Helicobacter pylon is a bacterium which has recently
attracted considerable attention in view of its relationship with
diseases of digestive organs. It has become an important factor to
be taken into consideration especially when diseases, particularly
ulcer, of stomach and duodenum is investigated.
[0004] As the drugs effective for inhibiting the growth of
Helicobacter pylori, antibiotic substances such as ampicillin,
amoxicillin, cephalexin and clarithromycin and synthetic
antibacterials such as of loxacin and ciprof loxacin have been
known. It has been clarified that Helicobacter pylori has
participated in recurrence of ulcer and, at the same time,
prevention of recurrence of ulcer by a combined use of those
antibacterial with antiulcer agent has been investigated.
[0005] Incidentally, when anitiobiotics or synthetic antibacterials
are administered, they are absorbed from digestive organs into
blood and arrive at the diseased site to achieve the effect while
some of them are excreted after passing through the digestive
organs and, when the drug passes through the intestinal tract, many
bacteria living in intestine are killed whereby enterobacterial
fleora is unbalanced. Accordingly, administration of such a drug
for long term is to be avoided.
[0006] Since Helicobacter pylori is a bacterium living in stomach,
there has been a demand for a compound having a property that its
activity to said bacterium is effectively achieved in stomach and,
after that, the antibacterial activity decreases when it moves to
duodenum and then to small intestine and finally said activity
disappears. Incidentally, there is a compound whose generic name is
benexate hydrochloride and it has been known that said compound has
an anti-Helicobacter pylori activity with MICs ranging from 25 to
50 .mu.g/ml ("Diseases of Digestive Organs and Helicobacter pylor",
page 91; published by Medical Review).
[0007] The present applicant previously found a compound showing an
effective antibacterial activity which is specific to Helicobacter
pylori although it was a result of the test in vitro (WO 96/06825)
and, in view of the fact that the result in vivo does not always
coincide with that in vitro, the mode of its use was investigated
whereby the present invention has been achieved.
DISCLOSURE OF THE INVENTION
[0008] The present invention is to provide a composition of
4-[4-(4-methylbenzyloxycarbonyl)phenyl]phenyl trans-4-guani
dinomethylcyclohexanecarboxylate or an acid addition salt thereof
(hereinafter, it will be referred to as "the present compound")
which is suitable for exterminating or eradicating Helicobacter
pylori in living body by the use of the present compound.
[0009] It has been known that, although the present compound
exhibits an antibacterial action quite specifically to Helicobacter
pylori according to an in vitro test (WO 96/06825), such an
antibacterial activity in vitro is not available in an in vivo test
as it is. As one of the causes, we took the solubility of the
present compound into consideration but, after various
investigations, it has been found that an improvement in solubility
only is not able to afford the effect of eradication or
extermination of Helicobacter pylori and, at the same time, we have
found that a combined use of the present compound with
cyclodextrin, particularly with .beta.-cyclodextrin, is effective
in eradication or extermination of Helicobacter pylori in living
body whereupon the present invention has been achieved.
[0010] The present invention provides a pharmaceutical composition
having an anti-Helicobacter pylori action which contains
4-[4-(4-methylbenzyloxy- carbonyl) phenyl]phenyl
trans-4-guanidinomethylcyclohexanecarboxylate or an acid addition
salt thereof and .beta.-cyclodextrin.
[0011] The present invention also provides a complex of
4-[4-(4methyl-benzyloxycarbonyl)phenyl]phenyl
trans-4-guanidinomethylcycl- ohexanecarboxylate or an acid addition
salt thereof with .beta.-cyclodextrin.
[0012] The composite which is an object of the present invention is
also an effective component of the pharmaceutical composition of
the present invention and it consists of the present compound and
.beta.-cyclodextrin and may be prepared by grinding the two
substances or by making them an aqueous solution followed by
subjecting to a treatment of spray-drying, freeze-drying or drying
by means of concentration.
[0013] Further, even in the case where they are just mixed, it is
effective in eradication or extermination of Helicobacter pylori
which is an object of the present invention provided that said
mixture is dissolved In water. Therefore, such a mixture is covered
by the present invention as well.
