Leena Christiansen

A novel method of producing a microcrystalline β-sitosterol suspension in oil

Abstract This paper describes a novel method of producing a microcrystalline oral suspension containing β-sitosterol in oil for the treatment of hypercholesterolaemia. β-Sitosterol pseudopolymorphs with different water contents were crystallized from acetone and acetone–water solutions. Structural analyses of the crystals were performed by Karl-Fisher titration, thermogravimetric analyses, X-ray diffraction and near infrared spectroscopy. The suspensions studied were composed of different concentrations of β-sitosterol, oil and water. Suspensions were prepared by crystallization of hot concentrated solution of β-sitosterol in oil by cooling with simultaneous agitation and water addition. The structural analyses of the suspensions were performed by X-ray diffraction, near infrared spectroscopy and optical microscopy. The viscosity of the suspensions was analysed as a function of shear stress. β-Sitosterol was observed to exist in three different forms: anhydrous, hemihydrated and monohydrated crystals. By changing both the β-sitosterol and the water concentration of the suspension, the crystal size and shape could be controlled. Addition of water resulted in the formation of monohydrated needle-shaped crystals instead of platy-like anhydrous crystals. Needle-shaped particles formed structured suspensions with shear thinning behaviour. By increasing the volume fraction of solid particles in suspension by increasing the water and/or sterol concentration, the viscosity increased. A high sterol concentration resulted in high supersaturation and thus formation of small crystals.

Determination of amorphous content in the pharmaceutical process environment.

The amorphous state has different chemical and physical properties compared with a crystalline one. Amorphous regions in an otherwise crystalline material can affect the bioavailability and the processability. On the other hand, crystalline material can function as nuclei and decrease the stability of an amorphous system. The aim of this study was to determine amorphous content in a pharmaceutical process environment using near infrared (NIR) and Raman spectroscopic techniques together with multivariate modelling tools. Milling was used as a model system for process‐induced amorphization of a crystalline starting material, α‐lactose monohydrate. In addition, the crystallization of amorphous material was studied by storing amorphous material, either amorphous lactose or trehalose, at high relative humidity conditions. The results show that both of the spectroscopic techniques combined with multivariate methods could be applied for quantitation. Preprocessing, as well as the sampling area, was found to affect the performance of the models. Standard normal variate (SNV) transformation was the best preprocessing approach and increasing the sampling area was found to improve the models. The root mean square error of prediction (RMSEP) for quantitation of amorphous lactose using NIR spectroscopy was 2.7%, when a measuring setup with a larger sampling area was used. When the sampling area was smaller, the RMSEPs for lactose and trehalose were 4.3% and 4.2%, respectively. For Raman spectroscopy, the RMSEPs were 2.3% and 2.5% for lactose and trehalose, respectively. However, for the optimal performance of a multivariate model, all the physical forms present, as well as the process environment itself, have to be taken into consideration.

Effect of β-sitosterol on precipitation of cholesterol from non-aqueous and aqueous solutions

The aim of the present work was to study the solubility and phase behaviour of the beta-sitosterol-cholesterol mixed crystals in the presence and absence of water. Cholesterol, beta-sitosterol and 3:1, 1:1 and 1:3 mixtures of these were co-precipitated from acetone and acetone-water solutions. Precipitated crystals were analysed using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), optical microscopy and Karl-Fischer titrimetry. The quantification of the sterols in solutions was preformed using GC-MS. The solubility of the sterols was mutually limiting. In the aqueous system, the solubility of both the sterols were significantly lower than in the absence of water, but the decrease in the solubility was considerably greater with the more hydrophobic beta-sitosterol. In the aqueous system, the total sterol solubility decreased with the increasing proportion of beta-sitosterol. The formation of new crystal structures, solid solutions of cholesterol and beta-sitosterol, was observed in non-aqueous as well as in aqueous environments except with the lowest cholesterol proportion in the system, in which case mixed crystals with eutectic behaviour were formed.

Influence of solvents on the variety of crystalline forms of erythromycin

The influence of the organic solvents widely used in the pharmaceutical industry (acetone, methylethylketone, ethanol, and isopropanol) both in the presence and in the absence of water on the crystallization behavior of erythromycin (Em), a clinically relevant antibiotic of the macrolide group, was investigated. It was observed that despite a high preference for water as a guest molecule, Em rather easily forms solvates with the organic solvents studied. Consequently, 4 distinct solvates of Em have been isolated by recrystallization: acetonate, methylethylketonate, ethanolate, and isopropanolate. It was established that in a pure organic solvent, or 1∶9 or 1∶1 water-organic solvent mixtures, the corresponding solvate is always crystallized. However, the recrystallization of erythromycin from 2∶1 water-organic solvent (excluding methylethylketone) mixture results in the formation of a crystal hydrate form. X-ray powder diffraction revealed the isostructurality of the solvates with acetone and methylethylketone. Thermogravimetric analysis showed that the loss of volatiles by all of the solvated crystals is nonstoichiometric. The desolvation behavior of the solvates with the organic solvents studied by means of variable-temperature x-ray powder diffraction indicates that in contrast to erythromycin dihydrate, they belong to a different class of solvates—those that produce an amorphous material upon desolvation.

