Note: Descriptions are shown in the official language in which they were submitted.
- 2 ~
" . ~ . "
These germs, which are distinct from Helicobacter pylori, are also further ` -
transferred from hurnan being to human being. Infestation with non~
Helicobacter ~lori bacteria must not be underestimated. Most of these
bacteria, lik0 Helicobacter pylori, live in the same ecological niche, that
is, on and in the mucosa of the stomach, under the protective mucus
layer. Those who keep dogs and cats are particularly at risk of infecting
themselves with these germs (for example, with Flexispira rappini).
Clinical pictures in which the bacteria mentioned above play a part arediscussed in the following publications: "Chronic Alcoholic Gastritis,"
Rajiv Uppal, MD; Syed K. Lateef, MD; Mark A. Korsten, MD; Fiorenzo ` `
Paronetto, MD; Charles S. Lieber, MD, Archives of Internal Medicine,
April 1991, Vol. 151, pages 760 -764; "Detection and identification of
Helicobacter pylori by the polymerase chain reaction," C. Clayton, K.
Kleanthous, S. Tabaqchali, J. Clin Pathol 1991, 44, pages 5t5-516. An
indication of a connection between Helicobacter pylori and stomach :
cancer is found in the essay "Helicobacter pylori and gastric carcinoma,"
R.J.L.F. Loffeld, I. Willems, J.A. Flendrig & Arends, J.W., Histopathology ~ -
1990, 17, pages 537-541.
Preparations that have been known thus far for treating the diseas0s .mentioned in the publications cited above exhibit an unfavorable
relationship o~ efficacy and side-effects. Due to the grave consequences
of an infection with these pathogens, see above, an effective prophylaxis
and treatment that poses no cause for concern is necessary.
The aforementioned di eases are treated according to therapeutic ~ ~
-' ~: ..
_2 1 4 8 2 ~
. . . ~ ':
~ `:,.... `
methods that havo been uGied thus fa;with preparations, whose pnncipal ~active ingredient is, for example, bisrnuth salts. Since a mono-therapy : "
with these salts proved to be insùfficient, by way of complement, a
complicated antibiotic combination therapy had to be conducted (for
exampls, with amoxicillin and metronidazole). Due to long years of the ~ ;
use of bismuth preparations, their profile of side-effects is sufficiently
well-known: neurotoxic symptoms were noted in the foreground. For ` ` -`
quits some time, the benefit-to-risk ratio of bismuth preparations has
been called to question. The spectrum of side-effects of the antibiotics ` : `
that have been used thus far is sufficiently well known.
: '.: ' . .:
Such methods of treatment are described, for example, in the essay ~.
"Diagnostic value of decreasing IgG, IgA, and IgM antibody titres after
eradication of Helicobacter pylori," Timo U. Kosunen, Kari Seppala,
Seppo Sarna, Pentti Sipponen, The Lancet, Vol 339, 11 April 1992, page :893, as well as in the survey article by J. Ormand and N. Talley~
"Helicobacter pylori: Controversies and an Approach to Management" -from Màyo Clin. Proc. 65: 414-426, 1990. ~ ;
Beyond that, symptomatic treatment involving antacids, H2-blockers, and `; ;`- -~
ATPase-inhibitors that has been used thus far generally leads to an `~ ~ -
ovsrabundance of bacteria and/or fungi in the stomach (fecal flora), since
the acidic protection of the hollow organ has been lost. In conjunction
with the acidic inhibition, a massive decrease in the secretion of
Iysozyme from the gastric`glands occurs. This anti-bacterially effective
enzyme of the body's own is essential for the repression of pathogenic `~
germs. To that extent, the suppression of acid favors certain
etiopathogenetic factors for diverse gastro-intestinal lesions (such as
gastric carcinoma and carcinoids). Apart from special indications, acid ~.
suppression is obsolete, and for ethical and economic reasons, it should ~:
;: ~
- 4 - ~ ~ ~
-:-. - . ~, ...
be r~placed by lantibiotic prophylaxis and therapy.
