Capsaicin-Sensitive Afferent Nerves and the Human Gastrointestinal Tract

Capsaicin activates the capsaicin (vanilloid) receptor expressed a subgroup of primary afferent nociceptive neurons (Szolcsányi, 2004). The capsaicin receptor has been cloned (Caterina et al., 1997) and turned out to be a cation channel. It is gated besides capsaicin and other capsaicinoids (some vanilloids) by low pH, noxious heat and various pains – producing endogenous and exogenous chemicals. Thus, these sensory nerve endings equipped with these ion channels are prone to be stimulated in gastric mucosa.


Introduction
Capsaicin is an active ingredient of red pepper and paprika. These plants are well known and used in every day of the culinary practice for about 9000-9500 years.
It was an important discovery that the capsaicin (capsaicin, dihydrocapsaicin, nordihydrocapsaicin and other capsaicinoids) specifically modify the function of certain nerves, later named to capsaicin sensitive afferent nerves (Jancsó et al., 1967;1968;1970).
Capsaicin activates the capsaicin (vanilloid) receptor expressed a subgroup of primary afferent nociceptive neurons (Szolcsányi, 2004). The capsaicin receptor has been cloned (Caterina et al., 1997) and turned out to be a cation channel. It is gated besides capsaicin and other capsaicinoids (some vanilloids) by low pH, noxious heat and various pains -producing endogenous and exogenous chemicals. Thus, these sensory nerve endings equipped with these ion channels are prone to be stimulated in gastric mucosa.
The action of capsaicin on the capsaicin sensitive afferent nerves is dose dependent (Szolcsányi and Barthó, 1981;Szolcsányi, 1984;1997;2004 The vagal nerve has a key-role in the development of GI mucosal damage and prevention (Mózsik et al., 1982). The potency of vagal nerve has been emphasized dominantly in the aggressive processes to GI mucosa (such as peptic ulcer disease, gastric mucosal damage, etc.) in both animal models and in human investigations. The "chemical" and "surgical" vagotomy widely used in the treatment of patients with peptic ulcer disease in the years up to middle of 1970 (Karádi and Mózsik, 2000). By the other words, the primary aims of this therapy were to decrease the activity of vagal nerve at the level of efferent nerves in the target organs.
The application of capsaicin in the animal experiments was used as a specific tool to approach the group of primary afferent nociceptive neurons (Szolcsányi, 2004;Buck and Burks, 1986;Holzer, 1988;1991;Szállasi andBlumberg, 1999, Holzer, 2013) involved in the different physiological, pathological processes and medical therapy in human healthy subjects and in patients with different GI disorders as well those treated with nonsteroidal anti-inflammatory drugs (NSAIDs). Szolcsányi and Barthó (1981) were the firsts, who clearly indicated the beneficial and harmful effect of capsaicin in the peptic ulcer disease in rats on dependence of applied doses of capsaicin. Later, Holzer started with a very extensive research work with capsaicin in the field of gastroenterology (Holzer, 1998;1999;Buck and Burks, 1986;Szállasi andBlumberg, 1999, Holzer, 2013). Our group also participated in the GI capsaicin research in animals experiments from 1980 (Mózsik et al., 1997) (the historical background see the chapter written by Szolcsányi, 2014).
The new and interesting results obtained with capsaicin application in animal experiments offered excellent tools to approach the different events of human GI physiology, pathology and pharmacology and to produce new drug or new drug combinations in human healthy subjects and patients with different GI and other diseases (myocardial infarction, thrombophylia, rheumatoid arthritis, chronic pain killer use).
These studies were carried out as propective, randomized and multiclinical studies of human healthy subjects and in patients with various gastrointestinal disorders including gastric mucosal damage produced by application of NSAIDs or H. pylori infection.
The aims of this review are: (1) to give a short summary on the actions of capsaicin on the human gastrointestinal tract (dominantly on the stomach), (2) to indicate some details of gastroprotective actions of capsaicin in human healthy subjects; (3) to demonstrate the capsaicin-induced gastric protective effects against the NSAID-(selective and non-selective COX inhibitor) induced gastric microbleedings in human healthy subjects; (4) to prove the independency of TRPV1 and CGRP expression from the presence of the H. pylori positive or negative chronic gastritis, the efficacy of successfully carried out eradication treatment in patients with H. pylori positive gastritis in patients; 5. to indicate the clinical pharmacological problems of plant origin capsaicin in humans (in term of drug processing); (6). to point out that the gastroprotective effects of capsaicin (given orally in stimulatory doses of capsaicin on the capsaicin-sensitive afferent nerves) to human healthy subjects, and as treatment to patients who are under chronic NSAID use (like patients with myocardial infarction, stroke, thrombophylia, rheumatic diseases, etc.).
In terms of classical pharmacology we would like to demonstrate clearly in the human observations that the applied doses of capsaicin stimulate the capsaicin-sensitive afferent nerve with a clear cut exclusion of the existence of capsaicin desensitation.
On the other hand, our research activity clearly demonstrates the different difficulties of capsaicin(oids) application as new gastroprotective drug for healthy subjects and for patients with different GI disorders and NSAIDs use in regards to toxicology plant cultivation, storage, chemical detection and standardization questions as well as permission requests from authorities needed prior the launch of industrial processing of plant-derived, orally applicable drug or drug combinations.
The human healthy subjects and patients were included into the groups of different randomized, prospective studies (see later).
The classical human clinical pharmacological phase I examinations were carried out in 15 healthy males in each protocol (additionally up today, 30 human healthy subjects for human phase I examinations for aspirin plus capsaicin and diclofenac plus capsaicin) (some parts of these observations are presented by the book chapter written Mózsik et al., 2014).
The physical, laboratory, and iconographic examinations were normal in the healthy human subjects, in patients with various GI disorders.
The healthy persons were randomized into different groups of prospective, randomized and prospective studies for evaluation the effects of capsaicin on: 7. indomethacin-induced gastric microbleedings without and with capsaicin (Fisher and Hunt, 1976).
The different doses of capsaicin (1 -8 μg/100 mL in saline solution) were intragastrically given to identify the ED 50 values on the gastric basal acid secretion. These doses of capsaicin were used to determine direct action of capsaicin on the gastric transmucosal potential difference (GTPD) (without and with intragastric administration of ethanol) and indomethacin-induced gastric microbleedings.
The all of the healthy volunteers received capsaicin in doses with random allocation. In the observations to study the gastric emptying, sugar loading test, the effect of the ED 50 value (400 μg intragastrically given) of capsaicin was tested.
The 178 patients with different GI disorders were studied by immunohistochemical examinations of biopsy samples. The histological diagnosis was established on the opinion of independent pathologist. The immunohistochemical examinations for capsaicin receptor, CGRP, SP were carried out on the same on paraffin-embedded tissues samples from which the classical histological diagnosis was established by the independent pathologist (Dömötör et al., 2005(Dömötör et al., , 2006. The patients with chronic gastritis (69 patients) were divided H. pylori positive and negative groups. Smaller group of patients with H. pylori infection were studied further after classical eradication treatment had been performed.
The observations were carried out according to the method of Good Clinical Practice (GCP).
The studies were carried out from 1997 up to now, which were permitted by the Regional Ethical Committee of University of Pécs, Hungary. Written informed consent was obtained from all participants.
The following main methods were used in the human observations:

