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PubMed2002

A 5' stem-loop and ribosome binding but not translation are important for the stability of Bacillus subtilis aprE leader mRNA.

The Bacillus subtilis aprE leader is a determinant of extreme mRNA stability. The authors examined what properties of the aprE leader confer stability on an mRNA. The secondary structure of the aprE leader mRNA was analysed in vitro and in vivo, and mutations were introduced into different domains of an aprE leader-lacZ fusion. The half-lives of the corresponding transcripts were determined and beta-galactosidase activities were measured. Removal of a stem-loop structure at the 5' end or diminishing the strength of the RBS reduced the half-lives from more than 25 min to about 5 min. Interfering with translation by abolishing the start codon or creating an early stop codon had no or little effect on mRNA stability. The authors conclude that a 5' stem-loop and binding of ribosomes are necessary for the stability of aprE leader mRNA. The present results, together with a number of other data, suggest that translation of a B. subtilis mRNA is generally not important for its stability; the situation seems different in Escherichia coli. It is further concluded that the calculated strength of a B. subtilis RBS cannot be used to predict the stability of the corresponding transcript.

5' Untranslated Regions↗

PubMed2000

Charge-charge interactions influence the denatured state ensemble and contribute to protein stability.

Several recent studies have shown that it is possible to increase protein stability by improving electrostatic interactions among charged groups on the surface of the folded protein. However, the stability increases are considerably smaller than predicted by a simple Coulomb's law calculation, and in some cases, a charge reversal on the surface leads to a decrease in stability when an increase was predicted. These results suggest that favorable charge-charge interactions are important in determining the denatured state ensemble, and that the free energy of the denatured state may be decreased more than that of the native state by reversing the charge of a side chain. We suggest that when the hydrophobic and hydrogen bonding interactions that stabilize the folded state are disrupted, the unfolded polypeptide chain rearranges to compact conformations with favorable long-range electrostatic interactions. These charge-charge interactions in the denatured state will reduce the net contribution of electrostatic interactions to protein stability and will help determine the denatured state ensemble. To support this idea, we show that the denatured state ensemble of ribonuclease Sa is considerably more compact at pH 7 where favorable charge-charge interactions are possible than at pH 3, where unfavorable electrostatic repulsion among the positive charges causes an expansion of the denatured state ensemble. Further support is provided by studies of the ionic strength dependence of the stability of charge-reversal mutants of ribonuclease Sa. These results may have important implications for the mechanism of protein folding.

Enzyme Stability↗

PubMed2004

Stabilization of a freeze-dried recombinant streptokinase formulation without serum albumin.

AIM: To evaluate the stability and content homogeneity of a new freeze-dried and albumin-free formulation of recombinant streptokinase (SKr) that has recently been approved by the Cuban National Center for the Quality Control of Medicaments. MATERIALS AND METHODS: The new formulation was stored at the intended recommended storage temperature of 4 degrees C, and under accelerated storage conditions (37 degrees C). The stability of the product was also examined after reconstitution and storage at room temperature (28 degrees C) for 24 h. Samples were periodically subjected to biological activity assays (S-2251 chromogenic-substrate method or in vitro clot-lysis assay), sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE), pyrogen and sterility testing, abnormal toxicity screening, organoleptic evaluation, and measurement of residual moisture and pH. RESULTS AND DISCUSSION: Accelerated storage (37 degrees C) data showed biochemical stability of SKr throughout the 6-month study with activity, remaining between 90 and 110% of its nominal value (0.75 x 10(6) IU/mL). SDS/PAGE-determined purity showed that SKr remained above 97 %. Furthermore, the formulation was non-pyrogenic, non-toxic, sterile and organoleptically acceptable. Real-time storage data confirmed the excellent biochemical long-term (30 months) stability of the new formulation of SKr. Comparison with other freeze-dried preparations showed that the new formulation was organoleptically better. The formulation was stable after reconstitution and storage at 28 degrees C for 24 h. The content homogeneity of this new formulation was also satisfactory. CONCLUSIONS: This study demonstrated the stability and the content homogeneity of this formulation, despite the absence of albumin as stabilizer.

Chemistry, Pharmaceutical↗

PubMed2002

Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity, protein processing, and genomic RNA dimer stability.

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.

Amino Acid Sequence↗

PubMed2001

Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA.

Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike alpha-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) alpha complex on its 3' untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human beta-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3' UTR of the beta-globin mRNA that exhibits many of the properties of the stability-enhancing alpha complex. The beta-globin mRNP complex is shown to contain one or more factors homologous to alphaCP, a 39-kDa RNA-binding protein that is integral to alpha-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3' UTR as the site of beta-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human beta-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the alpha- and beta-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.

