Stability-indicating LC Method for Quantification of Azelnidipine: Synthesis and Characterization of Oxidative Degradation Product

Objectives: In the work presented here, the degradation behavior of azelnidipine under diverse forced degradation conditions was studied. A stability-indicating liquid chromatographic method was established which could separate and resolve azelnidipine from its degradation products. Further, chemical kinetics under acidic and alkaline conditions were studied, and validation studies were performed. Materials and Methods: Using reversed-phase chromatography, azelnidipine and its formed degradants were resolved using phosphate buffer (pH 3.0) and methanol in a mixture of 10:90% v/v as a mobile phase at a flow rate of 1.0 mL/min. All eluents were detected at a wavelength of 256 nm. Results: Azelnidipine was degraded under acid, alkali, wet heat, and oxidized environment. The pH-dependent rate of hydrolysis of azelnidipine was studied under acidic and alkaline conditions and chemical kinetics were determined. Further, the oxidative degradation product of azelnidipine was synthesized and characterized as 3-(1-benzhydrylazetidin-3-yl) 5-isopropyl 2-amino-6-methyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate (dehydro-AZD). Conclusion: The susceptibility of azelnidipine to hydrolysis was attributed to the presence of ester at 3 and 5 positions of 1,4 dihydropyridine. Further, under oxidative conditions, the aromatization of 1,4 dihydropyrinine resulted in dehydro-AZD. Azelnidipine followed the first-order reaction under acid and alkali hydrolysis, and was more susceptible to degradation under acidic conditions. The synthesized and confirmed dehydro-AZD was found as one of the metabolites and impurities of azelnidipine. The evaluated validation parameters ascertained the practicality of the method for the quantification of azelnidipine tablets.

Development and Optimization of Proniosomal Gel Containing Etodolac: In-vitro, Ex-vivo and In-vivo Evaluation / Desarrollo y optimización del gel proniosomal que contiene etodolaco: evaluación in vitro, ex vivo e in vivo

Introducción: Etodolac se usa en el tratamiento del dolor agudo y la inflamación. Tiene baja solubilidad debido a la alta hidrofobia y se informa que tras la administración oral muestra alteraciones gástricas. Esto fomenta el desarrollo de formulación tópica en lugar de oral. Método: En este trabajo utilizamos el método de separación de fase de coacervación para el desarrollo del sistema vesicular cargado con etodolaco mediante el uso de tensioactivos no iónicos, colesterol y lecitina de soja. El diseño central compuesto (rotativo) se utilizó para optimizar las concentraciones de lecitina de soja, surfactante y colesterol. Las formulaciones preparadas se caracterizaron por análisis de tamaño de vesículas, potencial zeta, eficiencia de atrapamiento, permeación in vitro, permeación ex vivo y estudio antiinflamatorio. Resultados: Etodolac quedó atrapado con éxito en todas las formulaciones que tenían una eficiencia en el intervalo de 74,36% a 90,85%, siendo mayor a 4 ° C que a temperatura ambiente. Cuando se hidrata con agua, los niosomas se producen espontaneamente el rango de 54 a 141 (por mm cúbico). Los resultados del estudio de difusión in vitro revelaron que el etodolaco se liberó en un rango de 71,86 a 97,16% durante un período de 24 horas. El tamaño medio de vesícula de la formulación optimizada se encontró en 211,9 nm con un PDI de 0,5. Las respuestas observadas, es decir,% de eficacia de encapsulación y liberación de fármaco, fueron 74,12 y 95,08 respectivamente. El potencial zeta fue de -19,4 mV y reveló la estabilidad de la formulación, que fue confirmada adicionalmente por la ausencia de cambios en el contenido del fármaco y la liberación del fármaco después de los estudios de estabilidad. El% de inhibición en el volumen de la pata fue del 40,52% y del 43,61% para la prueba y el gel proniosómico comercializado. (AU)