Determination of gastric basal acid out (BAO)
Determination of gastric basal acid out (BAO) in human healthy subjects: After an overnight fasting, a nasogastric tube was introduced at 8.00 a.m., and the total gastric content was suctioned. Than the newly secreted gastric juice was suctioned every 15 min for 1 h (BAO Gastric acid secretion was measured by titration of gastric juice with 0.1 N NaOH to pH 7.0 (pH titrimeter, Radelkis, Budapest). The gastric acid outputs were expressed as mmol/h (means ± SEM).

Determination of affinity and intrinsic activity curves
Determination of affinity and intrinsic activity curves for drugs inhibiting BAO in healthy human subjects: The applied doses of drugs were expressed in molar values, which were used to determine the affinity and intrinsic activity curves of the different drugs by the method of Csáky (1969). For drawing the affinity and intrinsic activity curves the doses of various drugs were expressed in [-] molar values, which offered to analyze the drug actions on the BAO according to the classical molecular pharmacological methods. We identified the pD 2 values (necessary doses of drugs to produce 50 per cent inhibition on BAO values). The effect of atropine (α atropine =1.00) in case of identification of intrinsic activity curves for other drugs and capsaicin (Mózsik, 2006).

Chemical composition of gastric juice without and with capsaicin application
These observations were carried out in the same healthy human subjects. The concentrations of Na + , K + and calcium 2+ in the gastric juice were measured flamephotometrically. The concentration of Mg 2+ was measured by atomic absorption spectrophotometrically, the chloride concentration by colorimetric method, the protein content by the method of biuret reaction. The 400 μg of capsaicin (as ED 50 value) was used in these studies.

Calculation of "parietal" and " non-parietal" components of gastric secretory responses without and with capsaicin
The chloride linked to H + and sodium was calculated for the determination of "parietal" (chloride linked to H + ) and "non-parietal" (linked to sodium) components of gastric BAO (Hollander, 1934).

Measurement of gastric transmucosal potential difference (GTPD)
GTPD was measured during endoscopy. The exploring mucosal electrode was passed through the biopsy channel of gastroscope and the reference electrode was placed on the volar surface of the left forearm. The electrodes were connected to a digital voltmeter (Radelkis, Budapest, Hungary, OP 211/1). GTPD measurements were done at the greater curvature of the gastric body and the results were expressed in -mV (without and with intragastrically administration of different doses of capsaicin) (Hossennbocus et al., 1975;Mózsik et al., 2005). Five mL capsaicin (300 mL/L, diluted in saline) was intragastrically applied and only saline solution was given to identify the baseline in GTPD. The ∆GTPD values were expressed in -mV, ΔGTPD max was calculated at five minutes after intragastric application of capsaicin.

Effect of capsaicin on ethanol-induced changes GTPD changes
The GTPD baseline was identified. Then ethanol (5 mL, 300 mL/L) was intragastrically given. The GTPD change was determined after the ethanol had been passed through the biopsy channel of gastroscope without and with capsaicin administration (given in different doses in the same pathway after 1 min of ethanol administration).

Measurements of gastric microbleeding produced by acute application of indomethacin (without and with capsaicin administration) in healthy human subjects
Non-selective COX inhibitor, indomethacin (IND) was used to induce gastric microbleeding. The extents of IND-induced gastric microbleeding were measured in healthy human subjects by the method of hemoglobin concentration in the gastric juice respecting the value of gastric emptying rate (Fisher and Hunt, 1976). The details of this method were described in one previous paper (Mózsik et al., 2005).

Baseline of gastric mucosal microbleedings in acute observations with human healthy subjects
The baseline of gastric microbleeding was measured in the gastric juice without application of any drug and/or capsaicin. The hemoglobin concentration was determined. The extent of gastric emptying was measured with application of phenol red into the stomach (by the method of Fisher and Hunt 1976; Nagy et al., 1984). The extent of gastric microbleeding was expressed in mL/day (means ±SEM), and this value was taken as baseline (used as control for other observations).

Capsaicin-induced acute mucosal protection on the IND-induced acute gastric microbleeding in healthy human subjects
The healthy human subjects received IND (3x25 mg given orally for a day), and the forthcoming day the gastric microbleeding was measured on forthcoming day, when these healthy human subjects also received 25 mg IND orally. The extent of gastric microbleeding was measured as mentioned above, and its value was expressed as mL/ day (means ± SEM).

Measurements of gastric emptying
The gastric emptying measurements were performed on two consecutive days by the same protocol, without capsaicin on first day and with capsaicin (400 μg orally given, ED 50 value) on second day. The measurement procedure was the following. The healthy human subjects went on an overnight starvation and the observations were started at 8.00 a.m. In total, 100 mL of 13 C-octanoid acid (Izinta, Budapest, Hungary) was given for the gastric emptying measurements. This material was given in 200 mL physiological saline and 75 g glucose was added to the solution. The volunteers exhaled into a plastic bag with a volume of 0.5 L. The first air sample was considered as reference.

Sugar loading test in healthy human subjects
The glucose (75 g) was orally given in 100 mL water. The plasma level of glucose was measured enzymatical (Boehringer, Germany). The plasma levels of insulin (μIU/mL) (Biochem Immunsystem), C peptide and glucagon (pg/mL) (Byk-Sangtect Diagnostic GmbH) were measured by RIA. The all measurements were carried out before and after administration repeatedly in every 15 mins for 4 h period (Dömötör et al., 2006b).

