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PubMed2004

Interactions of phospholipase D and cytochrome P450 protein stability.

Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

Analysis of Variance↗
PubMed2006

Control of protein expression through mRNA stability in calcium signalling.

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.

3' Untranslated Regions↗
PubMed2005

Physical stability of the amorphous state of loperamide and two fragment molecules in solid dispersions with the polymers PVP-K30 and PVP-VA64.

The purpose of the present study was to investigate the impact of intermolecular forces on the stability of the amorphous state of loperamide and two of its fragment molecules (4-dimethylamino-N,N-dimethyl-2,2-diphenyl-butyramide (F1) and 4-(4-chlorophenyl)-4-piperidinol (F2)) in solid dispersions with PVP-K30 and PVP-VA64. The stability of originally homogeneous and amorphous dispersions was investigated under different storage conditions. The chemical stability of the compounds was evaluated with HPLC. TGA-analysis was used in order to assess the amount of water in the samples, whereas MT-DSC-measurements were performed to investigate changes in the physical state of the compounds caused by the storage procedure. TGA-analysis reveals a higher uptake of water in humid conditions of the dispersions with PVP-K30 in comparison to those with PVP-VA64, hereby reflecting the more hydrophilic nature of the former polymer. This water acts as a plasticizing agent resulting in an increased mobility and decreased glass transition temperature. Since the degree of supersaturation and the molecular mobility have an influence on the stability of the amourphous state, both parameters were assessed. With respect to the degree of supersaturation of the compounds in the dispersions, the materials seem to be very much alike. Therefore it was postulated that the induction of crystallization in the F1/polymer dispersions stored at high RH (52%) is due to higher molecular mobility of this compound in the dispersions in comparison to F2. The hydrogen bonds that are being formed between F2 and the polymers reduce its mobility and secure this compound from crystallization upon storage, thus indicating the importance of specific interactions with respect to stability issues of solid dispersions. No hydrogen bonds are formed between F1 and the polymers. As a result, the stability of the amorphous state of the compound is being compromised and crystallization takes place. Loperamide, that also does not form hydrogen bonds with the polymers, is less susceptible to crystallization due to its intrinsic good glass forming properties.

Calorimetry, Differential Scanning↗
PubMed2004

Improvement of stability and absorbability of dry insulin powder for inhalation by powder-combination technique.

The effect of pulmonary absorption enhancers on the stability of active ingredients is an important factor for successful inhalation therapy as well as the effect on pharmacological activity and safety. We examined the effect of pulmonary absorption enhancers on the stability of insulin in dry powders prepared by a spray-drying technique. Although the hypoglycemic effect was greatly improved when a dry insulin powder containing citric acid (MIC SD) was administered, insulin in the MIC SD was unstable compared with the other powders examined. Bacitracin and Span 85, which are potent pulmonary absorption enhancers of insulin formulated in solutions, showed no deteriorative effect on the stability of dry insulin powder. However, they did not improve the hypoglycemic effect of insulin in dry powders. We modified the insulin dosage form with citric acid to improve the insulin stability at room temperature without loss of hypoglycemic activity. MIC Mix was formulated as a combination of insulin powder (MI') and citric acid powder (MC). MIC Mix showed hypoglycemic activity comparable to MIC SD while the insulin stability was much better than that of MIC SD at a 60 degrees C/dry condition. However, moisture lowered the insulin stability and changed the particle morphology of MIC Mix with time at a 60 degrees C/75% relative humidity condition, suggesting that a package preventing moisture absorption was necessary for the MIC Mix powder.

Administration, Inhalation↗
PubMed2004

Stability of crystallised and spray-dried lysozyme.

Moisture and temperature promote protein degradation. The stabilities of commercial, crystallised and spray-dried lysozyme, a model protein, were assessed under these stresses to explore whether a crystalline protein had better storage stability than a conventionally produced one. Samples were maintained at different relative humidities (RH) and temperatures for 20 weeks and stabilities estimated in solid and aqueous states. Differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterised solid samples. Fourier transform Raman (FT-Raman) spectroscopy analysed solid material and aqueous solutions. High sensitivity differential scanning calorimetry (HSDSC) and enzymatic assays were used to monitor solutions. DSC and HSDSC data revealed that crystals maintained thermal stability at high RH; spray drying appreciably changed melting characteristics. These results correlated with enzymatic assays that demonstrated good activity retention for crystals but less so for spray-dried material (e.g. 95 and 87% relative to fresh samples after 20 weeks at 40 degrees C/75% RH). FT-Raman analysis showed that crystallised lysozyme better-maintained protein conformational integrity compared to spray-dried samples in accelerated stability studies. Based on TGA data, spray-dried protein absorbed water on storage under humid conditions, which induced instability. Thus, crystallisation enhanced storage stability of lysozyme with negligible loss of activity.

