WO1994009806A1

WO1994009806A1 - Preparation for therapeutic and especially prophylactic treatment of digestive tract disorders

Info

Publication number: WO1994009806A1
Application number: PCT/DE1993/001006
Authority: WO
Inventors: Rainer Denecke; Irmingard Gebert
Original assignee: Rainer Denecke; Irmingard Gebert
Priority date: 1992-10-29
Filing date: 1993-10-22
Publication date: 1994-05-11
Also published as: EP0666759A1; CA2148255A1; AU5174593A; JPH08504401A

Abstract

The preparation is used for therapeutic and prophylatic treatment of disorders of the digestive tract that occur when pathogenic and/or secondary-pathogenic bacteria are present, for example, in the presence of Helicobacter pylori. For this purpose a concentration of at least 5 νm of a polypeptide antibiotic per ml of liquid must reach the site of action. This is done by administering a defined dose.

Description

/

Preparation for therapy and especially prophylaxis of diseases of the digestive tract

The invention relates to a preparation for the therapy and in particular prophylaxis of diseases of the digestive tract which occur in the presence of pathogenic and / or facultative-pathogenic bacteria.

In this context, the disease-causing effect of Helicobacter pylori in humans is well known.

However, there are also a large number of bacteria that were originally transmitted from animals to humans (zoonoses), e.g. gastric spirils and Campylobacter-like-organisms (CLO), which have comparable or similar diseases / lesions as Helicobacter

Replacement blaff can trigger pylori. Proteus vulgaris should also be mentioned, which, like Helicobacter pylori, is urease-positive. Mixed infections are very common.

These germs different from Helicobacter pylori are also passed on from person to person. The infection with non-Helicobacter pylori bacteria should not be underestimated. Most of these bacteria, like Helicobacter pylori, live in the same ecological niche, i.e. on and in the gastric mucosa under the protective layer of mucus. Dog and cat owners are particularly at risk of becoming infected with these germs (e.g. with Flexispira rappini).

Diseases in which the above-mentioned bacteria are involved are discussed in the following publications: "Chronic Alcoholic Gastritis", Rajiv üppal, 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 Tabagchali, J Clin Pathol 1991, 44, pages 515-516. A reference to a connection between Helicobacter pylori and gastric cancer can be found in the article "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 known hitherto for the therapy of the diseases mentioned in the publications cited above have an unfavorable ratio of effectiveness and side effects. Due to the serious consequences of an infection with this pathogen, see above, is one

Ersatzb.atf effective and safe prophylaxis and therapy required.

The above-mentioned diseases are treated with preparations according to previously used therapeutic methods, the effective principle of which e.g. Bismuth salts are. Since a therapy with these salts alone was not sufficient, a complicated antibiotic combination therapy had to be carried out (e.g. with amoxicillin and metronidazole). Due to the long-term use of bismuth preparations, their side effect profile is well known: neurotoxic symptoms were observed in the foreground. The benefit-risk ratio of bismuth preparations has been questioned for some time. The spectrum of side effects of the antibiotics used hitherto is sufficiently known.

Such treatment methods are described, for example, in the article "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, April 11, 1992 , Page 893 and in the review by J. Ormand and N. Talley: "Helicobacter pylori: Controversies and an Approach to Management" from Mayo Clin. Proc. 65: 414-426, 1990.

In addition, the previous symptomatic therapy with antacids, H ₂ blockers and ATPase inhibitors generally leads to an overgrowth of the stomach with bacteria and / or fungi (faecal flora), since the acid protection of the hollow organ has been lost. Along with the acid inhibition, there is a massive decrease in secretion of lysozyme from the gastric glands. This is the body's own antibacterial enzyme

REPLACEMENT LEAF essential for the repression of pathogenic germs. In this respect, acid suppression favors certain aetiopathogenetic factors for various gastrointestinal lesions (eg gastric carcinoma and carcinoids). Apart from special indications, acid suppression is obsolete and should be replaced by lantibiotic prophylaxis and therapy for ethical and economic reasons.

