Mónika Kuzma

Production of Orally Applicable New Drug or Drug Combinations from Natural Origin Capsaicinoids for Human Medical Therapy

It is well known that the capsaicin stimulates (in small doses) or impairs (in high doses) the capsaicin-sensitive afferent nerves and the final effects of capsaicin depend on its applied doses. The effects of capsaicin were analyzed on the gastrointestinal mucosal protection and injury in animal experiments and in human beings (from 1980 up to now). From 2005 to 2008 an interdisciplinary group (21 researchers) participated in the production of orally applicable drug or drug combinations from capsaicin for human medical therapy of patients suffering from cardiovascular, degenerative joint and locomotor diseases, who received in their treatments non-steroidal anti-inflammatory compounds (NSAIDs). Our studies were based on the results of the NSAIDs-induced gastrointestinal side effects could be detected by application of small doses of capsaicin. Because natural (plant origin) capsaicin is chemically does not represent a uniform entity and used in the international research, consequently the authors met a lot of unpredictable scientific problems during the time of production of new capsaicin containing (alone or in combinations) drug before receiving official permissions from the different national and international authorities to start the classical human clinical pharmacological studies. This paper summarizes the different steps from the basic physiological and pharmacological notes (in animals), plant cultivation, chemistry of substance(s), animal (general and germinative) acute and chronic toxicology, human actions, basic clinical pharmacology of natural capsaicin (capsaicinoids) to introduce and to develop a new drug (or drug combinations) in the human medical therapy.

A validated HPLC-FLD method for analysis of intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in the rat

A sensitive and selective reverse-phase high performance liquid chromatographic method with fluorescence detection has been developed for determination of capsaicin (8-methyl-N-vanillyl-(trans)-6-nonenamid) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide) in samples generated in rat small intestine luminal perfusion experiments. The experiments were designed to study the biotransformation of capsaicinoids in the small intestine in the rat. The chromatographic separation was performed at room temperature on a ZORBAX Eclipse(®) XDB-C8 column using isocratic elution with a mobile phase consisting 0.05M orthophosphoric acid solution and acetonitrile (60:40, v/v; pH 3.0) with a flow rate of 1.5mL/min. Fluorescence detection was performed at excitation and emission wavelengths of 230 and 323nm, respectively. The method was evaluated for a number of validation characteristics (accuracy, repeatability and intermediate precision, limit of detection, limit of quantification and calibration range). The limit of detection (LOD) was 50ng/mL and the limit of quantification (LOQ) was 100ng/mL for both capsaicin and dihydrocapsaicin reference standards dissolved in blank perfusate. The method was successfully applied for investigation of intestinal absorption of capsaicin and dihydrocapsaicin while 30μg/mL standardized Capsicum extract - containing capsaicin and dihydrocapsaicin - was luminally perfused for a 90min period. The structure of the glucuronide metabolites of capsaicin and dihydrocapsaicin appeared in the perfusate was identified by mass spectrometry.

Further Aspects of Ochratoxin A-Cation Interactions: Complex Formation with Zinc Ions and a Novel Analytical Application of Ochratoxin A-Magnesium Interaction in the HPLC-FLD System

Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species. Since its mechanism of action is not fully understood yet, it is important to gain further insight into different interactions of OTA at the molecular level. OTA is found worldwide in many foods and drinks. Moreover, it can also be detected in human and animal tissues and body fluids, as well. Therefore, the development of highly sensitive quantitative methods for the determination of OTA is of utmost importance. OTA most likely forms complexes with divalent cations, both in cells and body fluids. In the present study, the OTA-zinc interaction was investigated and compared to OTA-magnesium complex formation using fluorescence spectroscopy and molecular modeling. Our results show that zinc(II) ion forms a two-fold higher stable complex with OTA than magnesium(II) ion. In addition, based on the enhanced fluorescence emission of OTA in its magnesium-bound form, a novel RP-HPLC-fluorescence detector (FLD) method was also established. Our results highlight that the application of magnesium chloride in alkaline eluents results in an approximately two-fold increase in sensitivity using the HPLC-FLD technique.

