CYCLOSPORIN A AFFECTS THE PROLIFERATION PROCESS IN NORMAL HUMAN DERMAL FIBROBLASTS.

Cyclosporin A is an immunosuppressant drug that is used not only in solid transplant rejection, but also in moderate and severe forms of psoriasis, pyoderma, lupus or arthritis. Serious side effects of the drug such as skin cancer or gingival hyperplasia probably start with the latent proliferation process. Little is known about the influence of cyclosporin A on molecular signaling in epidermal tissue. Thus, the aim of this study was to estimate the influence of cyclosporin A on the process of proliferation in normal human dermal fibroblasts. Fibroblasts were cultured in a liquid growth medium in standard conditions. Cyclosporin A was added to the culture after the confluence state. Survival and proliferation tests on human dermal fibroblast cells were performed. Total RNA was extracted from fibroblasts, based on which cDNA and cRNA were synthesized. The obtained cRNA was hybridized with the expression microarray HGU-133A_2.0. Statistical analysis of 2734 mRNAs was performed by the use of GeneSpring 13.0 software and only results with p < 0.05 were accepted. Analysis of variance with Tukey post hoc test with Benjamini-Hochberg correction for all three (8, 24, 48 h) culture stages (with and without cyclosporin A) was performed to lower the number of statistically significant results from 679 to 66, and less. Between statistically and biologically significant mRNAs down-regulated were EGRJ, BUBIB, MKI67, CDK1, TTK, E2F8, TPX2, however, the INSIG1, FOSL1, HMOX1 were up-regulated. The experiment data revealed that cyclosporin A up-regulated FOSL1 in the first 24 h, afterwards down-regulating its expression. The HMOX1 gene was up-regulated in the first stage of the experiment (CsA 8 h), however, after the next 16 h of culture time its expression was down-regulated (CsA 24 h), to finally increased in the later time period. The results indicate that cyclosporin A had a significant effect on proliferation in normal human dermal fibroblasts through the changes in the expression of genes related to the cell cycle and transcription regulation process.

EVALUATION OF LIPOPHILIC PROPERTIES OF BETAMETHASONE AND RELATED COMPOUNDS.

The lipophilicity (R(MW)) of betamethasone and its four related compounds: betamethasone-17,21-dipropionate, betamethasone-17-valerate, betamethasone-21-valerate and also betamethasone disodium phosphate was determined by reversed phase HPTLC and various mobile phase systems (methanol-water, dioxane-water and acetonitrile-water). The chromatographic lipophilicity parameters obtained for all examined compounds using abovementioned mobile phases onto three chromatographic plates (RP-2F254, RP-8F254, RP-18WF254) were compared with the theoretical partition coefficients which have been calculated by different computing programs: AlogPs, AClogP, AlogP, MlogP, KOWWIN, xlogP2, xlogP3, logP(ChemDraw) as well as with logP measured by shake-flask method. The results of this work demonstrate that regardless of applied method the greatest similarity in lipophilic properties show betamethasone-17-valerate, betamethasone-21-valerate and also betamethasone 17,21-dipropionate. The influence of solvent system as mobile phase on R(MW) values of examined compounds was observed. Among different mobile phases (organic modifier-water) proposed in this study, which allowed obtaining the reliable chromatographic lipophilicity parameters for all studied compounds is methanol-water mixture. The performance investigations showed that RP-HPTLC method has proved to be a rapid and cost effective analytical tool for describing the lipophilic properties of betamethasone and its related compounds.

LIPOPHILICITY ASSESSMENT OF SPIRONOLACTONE BY MEANS OF REVERSED PHASE LIQUID CHROMATOGRAPHY AND BY NEWLY DEVELOPED CALCULATION PROCEDURES.

