RESUMEN
The biological activity of three previously synthesized 17ß-carboxamide glucocorticoids (BG, BEG, and MPEA) was tested in vitro on mitogen stimulated and non-stimulated peripheral blood mononuclear cells (MNCs) and granulocytes from human healthy donors, and the results were compared to the conventional glucocorticoid dexamethasone. The tested 17ß-carboxamide glucocorticoids did not induce decreases in MNC viability and proliferation, while modulation of reactive oxygen species (ROS) synthesis in granulocytes was dependent on the cell donor. The obtained results indicate the possibility of avoidance of strong lymphocyte suppression, which is generally recognized during administration of conventional glucocorticoids. Furthermore, the metabolism of the tested derivatives was predicted in silico. The predicted metabolites were synthesized and the in silico results were confirmed by in vitro evaluation of the metabolism of BG, BEG, and MPEA in human serum and in cultures of peripheral blood MNCs. The results of the biological activity and metabolism evaluation and of previous in vivo evaluations of biological activity indicate the soft drug nature of BG, BEG, and MPEA. In order to be fully considered as soft glucocorticoids, further investigations on the toxicity and activity of the formed metabolites are required.
Asunto(s)
Glucocorticoides/farmacología , Granulocitos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Simulación por Computador , Dexametasona/farmacología , Glucocorticoides/química , Granulocitos/metabolismo , Humanos , Leucocitos Mononucleares/metabolismoRESUMEN
Soft glucocorticoids are compounds that are biotransformed to inactive and non-toxic metabolites and have fewer side effects than traditional glucocorticoids. A new class of 17ß-carboxamide steroids has been recently introduced by our group. In this study, local anti-inflammatory activity of these derivatives was evaluated by use of the croton oil-induced ear edema test. Glucocorticoids with the highest maximal edema inhibition (MEI) were pointed out, and the systemic side effects of those with the lowest EC50 values were significantly lower in comparison to dexamethasone. A 3D-QSAR model was created and employed for the design of 27 compounds. By use of the sequential combination of ligand-based and structure-based virtual screening, three compounds were selected from the ChEMBL library and used as a starting point for the design of 15 derivatives. Molecular docking analysis of the designed derivatives with the highest predicted MEI and relative glucocorticoid receptor binding affinity (20, 22, 24-1, 25-1, 27, VS7, VS13, and VS14) confirmed the presence of interactions with the glucocorticoid receptor that are important for the activity.
Asunto(s)
Antiinflamatorios/farmacología , Diseño Asistido por Computadora , Enfermedades del Oído/tratamiento farmacológico , Edema/tratamiento farmacológico , Glucocorticoides/farmacología , Animales , Antiinflamatorios/efectos adversos , Antiinflamatorios/química , Aceite de Crotón , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Enfermedades del Oído/inducido químicamente , Edema/inducido químicamente , Glucocorticoides/efectos adversos , Glucocorticoides/química , Masculino , Conformación Molecular , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad Cuantitativa , Ratas , Ratas WistarRESUMEN
Molecular docking studies were performed on 18 17ß-carboxamide steroids in order to select compounds with potential local anti-inflammatory activity. These derivatives are amides of cortienic acids (obtained from hydrocortisone, prednisolone, and methylprednisolone) with methyl or ethyl esters of six amino acids. Interactions with the glucocorticoid receptor (GR), binding energies and ligand efficiency values of these compounds were compared with dexamethasone and cortienic acid obtained from prednisolone (inactive metabolite). On the basis of molecular docking studies, seven compounds were selected and their binding affinities for the GR were predicted by use of the exponential model created in this study. Subsequently, selected compounds were synthesized in good yields by use of modified N,N'-dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) coupling procedure. Finally, the local anti-inflammatory activity of the synthesized compounds was examined by use of the croton oil-induced ear edema test. In vivo evaluation of systemic side effects as well as in silico prediction of metabolism were performed on the derivative with the best local anti-inflammatory activity. The combination of molecular docking studies and the exponential model for the GR binding affinity prediction could be used as an in silico tool for the rational design of novel 17ß-carboxamide steroids with potentially better biological profile than dexamethasone.
