Piracetam-induced neuroprotection in lipopolysaccharides-challenged EOC-20 cells and mouse brain via attenuating oxidative stress

Abstract Therapeutically, piracetam has been used for decades as a cognitive enhancer for memory- related neuronal disorders. The present study aimed to investigate the neuroprotective potential of piracetam on lipopolysaccharides (LPS)-induced neuronal deficit using both in-vitro and in-vivo experimental models. For the in-vitro analysis, EOC-20 murine microglial cells were induced with a neuronal toxicity of 100 µg/ml of LPS, and the formation of intracellular reactive oxygen species (ROS) and nitric oxide (NO) productions were determined. For in-vivo neuroprotective analysis, groups of mice were treated orally with two doses of piracetam (200 and 400 mg/kg) for 30 days. Neuronal toxicity was induced by four intraperitoneal injections of LPS (250 µg/kg/day). The malondialdehyde (MDA) level was measured for oxidative stress, and catalase reduced glutathione (GSH), glutathione reductase (GRD), and superoxide dismutase (SOD) levels were determined as the antioxidant parameters. The result of the cell viability study was that pre-treatment with piracetam significantly protected the LPS-induced cell loss, and attenuated the ROS generation and NO production in LPS-induced EOC-20 cells. Moreover, the treatment of piracetam significantly reduced the MDA levels and improved catalase, GSH, GRD, and SOD activities in LPS-induced mice brains. The overall results from this study supported the neuroprotective effects of piracetam against LPS-induced neuronal toxicity.

Development and validation of stability indicating chromatographic methods for simultaneous determination of citicoline and piracetam.

Citicoline and piracetam were subjected separately to different stress conditions as recommended by the international conference on harmonization. In addition, new stability indicating thin layer chromatographic and ultra high performance liquid chromatographic methods have been developed and validated for simultaneous determination of citicoline and piracetam in presence of their degradation products. Separation on the proposed thin layer chromatographic method was carried out using a developing system containing methanol:chloroform:ammonium chloride buffer (9:1:2, v/v/v) on silica gel plates at 230 nm. On the other hand, the mobile phase in the ultra high performance liquid chromatographic method was composed of water (containing 0.1% triethylamine):ethanol (92:8, v/v). The flow rate was 1 mL/min and ultraviolet detection was at 230 nm. Moreover, results of the developed methods were statistically compared to those obtained by the reported high-performance liquid chromatography method and no significant difference between them was found. The greenness profile of ultra high performance liquid chromatographic method was assessed and compared with those of the previously published high-performance liquid chromatography methods, it was noticed that the proposed ultra high performance liquid chromatographic method more environmentally friendly and greener than other methods.

Spectrofluorimetric approach for determination of citicoline in the presence of co-formulated piracetam through fluorescence quenching of eosin Y.

A simple, sensitive, and precise spectrofluorimetric method has been developed and validated for quantitation of citicoline in its pharmaceutical formulations. The proposed method based on quantitative quenching effect of citicoline on the native fluorescence of Eosin Y via developing of a binary complex reaction between the cited drug and Eosin Y in acidic medium using acetate buffer pH = 3.6. The quenching of the fluorescence of eosin was measured at 540 nm after excitation at 518 nm. Calibration graph was achieved in the range of 300-3000 ng/mL with 0.9996 as correlation coefficient and 291.0 and 93.86 ng/mL as quantitation and detection limits, respectively. The developed method considered as the first developed spectrofluorimetric one for quantitation of citicoline with high sensitivity and validated according to ICH guidelines. The selectivity of the proposed method was investigated by studying the interference of piracetam as co-formulated drug with CIT in pharmaceutical formulation, therefore the developed method could be used for routine quality control of citicoline in its pharmaceutical formulations either alone or in combination with piracetam.

Nootropic drugs: Methylphenidate, modafinil and piracetam - Population use trends, occurrence in the environment, ecotoxicity and removal methods - A review.