Capsaicin is a New Gastrointestinal Mucosal Protecting Drug Candidate in Humans — Pharmaceutical Development and Production Based on Clinical Pharmacology

Backgrounds. 1. The intact gastrointestinal mucosa is a result of excellently well regulated equilibrium between the aggressive (physical and other stress, xenobiotics, wide scale of drugs, chemicals, bacterial and viral infections) and defensive (bicarbonate secretion, mucus secre‐ tion, blood supply, prostaglandins, mucosal energy systems, etc.) factors, which are further controlled by different neural, hormonal and pharmacological mechanisms. 2. The vagal nerve takes an essential place both in the development of gastrointestinal mucosal damage and protection. 3. The physicians have widely been applied the nonsteroidal antiinflammatory drugs (such as aspirin, diclofenac, Naproxen, etc.) as antipyretic, anti-inflammatory, painkiller and platelet aggregation inhibitor agents in healthy humans and in patients with different disorders (such as myocardial infaction, different forms of thrombophylia, rheuma‐ toid arthritis and arthrosis or trauma) in the everyday medical practice. The administration of these drugs produces gastrointestinal complaints (mucosal damages, bleedings, perforations). So in one hand, the applications of these drugs are absolutely indicated, on the other hand, the

Analysis of microalbuminuria with immunonephelometry and high performance liquid chromatography. Evaluation of new criteria

INTRODUCTION Hypertension as well as type 2 diabetes mellitus is a major factor in population mortality. Both diseases damage the endothelium, the early sign of which is microalbuminuria, which can be screened by dipstick and can be diagnosed by using immuno-based and high performance liquid chromatography methods. Using high performance liquid chromatography, the non-immunoreactive albumin can be detected as well. AIMS The authors aimed at the examination of albuminuria in the case of immunonephelometrically negative patients with high performance liquid chromatography, in diabetic and hypertensive and non-diabetic hypertensive populations. The authors also wanted to compare the present (albumin-creatinine ratio: male: > or =2.5 mg/mmol, female: > or =3.5 mg/mmol) and a new criteria of the Heart Outcomes Prevention Evaluation study (patients without diabetes: immunological method, > or =0.7 mg/mmol; high performance liquid chromatography, > or =3.1 mg/mmol; individuals with diabetes: immunological method, > or =1.4 mg/mmol; high performance liquid chromatography, > or =5.2 mg/mmol) of microalbuminuria. METHODS Examination of fresh urines of 469 microalbuminuria negative patients by dipstick were performed by immunonephelometry. Patients, who were microalbuminuria negative by immunonephelometry as well, were further analyzed by high performance liquid chromatography using the Accumintrade mark Kit, based on size-exclusion chromatography. RESULTS Three times higher albuminuria were found with high performance liquid chromatography than with immunonephelometry. The intraindividual coefficient of variation did not differ in the two methods (37 +/- 31% vs. 40 +/- 31%, p = 0.869; immunonephelometry vs. high performance liquid chromatography; mean +/- standard deviation). Using the present criteria for microalbuminuria, 43% of immunonephelometrically negative patients proved to be microalbuminuric by high performance liquid chromatography. Using the new criteria of the Heart Outcomes Prevention Evaluation study, the rate of microalbuminuria positivity among the immunonephelometrically negative patients decreased to 14.5% by high performance liquid chromatography and the decrease in the number of microalbuminuria positive cases by high performance liquid chromatography could be observed mainly in the diabetic and hypertensive group (49% vs. 7.5%), while slighter decrease could be observed in the non-diabetic hypertensive group (37% vs. 26.5%). Applying the traditional criteria, the strongest predictor was the male gender by the logistic regression analysis. In 28% of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established using high performance liquid chromatography. CONCLUSIONS Almost in one-third of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established by high performance liquid chromatography for which diagnosis three constitutive urine examinations are still needed. New criteria determined by the Heart Outcomes Prevention Evaluation study can be used neither in case of diabetic and hypertensive patients, nor in the case of non-diabetic hypertensive patients. The gender as the most important predictor of microalbuminuria cannot be ignored.

