Methods for Developing a Process Design Space Using Retrospective Data.

Prospectively planned designs of experiments (DoEs) offer a valuable approach to preventing collinearity issues that can result in statistical confusion, leading to misinterpretation and reducing the predictability of statistical models. However, it is also possible to develop models using historical data, provided that certain guidelines are followed to enhance and ensure proper statistical modeling. This article presents a methodology for constructing a design space using process data, while avoiding the common pitfalls associated with retrospective data analysis. For this study, data from a real wet granulation process were collected to pragmatically illustrate all the concepts and methods developed in this article.

Development of a Carvedilol Oral Liquid Formulation for Paediatric Use.

Carvedilol (CARV) is an 'off-label' ß-blocker drug to treat cardiovascular diseases in children. Since CARV is nearly insoluble in water, only CARV solid forms are commercialized. Usually, CARV tablets are manipulated to prepare an extemporaneous liquid formulation for children in hospitals. We studied CARV to improve its aqueous solubility and develop an oral solution. In this study, we assessed the solubility and preliminary stability of CARV in different pH media. Using malic acid as a solubility enhancer had satisfactory results. We studied the chemical, physical, and microbiological stability of 1 mg/mL CARV-malic acid solution. A design of experiment (DoE) was used to optimize the CARV solution's preparation parameters. A 1 mg/mL CARV solution containing malic acid was stable for up to 12 months at 25 °C and 30 °C and 6 months at 40 °C. An equation associating malic acid with CARV concentrations was obtained using DoE. Microbiological data showed that the use of methylparaben was not necessary for this period of time. We successfully developed an aqueous CARV solution suitable for paediatrics and proven to be stable over a 12-month period.

SeDeM as a Tool to Validate Drug Substance Manufacturing Processes and Assess Scalability and Suitability for Direct Compression: Supplier Screening.

During the development of an oral solid form of a drug substance, a thorough understanding of the critical material attributes is necessary, as the physical properties of the active pharmaceutical ingredient (API) can profoundly influence the drug product's manufacturability, critical quality attributes, and bioavailability. The objective of this study was to validate the manufacturing process of the drug Linezolid from three different sources at both the pilot and industrial scale and to identify differences in critical material attributes between the API manufacturers. Furthermore, the scalability factor between the pilot and industrial scale and the suitability of a process for direct compression were also evaluated. In the present study, the different sources of API were characterized by SeDeM methodology, particle size distribution, and scanning electron microscopy determinations. The statistical analysis revealed that no statistically significant differences were found for any of the parameters under study for the same API source analyzed on both scales. On the other hand, for most of the parameters evaluated, statistical differences were observed between the different sources. It was concluded that SeDeM was able to successfully validate the API manufacturing process, assess scalability, and distinguish between sources. Therefore, it could be highly valuable in the formulation phase to select the best API source.

Palatability and Stability Studies to Optimize a Carvedilol Oral Liquid Formulation for Pediatric Use.

Carvedilol (CARV) is a blocker of α- and ß- adrenergic receptors, used as an "off-label" treatment for cardiovascular diseases in pediatrics. Currently, there is no marketed pediatric-appropriate CARV liquid formulation, so its development is necessary. Palatability (appreciation of smell, taste, and aftertaste) is a key aspect to be considered during the development of pediatric formulations since only formulations with good palatability also have adequate acceptability in this population. Consequently, the aim of this research was to assess the palatability and acceptability of different CARV formulations using an in vivo taste assessment (ID Number PR103/22) in order to select the highest palatability-rated CARV formulation. The preparation of CARV formulations was based on a reference 1 mg/mL CARV solution, which contains malic acid as a solubilizing agent. Subsequently, sucralose and flavoring agents were added and mixed until complete dissolution to the corresponding formulations. Adult volunteers participated in this study and evaluated the taste and odor of various CARV formulations through a questionnaire and a sensory test. The mean palatability score, measured on a 10-point scale, increased from 1.60 for the unflavored control to 7.65 for the highest-rated flavored formulation. Moreover, the bitterness of the optimized CARV formulation was reduced from 66.67% to 17.86%, and the taste pleasantness was increased from 25/100 to 73/100. This optimized CARV formulation contains a sweetening agent, sucralose, in addition to two flavoring agents at appropriate concentrations for pediatrics. Furthermore, the physicochemical and microbiological stability of the optimized CARV formulation were evaluated for 6 months at 25, 30, and 40 °C, in addition to in-use stability for 15 days at 25 °C, whose results were confirmed. Thus, we successfully developed a palatable CARV liquid solution that contains excipients appropriate for pediatrics and is stable under the studied conditions.

ncRNAs in Therapeutics: Challenges and Limitations in Nucleic Acid-Based Drug Delivery.