Immunohistochemical examinations in the gastric and large bowel mucosa in patients with various disorders
The classical pathological histological examinations were carried out by an independent pathologist for giving the histopathological diagnosis of patients (besides the classical laboratory, iconographic examinations).
The specific immunohistochemical examinations were used for the same biopsy specimens used for pathological examination. Specific antisera were used for detection of vanilloid (TRVP1) receptor (polyclonal anti-TRVP1, Abcam, Cambridge, UK), CGRP (polyclonal anti-CGRP, Abcam, Cambridge, UK) and SP (monoclonal anti SP, Abcam, Cambridge, UK).
The TRVP1 and CGRP positive and/or negativity was detected, meanwhile SP staining was evaluated by a semi-quantitave scale (Dömötör et. al., 2005).

Detection of Helicobacter pylori in patients with chronic gastritis
The presence of H. pylori was detected by 13 C-urea breath test (Izinta, Hungary) and with specific histological staining of biopsy specimens. The diagnosis of chronic gastritis was based on the classical pathological histology. The results of observations were expressed as means ± SEM. The unpaired and paired Student's t tests were used for the calculation of results between the identical observations. P value ≤ 0.05 was considered statistically significant.

Measurement of gastric microbleeding before and after 2 weeks capsaicin treatment
The baseline in gastric microbleeding was measured and carried out as those under the point of 7.1. The gastric microbleeding was expressed in mL/ day (means ±SEM).

Measurement of IND-induced acute gastric microbleeding before and after 2-week capsaicin treatment
These measurements were carried out in healthy human subjects as those were written under 7.2. These healthy subjects received 3x25 mg IND for one day and 25 mg IND on the next day before the measurement of the extent of gastric microbleeding. The gastric microbleeding was expressed in mL/day (means ±SEM).

Measurement of capsaicin-induced acute gastric mucosal protection against the IND-induced acute gastric microbleeding before and after 2-week capsaicin treatment
The observations were carried out under the observational circumstances mentioned in 12.2, however, different doses of capsaicin (200 and 400 μg given orally) were used. Two hundred and 400 μg capsaicin were applied given orally before the measurement of IND-induced acute gastric microbleeding before and after 2-week capsaicin treatment. The presence of H. pylori was determined by the methods mentioned above.
The eradication therapy was involved a seven days treatment with double dose proton-pump inhibitor consisting of PPI (pantoprazole 2x40 mg/day), amoxicillin (1000 mg twice daily) and clarithromycin (500 mg twice daily), according to European guidelines (Malfertheiner et al., 2007). Following this one week of eradication period, the patients further treated with normal dose of PPI for other another week.
The gastroscopies, gastric biopsies, general and special immuno-histochemical examinations were carried out at the time of entry of patients into the eradication treatment, after the eradication treatment (Lakner et al., 2011).

Statistical evaluation of results
The results were expressed as means ± SEM. The paired and unpaired Student's t test and ANOVA test were used for the statistical analysis of the results. The results were taken to be significant when the P value was found ≤ 0.05. Special mathematical programs were applied for the evaluation of results of human phase I. examinations.

Capsaicin-induced BAO in healthy human subjects
The capsaicin (given in doses

Affinity and intrinsic affinity curves for the capsaicin, muscarinic agents, H 2 -receptor antagonists and proton pump inhibitors on BAO in healthy human subjects
The action of the compounds inhibiting the gastric basal acid secretion is presented by Figure  3. The curve indicates that no competitive actions of these drugs exist on the gastric basal acid output. The pD 2 values were calculated from the affinity curves obtained in the molecular pharmacological studies. Inhibition  The intrinsic activity curves for the drugs inhibiting the gastric basal acid outputs in healthy human subjects were calculated. The intrinsic activity (α atropine =1.00) was taken to be equal to 1.00, and the values for other drugs were expressed to action of atropine ( Figure 4). The pA 2 values (50 % inhibition of intrinsic activity in [-] molar values) were calculated from the intrinsic activity curves.
For the molecular pharmacological understanding the background of the action of drugs, action the molecular weights, pD 2 values, of the intrinsic activity (in comparison to atropine action) and pA 2 values were calculated and presented in Table 1 Table 1 clearly indicate that the capsaicin acts in the smaller doses as those drugs acting on the muscarinic (atropine, Pirenzepine), H 2 R receptor antagonists (cimetidine, ranitidine, fomatidine, nizatidine) and proton pump inhibitors (Omeprazole, Esomeprazole).

Changes in the "parietal" and "non-parietal" components of gastric secretory responses in healthy human subjects
The measurements of cations (H + , sodium, potassium, calcium, magnesium) and of chloride offered the possibility to identify the "parietal" and "non-parietal" components of gastric secretion in humans without and with administration of different drugs (or compounds) (Hollander, 1934).
Using the Hollander's method for the calculation and evaluation of cations, chloride in the gastric juice indicated clearly the significant decrease of "parietal component" (∆-18±2 mmol/ L, P< 0.001) in association with significant increase of " non-parietal component" (∆+19±2 mmol/L, P < 0.001) of the gastric secretion after application of 400 μg (given orally) of capsaicin

Action of capsaicin on the GTPD in the healthy human subjects
The capsaicin (given ig. in doses of 100, 200, 400 and 800 μg) dose-dependently increased the GTPD alone [∆ value from to baseline 10 (

Action of ethanol on GTPD in healthy human subjects
The

Preventive action of capsaicin on the ethanol-induced decrease of GTPD in healthy human subjects
The intragastrically applied capsaicin (given in doses

Extent of IND-induced acute gastric mucosal damage in healthy human subjects
The baseline of blood losing was 2.0 ± 0.2 mL/ day (n=14) without application of IND, which was increased to 8.1 ± 0.2 mL/day (n= 14; P< 0.001) after application of indomethacin.

