Design of Lipid-Based Formulations for Oral Administration of Poorly Water-Soluble Drug Fenofibrate: Effects of Digestion

Lipid-based drug carriers are likely to have influence on bioavailability through enhanced solubilization of the drug in the gastrointestinal tract. The study was designed to investigate the lipid formulation digestibility in the simulated gastro intestinal media. Fenofibrate was formulated in representative Type II, IIIA, IIIB and IV self-emulsifying/microemulsifying lipid delivery systems (SEDDS and SMEDDS designed for oral administration) using various medium-chain glyceride components, non-ionic surfactants and cosolvents as excipients. Soybean oil was used only as an example of long-chain triglycerides to compare the effects of formulation with their counterparts. The formulations were subjected to in vitro digestion specifically to predict the fate of the drug in the gastro intestinal tract after exposure of the formulation to pancreatic enzymes and bile. In vitro digestion experiments were carried out using a pH-stat maintained at pH 7.5 for 30 min using intestinal fluids simulating the fed and fasted states. The digestion rate was faster and almost completed in Type II and IIIA systems. Most of the surfactants used in the studies are digestible. However, the high concentration of surfactant and/or cosolvent used in Type IIIB or IV systems lowered the rate of digestion. The digestion of medium-chain triglycerides was faster than long-chain triglycerides, but kept comparatively less drug in the post digestion products. Medium-chain mixed glycerides are good solvents for fenofibrate as rapidly digested but to improve fenofibrate concentration in post digestion products the use of long-chain mixed glycerides are suggested for further investigations.KEY WORDS: fenofibrate, in vitro lipolysis, lipid formulation classification system, self-emulsifying/microemulsifying drug delivery systems (SEDDS, SMEDDS)     

High Efficiency Lipid-Based siRNA Transfection of Adipocytes in Suspension

Background

Fully differentiated adipocytes are considered to be refractory to introduction of siRNA via lipid-based transfection. However, large scale siRNA-based loss-of-function screening of adipocytes using either electroporation or virally-mediated transfection approaches can be prohibitively complex and expensive.

Methodology/Principal Findings

We present a method for introducing small interfering RNA (siRNA) into differentiated 3T3-L1 adipocytes and primary human adipocytes using an approach based on forming the siRNA/cell complex with the adipocytes in suspension rather than as an adherent monolayer, a variation of “reverse transfection”.

Conclusions/Significance

Transfection of adipocytes with siRNA by this method is economical, highly efficient, has a simple workflow, and allows standardization of the ratio of siRNA/cell number, making this approach well-suited for high-throughput screening of fully differentiated adipocytes.     

Phase Equilibria in binary mixtures of dimyristoylphosphatidylcholine and cardiolipin

Paramagnetic resonance spectra of the spin-label 2,2,6,6-tetramethylpiperidinyl-l-oxy have been used to study phase separations in binary mixtures of dimyristoyl-phosphatidylcholine and cardiolipin. Two different samples of cardiolipin were used: (i) One sample contained calcium ions at a mole ratio of calcium:cardiolipin = 1:2; the experimental data support the view that cardiolipin is present in the bilayer membrane as calcium ion linked dimers, (CL)2 Ca2+. (ii) A calcium-free sodium cardiolipin sample yielded remarkable spin-label partition data that were quite different from those obtained in the presence of Ca2+. In both cases the spin-label data provide evidence for compound formation and for fluid-fluid immiscibility in the bilayer membrane.     

A Modified Gibbs Ensemble Method for Calculating Fluid Phase Equilibria

Abstract

A modification of the Gibbs ensemble Monte Carlo computer simulation method for fluid phase equilibrium is described. The modification, which is based on the assumption of a thermodynamic model for the vapor phase, reduces the computational time for the simulation as compared to the original Gibbs ensemble methods. Since the computational time is largely proportional to the number of particle-particle interactions, avoiding the direct simulation of the vapor phase typically leads to a thirty to forty percent reduction in computational time. For a pure Leonard-Jones-(12,6) fluid the results obtained at moderate reduced temperatures, T/T_c < 0.8, are in good agreement with the full Gibbs ensemble method.     

