Development of ezetimibe eutectic with improved biopharmaceutical and mechanical properties to design an optimized oral solid dosage formulation.

Eutectics are multicomponent systems which are an alternative to the conventional techniques for modulating the biopharmaceutical properties of a pharmaceutical. Ezetimibe (ETZ) is a hypocholesterolemic agent with limited dissolution, poor water solubility, and subsequently demonstrates low oral bioavailability. Additionally, ETZ exhibits poor mechanical properties, leading to difficulties in developing dosage forms through direct compression. The present work highlights the applicability of eutectics in the simultaneous improvement of physicochemical along with mechanical properties of ETZ. A pharmaceutical eutectic of ETZ with succinimide (SUC) was prepared by mechanochemical grinding and thoroughly characterized using thermoanalytical, X-ray diffraction, and spectroscopic methods. Intrinsic dissolution rate and pharmacokinetic analysis were also performed for ezetimibe-succinimide (ETZ-SUC) eutectic in contrast to pure ETZ. The eutectic demonstrated ∼2-fold increase in the solubility and dissolution rate. In pharmacokinetic studies, the area under the curve (AUC) for ETZ-SUC eutectic (28.03 ± 2.22 ng*h/mL) was found to be higher than ETZ (8.98 ± 0.36 ng*h/mL), indicating improved oral bioavailability for eutectics. Also, it was observed that enhanced material functionality aids in designing directly compressed tablets, where the eutectic formulation showed an improved dissolution profile over the ETZ formulation. The study demonstrates that eutectic conglomerates could be utilized to develop ideal oral solid dosage formulations.

Ezetimibe: A Review of Analytical Methods for the Drug Substance, Pharmaceutical Formulations and Biological Matrices.

Ezetimibe (EZM) is a selective inhibitor of the sterol transporter Niemann-Pick C1-Like 1 in the small intestine used as an adjunctive therapy to lower cholesterol levels in cases of hyperlipidemia. The goal of this work was to summarize the main physical-chemical, pharmacological and pharmacokinetic characteristics of EZM, as well as to describe the main analytical methodologies for the quantification of the drug. Methods described in the United States Pharmacopeia for EZM raw material and tablets were also presented. The drug has a large number of process-related impurities and degradation products and needs strict quality control of its impurities. Specific chiral methods for the evaluation of its chiral impurities are also a need for EZM. The main advantages and disadvantages of the compiled analytical methods were presented, as well as the limits of detection and quantitation. The fastest and most efficient methods were highlighted. Most methods for analyzing EZM used C8 or C18 stationary phases in gradient mode with binary mobile phases containing acetonitrile and an acidic buffer solution with ultraviolet detection. For analysis of EZM in biological matrices, liquid chromatography-tandem mass spectrometry is generally employed using electron spray ionization in negative ionization mode using multiple reaction monitoring. Different methods in the literature evaluate a large number of impurities for EZM, however new stability-indicating high-performance liquid chromatography methods for the drug are still needed.

Ternary Eutectic Ezetimibe-Simvastatin-Fenofibrate System and the Physical Stability of Its Amorphous Form.

In this study, the phase diagram of the ternary system of ezetimibe-simvastatin-fenofibrate was established. It has been proven that the ternary composition recommended for the treatment of mixed hyperlipidemia forms a eutectic system. Since eutectic mixtures are characterized by greater solubility and dissolution rate, the obtained result can explain the marvelous medical effectiveness of combined therapy. Considering that another well-known method for improving the aqueous solubility is amorphization, the ternary system with eutectic concentration was converted into an amorphous form. Thermal properties, molecular dynamics, and physical stability of the obtained amorphous system were thoroughly investigated through various experimental techniques compared to both: neat amorphous active pharmaceutical ingredients (considered separately) and other representative concentrations of ternary mixture. The obtained results open up a new way of selecting the therapeutic concentrations for combined therapies, a path that considers one additional variable: eutecticity.

Formulation and Evaluation of Linum usitatissimum Mucilage-Based Nanoparticles for Effective Delivery of Ezetimibe.