Introduction: Etodolac is used in the treatment of acute pain and inflammation. It has low solubility because of high hydrophobicity and it is reported that upon oral administration shows gastric disturbances. This encourages the development of topical vesicular formulation. Method: In this work we used coacervation-phase separation method for the development of etodolac loaded ve-sicular system by using non-ionic surfactants, cholesterol and soya lecithin. Central composite design (rotatble) was used to optimize the concentrations of soy lecithin, surfactant and cholesterol. The prepared formulations were characterized by number of vesicles formed, vesicle size, zeta potential, entrapment efficiency, in-vitro permeation, ex-vivo permeation and anti-inflammatory study. Results: Etodolac was successfully entrapped in all formulations having efficiency in the range of 74.36% to 90.85%, which was more at 4 °C than room temperature. When hydrated with water; niosome in the range of 54 to 141 (per cubic mm) were spontaneously produced. The results of in-vitro diffusion study revealed that etodolac was released in the range of 71.86 to 97.16% over a period of 24 hrs. The average vesicle size of optimized formulation was found 211.9 nm with PDI of 0.5. The observed responses i.e. % encapsulation efficiency and drug release were 74.12 and 95.08 respectively. The zeta potential was -19.4mV revealed the stability of formulation which was further confirmed by no changes in drug content and drug release after stability studies. The % inhibition in paw volume was 40.52% and 43.61% for test and marketed proniosomal gel. (AU)

AB2-type amphiphilic block copolymer containing a pH-cleavable hydrazone linkage for targeted antibiotic delivery.

A novel AB2-type amphiphilic block copolymer [OA-CN-NH-(PEG)2] with hydrazone linkage was synthesized and explored for pH-triggered antibiotic delivery. Vancomycin (VCM) loaded micelles of the polymer [OA-CN-NH-(PEG)2-VCM] were spherical in shape with size, polydispersity index, zeta potential and entrapment efficiency of 130.33 ± 7.36 nm, 0.163 ± 0.009, -4.33 ± 0.55 mV and 39.61 ± 4.01% respectively. The dilution stability study exhibited no significant change in the size distribution of OA-CN-NH-(PEG)2-VCM micelles up to 320-fold dilution. An in vitro drug release assay confirmed greater release of VCM from OA-CN-NH-(PEG)2-VCM at pH 6, compared to pH 7.4. An in vitro antibacterial activity evaluation of OA-CN-NH-(PEG)2-VCM showed 2-fold enhancement in antibacterial activity of VCM after 54 h of incubation against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) at acidic pH compared to physiological pH. An in vivo antibacterial activity of OA-CN-NH-(PEG)2-VCM further proved the enhanced activity of OA-CN-NH-(PEG)2-VCM against MRSA. In conclusion, micelles from pH-responsive OA-CN-NH-(PEG)2 could be utilized for site-specific delivery of VCM at the infection site.

Hydrazone linkages in pH responsive drug delivery systems.

Stimuli-responsive polymeric drug delivery systems using various triggers to release the drug at the sites have become a major focus area. Among various stimuli-responsive materials, pH-responsiveness has been studied extensively. The materials used for fabricating pH-responsive drug delivery systems include a specific chemical functionality in their structure that can respond to changes in the pH of the surrounding environment. Various chemical functionalities, for example, acetal, amine, ortho ester, amine and hydrazone, have been used to design materials that are capable of releasing their payload at the acidic pH conditions of the tumor or infection sites. Hydrazone linkages are significant synthons for numerous transformations and have gained importance in pharmaceutical sciences due to their various biological and clinical applications. These linkages have been employed in various drug delivery vehicles, such as linear polymers, star shaped polymers, dendrimers, micelles, liposomes and inorganic nanoparticles, for pH-responsive drug delivery. This review paper focuses on the synthesis and characterization methods of hydrazone bond containing materials and their applications in pH-responsive drug delivery systems. It provides detailed suggestions as guidelines to materials and formulation scientists for designing biocompatible pH-responsive materials with hydrazone linkages and identifying future studies.

Preparation and Optimization of Meropenem-Loaded Solid Lipid Nanoparticles: In Vitro Evaluation and Molecular Modeling.

