WO1998000119A2

WO1998000119A2 - Applications of active substances affecting the functions of non neuronal acetylcholine

Info

Publication number: WO1998000119A2
Application number: PCT/EP1997/003415
Authority: WO
Inventors: Ignaz Wessler
Original assignee: Boehringer Ingelheim Pharma Kg; Boehringer Ingelheim International Gmbh
Priority date: 1996-07-02
Filing date: 1997-07-01
Publication date: 1998-01-08
Also published as: ZA975804B; AU3263297A; DE19626373A1; WO1998000119A3

Abstract

The present invention relates to a process for determining the functional state of non-neuronal acetylcholine in human tissue or cells which are accessible either directly or by endoscopy. It also relates to the use of active ingredients which can affect the synthesis, release, inactivation and function of non-neuronal acetylcholine.

Description

Novel use of active ingredients that affect the functions of non-neuronal acetylcholine

The present invention relates to a novel use of active ingredients with which the functions of non-neuronal acetylcholine - e.g. epithelial acetylcholine - can be influenced.

Acetylcholine acts as a central neurotransmitter in the central, peripheral and enteral nervous system. Accordingly, acetylcholine can be detected in the corresponding neurons.

Surprisingly, it has now been found that acetylcholine does not only play a central role in neurons as a neurotransmitter, but that so-called non-neuronal acetylcholine plays an important role in human surface epithelium and is synthesized there by the enzyme choline acetyltransferase (ChAT).

For example, non-neuronal acetylcholine can be detected in the surface epithelium of the human respiratory tract, the gastrointestinal tract - such as the small intestine, colon, sigma and gallbladder - in the vaginal mucosa, in the epidermis of human skin and in many other cells.

The - surprisingly - widespread use of acetylcholine in the human epithelium could be demonstrated in several ways using experimentally different approaches: e.g.

Detection of the presence of the ChAT protein by immunohistochemistry and Western blot using poly- and monoclonal anti-ChAT antibodies;

CONFIRMATION KDPIE Detection of ChAT enzyme activity in isolated epithelial cells - and detection of acetylcholine in epithelial cells using HPLC analysis.

The detection of non-neuronal acetylcholine can be done by simply "wiping" the corresponding surfaces with a cotton swab. The cell material absorbed by the cotton contains so much acetylcholine that it is sufficient for analytical detection. With this method of sampling, for example, neither the basement membrane (bronchi) nor the lamina muscularis mucosae (intestinum) is damaged, which prevents contamination of the extracts obtained from the cotton wool with cholinergic neurons. In addition, it is known from the prior art that the surface epithelium of human respiratory tract - like the human epidernis - is not innervated by cholinergic neurons.

In addition to other convincing evidence, the so-called Western blot analysis provides impressive evidence of the presence of ChAT proteins in the epithelial cells mentioned. Among other things, ChAT proteins with molecular weights of approx. 45 and 51 kD are extracted from human bronchial epithelial cells.

A strong ChAT-related immunoreactivity in epithelial cells was demonstrated in the intestine. These immunopositive enterocytes embody brush border cells that have microvilli and that are involved in the absorption and secretion processes.

Epithelial acetylcholine was surprisingly found along the entire intestinal tract - starting in the oral mucosa to the small and large intestine and in the gallbladder. The same applies to the respiratory tract, with acetylcholine being detected in both large and small bronchi. Epidermal acetylcholine was also found in the area of almost the entire body surface (upper and lower extremities, thorax, abdomen and back). A particularly pronounced ChAT-corresponding immunoreactivity was found in the growth zone of the hair, the hair follicles. In addition, acetylcholine was also detected directly in the human body hair. Furthermore, acetylcholine was also used in epithelial cells to drain urine Pathways demonstrated, as well as in mesothelial cells (pleura, pericardium) as well as in certain immune cells (alveolar macrophages, mononuclear cells) and in glial lines.

In this context, the effects of acetylcholine on the function of epithelial / epidermal cells and in other cells should be demonstrated.