INTRODUCTION: The aim of current study was to prepare Linum usitatissimum mucilage (LUM) based nanoparticles, capable of encapsulating hydrophobic drug ezetimibe as nanocarriers. METHODS: Solvent evaporation and nanoprecipitation techniques were used to develop nanoparticles by encapsulating ezetimibe in the articulated matrix of polysaccharide fractions. Developed nanoparticles were characterized to determine the particle size, zeta potential, polydispersibility index (PDI), and entrapment efficiency (EE). Morphology and physicochemical characterization were carried out through SEM, FTIR, PXRD and thermal analysis. Saturation solubility and in vitro release studies were also performed. Safety assessment of ezetimibe loaded nanoparticles was evaluated via oral acute toxicity study. RESULTS: The mean particle size, zeta potential, PDI and EE for emulsion solvent evaporation were 683.6 nm, -28.3 mV, 0.39, 63.7% and for nanoprecipitation were 637.7 nm, 0.07, -27.1 mV and 80%, respectively. Thermal analysis confirmed enhanced thermal stability, whereas PXRD confirmed amorphous nature of drug. Saturation solubility (p-value <0.05) demonstrated improved solubility of drug when enclosed in linseed nanoparticles. Nanoprecipitation surpasses emulsion solvent evaporation in dissolution test by possessing smaller size. Acute oral toxicity study indicated no significant changes in behavioral, clinical or histopathological parameters of control and experimental groups. CONCLUSION: The in vitro release of ezetimibe was augmented by enhancing aqueous solubility through devised nanoparticles. Thus, linseed mucilage could act as biopolymer in the fabrication of nanoparticle formulation. The acute oral toxicological investigations provided evidence that LUMNs were safe after oral administration.

One-step modification to identify dual-inhibitors targeting both pancreatic triglyceride lipase and Niemann-Pick C1-like 1.

Pancreatic triglyceride lipase (PTL) and Niemann-Pick C1-like 1 (NPC1L1) have been identified as attractive therapeutic targets for obesity and hypercholesteremia, respectively. Obesity and hypercholesteremia usually co-exist, however no dual-inhibitors against PTL and NPC1L1 were reported for the treatment of obesity patients with hypercholesteremia so far. In this work, molecular hybridization-based one-step modification screening identified a potent dual-inhibitor against PTL and NPC1L1. Compound P1-11 has IC50 values of 2.1 µM against PTL through covalent binding, as well as significantly reduces cholesterol absorption in a non-competitive inhibitory manner. Molecule docking and molecular dynamics studies revealed the reason of its activity to both PTL and NPC1L1. Moreover, the gene and protein expression levels of PTL and NPC1L1 were also determined respectively after the treatment of P1-11. Development of dual-inhibitors against PTL and NPC1L1 could provide novel treatment options for obesity patients with hypercholesteremia. The results of current research would great support the development of dual-inhibitors against PTL and NPC1L1.

Co-processing of Atorvastatin and Ezetimibe for Enhanced Dissolution Rate: In Vitro and In Vivo Correlation.

Development of fixed dose combinations is growing and many of these drug combinations are being legally marketed. However, the development of these requires careful investigation of possible physicochemical changes during co-processing. This requires investigation of the effect of co-processing of drug combination in absence of excipients to maximize the chance of interaction (if any). Accordingly, the aim was to investigate the effect of co-processing of ezetimibe and atorvastatin on drugs dissolution rate. The objective was extended to in vitro in vivo correlation. Drugs were subjected to wet co-processing in presence of ethanol after being mixed at different ratios. The prepared formulations were characterized using FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry, scanning electron microscopy, and in vitro dissolution testing. These investigations proved the possibility of eutectic system formation after drugs co-processing. This was reflected on drugs dissolution rate which was significantly enhanced at dose ratio and 2:1 atorvastatin:ezetimibe molar ratio compared to the corresponding pure drugs. In vivo antihyperlipidemic effects of the co-processed drugs were monitored in albino mice which were subjected to hyperlipidemia induction using poloxamer 407. The results showed significant enhancement in pharmacological activity as revealed from pronounced reduction in cholesterol level in mice administering the co-processed form of both drugs. Besides, histopathological examinations of the liver showed marked decrease in hepatic vacuolation. In conclusion, co-processing of atorvastatin with ezetimibe resulted in beneficial eutexia which hastened the dissolution rate and pharmacological effects of both drugs.Graphical abstract.