Encapsulation of antibiotics into nanocarriers has the potential to overcome resistance and disadvantages associated with conventional dosage forms as well as increase half-life of an antibiotic. Encapsulation of meropenem (MRPN) into solid lipid nanoparticles (SLNs) remains unexplored among the limited work reported on nanoformulation incorporating MRPN. The study aimed to use an experimental design, to optimize MRPN-loaded SLNs, and to undertake in vitro and in silico evaluations. A Box-Behnken design (BBD) was used to optimize manufacturing conditions of glycerol monostearate (GMS) SLNs loaded with MRPN. The SLNs were prepared using hot homogenization and ultrasonication method. Optimized MRPN-SLNs showed particle size, zeta potential, and entrapment efficiency of 112.61 ± 0.66 nm, -20.43 ± 0.99 mV, and 89.94 ± 1.26%, respectively. The morphology of the SLNs revealed nearly spherical shaped particles. Differential scanning calorimetry and X-ray diffraction analysis showed that meropenem was present in amorphous form in the SLNs. Controlled in vitro MRPN release from SLNs was achieved and followed the Korsmeyer-Peppas model (R 2 = 0.9679). Prolonged in vitro antibacterial activity against Escherichia coli was also observed. The molecular modeling showed that both hydrophobic interactions and hydrogen bonding led to a stable MRPN-GMS complex formation, which was confirmed by its low heat of formation (-5536.13 kcal/mol). This stable complex could have contributed to the controlled release of MRPN from the SLNs and subsequent sustained antibacterial activity.

Knowledge, Attitude, and Behavioural Response of Corporate Employees in India towards Influenza: A Questionnaire Based Study.

OBJECTIVE: Understanding about influenza would be helpful in adapting preventive measures like social distancing, seeking medical attention and getting vaccinated. The objective of this study was to evaluate knowledge, attitude and practices (KAP) towards influenza among Indian corporate employees and assessment of their awareness through a questionnaire-based survey. METHODS: Indian corporate employees of either gender (≥18 years) were provided with a self-administered questionnaire with domains namely knowledge (21 questions), attitude (6 questions), and practices (6 questions). RESULTS: Overall, 500 corporate employees completed the survey from four zones. Total of 50.8% respondents had low level of knowledge of influenza, 34% had moderate knowledge, and 15.2% had high knowledge of influenza. Attitude domain revealed that 66% respondents were quiet concerned and 24% were extremely concerned regarding influenza. The practice domain demonstrated that 57.8% respondents took high level of precautions while 36.6% took moderate level precautions for the prevention of infection. CONCLUSIONS: Knowledge and attitude regarding importance of vaccine against influenza is not adequate. This study emphasizes the need for effective awareness programs for the management of influenza.

Development and Validation of Stability-Indicating Method for Estimation of Chlorthalidone in Bulk and Tablets with the Use of Experimental Design in Forced Degradation Experiments.

Chlorthalidone was subjected to various forced degradation conditions. Substantial degradation of chlorthalidone was obtained in acid, alkali, and oxidative conditions. Further full factorial experimental design was applied for acid and alkali forced degradation conditions, in which strength of acid/alkali, temperature, and time of heating were considered as independent variables (factors) and % degradation was considered as dependent variable (response). Factors responsible for acid and alkali degradation were statistically evaluated using Yates analysis and Pareto chart. Furthermore, using surface response curve, optimized 10% degradation was obtained. All chromatographic separation was carried out on Phenomenex HyperClone C 18 column (250 × 4.6 mm, 5 µ), using mobile phase comprising methanol : acetonitrile : phosphate buffer (20 mM) (pH 3.0 adjusted with o-phosphoric acid): 30 : 10 : 60% v/v. The flow rate was kept constant at 1 mL/min and eluent was detected at 241 nm. In calibration curve experiments, linearity was found to be in the range of 2-12 µg/mL. Validation experiments proved good accuracy and precision of the method. Also there was no interference of excipients and degradation products at the retention time of chlorthalidone, indicating specificity of the method.

Ultra-small lipid-dendrimer hybrid nanoparticles as a promising strategy for antibiotic delivery: In vitro and in silico studies.