[0014] Accordingly, the present invention provides a mixture or a
complex (inclusion compound) of 4-[4-(4-methylbenzyloxy
carbonyl)phenyl]phenyl trans-4-guanidinomethylcyclohexane
carboxylate or an acid addition salt thereof and
.beta.-cyclodextrin and also offers a composition containing the
same which has an anti-Helicobacter pylori action.
BRIEF EXPLANATION OF THE DRAWINGS
[0015] FIG. 1 shows the diffraction peak of the powder X-ray
diffraction of the complex of the present Invention. The charts of
SAMP-C and SAMP-D in FIG. 1 are those for the complex of the
present invention, that of SAMP-B is for .beta.-cyclodextrin and
that of SAMP-A is for a hydrochloride of the present compound.
[0016] FIG. 2 shows the result of the thermogravimetric
differential thermal analysis of the complex of the present
invention.
[0017] FIG. 3 shows the result of the thermogravimetric
differential thermal analysis of the present compound
(hydrochloride).
[0018] FIG. 4 shows the result of the thermogravimetric
differential thermal analysis of .beta.-cyclodextrin.
[0019] FIG. 5 shows the result of the thermogravimetric
differential thermal analysis of a mere 1:1 mixture of the present
compound (hydrochloride) with .beta.-cyclodextrin.
[0020] FIG. 6 shows a nuclear magnetic resonance spectrum of
hydrogen atoms which are directly bonded to carbon atoms of
.beta.-cyclodextrin.
[0021] FIG. 7 shows a nuclear magnetic resonance spectrum of
hydrogen atoms which are directly bonded to carbon atoms of a
.beta.-cyclodextrin moiety in the complex of the present
invention.
BEST MODES FOR CONDUCTING THE INVENTION
[0022] The present compound has been known already (WO 96/06825)
and can be manufactured by a known method. For example,
4-methylbenzyl 4-(4-hydroxyphenyl)benzoate is prepared by a
conventional method from 4-(4-hydroxyphenyl)benzoic acid and
4-methylbenzyl bromide and is made to react with
trans-4-guanidinomethylcyclohexanecarboxylic acid or a reactive
derivative thereof to give the present compound.
[0023] In addition, the present compound may be used as an acid
addition salt if necessary. Examples of the preferred salt are
salts with pharmaceutically acceptable organic and inorganic acids
such as hydrochloric acid, methanesulfonic acid, toluenesulfonic
acid, fumaric acid, oxalic acid, hydrobromic acid and sulfuric
acid.
[0024] With regard to cyclodextrin, that which is commercially
available may be used. Among the cyclodextrin, the use of
.beta.-cyclodextrin is particularly preferred. The amount of the
cyclodextrin used is 0.5-10 mole, preferably 1-4 moles or, more
preferably, 1-3 moles to one mole of the present compound. When the
molar ratio of cyclodextrin is less than 0.5, solubility decreases
and the chemical stability of the present compound which is a main
ingredient in a state of solution lowers. When the amount of
cyclodextrin used is too much, the content of the main ingredient
per unit weight becomes small whereby total amount to be
administered for giving a certain amount of the main ingredient
becomes large and that is not preferred.
[0025] The pharmaceutical composition of the present invention is
characterized in containing a complex of the present compound with
cyclodextrin, particularly with .beta.-cyclodextrin. If necessary,
various components and vehicles may be further added to the
pharmaceutical composition of the present invention.
[0026] The pharmaceutical composition of the present invention may
be made into a dosage form which is suitable for administration by
adding the above-mentioned compounded product and, if necessary,
various components and vehicles.
[0027] The compounded product of the present invention is offered
as powder which is prepared by grinding the mixture of the present
compound and .beta.-cyclodextrin in a ball mill device or prepared
by dissolving it in water with warming or by dissolving the present
compound by adding to .beta.-cyclodextrin dissolved in water with
warming followed by subjecting the resulting solution to
concentrating in vacuo, freeze-drying or spray-drying for
solidification.