Gastric mucosal preventive effect of capsaicin on the IND-induced acute gastric microbleeding in healthy human subjects
The

Increased glucose absorption from small intestine and of glucagon release by capsaicin during the glucose loading test in healthy human subjects
During glucose loading test we measured the glucose absorption in the proximal part of the small intestine, insulin, C peptide, glucagon using the plasma level of glucose as markers substance (Dömötör et al., 2006). The absorption of glucose from the small intestine and glucagon release increased by capsaicin administration; however no significant changes were obtained in neither insulin nor C peptide release under these observational circumstances ( Figure 15).
The plasma levels of glucose increased significantly 30 to 150 min and the plasma level of glucagon increased from 90 to 180 min after capsaicin administration in human healthy subjects given 75 g glucose orally. The plasma levels of insulin and C peptide increased from 75 to 165 min after glucose administration; however, levels did not differ significantly.

Demonstration of capsaicin receptors, CGRP and SP in the human gastric and large bowel mucosa in healthy human subjects
The results of these observations were obtained in " healthy human subjects", who had different functional complaints and the endoscopic examinations were carried out to exclude the presence of any histologically proven disease (the clinical histological examinations were carried out by the independent pathologist) and the opinion of pathologist gave normal histology.

Demonstration of capsaicin receptor, CGRP and SP in the gastric and large bowel mucosa in patients with different disorders
The preliminary results of these observations were published by Dömötör et al. (2005).
These patients went over the clinical endoscopic and pathological examinations. The original pathological diagnosis was given by an independent pathologist.
The capsaicin receptor, and released neuropeptides (CGRP and SP) could be detected in all types of patients with different disorders ( Table 3). The results of these preliminary clearly indicated the following: 1. capsaicin could be demonstrated in patients with superficial gastritis, erosive gastritis, stomach polyps, stomach cancer, inflammation of large bowel disease, colon polyps with severe dysplasia and colorectal cancers.
The CGRP could be demonstrated in most of the all of the above mentioned diseases; meanwhile the SP could not be demonstrated in these diseases (Table 3).   Figures 18 and 19)

Baseline before and after 2 weeks IND treatment without application of any drug and/or compound
The baseline of gastric microbleeding was detected 2.1 ± 0.1 vs. 2.0± 0.1 mL/day, before and after 2 weeks capsaicin treatment (without other drug /compound application).

Measurement of acutely applied capsaicin-induced gastric mucosal protective effect on the INDinduced acute gastric microbleeding before and after 2 weeks capsaicin treatment in healthy human subjects
The The symptoms of patient's suffering from chronic gastritis with or without H. pylori infection (20 H. pylori positive and 30 H. pylori negative) were nonspecific including epigastric discomfort, nausea, loss of appetite and vomiting. The patients underwent physical, laboratory, ultrasonographic, endoscopic and histological (including special immuno-histochemical) examinations. Twenty people with functional dyspepsia (all of them underwent the aforementioned medical, laboratory, iconographic and histological examinations and all of these examinations indicated negative results) were taken as healthy controls. The age of patients was 39 to 68 years; there were 22 males and 29 females with chronic gastritis, and ten males and ten females in the functional dyspepsia group.
The H. pylori infection was detected by 14 C urea breath test, rapid urease test, Warthinn-Starry silver staining and other specific histological examinations. The gastric tissue samples from the stomach and antrum were examined by an independent histologist and classified of chronic gastritis according to the Sydney System.
The immuno-histochemical studies were carried out on formalin fixed, paraffin embedded tissue samples of gastric mucosa using the peroxidase-labeled polymer method (Lab Vision Co., Fremont, USA). SP was detected by the NC1/34HL rat monoclonal antibody, the TRPV1 receptor and CGRP were labeled using polyclonal rabbit antisera (all from Alcam Ltd., UK, Cambridge).

Capsaicin-sensitive afferentation in H. pylori positive chronic gastritis before and after eradication treatment
These observations were carried out in 38 persons, including 20 healthy subjects and 18 patients with H. pylori positive gastritis. The age of persons with histologically intact gastric mucosa (controls) were 41 to 67 years (mean=52.2 years). The age of patients with chronic H. pylori positive gastritis (6 males, 12 females) was 39 to 68 years (mean=56.4 years).
The time period between the first and control gastroscopy was 6 weeks after the starting of the examinations. The biopsies were taken from the corpus and antrum of patients with chronic gastritis, before and after eradication treatment and from healthy subjects.
The H. pylori positive patients underwent 7-day eradication treatment with combination of double dose of PPI (pantoprazole 2x40 mg/ day), amoxicillin (1000 mg twice daily) and clarithromycin (500 mg twice daily) according to the European guidelines. After this one week combination treatment, the patients continued to take normal dose of PPI for another week.
The H. pylori infection was detected before and after treatment. The results of eradication treatment was successful in 89%, the gastric histology (by biopsy and by histology) indicated normal picture in 22% of cases, and 78 % of patients showed moderate gastritis.  Table 4).

Discussion
The vagal nerve plays a key role in the gastrointestinal physiology, pathology and pharmacology. The nerve fibres of vagus can be divided into afferent (about 90%) and efferent fibres (10 %) based on animal observations. About 9 per cent of afferent fibres are capsaicin sensitive afferent fibres (Gabella and Pease, 1973; Grijalva and Novin, 1990).
Many observations on the field of Gastroenterology were based on the modification of efferent fibres of vagal nerve, except the classical surgical vagotomy (both in the animal experiments and in human observations with peptic ulcer disease). This standpoint was emphasized during the research period performed by the application of different anticholinergic compounds acting at the level of muscarinic receptor.
Since histamine also plays an essential role in the gastric acid secretion and in many other immunological processes, various H 2 receptor antagonist compounds were developed like cimetidine, ranitidine, famotidine and nizatidine.
After recognition of the H + -K + -ATPase as biochemical structure of gastric acid secretion, the so-called proton pump inhibitors (H + -K + -ATPase inhibitors) were developed and studied in clinical practice with great efforts (Mózsik, 2006).  , 1965; 1967; 1969a; 1969b). These studied tried to reveal the details of absorption, metabolization and excretion of various anticholinergic agents in patients with peptic ulcer before the starting of chronic treatment, after the regular chronic treatment and after cessation of these drugs. The application of anticholinergic agents to patients was used to approach the cholinergic mediated processes both in the development and treatment of peptic ulcer diseases.
After the years of 1970, the H 2 R blocking compounds became deeply studied in human GI physiology, pathology and pharmacology. Many clinical pharmacological studies with H 2 R blockers have been carried out in patients with peptic ulcer disease (Mózsik et al., 1994;Patty et al., 1984;Tárnok et al., 1979;1983). In the last two decades the proton pump inhibitors were deeply studied. The results from animal experiments with capsaicin which is widely used in the every-day culinary practice, clearly indicated us that the stimulatory doses of capsaicin acting on capsaicin-sensitive fibres produce gastric mucosal defensive actions and they are able to prevent the NSAID-induced gastric mucosal damage. These scientifically carried out studies with capsaicin in animals also offered a new tool to approach the capsaicin sensitive afferent nerves in the healthy human beings, and to some extent in patients with different GI disorders.
Our studies with capsaicin in human healthy subjects and in patients with different gastrointestinal disorders have been stated from 1997. These studies were permitted by the Regional Ethical Committee of University of Pécs, Hungary, and these observations were carried out according to Good Clinical Practice respecting the Helsinki Declaration.
The human observations were carried out according to the basic laws of human clinical pharmacology (inclusion and exclusion criteria, randomization, prospective studies, generally self-controlled group of healthy human subjects, etc).
We had five aims in these studies with the capsaicin:

1.
To understand the main mechanisms of capsaicin sensitive afferent nerves in the gastric functions;

2.
To try to understand the potential role(s) of capsaicin sensitive afferent nerves in the development of human physiological, pathological and pharmacological events;

3.
To identify the dose range of capsaicin which stimulates only the capsaicin-sensitive afferent nerves and to identify the classical molecular pharmacological parameters (affinity and intrinsic activity curves, ED 50 and pA 2 values) in comparison the same parameters obtained in cases of every day used drugs;

4.
To exclude clearly the existence of desensitation of capsaicin-sensitive afferent nerves to capsaicin (under different observation circumstances, namely in acute administration, before and after two weeks capsaicin treatment, given in dose of 3 x 400 μg /day) to capsaicin;

5.
To process and even to produce a new capsaicin containing drug or drug combinations to modify the capsaicin-sensitive afferentation in human healthy subjects and to treat patients with GI mucosal damage against NSAIDs and H. pylori infection.
These aims determined us to use a significant number of the methodologies applied in the human studies. However, we had to use classical molecular pharmacological methods to compare and to understand some details of capsaicin-induced changes in the human physiological, pathological parameters.
The following main trends were applied in the capsaicin research:

1.
To determine the dose-response curves for the various drugs and capsaicin.

2.
To introduce the classical molecular pharmacological methods for understanding the capsaicin-induced action in comparison to others produced by anticholinergic drugs, H 2 R antagonists or proton pump inhibitors.

3.
The specific immunohistochemistry (for obtained morphological evidence) was incorporated into the research.

4.
Different parameters were simultaneously measured (e.g. plasma glucose, insulin, Cpeptide and glucagon were detected during the sugar loading test in the healthy human subjects).

5.
Capsaicin studies were carried out not only in acute administration and after a chronic capsaicin administration.

6.
The immunohistochemical studies were carried out in the GI mucosa of patients with different GI disorders (acute gastric mucosal damage, chronic inflammation, precancerous state and cancers in the stomach and in the large bowel);

7.
The human pathological diagnosis was given by an independent pathologist.

9.
To evaluate the possible role of capsaicin afferentation in the prevention or treatment of gastric mucosal damage produced by NSAIDs in healthy subjects and in patients who are treated with these drugs (as pain killers, platelet aggregation, anti-inflammatory compounds, etc).

Capsaicin-sensitive efferent nerves vs. gastric secretion in healthy human subjects
The capsaicin dose-dependent manner decreased the gastric basal secretion (BAO) . The capsaicin was applied in very small doses (200 to 800 μg orally), which stimulate the capsaicin-sensitive afferent nerves.
When we applied the molecular pharmacological approach the actions of capsaicin, anticholinergic agents, H 2 R antagonists or proton pump inhibitors, we were surprised that capsaicin was able to inhibit gastric acid secretion in smaller molar concentration than other clinically widely used drugs (Figures 3 and 4). Analyzing the intrinsic activity of these drugs and capsaicin by the molecular pharmacological methods (intrinsic activity of atropine was taken to be 1.00), we found capsaicin's action to be lesser than atropine's ( Figure 3).
The affinity curves of different drugs and capsaicin were molecular pharmacologically determined and given as pD 2 (the necessary doses of drugs and capsaicin to produce 50% inhibition of gastric acid secretion (basal acid output), which expressed in [-] molar value) and as intrinsic activity (pA 2 ) (the necessary doses of drugs and capsaicin to produce 50% inhibition, also expressed in [-] molar) values ( Table 1).
The results of these molecular pharmacological studies clearly indicated the sensitivity of the various regulatory targets of different drugs and capsaicin in comparison to possible physio-logical roles of the target organs and the drug actions influence their functional activities and states. There is no question that the stimulation of capsaicin-induced afferent sensitive nerves plays a very significant effect in regulatory processes important for the maintenance of gastric mucosal integrity in human beings (including in healthy subjects and patients with different GI disorders or treated with different drugs, especially with NSAIDs).
The decrease of gastric acid secretion was explained by the increased H + back diffusion after capsaicin application via the increased vasodilatator processes induced by the release of the CGRP and SP in animal observations or by the increase of somatostatin secretion. The CGRP and SP together with capsaicin receptor (TRVP1) can be detected by inmunohistological methods in the gastric mucosa around nerves, vascular spaces, parietal cells and in epithelial layer.
The increased gastric secretory responses are present along with increased gastric mucosal blood flow. On the other hand, the increased gastric acid (H + ) secretion is closely associated with the increase of K + , Mg 2+ , Ca + and albumin in gastric juice. However, the decrease of gastric acid secretion produced by antisecretory agents is associated with the decrease of H + , K + , Mg 2+ , Ca 2+ and albumin (Myren, 1968).
Human observations with capsaicin do suggest presence of increased H + back diffusion during capsaicin action (except when capsaicin was given in high doses):

The increased H + back diffusion suggests the decreased level of albumin in the gastric juice.
Our results cannot prove the existence of gastric H + back diffusion in human healthy subjects during the capsaicin action:

2.
We calculated the "parietal" and "non-parietal "components of gastric juice after Hollander's original observation (Hollander 1934). Our results clearly indicate that the decrease of gastric H + concentration (and output) is closely associated with the increased extent of "non-parietal component" during the capsaicin action in the healthy human subjects. The "non-parietal component" of the gastric juice is a buffering part, which cannot be obtained in circumstance of passive metabolic processes. Earlier, the significant increase of buffering ("non-parietal component") secretion was obtained in 2-10 days after cessation of a prolonged atropine treatment in patients with peptic ulcer disease (Antal et al., 1966).