The purpose of this study was to explore the preparation of a new lipid-dendrimer hybrid nanoparticle (LDHN) system to effectively deliver vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) infections. Spherical LDHNs with particle size, polydispersity index and zeta potential of 52.21±0.22 nm, 0.105±0.01, and -14.2±1.49 mV respectively were prepared by hot stirring and ultrasonication using Compritol 888 ATO, G4 PAMAM- succinamic acid dendrimer, and Kolliphor RH-40. Vancomycin encapsulation efficiency (%) in LDHNs was almost 4.5-fold greater than in lipid-polymer hybrid nanoparticles formulated using Eudragit RS 100. Differential scanning calorimetry and Fourier transform-infrared studies confirmed the formation of LDHNs. The interactions between the drug-dendrimer complex and lipid molecules using in silico modeling revealed the molecular mechanism behind the enhanced encapsulation and stability. Vancomycin was released from LDHNs over the period of 72 h with zero order kinetics and super case II transport mechanism. The minimum inhibitory concentration (MIC) against S. aureus and MRSA were 15.62 µg/ml and 7.81 µg/ml respectively. Formulation showed sustained activity with MIC of 62.5 µg/ml against S. aureus and 500 µg/ml against MRSA at the end of 72 and 54 h period respectively. The results suggest that the LDHN system can be an effective strategy to combat resistant infections.

Design, synthesis and molecular modeling studies of few chalcone analogues of benzimidazole for epidermal growth factor receptor inhibitor in search of useful anticancer agent.

In the present investigation, few 3-(substitutedphenyl)-1-[2-(1-hydroxy-ethyl)]-1H-benzimidazol-1-yl)prop-2-en-1-ones are EGFR antagonist are designed, by molecular docking analysis. The synthesized compounds were tested for their in vitro anticancer activity by propidium iodide fluorescent assay and Trypan blue viability assay against colorectal cancer cell lines (HCT116) and non-small cell lung cancer cell lines (H460). Human Epithelial Kidney cell lines (HEK) are used as normal cell lines for studying effect of drug on non-cancerous cells within human body. Evaluation of cytotoxic studies of synthesized compounds CHL(1-8) reveal that compound CHL1 [IC50=7.31 and 10.16 µM against HCT116 and H460 cell lines respectively, by PI assay] and CHL8 [IC50=12.52 and 6.83 against HCT116 and H460µM cell lines respectively] possess promising cytotoxic activity.

Solid lipid nanoparticles of clotrimazole silver complex: An efficient nano antibacterial against Staphylococcus aureus and MRSA.

New and effective strategies to transform current antimicrobials are required to address the increasing issue of microbial resistance and declining introduction of new antibiotic drugs. In this context, metal complexes of known drugs and nano delivery systems for antibiotics are proving to be promising strategies. The aim of the study was therefore to synthesize a silver complex of clotrimazole and formulate it into a nano delivery system for enhanced and sustained antibacterial activity against susceptible and resistant Staphylococcus aureus. A silver complex of clotrimazole was synthesized, characterized and further encapsulated into solid lipid nanoparticles to evaluate its antibacterial activity against S. aureus and methicillin-resistant S. aureus (MRSA). An in vitro cytotoxicity study was performed on HepG2 cell lines to assess the overall biosafety of the synthesized clotrimazole silver complex to mammalian cells, and was found to be non-toxic to mammalian cells (cell viability >80%). The minimum inhibitory concentrations (MIC) of clotrimazole and clotrimazole-silver were 31.25 and 9.76 µg/mL against S. aureus, and 31.25 and 15.62 against MRSA, respectively. Clotrimazole SLNs exhibited MIC values of 104 and 208 µg/mL against both MSSA and MRSA at the end of 18 and 36 h, respectively, but thereafter completely lost its antibacterial activity. Clotrimazole-silver SLNs had an MIC value of 52 µg/mL up to 54 h, after which the MIC value was 104 µg/mL against both strains at the end of 72 h. Thus, clotrimazole-silver SLNs was found to be an efficient nanoantibiotic.

Ion pairing with linoleic acid simultaneously enhances encapsulation efficiency and antibacterial activity of vancomycin in solid lipid nanoparticles.