Pharmaceuticals which originally were designed to treat people with neurological and psychiatric conditions, e.g. Alzheimer's disease or attention deficit hyperactivity disorder (ADHD), are nowadays often misused by students as a 'brain doping' substances. These substances are known as nootropic drugs, smart drugs or cognitive enhancers, as they increase memory, attention and concentration of healthy individuals. Since they are easily available illicitly, their consumption is observed to be growing. Currently, these pharmaceuticals started gaining researchers' attention, especially since they have been recently detected in wastewater, surface water and even drinking water. This review summarises the current state of knowledge on nootropic drugs in terms of their population use trends and ethics, occurrence in the environment and detection techniques, toxicity and removal methods, in example of methylphenidate, modafinil and piracetam - three most popular nootropics. It points out that the main sources of knowledge on cognitive enhancers illicit use are often inconsistent questionnaires, which are not supported by wastewater analysis to become more veracious. Simultaneously, the studies concerning toxicity and removal methods of nootropic drugs are still limited and in many cases environmentally irrelevant. Although the prescription rules has been subjected to more strict control in developed countries, regulatory frameworks with regard to their ecosystem occurrence are still lacking and should be introduced. Moreover, the use of environmentally relevant concentrations in toxicity studies should be a standard, leading to proper ecotoxicity risk assessment. Based on this review, it is recommended to routinely monitor nootropics and their metabolites in waste- and surface waters.

Quantifying API polymorphs in formulations using X-ray powder diffraction and multivariate standard addition method combined with net analyte signal analysis.

The direct quantification of Active Pharmaceutical Ingredients in solid formulations is a challenging open issue. A consolidated analytical technique based on X-ray Powder Diffraction is available, being the definitive test for the identification of polymorphs and crystal phases. However, its application for quantitative analysis is hindered by matrix effects: refinement methods (e.g. Rietveld method) require a complete knowledge of samples' composition, while univariate calibration methods require the matrix effect to be studied and severely suffer from the co-presence of crystalline and amorphous phases in the sample. Multivariate analysis is the only way to bypass problems affecting refinements procedures and univariate calibration. In particular, the multivariate standard addition method (SAM) is promising; however, it is straightforward only when the analytical blank (matrix devoid of analyte) is available: in that case SAM is applied by simply extrapolating the SAM model to the matrix experimental signal. In this work, the quantitative analysis of polymorphic forms of Active Pharmaceutical Ingredients based on X-ray Powder Diffraction is performed for the first time by a method based on multivariate standard addition method combined with net analyte signal procedure; it allows for reliable quantification of polymorphs of active principles in solid formulations, which are rapidly analyzed without any sample pre-treatment. Two test cases are presented: quantification of two polymorphs of piracetam in binary mixtures (forms II and III), and quantification of paracetamol (form I) in Tachifludec®.

Isolation of two Ochrobactrum sp. strains capable of degrading the nootropic drug-Piracetam.

Piracetam (2-oxo-1-pyrrolidine acetamide) is a popular cognitive enhancer, which has recently been detected in waste and drinking water. Nootropic drugs are designed to affect human metabolism and act on the nervous system, but their environmental effects have yet to be the subject of detailed studies. In this report, we present the efficient biodegradation of the cognitive enhancer, piracetam. Two bacterial strains capable of using this compound as the sole carbon source were isolated and later identified as Ochrobactrum anthropi strain MW6 and Ochrobactrum intermedium strain MW7. The compound's mineralization and the cleavage of the heterocyclic ring were shown in the experiments with 14C-labeled piracetam. This is also the first report of a pharmaceutical's degradation by the Ochrobactrum genus. This study presents model microorganisms that can be used in further investigation of piracetam's degradation pathways as well as enzymes and genes involved in the process.

Investigation of polymorphic transitions of piracetam induced during wet granulation.

