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At least 757 records · Page 42Linked to original sources
PubMed2001

Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active beta-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freeze-drying in phosphate buffers, even if there are large-scale changes in solution pH during freezing.

Buffers↗
PubMed2001

The effects of Tween 20 and sucrose on the stability of anti-L-selectin during lyophilization and reconstitution.

We have chosen an anti-L-selectin antibody as a model protein to investigate the effects of sucrose and/or Tween 20 on protein stability during lyophilization and reconstitution. Native anti-L-selectin secondary structure is substantially retained during lyophilization in the presence of sucrose (1 or 0.125%). However, aggregation of the protein during reconstitution of lyophilized protein powders prepared without sucrose is not reduced by the presence of sucrose in the reconstitution medium. Aggregate formation upon reconstitution is completely inhibited by freeze drying the protein with sucrose and reconstituting with a 0.1% Tween 20 solution. Tween 20 (0.1%) also partially inhibits loss of native anti-L-selectin secondary structure during lyophilization. However, upon reconstitution the formulations lyophilized with Tween 20 contain the highest levels of aggregates. The presence of Tween in only the reconstitution solution appears to inhibit the transition from dimers to higher order oligomers. Potential mechanism(s) for the Tween 20 effects were investigated. However, no evidence of thermodynamic stabilization of anti-L-selectin conformation (e.g., by Tween 20 binding) could be detected.

Antibodies, Monoclonal↗
PubMed2004

Retrospective statistical analysis of lyophilized protein formulations of progenipoietin using PLS: determination of the critical parameters for long-term storage stability.

Although certain criteria have become recognized as being essential for a stable lyophilized formulation, the relative importance of different stability criteria has not been demonstrated quantitatively. This study uses multivariate statistical methods to determine the relative importance of certain formulation variables that affect long-term storage stability of a therapeutic protein. Using the projection to latent structures (PLS) method, a retrospective analysis was conducted of 18 formulations of progenipoietin (ProGP), a potential protein therapeutic agent. The relative importance of composition, pH, maintenance of protein structure (as determined by infrared (IR) spectroscopy), and thermochemical properties of the glassy state (as measured by differential scanning calorimetry (DSC)) were evaluated. Various stability endpoints were assessed and validated models constructed for each using the PLS method. Retention of parent protein and the appearance of degradation products could be adequately modeled using PLS. The models demonstrate the importance of retention of native structure in the solid state and controlling the pH. The relative importance of T(g) in affecting storage stability was low, as all of the samples had T(g) values above the highest storage temperature (40 degrees C). However, other indicators of molecular mobility in the solid state, such as change in DeltaC(p) upon annealing, appear to be important, even for storage below T(g). For the first time, the relative importance of certain properties in controlling long-term storage stability could be assessed quantitatively. In general, the most important parameters appear to be pH and retention of native structure in the solid state. However, for some stability endpoints, the composition (concentration of protein or various excipients), as well as some DSC parameters, were found to be significant in predicting long-term stability.

Calorimetry, Differential Scanning↗
PubMed2005

Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation.

The objectives of this work were to determine the effects of nonionic surfactants (Tween 20 and Tween 80) on agitation-induced aggregation of the recombinant fusion protein, Albutropintrade mark (human growth hormone genetically fused to human albumin), and to characterize the binding interactions between the surfactants and the protein. Knowing the binding stoichiometry would allow a rational choice of surfactant concentration to protect the protein from surface-induced aggregation. Fluorescence spectroscopy and isothermal titration calorimetry (ITC) were employed to study Albutropin surfactant binding. Albutropin was agitated at 25 +/- 2 degrees C to induce aggregation, and samples were taken during a 96-h incubation. Size-exclusion chromatography (SEC-HPLC) (HPLC, high-performance liquid chromatography) was used to detect and quantify the extent of protein aggregation. The effect of surfactants on the protein's free energy of unfolding was determined using guanidine HCl as a denaturant. Tween 20 and Tween 80 had saturable binding to Albutropin with a molar binding stoichiometry of 10:1 and 9:1 (surfactant:protein), respectively. Binding of the surfactants to Albutropin increased the free energy of unfolding by over 1 and 0.6 kcal/mol, respectively. In protein samples that were agitated in the absence of surfactant, soluble aggregates were detected within 24 h, and there was almost complete loss of monomer to soluble aggregates by the end of the 96-h experiment. At the molar binding stoichiometry, Tween 20 and Tween 80 prevented the formation of soluble aggregates, even though the concentrations of surfactants were well below their critical micelle concentrations (CMC). Tween 20 and Tween 80 protected Albutropin against agitation-induced aggregation, even at concentrations below the CMC. Equilibrium unfolding data indicate that Tween confer protection by increasing the free energy of unfolding of Albutropin.