Formulation,Pharmacokinetic, and Efficacy Studies of Mannosylated Self-Emulsifying Solid Dispersions of Noscapine

Purpose

To formulate hydroxypropyl methylcellulose-stabilized self-emulsifying solid dispersible carriers of noscapine to enhance oral bioavailability.

Methods

Formulation of noscapine (Nos) self-emulsifying solid dispersible microparticles (SESDs) was afforded by emulsification using an optimized formula of Labrafil M1944, Tween-80, and Labrasol followed by spray-drying with hydroxypropyl methylcellulose (HPMC), with and without mannosamine (Mann-Nos_SESDs and Nos_SESDs respectively); self-microemulsifying liquid dispersions (SMEDDs) with and without mannosamine (Mann-Nos_SMEDDs and Nos_SMEDDs respectively) were also prepared. SMEDDs and SESDs were characterized for size, polydispersity, surface charge, entrapment efficiency, in vitro permeability, in vitro release kinetics, and oral pharmacokinetics in Sprague-Dawley rats (10 mg/kg p.o). The antitumor efficacy of Mann-Nos_SESDs on the basis of chemosensitization to cisplatin (2.0 mg/kg, IV) was investigated in a chemorefractory lung tumor Nu/Nu mouse model up to a maximal oral dose of 300 mg/kg.

Results

The oil/surfactant/co-surfactant mixture of Labrafil M1944, Tween-80, and Labrasol optimized at weight ratios of 62.8:9.30:27.90% produced stable self-microemulsifying dispersions (SMEDDs) at a SMEDD to water ratio of 1–3:7–9 parts by weight. SMEDDs had hydrodynamic diameters between 231 and 246 nm; surface charges ranged from -16.50 to -18.7 mV; and entrapment efficiencies were between 32 and 35%. SESDs ranged in size between 5.84 and 6.60 μm with surface charges from -10.62 to -12.40 mV and entrapment efficiencies of 30.96±4.66 and 32.05±3.72% (Nos_SESDs and Mann-Nos_SESDs respectively). Mann-Nos_SESDs exhibited saturating uptake across Caco-2 monolayers (P_app = 4.94±0.18 × 10⁻⁶ cm/s), with controlled release of 50% of Nos in 6 hr at pH 6.8 following Higuchi kinetics. Mann-Nos_ SESDs was 40% more bioavailable compared to Nos_SESDs; and was effective in sensitizing H1650 SP cells to Cisplatin in vitro and in an orthotopic lung tumor model of H1650 SP origin.

Conclusions

Mannosylated noscapine self-emulsifying solid dispersions (Mann-Nos_SESDs) are bioavailable and potentiate the antineoplastic effect of cisplatin-based chemotherapy in cisplatin-resistant NSCLC.     

Cell-Specific Targeting of Lipid-Based Carriers for ODN and DNA

It is well recognized that there is an urgent need for non-toxic systemically applicable vectors for biologically active nucleotides to fully exploit the current potential of molecular medicine in gene therapy. Cell-specific targeting of non-viral lipid-based carriers for ODN and DNA is a prerequisite to attain the concentration of nucleic acids required for therapeutic efficacy in the target tissue. In this review we will address the most promising approaches to selective targeting of liposomal nucleic acid carriers in vivo. In addition, the routes of entry and intracellular processing of these carrier systems are discussed as well as physiological factors potentially interfering with the biological and/or therapeutic activity of their nucleotide pay-load.     

Vapour-Liquid Phase Equilibria of n-alkanes by Direct Monte Carlo Simulations

Abstract

We report results of direct Monte Carlo simulations of n-pentane and n-decane at the liquidvapour interface for a number of temperatures. The intermolecular interactions are modeled using the last version of the anisotropic united atom model (AUA4). We have used the local long range correction energy and an algorithm allowing to select randomly with equal probability two different displacements. The liquid and vapour densities are in excellent agreement with experimental data and with those previously calculated using the GEMC method.     