. . . - .. -,
It is th~r~fore the task of ths present invention to indicatc a preparation
of the typ0 cit~d at thc outsot in such a way that It assures a high degree . ;
of efficacy without side-offects. '
This task is achieved according to the invention by virtue of the fact that
a defined dose of a polypeptide antibiotic is contained as the active ` -ingredient, as a result of the application of which an active ingredient -~. ; ;
concentration of at least 5 ~g/ml is achieved at the affected site.
Ths anti-bacterial effectiveness of lantibiotics, especially of nisin, is ~ d
already known. Use as a foodstuffs additive is described, for example, in
the European patent application EP-A-0 453 972. In addition, the anti-
bacterial effectiveness of nisin is also indicated in the United States `
patent 5 043 176. Th~ use of nisin in the cosmetic field is described in ~ ;
the published German patent application DE-OS 39 38 140.
. " '
The bacteria enumerated at the outset are the etiopathogenetic factor for ~ .chronic gastritis. The latter leads, over time, to atrophy of the mucosa,
and to intestinal metaplasia. The final stage of these processes is, as a ~ `
rule, a gastric tumor.
, ~
- ~,
As a result of timely prophylactic use of nisin, it would be possible to
achieve a drastic reduction in the incidence of these tumors. The
settlement of the stomach with pathogenic bacteria (such as
Gastrospirillum hominis or Helicobacter pylori) can give rise to a
weakening of the resistance of the mucosa, so that patients who are
compelled to take corticosteroids and non-steroidal antiphlogistics `~
(NSAIDS) on a regular basis develop peptic ulcers.
-. . . ~ ~. .
~, ~
~, . 21~82~jS ,
-5~
Even among this group of patients, a considerable reduction of this : `ailment can be achieved by tha timely prophylactic use of nisin, for
~xample. In this regard, some thought should also be devoted to the
addition of a nisinoid substance to alcoholic beverages, in order to ;; .
prevent stomach perforation, which is so feared among alcoholics, and ~ ;;
alcoholic gastritis. : ` `
, . . .
Approximately half of the world's population carries Helicobacter pylori. ; ~ ~ -
Due to suitable screening programs (serology, breath test, and PCR), ~:
groups at risk may be readily identified. In these casesj an economic
significance that should not be underestimated would be attached to the ~ "
prophylactic use of nisinoid substances.
- ~ .
The prophylactic use of nisin does not give cause for any medical
concern, since it is not absorbed, and since it is inactivated by pancreatic" ~-:
enzymes, especially alpha-chymotrypsin. ~: ;
The medication is applied until the treatment goal, namely, the ;~ ~ ` ;``
0radication of the germs, has been achieved. Since many of the
bacteria under discussion occur ubiquitously, reinfection must not be
precluded, however.
From the therapeutic standpointl the foliowing indications result~
- Duodenal ulcer
- Duodenal ulcerwith gastric metaplasia
- Gastriculcer ~- ~ p~
- Stomach lesions with intestinal metaplasia
- Erosions -
- chronically active gastritis ~ -
chronically inactive gastritis (e.g. atrophic) ~ ~
,
-2 ~ ~ 8 2 ~
. . . ~ ~ . .
,. ~ :.```, ~... ..
i i, . . .
;, ~. . ` ` ... ~
- 6-
- chronically inactive gastritis with metaplasia and/or dysplasia
- oth~r forms of gastritis `~
- acute gastritis -
- l~uodenitis -
- non-ulcerolls dyspepsia and functional dyspapsia
- other gastro-intestinal diseases
- Barrett's esophagus with dysplasia or metaplasia ` ~
- Diseasc on ths basis of haterotopic gastric mucosa in the digestive ~ -
- tract (e.g. in Meckel's diverticulum)
Other indicatlons include~
- Diseases on the basis of settlement of the efferent pathways ~ -
associated with the digestive tract by bacteria (Q~9. Brunner's glands) ~;
- endocrine disorders, such as hypergastrinemia and
hyperpepsinogenernia
- psychlatricdisorders (e~. depression)
- Ménétrier's disease
- Gastropathies, such as protein-losing hypertrophic gastropathy or
gastric Iymphomas.