Piracetam was investigated as a model API which is known to exhibit a number of different polymorphic forms. It is freely soluble in water so the possibility exists for polymorphic transformations to occur during wet granulation. Analysis of the polymorphic form present during lab-scale wet granulation, using water as a granulation liquid, was studied with powder X-ray diffraction and Raman spectroscopy as off-line and inline analysis tools respectively. Different excipients with a range of hydrophilicities, aqueous solubilities and molecular weights were investigated to examine their influence on these solution-mediated polymorphic transitions and experimental results were rationalised using molecular modelling. Our results indicated that as an increasing amount of water was added to the as-received piracetam FIII, a greater amount of the API dissolved which recrystallised upon drying to the metastable FII(6.403) via a monohydrate intermediary. Molecular level analysis revealed that the observed preferential transformation of monohydrate to FII is linked with a greater structural similarity between the monohydrate and FII polymorph in comparison to FIII. The application of Raman spectroscopy as a process analytical technology (PAT) tool to monitor the granulation process for the production of the monohydrate intermediate as a precursor to the undesirable metastable form was demonstrated.

Sensitive inexpensive spectrophotometric and spectrofluorimetric analysis of ezogabine, levetiracetam and topiramate in tablet formulations using Hantzsch condensation reaction.

Two highly sensitive, simple and selective spectrophotometric and spectrofluorimetric assays have been investigated for the analysis of ezogabine, levetiracetam and topiramate in their pure and in pharmaceutical dosage forms. The suggested methods depend on the condensation of the primary amino-groups in the three drugs with acetylacetone and formaldehyde according to Hantzsch reaction yielding highly fluorescent yellow colored dihydropyridine derivatives. The reaction products of ezogabine, levetiracetam and topiramate were measured spectrophotometrically at 418, 390 and 380nm or spectrofluorimetrically at λem/ex of 495/425nm, 490/415nm and 488/410nm, respectively. Various experimental conditions have been carefully studied to maximize the reaction yield. At the optimum reaction conditions, the calibration curves were rectilinear over the concentration ranges of 8-25, 60-180 and 80-200µg/mL spectrophotometrically and 0.02-0.2, 0.2-1.2 and 0.2-1.5µg/mL spectrofluorimetrically for ezogabine, levetiracetam and topiramate, respectively with good correlation coefficients. The suggested methods were applied successfully for the analysis of ezogabine, levetiracetam and topiramate in their commercial tablets with high percentage recoveries and negligible interference from various excipients in pharmaceutical dosage forms. The results were statistically analyzed and showed the absence of any significant difference between both developed and published methods. The procedures were validated and evaluated by the ICH guidelines revealing good reproducibility and accuracy. Therefore, the two proposed methods may be considered of high interest for practical and reliable analysis of ezogabine, levetiracetam and topiramate in pharmaceutical dosage forms.

Physicochemical stability and compatibility testing of levetiracetam in all-in-one parenteral nutrition admixtures in daily practice.