1. increase in proliferation, cell differentiation, regulation of the cell cycle;

2. Regulation of cell-cell communication with influencing the function of "tight junctions", "gap junctions" and desmosomes;

3. control of immune functions of mucous membranes and skin;

4. Increase in cilia activity;

5. regulation of chloride secretion;

6. secretion of phospholipids;

7. Regulation of absorption (e.g. choline);

8. Regulation of cell locomotion;

9. Trophic functions.

Indeed, experimental evidence has been provided that non-neuronal acetylcholine regulates important functions of non-neuronal cells (epithelial cells): cell-cell contact, barrier function, lymphatic drainage, proliferation. These new findings make it possible to apply the measures according to the invention to disease states which are associated with an adjustment of the non-neuronal acetylcholine system. Accordingly, substances which can have an effect on the function of non-neuronal acetylcholine can also achieve a positive therapeutic effect with respect to diseases of the gastrointestinal tract, the respiratory tract and the skin which are associated with a disturbed function of non-neuronal acetylcholine.

For example, the present invention opens up the possibility of the aqueous diarrhea - as e.g. when cholera is observed - to be able to treat with topically acting antagonists on muscarinic receptors (non-selective and M1 -selective antagonists). In addition, the therapy of inflammatory bowel diseases - such as ulcerative colitis - with topically effective agonists at muscarinic / nicotine receptors, as this barrier disrupts the mucosal barrier function.

With regard to respiratory diseases, the cystic fibrosis can be made possible according to the invention with topically acting agonists on muscarinic receptors, with non-selective or M1-selective agonists or topically active inhibitors of cholinesterase.

According to the invention, a therapeutic option for hypersensitivity (bronchial asthma) with topically acting antagonists on muscarinic receptors (non-subtype-selective M1 receptors) is opened.

Ultimately, the present invention in skin diseases - such as, for example, wound healing disorders with - drugs that have a stimulating effect on the function of the epidermal cholinergic system - such as agonists on muscarinic or nicotine receptors, inhibitors of cholinesterase, activators of epidermal acetylcholine, activators for the release and enhancement with respect to the expression of the synthesizing enzyme ChAT - the access to a positive therapeutic effect. In addition, the present invention enables the treatment of Alzheimer's disease by using activators of choline acetyl transferase (ChAT) in non-neuronal cells (glial cells). In primary cultures of rat glial cells 2 + 0.7 pmol per 10 ⁶ cells acetylcholine could be detected. On the other hand, according to the invention, successful treatment - for example atopic dermatitis, neurodermatitis, psorasis and, for example, cholinergic uterine artery - with active substances which have an inhibitory effect on the function of epidermal acetylcholine - such as antagonists on muscarinic or nicotine receptors , Inhibitors of choline acetyltransferase, inhibitors of epidermal acetylcholine release and reducers with regard to the expression of the synthesizing enzyme choline acetyltransferase.

Suitable topical agonists for the treatment of respiratory diseases are, for example, active substances such as those disclosed in German Offenlegungsschrift DE-OS 38 39 385 - in particular Wal 2014. In addition, for example Compounds such as carbachol or acetylcholine themselves are considered.

In addition, DMPP (dimethyl-4-phenylpiperazinium) can be used as a nicotinic antagonist.

As - among others In addition to tacrine and physostigmine, topically active inhibitors of cholinesterase are, for example, compounds such as those in European Offenlegungsschrift EP-A-0 296 650 - in particular donepezil and its acid addition salts or active substances such as those in German Offenlegungsschrift DE-OS 38 05 744 - in particular SDZ -ENA-713 - are disclosed.

As - among others - Topical antagonists on muscarinic receptors may be mentioned, for example - in addition to active ingredients as are known from the prior art - Atrovent, Propantelin, Buscopan, Alginor, Oxivent, Scopolamine and Atropine and compounds as are known from German Offenlegungsschrift 39 31 041 - in particular BA 679, BEA 2108 (bromide of endo-3 - [(hydroxy-di-2-thienylacetyl) oxy] -8,8-dimethyl-8-azonia-bicyclo [3.2.1] oct-6-ens) and BA 253 called.

With regard to topically effective antagonists on muscarinic or nicotine receptors for the treatment of skin diseases, reference is primarily made to nicotinic blockers, such as, for example, tubocurarine, alcuronium, galamine and decamethonium and pancuronium. such as tubocurarine, alcuronium, galamin and decamethonium and pancuronium.