The parameters of lipophilicity of spironolactone (a single member of steroids group), which is widely applied as diuretic and antihypertensive agent, were experimentally determined by reversed-phase TLC and HPLC methods as well as calculated using different computer programs and also by a novel mode based on topological indices. Various stationary phases, such as RP-18WFA4254, RP-2F254, RP-18F254 and also different binary solvent systems composed of organic modifier (e.g., methanol, dioxane, acetone) and water were used as mobile phases in order to predict the following chromatographic parameters: Rm, and logkw, respectively. LogP of examined spironolactone calculated with respective theoretical procedures: AlogPs, logPKOWWIN, xlogP2, xlogP3, AClogP, AlogP, MlogP and also logPaverage were obtained from online package software. The partition coefficients expressed as logP1, logP2 and logP3 were calculated by means of the formulae based on the numerical values of the following topological indices: , 0B, 1B, W, 0χv and IB, which was novelty of this study. A good agreement between logP calculated by new method and experimentally estimated lipophilicity parameters (by chromatography and by shake flask method) was found. The results confirmed applicability of the topological indices for calculating lipophilicity of spironolactone as alternative procedure to the experimental and other computed logP values.

Application of chemometric analysis based on physicochemical and chromatographic data for the differentiation origin of plant protection products containing chlorpyrifos.

The aim of this study was to investigate the similarities and dissimilarities between the pesticide samples in form of emulsifiable concentrates (EC) formulation containing chlorpyrifos as active ingredient coming from different sources (i.e., shops and wholesales) and also belonging to various series. The results obtained by the Headspace Gas Chromatography-Mass Spectrometry method and also some selected physicochemical properties of examined pesticides including pH, density, stability, active ingredient and water content in pesticides tested were compared using two chemometric methods. Applicability of simple cluster analysis and also principal component analysis of obtained data in differentiation of examined plant protection products coming from different sources was confirmed. It would be advantageous in the routine control of originality and also in the detection of counterfeit pesticides, respectively, among commercially available pesticides containing chlorpyrifos as an active ingredient.

Validation of an RPHPTLC-Densitometric Method Using Silica Gel 60 RP18WF254 for Simultaneous Determination of Nicotinamide in Selected Pharmaceutical Formulations.

This research study describes the applicability of silica gel 60 RPW18F254 plates for the development and validation of new, simple, economic, accurate, and precise RPHPTLC-densitometric method suitable for the quantification of nicotinamide (as Vitamin PP) in three marketed preparations. The mobile phase used was methanol-water in volume composition 3 : 7. Detection wavelength was 200 nm. The proposed method was validated according to ICH guidelines and also based on Ferenczi-Fodor and Konieczka reports. Results were found to be linear over a range of 1.00 to 2.00 µg/spot. Limit of detection (LOD) and limit of quantification (LOQ) were 0.15 µg/spot and 0.45 µg/spot, respectively. The percent content of nicotinamide in the investigated preparations was found to be 99.2% (Product 1), 99.3% (Product 2), and 99.4% (Product 3). Developed method is accurate and precise (CV < 3%) and may be successfully applied for the quality control of pharmaceutical formulations containing nicotinamide in the presence of its derivatives, such as N,N-diethylnicotinamide, N-methylnicotinamide, and nicotinic acid.

Application of various methods to determine the lipophilicity parameters of the selected urea pesticides as predictors of their bioaccumulation.

Different lipophilicity procedures including a newly developed (based on Oscik's equation) was applied in order to compare various urea pesticides with herbicidal and also insecticidal activity, such as monolinuron, chlorotoluron, diuron, isoproturon, linuron, dimefuron, diflubenzuron, teflubenzuron and lufenuron. Lipophilicity parameters (RMWS and RMW0) of nine examined pesticides were determined on the chromatographic plates RP-8F254 with the use of methanol-water as a mobile phase. Similarity analysis enabled to group all examined pesticides depending on their lipophilic character and allowed to perform a more objective comparison of different lipophilicity parameters obtained for investigated compounds by means of thin-layer chromatography and by the use of computational methods. It was stated that with the number of fluorine in examined pesticides, the lipophilic character of insecticides and also their tendency to bioaccumulation in the living systems increases noticeably. The results of this work confirmed that a new procedure for determining the lipophilicity parameter (RMW0) by Oscik's equation could be a suitable tool in the prediction of pesticide bioaccumulation in living system and may be used as an indicator in design of new urea pesticides, which will be safe for humans and the environment.