Drug Stability↗
PubMed2006

Effect of different surfactants in biorelevant medium on the secretion of a lipophilic compound in lipoproteins using Caco-2 cell culture.

The impact of a pharmaceutical relevant metabolizable, ionic surfactant or two synthetic, nonionic surfactants on the absorption and lipoprotein incorporation of a lipophilic drug, retinol, was studied in the Caco-2 cell culture. Filter-grown monolayers of Caco-2 cells were incubated for 20 h with (3)H-retinol and (14)C-oleic acid and with increasing concentrations of lyso-phosphatidylcholine (lyso-PC), Cremophor RH40, or Tween 80. The concentration of (3)H-retinol and (14)C-lipid was measured in the apical, intracellular, and basolateral compartments. The basolateral medium was ultracentrifugated into different lipoprotein classes and their (3)H-retinol and (14)C-lipid concentrations were determined. The cells incubated with lyso-PC and Tween 80 increased the incorporation of (3)H-retinol and (14)C-lipid into chylomicrons and very low density lipoproteins (VLDL). The explored surfactants impacted the incorporation of (3)H-retinol and (14)C-lipid in chylomicrons and VLDL in a concentration-dependent manner. As these surfactants interfere with the intestinal lipoprotein secretion, inclusion of high concentrations of the surfactants in lipid-based formulations of poorly aqueous soluble drugs might impact the degree of intestinal lymphatic transport of the drug after oral administration.

Caco-2 Cells↗
PubMed2006

Solubilization of two structurally related anticancer drugs: XK-469 and PPA.

The efficiency of a solubilization technique is determined by the physical-chemical properties of the drug. This study investigates the solubilization on two structurally related anticancer drugs, XK-469 and PPA. XK-469 is much less polar than PPA with an intrinsic solubility of 0.000274 mg/mL, which is about 10,000 fold less than that of PPA. Fortunately, its physical-chemical properties make it much more formulatable. An ionizable drug can be solubilized by pH adjustment with cosolvency, micellization, or complexation. Both XK-469 and PPA are weak acids with pKa values of 2.7 and 2.9, respectively. Thus, they can be solubilized by pH adjustment. At pH 4.55, neither cosolvency, micellization nor complexation has much effect on the solubility of PPA. However, these techniques can significantly increase the solubility of XK-469. In fact, the solubility of XK-469 in 20% HPbetaCD at pH 4.55 is 5.85 mg/mL, which is more than 20,000 times greater than its intrinsic solubility. With the solubilization descriptors obtained from the experimental data for both unionized and ionized drug species at pH 1.0 and pH 4.55, the solubility of each drug at any pH and excipient concentration can be estimated. Then, a solubilization technique can be chosen for preparing a desired final drug concentration.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed2006

A novel peroxy radical based oxidative stressing system for ranking the oxidizability of drug substances.

A novel oxidative stressing system is described which generates high levels of peroxy radicals in solution at room temperature, without the use of azonitrile initiators. The oxidative stressing system is composed of a 10% solution of Tween 80 in water to which FeCl3 x 6H2O is added. The Tween 80 acts as a solubilizing agent for drug compounds, and also contains substantial amounts of organic hydroperoxides. It is shown that the Fe III/ Fe II couple operates on the hydroperoxide concentration to effectively generate new peroxy radicals, which then propagate in the Tween 80 solution. Key features of the Tween 80/Fe III system are investigated, and the oxidizability of seven known compounds and ten developmental compounds are examined. Relative reaction rates span a 300-fold range, from benzoic acid (nonreactive, defined as <0.5% reacted per day) to Vitamin D3 (7% reacted per hour). Oxidizability "rankings" thus generated are shown to agree well with azonitrile initiated oxidative stress. The potential for general correlations between this type of oxidizability data and actual oxidative performance in LFC and solid oral dosage forms is discussed.