The invention described will now be illustrated by the following examples. Various, other configurations will become apparent to those skilled in the art from the present description. It is expressly pointed out, however, that the examples and the description are provided for illustration only and are not to be regarded as a limitation of the invention.

To determine the functional state of non-neuronal acetylcholine in directly or endoscopically accessible human tissues or cells, it should be noted in advance that this can generally be done by taking a sample from these tissues and the content in this sample in a manner known per se determined on acetylcholine.

Sampling can be done, for example, by wiping the surface with a cotton swab. In addition - for example to determine the function of epithelial acetylcholine within the respiratory, gastrointestinal and urogenital tract - the sample can be taken in such a way that the sample is obtained with a cotton wool roll, which may be moistened with a test liquid.

Furthermore, the so-called "cup" technique can be used to determine the acetylcholine from the skin, the oral mucosa and the vaginal mucosa. For this purpose, a vessel suitable for sampling and charged with a liquid containing the acetylcholine is placed on the skin in such a way that the acetylcholine can diffuse into the device. In addition, stimulation solutions known from the prior art can be used to determine the functionality of non-neuronal acetylcholine - such as solutions of nicotine, citric acid or β-adrenoreceptor agonists. Examples

Methods

Separation of the epithelial cells

Human tissue (bronchi, intestinal tissue) was obtained from surgical material that was obtained in the context of tumor and stone surgery.

Small pieces of human skin (10 to 50 mg) were also obtained from surgical material. Only - macroscopic - tumor-free tissue was analyzed. Human bronchial tubes (up to 4 mm in diameter) or intestinal tissues (stomach, small intestine and large intestine as well as gallbladder) were carefully removed from attached tissue immediately after dissection and on their way from the hospital to the laboratory in an oxygenated saline solution (composition in mmol = 125 NaCI, 23.8 NaHCθ3, 5.05 glucose, 2.68 KCI, 1.80 CaCl2, 0.539 NaH2Pθ4, 0.0567 ascorbic acid and 0.001 choline chloride).

Acetylcholine and ChAT activity was determined on mechanically or enzymatically isolated epithelial cells. For mechanical isolation, the luminal surface of the respective tissue was carefully wiped off with a cotton swab (Q-Tip).

For example, human bronchi or jejenum were fixed in a petri dish with the luminal side up. The luminal surface was wiped off with a cotton swab (wiping time approx. 5 seconds), the cotton swab never penetrating the epithelial surface to the basement membrane, i.e. the underlying lamina propria remained undamaged. This was checked histologically several times.

The epithelial surface of intestinal tissue was examined in the same way, taking great care to ensure that the lamina muscularis mucosa with the underlying cholinergic, submucosal, plexus remained intact. A histological check was also carried out on this. Analogously, samples were taken from the epithelial surface of the oral mucosa or the vaginal mucosa.

After wiping, the cotton swabs were extracted with 1 ml of a vol. 15% formic acid solution in acetone. The acetylcholine was then detected from this extract.

Epithelial cells of the human bronchi and small intestine were isolated enzymatically - by incubation (2 h at 36 ° C or 24 h at 4 ° C) with 0.1% pronase in a DMEM / F12 medium to isolate the epithelial cells and subsequent measurement of enzyme activity or for "western blot analysis" treated. Isolated epithelial cells were checked for their epithalial origin by staining with an antibody against pan-cytokeratin.

Measurement of endogenous acetylcholine

The acetylcholine content was determined in cotton swab extracts from surface epithelia and in homogenized human skin. The skin was placed in 1 ml of a 15% by volume solution of formic acid in acetone and crushed with a knife.