Validation of a thin-layer chromatography for the determination of hydrocortisone acetate and lidocaine in a pharmaceutical preparation.

A new specific, precise, accurate, and robust TLC-densitometry has been developed for the simultaneous determination of hydrocortisone acetate and lidocaine hydrochloride in combined pharmaceutical formulation. The chromatographic analysis was carried out using a mobile phase consisting of chloroform+acetone+ammonia (25%) in volume composition 8:2:0.1 and silica gel 60F254 plates. Densitometric detection was performed in UV at wavelengths 200 nm and 250 nm, respectively, for lidocaine hydrochloride and hydrocortisone acetate. The validation of the proposed method was performed in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and robustness. The applied TLC procedure is linear in hydrocortisone acetate concentration range of 3.75÷12.50 µg·spot(-1), and from 1.00÷2.50 µg·spot(-1) for lidocaine hydrochloride. The developed method was found to be accurate (the value of the coefficient of variation CV [%] is less than 3%), precise (CV [%] is less than 2%), specific, and robust. LOQ of hydrocortisone acetate is 0.198 µg·spot(-1) and LOD is 0.066 µg·spot(-1). LOQ and LOD values for lidocaine hydrochloride are 0.270 and 0.090 µg·spot(-1), respectively. The assay value of both bioactive substances is consistent with the limits recommended by Pharmacopoeia.

Detection progress of selected drugs in TLC.

This entry describes applications of known indicators and dyes as new visualizing reagents and various visualizing systems as well as photocatalytic reactions and bioautography method for the detection of bioactive compounds including drugs and compounds isolated from herbal extracts. Broadening index, detection index, characteristics of densitometric band, modified contrast index, limit of detection, densitometric visualizing index, and linearity range of detected compounds were used for the evaluation of visualizing effects of applied visualizing reagents. It was shown that visualizing effect depends on the chemical structure of the visualizing reagent, the structure of the substance detected, and the chromatographic adsorbent applied. The usefulness of densitometry to direct detection of some drugs was also shown. Quoted papers indicate the detection progress of selected drugs investigated by thin-layer chromatography (TLC).

TLC--densitometric method for qualitative analysis of betamethasone and its related compounds in pharmacautical preparations.

A new simple and rapid TLC-densitometric procedure for the separation and identification of betamethasone and its related substances, betamethasone-17,21-dipropionate, betamethasone-17-valerate, betamethasone-21-valerate and also betamethasone disodium phosphate was developed. One of the chromatographic systems proposed in this study, which has been satisfactory applied in separation of four pairs of examined compounds was silica gel 60F254 (E. Merck, Art. 1.05554) and a mixture containing chloroform-methanol-acetic acid (99.5%) in volume composition 28:5:0.5. Densitometric measurements were done using densitometer TLC Scanner 3 at 246 nm. The proposed method was checked in terms of its specificity for the determination of betamethasone-17,21-dipropionate and betamethasone disodium phosphate in commercially available products containing both compounds, separately, as active ingredients. The results showed that the method is suitable for qualitative analysis of betamethasone derivatives in simple and combined pharmaceuticals in various dosage forms e.g., lotion and injection solution. It also can be applied in quality control of pharmaceutical formulations of betamethasone and its related compounds in form of salts and esters.

Application of TLC, HPLC and GC methods to the study of amino acid and peptide enantiomers: a review.