Asunto(s)
Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Diseño de Fármacos , Edema/prevención & control , Glucocorticoides/síntesis química , Glucocorticoides/farmacología , Inflamación/prevención & control , Animales , Antiinflamatorios/metabolismo , Biotransformación , Aceite de Crotón , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Glucocorticoides/metabolismo , Hidrocortisona/análogos & derivados , Hidrocortisona/síntesis química , Hidrocortisona/metabolismo , Hidrocortisona/farmacología , Inflamación/inducido químicamente , Ligandos , Metilprednisolona/análogos & derivados , Metilprednisolona/síntesis química , Metilprednisolona/metabolismo , Metilprednisolona/farmacología , Modelos Biológicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Prednisolona/análogos & derivados , Prednisolona/síntesis química , Prednisolona/metabolismo , Prednisolona/farmacología , Ratas , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Relación Estructura-ActividadRESUMEN
Apart from cytotoxicity, inhibitors of the COX-2 enzyme have demonstrated additional effects important for cancer treatment (such as radiosensitization of tumor cells and cell antimigratory effects); however, the relationship between the inhibition of other inflammation-related enzyme 5-LOX inhibitors and anticancer activity is still not well understood. In our study, the cytotoxicity of thirteen COX-2 and 5-LOX inhibitors previously presented by our group (1-13) was tested on three cancer cell lines (HCT 116, HT-29 and BxPC-3) and one healthy cell line (MRC-5). Compounds 3, 5, 6 and 7 showed moderate cytotoxicity, but good selectivity towards cancer cell lines. IC50 values were in the range of 22.99-51.66 µM (HCT 116 cell line), 8.63-41.20 µM (BxPC-3 cell line) and 24.78-81.60 µM (HT-29 cell line; compound 7 > 100 µM). In comparison to tested, commercially available COX-2 and 5-LOX inhibitors, both cytotoxicity and selectivity were increased. The addition of compounds 6 and 7 to irradiation treatment showed the most significant decrease in cell proliferation of the HT-29 cell line (p < 0.001). The antimigratory potential of the best dual COX-2 and 5-LOX inhibitors (compounds 1, 2, 3 and 5) was tested by a wound-healing assay using the SW620 cell line. Compounds 1 and 3 were singled out as compounds with the most potent effect (relative wound closure was 3.20% (24 h), 5,08% (48 h) for compound 1 and 3.86% (24 h), 7.68% (48 h) for compound 3). Considering all these results, compound 3 stood out as the compound with the most optimal biological activity, with the best dual COX-2 and 5-LOX inhibitory activity, good selectivity towards tested cancer cell lines, significant cell antimigratory potential and a lack of toxic effects at therapeutic doses.
RESUMEN
DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests - parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.
Asunto(s)
Absorción Gastrointestinal , Relación Estructura-Actividad Cuantitativa , Inhibidores de Topoisomerasa II , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacocinética , Micelas , Modelos Lineales , Membranas Artificiales , Girasa de ADN/metabolismo , Girasa de ADN/química , Humanos , Topoisomerasa de ADN IV/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/químicaRESUMEN
Electrochemical oxidation of newly synthesized acridine derivatives (ADs): PG, PA, EG and EA was studied using square wave voltammetry at a glassy carbon electrode. Oxidation occurs as an irreversible process for all ADs. The ADs interaction with DNA was investigated using multi-layer DNA electrochemical biosensor. The shift of dA peak in positive direction indicated that the type of interaction is most likely an intercalation. PG showed the widest range of concentration capable to interact with DNA (1 × 10-7 M - 2.5 × 10-4 M). Analysing logIcomplexIDNA-IcomplexvslogcAD plots, the binding constants were determined. For the lowest PG concentrations, obtained K value close to 106 M-1 refers to strong binding. The values of K for PA may be classified as medium strength, while EG and EA showed low K values. Our results unequivocally showed that the characteristics of association complexes may vary depending on the concentration of the interacting substance. The negative ΔG value for all ADs, (- 21 to - 34 kJ mol-1), confirms the process spontaneity. The best result, indicating the most stable formed complex with DNA adsorbed at the electrode surface, showed PG when present in low concentration, order of 10-7 M. The intercalation of ADs into DNA was supported by molecular docking analysis.