Crystallization↗
PubMed2004

Stability study of losartan/hydrochlorothiazide tablets.

The purpose of stability testing is to investigate how the quality of a drug product changes with time under the influence of environmental factors, to establish a shelf life for the product and to recommend storage conditions. Stability study of losartan/hydrochlorothiazide tablets is presented in this paper. Losartan (angiotensin II receptor antagonist) and hydrochlorothiazide (diuretic) are successfully used in association in the treatment of hypertension. Stability study of losartan/hydrochlorothiazide tablets consisted of three steps: stress test (forced degradation study), preliminary testing (selection of packaging) and formal stability testing. The results of stress test suggested that losartan/hydrochlorothiazide tablets are sensitive to moisture. It was demonstrated that the developed analytical methods are stability indicating. Additional preliminary testing was performed in order to select appropriate packaging for losartan/hydrochlorothiazide tablets. OPA/Al/PVC//Al blisters were found to provide adequate protection for the product. Based on the first 12 months of the formal stability study, a shelf life of 24 months was proposed. Losartan/hydrochlorothiazide tablets in OPA/Al/PVC//Al blisters are demonstrated to be chemically, physically and microbiologically stable.

Drug Combinations↗
PubMed2006

Influence of sodium metabisulfite and glutathione on the stability of vitamin C in O/W emulsion and extemporaneous aqueous gel.

Vitamin C exerts several functions on skin as collagen synthesis, depigmentant and antioxidant activity. Vitamin C is unstable in the presence of oxygen, luminosity, humidity, high temperatures and heavy metals, which presents a significant challenge to the development of cosmetic formulations. Therefore, the utilization of an effective antioxidant system is required to maintain the vitamin C stability. The purpose of this research work was to develop prototypes of cosmetic formulations, as O/W emulsion and extemporaneous aqueous gel, containing vitamin C and to evaluate the influence of sodium metabisulfite (SMB) and glutathione (GLT), as antioxidants, on the stability of the active substance. A HPLC stability-indicating method was developed and validated for this study and stability assays were performed in 90 and 26 days and storage conditions were 5.0+/-0.5, 24+/-2 and 40.0+/-0.5 degrees C. The HPLC stability-indicating method showed linearity (r(2)>0.99), specificity, R.S.D.<1.22% and accuracy/recovery ranging from 95.46 to 101.54%. Preparations with SMB or GLT and the antioxidant-free presented results statistically distinct, demonstrating the necessity of the antioxidant system addition. O/W emulsions with SMB or GLT retained the vitamin C content >90.38% stored at 5.0+/-0.5 and 24+/-2 degrees C. For the aqueous gel with SMB or GLT, the active substance concentration was maintained >94.03%. Considering the vitamin C stability, the SMB and the GLT showed to be statistically adequate, as antioxidants, for the cosmetic formulations.

Antioxidants↗
PubMed2005

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports.

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.

Adsorption↗
PubMed2005

Formation and stability of polychlorinated biphenyl Pickering emulsions.