BACKGROUND: Parenteral antiepileptic drugs are frequently used in critically ill patients for seizure control therapy or prevention. Many of these patients require additional parenteral nutrition (PN). Therefore, a parallel infusion of the frequently used antiepileptic drug levetiracetam (LEV) is interesting in terms of the restricted i.v. lines (e.g., neonates). The potential interactions of the complex PN admixture with the drug product and the appropriate admixing of a drug at effective dosages require physicochemical lab assessments to obtain specific and reliable pharmaceutical documentation for the intended admixing. AIM: To assess the of compatibility and stability of LEV, a neutral and hydrophilic drug, in commercial all-in-one (AiO) PN admixtures using simple validated tests to provide necessary data in a timely manner and to allow convenient, documented and safe treatment with PN as the drug vehicle. METHODS: Different concentrations of LEV were injected into two different AiO PN admixtures with no further additives. Stability and compatibility tests for the drug and the PN admixtures were performed over seven days at +4°C, +23±1°C and +37°C without light protection. Stability and sample characteristics were observed by visual inspection and the validated light microscope method. Moreover, the pH level of the admixture was checked, as were the concentrations of LEV over time in the PN admixtures, using an established LC-MS/MS method. RESULTS: The stability controls of LEV at different temperatures were within absolute ±20% of the theoretical value in a concentration range of 98.91-117.84% of the initial value. No changes in pH occurred (5.55±0.04) and no microscopic out of specification data or visual changes were observed. The mean value of the largest lipid droplet in each visual field over seven days was 2.4±0.08µm, comparable to that of the drug-free AiO admixture. Samples stored at +37°C showed yellowish discolorations after 96h of storage. CONCLUSION: LEV showed compatibility and stability over seven days in the selected PN admixtures, and the described methods represented a valuable and timely approach to determine the stability and compatibility of the highly hydrophilic, not dissociated LEV in AiO admixtures under conditions of use. Further studies with clinically relevant and representative examples of physicochemically different drug classes are needed.

Stability-Indicating TLC-Densitometric and HPLC Methods for the Simultaneous Determination of Piracetam and Vincamine in the Presence of Their Degradation Products.

Newly established TLC-densitometric and RP-HPLC methods were developed and validated for the simultaneous determination of Piracetam (PIR) and Vincamine (VINC) in their pharmaceutical formulation and in the presence of PIR and VINC degradation products, PD and VD, respectively. The proposed TLC-densitometric method is based on the separation and quantitation of the studied components using a developing system that consists of chloroform-methanol-glacial acetic acid-triethylamine (8 + 2 + 0.1 + 0.1, v/v/v/v) on TLC silica gel 60 F254 plates, followed by densitometric scanning at 230 nm. On the other hand, the developed RP-HPLC method is based on the separation of the studied components using an isocratic elution of 0.05 M KH2PO4 (containing 0.1% triethylamine adjusted to pH 3 with orthophosphoric acid)-methanol (95 + 5, v/v) on a C8 column at a flow rate of 1 mL/min with diode-array detection at 230 nm. The developed methods were validated according to International Conference on Harmonization guidelines and demonstrated good accuracy and precision. Moreover, the developed TLC-densitometric and RP-HPLC methods are suitable as stability-indicating assay methods for the simultaneous determination of PD and VD either in bulk powder or pharmaceutical formulation. The results were statistically compared with those obtained by the reported RP-HPLC method using t- and F-tests.

Comparison of piracetam measured with HPLC-DAD, HPLC-ESI-MS, DIP-APCI-MS, and a newly developed and optimized DIP-ESI-MS.

The direct inlet probe-electrospray ionization (DIP-ESI) presented here was based on the direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) developed by our group. It was coupled to an ion trap mass spectrometer (MS) for the detection of more polar compounds such as degradation products from pharmaceuticals. First, the position of the ESI tip, the gas and solvent flow rates, as well as the gas temperature were optimized with the help of the statistic program Minitab® 17 and a caffeine standard. The ability to perform quantitative analyses was also tested by using different concentrations of caffeine and camphor. Calibration curves with a quadratic calibration regression of R (2) = 0.9997 and 0.9998 for caffeine and camphor, respectively, were obtained. The limit of detection of 2.5 and 1.7 ng per injection for caffeine and camphor were determined, respectively. Furthermore, a solution of piracetam was used to compare established analytical methods for this drug and its impurities such as HPLC-diode array detector (DAD) and HPLC-ESI-MS with the DIP-APCI and the developed DIP-ESI. With HPLC-DAD and 10 µg piracetam on column, no impurity could be detected. With HPLC-ESI-MS, two impurities (A and B) were identified with only 4.6 µg piracetam on column, while with DIP-ESI, an amount of 1.6 µg piracetam was sufficient. In the case of the DIP-ESI measurements, all detected impurities could be identified by MS/MS studies. Graphical Abstract Scheme of the DIP-ESI principle.