After storing on ice for 30 minutes, the extraction medium was centrifuged (10 min. 4000 rpm) and the supernatant was evaporated to dryness in a stream of nitrogen. The sediment of the dried sample was resuspended in 300-500 μl of the mobile phase for the HPLC analysis. A volume of 20 μl was injected, the acetylcholine being determined by cation exchange HPLC using electrochemical detection; the "BAS 481 Microbore System" was used for this. Choline and acetylcholine were chromatographed on a Sep Stik column (1 x 530 mm) with a mobile phase of a 40 mM phosphate buffer and 0.3 mM EDTA (adjusted to pH 8.5). The analytical column was followed by a reaction step with an immobilized enzyme (Sep Stik IMER 2 / pkg), in which the acetylcholine was hydrolyzed. The subsequent reaction with choline oxidase produced hydrogen peroxide, which washes around a platinum electrode (reference electrode Ag / AgCI, 0.5 V). The resulting current is proportional to the amount of acetylcholine formed [Reinheimer, T., P. Bernedo, H. Klapproth, H. Oelert, B. Zeiske, K. Racke, and I. Wessler, 1996. Acetylcholine in isolated airways of rat, guinea-pig, and human: species differences in the role of airway mucosa, Am. J. Physiol. 14, L722-728], the detection limit for acetylcholine being 10 fmol per 20 μl injection volume [Ricny J., K.-D. Höhle, K. Racke and I. Wessler, 1995. Effect of inhaled steroids on cholinergic transmission in human isolated bronchi. Eur. Respir. J. 8: 587-589]. In order to rule out contamination during the extraction process, cotton swabs that had not come into contact with human tissue were extracted under analogous conditions. The extracts obtained in this way gave no signal for acetylcholine.

Immunohistochemistry and "Western Blot Analysis"

The ChAT protein could be detected immunohistochemically and by "Western blot analysis" using a polyclonal anti-ChAT antibody [Schemann, M., H. Sann, C. Schaaf, and M. Mäder, 1993, Identification of cholinergic neurons in enteric nerves by antibodies against choline acetyltransferase, Am. J. Physiol. 265, G1005-G1009]. A monoclonal anti-ChAT antibody was also used.

To carry out immunohistochemistry, tumor-free, human tissue (bronchi, small intestine and stomach, skin) was flash-frozen with isopentane immediately after the operative isolation and then stored in liquid nitrogen. Frozen sections (4 μm) were made - each over a period of 15 min. - Permeabilized with 4% formaldehyde solution and by incubation with 0.1% Triton X-100 solution.

Primary antibodies and then secondary antibody conjugates were used to detect the ChAT antigen. For the "Western blot analysis" isolated epithelial cells or - for comparison - homogenized rat brain were lysed by enzymatic means and the proteins of the cytosol were extracted by means of ultracentrifugation. 100 μg of the protein were separated (SDS-PAGE) and applied to nitrocellulose.

The visualization was carried out using the anti-ChAT antibody and the anti-rabbit IgG antibody-phosphatase conjugate.

Measurement of ChAT activity

The determination of the ChAT enzyme activity was carried out in extracts of cotton swabs from smears of the intestinal surface or in extracts from enzymatically isolated epithelial cells (from human bronchi and small intestine) or from extracts of human skin.

The ChAT assay was carried out according to methods which are known from the prior art [Reinheimer, T., P. Bemedo, H. Klapproth, H. Oelert, B. Zeiske, K. Racke, and I. Wessler, 1966. Acetylcholine in isolated airways of rat, guinea-pig, and human: species differences in the role of airway mucosa, Am. J. Physiol. In press and Ricny J., K.-D. Höhle, K. Racke and I. Wessler, 1995: Effect of inhaled steroids on cholinergic transmission in human isolated bronchi. Eur. Respir. J. 8: 587-589],

Enzymatically isolated epithelial cells were prepared from a DMEM / F12 medium by careful centrifugation (5 min, 200 rpm) and subsequent washing of the pellets thus obtained three times with 3 ml of phosphate-buffered saline solution. The cell pellet or cotton swab was brought into contact with 0.5 1 ml of extraction buffer (10 mM Na2HP04, 100 NaCl, 2 mM EDTA, 0.5% (v / v) Triton X-100). After 15 minutes of ice cooling, the samples were centrifuged (3 min; 12,000 rpm; 0 ° C). An aliquot (20 ul) of the supernatant solution was added to a buffer containing 8 mM choline chloride, 0.1 mM physostigmine and 0.2 mM [ ³ H] AcCoA. The enzymatic reaction was stopped after 30 minutes and the synthesized [ ³ H] acetylcholine was extracted by ion pair extraction isolated and the tritium content was determined using liquid scintillation spectrometry, [Ricny J., K.-D. Höhle, K. Racke and I. Wessler, 1995. Effect of inhaled steroids on cholinergic transmission in human isolated bronchi. Eur. Respir. J. 8: 587-589]. In order to be able to determine the specific ChAT activity, the selective inhibitor bromoacetylcholine was added to the assay buffer. The protein content was determined according to Smith et al. [Smith, PK, R. Krohn, GT Hermanson, AK Mallia, FH Gärtner, MD Provenzano, EK Fujimoto, NM Goeke, BJ Olson, and DC Klenk (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85].