Non-coding RNAs (ncRNAs) are emerging therapeutic tools but there are barriers to their translation to clinical practice. Key issues concern the specificity of the targets, the delivery of the molecules, and their stability, while avoiding "on-target" and "off-target" side effects. In this "ncRNA in therapeutics" issue, we collect several studies of the differential expression of ncRNAs in cardiovascular diseases, bone metabolism-related disorders, neurology, and oncology, and their potential to be used as biomarkers or therapeutic targets. Moreover, we review recent advances in the use of antisense ncRNAs in targeted therapies with a particular emphasis on their basic biological mechanisms, their translational potential, and future trends.

Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease.

Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics.

Robustness Optimization of an Existing Tablet Coating Process Applying Retrospective Knowledge (rQbD) and Validation.

The objective of these studies is to verify and validate the improvement in the inter-tablet coating uniformity for an industrially commercialized coated tablet, without involving changes in the approved registration dossier. Using the CPP (critical process parameters) determined from previous retrospective statistical analysis, the recommended working ranges are identified. Retrospective analysis showed that the design of experiments (DoE) provided an improved process variable configuration. Therefore, it is decided to study two critical parameters: Product temperature and drum speed, with an additional 22 experimental design. The quality results of the samples analyzed show that the aesthetic defects of the batches made with the new working ranges have been reduced. These results have also been corroborated with the 42 industrial batches manufactured with the new ranges. With the optimized parameters, tablets have been coated and the suitability of the model determined. The results demonstrated the overall reliability and effectiveness of the proposed Quality by Design approach and provides a useful tool to help optimize the industrial coating process. This study confirms that it is possible to optimize and validate the manufacturing process of an existing commercial product by means of a DoE with retrospective data. Therefore, no variation in the dossier is required.

Formulation of Sustained Release Hydrophilic Matrix Tablets of Tolcapone with the Application of Sedem Diagram: Influence of Tolcapone's Particle Size on Sustained Release.

Hydrophilic matrix tablets are a type of sustained release dosage form characterized by distributing a drug in a matrix that is usually polymeric. Tolcapone is a drug that inhibits the enzyme catechol-O-methyl transferase. In recent years, it has been shown that tolcapone is a potent inhibitor of the amyloid aggregation process of the transthyretin protein, and acts by stabilizing the structure of the protein, reducing the progression of familial amyloid polyneuropathy. The main objective of this study was to obtain a sustained release tablet of tolcapone for oral administration with a preferred dosage regimen of 1 administration every 12 or 24 h and manufactured, preferably, by direct compression. The SeDeM Diagram method has been used for the formulation development of hydrophilic matrix tablets. Given the characteristics of tolcapone, the excipient selected for the formation of the polymeric matrix was a high viscosity hydroxypropylmethylcellulose (Methocel® K100M CR). A decrease in the particle size of tolcapone resulted in a slower dissolution release of the formulation when the concentration of the polymer Methocel® K100M CR was below 29%. These surprising and novel results have given rise to patent number WO/2018/019997.

Phytosterolaemia associated with parenteral nutrition administration in adult patients.