Randomized Comparative Bioavailability of a Novel Three-Dimensional Printed Fast-Melt Formulation of Levetiracetam Following the Administration of a Single 1000-mg Dose to Healthy Human Volunteers Under Fasting and Fed Conditions.

BACKGROUND: Rapidly disintegrating or 'fast-melt' oral formulations have been developed recently to facilitate drug intake among patients. Even though these formulations have helped to improve therapy adherence, some of their limitations include: the dissolution time, their facility to be swallowed, and the dosage strengths that may be accommodated. To overcome these limitations, a novel, porous, quickly disintegrating, and easier-to-swallow fast-melt formulation based on powder-liquid, three-dimensional printing (3DP) technology has been developed. OBJECTIVE: To determine and compare the relative bioavailability of a novel 3DP fast melt containing levetiracetam in healthy male and female subjects. METHODS: This study included 33 subjects in a three-way crossover design. The 3DP fast-melt formulation was compared against the conventional immediate-release tablet of levetiracetam (Keppra(®)) after a single 1000-mg dose administration under fasting conditions following the bioequivalence criteria used by the US Food and Drug Administration. This study also evaluated the food effect on the bioavailability of the levetiracetam 3DP fast melt. A small sip of liquid was used to administer the fast-melt formulation. RESULTS: The novel 3DP fast melt showed rapid oral disintegration (mean duration of 11 s from a sip of water to completion of swallowing) following its administration, and did not affect the pharmacokinetic profile of levetiracetam. A lower absorption peak was observed after administration of the 3DP fast melt under fed conditions, as expected. In addition, time of maximum measured plasma concentration was delayed by approximately 3.5 h under fed conditions. These effects are unlikely to be of clinical significance with long-term administration, and may help reduce the adverse events and facilitate compliance. Finally, no change in the oral mucosa was observed with the 3DP fast melt while being as safe and well tolerated as the standard levetiracetam tablet. CONCLUSION: This study quantified the rapid disintegration of the 3DP levetiracetam fast melt and confirmed its equivalent rate and extent of absorption to the conventional immediate-release tablet in the fasted state, using standard bioequivalence criteria.

Rapid Enantiomeric Separation and Quantitation of Levetiracetam on α-Acid Glycoprotein (AGP) Chiral Stationary Phase by High-Performance Liquid Chromatography.

A new, simple, and rapid chiral HPLC method was developed for enantioselective analysis of levetiracetam and its enantiomer [(R)-α-ethyl-2- oxo-pyrrolidine acetamide] in a pharmaceutical formulation and bulk material. Enantiomeric separation was achieved on a chiral-α1-acid glycoprotein (AGP) column (150×4.0 mm, 5 µm) using an isocratic mobile phase of phosphate buffer (pH=7) at a flow rate of 0.7 mL/min. The UV detector was set at 210 nm. Calibration curves were linear in the range of 1-100 µg/mL and 0.4-20 µg/mL for levetiracetam and the (R)-enantiomer, respectively. LOD and LOQ for the (R)-enantiomer were 0.1 and 0.4 µg/mL, respectively. The run time of analysis was less than 5.0 min.

Low-content quantification in powders using Raman spectroscopy: a facile chemometric approach to sub 0.1% limits of detection.

A robust and accurate analytical methodology for low-content (<0.1%) quantification in the solid-state using Raman spectroscopy, subsampling, and chemometrics was demonstrated using a piracetam-proline model. The method involved a 5-step process: collection of a relatively large number of spectra (8410) from each sample by Raman mapping, meticulous data pretreatment to remove spectral artifacts, use of a 0-100% concentration range partial least-squares (PLS) regression model to estimate concentration at each pixel, use of a more accurate, reduced concentration range PLS model to calculate analyte concentration at each pixel, and finally statistical analysis of all 8000+ concentration predictions to produce an accurate overall sample concentration. The relative prediction accuracy was ∼2.4% for a 0.05-1.0% concentration range, and the limit of detection was comparable to high performance liquid chromatography (0.03% versus 0.041%). For data pretreatment, we developed a unique cosmic ray removal method and used an automated baseline correction method, neither of which required subjective user intervention and thus were fully automatable. The method is applicable to systems which cannot be easily analyzed chromatographically, such as hydrate, polymorph, or solvate contamination.