Results

Endogenous acetylcholine in human epithelial cells

The epithelial cells were obtained by gently rubbing the luminal surface of isolated bronchial tubes with a cotton swab without penetrating the basement membrane. In the extracts of the epithelial cell layer on the surface, significant amounts of acetylcholine (33 + 10 pmol / g bronchi (n = 15) could be detected by HPLC-EC. The specificity of the peak, which was assigned to the acetylcholine in the chromatogram, could be determined. be backed up several times.

The detection of acetylcholine in epithelial cells of the human respiratory tract indicates that important epithelial functions are regulated by non-neural acetylcholine (secretion, mucociliary clearance, barrier function, cell proliferation).

This surprising effect thus opens up a new therapeutic option for the treatment of respiratory diseases - such as cystic fibrosis - with topical agonists on muscarinic receptors (non-selective or M1-selective agonists) or topically active inhibitors or cholinesterinase or bronchial asthma with antagonists on muscarinic receptors (non-subtype selective or selective M1 receptors. In a comparable way, human epithelial cells could be obtained from the intestinal tract. Tumor-free segments of the gastric jejunum of the ileum, the colon and the sigma of patients with tumors in the intestinal area were examined. Optical microscopic images of the specimens show that only a few villi were removed after rubbing off the luminal surface, whereas the lamina muscularis mucosa with the underlying submucosal cholinergic plexus remained completely intact.

The extracts from epithelial cells of the human small intestine contained about 1 nmol / g jejunum. In contrast, significantly less acetylcholine was detected in the corresponding epithelial extracts of the colon or sigma (1 - 50 pmol / g). In contrast, no epithelial acetylcholine could be detected in the corresponding extracts from epithelial cells of the gastric mucosa (pyloric part). On the other hand, significant amounts of acetylcholine were found in epithelial extracts of the gallbladder (12 + 5 pmol / g, n = 5).

This unexpected detection of epithelial acetylcholine in the intestinal tract and the observed cellular effects of acetylcholine open up a new therapeutic route for the treatment of inflammatory diseases of the gastrointestinal tract - such as ulcerative colitis - with topically active agonists on muscarinic / nicotine receptors, for example, or the watery diarrhea when cholera is observed - to be able to treat with topically acting antagonists on muscarinic receptors (non-selective and M1-selective type).

In addition to the previously mentioned epithelial cells of the respiratory tract and intestine, acetylcholine could be detected in the epithelial cells of the oral mucosa and the mucosa of the vagina. The epithelial cells of male adults had 8 + 2 pmol (n = 5) acetylcholine per extract. The acetylcholine content of the epithelial cells of mucous membranes of vaginal origin was 6 + 2 pmol acetylcholine (n = 4).

In addition, significant amounts of acetylcholine were found in sections of human skin (epidermis / dermis) (1000 + 300 pmol / g, n = 7). The acetylcholine content of the hairy scalp hair was 1.5 + 0.2 nmol / g (n = 3). In all In some cases, the specificity of the acetylcholine peak from all of the samples mentioned could be ensured as part of the HPCL.