Amino acids are very important organic compounds in nature. The biological activity of amino acids depends mainly on their stereoisomeric configuration (d- or l-). Thus, the stereochemical analysis of amino acids and peptides is an important aspect of their characterization. Owing to the increasing role of amino acid configuration in biomedical and pharmaceutical studies, numerous analytical methods have been described in the literature. Among a wide range of analytical techniques available for the steroselective separation of different amino acids, which were obtained from plants or biological samples, chromatographic methods such as thin-layer chromatography, high-performance chromatography and also gas chromatography are very useful. This review presents such systems developed for direct stereoisomeric separation and quantitative determination of amino acid and peptide enantiomers with emphasis on selected literature published during two last decades. Almost all aspects, including sample preparation prior chromatographic analysis, stationary phase, solvent system and detection system, are discussed. New possibilities in chiral amino acid analysis have been opened up by the application of mass spectrometry and infrared detection in thin-layer chromatography and mass spectrometry-selected ion monitoring detection system in gas chromatography. A modern trend in high-performance chromatography is two-dimensional chromatography. These innovations have led to decreased development time and increased amino acid resolution and detectability.

Validation thin layer chromatography for the determination of acetaminophen in tablets and comparison with a pharmacopeial method.

Adsorption thin layer chromatography (NP-TLC) with densitometry has been established for the identification and the quantification of acetaminophen in three leading commercial products of pharmaceutical tablets coded as brand: P1 (Product no. 1), P2 (Product no. 2), and P3 (Product no. 3). Applied chromatographic conditions have separated acetaminophen from its related substances, namely, 4-aminophenol and and 4'-chloroacetanilide. UV densitometry was performed in absorbance mode at 248 nm. The presented method was validated by specificity, range, linearity, accuracy, precision, detection limit, quantitative limit, and robustness. The TLC-densitometric method was also compared with a pharmacopeial UV-spectrophotometric method for the assay of acetaminophen, and the results confirmed statistically that the NP-TLC-densitometric method can be used as a substitute method. It could be said that the validated NP-TLC-densitometric method is suitable for the routine analysis of acetaminophen in quantity control laboratories.

Topological indexes and QSRR methodology in pharmaceutical and chemical analysis.

This paper presents an assessment of the possibility of using topological indexes and related structural descriptors in TLC, HPLC, GC, micellar electrokinetic capillary chromatography (MEKC), and capillary zone electrophoresis (CZE) techniques. Every work quoted is a new aspect of the use of topological indexes. Data from the scientific literature indicate that the topological indexes discussed can be used to predict and describe retention and lipophilic parameters obtained by TLC, HPLC, GC, MEKC, and CZE for different classes of organic compounds including drugs. The results presented indicate that the topological indexes and other structural descriptors have significance in analytical investigations of organic compounds. This review indicates that further investigations on the application of the topological indexes in TLC, HPLC, GC, MEKC, and CZE are justifiable.

Lipophilicity of thiobarbiturates determined by TLC.

Lipophilicty of 29 thiobarbituric acid derivatives were assessed by reversed-phase thin-layer chromatography using methanol: water mixtures as a mobile phase. A linear relationship was found between RM values and methanol concentrations in the mobile phase. The retention parameter, RM0, was related to theoretical partition coefficients calculated by means of different theoretical procedures (AlogPs, IAlogP, miLogP, logPKowwin, xlogP).

Densitometric determination of arbutin in cowberry leaves (Vaccinium vitis idaeae).

Densitometry was used for quantitative determination of arbutin (Vaccinium vitis idaeae) in leaves of cowberry collected from region of Suwalszczyzna, Poland. Arbutin was extracted using methanol. Chromatography was performed on glass TLC plates with layers of silica gel. The quantitative densitometric analysis was performed using internal standard solution method. On the base of densitometric analysis it was shown that the band characteristic for absorption maximum of arbutin is placed at lambda(max) = 285 nm. The second absorption band is at lambda = 225 nm. It was stated that contents of arbutin are ca. 35 mg and 47 mg in 1 g of herbs, in cowberry leaves coming from collections in 2005 and 2006 year, respectively. The presented method is accurate, selective, and precise, and can be used for routine quality control analysis and quantitative determination of arbutin in cowberry leaves.