Nanocrystals of zeolite act as enhanced sensing interface for biosensing of leviteracetum.

In present work, nanocrystals of zeolites were employed as sensing interface for augmented electrochemical sensing of antiepileptic drug, that is, leviteracetum (LEV). Electrochemical sensing method was developed by depositing nanocrystals of zeolites and horseradish peroxidase (nanocrys zeolites-HRP) onto indium tin oxide (ITO)-coated glass surface for LEV detection. Various stages of biosensor fabrication were characterized by Transmission electron microscopy, X-ray diffraction, scanning electron microscopy, and electrochemical impedance spectroscopy. The fabricated nanocrys zeolites-HRP-modified sensor exhibited wide linear range (10-500 µM) and a low detection limit of 0.01 µM. The biosensor also showed a short response time (within 2 s). In addition, the biosensor exhibited high reproducibility, good storage stability, and anti-interference ability. The applicability of the nanocrys zeolites-HRP-modified sensor is to determine LEV level in spiked serum samples.

Development of new method for simultaneous analysis of piracetam and levetiracetam in pharmaceuticals and biological fluids: application in stability studies.

RP-HPLC ultraviolet detection simultaneous quantification of piracetam and levetiracetam has been developed and validated. The chromatography was obtained on a Nucleosil C18 column of 25 cm×0.46 cm, 10 µm, dimension. The mobile phase was a (70:30 v/v) mixture of 0.1 g/L of triethylamine and acetonitrile. Smooth flow of mobile phase at 1 mL/min was set and 205 nm wavelength was selected. Results were evaluated through statistical parameters which qualify the method reproducibility and selectivity for the quantification of piracetam, levetiracetam, and their impurities hence proving stability-indicating properties. The proposed method is significantly important, permitting the separation of the main constituent piracetam from levetiracetam. Linear behavior was observed between 20 ng/mL and 10,000 ng/mL for both drugs. The proposed method was checked in bulk drugs, dosage formulations, physiological condition, and clinical investigations and excellent outcome was witnessed.

Separation of piracetam derivatives on polysaccharide-based chiral stationary phases.

High-performance liquid chromatography was used for the enantiomeric separation of two chiral piracetam derivatives. The suitability of six commercially available polysaccharide-based chiral stationary phases (CSPs) under normal phase mode for direct enantioseparation has been investigated. The influence of the CSPs as well the nature and content of an alcoholic modifier in the mobile phase on separation and elution order was studied. It was established that CSP Lux Amylose-2 shows high chiral recognition ability towards 4-phenylsubstituted piracetam derivatives.

Optimized and validated flow-injection spectrophotometric analysis of topiramate, piracetam and levetiracetam in pharmaceutical formulations.

Application of a sensitive and rapid flow injection analysis (FIA) method for determination of topiramate, piracetam, and levetiracetam in pharmaceutical formulations has been investigated. The method is based on the reaction with ortho-phtalaldehyde and 2-mercaptoethanol in a basic buffer and measurement of absorbance at 295 nm under flow conditions. Variables affecting the determination such as sample injection volume, pH, ionic strength, reagent concentrations, flow rate of reagent and other FIA parameters were optimized to produce the most sensitive and reproducible results using a quarter-fraction factorial design, for five factors at two levels. Also, the method has been optimized and fully validated in terms of linearity and range, limit of detection and quantitation, precision, selectivity and accuracy. The method was successfully applied to the analysis of pharmaceutical preparations.