Ion pairing of a fatty acid with an antibiotic may be an effective strategy for formulation optimization of a nanoantibiotic system. The aim of this study was therefore to explore the potential of linoleic acid (LA) as an ion pairing agent to simultaneously enhance encapsulation efficiency and antibacterial activity of triethylamine neutralized vancomycin (VCM) in solid lipid nanoparticles (SLNs). The prepared VCM-LA2 conjugate was characterized by Fourier transform-infrared (FT-IR) spectroscopy, logP and binding energy calculations. The shifts in the FT-IR frequencies of COOH, NH2 and CO functionalities, an increase in logP value (1.37) and a lower interaction energy between LA and VCM (-125.54 kcal/mol) confirmed the formation of the conjugate. SLNs were prepared by a hot homogenization and ultrasonication method, and characterized for size, polydispersity index (PI), zeta potential (ZP), entrapment efficiency (%EE), surface morphology and physical stability. In vitro antibacterial activity studies against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) were conducted. Size, PI and ZP for VCM-LA2_SLNs were 102.7±1.01, 0.225±0.02 and -38.8±2.1 (mV) respectively. SLNs were also stable at 4 °C for 3 months. %EE for VCM-HCl_SLNs and VCM-LA2_SLNs were 16.81±3.64 and 70.73±5.96 respectively, indicating a significant improvement in encapsulation of the drug through ion pairing with LA. Transmission electron microscopy images showed spherical nanoparticles with sizes in the range of 95-100 nm. After 36 h, VCM-HCl showed no activity against MRSA. However, the minimum inhibitory concentration for VCM-HCl_SLNs and VCM-LA2_SLNs were 250 and 31.25 µg/ml respectively against S. aureus, while against MRSA it was 500 and 15.62 µg/ml respectively. This confirms the enhanced antibacterial activity of VCM-LA2_SLNs over VCM-HCl_SLNs. These findings therefore suggest that VCM-LA2_SLNs is a promising nanoantibiotic system for effective treatment against both sensitive and resistant S. aureus infections.

Development and validation of RP-HPLC method for estimation of eplerenone in spiked human plasma.

A rapid and simple high performance liquid chromatography (HPLC) method with a UV detection (241 nm) was developed and validated for estimation of eplerenone from spiked human plasma. The analyte and the internal standard (valdecoxib) were extracted with a mixture of dichloromethane and diethyl ether. The chromatographic separation was performed on a HiQSil C-18HS column (250 mm×4.6 mm, 5 µm) with a mobile phase consisting of acetonitrile:water (50:50, v/v) at flow rate of 1 mL/min. The calibration curve was linear in the range 100-3200 ng/mL and the heteroscedasticity was minimized by using weighted least squares regression with weighting factor 1/X.

Optimization of forced degradation using experimental design and development of a stability-indicating liquid chromatographic assay method for rebamipide in bulk and tablet dosage form.

A novel stability-indicating RP-HPLC assay method was developed and validated for quantitative determination of rebamipide in bulk and tablet dosage form. Rebamipide (drug and drug product) solutions were exposed to acid and alkali hydrolysis, thermal stress, oxidation by hydrogen peroxide and photodegradation. Experimental design has been used during forced degradation to determine significant factors responsible for degradation and to obtain optimal degradation conditions. In addition, acid and alkali hydrolysis was performed using a microwave oven. The chromatographic method employed the HiQ sil C-18HS (250 × 4.6 mm; 5 µm) column with mobile phase consisting of 0.02 M potassium phosphate (pH adjusted to 6.8) and methanol (40:60, v/v) and the detection was performed at 230 nm. The procedure was validated for specificity, linearity, accuracy, precision and robustness. There was no interference observed of excipients and degradation products in the determination of the active pharmaceutical ingredient. The method showed good accuracy and precision (intra and inter day) and the response was linear in a range from 0.5 to 5 µg mL(-1). The method was found to be simple and fast with less trial and error experimentation by making use of experimental design. Also, it proved that microwave energy can be used to expedite hydrolysis of rebamipide.