Vegetable lipid emulsions (LE) contain non-declared phytosterols (PS). We aimed to determine PS content depending on the brand and LE batch, and in adult hospitalised patients treated with parenteral nutrition (PN), to establish the association between plasma and administered PS. Part I was the LE study: totals and fractions of PS in three to four non-consecutive batches from six LE were analysed. Part II was the patient study: patients with at least 7 previous days of PN with 0·8 g/kg per d of an olive/soyabean (O/S) LE were randomised (day 0) 1:1 to O/S or 100 % fish oil (FO) at a dose of 0·4 g/kg per d for 7 d (day 7). Plasma PS, its fractions, total cholesterol on days 0 and 7, their clearance and their association with PS administered by LE were studied. In part I, LE study: differences were found in the total PS, their fractions and cholesterol among different LE brands and batches. Exclusive soyabean LE had the highest content of PS (422·36 (sd 130·46) µg/ml). In part II, patient study: nineteen patients were included. In the O/S group, PS levels were maintained (1·11 (sd 6·98) µg/ml) from day 0 to 7, while in the FO group, significant decreases were seen in total PS (-6·21 (sd 4·73) µg/ml) and their fractions, except for campesterol and stigmasterol. Plasma PS on day 7 were significantly associated with PS administered (R2 0·443). PS content in different LE brands had great variability. PS administered during PN resulted in accumulation and could be prevented with the exclusive administration of FO LE.

Development and Validation of a New High-Performance Liquid Chromatography Method for the Simultaneous Quantification of Coenzyme Q10, Phosphatidylserine, and Vitamin C from a Cutting-Edge Liposomal Vehiculization.

A high-performance liquid chromatography (HPLC) method was developed to simultaneously quantify three widely used active substances such as coenzyme Q10, phosphatidylserine, and vitamin C. This new method optimizes current timing and costs in the analyses of these three active substances. Additionally, since the analyzed compounds were encapsulated on a cutting-edge liposomal formulation, further processing was necessary to be developed prior to HPLC analyses. The technique was studied and adequately validated in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) regarding selectivity, linearity, accuracy, precision, and robustness. After data treatment of results, linear regressions for all active substances showed an optimal linearity with a correlation coefficient of >0.999 in the concentration range between 70 to 130% of the liposomal formulation and less than a 3% relative standard deviation (RSD) in accuracy and precision.

Formulation of Direct Compression Zidovudine Tablets to Correlate the SeDeM Diagram Expert System and the Rotary Press Simulator Styl'ONE Results.

The SeDeM diagram expert system has been applied to study Zidovudine and some excipients. From the obtained diagrams, a pharmaceutical formula has been designed. SeDeM diagram ascertains the critical parameters that are suitable for a direct compression. The formula is compressed using a rotary tablet press simulator which emulates rotary tablet press' compression profiles. From these compressions, we study the formula behavior under different industrial production conditions but saving a huge amount of material. The study is done at different compression forces and compression speeds and taking into account the influence of the pre-compression force. The differences observed between the compression profiles are hereby described. The results indicate that the formulation is able to be compressed adequately with the emulated compression profiles and no differences are observed between the final products. Therefore, we can assure that the SeDeM diagram expert system is accurate and robust. Moreover, its results are comparable with the compression results in a rotary tablet press, which has never been described in the pharmaceutical literature before. From the obtained results, it is possible to select the best rotary press to scale-up this formulation.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system.

Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed. Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity. These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.

Determinación de fitoesteroles en emulsiones lipídicas para nutrición parenteral / Phytosterol determination in lipid emulsions for parenteral nutrition

Objetivo: La presencia de fitoesteroles en emulsiones lipídicas de origen vegetal se ha relacionado con la aparición de alteraciones de los parámetros de la función hepática. El objetivo es determinar la presencia de fitoesteroles en las emulsiones registradas en el mercado farmacéutico. Método: Se analizaron tres-cuatro lotes no consecutivos de seis marcas distintas de emulsiones lipídicas (Clinoleic(R), Intralipid(R), Lipofundina(R), Lipoplus(R), Omegaven(R) y Smoflipid(R)) y las diferencias en contenido de fitoesteroles totales entre marcas y entre lotes se estudiaron estadísticamente (ANOVA de un factor, aproximación no paramétrica de Kruskal-Wallis y análisis post hoc Scheffé; p < 0,05). Resultados: Se encontró ausencia de fitoesteroles en el preparado Omegaven(R) con aceite de pescado. El contenido más alto de fitoesteroles (422,4 ± 130,5 µg/mL) coincidió con el porcentaje más alto de aceite de soja (Intralipid(R)). En el resto de las emulsiones se detectaron concentraciones de fitoesteroles entre 120 y 210 µg/mL, relacionadas con el contenido de aceite de soja. Se observaron diferencias estadísticamente significativas entre todas las marcas de emulsiones lipídicas (F = 42,97; p = 0,000) y entre lotes no consecutivos. Clinolenic(R) (F = 23,59; p = 0,000); Intralipid(R) (F = 978,25; p = 0,000); Lipofundina(R) TCL/TCM (F = 5,43; p = 0,045); Lipoplus(R) (F = 123,53; p = 0,000),; y Smoflipid(R) (16,78; p = 0,000). Excepto en el caso de la Lipofundina(R) TCL/TCM las diferencias entre lotes fueron marcadas. Conclusiones: Las emulsiones lipídicas registradas en el mercado farmacéutico español contienen cantidades variables de fitoesteroles en función de la marca comercial y el lote. La determinación del contenido de fitoesteroles, actualmente no declarados, permitiría desarrollar estrategias para prevenir o tratar la aparición de estas alteraciones