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Técnicas Biosensibles , ADN , Simulación del Acoplamiento Molecular , ADN/química , Técnicas Biosensibles/métodos , Electrodos , AminoácidosRESUMEN
Various dual inhibitors of COX-2 and 5-LOX enzymes have been developed so far in order to obtain more effective and safer anti-inflammatory drugs. The aim of this study was to design and synthesize new dual COX-2 and 5-LOX inhibitors, and to evaluate their enzyme inhibition potential and redox properties. Thirteen compounds (1-13) were designed taking into account structural requirements for dual COX-2 and 5-LOX inhibition and antioxidant activity, synthesized, and structurally characterized. These compounds can be classified as N-hydroxyurea derivatives (1, 2 and 3), 3,5-di-tert-butylphenol derivatives (4, 5, 6, 7 and 13), urea derivatives (8, 9 and 10) and "type B hydroxamic acids" (11 and 12). COX-1, COX-2 and 5-LOX inhibitory activities were evaluated using fluorometric inhibitor screening kits. The evaluation of the redox activity of newly synthesized compounds was performed in vitro in the human serum pool using redox status tests. The prooxidative score, the antioxidative score and the oxy-score were calculated. Seven out of thirteen synthesized compounds (1, 2, 3, 5, 6, 11 and 12) proved to be dual COX-2 and 5-LOX inhibitors. These compounds expressed good COX-2/COX-1 selectivity. Moreover, dual inhibitors 1, 3, 5, 11 and 12 showed good antioxidant properties.
RESUMEN
The permeation properties of twenty newly synthesized α-alkoxyalkanoyl and α-aryloxyalkanoyl C-21 esters of standard corticosteroids: Fluocinolone acetonide, dexamethasone, triamcinolone acetonide and hydrocortisone were established using a PAMPA assay (70% silicone oil and 30% isopropyl myristate). The data were compared with parent corticosteroids with addition of mometasone furoate and hydrocortisone acetate. All newly synthesized corticosteroid C-21 esters have effective permeability coefficients higher then -6, mostly followed with high values of retention factors and low permeation. The examined compounds were grouped through relationship between obtained retention factors and permeation parameters (groups I-III). The classification confirmed group I (membrane retentions as well as permeation lower then 30%) for all corticosteroid standards except mometasone furoate, a potent topical corticosteroid which, with high membrane retention (81%) and low permeation (7.7%) fits into group III. The largest number of new synthesized corticosteroids C-21 esters, among them all fluocinolone acetonide C-21 esters, have high membrane retentions (32.4%-86.5%) and low permeations (1.3%-27.1%), fitting in group III. The classification was related to previously obtained anti-inflammatory activity data for the fluocinolone acetonide C-21 esters series. According to the PAMPA results the new synthesized esters could be considered as potential new prodrugs with useful benefit/risk ratio.
Asunto(s)
Ésteres/química , Membranas Artificiales , Modelos Biológicos , Pregnanos/química , Profármacos/química , Ésteres/síntesis química , Ésteres/normas , Humanos , Miristatos/química , Permeabilidad , Pregnanos/síntesis química , Pregnanos/normas , Profármacos/síntesis química , Profármacos/normas , Estándares de Referencia , Aceites de Silicona/química , Absorción CutáneaRESUMEN
In this study the solvolysis of newly synthesized fluocinolone acetonide C-21 esters was analysed in comparison with fluocinonide during a 24-hour period of time. The solvolysis was performed in an ethanol-water (90:10 v/v) mixture using the excess of NaHCO3. The solvolytic mixtures of each investigated ester have been assayed by a RP-HPLC method using isocratic elution with methanol-water (75:25 v/v); flow rate 1 mL/min; detection at 238 nm; temperature 25 °C. Solvolytic rate constants were calculated from the obtained data. Geometry optimizations and charges calculations were carried out by Gaussian W03 software. A good correlation (R = 0.9924) was obtained between solvolytic rate constants and the polarity of the C-O2 bond of those esters. The established relation between solvolytic rate constant (K) and lipophilicity (cLogP) with experimental anti-inflammatory activity could be indicative for topical corticosteroid prodrug activation.