An emulsion stabilized by colloidal suspensions of finely divided solids is known as a Pickering emulsion. The potential for polychlorinated biphenyls (PCBs) to form Pickering emulsions ex situ when in contact with powdered solids, such as clays and metal oxides, is investigated here. Bentonite, iron oxide and magnesium oxide dispersions proved to be robust Pickering emulsion stabilizers, whereas manganese oxide dispersions were not. Batch experiments revealed that emulsions can be formed using a moderately low energy input and can be stabilized with solid concentrations as low as 0.5 wt.%. For the base conditions (volumetric oil fraction (phi(oil))=30 vol.%; solid concentration (chi)=2 wt.%), the formed emulsions were indefinitely stable and the initial average droplet diameters varied from 80 to 258 mum, depending on the solid used in the colloidal dispersion. The average droplet size varied at early time, but for most conditions stabilized to a steady-state value 1 week after preparation. The effect of Ostwald ripening was limited. At greater than 0.5 wt.% concentration, the efficiency of the solid dispersion as a stabilizer was dependant on the volumetric oil fraction but not on the solid concentration. Generally, systems with volumetric oil fractions outside of the 20-70 vol.% range were unstable. The emulsions' droplet stability, average droplet size and size distribution were observed to vary as a function of the amount of energy provided to the system, the volumetric oil fraction, and the concentration of the solid in the aqueous dispersion. It is hypothesized that drilling through fractured rock in the immediate vicinity of dense, non-aqueous phase liquid (DNAPL) PCBs may provide both the energy and solid material necessary to form Pickering emulsions.

Bentonite↗
PubMed2004

Kinetic stability and crystal structure of the viral capsid protein SHP.

SHP, the capsid-stabilizing protein of lambdoid phage 21, is highly resistant against denaturant-induced unfolding. We demonstrate that this high functional stability of SHP is due to a high kinetic stability with a half-life for unfolding of 25 days at zero denaturant, while the thermodynamic stability is not unusually high. Unfolding experiments demonstrated that the trimeric state (also observed in crystals and present on the phage capsid) of SHP is kinetically stable in solution, while the monomer intermediate unfolds very rapidly. We also determined the crystal structure of trimeric SHP at 1.5A resolution, which was compared to that of its functional homolog gpD. This explains how a tight network of H-bonds rigidifies crucial interpenetrating residues, leading to the observed extremely slow trimer dissociation or denaturation. Taken as a whole, our results provide molecular-level insights into natural strategies to achieve kinetic stability by taking advantage of protein oligomerization. Kinetic stability may be especially needed in phage capsids to allow survival in harsh environments.

Bacteriophage lambda↗
PubMed2005

Asp79 makes a large, unfavorable contribution to the stability of RNase Sa.

The two most buried carboxyl groups in ribonuclease Sa (RNase Sa) are Asp33 (99% buried; pK 2.4) and Asp79 (85% buried; pK 7.4). Above these pK values, the stability of the D33A variant is 6kcal/mol less than wild-type RNase Sa, and the stability of the D79A variant is 3.3kcal/mol greater than wild-type RNase Sa. The key structural difference between the carboxyl groups is that Asp33 forms three intramolecular hydrogen bonds, and Asp79 forms no intramolecular hydrogen bond. Here, we focus on Asp79 and describe studies of 11 Asp79 variants. Most of the variants were at least 2kcal/mol more stable than wild-type RNase Sa, and the most interesting was D79F. At pH 3, below the pK of Asp79, RNase Sa is 0.3kcal/mol more stable than the D79F variant. At pH 8.5, above the pK of Asp79, RNase Sa is 3.7kcal/mol less stable than the D79F variant. The unfavorable contribution of Asp79 to the stability appears to result from the Born self-energy of burying the charge and, more importantly, from unfavorable charge-charge interactions. To counteract the effect of the negative charge on Asp79, we prepared the Q94K variant and the crystal structure showed that the amino group of the Lys formed a hydrogen-bonded ion pair (distance, 2.71A; angle, 100 degrees ) with the carboxyl group of Asp79. The stability of the Q94K variant was about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged. Differences in hydrophobicity, steric strain, Born self-energy, and electrostatic interactions all appear to contribute to the range of stabilities observed in the variants. When it is possible, replacing buried, non-hydrogen bonded, ionizable side-chains with non-polar side-chains is an excellent means of increasing protein stability.

Aspartic Acid↗
PubMed2002

Nonisothermal stability tests of famotidine and nizatidine.

Nonisothermal stability tests have been proposed as an attractive and alternative method to the conventional isothermal stability tests. The stability and the degradation properties of famotidine and nizatidine were investigated using both isothermal and nonisothermal stability test techniques. Linear and logarithmic temperature programs were used and the degradation rate constant and activation energies were calculated using a computer program, which was written in BASIC. Also the advantages and disadvantages of these temperature programs are compared. The method to estimate parameters is based on nonlinear curve fitting the nonisothermal concentration-time-temperature curve equation. The nonisothermal stability test results were compared with the results of isothermal stability tests and similar results were obtained.

