Separation and characterization of modified pregabalins in terms of cyclodextrin complexation,using capillary electrophoresis and nuclear magnetic resonance

The (S)-(+)-isomer of 3-isobutyl-GABA (pregabalin), the blockbuster drug in the treatment of neuropathic pain has been separated from its R isomer by cyclodextrin modified capillary zone electrophoresis (CZE) using uncoated fused-silica capillary. Derivatization of the single isomer and the racemate with tosyl- and dansyl-chloride was carried out to introduce strong UV chromophores of different size. CE-pH titrations were performed to determine the dissociation constants for both derivatives. 30 cyclodextrin (CD) derivatives as chiral agents were used at four different pH values to study the enantioseparation of the differently protonated guest molecules. The separation was optimized as a function of CD concentration, buffer type and concentration, pH and applied voltage. For the tosylated derivate the best resolution (R_s = 2.76) was found with 6-monodeoxy-6-mono-(3-hydroxy)-propylamino-beta-cyclodextrin hydrochloride (PA-β-CD) at pH 6.8, while with the same selector at pH 7.2 enantioseparation with an R_s value of 4.32 could be achieved for the dansylated pregabalin. At pH 2.5 for the dansylated derivative trimethylated alpha- and beta-CD systems resulted the most significant separation (R_s = 7.38 and R_s = 7.74, respectively). Experiments with dual CD systems were carried out as well. The stoichiometry of the complexes was determined using the Job plot method and resulted in a 1:1 complex in both cases. The structures of the inclusion complexes were elucidated using 2D ROESY NMR experiments.     

The aqueous stability of bupropion

In this study the aqueous stability of bupropion was determined and the pH-degradation profile was obtained. The effects of hydrogen ion, solvent and hydroxide ion concentration are discussed with particular emphasis on the kinetics of degradation of bupropion. Kinetics and degradation of bupropion were determined by HPLC-UV and LC–MS analysis both utilising high pH chromatographic methods. Degradation of bupropion in aqueous solutions follows first-order reaction kinetics. The pH-degradation profile was determined using non-linear regression analysis. The micro- and macro-reaction constants for degradation are presented. Bupropion was most stable in aqueous solutions below pH 5. Degradation was catalyzed by water but mainly by hydroxide ion on the unionised form of bupropion. The energy of activation for decomposition in aqueous solution pH 10.7 I = 0.055 was determined to be 53 kJ mol⁻¹ with a frequency factor of 6.43 × 10¹⁰ s⁻¹. The degradants of bupropion were positively identified and a mechanism of degradation is proposed. The inherent instability of bupropion above pH 5 has implications for its therapeutic use, formulation, pharmacokinetics and use during analysis and storage.     

Pharmacokinetic/pharmacodynamic modelling of the bactericidal activity of free lung concentrations of levofloxacin and gatifloxacin against Streptococcus pneumoniae

The aim of this work was to compare the pharmacological properties of levofloxacin and gatifloxacin against Streptococcus pneumoniae by pharmacokinetic/pharmacodynamic (PK/PD) modelling of the time-kill curves employing an E_max model. An in vitro infection model was used to simulate free pulmonary fluctuating concentrations expected after multiple dosing regimens of both drugs in humans or constant multiples of the minimum inhibitory concentration. PK/PD parameters and PK/PD indices of the simulated dosing regimens were compared. The levofloxacin EC₅₀ (concentration producing 50% of the maximum effect) (mean ± standard deviation 3.57 ± 2.16 mg/L) was higher than that for gatifloxacin (0.95 ± 0.56 mg/L) when simulating multiple dosing regimens as well as constant concentrations (EC_{50,levofloxacin} = 2.75 ± 0.45 mg/L; EC_{50,gatifloxacin} = 1.03 ± 0.52 mg/L). The maximum killing rate constant (k_max) was not statistically different for both drugs independent of fluctuating (k_{max,levofloxacin} = 0.40 ± 0.19 h⁻¹; k_{max,gatifloxacin} = 0.48 ± 0.15 h⁻¹) or constant concentrations (k_{max,levofloxacin} = 0.34 ± 0.06 h⁻¹; k_{max,gatifloxacin} = 0.39 ± 0.23 h⁻¹). The PK/PD model was able to describe the effect of levofloxacin and gatifloxacin against S. pneumoniae in vitro for all the simulations investigated. Gatifloxacin was more potent than levofloxacin, but both presented equivalent efficacy. The model can be used for simulating alternative regimens and optimising therapy to treat streptococcal infextions.     