.. . - :~
The use of a lantibiotic, especially of a nisinoid active ingredient, proves
to be an extremely effective measure for killing off aggressive gastric
bactsria~ Nisinoid substances are resistant to gastric juice, because they
: . . .
contain lanthionine and/or methyl lanthionine, and they can even develop
their optimal effect in a pH of 1-2, which is typically extant in the
stomach.
The use of nisinoid substances is also indicated in the case of pets and
farm animals, as well as laboratory and zoo animals. In these animals,
the ~xistence of bacteria that occur in the same ecological niche as - - ~
~,
2 ~ -8 ~
- 7-
Heticobacter pylori in human beings was shown. Undar normal
conditions, Helicobacter pylori is human-specific, and, with the exception
of primates, it does not occur in the aforementioned animals. " ~
Given the lack of sufficient study findings and taxonomic turbulences in ` ~ ~ .
this special sector, these spiral-shapad organisms are currently being
classified as in the publication "An Uncultured Gastric Spiral Organism Is
a Newly Identified Helicobacter in Humans," Jay V. Solnick, Jani
O'Rourke, Adrian Lee, Bruce J. Paster, Floyd E. Dewhirst and Lucy S.
Tomkins! The Journal of Infectious Diseases, 1993, 168, pages 379 ff.
The following bacteria were presented~
- Hclicobacterfelis DS3
- Helicobacterfelis CS1
- (Gaslrospirillum hominis) 2
- (Gastrospirillum hominis) 1 `~
- Helicobacterpylori . ~
- Helicobacteracinonyx : ~;: .`
- Helicobactersp. CL03
- Helicobacter mustelae
- Helicobacier fennelliae :
- Helicobacter muridarum
- Helicobacter cinaedi
- (Flexispira rappini)
- Wolinella succinogenes
- Campylobacter fetus ss fetus ;~
- Campylobacter largi
- Campylobactercoli ; :~
- Campylobacter jejuni
,. . ,., ~, ~ ~". ~ .
~ ~ 4 8 ~
- 8~
The bacteria of the Campylbacter-H01icobacter complex that are isolated
from animals induce gastro-intestinal lesions that are comparable to
those in human beings, so that here, too, the use of nisin seems to make `
economic sense. In addition, they are frequently transferred to human
beings, and then produce the well known picture of Helicobacter pylori.
This germ spectrum, as well, should be attacked with nisinoid
substances.
The most recent knowledge points to a bacterial etio-pathogenesis of
Crohn's disease and ulcerous colitis (chronic inflammatory intestinal
diseases). Here, too, some thought might be given to the use of
lantibiotics, especiallythat of nisinoid substances.
In ~ha production and use of the preparation, nisin's solubility in water
can be exploited. Solubility in water depends upon the pH value that is ;;
present in each case, and, for a quantity of water of 100 mill~liters, it is
12 grams in the cass of a pH value of 2.4, 1 gram in the case of a pH
value of 5.6, and 0.075 grams In the case of a pH value of 7Ø Thus, in
the case of low pH values in particular, a high degree of water-solubility` ~
is given. In non-polar solvents, by contrast, a comparably pronounced `
Insolubility is given.
" , ~ ...
Nisin belongs to the group of polypeptide antibiotics, sub-group - - ~
lantibiotics, and it consists of 34 amino acids. This polypeptide is
produced by certain strains of Lactococcus lactis ssp. `
Lantibiotics, whose preferred embodiment may be seen in subtilin, nisin,
and nisinoid substances, are taken to mean, amon3 other things, those
bactenocines as well, that contain lanthionine and/or methyl lanthionine. I
" '~ -;'~"'
---.. _ .. ....... .
2~5 :~
It is characteristic that within the nisin molecule, five ring structures are
prss~nt, each of which exhibits a different number of amino acids. The .
molecular weight is approximately 3500. However, there are also ~ :;
molecular structures, especially dimers or multimers, that have become
known, that exhibit molecular weights of up to 14000. This is indicated,
for example, by B. Jarvis, Jeffsoat, Joan & Cheeseman, Bioch. Biophys.
Acta, 1968, 168 (1), page 153.
Production of an active ingredient with tried and true means can be ~ ` -
accomp!ished by virtus of the fact that th~e nisinoid substance is
embodied as nisin.