Objective: The presence of phytosterols in vegetal lipid emulsions has been associated with alterations of liver function tests. Determination of phytosterols content, currently undeclared, would allow the development of strategies to prevent or treat these alterations. Method: 3-4 non-consecutive batches of 6 lipid emulsions from different providers (Clinoleic(TM), Intralipid(TM), Lipofundina(TM), Lipoplus(TM), Omegaven(TM) and Smoflipid(TM)) were analyzed. Differences in total phytosterol assay between providers and batches were statistically studied by a one-way ANOVA and Kruskal-Wallis non-parametric approximation and post hoc Scheffé test (p < 0.05). Results: The absence of phytosterols was confirmed in Omegaven(TM), emulsion based on fish oil. The highest assay of phytosterols (422.4 ± 130.5 µg/mL) has been related with the highest percentage of soya bean oil in Intralipid. In the remaining emulsions, concentrations were from 120 to 210 µg/mL related to the percentage of soya bean oil. Statistically significant differences of phytosterol content in lipid emulsions were observed among different providers (F = 23.59; p = 0.000) as well as among non-consecutive batches. Clinolenic(TM) (F = 23.59; p = 0.000), Intralipid(TM) (F = 978.25; p = 0.000), Lipofundina(TM) TCL/TCM (F = 5.43; p = 0.045), Lipoplus(TM) (F = 123.53; p = 0.000) and Smoflipid(TM) (16.78; p = 0.000). Except for Lipofundina(TM) TCL/TCM, the differences between batches were marked. Conclusions: Lipid emulsions, registered on Spanish pharmaceutical market, contain variable quantities of phytosterols dependent on commercial brand and batch

Phytosterol determination in lipid emulsions for parenteral nutrition.

OBJECTIVE: The presence of phytosterols in vegetal lipid emulsions has been  associated with alterations of liver function tests. Determination of phytosterols  content, currently undeclared, would allow the development of strategies to  prevent or treat these alterations. METHOD: 3-4 non-consecutive batches of 6 lipid emulsions from different providers (Clinoleic, Intralipid, Lipofundina, Lipoplus, Omegaven and Smoflipid) were analyzed. Differences in total phytosterol assay between providers and batches were statistically studied by a one-way ANOVA and Kruskal-Wallis non-parametric approximation and post hoc Scheffé test (p < 0.05)Results: The absence of phytosterols was confirmed in Omegaven, emulsion  based on fish oil. The highest assay of phytosterols (422.4 ± 130.5 µg/mL) has  been related with the highest percentage of soya bean oil in Intralipid. In the  remaining emulsions, concentrations were from 120 to 210 µg/mL related to the  percentage of soya bean oil. Statistically significant differences of phytosterol  content in lipid emulsions were observed among different providers (F = 23.59;  p = 0.000) as well as among non-consecutive batches. Clinolenic (F = 23.59; p  = 0.000), Intralipid (F = 978.25; p = 0.000), Lipofundina TCL/TCM (F = 5.43; p  = 0.045), Lipoplus (F = 123.53; p = 0.000) and Smoflipid (16.78; p = 0.000).  Except for Lipofundina TCL/TCM, the differences between batches were marked. CONCLUSIONS: Lipid emulsions, registered on Spanish pharmaceutical market,  contain variable quantities of phytosterols dependent on commercial brand and  batch.