3' Untranslated Regions↗

PubMed1999

Stabilities of uncomplemented and complemented M15 beta-galactosidase (Escherichia coli) and the relationship to alpha-complementation.

M15 beta-galactosidase (Escherichia coli) is a mutant form of beta-galactosidase having residues 11-41 deleted. It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues. The activities of uncomplemented and complemented M15 beta-galactosidases decreased starting at 42 degrees C--uncomplemented over a narrow temperature range, complemented over a broad range. This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures. The effects of added components on stability and alpha-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation. Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10x less Mg2+ was needed for complemented protein). Alpha-complementation increased somewhat at low Mg2+ but decreased at high Mg2+. These effects can be explained by differential Mg2+ binding to the native and susceptible forms. The enhancement of both stability and alpha-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins. Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 beta-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 beta-galactosidase. Alpha-complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and alpha-complementation at low 2-mercaptoethanol owing to interactions with native forms. The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol. In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small. This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation.

Enzyme Stability↗

PubMed1993

Structural and genetic analysis of protein stability.

One very encouraging development has been the freedom with which amino acid replacements can be introduced in a protein of interest. This has made it possible to obtain detailed structural and thermodynamic data on a wide variety of mutants that modify protein stability. Substitutions of solvent-exposed amino acids on the surfaces of proteins are seen to have little if any effect on protein stability or structure, leading to the view that it is the rigid parts of proteins that are critical for folding and stability. There is every reason to expect that it will be possible to rationalize the stabilities of mutant proteins from accurate knowledge of their structures. Substantial progress is being made in quantitating the interactions that determine and stabilize protein structures. Although not specifically the subject of this review, substantial progress is also being made in developing methods to engineer proteins of enhanced stability.

Drug Stability↗

PubMed2005

Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface.

BACKGROUND: Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B. RESULTS: Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3-4 degrees C higher over wild type. All mutants showed 2-fold higher thermal stability at 55 degrees C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities. CONCLUSIONS: We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity.

Actinobacteria↗

PubMed2004

Dexamethasone enhances LPS induction of tissue factor expression in human monocytic cells by increasing tissue factor mRNA stability.

Glucocorticoids, such as dexamethasone (Dex), are used clinically in the treatment of various inflammatory diseases. Dex acts by inhibiting the expression of inflammatory mediators, such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1). It is surprising that Dex enhances bacterial lipopolysaccharide (LPS) induction of tissue factor (TF) expression in human monocytic cells. TF is a transmembrane glycoprotein that activates the coagulation protease cascade. In this study, we analyze the mechanism by which Dex enhances LPS-induced TF expression in human monocytic cells. We found that Dex reduced LPS-induced TF gene transcription but increased the stability of TF mRNA. Dex decreased the stability of MCP-1 mRNA and did not affect TNF-alpha mRNA stability. Finally, we showed that Dex increased the stability of a transcript consisting of the final 297 nucleotides of the TF mRNA in in vitro decay assays. This region contains AU-rich elements that regulate mRNA stability and may mediate the Dex response. Therefore, despite an inhibition of TF gene transcription, Dex enhances TF expression in human monocytic cells by increasing the stability of TF mRNA.

Base Sequence↗

PubMed2003

Rheology and stability of water-in-oil-in-water multiple emulsions containing Span 83 and Tween 80.

Multiple emulsions are often stabilized using a combination of hydrophilic and hydrophobic surfactants. The ratio of these surfactants is important in achieving stable multiple emulsions. The objective of this study was to evaluate the long-term stability of water-in-oil-in-water (W/O/W) multiple emulsions with respect to the concentrations of Span 83 and Tween 80. In addition, the effect of surfactant and electrolyte concentration on emulsion bulk rheological properties was investigated. Light microscopy, creaming volume, and rheological properties were used to assess emulsion stability. It was observed that the optimal surfactant concentrations for W/O/W emulsion long-term stability were 20% wt/vol Span 83 in the oil phase and 0.1% wt/vol Tween 80 in the continuous phase. Higher concentrations of Tween 80 had a destructive effect on W/O/W emulsion stability, which correlated with the observation that interfacial film strength at the oil/water interface decreased as the Tween 80 concentration increased. High Span 83 concentrations increased the storage modulus G' (solidlike) values and hence enhanced multiple emulsion stability. However, when 30% wt/vol Span 83 was incorporated, the viscosity of the primary W/O emulsion increased considerably and the emulsion droplets lost their shape. Salt added to the inner aqueous phase exerted an osmotic pressure that caused diffusion of water into the inner aqueous phase and increased W/O/W emulsion viscosity through an increase in the volume fraction of the primary W/O emulsion. This type of viscosity increase imposed a destabilizing effect because of the likelihood of rupture of the inner and multiple droplets.