The structure of nisin, in principle, as well as possibilities for the
production of nisin, result from the patents GB [Great Britainl 844 782,
and DE [Germanyl 11 95 434. Additional production methods are , ;
indicated, for example, in the patents DE 16 17 580 as well as DE 20 00 ``
818. Nisinoid substances (anti-microbially effective peptide analogs) can
be manufactured by bio-technological means, or produced synthetically.
By "genetic sngineering" or carefully directed mutagenesis, mutants that
are superior to the natural substance can be produced. In particular, it is
possible, by these means, to undertake an alteration of the chemical
configuration, for example, by the introduction of new amino acids, or the
substitution or deletion of amino acids, or certain chemical groups.
A combination of the nisinoid substance with preparations that lead to a
reduction of the mucosal barrier enhances the effectiveness (for
example, Iysezyme, cyclo-oxygenase inhibitors, cationic detergents, ~ `
chelators, etc.). The Iysozyme, for example, not only has a synergistic - ` -
effect, it also causes a delay in the development of resistance, and, like
- i, ,' ',
-: .... . .
` 21482
-~lo-
nisin, it is resistant to gastric juice. In particular, thought is given to a ~ -
combination with permaabilizing agents.
` . . ', ~':~,"', ;~
The high relapse quotient, or exacerbation of gastro-intestinal ailments
may be attributed, based upon our own investigations, to the fact, for
example, that Helicobacter pylori persists intracellularly in the stomach's ~;
~pith~lial c~lls. These intracellular germs could not be reached`with thc
methods of therapy used to data. With the aid of liposomes or proteo-
liposomes, it is possible to sluice nisin into affected cells without any
difficulties, leading, in this way, to a killing off of the bacteria. Thus, the
danger of recidivism is eliminated.
In order to render the ingestion of the preparation easier, it is suggested
that the dose to be administered be cmbodied as the filling of a capsule.
The amount of the dosa can be realized in the case of an arrangement
within the capsule in such a way, for exa`mple, that the filling is embodied
as a substance in the form of a powder. It is also possible, however, for
the filllng to be embodied as a solution of the substances, or, for a
dispersion (suspansion or emulsion) to be extant. `
- ~ ~
According to another preparation, it is possible for the amount of the
dose to be provided within the area of a tablet. A delayed release of the
preparation out of a special galenic formulation is conceivable. To this
and, for example, liposomes or hydrogels that are in keeping with the
publication "Hydrogels: Swelling, Drug Loading, and Release," S.W. Kim ~;
et al., Pharmaceutical Research, 9, 1992, pages 283-290, can be used,
as can bio-aclhesive materials of natural and synthetic origin. Additional
galenic formulations in this respect are described in the publication
"Formulations releasing the drug proximal to the pylorus in the dog," J. `
. .:
~l~g2~
Hein~m~ki et al., International Journal of Pharmaceutics, 48 ~1988), ~ -
pages 51-61.
. :
In order to render the development of the active ingredien~ in the area of ` ~ ~ `
the stomach possible, soma thought is also dcvoted to the fact that the ~;
quantity of the dose is contained in a dosable fluid, and that it is
administered orally. A solution or a dispersion may be extant. The oral ;~ `
application is, as a matter of principle, used to good purpose in the case
of gastric applications and applications to the small intestine. In
applications in the region of the large intastine, application should be
accomplished rectally. - ;
, ;`','~
The various possibilities for compounding the preparation can be
complemented by the following inactive ingredients, such as, for
exampla, preservative substances, buffer substances, carriers, flavor
correctors, dyestuffs, binding agents, adhesives, lubricants, adsorbing
agents, thickening agents, and thinners. As a result of use over decades
in the preservation of foodstuffs without any cause for concern, it seems
to be to good purposa to administer the composition of the active
ingredient with acidic beverages (for example, fruit juices, colas), which
would increase the compliance. In addition, other foodstuffs having a
broad acceptance in the population are conceivable as carriers of the`~
preparation (for axample, milk). `
An additional fascinating possibility for the elimination of the bacteria
enumerated at the outset would be Drobiotic therapy. In this process, ~ -
living, bacteriocin-producing bacteria, such as, for example, lactobacilli,
are used, which are, themselves, harmless, but which, as a result of
competitive and bactericidal mechanisms, lead to an exclusion of the
pathogenic/faculta1ive-pathogenic germs. ::
. .