[0028] Among the compounded product in accordance with the present
invention, chemical structure of the product prepared by grinding
with a ball mill device or the complex of the present compound with
.beta.-cyclodextrin obtained by dissolving with warming followed by
solidifying has not been definitely clarified but it will be
understood from the data of the physical properties measured which
will be mentioned later that the present compound and
.beta.-cyclodextrin act each other forming a united state (such as
an inclusion compound).
[0029] Thus, as shown in FIG. 1 where charts of results of powder
X-ray diffraction of the hydrochloride of the present compound
(SAMP-A), .beta.-cyclodextrin (SAMP-B) and the complex of the
present invention (SAMP-C and SAMP-D) are given, it is apparent
that there is no diffraction peak derived from crystals of the
present compound and of .beta.-cyclodextrin in the case of the
complex of the present invention, that they are in an amorphous
form in the complex of the present invention and accordingly that
there is a clear difference between the two.
[0030] Further, as shown in FIGS. 2-5 where the result of
thermogravimetric differential thermal analysis of the complex of
the present invention (FIG. 2), that of the present compound (FIG.
3), that of .beta.-cyclodextrin (FIG. 4), and that of a mere
mixture of the present compound and .beta.-cyclodextrin (FIG. 5)
are given, the present compound shows endothermic peaks at
151.5.degree. C. and 184.0.degree. C. (FIG. 3), .beta.-cyclodextrin
shows an endothermic peak at 90.degree. C. (FIG. 4; incidentally,
in FIG. 4, a loss curve per minute in the center of the figure as
seen in FIGS. 2, 3 and 5 is not shown), and a mere mixture of them
shows endothermic peaks at the three points corresponding to the
components thereof (FIG. 5). On the contrary, the complex of the
present invention shows only a gentle and single endothermic peak
at 53.7.degree. C. whereby it is apparent from the results of
thermogravimetric differential thermal analysis that the complex of
the present invention is not a mere mixture but is a complex
wherein both act each other.
[0031] Further, in FIGS. 6-7, nuclear magnetic resonance spectrum
of hydrogen atoms which are directly bonded to carbon atoms of
.beta.-cyclodextrin (FIG. 6) and that of hydrogen atoms at the same
positions of the complex of the present invention (FIG. 7) are
shown. The thing which is to be particularly noted in the spectra
is the peak which appears at 3.8-4.0 ppm and that is the
characteristic values of hydrogen atoms bonded to carbon atoms of
3-, 5- and 6-positions of .beta.-cyclodextrin.
[0032] In the complex of the present invention, said characteristic
value is shifted to the higher magnetic field side to an extent of
0.1 ppm and is available at 3.7-3.9 ppm and said fact supports that
there is an interaction between the present complex and
.beta.-cyclodextrin.
[0033] As such, it is believed that the complex of the present
invention is not a mere mixture of the present compound and
.beta.-cyclodextrin but is a complex wherein both act each
other.
[0034] The present invention will be further illustrated by way of
specific examples as hereunder although the present invention is
not limited thereto.
[0035] Referential Example (Manufacture of the Present
Compound)
[0036] 4-[4-(4-Methylbenzyloxycarbonyl)phenyl]phenyl
trans-4-guanidinomethyloyclohexanecarboxylate hydrochloride which
is used in the present invention was synthesized as follows. Thus,
1.11 g of thionyl chloride was dropped into a mixture of 5.95 g of
4-methylbenzyl 4-(4-hydroxyphenyl)benzoate [m.p. 128-130.degree.
C.; which was synthesized by a conventional method from
4-(4-hydroxyphenyl)benzoic acid and 4-methylbenzyl bromide], 1.48 g
of pyridine and 30 ml of dry tetrahydrofuran under ice-cooling.
This was stirred for three hours at room temperature, crystals
separated out therefrom were filtered off and the filtrate was
concentrated in vacuo. Dry pyridine (30 ml) was added thereto, the
mixture was cooled with ice and 2.00 g of
trans-4-guanidinomethylcyclohexanecarboxylic acid hydrochloride was
added thereto. The mixture was stirred overnight at room
temperature and concentrated In vacuo and the crystals separated
out therefrom by addition of acetone were collected by filtration
and well washed with isopropanol to give
4-[4-(4-Methylbenzyloxycarbonyl)phenyl]phenyl
trans-4-guanidinomethylcyclohexanecarboxylate hydrochloride.