The object of the present invention is therefore to provide a preparation of the type mentioned in the introduction in such a way that it ensures high effectiveness without side effects.

This object is achieved according to the invention in that a defined dose of a polypeptide antibiotic is contained as the active substance, the application of which achieves an active substance concentration of at least 5 μg / ml at the site of action.

The antibacterial activity of lantibiotics, in particular nisin, is already known. A use as a food additive is described for example in EP-A-0 453 972. The antibacterial activity of nisin is also specified in US Pat. No. 5,043,176. The use of nisin in the cosmetic field is described in DE-OS 39 38 140.

The bacteria mentioned at the beginning are the etiopathogenetic factor for chronic gastritis. This leads to mucosal atrophy and intestinal metaplasia over time. The end stage of these processes is usually a stomach tumor.

The timely prophylactic use of nisin would drastically reduce the incidence of these tumors

REPLACEMENT LEAF reduce. Colonization of the stomach with pathogenic bacteria (e.g. Gastrospirillum hominis or Helicobacter pylori) can lead to a weakening of the mucous membrane resistance, so that patients who regularly take corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDS) are dependent to develop peptic ulcers.

With this patient material too, the timely prophylactic use of e.g. A significant reduction in this disease can be achieved. In this context, consideration should also be given to the addition of a nisinoid substance to alcoholic beverages in order to prevent the gastric breakthrough so feared by alcoholics and alcoholic gastritis.

About half of the world's population is populated with Helico-bacter pylori. Suitable screening programs (serology, breath test and PCR) make it easy to identify risk groups. In these cases, the prophylactic use of nisinoid substances would have an economic importance that should not be underestimated.

The prophylactic use of nisin is medically harmless since it is not absorbed and is inactivated by pancreatic enzymes, in particular alpha-chymotrypsin.

The medication is applied until the aim of the treatment, namely germination, has been achieved. Since many of the bacteria discussed occur ubiquitously, reinfection cannot be ruled out.

REPLACEMENT LEAF From a therapeutic point of view, the following indications result:

Duodenal ulcer

Duodenal ulcer with gastric metaplasia

Ventricular ulcer

Gastric lesions with intestinal metaplasia

Erosions chronically active gastritis chronically inactive gastritis (e.g. atrophic) chronically inactive gastritis with metaplasia and / or dyplasia other gastritis acute gastritis

Duodenitis non-ulcerative dyspepsia and functional dyspepsia other gastrointestinal diseases

Barrett's esophagus with dysplasia or metaplasia

Disease based on heterotopic gastric mucosa in the digestive tract (e.g. in Meckelschen

Diverticulum)

Other indications are:

Diseases based on colonization of the digestive tract associated with

Bacteria (e.g. Brunner's glands) endocrine disorders such as hypergastrinemia and

Hyperpepsinogenemia mental disorders (e.g. depression)

Menetrier's disease

Gastropathies, e.g. protein-losing hypertrophic gastropathy or gastric lymphoma

REPLACEMENT LEAF The use of a lantibiotic, in particular a nisinoid active ingredient, has proven to be an extremely effective measure for killing aggressive gastric bacteria. Nisinoid substances are resistant to gastric juice because they contain lanthionine and / or methyllathionine and can even exert their optimal effect at a pH of 1-2, which is typically present in the stomach.

The use of nisinoid substances is also indicated in domestic and farm animals as well as laboratory and zoo animals. Bacteria were found in these animals that occur in the same ecological niche as Helicobacter pylori in humans. Helicobacter pylori is human-specific under normal conditions and, with the exception of the monkey, does not occur in the animals mentioned above.

In the absence of sufficient investigation results and taxonomic turbulence in this special sector, these spiral organisms are currently described 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. Tompkins, The Journal of Infections Diseases, 1993, 168, page 379 ff.