Formulation of Vaccine Adjuvant Muramyldipeptides (MDP). 2. The Thermal Reactivity and pH of Maximum Stability of MDP Compounds in Aqueous Solution

The degradation of muramyldipeptides (MDPs) in aqueous solution obeys the rate law k _obs = k _H+_H+ + k _o + k _HO- a _HO- and the Arrhenius equation. For example, the rate constants for degradation of N-acetylmuramyl-L-threonyl-D-isoglutamine, 3, at 25°C are k _H+ = 2.3 × 10^–6 M ^–1 sec^–1, k _o = 8.2× 10^–10 sec^–1, and k _HO– = 0.19 M ^–l sec^–1. The degradation rates are dependent on the side-chain substituents; it is predicted that sterically hindered MDP compounds will show an extended shelf life in aqueous solution. Product studies in the weakly acid pH region (where the pH of maximum stability occurs) show that MDP compounds degrade largely by hydrolysis of the dipeptide side chain. These data show that MDP 3 exhibits a shelf life (t ₉₀) of greater than 2 years in aqueous solutions of pH 4–4.5, the pH of maximum stability.     

Separation and determination of nimesulide related substances for quality control purposes by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed and validated for the determination of nimesulide related compounds in pharmaceutical formulations. Electrophoretic separation of six European Pharmacopoeia (EP) impurities (A–F) was performed using a fused silica capillary (L_eff. = 50 cm, L_tot. = 57 cm, 50 μm i.d.) with a background electrolyte (BGE) containing 25 mM borate buffer (pH 9.5), 30 mM sodium dodecyl sulphate and φ = 3% (v/v) acetonitrile. The influence of several factors (surfactant and buffer concentration, pH, organic modifier, applied voltage, capillary temperature and injection time) was studied. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. The calibration curves obtained for the six compounds were linear over the range 5–12 μg ml⁻¹ (0.05–0.12%). The relative standard deviations (s_r) of intra- and inter-day experiments were less than 5.0%. The detection limits ranged between 0.7 and 1.6 μg ml⁻¹ depending on the impurity. The proposed method was applied successfully to the quantification of nimesulide impurities in its pharmaceutical formulation.     

In Vitro Characterization of the Erythrocyte Distribution of Methazolamide: A Model of Erythrocyte Transport and Binding Kinetics

The rate and extent of binding of methazolamide to human erythrocytes was studied in vitro. All experiments were carried out at physiological temperature (37C) and pH (7.4). Methazolamide (MTZ) buffer concentrations were analyzed by HPLC. Distributional equilibrium between buffer and washed red blood cells was achieved after 1 hr. Results of equilibrium studies were consistent with two classes of binding sites for MTZ within the erythrocyte: a low affinity, high capacity site (CA-I) and a high affinity, low capacity site (CA-II). A two-binding site model was fitted to experimental data generating estimates for binding parameters Ka₁ (0.0017 ± 0.00022 M ^–1 ) nM₁ (636 ± 5.23 M), Ka₂ (0.46 ± 0.0083 M ^–1 ), and nM₂ (80.9 ± 0.389 M). Based upon these findings, kinetic studies were performed in order to characterize the rate of drug distribution. The rate of erythrocyte uptake of MTZ was mathematically modeled using a series of differential equations describing drug diffusion across the red blood cell membrane and subsequent complexation with intracellular binding sites. The model assumed that penetration of MTZ into the red blood cells was passive but drug binding to the carbonic anhydrase isozymes was not instantaneous. Using a novel curve fitting technique, parameter estimates of RBC membrane permeability (0.0102 ± 0.000618 cm/min), and binding rate constants k_–1 (0.254 ± 0.0213 min ^–1 ), k₁ (0.0022 ± 0.00020 ml/g-min), k_–2 (1.59 ± 0.0358 min ^–1 ), and k₂ (3.1 ± 0.035 ml/g-min) were obtained. The model characterized the observed biphasic decline of MTZ buffer concentrations over time and may help explain the prolonged residence of MTZ in vivo.     

Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC

A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab^® software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C₁₈ XTerra column using a mobile phase of (water + triethylamine (2%, v/v)): acetonitrile = 90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5 ml/min and UV detection at 290 nm was employed. The column was thermostated at 45 °C. The resolution between the two least resolved impurity peaks was in average, R_s,min > 1.5. Method validation parameters indicate linear dynamic range 0.2–2.0 μg/ml with LOQ ca. 0.20 μg/ml and LOD ca. 0.05 μg/ml for all analytes.     

Electrochemical behavior of paclitaxel and its determination at glassy carbon electrode

The electrochemical behavior of paclitaxel drug was studied at a glassy carbon electrode in phosphate buffer solutions using cyclic and differential-pulse voltammetric techniques. The oxidation process was shown to be irreversible over the pH range (3.0–10.4) and was diffusion controlled. Effects of anodic peak potential (E_p), anodic peak current (I_pa), scan rate, pH, heterogeneous rate constant (k⁰), etc have been discussed. A possible electro-oxidation mechanism was proposed. An analytical method was developed for the determination of paclitaxel in phosphate buffer solution at pH = 7.0 as a supporting electrolyte. The anodic peak current varied linearly with paclitaxel concentration in the range 1.0 × 10⁻⁶ M to 1.0 × 10⁻⁵ M with a limit of detection (LOD) of 1.23 × 10⁻⁸ M and limit of quantification (LOQ) of 4.10 × 10⁻⁸ M. The proposed method was successfully applied to the determination of paclitaxel in pure and real samples.     

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-d-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC₅₀ around 1 μM at −70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca²⁺ responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells.Memantine was confirmed to be a moderate affinity (IC₅₀ at −70 mV of 0.79 ± 0.02 μM, Hill = 0.92 ± 0.02), strongly voltage-dependent (δ = 0.90 ± 0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 μM - onset τ_fast = 273 ± 25 ms (weight 69%), onset τ_slow = 2756 ± 296 ms, offset τ_fast = 415 ± 82 ms (weight 38%) offset τ_slow = 5107 ± 1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC₅₀ at −70 mV of 0.71 ± 0.03 μM, Hill = 0.84 ± 0.02) but somewhat less pronounced voltage-dependency (δ = 0.79 ± 0.04), slower, single exponential kinetics (ketamine: k_on = 0.15 ± 0.05 × 10⁶ M⁻¹ s⁻¹, k_off = 0.22 ± 0.05 s⁻¹ c.f. memantine following normalization k_on = 0.32 ± 0.11 × 10⁶  M⁻¹ s⁻¹, k_off = 0.53 ± 0.10 s⁻¹) and was fully trapped.The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.     

Chiral HPLC analysis of formoterol stereoisomers and thermodynamic study of their interconversion in aqueous pharmaceutical formulations

A chiral HPLC method was validated and successfully applied for the determination of formoterol stereoisomers and their inversion products in an aqueous matrix stored at 5–70 °C up to 3 weeks. Analysis was performed on a Chiral-AGP column (100 × 4-mm, 5-μm) using a variable mixture of mobile phase A (50-mM sodium phosphate buffer, pH 7.0) and B (10% v/v IPA) at a flow rate of 1.3 ml min⁻¹, and UV detection at 242 nm. All four formoterol stereoisomers were adequately resolved with acceptable detection and quantitation limits varying from 0.01–0.04 μg/ml and 0.04–0.1 μg/ml, respectively. The method showed acceptable accuracy (≥88%), precision (RSD ≤ 8.5%) and good linearity (r² ≥ 0.9999) over the concentration range investigated. While interconversion at 5 ± 3 °C and 25 ± 2 °C/60% RH ±5% RH was too low to be determined accurately within the study period, chiral inversion of formoterol stereoisomers measured at high temperatures followed the first order rate kinetics and occurred at a single chiral center, resulting in the reversible formation of diastereoisomers, (R,R) ↔ (S,R) and (S,S) ↔ (R,S). No enantiomerization or diastereomerization occurred. There was no significant difference in inversion of the active components in racemic (R,R/S,S)-formoterol fumarate and the single isomer (R,R)-arformoterol tartrate drug formulations, and both drugs are expected to maintain their stereochemical integrity throughout the proposed shelf-life at the recommended storage condition (5 ± 3 °C).     