Chlorides↗
PubMed2007

Diffusion of nutrients molecules and model drug carriers through mucin layer investigated by magnetic resonance imaging with chemical shift resolution.

Magnetic resonance imaging (MRI) with chemical shift resolution is a recent extension of MRI and it provides information about species resolved molecular transport on the macroscopic scale in complex systems. In this contribution, we show that by using this novel method, one can predict the behavior of drug and food molecules when they are in contact with the mucosal layer in the gastrointestinal tract. For the first time, the transport properties of a mixture of nutrients (i.e., a solution of ethanol and glucose) and of a model drug carrier (i.e., an equimolar solution of cationic and nonionic surfactants) through a mucin gel have been investigated. This study shows that transport properties of the diffusing molecules through a mucin gel are dependent on their size and physicochemical properties. In addition, we show that mucin gel acts as an efficient selective barrier. It favors the disintegration of mixed micelles of nonionic and cationic surfactants by stopping the diffusion of cationic surfactants with slightly affecting the diffusion of the nonionic surfactants.

Deuterium Oxide↗
PubMed2007

Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer.

This article describes the design, performance testing, and application of a controlled humidity mini-freeze-dryer in studying the physical stability of lactate dehydrogenase during lyophilization. Performance evaluation of the mini-freeze-dryer was conducted with tests, namely water sublimation, radiation heat exchange, lowest achievable temperature, and leak testing. Protein stability studies were conducted by comparing protein activity at various stages of lyophilization with the initial activity. The shelf and condenser temperature were stable at <-40 degrees C, wall temperature was within 2 degrees C of the shelf temperature, and the leak rate was small. The chamber pressure was controlled by the ice on the condenser and the product temperature during sublimation was equal to the shelf temperature. Addition of Tween 80 prevented activity loss in solution and after freeze-thaw. No activity loss was observed after primary-drying even in absence of lyoprotectants and with collapse of cake structure. Five percent (w/w) sucrose concentration was required to achieve full stabilization. In conclusion, performance testing established that the mini-freeze-dryer was suitable for mechanistic freeze-drying studies. Secondary-drying was the critical step for protein stability. The concentration of sucrose required to stabilize the protein completely was several orders of magnitude higher than that required to satisfy the direct interaction requirement of the protein.

Animals↗
PubMed1976

Enhancement of dissolution rates of poorly water-soluble drugs by crystallization in aqueous surfactant solutions I: Sulfathiazole, prednisone, and chloramphenicol.

Effects of crystallization of poorly water-soluble drugs in aqueous surfactant solution on in vitro dissolution rates were investigated. Marked enhancement was observed for chloramphenicol, sulfathiazole, and prednisone. Differential thermal analysis studies indicated the presence of small amounts of surfactant in surfactant-treated crystals. Possible mechanisms of dissolution enhancement are discussed.

Chemistry, Pharmaceutical↗
PubMed1995

Effect of surfactants on the physical stability of recombinant human growth hormone.

The physical stability of a human growth hormone (hGH) formulation upon exposure to air/water interfaces (with vortex mixing) and to nonisothermal stress [determined by differential scanning calorimetry (DSC)] was investigated. The effect of these stresses on the formation of soluble and insoluble aggregates was studied. The aggregates were characterized and quantified by size exclusion-HPLC and UV spectrophotometry. Vortex mixing of hGH solutions (0.5 mg/mL) in phosphate buffer, pH 7.4, for just 1 min caused 67% of the drug to precipitate as insoluble aggregates. These aggregates were noncovalent in nature. Non-ionic surfactants prevented the interfacially induced aggregation at their critical micelle concentration (cmc) for Pluronic F-68 (polyoxyethylene polyoxypropylene block polymer) and Brij 35 (polyoxyethylene alkyl ether) and above the cmc for Tween 80 (polyoxyethylene sorbitan monooleate). However, the same surfactants failed to stabilize hGH against thermal stress in DSC studies. Higher concentrations of surfactants actually destabilized hGH as evidenced by the decrease in the onset temperature for the denaturation endotherm.

Chromatography, High Pressure Liquid↗
PubMed2004

Toxicity of the entomopathogenic fungus, Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) to adult females of the blowfly Lucilia sericata (Diptera: Calliphoridae).

