RESUMEN
The protection of lactate dehydrogenase (LDH) by low concentrations of the non-surface-active polyethylene glycol (PEG 6000) or the non-ionic surfactant PEG dodecyl ether (Brij 35) was investigated during freeze-thawing. The freeze-thawing process was performed with a controlled temperature history, and the protective mechanisms were elaborated. The systems were examined by differential scanning calorimetry (DSC), fluorescence spectroscopy and surface tension measurements. LDH activity assays were performed spectrophotometrically. Very low concentrations of PEG 6000 (8 x 10(-5) mM) or Brij 35 (4 x 10(-3) mM) protected LDH during freeze-thawing with a low cooling rate. With an increased freezing rate, higher concentrations of the additives were needed for full protection. No interaction was detected between LDH and Brij molecules. The strong interaction between LDH and PEG molecules disappeared with a small change in the protein structure, using a hybrid of LDH. The protein was nevertheless completely protected. The amount of Brij required for complete protection at high cooling rates correlated with the created ice surface area. The protection by PEG indicated a certain correlation with the ice crystal size and with the formation of a PEG hydrate. Brij or PEG hydrate molecules might compete with the protein for adsorption at the ice surface and thereby protect the protein during freeze-thawing.
Asunto(s)
L-Lactato Deshidrogenasa , Polietilenglicoles/farmacología , Tecnología Farmacéutica/métodos , Animales , Cristalización , Congelación , Calefacción , Polidocanol , ConejosRESUMEN
PURPOSE: The weak aqueous interaction between the protein lactate dehydrogenase (LDH) and the nonionic surfactant Tween 80 has been investigated, because weak protein-amphiphile interactions are of significant importance in pharmaceutical formulations, but are experimentally hard to determine. The system LDH/sodium dodecyl sulphate (SDS) was used as reference because SDS, by its strong protein binding, denatures LDH completely. METHODS: Fluorescence spectroscopy with pyrene and 1,3-bis(lphenyl)propane (P3P) as probes, intrinsic protein fluorescence and NMR spectroscopy have been used. RESULTS: The fluorescence probe pyrene monitors a weak Tween-LDH interaction, detectable below the critical micelle concentration of ordinary Tween micelles. The microviscosity probe P3P shows a surfactant-induced denaturation in the case of LDH/SDS but not in the case of LDH/Tween 80. Intrinsic LDH fluorescence verifies this behavior. Pulsed-gradient spin-echo NMR was also used to verify the weak LDH-Tween 80 interaction. CONCLUSIONS. A weak interaction between LDH and Tween 80 occurs at hydrophobic zones of the protein, but it is not strong enough to denature LDH. The experimental outline used here provides a useful approach for mapping the very weak protein-amphiphile interactions often present in pharmaceutical formulations.
Asunto(s)
L-Lactato Deshidrogenasa/química , Polisorbatos/química , Agua/química , Animales , Rastreo Diferencial de Calorimetría/métodos , Interacciones Farmacológicas , L-Lactato Deshidrogenasa/farmacocinética , Soluciones Farmacéuticas/química , Soluciones Farmacéuticas/farmacocinética , Polisorbatos/farmacocinética , Pirenos/farmacocinética , Conejos , Espectrometría de Fluorescencia/métodos , ViscosidadRESUMEN
The purpose of the study was to investigate the protective mechanism of a non-ionic surfactant, Tween 80, at freeze-thawing with controlled temperature history of a model protein, lactate dehydrogenase (LDH). The system was examined by differential scanning calorimetry (DSC) and infrared spectroscopy (IR). LDH activity assays were performed spectrophotometrically. In all samples, independent of temperature history and addition of surfactant, all water was crystallized to polycrystalline ice at temperatures below -20 degrees C. The size and perfection of the ice crystals could be varied by a range of cooling rates giving different degrees of undercooling. At Tween concentrations below the cmc at crystallization, lower concentrations were required at low cooling rates compared to higher cooling rates to protect LDH. Concentrations above cmc of Tween reduced the protection at a cooling rate of 5 degrees C min(-1) and at quenching in N(2)(l). The amount of Tween needed for complete protection correlated to the surface area of the ice crystals at a certain temperature history. Tween 80 protects LDH from denaturation at freeze-thawing by hindering its destructive interaction with the ice crystals. The protective effect might be obtained when Tween molecules compete with the protein for sites on the ice surface. The optimum concentration of Tween needed for complete protection is dependent on the temperature history.
Asunto(s)
Excipientes/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Polisorbatos/metabolismo , Frío/efectos adversos , Excipientes/química , Congelación , Polisorbatos/química , Proteínas/metabolismoRESUMEN
Various solid dispersions of alpha-, beta- and gamma-cyclodextrin (CD) in PEG 6000 with and without the addition of 5% w/w indomethacin were prepared by the melting method using the original components. The samples were investigated by solid-state (13)C NMR, and the interactions between the drug and the cyclodextrins were evaluated. The indomethacin-gamma-CD phase with tetragonal symmetry found in a previous X-ray study gave chemical shifts which suggested that this phase is a complex between indomethacin and gamma-CD. Evidence of an indomethacin-beta-CD complex were found. A distribution of the chemical shifts for beta-CD was attributed to the possible formation of different types of complexes between indomethacin and beta-CD. No complex formation was found in the alpha-CD system. The degree of relative crystallinity of the samples in the gamma-CD system was measured by (1)H NMR, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), and modulated-temperature DSC (MTDSC). The results obtained by the NMR, XRD, and DSC techniques showed that the dispersions were less crystalline than the pure polymer carrier, and the dispersion containing the indomethacin-gamma-CD complex had the lowest degree of crystallinity. By the MTDSC method a deviation was found for the PEG 6000/indomethacin dispersion. This emphasizes that the different techniques give specific information on the crystallinity.