Studies of the Formation and Stability of Ezetimibe-Cyclodextrin Inclusion Complexes.

In the presented studies, the interactions between ezetimibe (EZE) and selected cyclodextrins were investigated. α-Cyclodextrin (αCD), ß-cyclodextrin (ßCD) and its modified derivatives, hydroxypropyl-ß-cyclodextrin (HPßCD) and sulfobutylether-ß-cyclodextrin (SBEßCD), were selected for the research. Measurements were carried out using calorimetric and spectroscopic methods. Additionally, the Hirshfeld surface and biochemical analysis were achieved. As a result of the study, the inclusion complexes with 1:1 stoichiometry were obtained. The most stable are the complexes of ß-cyclodextrin and its derivatives. The comparison of ßCD with its derivatives shows that the modifications have an affect on the formation of more durable and stable complexes.

Determining the Regiochemistry and Relative Stereochemistry of Small and Druglike Molecules Using an Alignment Algorithm for Infrared Spectra.

The relative stereochemistry and isomeric substitution pattern of organic molecules is typically determined using nuclear magnetic resonance spectroscopy (NMR). However, NMR spectra are sometimes nonconclusive, e.g., if spectra are extremely crowded, coupling patterns are not resolved, or if symmetry reasons preclude interpretation. Infrared spectroscopy (IR) can provide additional information in such cases, because IR represents a molecule comprehensively by depiction of the complete set of its normal vibrations. The challenge is thereby that diastereomers and substitution isomers often give rise to highly similar IR spectra, and visual distinction is insufficient and may be biased. Here we show the adaptation of an alignment algorithm, originally developed for vibrational circular dichroism (VCD) spectroscopy, to automatically match IR spectra and provide a quantitative measure of the goodness of fit, which can be used to distinguish isomers. The performance of the approach is demonstrated for different use cases: diastereomers of flexible druglike molecules, E/Z-isomers, and substitution isomers of pyrazine and naphthalene. It can be applied to IR spectra measured both in the gas phase (gas chromatography IR) and in solution.

Dual drug nanocrystals loaded microparticles for fixed dose combination of simvastatin and ezetimibe.

Dual drug nanocrystals loaded nano embedded microparticles (DNEMs) were prepared for fixed dose combination of simvastatin (SIM) and ezetimibe (EZE) using NanoCrySP technology. The purpose was to generate nanonized SIM and EZE dispersed in matrix of single crystallization inducing excipient and investigate their in vitro performance. DNEM were prepared using mannitol (MAN) as crystallization inducer (active pharmaceutical ingredients (APIs)/MAN = 3:7 w/w) using spray drying. TPGS (0.1% w/v) was used as surfactant for stabilization of nanocrystals. Crystallinity of DNEM was confirmed by solid-state characterization using DSC and PXRD. Particle size analysis was carried out using Zetasizer® and the Scherrer equation as primary techniques and SEM and TEM as orthogonal techniques. Size of both SIM and EZE in DNEM was close to 600 nm. In vitro performance was assessed using USP apparatus II in 0.025% SLS containing sodium phosphate buffer. Powder dissolution of DNEM increased 1.45 times for SIM and 1.65 times for EZE as compared to their physical mixture in discriminatory medium. MAN did not plasticize SIM or EZE by virtue of its immiscibility with the two drugs. However, MAN helped in inducing crystallization via heterogeneous nucleation. The generated DNEM were stable in terms of assay, polymorphic form and dissolution for 90 days of accelerated storage at 40 °C/75% RH.

Central composite designed ezetimibe solid dispersion for dissolution enhancement: synthesis and in vitro evaluation.

Aim: The current research is focused on increasing aqueous solubility and dissolution of BCS class II drug by using modified solvent evaporation technique to produce solid dispersions of ezetimibe (EZSD) using gelucire 50/13 and polyvinyl pyrollidone K30. Methodology & results: Central composite design analyzed the effect of gelucire 50/13 and polyvinyl pyrollidone K30 on the percentage of drug released in 5 and 30 min. Ezetimibe (EZ) aqueous saturation solubility (4.56 ± 0.94 µg/ml) was increased 25-fold in EZSD (115 ± 3.41 µg/ml). Cumulative drug release from EZ and optimized EZSD were observed 24.67 and 87.54% within 1 h, respectively. Conclusion: Manufacturing EZSD using modified solvent evaporation technique using rotary evaporator holds great promise for enhancing EZ's solubility and dissolution.