Drug Stability↗

PubMed2000

Analysis of the stability and degradation products of triptolide.

Triptolide is the major active ingredient of the Chinese herbal remedy Tripterygium wilfordii Hook F. (TwHF). As triptolide content is used to estimate the potency of preparations of TwHF, assessment of its stability is warranted. The accelerated stability of triptolide was investigated in 5% ethanol solution in a light-protected environment at pH 6.9, within a temperature range of 60-90 degrees C. The observed degradation rate followed first-order kinetics. The degradation rate constant (K25 degrees C) obtained by trending line analysis of Arrhenius plots of triptolide was 1.4125 x 10(-4) h(-1). The times to degrade 10% (t1/10) and 50% (t1/2) at 25 degrees C were 31 and 204 days, respectively. Stability tests of triptolide in different solvents and different pH conditions (pH4-10) in a light-protected environment at room temperature demonstrated that basic medium and a hydrophilic solvent were the major factors that accelerated the degradation of triptolide. Triptolide exhibited the fastest degradation rate at pH 10 and the slowest rate at pH 6. In a solvent comparison, triptolide was found to be very stable in chloroform. The stability of triptolide in organic polar solvents tested at both 100% and 90% concentration was greater in ethanol than in methanol than in dimethylsulphoxide. Stability was also greater in a mixture of solvent:pH6 buffer (9:1) than in 100% solvent alone. An exception was ethyl acetate, which is less polar than the other solvents tested, but permitted more rapid degradation of triptolide. Two of the degradation products of triptolide were isolated and identified by HPLC and mass spectroscopy as triptriolide and triptonide. This suggested that the decomposition of triptolide occurred at the C12 and C13 epoxy group and the C14 hydroxyl. The opening of the C12 and C13 epoxy is an irreversible reaction, but the reaction occurring on the C14 hydroxyl is reversible. These results show that the major degradation pathway of triptolide involves decomposition of the C12 and C13 epoxy group. Since this reaction is very slow at 4 degrees C at pH 6, stability is enhanced under these conditions.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMedDate not supplied

Stability of methylprednisolone sodium succinate in autodose infusion system bags.

OBJECTIVE: To evaluate the physical and chemical stabilities of methylprednisolone sodium succinate solutions packaged in sterile AutoDose Infusion System bags. SETTING: Laboratory. INTERVENTIONS: The test samples were prepared by reconstituting the methylprednisolone sodium succinate, adding the required amount of drug to the AutoDose Infusion System bags, and diluting to the target concentrations of 100 mg/100 mL and 1 gram/100 mL with 0.9% Sodium Chloride Injection. MAIN OUTCOME MEASURES: Physical stability and chemical stability based on drug concentrations initially and at appropriate intervals over periods up to 3 days at 23 degrees C and 30 days at 4 degrees C. RESULTS: The admixtures initially were clear when viewed in normal fluorescent room light and with a Tyndall beam. Measured turbidity and particulate content were low initially and exhibited little change. All samples were essentially colorless throughout the study. High-performance liquid chromatography analysis revealed some decomposition in the samples. Methylprednisolone sodium succinate exhibited about 8% loss after 2 days and about 13% loss after 3 days at 23 degrees C. In the samples stored at 4 degrees C, methylprednisolone sodium succinate exhibited acceptable stability through 21 days of storage, but losses exceeded 10% after 30 days. CONCLUSION: Methylprednisolone sodium succinate exhibited physical and chemical stabilities consistent with those found in previous studies. The AutoDose Infusion System bags did not adversely affect the physical or chemical stability of this drug.

Chromatography, High Pressure Liquid↗

PubMed2001

Stability of piperacillin and ticarcillin in AutoDose infusion system bags.