Is there any association between imidapril hydrochloride stability profile under dry air conditions and cancer initiation?

Stability study for imidapril hydrochloride (IMD) was performed under stress conditions of increased temperature (T = 373 K) and decreased relative air humidity (RH = 0%) in order to obtain and identify its degradation product. The degradation sample stored for 15 days under the above environmental conditions was analyzed by LC–MS technique and it was found that the only degradation impurity formed in the course of the investigated drug degradation was IMD diketopiperazine derivative (DKP) which was produced by dehydration and intramolecular cyclization. The kinetics of its formation was analyzed by a revalidated RP-HPLC method and the kinetic model of this reaction was established. It was concluded that the DKP formation follows Prout–Tompkins kinetics with the rate constant k ± Δk = 2.034 ± 0.157 × 10⁻⁶ [s⁻¹]. The obtained degradation impurity was further assessed with respect to its mutagenic potential using commercial Ames MPF 98/100 microplate format mutagenicity assay kit equipped with Salmonella typhimurium strains TA 98 and TA 100. Both strains were exposed to six concentrations (in a range of 0.16–5.0 mg/mL) of DKP in the presence and absence of metabolic activation system. No mutagenic effect was observed confirming that the presence of DKP in IMD final dosage form has no impact on cancer initiation.     

Novel inclusion complex of ibuprofen tromethamine with cyclodextrins: Physico-chemical characterization

Guest–host interactions of ibuprofen tromethamine salt (Ibu.T) with native and modified cyclodextrins (CyDs) have been investigated using several techniques, namely phase solubility diagrams (PSDs), proton nuclear magnetic resonance (¹H NMR), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffractometry (XRPD), scanning-electron microscopy (SEM) and molecular mechanics (MM). From the analysis of PSD data (A_L-type) it is concluded that the anionic tromethamine salt of ibuprofen (pK_a = 4.55) forms 1:1 soluble complexes with all CyDs investigated in buffered water at pH 7.0, while the neutral form of Ibu forms an insoluble complex with β-CyD (B_S-type) in buffered water at pH 2.0. Ibu.T has a lower tendency to complex with β-CyD (K₁₁ = 58 M⁻¹ at pH 7.0) compared with the neutral Ibu (K₁₁ = 4200 M⁻¹) in water. Complex formation of Ibu.T with β-CyD (ΔG° = −20.4 kJ/mol) is enthalpy driven (ΔH° = −22.9 kJ/mol) and is accompanied by a small unfavorable entropy (ΔS° = −8.4 J/mol K) change. ¹H NMR studies and MM computations revealed that, on complexation, the hydrophobic central benzene ring of Ibu.T and part of the isobutyl group reside within the β-CyD cavity leaving the peripheral groups (carboxylate, tromethamine and methyl groups) located near the hydroxyl group networks at either rim of β-CyD. PSD, ¹H NMR, DSC, FT-IR, XRPD, SEM and MM studies confirmed the formation of Ibu.T/β-CyD inclusion complex in solution and the solid state.     