The following bacteria are listed:

Helicobacter felis DS3 Helicobacter felis CS1 (Gastrospirillum hominis) 2 (Gastrospirillum hominis) 1 Helicobacter pylori Helicobacter acinonyx

Ers Helicobacter sp. CL03 Helicobacter mustelae Helicobacter fennelliae Helicobacter muridarum Helicobacter cinaedi (Flexispira rappini) Wolinella succinogenes Campylobacter fetus ss fetus Campylobacter largi Campylobacter coli Campylobacter jejuni

The bacteria of the Campylobacter-Helicobacter complex isolated from animals induce gastrointestinal lesions which are comparable to humans, so that the use of nisine also appears to be economically sensible here. In addition, they are often transferred to humans and then generate the image known from Helicobacter pylori. This spectrum of germs should also be tackled with nisinoid substances.

The latest findings indicate a bacterial etiopathogenesis of Crohn's disease and ulcerative colitis (chronic inflammatory bowel diseases). Here too, one could think of using lantibiotics, in particular nisinoid substances.

The water solubility of nisin can be used in the manufacture and use of the preparation. The water solubility is dependent on the respective pH value and is 12 grams for a quantity of water of 100 milliliters at a pH of 2.4, 1 gram at a pH of 5.6 and 0.075 grams at a pH of Value of 7.0. This means that water solubility is particularly high at low pH values. In non-polar

REPLACEMENT LEAF Solvents, on the other hand, have a comparatively pronounced insolubility.

Nisin belongs to the group of polypeptide antibiotics, subgroup Lantibiotica, and consists of 34 amino acids. This polypeptide is used by certain strains of Lactococcus lactis ssp. produced.

Lantibiotics, the preferred formation of which can be seen in subtilin, nisin and nisinoid substances, are also understood to mean, inter alia, bacteriocins which contain lanthionine and / or methyl lanthionine.

It is characteristic that five ring structures are present within the nisin molecule, each of which has a different number of amino acids. The molecular weight is about 3500. However, molecular structures, in particular dimers or multimers, have also become known which have molecular weights of up to 14000. This is the case, for example, with B. Jarvis, Jeffsoat, Joan & Cheeseman, Bioch. Biophys. Acta, 1968, 168 (1), page 153.

Active ingredient production with proven means can take place in that the nisinoid substance is formed as nisin.

The basic structure of nisin and possibilities for the production of nisin result from the patent specifications GB 844 782 and DE 11 95 434. Further production processes are given, for example, in the patent specifications DE 16 17 580 and DE 20 00 818. Nitinoid substances (antimicrobial peptide analogs) can be produced biotechnologically or generated synthetically.

ER _SA TZBLATT Through genetic engineering or targeted mutagenesis, mutants superior to the natural substance can be produced. In particular, this makes it possible to change the chemical configuration, for example by introducing new amino acids or replacing or deleting amino acids or certain chemical groups.

A combination of the nisinoid substance with preparations that lead to a reduction of the mucosal barrier potentiates the effectiveness (e.g. lysozyme, cyclooxygenase inhibitor, cationic detergents, chelators, etc.). The lysozyme acts e.g. not only synergistic, it also delays the development of resistance and, like nisin, is enteric-resistant. In particular, a combination with permeabilizers is intended.

The high relapse rate or exacerbation of gastrointestinal diseases can be attributed to the fact that, for example, Helicobacter pylori persists intracellularly in gastric epithelial cells. These intracellular germs could not be reached with previous therapy methods. With the help of liposomes or proteoliposomes, nisin can be easily infiltrated into the infected cells and thus kills the bacteria. This eliminates the risk of recurrence.

To make it easier to take the preparation, it is proposed that the dose to be administered be designed as a capsule filling.

The dosing amount can be realized in an arrangement within a capsule, for example, such that the filling is in the form of a powdery substance

ER ^SA TZBLATT is. But it is also possible that the filling is designed as a solution of the substances or that a dispersion (suspension or emulsion) is present.