A comparison of theoretical methods of calculation of partition coefficients for selected drugs.

Hundred ninety three drugs of different pharmacological activity were studied. Lipophilicity of a drug is one of the parameters, which influence its biological activity. The n-octanol-water partition coefficients were calculated for these compounds by use of different theoretical procedures (AlogPs, IAlogP, miLogP, ClogP, logP(Kowwin), and xlogP). Particular theoretical partition coefficients were compared with experimental n-octanol-water partition coefficients (logP(exp)) for all studied drugs. It was shown that experimental partition coefficients correlate the best with theoretical partition coefficients calculated by use of logP(Kowwin) and AlogPs methods. It was shown that it exists the possibility of the prediction of experimental n-octanol-water partition coefficients on the basis of logP(Kowwin), AlogPs, and ClogP for fifteen drugs (adrenalin, clobazam, 5,5-dimethylbarbituric acid, ethyl nicotinate, fluphenazine, ibuprofen, methyllorazepam, pimozide, prednisolone, promethazine, spironolactone, surital, theophylline, triamterene, and trimethoprim).

Application of structural descriptors for the evaluation of some physicochemical properties of selected bile acids.

Selected bile acids such as: cholic (C), glycocholic (GC), glycodeoxycholic (GDC), chenodeoxycholic (CDC), deoxycholic (DC), lithocholic (LC) and glycolithocholic acids (GLC) were separated by TLC technique. Selected topological indices, based on adjacency and distance matrices, and electrotopological states were calculated for the studied bile acids. Different possibilities of application of structural descriptors to calculate specified physicochemical data of the examined bile acids were found (QSPR). Usability of structural descriptors for the estimation of chromatographic separations of the examined bile acids was shown only for optimal separation conditions. Of all topological indices, only the index C allows to describe the order of adsorption of the examined bile acids (QSRR). Whereas, Gutman's index Mv was proved to be most useful for the estimation of lipophilicity of the examined bile acids (QSAR). The studies indicate that the range of applicability of given structural descriptors is limited for certain analytical and physicochemical problems.

A comparison of chromatographic separation of selected nicotinic acid derivatives by TLC and HPLC techniques.

The separations of nicotinic acid and its derivatives analyzed by adsorption and reversed phase TLC and HPLC were compared. Results were presented as the dependencies of chromatographic retention parameters of studied compounds on the volume composition [%] of mobile phases. It was shown that there exists the possibility of the separation of compounds into specified groups (adsorption TLC and HPLC) and particular substances within individual groups (reversed phase TLC).

Application of topological indexes for evaluation of the TLC separation of selected essential oil components.

The selected essential oil components (menthol, (+)bomeol, geraniol, linalool, carvone, camphor, (IR)-(-)fenchone) were separated by adsorption thin-layer chromatography using benzene as mobile phase. Investigated terpenes were characterized by selected topological indexes based on the adjacency and distance matrix. From among all the topological indexes counted only Si(o) index (the sum of the distance between the oxygen atom and all the remaining atoms in graph) as well as cluster Randic indexes (4chi(c), and 4chi(v)c) allow for estimation of the chromatograms obtained. With the fact mentioned above, there exists the possibility of prediction of relative situation on a chromatogram of the terpenes investigated. For the rest of topological indexes that possibility was not stated.

Application of topological indices for prediction of the biological activity of selected alkoxyphenols.

The topological indices based on adjacency and distance matrices, and electrotopological states were calculated for selected meta and para alkoxyphenols. The toxicities of alkoxyphenols on gram-positive M. pyogenes var. aurens and gram-negative S. typhosa bacteria were calculated on the basis of linear or parabolic equations with one topological index or hydrophobic constants. It was affirmed, that structural descriptors describe better the toxic properties of studied alkoxyphenols than the hydrophobic constants.