Optimization and validation of a high performance liquid chromatography method for rapid determination of sinafloxacin,a novel fluoroquinolone in rat plasma using a fused-core C18-silica column

A novel, simple and rapid high performance liquid chromatographic method has been developed and validated for the determination of sinafloxacin, a new fluoroquinolone, in rat plasma using 96-well protein precipitation, fused-core C₁₈-silica column (4.6 mm × 50 mm, 2.7 μm) packed with a new solid support, which is made of 2.7 μm particles that consist of a 1.7 μm solid core covered with a 0.5 μm thick shell of porous silica.The chromatographic separation was achieved with a mobile phase of 20:80 (v/v) of acetonitrile and phosphate buffer (pH = 3.0) at a flow rate of 1 ml min⁻¹. Fluorescence detection was employed with λ_ex 295 nm and λ_em 505 nm. Lomefloxacin was used as internal standard (IS). The total analysis time was as short as 3 min. The method was sensitive with a limit of detection (LOD) of 2 ng ml⁻¹, with good linearity (R² = 0.9996) over the linear range of 5–500 ng ml⁻¹. The intra-day and inter-day precision was less than 5.8% and accuracy ranged from 100.3% to 103.5% for quality control (QC) samples at three concentrations of 10, 50 and 400 ng ml⁻¹.The fused-core C₁₈-silica column method offered high sample throughput, low injection volume and low consumption of organic solvents. The method was successfully employed in the pharmacokinetic study of sinafloxacin formulation product after tail vein injection to healthy rats.     

Development and validation of a stability-indicating HPLC-UV method for the determination of alizapride and its degradation products

A stability-indicating high-performance liquid chromatography procedure has been developed for the determination of alizapride (AL) and its main degradation products alizapride carboxylic acid (AL-CA) and alizapride N-oxide (AL-NO2) in drug substance and product. The method was developed based on forced degradation data obtained by HPLC–MS analysis. Indeed AL underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation the main degradation products being AL-CA and AL-NO2 respectively. The separation and quantisation were achieved on a 150-mm reverse phase column with a hydrophilic linkage between silica particles and hydrophobic alkyl chains. The mobile phase was constituted (flow rate 1.5 mL min⁻¹) of eluant A: aqueous acetate buffer (pH 4.0; 20 mM) and eluant B: CH₃OH using a gradient elution and detection of analytes at 225 nm. The method showed good linearity for the AL, AL-CA, AL-NO2 mixture in the 25–75, 1–15 and 1–15 μg mL⁻¹ ranges respectively, being all the square of the correlation coefficients greater than 0.999. The precision, determined in terms of intra-day and inter-day precisions and expressed as RSDs were 0.8, 1.3 and 2.1% and 1.0, 1.7, 4.8% for AL, AL-CA and AL-NO2 respectively. The method demonstrated also to be accurate; indeed the average recoveries, at 100% and 0.2% of the target assay concentration, were 100.5, 98.6, and 96.8% for AL, AL-CA and AL-NO2 respectively. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.     

Development and validation of a stability-indicating LC method for determining palonosetron hydrochloride, its related compounds and degradation products using naphthalethyl stationary phase

A selective and simple reversed phase HPLC method using naphthalethyl stationary phase was developed and validated for the quantitative determination of Palonosetron hydrochloride (PALO), its related compounds and degradation products. Chromatographic separation (R_s > 2) was achieved with linear gradient mode of elution at a flow rate of 1 mL/min and with UV detection at 210 nm. The intra and inter-day coefficients of variation were less than 1.0% (RSD). Consistent recoveries were obtained for PALO (99.2-100.5%) and its impurities (90.0-104.8%). All the analytes exhibited excellent linearity with R² value greater than 0.998. Limit of detection (LOD) and limit of quantification (LOQ) were determined to be in the range 0.011-0.013 μg/mL and 0.035-0.046 μg/mL respectively. The test solution was found to be stable up to 5 days. Induced degradation methods were applied to study the degradation behavior of the drug. LC-MS was used to analyze the degraded samples and possible structural identifications were assigned based upon known reactivity of the drug and molecular weights. The m/z values matched with the hydroxylated, keto and N-oxide metabolites of PALO. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.9%.     

Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(−) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (k_cat/K_M = 5 × 10⁶ M⁻¹ min⁻¹). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5 mg kg⁻¹ i.v.) 5 min after s.c. challenge with VX (∼2LD₅₀) prevented systemic toxicity. A lower C23 dose (2 mg kg⁻¹) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15 min post VX) with 5 mg kg⁻¹ C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(−)-VX enantiomer that correlates with the C23 blood levels and its k_cat/K_M value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.     