According to another preparation, it is possible for the metered amount to be arranged in the area of a tablet. A delayed release of the preparation from a special galenical formulation is conceivable. For this purpose, for example, liposomes or hydrogels can be used in accordance with the publication "Hydrogels: Swelling, Drug Loading, and Release", SW Kim et al. , Pharmaceutical Research, 9, 1992, pages 283-290, bioadhesive materials of natural and synthetic origin are also used. Further galenical formulations in this regard can be found in ^{1 of} the publication "Formulations releasing the drug proximal to the pylorus in the dog", J. Heinämäki et al. , International Journal of Pharmaceuticals, 48 (1988), pages 51-61.

In order to enable the active ingredient to develop in the area of the stomach, it is also contemplated that the metered amount is contained in a meterable liquid and is administered orally. For example, there may be a solution or dispersion. Oral administration is generally expedient in gastric and small intestine applications. For applications in the colon area, the application should be rectal.

The various options for the composition of the preparation can be supplemented by the following auxiliaries, e.g. Preservatives, buffer substances, carriers, flavoring agents, colorants, binders, adhesives, lubricants, adsorbents, thickeners and thinners. Due to the decades-long, safe use in food preservation, it seems appropriate to include the active ingredient composition

satebϊatt to administer acidic beverages (e.g. fruit juices, cola drinks), which would increase compliance. Other foods with broad acceptance among the population are also conceivable as carriers of the preparation (eg milk).

Another fascinating way to remove the bacteria mentioned in the introduction would be probiotic therapy. Living bacteria, producing bacteriocins, e.g. Lactobacilli used, which are harmless themselves, but lead to an exclusion of the pathogenic / facultative-pathogenic germs by means of competitive and bactericidal mechanisms.

This approach can of course also be used prophylactically.

An elegant method would be e.g. for the gastrointestinal area, the enrichment of yogurt with nisin-producing lactococci; with appropriate preparation and with additive auxiliaries, the compliance problems of some patients and their aggressive bacteria can be eliminated.

In this or a similar way, the criteria of an OTC medicinal product listed by WHO 1988 and H. Cranz are ideally met; described in the article: Cranz, H. Over-the-counter-drugs: the issues. Drug Safety 5 (Suppl. 1): pp. 120-125, 1990.

Diseases of the digestive tract are understood to mean diseases of the intestine, stomach and esophagus.

Replacement_? B.at £ NISIN - sensitivity test

An experimental test of the nisin effect on Helicobacter pylori was carried out according to the table below.

1. Test strength

Helicobacter pylori I, origin MHH, 10.8.92 Helicobacter pylori II, MHH, 17.8.92 The strains were stored as frozen foods (10% glycerine additive) or on transport medium carriers (Transwab) at -172 ° C.

2. Culture medium

Columbia agarbase (Merck) with the addition of 10% horse blood (prepared as a blood plate)

3. Culture conditions

Cultivation in anaerobic pot according to Brewer using BBL CampyPac Plus from Becton Dickinson (standardized H ", CO_ atmosphere)

4. Preparation of the test solution

Pure nisin from "Apiin & Barrett LTD" was used as the test substance. The substance was tested at 2 different solvent pH, pH 4.43 and pH 2.0

The following concentrations were tested in the first experiment:

REPLACEMENT LEAF 250 micrograms / ml 1,000 micrograms / ml 10,000 micrograms / ml.

Since there was no evidence of a different effect at pH 2.0 and pH 4.42 from the first test, the repeat test was carried out with the dilution solution adjusted to pH 2.0 and with the following concentrations:

500 micrograms / ml

1,000 micrograms / ml

2,500 micrograms / ml

5,000 micrograms / ml

10,000 micrograms / ml.

5. Sensitivity test according to DIN 58940, part 6 agar dilution method

Exception to this test procedure: Testing not on Mueller-Hinton culture medium (blood and serum-free), but on 10% horse-boiled blood agar, since the germs had not previously been grown on serum-free culture media.