[0037] Yield : 3.19 g.
[0038] m.p.: 171-173.degree. C.
[0039] A. Preparation of Drugs to be Tested and Solution to be
Administered
[0040] Example 1
[0041] .beta.-Cyclodextrin (8.469 g) was dissolved in 120 ml of
water with warming, 4 g of the present compound (hydrochloride) was
added thereto (molar ratio thereof being 1:1) and the mixture was
heated at 60-70.degree. C. for one hour to dissolve. This was
freeze-dried.
[0042] Solubility of this product at 25.degree. C. was as
follows.
[0043] In water: 408.mu.g/ml
[0044] In the first fluid (The Japanese Pharmacopoeia) (pH 1.2): 9
82 g/ml
[0045] In 0.1 M acetate buffer (pH 4.0): 158 .mu.g/ml
[0046] In 0.05 M phosphate buffer (pH 6.5): 9 .mu.g/ml
[0047] This was dissolved in distilled water with warming to make
the amount of the present compound (hydrochloride) 100 mg/10 ml and
subjected to administration.
[0048] Example 2
[0049] The present compound (hydrochloride) (100 mg) and 212 mg of
.beta.-cyclodextrin were dissolved In 10 ml of distilled water with
warming and subjected to administration.
[0050] Example 3
[0051] The mixture of the present compound (hydrochloride) (5 g)
and 11.7 g of .beta.-cyclodextrin (molar ratio being 1:1) was
ground for seven hours using CENTRIFUGAL BALL MILL (manufactured by
Retsch) under the conditions where three balls were used and speed
scale was adjusted to 70.
[0052] The resulting powder was subjected measurements for powder
X-ray diffraction and thermogravimetric differential thermal
analysis and the state was investigated.
[0053] The data were compared with those of the present compound
per se, .beta.-cyclodextrin and a mere 1:1 mixture (molar ratio) of
them.
[0054] In the powder X-ray diffraction, no diffraction peak derived
from the crystals of the present compound was noted and, therefore,
it is apparent that the present invention was made into amorphous
(refer to FIG. 1).
[0055] In the infrared absorption spectrum, the characteristic
absorptions derived from the two types of ester bond near 1742 cm
.sup.-1 and 1702 cm.sup.-1 noted in the present compound moved to
the higher wave number side. This shows that some physicochemical
interaction took place between the present compound and
.beta.-cyclodextrin.
[0056] It was further noted by the thermogravimetric differential
thermal analytical data that the endothermic peaks shown in FIGS.
3-5 which were noted in the present compound per se (FIG. 3),
.beta.-cyclodextrin (FIG. 4) and a mere mixture (molar ratio being
1:1) of them (FIG. 5) disappeared in the powder resulted here
(refer to FIG. 2). In addition, almost no change was noted in
hydrogen atom signals at the positions of C.sub.1, C.sub.2 and
C.sub.4 in the nuclear magnetic resonance spectrum (.sup.1H
resonance frequency: 400 MHz; concentration for the measurement:
2.8 mmol/liter; solvent: heavy water; external standard substance:
TSP; temperature for the measurement: 303.degree. K.). On the other
hand, the hydrogen atom signals which are the characteristic values
for .beta.-cyclodextrin appearing at 3.8-4.0 ppm for C.sub.3,
C.sub.5 and C.sub.6 derived from the hexose structure shifted to
3.7-3.9 ppm. When the bonding direction of hydrogen atoms at the
above-mentioned positions in hexose which constitutes
.beta.-cyclodextrin is taken into consideration, such a change in
said spectrum strongly suggests that inclusion takes place between
.beta.-cyclodextrin and the present compound.
[0057] Solubility of this product in water was 4 mg/ml (25.degree.
C.).
[0058] The product was dissolved in distilled water to make the
content of the present compound (hydrochloride) 100 mg/10 ml and
subjected to administration.
