Unraveling protein stabilization mechanisms: vitrification and water replacement in a glass transition temperature controlled system.

The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominantly determines stability at higher glass transition temperatures.

Evaluation and optimization of a force field for crystalline forms of mannitol and sorbitol.

Two force fields, the GROMOS53A5/53A6 (united atom) and the AMBER95 (all atom) parameter sets, coupled with partial atomic charges derived from quantum mechanical calculations were evaluated for their ability to reproduce the known crystalline forms of the polyols mannitol and sorbitol. The force fields were evaluated using molecular dynamics simulations at 10 K (which is akin to potential energy minimization) with the simulation cell lengths and angles free to evolve. Both force fields performed relatively poorly, not being able to simultaneously reproduce all of the crystal structures within a 5% deviation level. The parameter sets were then systematically optimized using sensitivity analysis, and a revised AMBER95 set was found to reproduce the crystal structures with less than 5% deviation from experiment. The stability of the various crystalline forms for each of the parameter sets (original and revised) was then assessed in extended MD simulations at 298 K and 1 bar covering 1 ns simulation time. The AMBER95 parameter sets (original and revised) were found to be effective in reproducing the crystal structures in these more stringent tests. Remarkably, the performance of the original AMBER95 parameter set was found to be slightly better than that of the revised set in these simulations at 298 K. The results of this study suggest that, whenever feasible, one should include molecular simulations at elevated temperatures when optimizing parameters.

A novel bottom-up process to produce drug nanocrystals: controlled crystallization during freeze-drying.

To improve the dissolution behavior of lipophilic drugs, a novel bottom-up process based upon freeze drying which allows for the production of nanocrystalline particles was developed: "controlled crystallization during freeze drying". This novel process could strongly increase the dissolution behavior of fenofibrate. For example at a drug load of 30% w/w, 80% of the drug dissolved within 10 min from tablets prepared from the controlled crystallized dispersions, while from tablets prepared from the physical mixture only 50% was dissolved after 120 min. Furthermore it was found that faster freezing or using a solution with a lower water/tertiary butyl alcohol (TBA) ratio resulted in faster dissolution, indicating that the crystalline dispersions contained smaller crystals. Crystallization of the drug could occur during freezing or during drying. When crystallization occurs during freezing, faster freezing or using solutions with a lower water/TBA ratio results in the formation of more nuclei and consequently smaller crystals. When crystallization occurs during drying, faster freezing or using solutions with a higher water/TBA ratio results in the formation of smaller solvent crystals and therefore smaller interstitial spaces which contain the freeze-concentrated fraction. Since crystallization occurs in the freeze-concentrated fraction and the size of the crystals are limited to the size of the interstitial spaces, smaller crystals are formed in these situations.

Unexpected differences in dissolution behavior of tablets prepared from solid dispersions with a surfactant physically mixed or incorporated.

In a previous study, it was shown that the incorporation of poorly soluble drugs (BCS class II) in sugar glasses could largely increase the drug's dissolution rate [van Drooge, D.J., Hinrichs, W.L.J., Frijlink, H.W., 2004 b. Anomalous dissolution behaviour of tablets prepared from sugar glass-based solid dispersions. J. Control. Release 97, 441-452]. However, the application of this technology had little effect when high drug loads or fast dissolving sugars were applied due to uncontrolled crystallization of the drug in the near vicinity of the dissolving tablet. To solve this problem a surfactant, sodium lauryl sulphate (SLS), was incorporated in the sugar glass or physically mixed with it. Diazepam and fenofibrate were used as model drugs in this study. The dissolution behavior of tablets prepared from solid dispersions in which SLS was incorporated was strongly improved. Surprisingly, the dissolution rate of tablets prepared from physical mixtures of SLS and the solid dispersion was initially fast, but slowed down after about 10 min. The solid dispersions were characterized by DSC to explain this unexpected difference. These measurements revealed the existence of interaction of SLS with both the drug and the sugar in the solid dispersion when SLS was incorporated. It is hypothesized that due to this interaction, the dissolution of SLS was slowed down by which a high solubility of the drug in the near vicinity of the dissolving tablet is maintained during the whole dissolution process. Therefore, uncontrolled crystallization is effectively prevented.

Habitual dietary calcium intake and cortical bone loss in perimenopausal women: a longitudinal study.

During an 8-year follow-up study, the effect of habitual dietary calcium intake on cortical bone loss in 154 healthy perimenopausal women was examined. Dietary calcium intake, determined by the cross-check dietary history method, and cortical bone mineral content of the radius were measured annually. Habitual dietary calcium intake was calculated as the mean of the estimated daily dietary calcium intake during the follow-up period. The women were classified according to their habitual calcium intake: those with an intake below 800 mg/day (n = 28), between 800 and 1350 mg/day (n = 95), and above 1350 mg/day (n = 31). The results show a continuous significant loss of cortical bone in all groups, amounting yearly to 1.3 +/- 0.25, 1.5 +/- 0.10, and 1.9 +/- 0.23% (mean +/- SE) for the groups with a low, medium, and high habitual calcium intake, respectively (P less than 0.01). The differences among the three groups did not reach statistical significance (P = 0.11). Body mass index was found to be positively correlated with the negative changes in cortical bone mineral density (r = 0.32, P less than 0.01), even after adjustments had been made for confounding factors. It is concluded that a habitual calcium intake exceeding 800 mg/day (the current Recommended Daily Allowance for adults) is ineffective in preventing cortical bone loss during early menopause. Body mass index is of major importance for the perimenopausal bone loss.

Relation of axial bone mass to habitual calcium intake and to cortical bone loss in healthy early postmenopausal women.