Dissolution rate enhancement of new co-crystals of ezetimibe with maleic acid and isonicotinamide.

Ezetimibe (EZT) is a selective cholesterol absorption inhibitor with poor aqueous solubility (0.012mg/ml 23oC) and low oral bioavailability (about 35-65% for a once 10mg dose). The present study illustrates the preparation and characterization of two new co-crystals of ezetimibe using maleic acid and isonicotinamide as the coformers by solid grinding method. The co-crystal structures were characterized by X-ray powder diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (IR) techniques. Crystallinity and surface morphological characteristics of these prepared co-crystals were observed by scanning electron microscope (SEM). Dissolution rate tests demonstrated that both of the new co-crystals showed significant improvement in sodium lauryl sulfate -sodium acetate buffer solution (PH=4.5) at 15min and 20min. This study enriched the types of EZT co-crystals and identified that pharmaceutical co-crystal engineering technique play an important role in the dissolution rate enhancement of ezetimibe.

Theoretical Model for the Structural Relaxation Time in Coamorphous Drugs.

We propose a simple approach to investigate the structural relaxation time and glass transition of amorphous drugs. Amorphous materials are modeled as a set of equal sized hard spheres. The structural relaxation time over many decades in hard-sphere fluids is theoretically calculated using the elastically collective nonlinear Langevin equation theory associated with Kramer's theory. Then, new thermal mapping from a real material to an effective hard-sphere fluid provides temperature-dependent relaxation time, which can be compared to experiments. Numerical results quantitatively agree with previous experiments for pharmaceutical binary mixtures having different weight ratios. We carry out experiments to test our calculations for an ezetimibe-simvastatin-Kollidon VA64 mixture. Our approach would provide a simple but comprehensive description of glassy dynamics in amorphous composites.

Synthesis of magnetic nanoparticle-based molecularly imprinted polymer as a selective sorbent for efficient extraction of ezetimibe from biological samples.

In the present study, a novel and efficient adsorbent constructed of molecularly imprinted polymer on the surface of modified magnetic nanoparticles with oleic acid (MNPs) was applied for the selective extraction of ezetimibe. The magnetic molecularly imprinted polymer (MMIP) was polymerized at the surface of modified MNPs using methacrylic acid as functional monomer, ezetimibe as template and ethylene glycol dimethacrylate as cross-linker. The resulting MMIP showed high adsorption capacity, good selectivity and fast kinetic binding for the template molecule. It was characterized by Fourier transform infrared analysis, scanning electron microscopy and transmission electron microscopy methods. The maximum adsorption capacity of MMIP was obtained as 137.1 mg g-1 and it took about 20 min to achieve the equilibrium state. The adsorption model of the adsorbent was fitted with the Freundlich and Langmuir isotherm equations. The assay exhibited a linear range of 0.003-20.000 mg L-1 for ezetimibe with a correlation coefficient of 0.995. The relative standard deviations for the recoveries were <5.2. The method was also examined for the analysis of ezetimibe in the biological samples.

Combined spectroscopic and quantum chemical approach to study the effect of hydrogen bonding interactions in ezetimibe.

Molecular structure, chemical and physical reactivity, spectroscopic behavior, intermolecular interactions play an important role in understanding the biological nature of pharmaceutical drugs. The objective of the study is to combine the spectroscopic and computational methodology for the investigation of structural behavior of ezetimibe (EZT). Computational study was done on monomeric, dimeric and trimeric models of EZT using B3LYP/6-311G(d,p). Hydrogen bond interactions were taken into consideration to validate the theoretical results with the experimental one. Results obtained for trimeric model were better than monomer and dimer. HOMO-LUMO energy band gap shows that the chemical reactivity calculated using dimeric and trimeric model is higher than that of monomeric model. Higher value of electrophilicity index (ω = 2.5654 eV) also confirms that trimer behaves as a strong electrophile in comparison with monomer and dimer. To examine the hyperconjugation interactions and the stability of the molecule, natural bond analysis (NBO) was done on dimer and trimer of EZT. Nature and the strength of hydrogen bonds were examined by quantum theory of atoms in molecules (QTAIM). Binding energy calculated from counterpoise method was -7.40 kcal/mol for dimer and -21.47 kcal/mol for trimer.

