Thomas Österberg

Theoretical Calculation and Prediction of Caco-2 Cell Permeability Using MolSurf Parametrization and PLS Statistics

AbstractPurpose. To statistically model the permeability across Caco-2 cell monolayers using theoretically computed molecular descriptors and multivariate statistics. Methods. Seventeen structurally diverse compounds were investigated. The program MolSurf was used to compute theoretical molecular descriptors related to physico-chemical properties such as lipophilicity, polarity, polarizability and hydrogen bonding. The multivariate Partial Least Squares Projections to Latent Structures (PLS) method was used to delineate the relationship between the permeability across Caco-2 cell monolayers and the theoretically computed molecular descriptors. Results. Excellent statistical models were derived. Properties associated with hydrogen bonding had the largest impact on diffusion through the monolayers and should be kept at a minimum to promote high permeability. High lipophilicity and the presence of surface electrons, i.e. valence electrons, which are not tightly bonded to the molecule, were also found to have a favorable influence to achieve high permeability. Conclusions. The results indicate that theoretically computed molecular MolSurf descriptors in conjunction with multivariate statistics of PLS type can be used to successfully model permeability across Caco-2 cell monolayers and, thus, differentiate drugs with poor permeability from those with acceptable permeability at an early stage of the pre-clinical drug discovery process.

Immobilized liposome and biomembrane partitioning chromatography of drugs for prediction of drug transport

Drug partitioning into lipid bilayers was studied by chromatography on liposomes and biomembranes immobilized in gel beads by freeze–thawing. The drug retention volume was expressed as a capacity f ...

Theoretical calculation and prediction of intestinal absorption of drugs in humans using MolSurf parametrization and PLS statistics

A method for modeling and prediction of the intestinal absorption of drugs in humans using theoretically computed molecular descriptors and multivariate statistics has been investigated using 20 diverse drug-like compounds. The program MolSurf was used to compute theoretical molecular descriptors related to physicochemical properties such as lipophilicity, polarity, polarizability and hydrogen bonding. The multivariate Partial Least Squares Projections to Latent Structures (PLS) method was used to delineate the relationship between the intestinal absorption of drugs in humans and the theoretically computed molecular descriptors.Good statistical models were derived. Properties associated with hydrogen bonding had the largest impact on absorption and should be kept to a minimum to promote high absorption. High charge-transfer properties and the presence of surface electrons, i.e. valence electrons, which are not tightly bonded to the molecule, were also found to promote high absorption.

Development of a Freeze-Dried Albumin-Free Formulation of Recombinant Factor VIII SQ

AbstractPurpose. To develop a stable freeze-dried formulation of recombinant factor VIII-SQ (r-VIII SQ) without the addition of albumin. Methods. Different formulations were evaluated for their protective effect during sterile filtration, freeze-thawing, freeze-drying, reconstitution and long term storage. Factor VIII activity (VIII:C), visual inspection, clarity, solubility, moisture content and soluble aggregates and/or fragments were assayed. Results. A combination of non-crystallising excipients (L-histidine and sucrose), a non-ionic surfactant (polysorbate 80) and a crystalline bulking agent (sodium chloride) was found to preserve the factor VIII activity during formulation, freeze-drying and storage. Calcium chloride was included to prevent dissociation of the heavy and light chains of r-VIII SQ. Sodium chloride was chosen as the primary bulking agent since the concentration of sodium chloride necessary for dissolution of r-VIII SQ in the buffer will inhibit the crystallization of many potential cake formers. It was found that L-histidine, besides functioning as a buffer, also protected r-VIII SQ during freeze-drying and storage. A pH close to 7 was found to be optimal. Some potential macromolecular stabilisers, PEG 4000, Haes®-steril and Haemaccel®, were evaluated but they did not improve the recovery of VII:C. The freeze-dried formulation was stable for at least two years at 7°C and for at least one year at 25°C. The reconstituted solution was stable for at least 100 hours at 25°C. Conclusions. The albumin-free formulation resulted in consistently high recovery of VII:C, very low aggregate formation and good storage stability. The stability of the reconstituted solution makes the formulation suitable for continuous administration via infusion pump. The formulation strategy described here may also be useful for other proteins which require a high ionic strength.