Optimizing the crystal size and habit of β-sitosterol in suspension

The aim of this work was to survey how processing parameters affect the crystal growth of β-sitosterol in suspension. The process variables studied were the cooling temperature, stirring time and stirring rate during recrystallization. In addition, we investigated the effect a commonly used surfactant, polysorbate 80, has on crystal size distribution and the polymorphic form. This study describes the optimization of the crystallization process, with the object of preparing crystals as small as possible. Particle size distribution and habit were analyzed using optical microscopy, and the crystal structure was analyzed using X-ray diffractometry. The cooling temperature had a remarkable influence on the crystal size. Crystals with a median crystal length of ≈23 μm were achieved with a low cooling temperature (<10°C); however, a fairly large number of crystals over 50 μm appeared. Higher cooling temperatures (>30°C) caused notable crystal growth both in length and width. Rapid (250 rpm), continuous stirring until the suspensions had cooled to room temperature created small, less than 50 μm long (median <20 μm), needle-shaped crystals. The addition of surfactant slightly reduced the size of the initially large crystals. Both hemihydrate and monohydrate crystal forms occurred throughout, regardless of the processing parameters. By using an optimized process, it was possible to obtain a microcrystalline suspension, with a smooth texture.

A comparison of the effect of medium- vs. long-chain triglycerides on the in vitro solubilization of cholesterol and/or phytosterol into mixed micelles.

Despite clinical evidence of the cholesterol-lowering effects of phytosterols, the exact mechanisms involved are still unclear. Displacement of cholesterol by phytosterols from mixed micelles, which is due to their greater hydrophobicity, is one of the hypotheses for the lumenal effects contributing to the reduction of intestinal cholesterol absorption. In this study a dynamic in vitro lipolysis method was used to examine the solubilization behavior of cholesterol and/or phytosterols during lipolysis to probe the efficacy of cholesterol displacement from mixed micelles by phytosterols. The effects of lipid chain length on sterol solubilization were studied by using microcrystalline suspensions containing 17% phytosterol or cholesterol, formulated in long-chain TG (LCT) and medium-chain TG (MCT). When digesting cholesterol suspended in LCT, the entire cholesterol dose was incorporated into the micellar phase. For the cholesterol formulation suspended in MCT, 50.3% of the initial dose was recovered in the micelles. Under the respective conditions, we observed lower solubilization of phytosterols than of cholesterol (roughly fourfold). Only 25% of the initial phytosterol dose was solubilized from suspensions formulated with LCT, and 13% was solubilized from MCT formulations. Co-administration of phytosterol and cholesterol suspensions showed a significant reduction of cholesterol solubilization, particularly when dosed in MCT, with ≈25% of the cholesterol dose solubilized. Insignificant amounts of cholesterol were displaced by phytosterols when cholesterol was presolubilized in the mixed micelles. The results show that, compared with LCT, mixed micelles containing MCT lipolysis products have a reduced solubilizing capacity for cholesterol, which adds to the effectiveness of the phytosterols in displacing cholesterol. This suggests potential benefits of using medium chain length lipids in cholesterol-lowering phytosterol products.

Physical changes of β-sitosterol crystals in oily suspensions during heating

The aim of this research was to describe the thermal behavior of β-sitosterol crystals in oil-suspensions with a focus on the role of water during heating. The suspensions were prepared by recrystallization in order to achieve a microcrystalline particle size. The structural changes together with the mechanical properties of the suspensions during heating were studied by using variable temperature X-ray powder diffractometry (VT-XRPD), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). Hydrated β-sitosterol crystals in an oil-suspension, dehydrated, despite the composition of the suspensions, at low temperatures. At high β-sitosterol concentration, the monohydrate crystal form changed partially to a hemihydrated form, and when only a small amount of water was initially incorporated, the hemihydrate crystal form dehydrated to a mostly anhydrate crystal form. The released water, which was immiscible in the surrounding oil, caused the recrystallization of hydrated β-sitosterol during cooling. This procedure indicated a reversible dehydration process. Structural and thermal analysis of β-sitosterol crystals in suspensions, together with mechanical analysis made it possible to understand various physical changes during heating.

Effects of microcrystalline plant sterol suspension and a powdered plant sterol supplement on hypercholesterolemia in genetically obese Zucker rats

Because dietary fat appears to be an effective vehicle for dispensing plant sterols into the diet, a special plant‐sterol‐containing ingredient has recently been developed. This ingredient is a plant sterol suspension in oil in which the sterols are in microcrystalline form. The objective of the present study was to analyse the cholesterol‐lowering effects and safety of two different plant sterol preparations, an orally administered microcrystalline plant sterol suspension (MPS) in rapeseed oil and a powdered plant sterol supplement, in obese Zucker rats. Dietary plant sterol supplements (0.5%, w/w) were given concurrently with a high cholesterol diet (HCD, 1% cholesterol and 18% fat, w/w). No significant changes in serum triglyceride, blood glucose, serum glutamate oxaloacetic transaminase and glutamic pyruvic transaminase values or body and liver weights were observed. The powdered plant sterol supplement lowered the serum cholesterol by 25% (P< 0.05) and the MPS diet by 35% (P< 0.001) compared with HCD by the end of the 12‐week experiment. Interestingly, the plant sterol supplements also produced a marked reduction in serum ubiquinone levels, suggesting a possible effect on isoprene synthesis. Unlike the powdered plant sterol, both MPS and plain rape‐seed oil decreased the serum baseline diene conjugation values, suggesting that they protect against oxidative stress‐induced lipid peroxidation in rats. This lipid peroxidation diminishing effect is probably due to some antioxidative components in rapeseed oil. These findings indicate that an unesterified plant sterol, such as the microcrystalline suspension in oil, effectively prevents cholesterol absorption in obese Zucker rats.