According to the invention, the detection of epidermal acetylcholine and the observed cellular effects open up a new approach to the treatment of skin diseases - such as wound healing disorders - with pharmaceutical active substances which have a stimulating effect on the function of the epidermal, cholinergic system - such as e.g. Agonists on muscarinic or nicotine receptors, cholinesterase inhibitors, activators of epidermal acetylcholine, activators for the release and enhancement with regard to the expression of the synthesizing enzyme choline acetyltransferase. The same applies to disorders of hair growth or hair loss:

Examinations of skin biopsies from patients with neurodermatitis or psoriasis show a clear adjustment of the epithelial cholinergic system. - So the content of epidermal acetylcholine in 4 mm skin punches was 156 + 33 pmol per gram dry weight (n = 14). A content of 1900 + 670 pmol per gram of dry weight was found in patients with neurodermatitis (n = 7, p <0.01) and in patients with psoriasis vulgaris the level was 630 + 210 pmol per gram of dry weight (n = 8). These pronounced changes suggest a pathophysiological significance of the epidermal system in the diseases described.

It was noticeable that in the patients with neurodermatitis there was also an upregulation of epithelial acetylcholine in the area of the oral mucosa. A 1% solution of citric acid in water can be used to release 1.6 + 0.6 pmol / ml (n = 9) acetylcholine from the oral mucosa from a defined surface within one minute, while in patients with neurodermatitis 4.0 + 0.9 pmol / ml (n = 11; p <0.05) acetylcholine was found. This observation indicates that epithelial acetylcholine is up-regulated in allergic-inflammatory diseases. As a further confirmation of this conclusion, it was found that the epithelial acetylcholine in the trachea was increased in guinea pigs which were given a classic inhalation sensitization protocol.

Furthermore, the unexpected findings described enable a new approach to the successful treatment of skin diseases - such as atopic dermatitis, neurodermatitis, psoriasis and cholinergic utricaria - with active substances that can inhibit the function of epidermal acetylcholine - such as Antagonists on muscarinic or nicotine receptors, inhibitors of choline acetyltransferase, inhibitors of epidermal acetylcholine release and inhibitors with regard to the expression of the synthesizing enzyme choline acetyltransferase.

Immunohistochemistry for the ChAT protein

The question of whether the enzyme ChAT, which is responsible for the synthesis of acetylcholine, is expressed in the epithelial cells on the surfaces of the bronchi and intestine was clarified in further experiments. In fact, a specific and intense ChAT immunoreactivity in the surface epithelium of human bronchial tubes - especially in cilia-bearing cells - and to a much greater extent in the human small intestine - was not found in the mucosa of the pyloric part of the human stomach. A specific ChAT-like immunoreactivity was also found in epithelial cells of the human colon and gallbladder and in human skin - especially in basal cells.

The expression of the ChAT protein could be detected with both a poly and a monoclonal antibody. A positive ChAT immunoactivity was also detected in mesothelial cells (eg pleura, pericardium). "Western blot analysis" for the ChAT protein in epithelial cells of human bronchi

"Western blot analysis" of the protein, which was enzymatically isolated from epithelial cells of human bronchi, provided further evidence of the presence of the ChAT protein. For comparison, a rat brain homogenate was also examined.

In the sample from the brain of the rat, the prominent band corresponds to the previously known 68 kd "neuronal" ChAT protein; in contrast, ChAT proteins with a lower molecular weight of 41 and 54 kd are found in epithelial cells of bronchial origin.

ChAT enzyme activity in epithelial cells

A further proof was provided by the direct measurement of the ChAT enzyme activity in the extracts of the intestinal epithelium, which were obtained with cotton swabs or in extracts, which were obtained enzymatically from epithelial cells.

ChAT activities of 3.5 + 1.3 (n = 5) and 28 + 11 (n = 5) nmol / mg protein / h were determined in a material isolated from human bronchi and small intestine. ChAT enzyme activity was also detected in epithelial cells cultured for 24 hours.

Bromoacetylcholine (30 uM), which is a specific ChAT inhibitor, reduced the enzyme activity by 80-90%.

Studies on the function of non-neuronal acetylcholine

Examinations on human skin

Small (1 x 0.4 cm) pieces of skin were obtained from fresh surgical material and placed in an oxygenated nutrient solution. The nutrient solution contained either the Addition of 1 γM atropine (blockade of muscarinic receptors) or 30 γM tubocurarine (blockage of nicotine receptors); the control was carried out without the addition of any medication. After 30 minutes and 2.5 hours, pieces of skin were removed from the medium and processed according to the prior art for electron microscopy.