Recombinant factor VIII SQ—influence of oxygen, metal ions, pH and ionic strength on its stability in aqueous solution

Recombinant factor VIII SQ (r-VIII SQ) is a derivative of human factor VIII in which the B-domain has been deleted. It corresponds to the smallest active form, a metal ion-linked 80+90 kDa heterodimer, present in therapeutic factor VIII concentrates. The stability of r-VIII SQ was investigated in aqueous solution, without albumin (human) as a stabiliser. Activity assay (VIII:C), visual inspection and gel filtration were performed after storage at different temperatures. The influence of oxygen, metal ions, pH and ionic strength was studied. The thermal stability was investigated using differential scanning calorimetry (DSC). There was a rapid loss of activity when r-VIII SQ was stored in solution in vials containing air in the headspace. The stability was markedly improved by reducing the oxygen content. pH 6.5–7.0 was optimal for stability at both low and high ionic strengths. The best results were obtained at high ionic strengths, since r-VIII SQ precipitated at sodium chloride concentrations below 5 mg/ml. The loss of VIII:C correlated with dissociation of the light (80 kDa) and heavy (90 kDa) chain complex. The separation of these chains was partly prevented by addition of calcium or strontium ions in the concentration range 1–10 mM. The transition point for heat denaturation (Tm′) was 64±0.2°C in a formulation containing 9500 IU r-VIII SQ/ml, 18 mg/ml sodium chloride, 0.5 mg/ml calcium chloride dihydrate, 3 mg/ml l-histidine and 0.2 mg/ml polysorbate 80. The stability results presented here show promise and prompt further investigations into the development of a stable solution of r-VIII SQ.

Physical state of L-histidine after freeze-drying and long-term storage

Liquid samples of L-histidine of varying pH values and mixed with salt, metal ions, polysorbate 80 and sucrose have been analysed by differential scanning calorimetry to evaluate the influence of these additives on the glass transition temperature and crystallisation of L-histidine during freezing and thawing. L-Histidine solutions of varying pH were freeze-dried with and without a thermal cycle and the physical state of the freeze-dried cakes, following long-term storage, were studied by powder X-ray diffraction. Amorphous L-histidine crystallised when it was exposed to moisture, and the identity of the crystalline materials is reported. The crystallisation of L-histidine during freezing and thawing is dependent on the pH of the solution and is shown to be at a minimum at pH 6, which coincides with the pK(a) of the imidazoline function. Sucrose inhibited the crystallisation of L-histidine during thawing, while sodium chloride or polysorbate 80 did not. The addition of metal ions (Ca2+ and Mg2+) up to 10% (w/w) did not depress the glass transition temperature significantly, while the addition of Zn2+ increased it. The physical state of L-histidine after freeze-drying is shown to be dependent on both the pH of the solution and the freezing cycle. The risk of crystallisation of amorphous L-histidine is low if the freeze-dried material is protected from moisture.

Recombinant factor VIII SQ--inactivation kinetics in aqueous solution and the influence of disaccharides and sugar alcohols.

AbstractPurpose. To investigate the influence of various nonreducing disaccharides and sugar alcohols on the inactivation kinetics of recombinant factor VIII SQ (r-VIII SQ) in aqueous solution not containing albumin as a stabiliser. Methods. The stability of r-VIII SQ was followed using measurement of activity (VIII: C) and HPLC gel filtration at different temperatures. The thermal stability was investigated using differential scanning calorimetry (DSC). Results. The decline in VIII:C followed pseudo-first order kinetics. However, the Arrhenius plot was not linear for formulations without carbohydrate, demonstrating a distinct, reproducible curvature. The reaction rate at 5°C was faster than expected from the Arrhenius kinetics. The energy of activation (Ea) for formulations without added carbohydrates, derived from the linear part of the Arrhenius plot, varied between 77 and 86 kJ/mole in the temperature range 20−37°C. The addition of 600 mg/ml sucrose increased the Ea to 104 kJ/mole. DSC measurements showed thatTm′ was 64.2 ± 0.2°C for r-VIII SQ without stabiliser. This value increased linearly with increasing concentrations of carbohydrate. This stabilising effect is most probably explained by the theory of preferential hydration. Conclusions. The inactivation kinetics of r-VIII SQ in aqueous solution without addition of carbohydrates followed pseudo-first order kinetics but the Arrhenius plot was nonlinear. Sucrose and sorbitol both had highly stabilising effects on r-VIII SQ at concentrations above 300 mg/ml. The preparation containing 600 mg/ml sucrose was stable for at least 12 months at 5°C and 6 months at 25°C.