Chromatography, High Pressure Liquid↗
PubMed2002

Structural bases of stability-function tradeoffs in enzymes.

The structures of enzymes reflect two tendencies that appear opposed. On one hand, they fold into compact, stable structures; on the other hand, they bind a ligand and catalyze a reaction. To be stable, enzymes fold to maximize favorable interactions, forming a tightly packed hydrophobic core, exposing hydrophilic groups, and optimizing intramolecular hydrogen-bonding. To be functional, enzymes carve out an active site for ligand binding, exposing hydrophobic surface area, clustering like charges, and providing unfulfilled hydrogen bond donors and acceptors. Using AmpC beta-lactamase, an enzyme that is well-characterized structurally and mechanistically, the relationship between enzyme stability and function was investigated by substituting key active-site residues and measuring the changes in stability and activity. Substitutions of catalytic residues Ser64, Lys67, Tyr150, Asn152, and Lys315 decrease the activity of the enzyme by 10(3)-10(5)-fold compared to wild-type. Concomitantly, many of these substitutions increase the stability of the enzyme significantly, by up to 4.7kcal/mol. To determine the structural origins of stabilization, the crystal structures of four mutant enzymes were determined to between 1.90A and 1.50A resolution. These structures revealed several mechanisms by which stability was increased, including mimicry of the substrate by the substituted residue (S64D), relief of steric strain (S64G), relief of electrostatic strain (K67Q), and improved polar complementarity (N152H). These results suggest that the preorganization of functionality characteristic of active sites has come at a considerable cost to enzyme stability. In proteins of unknown function, the presence of such destabilized regions may indicate the presence of a binding site.

Amino Acid Substitution↗
PubMed1999

Microencapsulation of DNA using poly(DL-lactide-co-glycolide): stability issues and release characteristics.

The design of DNA vaccination delivery systems for the targeting of professional antigen presenting cells could be an interesting approach to elicit cytotoxic T-cell responses to fight viral infections and in cancer therapy. Stability studies with linear high and low molecular DNA and supercoiled plasmid DNA were performed in order to check their ability to withstand stress conditions applied during formulation processes. DNA was tested for integrity by the PicoGreen assay and transfectivity was assessed in cell culture transfection experiments. Double-stranded DNA is extremely stable under physiological conditions in vitro but is rapidly degraded under acidic conditions and high shear forces. Thereby, different stress factors resulted in distinct degradation patterns such as fragmentation and strand separation possibly followed by further decomposition of single-stranded DNA. DNA containing PLGA microparticles as a potential delivery system was prepared by spray-drying. Encapsulation efficiency, DNA stability and burst release varied significantly depending on the different parameters explored in this study. The microencapsulation process was altered to achieve maximal stability of encapsulated DNA by reducing exposure to shear forces and by the addition of NaHCO(3) which acts as a buffering agent and furthermore stabilizes dsDNA against mechanical degradation. Stability of DNA is maintained during the burst release phase, but massive degradation occurred during the second release phase possibly due to acidic catalyzed decomposition. In summary, we feel that microencapsulation of DNA vaccines by spray-drying offers manifold possibilities to design suitable delivery systems in terms of optimizing phagocytosis by APCs and maintaining stability of DNA in phagosomes.

Animals↗
PubMed2001

Stabilization of minodronic acid in aqueous solution for parenteral formulation.

The composition, concentration, and buffer pH of potential minodronic acid formulations were evaluated for their drug stability and for their tendency to generate particles after storage for up to 4 weeks at 60 degrees C. The results indicate that citrate and tartrate buffers maintain drug stability and inhibit the formation of particles. The stability of minodronic acid in these solutions increased slightly as the buffer concentration increased, exhibiting less particle formation than in other buffers. Since citrate buffer was considered the most promising stabilizer for minodronic acid, the pH-stability relationship in 100 mM citrate with pH ranging from 3 to 7 was evaluated during storage for 4 weeks at 60 degrees C. The results demonstrate that solution pH of 3-5 result in optimal stability of minodronic acid with no formation of precipitates. A white precipitate was observed in citrate-containing sample solutions with pH of 6 and 7. Analysis of the isolated precipitate provided support for the hypothesis that the precipitate is a complex between minodronic acid and aluminum ions apparently leached from the glass of the ampoules.