Phase Equilibria of Quadrupolar Fluids by Simulation in the Gibbs Ensemble

Abstract

Vapour-liquid phase diagrams for pure fluids and mixtures of molecules with Lennard-Jones plus quadrupole-quadrupole interaction potentials were determined by Monte Carlo simulation in the Gibbs ensemble [1]. This is the first reported application of the method to molecular fluids. We have demonstrated that the Gibbs method works reliably for strongly interacting molecular fluids at liquid densities. Pure fluid calculations were performed for reduced quadrupole strengths, Q* = Q/(εσ⁵)^1/2 equal to 1 and √2, typical of molecules like C₂H₂ and C₂H₄. It was found that the critical temperature of the quadrupolar fluid increased rapidly with increasing quadrupolar strength, in good agreement with previous computer simulation and theoretical results. A single mixture with components characterized by identical Lennard-Jones parameters and Q*₁ = + 1, Q*₂ = - 1 was studied at three temperatures. A negative azeotrope was observed at the lowest temperature studied, as seen experimentally in the CO₂/C₂H₂ mixture. The perturbation theory calculations are in good agreement with the simulation results for all properties except coexisting liquid densities. The results illustrate some of the strengths and limitations of perturbation theories based on the Padé approximant for the free energy of polar fluids.     

An Alternative Approach for the Assessment of Bioequivalence Between Two Formulations of a Drug

The problem of the assessment of bioequivalence between a test formulation (T) and a reference formulation (R) of a drug using a two-way crossover experiment is considered. To claim bioequivalence between two formulations, it is required by the United States Food and Drug Administration (FDA) to demonstrate that the true ratio of means μT/μR of pharmacokinetic parameters of concern falls within some reasonable limits (e.g., (80%, 120%)) with certain assurance. A commonly used approach is to construct an approximate 90% confidence interval for μT/μR and compare it with (80%, 120%). In this paper, an exact approach according to the FDA's criteria is proposed. The proposed procedure is derived by constructing an exact confidence region (an ellipse) for (μR, μT) and comparing it with the region bounded by μT = 0.8 μR and μT = 1.2 μR. Bioequivalence is concluded if the ellipse is within the critical region.     

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO₂ system was studied at 25 and 40 °C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO₂ pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)^1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.     

Efficiency of Original versus Generic Intravenous Iron Formulations in Patients on Haemodialysis

Aims

The appropriate use of intravenous (IV) iron is essential to minimise the requirements for erythropoiesis-stimulating agents (ESAs). The clinical efficacy of generic IV iron compared to the original formulation is controversial. We evaluated the changes that were induced after switching from a generic IV iron to an original formulation in a stable, prevalent haemodialysis (HD) population.

Methods

A total of 342 patients were included, and the follow-up period was 56 weeks for each formulation. Anaemia parameters and doses of ESA and IV iron were prospectively recorded before and after the switch from generic to original IV iron.

Results

To maintain the same haemoglobin (Hb) levels after switching from the generic to the original formulation, the requirements for IV iron doses were reduced by 34.3% (from 52.8±33.9 to 34.7±31.8mg/week, p<0.001), and the ESA doses were also decreased by 12.5% (from 30.6±23.6 to 27±21μg/week, p<0.001). The erythropoietin resistance index declined from 8.4±7.7 to 7.4±6.7 IU/kg/week/g/dl after the switch from the generic to the original drug (p = 0.001). After the switch, the transferrin saturation ratio (TSAT) and serum ferritin levels rose by 6.8%(p<0.001) and 12.4%(p = 0.001), respectively. The mortality rate was similar for both periods.

Conclusions

The iron and ESA requirements are lower with the original IV iron compared to the generic drug. In addition, the uses of the original formulation results in higher ferritin and TSAT levels despite the lower dose of IV iron. Further studies are necessary to analyse the adverse effects of higher IV iron dosages.     