3.
There are other arguments also exist against the existence of the passive H + back diffusion in the stomach during capsaicin action in the healthy human subjects. When the capsaicin was directly given to gastric mucosa (using gastroscope), then GTPD increased in a dose dependent manner (Mózsik et al., 2005).
If ethanol was intragastrically given (using the biopsy channel), then GTPD immediately decreased, which could be reversed by the topical application of capsaicin. This action is also dose-dependent from capsaicin after ethanol application in the healthy human subjects (Mózsik et al., 2005).
The capsaicin action on the gastric secretory responses can be explained by different ways: chlorideproteins (albumin) Typical changes to decrease of inhibition of BAO in human healthy subjects Figure 23. Changes in the contents of electrolytes of gastric juice with increase and decrease of H + output, without (upper part) and with (below part) capsaicin application.

the capsaicin (given in doses producing the stimulation of capsaicin-sensitive afferent nerves) results directly neural (or hormonal) influences on the gastric mucosa;
4. other yet not known mechanism(s) existing in the regulation of the human gastric secretion.
The capsaicin given in ED 50 increased the gastric emptying Mózsik et al., , 2004b. This action of capsaicin on the gastric emptying can be explained at least by two pathways: 1. decrease of gastric acid secretion; 2. direct action on muscular function of the stomach (pylorus).
Up to now, the acute action of capsaicin was evaluated dominantly by the measuring one or two parameters.

Capsaicin action of glucose absorption from small intestine in human healthy subjects and its hormonal and metabolic backgrounds
The sugar loading test was applied for measuring absolutely different physiological events, e.g. glucose absorption from the proximal part of the small intestine and consequently the produced hormonal regulations during the glucose loading test.
The response to glucose loading in healthy human subject can be divided into three different periods on basis of physiological events after orally applied glucose in human healthy persons: 1. absorption (first period, from 30 to 90 min); 2. insulin (and other hormones) release (second period, from 60 to 150 min); 3. glycogen mobilization by the liver (third period, from 150 to 180 min) under adrenergic neural influences.
The glucose can be absorbed from the proximal part of the small intestine by active transport mechanism (in presence of Mg 2+ , mitochondrial ATP breakdown into ADP) (Dömötör et al., 2006b). The monitoring of the glucose level was used as biological marker for the equilibrium of the different physiological events. In the first period the plasma glucose level depends only on the glucose absorption; in the second period the plasma level of glucose represents the equilibrium between the absorption and hormone release; and in the third the glucose level represents the mobilization of glucose by the liver in healthy human subjects. It is also important that the insulin and glucagon act contra regulatory on glucose utilization in the serum.
By studying the time sequence of changes in plasma levels of glucose, insulin, C-peptide and glucagon after glucose loading in healthy human subjects without and with capsaicin (400 μg orally), we found the followings: 1. The plasma level of glucose (from 30 to 150 min) and the glucagon (from 90 to 180 min) increased significantly after glucose plus capsaicin administration.

2.
The plasma levels of insulin and C-peptide were increased from 90 to 165 min, however, no significant changes were observed between subjects without and with capsaicin.

3.
No significance in timing of insulin and glucagon release was observed, which clearly excludes the existence of antagonism between insulin and glucagon release (short time).

4.
The plasma level of glucagon was high for longer period than it was in case of insulin. It should be noted that capsaicin increased the glucagon level.
The results clearly indicate that capsaicin sensitive afferent nerves have a key-role in the regulation of glucose absorption from the small intestine (due to a local increase of blood flow), glucose utilization, and release of neuropeptide (presently the glucagon release). This human observation proved clear-cut the active participation of capsaicin sensitive afferent nerves in the carbohydrate metabolism (by the ways of modification of sugar absorption, hormone release). Up till now only the somatostatin release induced by capsaicin has been known (Szolcsányi, 2004).

Capsaicin given orally in small doses prevents with IND-induced gastric microbleedings in human healthy subjects
The IND was used in these studies as NSAID, which is a non-selective COX blocker (the ratio of ED 50 of IND on COX-1 /COX-2 = 0.30) (Kawai et al., 1998) ( Table 5). The extent of gastric microbleeding appears as consequence of COX-1 and COX-2 inhibition.
The results of our observations (Sarlós et al., 2003;Mózsik et al., 2003 the capsaicin (ig. given) dose dependently prevented the IND-induced gastric microbleeding before and after 2 weeks capsaicin treatment.
Under the results of Kawai et al. (1998), we calculated the extents of gastric microbleeding depending on COX-1 and COX-2 enzyme activity (Table 6). It was found that the capsaicininduced gastric mucosal protection remained unchanged -before and after 2 weeks capsaicin treatment -after both COX-1 and COX-2 enzyme inhibition.

Facts to exclude the presence of the desensibilization of capsaicin receptor to applied doses of capsaicin in acute observation circumstances
When we applied the capsaicin twice in doses of 800 μg ig. after 5 min interval, then we received the same extents of increase in difference of GTPD, indicating a very active metabolic action under these observation circumstances.
The same extent gastric mucosal protection was obtained in the IND-induced gastric mucosal damage in human healthy subjects. The actions of gastric mucosal prevention was dose-dependent on the IND-induced gastric microbleedings (please note that these human observations were carried out in prospective, randomized and multiclinical studies) (Figures 18 and 19).
The results of these mentioned observations clearly indicated that the doses of capsaicin in our studies could not produce desensibilization of capsaicin receptors (these doses of capsaicin only were able to stimulate the capsaicin-sensitive afferent nerve).