[0059] Example 4
[0060] The present compound (hydrochloride) (4 g) and 18.7 g of
.beta.-cyclodextrin (molar ratio being 1:2) were treated by the
same manner as in Example 3 to give powder. The resulting powder
showed the same physical property as that of the powder of Example
3 except the solubility (8.9 mg/ml).
[0061] The resulting powder was dissolved in distilled water to
make the content of the present compound (hydrochloride) 100 mg/ml
and subjected to administration.
[0062] Example 5
[0063] The present compound (2 g) and 18.7 g of .beta.-cyclodextrin
(molar ratio being 1:4) were treated by the same manner as in
Example 3 to give powder. The resulting powder showed the same
physical property as that of the powder of Example 3 except the
solubility (9.4 mg/ml).
[0064] The resulting powder was dissolved in distilled water to
make the content of the present compound (hydrochloride) 100 mg/ml
and subjected to administration.
[0065] Example 6
[0066] The present compound (2 g) and 14.0 g of .beta.-cyclodextrin
(molar ratio being 1:3) were dissolved in 200 ml of warm water and
spray-dried (temperature at inlet: 130.degree. C.; air flow rate: 6
ml/minute) to give powder. The resulting powder showed the same
physical property as that of the powder of Example 3 except the
solubility (8.0 mg/ml).
[0067] The resulting powder was dissolved in distilled water to
make the content of the present compound (hydrochloride) 100 mg/ml
and subjected to administration.
[0068] Example 7
[0069] .gamma.-Cyclodextrin (9.678 g) was dissolved in 100 ml of
water with warming, 4 g of the present compound (hydrochloride) was
added to the resulting solution (molar ratio being 1:1) and the
mixture was heated at 60-70.degree. C. for one hour followed by
freeze-drying.
[0070] Solubility of the product at 25.degree. C. was as
follows.
[0071] In water: 729 .mu.g/ml
[0072] In the first fluid (The Japanese Pharmacopoeia) (pH 1.2): 15
.mu.g/ml
[0073] In 0.1 M acetate buffer (pH 4.0): 431 .mu.g/ml
[0074] In 0.05 M phosphate buffer (pH 6.5): 2 .mu.g/ml This was
dissolved in distilled water with warming to make the amount of the
present compound (hydrochloride) 100 mg/10 ml and subjected to
administration.
[0075] Example 8
[0076] The present compound (hydrochloride) (2 g) and 14.0 g of
.beta.-cyclodextrin (molar ratio being 1:3) were treated by the
same manner as in Example 3 to give powder. The resulting powder
showed the same physical property as that of the powder of Example
3 except the solubility (9.0 mg/ml).
[0077] The resulting powder was dissolved in distilled water to
make the content of the present compound (hydrochloride) 100 mg/ml
and subjected to administration.
[0078] Comparative Example 1
[0079] The present compound (hydrochloride) per se was administered
as well and its solubility at 25.degree. C. was as follows.
[0080] In water: 24 .mu.g/ml
[0081] In the first fluid (The Japanese Pharmacopoeia) (pH 1.2):
<0.1 .mu.g/ml
[0082] In 0.1 M acetate buffer (pH 4.0); <0.1 .mu.g/ml
[0083] In 0.05 M phosphate buffer (pH 6.5): <0.1 .mu.g/ml
[0084] This was dissolved in distilled water containing 0.5% of
methyl cellulose to make the content of the present compound
(hydrochloride) 100 mg/ml and subjected to administration.
[0085] Comparative Example 2
[0086] A mixture of 3 g of the present compound (hydrochloride) and
6 g of polyvinylpyrrolidone K30 was dissolved in methanol and the
solution was evaporated to dryness in vacuo to give powder. Its
solubility at 25.degree. C. was as follows.
[0087] In water: 761 .mu.g/ml
[0088] In the first fluid (The Japanese Pharmacopoeia) (pH 1.2):253
.mu.g/ml
[0089] In 0.1 M acetate buffer (pH 4.0): 320 .mu.g/ml
[0090] In 0.05 M phosphate buffer (pH 6.5): 2 .mu.g/ml
[0091] This was dissolved in distilled water to make the content of
the present compound (hydrochloride) 100 mg/ml and subjected to
administration.