Therapeutic efficacies of Coriandrum sativum aqueous extract against metronidazole-induced genotoxicity in Channa punctatus peripheral erythrocytes

Metronidazole (MTZ), a nitroimidazole drug, is primarily used as an anti-protozoan or an anti-bacterial agent in humans, although its genotoxic and carcinogenic effects have been widely reported, particularly in aquatic organisms. MTZ may induce DNA damages through single-strand breaks, modification of bases, DNA–DNA and DNA–protein cross-links, ultimately leading to apoptosis or necrosis. Here, we have assessed the genotoxicity of MTZ in the peripheral erythrocytes of Channa punctatus, using micronucleation (MN) and binucleation (BN) as genotoxicity markers. The therapeutic potential of aqueous extract of Coriandrum sativum against MTZ-induced genotoxicity has also been examined. The results show significant (P < 0.05) increase in both MN and BN formation due to MTZ treatment. Such aberrations were higher in smaller fish samples for a particular dosage of MTZ, as established by correlation analysis between fish body weight and MN/BN count at P < 0.05. However, such degenerative damages were found to be alleviated by a great extent due to treatment with C. sativum leaf extract. Hence, we establish that MTZ can produce considerable degrees of micronucleus and binucleus formation in peripheral erythrocytes of C. punctatus, and such deleterious effect of MTZ treatment can be mitigated by aqueous extract of C. sativum leaves.     

Antioxidant activity of propolis extracts from Serbia: A polarographic approach

Antioxidant activity (AO) of commercial propolis extracts (PEs), available on Serbian market, was determined by direct current (DC) polarography. Polarographic anodic current of 5.0 mmol L⁻¹ alkaline solution of H₂O₂ was recorded at potentials of mercury dissolution. Decrease of the current was plotted against the volume of gradually added PEs. The volume of PE causing 20% current decrease was determined from the linear part of the plot. Antioxidant activity was expressed in H₂O₂ equivalent (HPEq), representing the volume of PE that corresponds to 1.0 mmol L⁻¹ H₂O₂ decrease. Resulting HPEq ranged between 1.71 ± 0.11 and 8.00 ± 0.18 μL. Range of 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity was from 0.093 ± 0.004% to 0.346 ± 0.006%. Total phenolic content (TCP) of PE with superior AO activity was 5.31 ± 0.05%g GAE, while the extract with the lowest activity contained 1.45 ± 0.02%g GAE. Antioxidant activity, determined by polarographic method, was correlated with DPPH scavenging activity (R² = 0.991) and TCP (R² = 0.985). Validity of obtained results was further confirmed using ANOVA and post hoc Tukey HSD test.     

Improved photostability and reduced skin permeation of tretinoin: Development of a semisolid nanomedicine

The aims of this work were to increase the photostability and to reduce the skin permeation of tretinoin through nanoencapsulation. Tretinoin is widely used in the topical treatment of various dermatological diseases such as acne, psoriasis, skin cancer, and photoaging. Tretinoin-loaded lipid-core polymeric nanocapsules were prepared by interfacial deposition of a preformed polymer. Carbopol hydrogels containing nanoencapsulated tretinoin presented a pH value of 6.08 ± 0.14, a drug content of 0.52 ± 0.01 mg g⁻¹, pseudoplastic rheological behavior, and higher spreadability than a marketed formulation. Hydrogels containing nanoencapsulated tretinoin demonstrated a lower photodegradation (24.17 ± 3.49%) than the formulation containing the non-encapsulated drug (68.64 ± 2.92%) after 8 h of ultraviolet A irradiation. The half-life of the former was seven times higher than the latter. There was a decrease in the skin permeability coefficient of the drug by nanoencapsulation, independently of the dosage form. The liquid suspension and the semisolid form provided K_p = 0.31 ± 0.15 and K_p = 0.33 ± 0.01 cm s⁻¹, respectively (p ? 0.05), while the samples containing non-encapsulated tretinoin showed K_p = 1.80 ± 0.27 and K_p = 0.73 ± 0.12 cm s⁻¹ for tretinoin solution and hydrogel, respectively. Lag time was increased two times by nanoencapsulation, meaning that the drug is retained for a longer time on the skin surface.