Facts to exclude the presence of desensibilization to capsaicin receptor by applied doses of capsaicin during 2 weeks capsaicin treatments
The capsaicin exerts gastroprotective effect remained unchanged on IND-induced gastric mucosal microbleeding before and after 2-week capsaicin (3x 400μg orally given) treatment (Figures 19 and 24. By other words, the gastric baseline microbleeding remained unchanged after the application of different doses of capsaicin (200, 400 and 800 μg orally) induced gastric mucosal prevention.
The registration of these facts are very important, since a very wide scale of capsaicin doses applied in human observations resulted very contradictory data (capsaicin were applied in stimulatory doses to capsaicin sensitive afferent nerve and much higher doses producing reversible and irreversible inhibitory actions in various animal experiments).
The capsaicin acts at the level of capsaicin receptor, which has been cloned (Caterina et al., 1997). It is true that the capsaicin action depends on its doses proved by animal experiments (Szolcsányi, 1984;Mózsik et al., 2001). The small dose (400 μg=ED 50 ) was applied for two weeks (in dose of 400 μg x 3 orally) in healthy subjects. The gastric mucosal microbleeding was produced by orally given IND ( Figure 19 and Table 6.
The following results were obtained: 1. The extent of baseline gastric microbleeding before and after 2 weeks capsaicin (without application of any drug) remained unchanged.

2.
The extent of acutely given IND-induced gastric microbleeding was also unchanged.

3.
The extent of capsaicin-induced gastric mucosal protection was also found to be the same (and dose dependent) before and after 2 weeks capsaicin treatment.
It has been concluded from these observations that the sensitivity of capsaicin receptor (TRVP1) unchanged, and on the other hand, the orally applied capsaicin is capable to exert gastric mucosal protection against IND.
Probably the applied small dose of capsaicin -used in our present studies -could not modify the TRVP1 receptor sensitivity. We have no knowledge in this field by using higher dose(s) of capsaicin in human healthy subjects.
These facts are very important from the point of selection of capsaicin containing drug, because these dosages of capsaicin could exert beneficial effect (given orally in small doses), meanwhile the capsaicin can produce reversible or irreversible damage on the human gastric mucosa.
Earlier similar types of observations were carried out with atropine and other parasympatholytics 1967; 1969 a,b) and cimetidine (Wildersmith et al., 1990). Tolerance developed to the drugs applied chronically in patients with peptic ulcer disease, and the "pharmacological denervation hypersensibility" occurred with a clinically detectable tolerance (Mózsik et al., 1966;1967;1969). These clinical pharmacological examinations modified the periodicity and dosage of applicable drugs (used in chronic treatment).
The careful analysis of the results with capsaicin actions, COX-1 and COX-2 systems and gastric microbleedings produced by IND in human healthy subjects before and after two weeks capsaicin (3 x 400 μg orally) treatment offered an excellent possibility to approach the capsaicin actions on the COX-1 and COX-2 enzyme system (Table 6).

Gastric (gastrointestinal) cytoprotection vs. capsaicin actions (given in stimulatory doses of capsaicin-sensitive afferent nerves)
Observations with different chemical agents (drugs, mediators, nutritive agents like retinoids, prostaglandins and others) indicated a special gastric mucosal protection, which did not depend on the gastric acid inhibition (see the reviews Mózsik et al. 2010, 2011). Later on, many observations indicated that the so-called gastric cytoprotective agents are able to prevent the development of injuries of numerous organs (produced by different actions) and they can accelerate healing processes.
The existence of gastric cytoprotection was earlier proved in patients with peptic ulcer (Mózsik et al., , 2011 as it was nominated by André Robert (1979).
Only few points are interesting in our present position, namely: 1. the capsaicin-induced gastric mucosal protection in human healthy subjects is accompanied by the decrease of gastric acid secretion (that was one of the criteria differed from "classic" gastric cytoprotection); 2.both "classical cytopotective effects" as well as capsaicin-produced gastric mucosal protection disappear after surgical vagotomy (but not after chemical vagotomy); 3. gastric cytoprotective effect produced by different agents remained to be the same besides application of capsaicin (given in doses to produce stimulation of capsaicin-sensitive afferent nerves) (Mózsik et al., 1997). These results offer us to suggest that: 1. some part a gastric mucosal (cyto)protection is very closely associated with the functional activity of capsaicin-sensitive afferent nerves; 2.

Distribution of capsaicin receptor, CGRP and SP in the gastric and large bowel mucosa of patients with different gastrointestinal disorders
The use of specific antisera against TRVP1 receptor, CGRP and SP released by the stimulation of capsaicin sensitive afferent nerve can immunohistochemical demonstrate their presence in the GI mucosa. Please note, that mucosal biopsy cannot be performed in healthy human subjects due to ethical regulations. So "healthy subjects"are represented by human subjects with functional disorders having no endoscopic abnormalities and diagnosed by clinicians and independent pathologist. There is another problem, namely the regular biopsy can be offered a small tissue sample for its regular pathological histological evaluation. Our specific immunohistochemical observations could be done only after successful routine histological examination.
We studied l78 patients with different gastrointestinal disorders (complaints), and the persons, who had normal histology based on the opinion of independent pathologist, were used as normal (healthy) human subjects (Dömötör et al., 2007;Mózsik et al., 2007).
The TRVP1 receptor, CGRP and SP neuropeptides released by the stimulation of capsaicin receptor can be shown by immonohistochemistry both in the human gastric and colon mucosa.
The TRVP1 receptors and neuropetides (CGRP and SP) could be demonstrated in the GI mucosa around the nerve endings, vascular elements, parietal cells, and also in epithelial layer.
The capsaicin application and the immunohistochemical demonstration of TRVP1 receptor in the GI tract newly met in the healthy human subjects. We studied the immunohistochemical distribution of TRVP1, CGRP and SP in the GI mucosa of patients with different disorders. The TRVP1 receptor, CGRP and SP could be detected practically in all patients with different acute, chronic diseases (including benignant, precancerous and cancerous diseases) (Dömötör et al., 2005). The results of these observations can be taken only as preliminary results. Their expression differed significantly in the GI mucosa of patients with acute and chronic disorders. Of course, the critical evaluation of these immunohistochemical observations is extremely hard, since we have only very limited information on the origin, stages, time periods of diseases, drug therapies and suggested etiological backgrounds. The studies dealing with the changes of individual patients with chronic GI disorders -in this research respect -are in progress. We have to emphasize two different main points in these studies, namely 1. The changes of the expressions of TRVP1, CGRP and SP are helpful from the point of development of GI mucosal injury and prevention, and 2. probably the participations of TRVP1, CGRP and SP differ in these GI pathological circumstances.