[0092] Incidentally, the method for measuring the solubility in
each example is as follows. Thus, an excessive amount of the
present compound was added to a certain amount of solvent, the
mixture was shaken for one hour, the supernatant liquid was removed
and the amount of the present compound contained therein was
measured wherefrom the solubility was calculated.
[0093] Test Example
[0094] (1) Preparation of Animals Infected by Helicobacter
pylori
[0095] Helicobacter pylori ATCC 43504 (100 .mu.l) was inoculated
into Brain heart infusion broth containing 10% calf serum and
incubated at 37.degree. C. for 24 hours in a microaerophilic
atmosphere. The culture broth (0.5 ml containing about
3.times.10.sup.6 CFU/ml) was administered orally to Mongolian
gerbils (MON/Jms/Gba Slc; male of six weeks age) after fasting for
24 hours, then neither feed nor water was given for four hours and,
after that, the animals were given with feed and water for ten
days.
[0096] (2) Administration of the Drug to be Tested
[0097] Three infected animals as prepared in (1) were used per
group. They were fasted from 20 hours before the 11th day (although
water was made freely taken by them) and, during the fasted stage,
10 ml/kg each of the test drugs for administration prepared in
Examples and Comparative Examples were orally given to them at
9:00, 13:00 and 17:00 o'clock on the 11th day. Feeding was started
as from 21:00 o'clock and, after 20 hours from the final
administration, stomach of the animals was removed and Helicobacter
pylori living therein was subjected to a viable cell counting.
[0098] (3) Method of Counting the Viable Cells
[0099] The stomachs were cut open and the contents were removed (no
washing conducted) and homogenized in 20 ml of 0. 1 M phosphate
buffer (pH 7.2) to give a sample for viable cell counting. This
sample was diluted with 0.1 M phosphate buffer (pH 7.2) (0, -1, -2,
-3), each 0.1 ml thereof was smeared on a selective medium,
incubated at 37.degree. C. in a microaerophilic atmosphere. The
growing colonies were counted after 6 days.
[0100] (4) Composition of the selected medium used here was as
follows. Thus, it consisted of 43 g of brucella agar, 100 ml of
horse blood, 10 mg of vancomycin, 2500 U of polymyxin B, 5 mg of
trimethoprim, 2 mg of amphotecillin B, 50 mg of
2,3,5-triphenyltetrazolium chloride and 900 ml of distilled water
and its pH was 7.2 .+-.0.2.
[0101] The case where no test drug was administered was used as a
control (administered only with distilled water containing 0.5% of
methyl cellulose) and the resulting effect was compared with that
where the test drug was administered using the eradication rate.
The result is given in Table 1.
[0102] Incidentally, the eradication rate was calculated according
to the following calculating expression.
[0103] Eradication Rate={[(Viable Cell Numbers in Control)--(Viable
Cell Numbers in Drug-Administered Group)]/[Viable Cell Numbers in
Control ]}.times.100
[0104] The viable cell numbers in stoach of infected Mongolian
gerblis in the control group were 3.7.times.10.sup.4 CFU/stomach
average of three animals).
1TABLE 1 Result of Measurement of Eradication Rate Drug Tested
Eradication Rate (%) Example 1 1 99.1 2 >99.9 3 >99.9 Example
2 1 99.4 2 99.4 3 99.7 Example 3 1 95.8 2 97.2 3 >99.9 Example 4
1 99.1 2 >99.9 3 99.2 Example 5 1 99.9 2 >99.9 3 99.99
Example 6 1 >99.9 2 98.2 3 98.7 Example 7 1 78.9 2 91.4 3 99.5
Example 8 1 99.8 2 99.3 3 99.8 Comp. Ex. 1 1 95.7 2 62.2 3 0 Comp.
Ex. 2 1 87.0 2 89.2 3 62.2 Viable cell numbers in the control: 3.7
.times. 10.sup.4 CFU/stomach (average of three animals)
* * * * *