A group of 60 healthy early postmenopausal women participating in an ongoing study on the effect of habitual calcium intake on the rate of cortical bone loss at the radius, were subjected to additional skeletal measurements at the lumbar spine and femoral neck. The women were between 58 and 64 years of age, and 3 to 10 years postmenopausal. No correlations were found between habitual calcium intake (range 560 to 2580 mg/day) and either bone mineral content of the radius, the lumbar spine and the femoral neck, or spine deformity index. Body mass index was found to be positively correlated with bone mass indices of the radius (decrease of BMD and BMD) and femoral neck (BMC), but not with of the lumbar spine (BMC, BMD and SDI), even after adjustments had been made for confounding factors. Although the rate of cortical bone loss at the radius correlated significantly with bone mineral content of lumbar spine and femoral neck, the error in predicting bone mass of the lumbar spine or the femoral neck from longitudinal measurements of cortical bone at the radius was high. The rate of cortical bone loss did not correlate with the spine deformity index. We conclude that in healthy women in early menopause, the bone mineral content of both the appendicular and the axial skeleton are not influenced by habitual calcium intake. A higher body mass index has a protective effect on the appendicular skeleton but appears to be less protective to the axial skeleton. Longitudinal measurements of cortical bone mass are of limited value to predict bone density of the appendicular and axial skeleton.

Contributions of ovarian failure and aging to blood pressure in normotensive perimenopausal women: a mixed longitudinal study.

Epidemiologic studies have shown that blood pressure increases more rapidly in middle-aged women than in middle-aged men. Whether or not ovarian failure contributes to this rapid rise is still not clear. In a follow-up study begun in 1979 and to continue for 10 years, the blood pressure of 193 healthy normotensive perimenopausal women, who lived in the mixed rural/industrial community of Ede, the Netherlands and who were initially aged between 49 and 56 years, was measured annually. During the course of the study, the onset of menopause of each participant could be established. Because of the mixed longitudinal design of the study, it was possible to evaluate the effects of both chronologic aging and time pre- or postmenopause on blood pressure. After the first seven years of follow-up, it was demonstrated that blood pressure did not increase in 168 women whose body weight was relatively stable. After multivariate analyses, systolic as well as diastolic pressure showed a significant negative relation (slope, 1.34 mmHg per year and 0.63 mmHg per year, respectively) with the years since menopause. On the other hand, the observed positive relation (slope, 0.81 mmHg per year) of systolic pressure with chronologic aging was not significant. No consistent association was found between diastolic pressure and chronologic aging. It is concluded that menopause cannot be regarded as a cause of hypertension; on the contrary, ovarian failure appears to have a protective effect on the increase in blood pressure as a result of chronologic aging. on the increase in blood pressure as a result of chronologic aging.

Oral calcium and blood pressure: a controlled intervention trial.

In a double-blind, placebo-controlled trial with 58 normotensive female students, the effect of oral-calcium supplementation (1500 mg Ca++/day for 6 wk) on diastolic and systolic blood pressure was studied while students were consuming a low-calcium diet (500 mg Ca++/day) by restricting the intake of dairy products. Results show that, in both the calcium- and placebo-supplemented groups, blood pressure values decreased slightly and no effect of oral-calcium supplementation on blood pressure could be demonstrated. In addition, at baseline neither systolic nor diastolic blood pressure correlated with habitual calcium intake. Diastolic but not systolic blood pressure correlated significantly with body mass index (r = 0.31, p = 0.01). It is concluded that oral-calcium supplementation for 6 wk does not influence blood pressure in young, healthy normotensive females consuming low-calcium diet.

Design and data quality of a mixed longitudinal study to elucidate the role of dietary calcium and phosphorus on bone mineralization in pre-, peri-, and postmenopausal women.

The study design and data quality control of an ongoing study (10 yr duration) in a few hundred women are presented. Good variables with respect to their longitudinal usefulness are: body weight, body height, and span-width. Reasonable variables are the bone parameters of the radius (BMC, BW, and BMC/BW). Poor variables are: dietary calcium and phosphorus intake, dietary calcium-to-phosphorus ratio, urinary calcium-to-creatinine ratio, urinary sodium-to-creatinine ratio, hematocrit, serum alkaline phosphatase activity, serum gamma-GT activity, and serum parathyroid-hormone concentration. Bad variables are: urinary phosphorus-to-creatinine ratio, urinary hydroxyproline-to-creatinine ratio, creatinine clearance, hemoglobin, MCHC, serum calcium, serum ionized calcium, serum phosphorus, serum total protein, serum albumin, and serum creatinine. In conclusion, it is possible to relate bone loss to food intake and to changes in anthropometric variables on an individual basis. However, quantification of the metabolic process is not possible.

Casein-induced hypercholesterolaemia in rabbits is calcium-dependent.

Young male rabbits were fed semi-purified diets containing either casein or soy protein, both at a normal (0.84%, w/w) and a high (1.44%, w/w) level of dietary calcium. At the normal calcium level, casein, as compared with soy protein, increased the concentration in serum of total and free cholesterol and the ratio of free cholesterol to phospholipid. Also, casein increased the intestinal phosphate absorption and decreased the faecal fat excretion. The hypercholesterolaemic response of the rabbits on the casein diet was significantly correlated with both phosphate absorption (r = +0.744) and fat excretion (r = -0.701). The increased amount of dietary calcium inhibited the casein-specific effects on both the intestinal and the serum lipid parameters. In contrast, calcium did not change these parameters in rabbits fed the soy protein diet. These results support the hypothesis that the degree of phosphorylation of casein is involved in the mechanism of the casein-induced hypercholesterolaemia by means of its effect on the enterohepatic cycle of bile acids.