It was found that the treatment with Atropiπ after 30 minutes led to an expansion of the intercellular space, a loosening of keratin filaments and desmosomes. The gap was measured morphometrically (NIH image program). The mean distance between two neighboring cells was 0.781 + 0.06 γM (20 interfaces) in the control, 1.06 + 0.006 γM (20 interfaces; p <0.01) after atropine treatment and 0.756 + 0.035 after tubocurarine (Counting 16 interfaces). After 2.5 hours of exposure, there was also an expansion after treatment with tubocurarine (control: 0.728 + 0.037 γm, count of 20 interfaces; tubocurarine: 0.926 + 0.118 γm, count of 20 interfaces, ρ <0.001).

Investigations on human small intestine

Small intestine pieces were obtained from fresh surgical material and incubated in an oxygenated nutrient solution in a manner similar to that described for skin. Instead of an electron microscopic examination, light microscopy was carried out after prior HE staining. It was found that atropine caused a significant expansion of small lymphatic vessels and lymphedema in the villous stroma compared to the control after 30 minutes of exposure. It is known that an intact function of "gap-juction" is necessary for the orderly contractile activity of small lymph vessels (Zawieja et al., Am J. Physiol 264, H1283-91, 1993). By blocking muscarinic receptors, ie by switching off the function of acetylcholine, a disturbance in the lymphatic flow was triggered. I ⁷

Studies on cultivated epithelial cells in human bronchi

Bronchia were isolated from the surgical material and epithelial cells were obtained according to the state of the art (Reinheimer et al, Am J Physiol 270, L 722-8, 1996). A standardized measuring method, the MTT method, was used to measure cell proliferation (Bagge et al , J Immunol Meth 119, 203-10, 1989) It was found that added acetylcholine triggered a concentration-dependent increase in cell proliferation (0 1 nM - 10 μM), while bromoacetylcholine, an inhibitor of acetylcholine synthesis, causes antagonists of nicotine and antagonists Muscarinic receptors lead to an antiproliferative effect. These results show that non-neuronal acetylcholine is involved in the regulation of cell proliferatives

³ H-thyme incorporation was used as a further method for measuring cell proliferation. This also showed that acetylcholine mediates an increase in profiling activity, while synthesis inhibitors reduce the nitrous rate

Examination of immune cells

Acetylcholine and ChAT enzyme activity were found in isolated human mononuclear cells (0 4 + 0 07 pmol / 10 ⁶ cells, n = 6) and in human alveolar macrophages

Regarding the interaction between the cholinergic system and immune cells, it could be demonstrated that exogenous and endogenous acetylcholine inhibits the activation of mucosal mast cells in human bronchial tubes.However, this inhibitory control function takes place in a narrow concentration window, under the conditions of a highly regulated cholinergic system the functional control can be lost pro-inflammatory effect This can be enhanced by a stimulatory effect of acetylcholine on the synthesis and release of the pro-inflammatory cytokine GM-CSF from human bronchial epithelial cells. Exogenously applied acetylcholine increases the GM-CSF release from Pπmark cultures more human bronchial epithelial cells two to three times. In an experiment on isolated human macrophages, it was found that after the blocking of nicotine and muscarinic receptors, an explosive migration with accompanying lysis of the cells occurred.

This means that non-neuronal acetylcholine is involved in the control of macrophage migration and phagocytosis.

In addition, reference is made in full to the disclosure of the German patent application No. 196 26 373.5, the priority of which this application claims.

Claims

claims

1. Use of a topical pharmaceutical active ingredient for the manufacture of a medicament for the treatment of disease states in the area of the skin, the respiratory tract and the gastrointestinal tract, which are caused by the action of non-neuronal acetylcholine.

2. A method for the treatment of disease states of the skin, the respiratory tract and the gastrointestinal tract of an individual, which are caused by the action of non-neuronal acetylcholine, characterized in that the individual is topically administered a pharmaceutically active substance in a pharmaceutically effective dose.

3. Use or method according to claim 1 or 2 for the treatment of the underfunction of non-neuronal acetylcholine in the area of the skin, the respiratory tract and the gastrointestinal tract.