Development of a Simple, Highly Selective RP-LC Method for the Quantification of Diastereomers and Other Related Substances of Ezetimibe Using Multivariate Analysis.

A simple reverse phase method for the selective quantification of ezetimibe (EZM), its diastereomers and other related substances was developed. The method demonstrated an excellent separation between each of the 14 impurities (including diastereomers, specified impurities and degradation products) and EZM within a runtime of 45 min. The developed method was evaluated against the reported USP method, other literature methods found that none of them was able to separate/show the absence of all the diastereomers and degradation products. The critical method parameters were optimized using central composite design. Forced degradation studies proved the method to be highly specific and the structure of all the major degradation products were confirmed by LC-MS study. The results of validation proved the method to be precise (% RSD < 4), accurate (recoveries in range of 100 ± 6%), linear (R2 > 0.999) and sensitive (LOQ ≤ 0.04% and LOD ≤ 0.01%) for all the impurities and drug. The method is suitable for both drug substance and oral solid dosage form.

Development of a joint population pharmacokinetic model of ezetimibe and its conjugated metabolite.

Ezetimibe (EZE) is an extensively used antihyperlipidemic drug with an important cholesterol lowering activity. It undergoes extensive first-pass metabolism to form its active glucuronide metabolite (EZEG). Both drugs exhibit complex pharmacokinetic profiles attributed mainly to repetitive enterohepatic kinetics. The aim of the present study was the investigation of EZE and EZEG pharmacokinetics (PK), through the development of a joint population pharmacokinetic model able to characterize their kinetic processes and enterohepatic recirculation simultaneously. Concentration-time data derived from a bioequivalence study in 28 healthy subjects were used for the analysis. Population PK modeling was performed on the obtained data using nonlinear mixed effect modeling approach, where different methodologies were applied for the description of the complex metabolism and recirculation processes of the two compounds. EZE and EZEG concentrations were best described by a population PK model incorporating first-pass metabolism and an enterohepatic recirculation loop, accounting for the recycling process of the two moieties. This is the first joint population pharmacokinetic model describing the kinetics of both EZE and EZEG.

Simultaneous quantitation of rosuvastatin and ezetimibe in human plasma by LC-MS/MS: Pharmacokinetic study of fixed-dose formulation and separate tablets.

A simple, high-throughput and highly sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous estimation of rosuvastatin and free ezetimibe. Liquid-liquid extraction was carried out using methyl-tert butyl ether after prior acidification from 300 µL human plasma. The recovery for both the analytes and their deuterated internal standards (ISs) ranged from 95.7 to 99.8%. Rosuvastatin and ezetimibe were separated on Symmetry C18 column using acetonitrile and ammonium formate buffer, pH 3.5 (30:70, v/v) as the mobile phase. The analytes were well resolved with a resolution factor of 3.8. Detection and quantitation were performed under multiple reaction monitoring using ESI(+) for rosuvastatin (m/z 482.0 → 258.1) and ESI(-) for ezetimibe (m/z 407.9 → 271.1). A linear response function was established in the concentration ranges of 0.05-50.0 ng/mL and 0.01-10.0 ng/mL for rosuvastatin and ezetimibe, respectively, with correlation coefficient, r2 ≥ 0.9991. The IS-normalized matrix factors for the analytes ranged from 0.963 to 1.023. The developed method was successfully used to compare the pharmacokinetics of a fixed-dose combination tablet of rosuvastatin-ezetimibe and co-administered rosuvastatin and ezetimibe as separate tablets to 24 healthy subjects. The reliability of the assay was also assessed by reanalysis of 115 subject samples.

Development of an LC-MS Method for 4-Fluoroaniline Determination in Ezetimibe.