In the agar dilution method, the concentrations to be tested were added to the still liquid culture medium as a 10-fold concentrated solution and mixed thoroughly with the culture medium in a ratio of 1:10. After the agar had solidified, the test strains were inoculated onto the agar plates of different nisin concentration as a solid inoculum and, as required in the DIN, as a germ suspension. The result was read after 4 days of incubation at 37 °.

ER ^S A ^T ZBLATT 6. Result of the test

6.1 Result of the first orienting test with the nisin pure substance

Tab. 1 Growth after 4d 37 ° C Helicobacter pylori I Helicobacter pylori II Massive dilution Massive dilution sowed sowed

pH 4.42

+++ +++

Control +++ +++ +++ +++ pH neutral

Here +++ means confluent growth, + / -, +, ++ reduced growth, - no growth.

Spare sheet 6.2 Retest

Tab. 2 Growth after 4d 37 ° C Helicobacter pylori I Helicobacter pylori II Massive dilution Massive dilution sowed

PH 2.0

+++ +++

Control +++ +++ +++ +++ pH neutral

7. Summary:

H.p.I: The substance effect starts at 1,000 μg / ml, the inhibition is completely at 2,500 μg / ml, H.p.II: The sensitivity of this strain or its minimum inhibitory concentration is <250 μg / ml.

REPLACEMENT LEAF When this substance was tested beforehand (preparation SIGMA, no pure substance), an effect of the substance at 250 μg / ml was indicated in all 5 strains tested (complete inhibition was not achieved).

Overall, depending on the specific species of the bacteria and the specific implementation of the lantibiotic, in particular the nisinoid substance, an effect can be achieved in a pharmaceutical composition from an active substance concentration of 5 μg / ml.

The permeabilizers already mentioned by way of example for reducing the mucosal barrier and as an active ingredient booster can basically be divided into the following groups:

1. lysine polymers and protamines,

2. Polycationic peptides, for example lactoferrin, lysozyme, cathepsin G,

3. aminoglycosides, carboxylic ionophores,

4. cationic, anionic and amphoteric detergents, for example benzalkonium chloride or SDS,

5. polymyxins, PMBM and corresponding derivatives,

6. Chelators, for example various EDTA's, HMP, NTA, citrate

7. surfactants, for example tweens, tritons, glycerides,

REPLACEMENT LEAF 8. general permeabilizers, for example Tris, C _., A 2+, M_, g 2+, N _xτ a +,

9. carbohydrate fatty acid esters, for example sucrose mono fatty acid esters,

10. bioadhesive substances,

11. liposomes for the intracellular killing of internalized bacteria,

12. Cyclooxygenase inhibitors such as 5-ASA,

13. Protective antioxidants such as Vitamin C,

14. Other substances which reduce the hydrophobicity of the gastric mucus.

In principle, any combination of two or more permeabilizers from the groups given above can be used instead of an isolated permeabilizer, the particular combination being dependent on the conditions of use.

Spare sheet

Claims

P a t e n t a n s r u c h e

Preparation for the therapy and in particular prophylaxis of diseases of the digestive tract which occur in the presence of pathogenic and / or facultative-pathogenic bacteria, characterized in that the active ingredient contains a defined dose of a polypeptide antibiotic, the application of which gives an active ingredient concentration of at least 5 μg / ml is reached at the site of action.

Preparation according to claim 1, characterized in that the polypeptide antibiotic is designed as a lantibiotic.

Spare sheet 3. Preparation according to claim 1 or 2, characterized gekenn¬ characterized in that the lantibioticum is designed as a nisinoid substance.

4. Preparation according to claim 3, characterized in that the nisinoid substance is designed as a nisin.

5. Preparation according to claim 3 or 4, characterized gekenn¬ characterized in that the nisinoid substance is formed from a mixture of at least two polypeptides.