4. Use according to claim 1 or 2, characterized in that the active ingredient is an agonist on muscarinic / nicotine receptors, an inhibitor of cholinesterase or an activator of the synthesis or release of non-neuronal acetylcholine.

5. Use or method according to claim 1 or 2 for the treatment of an hyperfunction of non-neuronal acetylcholine in the area of the skin, the respiratory tract and the gastrointestinal tract.

6. Use or method according to claim 1 or 2, characterized in that the active ingredient is an antagonist of muscarinic / nicotine receptors, an inhibitor of the release of acetylcholine and / or an inhibitor of the synthesis of acetylcholine.

7. Use or method according to one of claims 1 to 4, characterized in that the active ingredient is a topically acting agonist on muscarinic receptors for the treatment of respiratory diseases.

8. Use or method according to claim 7 for treatment or cystic fibrosis.

9. Use or method according to one of claims 1 to 4, characterized in that the active ingredient is a topically acting inhibitor of cholinesterase for the treatment of respiratory diseases.

10. Use or method according to claim 9 for the treatment of cystic fibrosis.

11. Use or method according to any one of claims 1 to 4, characterized in that the active ingredient is a stimulant of the epidermal cholinergic system for the treatment of skin diseases.

12. Use or method according to claim 11 for the treatment of pemphigus, wound healing disorders, hair loss or disorders of hair growth.

13. Use or method according to one of claims 1, 2, 5 or 6, characterized in that the active ingredient is an inhibitor of choline acetyl transferase for the treatment of skin diseases.

14. Use or method according to one of claims 1, 2, 5 or 6, characterized in that the active substance is an antagonist of muscarinic receptors for the treatment of respiratory diseases.

15. Use or method according to claim 14 for the treatment of bronchial asthma.

16. Use or method according to any one of claims 1 to 6, characterized in that the active ingredient is an agonist or antagonist of muscarinic or nicotine receptors for the treatment of inflammatory diseases of the gastrointestinal tract.

17. Use or method according to claim 16 for the treatment of ulcerative colitis.

18. Use or method according to claim 16, characterized in that the active substance is an antagonist of muscarinic receptors for the treatment of diseases of the gastrointestinal tract.

19. Use or method according to claim 18 for the treatment of aqueous diarrhea.

20. Use or method according to one of claims 1, 2, 5 or 6, characterized in that the active ingredient is an inhibitor of the epidermal cholinergic system for the treatment of skin diseases.

21. Use or method according to one of claims 1, 2, 5 or 6, characterized in that the active ingredient is an antagonist of muscarinic and / or nicotine receptors for the treatment of inflammatory skin diseases.

22. The method or use according to claim 21 for the treatment of neurodermatitis, atopic dermatitis or contact dermatitis.

23. The method or use according to claim 21 for the treatment of psoriasis.

24. The method or use according to claim 21 for the treatment of cholinergic utricia.

25. The method or use according to any one of claims 1, 2, 3 or 4, characterized in that the active ingredient is an activator of choline acetyl transferase or stimulates the release of acetylcholine from non-neuronal cells (glial cells) for the treatment of Alzheimer's disease.

26. A method for determining the functional state of non-neuronal acetylcholine in direct or endoscopically accessible human tissues or cells, characterized in that a sample is taken from these tissues and the content of acetylcholine in this sample is determined in a manner known per se.

27. The method according to claim 26, characterized in that the sampling is carried out by wiping the surface with a cotton swab.

28. The method according to claim 26 or 27, characterized in that the sampling is carried out with a moistened cotton roll in order to determine the content of epithelial acetylcholine within the mucous membranes of the respiratory tract, gastrointestinal tract or the urogenital tract.

29. The method according to claim 26, characterized in that the cup technique is used to determine the acetylcholine from the skin, the oral mucosa and the vaginal mucosa.

30. The method according to any one of claims 26 to 29, characterized in that stimulation solutions are used to determine the functionality of non-neuronal acetylcholine.

31. The method according to claim 30, characterized in that solutions of nicotine, citric acid or ß-adrenoreceptor agonists are used.