A rapid and sensitive high-performance liquid chromatography-mass spectrometry method was developed and validated to determine 4-fluoroaniline concentration in ezetimibe. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 µm) maintained at 30°C. The liquid chromatography system was operated in gradient mode with an injection volume of 20 µL at a flow rate of 1 mL/min. Mobile phase A was water and mobile phase B consisted of acetonitrile with 0.05% acetic acid. The detection was performed using a single quadrupole mass spectrometer in single ion monitoring mode by using positive ionization. An m/z value of 112 was selected for monitoring 4-fluoroaniline. The method showed good linearity over the concentration range of 0.94-30.26 ng/mL. The limit of quantification and limit of detection were 0.19 and 0.94 ng/mL, respectively. The precision relative standard deviations were less than 8.7% (n = 12), and the accuracy values were within 92-99%. A standard solution of 4-fluoroaniline was stable for at least 24 h at 25°C. Small changes in the organic phase acidity of the mobile phase, flow rate, column temperature, and the instrument parameters had no significant effect on the results for 4-fluoroaniline.

Investigation of supersaturation and in vitro permeation of the poorly water soluble drug ezetimibe.

The interplay between supersaturation, precipitation and permeation characteristics of the poorly water-soluble drug ezetimibe (EZ) was investigated. Supersaturation and precipitation characteristics of EZ in the presence of Caco-2 cells were compared to those in a cell-free environment. The effect of the water-soluble polymer polyvinyl pyrrolidone (PVP-K30) on the supersaturation, precipitation and transport of EZ was also investigated and the amount of drug taken up by Caco-2 cells was quantified. A one-compartment setup without Caco-2 cells (i.e. in the wells of cell-culture plates) was used to mimic a non-sink in vitro dissolution chamber. The two-compartment Caco-2 cell monolayer setup (with apical and basolateral compartments) was used to investigate how the absorption of EZ affects supersaturation. EZ in varying degrees of supersaturation (DS; 10, 20, 30 and 40) was introduced into the one-compartment setup or the apical chamber of the two-compartment setup. Samples were collected at specific times to determine supersaturation, precipitation and permeation. At the end of the study, Caco-2 cells were lysed and the intracellular amount of EZ was quantified. In the one-compartment setup, a high DS was associated with rapid precipitation. Supersaturation was maintained for longer time periods and precipitation was lower in the presence of Caco-2 cells. There were no significant differences in the absorption rate of the drug, even at high concentrations on the apical side. Permeability coefficients for all supersaturated solutions (i.e. DS 10-40) were significantly (p < 0.05) different from those when EZ was present in crystalline form. Both concentrations of PVP-K30 (i.e. 0.05% and 0.1% w/v) improved solubility and supersaturation of EZ when added to the apical side, however, the increase in absorption at the higher concentration was not proportional. The amount of intracellular EZ increased with increasing DS in the apical side, until the saturation limit was reached in the cells (i.e. at DS 30 and higher). This study demonstrated that precipitation of EZ could be overestimated when supersaturation was investigated without the implementation of an absorption compartment in vitro, both in the absence and in the presence of polymer.

Chemometric-Assisted Spectrophotometric Method for the Simultaneous Quantitative Determination of Ezetimibe and Simvastatin in Their Combined Dosage Forms.

The multivariate method, partial least-squares (PLS), was used as a calibration procedure for the simultaneous UV spectrophotometric determination of ezetimibe and simvastatin in their pharmaceutical forms. The method was developed and satisfactorily validated according to International Conference on Harmonization guidelines with respect to specificity, linearity, precision, accuracy, and robustness. In this study, the PLS algorithms are based on the absorption spectra of 25 different mixtures of drugs obtained by a multilevel factorial design. The method was linear in the concentration range of 2-8 µg/mL for ezetimibe and 4-16 µg/mL for simvastatin (r2 > 0.99; n = 7) at wavelengths of 238 and 247 nm, respectively. The LOD and LOQ were 0.28 and 0.93 µg/mL for ezetimibe and 0.16 and 0.53 µg/mL for simvastatin, respectively. Precision and accuracy data, evaluated by RSD, were lower than 2%. The method, which proved to be robust, was performed with a 2n full-factorial design. The validated method is simple and low cost, has a low use of polluting reagents, and is environmental friendly. Therefore, the proposed method was successfully applied for the simultaneous quantitative analysis of ezetimibe and simvastatin in commercial formulations.