6. Preparation according to one of claims 1 to 5, characterized in that active multimers with molecular weights between 7,000 and 14,000 are formed from the nisinoid substance.

7. Preparation according to one of claims 1 to 6, characterized in that the dose is designed as a capsule filling.

8. A preparation according to claim 7, characterized in that the filling is designed as a powdery substance.

9. A preparation according to claim 7, characterized in that the filling is formed as a substance dispersion.

10. Preparation according to one of claims 1 to 6, characterized in that the dose is arranged in the region of a tablet.

REPLACEMENT LEAF 11. Preparation according to one of claims 1 to 6, characterized in that the dose is dissolved in a meterable liquid.

12. Preparation according to one of claims 1 to 6, characterized in that the dose is dispersed in a liquid. ^s

13. Preparation according to one of claims 1 to 12, characterized in that the dose is arranged in a special galenical formulation with delayed active ingredient release and / or extended retention.

14. Preparation according to one of claims 1 to 10, characterized in that an active substance concentration of at least 50 μg / ml is achieved at the site of action by the applied dose.

15. Preparation according to one of claims 1 to 13, characterized in that an active substance concentration of at least 250 μ g / ml is achieved at the site of action by the dose applied.

16. Preparation according to one of claims 1 to 13, characterized in that an active substance concentration of at least 1000 μg / ml is achieved at the site of action by the dose applied.

17. Preparation according to one of claims 1 to 16, characterized in that the dose is combined with an active ingredient amplifier.

REPLACEMENT LEAF 18. Preparation according to one of claims 1 to 17, characterized in that the active substance amplifier is designed as a permeabilizer to reduce the mucosal barrier.

19. Preparation according to one of claims 1 to 18, characterized in that the permeabilizer is designed as a chelator.

20. Preparation according to one of claims 1 to 18, characterized in that the permeabilizer is designed as an EDTA-Tris.

21. Preparation according to one of claims 1 to 18, characterized in that the permeabilizer is designed as a cyclo-oxygenase inhibitor.

22. A preparation according to claim 21, characterized in that the cyclooxygenase is formed as acetylsalicylic acid.

23. A preparation according to claim 21, characterized in that the cyclooxygenase inhibitor is designed as 5-ASA.

24. A preparation according to one of claims 1 to 18, characterized in that the permeabilizer is designed as a surfactant.

25. A preparation according to any one of claims 1 to 24, characterized in that an indication in the veterinary area is provided.

REPLACEMENT LEAF 26. Preparation according to one of claims 1 to 24, characterized in that an indication is provided for the treatment of diseases of the digestive tract which are caused by non-Helicobacter pylori bacteria.

27. A preparation according to claim 26, characterized in that the bacteria are transferred from animals to humans.

28. A preparation according to claim 26, characterized in that the bacteria are transmitted from humans to the animals.

29. A preparation according to claim 26, characterized in that the bacteria are transmitted from humans to humans.

30. A preparation according to any one of claims 1 to 29, characterized in that a combination of active ingredients with a pH reducer is provided in the intestinal area to provide an acidic medium at the site of action.

31. A preparation according to claim 30, characterized in that the pH reducer is formed as 5-aminosalicylic acid.

32. Preparation according to one of claims 1 to 31, characterized in that an indication is provided for the treatment of Crohn's disease.

33. Preparation according to one of claims 1 to 32, characterized in that an indication for the treatment of ulcerative colitis is provided.

Replacement Blade 34. Preparation according to one of claims 1 to 33, characterized in that an indication is provided for the treatment of certain hypergastrinemias in which bacteria are involved.

35. Preparation according to one of claims 1 to 34, characterized in that an indication is provided for the treatment of certain mental disorders which are endocrinological or toxic.

36. Preparation according to one of claims 1 to 35, characterized in that at least part of the active ingredient is limited to enable cell membrane penetration by liposomes.

37. Preparation according to one of claims 1 to 36, characterized in that at least part of the active substance is designed as a lysozyme.

Spare sheet