Objetivo: La presencia de fitoesteroles en emulsiones lipídicas de origen vegetal se ha relacionado con la aparición de alteraciones de los parámetros de la  función hepática. El objetivo es determinar la presencia de fitoesteroles en las  emulsiones registradas en el mercado farmacéutico.Método: Se analizaron tres-cuatro lotes no consecutivos de seis marcas  distintas de emulsiones lipídicas (Clinoleic®, Intralipid®, Lipofundina®,  Lipoplus®, Omegaven® y Smoflipid®) y las diferencias en contenido de  fitoesteroles totales entre marcas y entre lotes se estudiaron estadísticamente  (ANOVA de un factor, aproximación no paramétrica de Kruskal-Wallis y análisis  post hoc Scheffé; p < 0,05).Resultados: Se encontró ausencia de fitoesteroles en el preparado Omegaven® con aceite de pescado. El contenido más alto de fitoesteroles (422,4 ± 130,5  µg/mL) coincidió con el porcentaje más alto de aceite de soja (Intralipid®). En el resto de las emulsiones se detectaron concentraciones de fitoesteroles entre 120  y 210 µg/mL, relacionadas con el contenido de aceite de soja. Se  observaron diferencias estadísticamente significativas entre todas las marcas de  emulsiones lipídicas (F = 42,97; p = 0,000) y entre lotes no consecutivos.  Clinolenic® (F = 23,59; p = 0,000); Intralipid® (F = 978,25; p = 0,000);  Lipofundina® TCL/TCM (F = 5,43; p = 0,045); Lipoplus ® (F = 123,53; p =  0,000),; y Smoflipid® (16,78; p = 0,000). Excepto en el caso de la  Lipofundina® TCL/TCM las diferencias entre lotes fueron marcadas.Conclusiones: Las emulsiones lipídicas registradas en el mercado farmacéutico español contienen cantidades variables de fitoesteroles en función  e la marca comercial y el lote. La determinación del contenido de fitoesteroles, actualmente no declarados, permitiría desarrollar estrategias para prevenir o tratar la aparición de estas alteraciones.

Development and validation of a simple high-performance liquid chromatography analytical method for simultaneous determination of phytosterols, cholesterol and squalene in parenteral lipid emulsions.

A simple analytical method for simultaneous determination of phytosterols, cholesterol and squalene in lipid emulsions was developed owing to increased interest in their clinical effects. Method development was based on commonly used stationary (C18 , C8 and phenyl) and mobile phases (mixtures of acetonitrile, methanol and water) under isocratic conditions. Differences in stationary phases resulted in peak overlapping or coelution of different peaks. The best separation of all analyzed compounds was achieved on Zorbax Eclipse XDB C8 (150 × 4.6 mm, 5 µm; Agilent) and ACN-H2 O-MeOH, 80:19.5:0.5 (v/v/v). In order to achieve a shorter time of analysis, the method was further optimized and gradient separation was established. The optimized analytical method was validated and tested for routine use in lipid emulsion analyses.

Optimization and Validation of a Fast UPLC Method for Simultaneous Determination of Hydroquinone, Kojic Acid, Octinoxate, Avobenzone, BHA, and BHT.

A previously published HPLC method for the simultaneous determination of six major components (hydroquinone, kojic acid, octinoxate, avobenzone, butylated hydroxyanisole, and butylated hydroxytoluene) in a skin-whitening cream was transferred and optimized to an ultra-performance LC system. Separation was achieved in a ZORBAX SB-Phenyl Rapid-Resolution High Throughput column (2.1 × 100 mm, 1.8 µm), using a mobile phase consisting of water with 0.1% acetic acid and acetonitrile at a flow rate of 0.7 mL/min. The column was maintained at 40°C, and detection was carried out at 230 nm using a diode-array detector. These chromatographic conditions allow the separation of the six compounds in 3 min instead of 14 min. The extraction procedure was optimized, reducing the time and demonstrating its suitability. The method was validated according to International Conference on Harmonization guidelines, with respect to specificity, precision, accuracy, and linearity. Selectivity was found to be satisfactory. Linear regression analysis data for all compounds showed a good linear relationship, with r2 > 0.999 in the concentration range of 50-120% of the label claim for each compound. The RSD for precision and accuracy of the method was found to be less than 2% for all compounds. Comparison of system performance with the previously published HPLC method was made with respect to analysis time, efficacy, and resolution. The proposed method is faster and consumes less solvent and was applied in the determination of six major compounds in batches of skin-whitening cream manufactured during the validation process.