The fungus Metarhizium anisopliae (Metchnikoff) (Deuteromycotina: Hyphomycetes) is a natural component of soil flora world-wide and is a causal agent of the green muscardine diseases of insects. The use of this pathogen as a potential biocontrol agent against adult females of the blowfly, Lucilia sericata (Meigen) (Diptera: Calliphoridae) was considered. The blowfly L sericata is an economically important agent of cutaneous myiasis of sheep, particularly in northern Europe. Exposure of flies to suspensions of fungal spores by immersion, topical application or tarsal contact with treated surfaces all resulted in high levels of fatal infection. Spores suspended in silicone oil resulted in higher levels of infection (50-70%) than those formulated in a 0.3 glitre(-1) solution of the detergent Tween 80 (10-20%). Spore concentration had a significant effect on levels of infection of flies, with the highest levels of mortality (64%) resulting from a suspension of 1 x 10(7) conidia ml(-1). The duration of tarsal contact had a significant effect on levels of infection. Mean infection levels of 30% were observed following exposure of free-flying adults to a single spore-treated, 5 x 15 cm2 cloth surface suspended from the roof of a cage (30 cm cube). The implications of the results for the potential use of M anisopliae in the biocontrol of blowflies are discussed.

Animals↗
PubMed2006

In vitro anthelmintic activity of Melia azedarach naturalized in Argentina.

The anthelmintic activity of the drupe extracts of Melia azedarach L. (Meliaceae) growing in Argentina was tested against tapeworms, hookworms, nodular worms and earthworms, and was shown to be better than the standards piperazine phosphate and hexylresorcinol against tapeworms and hookworms, respectively.

Ancylostomatoidea↗
PubMed1977

Non-confirmation of thalidomide induced teratogenesis in rats and mice.

It has been reported that thalidomide, dissolved in a 1:3 mixture of Tween 20 and physiological saline and administered intraperitoneally to pregnant mice and rats induces a variety of malformations, including limb deformaties, characteristic of the primate syndrome. The studies reported herein attempted to confirm these findings without success although the rodent strains used were not the same. A low level of non-specific malformations was observed in the fetuses of both species at dose levels reported to cause a 47 percent and 92 percent rate of malformation in mice and rats respectively. One possible source of difference was Tween 20 which was toxic to the point of lethality in these studies at dose levels reported to be non-toxic in the earlier studies.

Abnormalities, Drug-Induced↗
PubMed1993

The effect of nonionic detergents on the activity and/or stability of rat brain nitric oxide synthase.

The results of this study indicate that the addition of low concentrations of a nonionic detergent such as those represented by the Tween, Brij, or Triton classes causes an apparent activation of nitric oxide synthase. It is possible that this apparent activation is due to the ability of these detergents to stabilize the protein. The stabilizing influence of the detergents may be a result of inhibiting the dissociation of the dimeric protein into monomers or the dissociation of an essential cofactor or prosthetic group from the active enzyme. Regardless of the mechanism of action, the addition of low concentrations of nonionic detergents results in longer and increased nitric oxide synthase activity and may be an important tool for those involved in enzymological studies of nitric oxide synthase.

Amino Acid Oxidoreductases↗
PubMed1998

Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid.

Freeze-drying is often used to improve storage stability of therapeutic proteins. In order to obtain a product with optimal storage stability it is important to understand the mechanisms by which solutes protect the protein against freeze-drying-induced stresses and also against damage induced during subsequent storage. The objective of the current study was to examine the importance of various mechanisms proposed to account for acute and long-term storage stability using recombinant human Factor XIII (rFXIII)4 as a model protein. Initially, for acute stability during freeze-drying, it was found that solutes which formed an amorphous phase stabilized rFXIII to a greater degree than solutes which crystallized during freeze-drying. However, only amorphous solutes which were able to hydrogen bond to the protein, and thus preserve the native protein structure in the dried solid, provided optimal acute stability. Thus, in addition to forming an amorphous phase, it was also important to possess the ability to hydrogen bond to the protein. Long-term storage stability was found to be optimal in the presence of solutes which formed and maintained amorphous phases with Tg values above the storage temperature and which also preserved the native protein structure during freeze-drying. Solute crystallization during storage compromised storage stability.

Dextrans↗