, ; ~: . .
. . .
:.`.`
-1 2 -
This principle can, obviously, also be exploited in a prophylactic manner.
An elegant method, for example, would be, in the case of the gastro~
intestinal region, to enrich yoghurt with lactococei that produce nisin; in
correspondin~ preparation with additive inactive ingredients, the ~ -
compliance problems of some patients, and their aggressive bacteria,
can be eliminated.
- ,,
In this or some similar manner, the criteria established in 1988 by the - ~;
WHO and listed by H. Cranz for an OTC ~over the counter] drug are met
in an ideal way; they are described in the essay: Crantz, H. Over-the-
counterdrugs: the issues. Drug Safety 5 (Suppl. 1): pages l20-125,
1 990.
"~
Ailments of ths digestive tract are taken to mean ailments of the gut, the
stomach, and the esophagus.
~; .'~ ' .`''-,,,
: . ~ . , ' 1 , .. :
2 ~ 3 ~ ~ ~
', " "~..
- 13-
NISIN - Sensitivity Testing
An ~xperimental test of the effects of nisin upon Helicobacter pylori took
plac~ as follows.
1. Test strains
Helicobacter pytori 1, origin MHH, ~110/92 ~ ` `
Helicobacter pylori ll, MHH, 8/17192
The strains were stored as frozen preserves (10% ~Iycerine added),
or, they were stored in transport medium carrisrs (Transwab) at
172C. `
2. Nutritiva madia
Columbia agar base (tha firm of Merck), with the addition of 10%
horse's blood (manufactured as a chocolate agar plate).
3. Culture conditions
Culture in anaerobic pot after Brewer, using BBL CampyPae Plus of
the firm of Becton Dickinson (standardized H2-, CO2- atmosphere)
4. Production ofthe Test Solution
Pure nisin, made by the firm of "Aplin & Barrett LTD~' was used as
the test substance. The testing of the substance took place at two
different solvent pH values, pH 4.43 and pH 2Ø
~;.'`,.:,',~".
- 14
The following concentrations were used in the first trial batch~
260 micrograms/ml
1,000 micrograms/ml
10,000 micrograms/ml. ~ ; ;;
Since the first trial yielded no refsrence point for a varied effect in the
case of plt 2.0 or pH 4.42, the repeat trial was conducted with the
diluent, which had been adjusted to 2.0, and with the following
concentrations:
500 micrograms/ml
1,000 micrograms/ml :
2,600 microgramslml
5,000 micrograms/ml ~ ~
10,000 micrograms/ml. ~ ;
' ~
5. Sensitivity testing according to DIN [German Industrial Norm] 58940, ~ ~ -
Part 6, agar dilution method
......
The exception to the performance of this test: the test was not
conducted on Mueller-Hinton nutritive medium (free of blood and
s~rum), but rather on 10% horse's blood chocolate agar, because
thus far, the germs could not be cultured on serum-free nutritive
media.
In the agar dilution method, the concentraUons to be tested were ;
added to th~ as yet liquid nutritive media as 10-fold concentrated
solution, and carefully mixed with the nutritive medium in a ratio of
1:10. Aftsr tho solidification of the agar, the test strains, as a
~ ~823~ ~
.. ...
-15- ~ ~`
massive inoculum, were inoculated onto the agar plates, and, as the
DIN demands, they were inoculated onto the agar plates of various
nisin concentrations. The results were read after 4 days of
incubation at 37 [presumably Centigrade].
6. Test Results
6.1 Results of the first orienting t~st with the pure nisin substance ~ -
, , ~., ~.,
Table 1 Gro~h after4 days, 37C ~-
Helicobacter pylori I Helicobacter pylori ll
Massive Dilution Massive `~
......... ................. ......................... .... ... ........ ..
Dilution
sowing sowing ` ~.
... ... .. . '.. ' ':~ . ,
pH 4.42
Control +++ +++ +++ +++ .