Asunto(s)
Fluocinolona Acetonida/química , Profármacos/farmacocinética , Ésteres , Cinética , SolventesRESUMEN
The electrochemical behavior of 9-chloroacridine (9Cl-A), a precursor molecule for synthesis of acridine derivatives with cytostatic activity, is a complex, pH-dependent, diffusion-controlled irreversible process. Oxidation of 9Cl-A initiates with the formation of a cation radical monomer, continues via the formation of a dimer subsequent oxidation to new cation radical. Reduction of 9Cl-A produces radical monomers which are stabilized by dimer formation. The investigation was performed using cyclic, differential pulse and square wave voltammetry at a glassy carbon electrode. The interaction between 9Cl-A and double-stranded DNA (dsDNA) was investigated using a multilayer dsDNA-electrochemical biosensor and 9Cl-A solutions from 1.0×10-7M (the lowest 9Cl-A concentration whose interaction with DNA was possible to detect) up to 1×10-4M. These allowed the binding constant, K=3.45×105M-1 and change in Gibbs free energy of the formed adsorbed complex to be calculated. Complex formation was a spontaneous process proceeding via 9Cl-A intercalation into dsDNA inducing structural changes. The intercalation of 9Cl-A into dsDNA was supported by molecular docking analysis. The combination of simple methodology and the use of biosensors to investigate DNA interactions is a powerful tool to offer insight into aspects of drug design during pharmaceutical development.
Asunto(s)
Acridinas/química , ADN/química , Técnicas Electroquímicas/métodos , Electrodos , Simulación del Acoplamiento Molecular , Oxidación-ReducciónRESUMEN
A series of eleven 9-acridinyl amino acid derivatives were synthesized using a two-step procedure. Cytotoxicity was tested on the K562 and A549 cancer cell lines and normal diploid cell line MRC5 using the MTT assay. Compounds 6, 7, 8 and 9 were the most active, with IC50 values comparable to or lower than that of chemotherapeutic agent amsacrine. 8 and 9 were especially effective in the A549 cell line (IC50 ≈ 6 µM), which is of special interest since amsacrine is not sufficiently active in lung cancer patients. Cell cycle analysis revealed that 7 and 9 caused G2/M block, amsacrine caused arrest in the S phase, while 6 and 8 induced apoptotic cell death independently of the cell cycle regulation. In comparison to amsacrine, 6, 7, 8, and 9 showed similar inhibitory potential towards topoisomerase II, whereas only 7 showed DNA intercalation properties. In contrast to amsacrine, 6, 7, 8 and 9 showed a lack of toxicity towards unstimulated normal human leucocytes.
RESUMEN
In this study, the interaction of the anion of quinapril (QUIN), angiotensin converting enzyme (ACE) inhibitor, with cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of cationic micelles on the spectroscopic and acid-base properties of QUIN was studied at pH 8. The binding of QUIN anion to CTAB micelles implied a shift in drug acidity constant (pK(a)(water)-pK(a)(micelle)=1.39) proving the great affinity of negatively charged QUIN ion for the positively charged CTAB micelle surface. The strong dependence of the partition coefficient K(x) on QUIN concentration, obtained by using pseudo-phase model, is consistent with an adsorption-like phenomenon. From the dependence of differential absorbance at lambda=272 nm on CTAB concentration, by using mathematical model that treats the solubilization of QUIN anion as its binding to specific sites in the micelles (Langmuir adsorption isotherm), the binding constant K(b)=(2.3+/-0.4)x10(3) mol(-1)dm(3) was obtained. QUIN-CTAB binding constant was also calculated from micellar liquid chromatography (MLC) and this method was found to be not accurate enough for its determination.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Compuestos de Cetrimonio/química , Micelas , Tetrahidroisoquinolinas/química , Adsorción , Aniones/química , Cationes/química , Cetrimonio , Modelos Químicos , Estructura Molecular , Quinapril , Análisis Espectral , Propiedades de SuperficieRESUMEN
A capillary zone electrophoresis (CZE) and a high-performance liquid chromatography (HPLC) method have been developed for identification and determination of ketoconazole, an imidazole antifungal, in pharmaceutical preparations. The suitabilities of both methods for quantitative determination of ketoconazole were approved through validation specification such as linearity, precision, accuracy, limit of detection and quantification. The proposed methods were used for determination of ketoconazole in commercial pharmaceutical dosage forms (tablets and creams). Under described conditions, CZE method is more selective, while the HPLC method is more sensitive. Both methods are rapid (tR(CZE)=5.14 min and tR(HPLC)=2.66 min), which is important for routine application. However, the HPLC method provides a repeatability of the quantitative analysis of ketoconazole in drug formulations below 1.5% relative standard deviation (R.S.D), while the repeatability of the CZE method is in the order of 2-3% R.S.D.