Efficiency of a Two-Stage Procedure for Survival Studies

A two-stage procedure using occurrences of a surrogate endpoint regarding a true clinical endpoint has been proposed (FLANDRE, O'QUIGLEY and BROOKMEYER, 1994). The procedure allows estimation of the survival function S(t) reliably and comparison of treatment groups. The aim of this paper is to investigate the efficiency of such a two-stage procedure compared to a one stage procedure, i.e. a classical study. The efficiency is estimated by the ratio of the standard derivation of S(t) for the classical procedure to the standard derivation of S(t) for the two-stage procedure and represents the precision on the estimation of S(t). Standard deviation of S(t) is approximate by the delta-method. Using the exponential model. some numerical results are presented.     

Nano-extrusion: a One-Step Process for Manufacturing of Solid Nanoparticle Formulations Directly from the Liquid Phase

This paper presents a novel one-step process for converting a liquid stabilized nano-suspension into a solid formulation via hot-melt extrusion combined with an internal devolatilization process (nano-extrusion, NANEX). A polymer (Soluplus®) was fed into the extruder and molten, after which a stable nano-suspension was added via side-feeding devices. The solvent (water) was removed by devolatilization and the polymer solidified at the outlet. The solid material can be tableted or filled in a capsule directly. The results showed that the obtained extrudates comprised nanocrystals in the de-aggregated form, confirming that a solid nano-formulation was prepared. This method is capable of overcoming many of the problems associated with other processes involving solid nano-dosage forms and poses a straightforward approach towards manufacturing such products.     

Improving the Flexibility and Efficiency of Phase II Designs for Oncology Trials

Summary Phase II trials in oncology are usually conducted as single-arm two-stage designs with binary endpoints. Currently available adaptive design methods are tailored to comparative studies with continuous test statistics. Direct transfer of these methods to discrete test statistics results in conservative procedures and, therefore, in a loss in power. We propose a method based on the conditional error function principle that directly accounts for the discreteness of the outcome. It is shown how application of the method can be used to construct new phase II designs that are more efficient as compared to currently applied designs and that allow flexible mid-course design modifications. The proposed method is illustrated with a variety of frequently used phase II designs.     

Development of Lipid-Based Nanoparticles for Enhancing the Oral Bioavailability of Paclitaxel

The current research work investigates the potential of solid lipid nanoparticles (SLNs) in improving the oral bioavailability of paclitaxel. Paclitaxel-loaded SLNs (PTX-SLNs) were prepared by modified solvent injection method using stearylamine as lipid, soya lecithin and poloxamer 188 as emulsifiers. SLNs were characterized in terms of surface morphology, size and size distribution, surface chemistry and encapsulation efficiency. Pharmacokinetics and bioavailability studies were conducted in male Swiss albino mice after oral administration of PTX-SLNs. SLNs exhibited spherical shape with smooth surface as analyzed by transmission electron microscopy (TEM). The mean particle size of SLNs was 96 ± 4.4 nm with a low polydispersity index of 0.162 ± 0.04 and zeta potential of 39.1 ± 0.8 mV. The drug entrapment efficiency was found to be 75.42 ± 1.5% with a loading capacity of 31.5 ± 2.1% (w/w). Paclitaxel showed a slow and sustained in vitro release profile and followed Higuchi kinetic equations. After oral administration of the PTX-SLNs, drug exposure in plasma and tissues was ten- and twofold higher, respectively, when compared with free paclitaxel solution. PTX-SLNs produced a high mean C _max (10,274 ng/ml) compared with that of free paclitaxel solution (3,087 ng/ml). The absorbed drug was found to be distributed in liver, lungs, kidneys, spleen, and brain. The results suggested that PTX-SLNs dispersed in an aqueous environment are promising novel formulations that enhanced the oral bioavailability of hydrophobic drugs, like paclitaxel and were quite safe for oral delivery of paclitaxel as observed by in vivo toxicity studies.