Buffers↗
PubMed2001

Stability of ascorbyl palmitate in topical microemulsions.

Ascorbyl palmitate and sodium ascorbyl phosphate are derivatives of ascorbic acid, which differ in stability and hydro-lipophilic properties. They are widely used in cosmetic and pharmaceutical preparations. In the present work the stability of both derivatives was studied in microemulsions for topical use as carrier systems. The microemulsions were of both o/w and w/o types and composed of the same ingredients. The stability of the less stable derivative ascorbyl palmitate was tested under different conditions to evaluate the influence of initial concentration, location in microemulsion, dissolved oxygen and storage conditions. High concentrations of ascorbyl palmitate reduced the extent of its degradation. The location of ascorbyl palmitate in the microemulsion and oxygen dissolved in the system together significantly influence the stability of the compound. Light accelerated the degradation of ascorbyl palmitate. In contrast, sodium ascorbyl phosphate was stable in both types of microemulsions. Sodium ascorbyl phosphate is shown to be convenient as an active ingredient in topical preparations. In the case of ascorbyl palmitate, long-term stability in selected microemulsions was not adequate. To formulate an optimal carrier system for this ingredient other factors influencing the stability have to be considered.

Administration, Topical↗
PubMed1999

Stabilization of aerosolized IFN-gamma by liposomes.

Aerosolized IFN-gamma is very unstable. We have improved the stability of IFN-gamma in the jet nebulizer by adding small liposomes. Aerosolized IFN-gamma was recovered in PBS solution by bubbling and its concentration was determined. After nebulization for 30 min, aerosolized IFN-gamma was detected only 0.4+/-0.2% of the initial amount in the PBS solution and 3.1+/-0.7% in the reservoir. On the other hand, the addition of small liposomes (HSPC/DSPG=10/1 (molar ratio), 45+/-24 nm) in the nebulizer increased the stability of IFN-gamma, 27.2+/-4.7% of the initial amount in the PBS solution and 25.7+/-12.6% in the reservoir. The present study also examined the effects of composition and concentration of liposomes on the stabilization of aerosolized IFN-gamma. Liposome prepared from distearoyl phosphatidylcholine (DSPC) or hydrogenated soy phosphatidylcholine (HSPC) was very effective for stabilization of aerosolized IFN-gamma (DSPC/DPPG=10/1, HSPC/DSPG=10/1). HSPC/DSPG liposome was efficient at the concentration higher than 12.5 micromols/ml for the stabilization of 5x10(5) JRU/ml of IFN-gamma. In considering the mechanism of this stabilization, the results of gel filtration chromatography suggest that IFN-gamma is inactivated by polymerization or aggregation in nebulization, while the inactivation is suppressed by liposomes due to their adsorption to IFN-gamma.

Aerosols↗
PubMed2002

Fortum stability in different disposable infusion devices by pyridine assay.

The stability of ceftazidime in 5% dextrose injection and 0.9% sodium chloride injection when stored in a different disposable infusion device was determined. Solutions of ceftazidime 40 mg/ml were used to fill the drug administration devices. Stability was determined for both 5% dextrose injection and 0.9% sodium chloride injection solutions at 37 degrees C in four disposable infusion devices. Ceftazidime and its mean degradation product, pyridine, were simultaneously assayed in triplicate by a stability-indicating high-performance liquid chromatographic (HPLC) method. This method was simple, sensitive (limit of quantitation (LOQ), 2 ng injected for both compounds), rapid (run time was 7 min) and precise (mean recovery was 100.5+/-2.9 and 103.6+/-1.9% for pyridine and ceftazidime, respectively). The ceftazidime stability in the 5% dextrose solution was lower than in the 0.9% sodium chloride solution. When stored at 37 degrees C in a disposable infusion device, the stability of the ceftazidime is included in large hourly range, depending strongly on the manufacturer. The stability of ceftazidime exceed 19 h in none studied cases. The pyridine formed in 24 h was in the range of 100-400 mg depending on devices and infusions.

Ceftazidime↗