Asunto(s)
Antifúngicos/análisis , Cromatografía Líquida de Alta Presión/métodos , Electroforesis Capilar/métodos , Cetoconazol/análisis , Química Farmacéutica , Formas de Dosificación , Imidazoles/análisis , Cetoconazol/química , Reproducibilidad de los ResultadosRESUMEN
In this paper, human skin and corneal permeability of twenty-two newly synthesized 17ß-carboxamide steroids was predicted using biopartitioning micellar chromatography (BMC). These compounds are potential soft glucocorticoids with local anti-inflammatory activity when applied to the skin or eye. BMC systems are used to simulate physicochemical properties of human skin (BMC-skin) and cornea (BMC-cornea). Micellar mobile phase, consisted of 0.04 M solution of polyoxyethylene (23) lauryl ether (Brij 35), was prepared at different pH values - 5.50 (BMC-skin) and 7.50 (BMC-cornea). Retention factors (k), obtained by use of BMC, were calculated for all newly synthesized 17ß-carboxamide steroids as well as for parent glucocorticoids (hydrocortisone, prednisolone, methylprednisolone, dexamethasone and betamethasone). Good correlation was obtained between BMC-skin retention factors and permeability coefficients calculated by use of the artificial membrane that simulates stratum corneum of the human skin. Quantitative structure-retention relationship (QSRR) study was performed in order to explain retention factors of these compounds in the tested BMC systems. ANN-QSRR(k), PLS-QSRR(k) and MLR-QSRR(k) models, created by use of BMC-skin retention data, were compared and optimal model (PLS-QSRR(k)) was selected. Molecular descriptors of the selected model indicate that lipophilicity and number of short C-C fragments of tested compounds have the strongest influence on the retention in the BMC-skin system and presumably on their in vivo permeability through human skin. The same model can be applied to the BMC-cornea system and the same conclusion can be drawn for corneal permeability. This model could be used as a predictive tool for the synthesis of novel 17ß-carboxamide steroids with desirable permeability through human skin or cornea, depending on their potential pharmacological application.