250 ~lg/ml +++ +++
1,000,ug/ml +
10,000 ~Ig/ml
DH 2.0 `: `
Control +++ +++ ++~ +++
250 ,ug/ml ++-+++ ++-+++
~,000 llg/ml +-++ +/-
10,000 ~g/ml
Control ~++ +++ +++ +++
pH neutral . ~
. ~.... ..
~ . - . ,. - ~ ,~
" :~ '` :",
2-14 8 ?. ~ 5
- 16~
In this case :; -
+++ signifies confluence of growth, :~
+/-, +, ~+ si~nifies reduced growth
- signifies no growth.
~'` '``
6.2 Repeat test ` ~:
Table 2 Growth after 4 days, 37C
Helicobacter pylori I Helicobacter pylori ll . : ` " `- '
Massive Dilution Massive Dilution
sowing sowing
pH 2.0
Control +++ +++ +++ ~++
500~Ig/ml ++++ ++~
1,000 ~g/ml + +/~
2,500 ~,Ig/ml - - - - :
5,000 llg/rnl - - - - . : .
10,000 ~lg/ml ~ n
~ ~.
Control +++ +++ +++ +++
pH neutral
7. Summary
H.p. I: The substance's effect begins at 1,000 Ilg/ml, inhibition is
complete at 2,500 llg/ml, :: .
H.p. Il: The sensitivity of this strain, or rather, its minimal inhibiting :: .
concentration, lies at ~ 250 ~,lg/ml. -
""
',`,"' '~ '~'~.'`
- - 2 1 ~ ~ 2 ~
~ ` .. - .
- 17~
In a test of this substance that had taken place previously (SIGMA
preparation, not ~he Pure substance~, there were indications that the
. . -, - ~
substance worked on all 5 strains that were tested at 250 llg/ml i
(complete inhibition was not achieved).
~, ~
.
Overall, depending upon the species of bacteria that are extant in
concrete terms, and, depending upon the concrete realization of the
-, ~
lantibiotic, and especially of the nisinoid substance, an effect could `
be achi~ved in a pharmaceutical composition beginning at a
concentration of active ingredient of 5 ~g/ml. -
: ~: , ...:. . ~
~...... ... ... :
The permeabilizing agents for reducing the mucosal barrier and for
enhancing the active ingredient that hava already been mentioned in ` `
examplary fashion, may be divided into the following groups in : ` "i `
principle~
~ . ~
1. Lysine polymers and protamines, `~
.~ , . . .i -
. . - .
- .:
2. Polycationic peptides, such as, for example, lactoferrin, Iysozyme,
cathepsin G,
,: : ~.:,~
3. Amimoglycosides, carboxylic ionophors,
4. cationic, anionic, and amphoteric detergents, such as, for
example, benzalkonium chloride or SDS,
5. polymyxins, PMBM, and corresponding derivatives, ~ `
6. chelating agents, for example, various EDTA's, HMP, NTA, citrate
-::. , -, ~.,~
.... ,. ~...
2 1 ~
-18 - 1 -~
7. surfactants, such as Tweens, Tritons, glycerides, for example,
.
~. gen~ral permeabilizers, for example, Tris, Ca~2, Mg~2, Na+, `
9. Carbohydrate fatty acid esters, for example, saccharose mono ~ -
fattyacid ester, :
10. bioadhesive substances,
.. - . .~. ~,~
11. Iiposomes for the intracellular killing of internalized bacteria, ~ ~ -
'
12. cyclo-oxygenase inhibitors, such as 5-ASA, for example,
; ~.
13. prot~ctive antioxidants, such as vitamin C, for example, ~ `
14. other substances that reduce the hydrophobicity of the stomach
mucosa.
As a matter of principle, instead of an isolated permeabilizer, random
combinations of two or more permeabilizers from among the groups
Iisted above may be used, but the combination in question depends upon
- -
the underlying conditions surrounding their use. ~ . -
. . .:. -:.: .
,..." :,.;,."......
'., ~ .~'.' ' ,".,
- ... . - -. -
,,. . ~,~
. :- -:~ .,..,. :,....