Authentication of gold nanoparticle encoded pharmaceutical tablets using polarimetric signatures.

The counterfeiting of pharmaceutical products represents concerns for both industry and the safety of the general public. Falsification produces losses to companies and poses health risks for patients. In order to detect fake pharmaceutical tablets, we propose producing film-coated tablets with gold nanoparticle encoding. These coated tablets contain unique polarimetric signatures. We present experiments to show that ellipsometric optical techniques, in combination with machine learning algorithms, can be used to distinguish genuine and fake samples. To the best of our knowledge, this is the first report using gold nanoparticles encoded with optical polarimetric classifiers to prevent the counterfeiting of pharmaceutical products.

Development and Validation of a Stability Indicating RP-HPLC Method for Hydrocortisone Acetate Active Ingredient, Propyl Parahydroxybenzoate and Methyl Parahydroxybenzoate Preservatives, Butylhydroxyanisole Antioxidant, and Their Degradation Products in a Rectal Gel Formulation.

A stability indicating method was established through a stress study, wherein different methods of degradation (oxidation, hydrolysis, photolysis, and temperature) were studied simultaneously to determine the active ingredient hydrocortisone acetate, preservatives propyl parahydroxybenzoate, and methyl parahydroxybenzoate, antioxidant butylhydroxyanisole (BHA), and their degradation products in a semisolid dosage gel form. The proposed method was suitably validated using a Zorbax SB-Phenyl column and gradient elution. The mobile phase consisted of a mixture of methanol, acetonitrile, and water in different proportions according to a planned program at a flow rate of 1.5 mL/min. The diode array detector was set at 240 nm for the active substance and two preservatives, and 290 nm for BHA. The validation study was conducted according to International Conference on Harmonization guidelines for specificity, linearity, repeatability, precision, and accuracy. The method was used for QC of hydrocortisone acetate gel and for the stability studies with the aim of quantifying the active substance, preservatives, antioxidant, and degradation products. It has proved to be suitable as a fast and reliable method for QC.

Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method.

BACKGROUND: A new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride. METHODS: For RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 µm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K2HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min (-1). Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification. RESULTS: The method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products. Regression analysis showed r(2) > 0.999 for cetirizine dihydrochloride in the concentration range of 650 µg mL (-1) to 350 µg mL(-1) for drug substance assay and a r(2) > 0.999 in the concentration range of 0.25 µg mL(-1) to 5 µg mL(-1) for degradation products. The method presents a limit of detection of 0.056 µg mL (-1) and a limit of quantification of 0.25 µg mL(-1). The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method. CONCLUSIONS: The proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.

A new optimized formulation of cationic solid lipid nanoparticles intended for gene delivery: development, characterization and DNA binding efficiency of TCERG1 expression plasmid.

Solid lipid nanoparticles (SLNs) are being considered as a new approach for therapeutics for many known diseases. In addition to drug delivery, their use as non-viral vectors for gene delivery can be achieved by the inclusion of cationic lipids, which provide a positive surface potential that favours binding to the DNA backbone. This work is based on the idea that the optimization of the components is required as the first step in simplifying the qualitative and quantitative composition of SLNs as much as possible without affecting the essential properties that define SLNs as optimal non-viral vectors for gene delivery. We selected the best lipids and surfactants in terms of particle size and zeta potential and characterized the properties of the resulting nanoparticles using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The SLNs had a particle size of approximately 120 nm and a positive surface charge of 42 mV. In addition, we analysed the main physicochemical characteristics of the bulk components of the nanoparticles using X-ray diffraction (XRD), differential scanning calorimetry (DSC) and mass spectrometry (MS). The suitability of the optimized SLNs for DNA binding was evaluated after the lyophilisation process using a carboxyl-terminal region of the TCERG1 gene, a human factor that has been implicated in several diseases. We show that the SLNs presented high efficiency in the binding of DNA, and importantly, they presented no toxicity when assayed in an in vivo system.