Asunto(s)
Amidas/metabolismo , Córnea/metabolismo , Piel/metabolismo , Esteroides/metabolismo , Amidas/química , Cromatografía/métodos , Humanos , Micelas , Permeabilidad , Relación Estructura-Actividad Cuantitativa , Absorción Cutánea , Esteroides/químicaRESUMEN
In this paper, twenty-two 17ß-carboxamide steroids were synthesized from five corticosteroids (hydrocortisone, prednisolone, methylprednisolone, dexamethasone and betamethasone) in two steps. The first step was periodic acid oxydation of these corticosteroids to corresponding cortienic acids and the second step was amidation of thus obtained cortienic acids with esterified l-amino acids. These compounds are potential soft corticosteroids with local anti-inflammatory activity in the skin. Parallel artificial membrane permeability assay (PAMPA) was applied in order to predict permeability and retention of these compounds in human skin. Comparison of permeability and retention parameters between 17ß-carboxamide steroids and corresponding corticosteroids was performed. Compounds with significantly higher retention were identified and the derivative that does not have significantly higher permeability was underlined. Molecular structures of all compounds were optimized by use of Gaussian semiempirical/PM3 method. Geometrical, thermodynamic, physicochemical and electronical molecular parameters of the optimized structures were calculated and quantitative structure-property relationship (QSPR) analysis was performed in order to explain permeability and retention of these compounds. ANN-, PLS- and MLR-QSPR models were created. Quality of these models was evaluated by commonly used statistical parameters and the most reliable models were selected. Analyzing descriptors in the selected models, main molecular properties that influence permeability and retention in the PAMPA artificial membrane were identified. Based on these data, further structural modifications could be applied in order to increase retention without significant increase of permeability, which can positively affect potential local anti-inflammatory activity of these compounds. Selected QSPR models could be used as in silico tool for predicting human skin permeability and retention of novel 17ß-carboxamide steroids without performing PAMPA experiments.
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Corticoesteroides/metabolismo , Amidas/metabolismo , Membranas Artificiales , Absorción Cutánea , Corticoesteroides/química , Amidas/química , Androstenodioles/química , Humanos , Permeabilidad , Relación Estructura-Actividad Cuantitativa , Piel/metabolismoRESUMEN
Series of twelve chalcone and propafenone derivatives has been synthesized and evaluated for anticancer activities against HeLa, Fem-X, PC-3, MCF-7, LS174 and K562 cell lines. The 2D-QSAR and 3D-QSAR studies were performed for all compounds with cytotoxic activities against each cancer cell line. Partial least squares (PLS) regression has been applied for selection of the most relevant molecular descriptors and QSAR models building. Predictive potentials of the created 2D-QSAR and 3D-QSAR models for each cell line were compared, by use of leave-one-out cross-validation and external validation, and optimal QSAR models for each cancer cell line were selected. The QSAR studies have selected the most significant molecular descriptors and pharmacophores of the chalcone and propafenone derivatives and proposed structures of novel chalcone and propafenone derivatives with enhanced anticancer activity on the HeLa, Fem-X, PC-3, MCF-7, LS174 and K562 cells.
Asunto(s)
Antineoplásicos/síntesis química , Propiofenonas/síntesis química , Propiofenonas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chalcona/síntesis química , Chalcona/química , Chalcona/farmacología , Células HeLa , Humanos , Concentración 50 Inhibidora , Células K562 , Células MCF-7 , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Propafenona/síntesis química , Propafenona/química , Propafenona/farmacología , Propiofenonas/química , Relación Estructura-Actividad CuantitativaRESUMEN
A novel topical corticosteroid FA-21-PhP, 2-phenoxypropionate ester of fluocinolone acetonide, has been synthesized in order to investigate the possibility of decreasing systemic side effects. In this study model system for in vitro solvolytic reaction of FA-21-PhP has been analyzed in ethanol/water (90:10, v/v) with excess of sodium hydrogen carbonate. The selected conditions have been used as in vitro model for activation of corticosteroid C-21 ester prodrug. The second-order derivative spectrophotometric method (DS) using zero-crossing technique was developed for monitoring ternary mixture of solvolysis. Fluocinolone acetonide (FA) as a solvolyte was determined in the mixture in the concentration range 0.062-0.312 mM using amplitude (2)D(274.96). Experimentally determined LOD value was 0.0295 mM. The accuracy of proposed DS method was confirmed with HPLC referent method. Peak area of parent ester FA-21-PhP was used for solvolysis monitoring to ensure the initial stage of changes. Linear relationship in HPLC assay for parent ester was obtained in the concentration range 0.054-0.54 mM, with experimentally determined LOD value of 0.0041 mM. Investigated solvolytic reaction in the presence of excess of NaHCO(3) proceeded via a pseudo-first-order kinetic with significant correlation coefficients 0.9891 and 0.9997 for DS and HPLC, respectively. The values of solvolysis rate constant calculated according to DS and HPLC methods are in good accordance 0.038 and 0.043 h(-1), respectively.