Development and Validation of an HPLC-DAD Analysis for Pharmacopoeial Qualification of Industrial Capsicum Extracts

A reverse-phase high-performance liquid chromatography method was developed to quantify capsaicin (trans-8-methyl-N-vanillyl-6-nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide) and the main capsaicinoid contents of Capsicum extracts. The chromatographic separation was carried out on a C8 column using isocratic mobile phase consisting of 40% (v/v) acetonitrile and 60% (v/v) orthophosphoric acid solution with flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at wavelength of 281 nm. The method was evaluated for number of validation characteristics (selectivity, accuracy (confidence intervals <1%), repeatability and intermediate precision (RSD% < 2.5%), limit of detection (LOD), limit of quantification (LOQ) and calibration range). The LOD was 0.25 µg/mL and the LOQ was 0.5 µg/mL. Using methanolic solutions of United States Pharmacopoeia (USP) Capsaicin and Dihydrocapsaicin Reference Standards, the method was linear over the concentration range 0.0005-0.5000 mg/mL for both capsaicinoids. The method was applied to qualify capsaicinoid content of two industrial capsicum extracts according to the USP 29.

Interactions of zearalenone and its reduced metabolites α-zearalenol and β-zearalenol with serum albumins: species differences, binding sites, and thermodynamics

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species. ZEN mainly appears in cereals and related foodstuffs, causing reproductive disorders in animals, due to its xenoestrogenic effects. The main reduced metabolites of ZEN are α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL). Similarly to ZEN, ZELs can also activate estrogen receptors; moreover, α-ZEL is the most potent endocrine disruptor among these three compounds. Serum albumin is the most abundant plasma protein in the circulation; it affects the tissue distribution and elimination of several drugs and xenobiotics. Although ZEN binds to albumin with high affinity, albumin-binding of α-ZEL and β-ZEL has not been investigated. In this study, the complex formation of ZEN, α-ZEL, and β-ZEL with human (HSA), bovine (BSA), porcine (PSA), and rat serum albumins (RSA) was investigated by fluorescence spectroscopy, affinity chromatography, thermodynamic studies, and molecular modeling. Our main observations are as follows: (1) ZEN binds with higher affinity to albumins than α-ZEL and β-ZEL. (2) The low binding affinity of β-ZEL toward albumin may result from its different binding position or binding site. (3) The binding constants of the mycotoxin-albumin complexes significantly vary with the species. (4) From the thermodynamic point of view, the formation of ZEN-HSA and ZEN-RSA complexes are similar, while the formation of ZEN-BSA and ZEN-PSA complexes are markedly different. These results suggest that the toxicological relevance of ZEN-albumin and ZEL-albumin interactions may also be species-dependent.

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat.

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 μm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC-DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5-50 μg/mL. After liquid-liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC-MS. The method was linear over the concentration range 0.25-5.0 μg/mL. Two hydroxylated metabolites of salicylic acid (2,5-dihydroxybenzoic acid and 2,3-dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3-DHB, 72.5% for 2,5-DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.

Oxidation of Hydroxy- and Dihydroxybenzoic Acids Under the Udenfriend's Conditions. An HPLC Study

Background: Non-enzymatic hydroxylation of aromatic compounds to the respective phenolic derivatives is a possible metabolic pathway of xenobiotics. The formed metabolites can undergo consecutive oxidative reactions with free radicals to form potential toxic molecules. Objective: Development of HPLC methods to separate, identify and quantitate the main products formed from salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid under in vitro hydroxylation conditions (Udenfriends system). Method: An RP-HPLC-UV-Vis method was developed to separate salicylic acid and isomeric dihydroxybenzoic acids formed in the Udenfriends system. Confirmation of structures of the oxidized products of salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was performed by HPLC-ESI-MS/MS method. Results: The HPLC-UV-Vis method was evaluated for a number of validation characteristics (selectivity, repeatability and intermediate precision, LOD, LOQ and calibration range). It was found that oxidation of salicylic acid resulted in the formation of 2,3- and 2,5-dihydroxybenzoic acids. Furthermore, the hydroxylated metabolites can be further metabolized under the Udenfriend’s conditions. Conclusion: The results give evidence for possible involvement of the oxidized metabolites of salicylic acid in the development of biological action of salicylates at the site of inflammation, where high hydroxyl radical level can be detected.

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Abstract Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.