Immunohistochemical examinations in patients with chronic H. pylori positive and negative gastritis
In model human observations, the possible participation of TRVP1, CGRP and SP were studies in patients with chronic gastritis. The laboratory tests (quick urease test, urea breath test) and specific histological staining widely used in every day medical practice for demonstration of the presence of H. pylori infection were used, which suggested the presence of bacteria as the etiological for chronic gastritis.
Our studies were carried out in patients with H. pylori positive and negative chronic gastritis. The expression of TRVP1, CGRP and SP increased significantly in the gastric mucosa with chronic gastritis; however, no difference was obtained their expression in patients with H. pylori positive and negative chronic gastritis (Dömötör et al., 2006a).
The inflammation of the tissues does not represent a specific tissue reaction to inducing agents. This statement can be concluded from our results obtained in patients with H. pylori positive and negative chronic gastritis due the potential role of capsaicin sensitive afferent nerves. There is no question that the so-called "neurogenic inflammation" participated in the "general inflammatory processes" in patients with different gastrointestinal disorders.

Capsaicin sensitive afferentation in patients with chronic H. pylori positive gastritis before and after eradication treatment
The last step of our observations indicated that capsaicin-sensitive afferentation did not differ before and 6 weeks after successful eradication treatment in patients with chronic H. pylori positive gastritis (meanwhile the control biopsy was normal in 22 %, in 78% of cases it indicated a moderate improvement of the histological picture of gastritis) ( After careful analysis of these results, it can be stated that the capsaicin-sensitive afferentation represents as an essential pathway in the healing of chronic gastritis (probably without and with H. pylori infection). These results suggest an independent mechanism in the healing of chronic gastritis which differs from the eradication treatment. By other words, besides the H. pylori infection other factors might exist. The H. pylori infection can be taken as one (but probably most important) exogenous factor, however, the capsaicin-sensitive afferent fibres of vagal nerve represents an endogenous factor in the injury and protection of gastric mucosa (Mózsik et al., 2013).
Our results with capsaicin (in healthy human subjects and in patients with different gastrointestinal disorders) are summarized in Figure 21. The vagal nerve is able to modify the GI tract by regulatory steps at the central nervous system and at the level of target organ. The peripheral action of capsaicin is a dose dependent action (Szolcsányi, 1984; Mózsik et al., 2001) ( Table 5) There were a few very surprising matters due to capsaicin from the point of human classical clinical pharmacology prior our observations: 1. no permitted orally applicable capsaicin preparation was available for humans; 2. no chronic toxicology was known to capsaicin by animal observations; 3. no direct clinical pharmacological study had been performed to determine germinative function; 4. the capsaicin (Sigma-Aldrich, USA) chemically is not uniform due to its varying content of dihydrocapsaicin, nordihydrocapsaicin and some other capsaicinoids; 5. no classical human clinical pharmacological study (human phase I and phase II) was found in the literature; 6. no correct Drug Master File (DMF) for capsaicin preparation were developed for the commercially obtained capsaicin (exception of a certain capsaicin preparation obtained from India and used by us); 7. no human pharmacokinetic observations were available for capsaicin (please remember that our capsaicin studies in human healthy subjects were started in 1997). Bernard et al. (2008) were unable to create pharmacokinetic profile of capsaicinoids after administration of 15 and 30 mg of capsaicin/person; meanwhile, the detection limit was 10 ng/ ml. Chaiyasit et al. (2009) after the application of 5 gram of C. frurenscens orally (equivalent to 26.6 mg pure capsaicin) found that the capsaicin could be detected in plasma after 10 min, and the peak concentration (C max ) was 2.47 ± 0.13 ng/mL, t max was 47.1 ± 2.0 min and t ½ was 24.9 ± 5.0 min. After 90 min, capsaicin could not be detected in the plasma. Chaiyasit  We also received new information from the chronic toxicological studies in Beagle dogs (2008). These animals were treated with different doses (0.1, 0.3 and 0.9 mg/kg body weight/day orally given) of capsaicin(noids) for one month. No toxic effects were observed in these dogs during the whole treatment periods. We noticed surprisingly that capsaicin and dihydocapsaicin could not be detected in the sera of Beagle dogs by either high pressure chromatography with the detection limit of 20 ng/mL serum or liquid chromatography-mass spectrometry with the detectation was 26 fg/mL in the serum at any time after the oral administration (

Main conclusion from our obervations on capsaicin application in human healthy subjects and in patients with different disorders
1. The capsaicin (as a specific agent to stimulate the capsaicin-sensitive afferent nerves) plays a special role in the regulation the gastric functions (decrease of gastric acid secretion, increase of gastric emptying, increase in "buffering part" of the gastric secretion, increase of GTDP), absorption of glucose (and in its metabolism as well as in increase of glycagon release) in human healthy subjects, when it is applied in stimulatory doses;

2.
The capsaicin given orally in stimulatory doses protects against the alcohol-and NSAIDinduced gastric mucosal damage in healthy human subjects;.

3.
If the capsaicin is given in stimulatory doses to capsaicin-sensitive afferent nerves in human healthy subjects, it will not produce desensibilization of its receptor (neither in acute administrations nor in chronic administrations) in human healthy subjects ;

4.
The presence of capsaicin receptor (also CGRP and SP, which are very close in physiological relation) can be shown by specific immuno-histochemical methods under various GI disorders ;

5.
The capsaicin-induced gastroprotection differs from the eradication treatment in patients with chronic H. pylori positive gastritis (and probably the roles of capsaicin receptor and CGRP differ from SP);

6.
Very important to note that these gastroprotective actions can be obtained only by administration of capsaicin in stimulatory doses to its sensitive afferent nerves.
After looking of this conclusion list, there are clear evidences that the capsaicin is an orally applicable drug either alone or in combinations with different NSAIDs to human healthy subjects and to patients. Its indication is wide from prevention against drug-induced gastric mucosal damage, from patients under long term aspirin treatment (due to cardiovascular disease, thrombophylia, rheumatoid arthritis), pain killer treatment, patients having chronic H. pylori positive gastritis and to patient with different carbohydrate disorders.
We are of the opinion that we scientifically put down the clinical pharmacological bases of the oral capsaicin or drug combination use. We are in need of competent pharmaceutical partners for commercial introduction of these drug candidates into the everyday medical treatment (Mózsik, 2014; Mózsik et al., 2014).
Of course, we are aware that many other observations are need to be carried out in according to the regulations of international drug development, production and marketing for a capsaicin containing drug to licensed for everyday medical treatment.