OBJECTIVE: To evaluate the physical and chemical stability of piperacillin sodium 3 and 4 g/100 mL and ticarcillin disodium 3 g/100 mL each admixed in NaCl 0.9% injection packaged in AutoDose Infusion System bags. DESIGN: Triplicate test samples of the penicillin antibiotics in NaCl 0.9% injection were packaged in ethylene vinyl acetate (EVA) plastic containers, AutoDose bags, designed for use in the AutoDose Infusion System. Samples were stored protected from light and evaluated at appropriate intervals for up to seven days at 23 degrees C and up to 30 days at 4 degrees C. Physical stability was assessed using turbidimetric and particulate measurement as well as visual inspection. Chemical stability was assessed by HPLC. RESULTS: All of the penicillin admixtures initially were clear when viewed in normal fluorescent room light and with a Tyndall beam. Measured turbidity and particulate content were low initially and exhibited little change in the ticarcillin disodium samples throughout the study. The piperacillin sodium samples exhibited small increases in measured haze throughout the study, but no accompanying increase in particulates > or = 1.04 microm was found. All samples were essentially colorless throughout the study. HPLC analysis found some decomposition in the samples. Piperacillin sodium exhibited a 9-10% loss after five days at 23 degrees C. In the samples stored at 4 degrees C, piperacillin sodium exhibited acceptable stability through 21 days of storage, but losses exceeded 10% after 30 days. Ticarcillin disodium exhibited a 7% loss after three days, but 14% after five days at 23 degrees C. Less than 7% loss occurred after 21 days, but 12% loss after 30 days at 4 degrees C. CONCLUSIONS: Piperacillin sodium and ticarcillin disodium exhibited physical and chemical stability consistent with previous studies on these drugs. The AutoDose Infusion System bags were not found to adversely affect the physical and chemical stability of these penicillin antibiotics.

Chromatography, High Pressure Liquid↗

PubMed2000

Hormonal regulation of mRNA stability and RNA-protein interactions in the pituitary.

Regulating gene expression from DNA to protein is a complex multistage process with multiple control mechanisms. Transcriptional regulation has been considered the major control point of protein production in eukaryotic cells; however, there is growing evidence of pivotal posttranscriptional regulation for many genes. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, with individual mRNAs displaying a wide range of stability that has been linked to discrete sequence elements and specific RNA-protein interactions. This review will focus on current knowledge of the determinants of mRNA stability and RNA-protein interactions in the pituitary. This field is rapidly expanding with the identification of regulated cis-acting stability-modifying elements within many mRNAs, and the cloning and characterization of trans-acting proteins that specifically bind to their cognate cis elements. We will present evidence for regulation of multiple pituitary genes at the level of mRNA stability and some examples of the emerging data characterizing RNA-protein interactions.

Animals↗

PubMed2003

Metabolic stability for drug discovery and development: pharmacokinetic and biochemical challenges.

Metabolic stability refers to the susceptibility of compounds to biotransformation in the context of selecting and/or designing drugs with favourable pharmacokinetic properties. Metabolic stability results are usually reported as measures of intrinsic clearance, from which secondary pharmacokinetic parameters such as bioavailability and half-life can be calculated when other data on volume of distribution and fraction absorbed are available. Since these parameters are very important in defining the pharmacological and toxicological profile of drugs as well as patient compliance, the pharmaceutical industry has a particular interest in optimising for metabolic stability during the drug discovery and development process. In the early phases of drug discovery, new chemical entities cannot be administered to humans; hence, predictions of these properties have to be made from in vivo animal, in vitro cellular/subcellular and computational systems. The utility of these systems to define the metabolic stability of compounds that is predictive of the human situation will be reviewed here. The timing of performing the studies in the discovery process and the impact of recent advances in research on drug absorption, distribution, metabolism and excretion (ADME) will be evaluated with respect to the scope and depth of metabolic stability issues. Quantitative prediction of in vivo clearance from in vitro metabolism data has, for many compounds, been shown to be poor in retrospective studies. One explanation for this may be that there are components used in the equations for scaling that are missing or uncertain and should be an area of more research. For example, as a result of increased biochemical understanding of drug metabolism, old assumptions (e.g. that the liver is the principal site of first-pass metabolism) need revision and new knowledge (e.g. the relationship between transporters and drug metabolising enzymes) needs to be incorporated into in vitro-in vivo correlation models. With ADME parameters increasingly being determined on automated platforms, instead of using results from high throughput screening (HTS) campaigns as simple go/no-go filters, the time saved and the many compounds analysed using the robots should be invested in careful processing of the data. A logical step would be to investigate the potential to construct computational models to understand the factors governing metabolic stability. A rational approach to the use of HTS assays should aim to screen for many properties (e.g. physicochemical parameters, absorption, metabolism, protein binding, pharmacokinetics in animals and pharmacology) in an integrated manner rather than screen against one property on many compounds, since it is likely that the final drug will represent a global average of these properties.

Animals↗

PubMed2004

Common structural stability properties of 4-helical bundle cytokines: possible physiological and pharmaceutical consequences.

Biological activity and clinical efficacy of a therapeutic protein are contingent upon the structural stability, bioavailability, and clearance rates of the protein. In this review, we examine the class of 4-helical bundle cytokines for common stability properties that may affect biological structure and efficacy. Three critical stability features that are hallmarks of this class of cytokines are the pH dependence of structural stability, the presence of folding intermediates, and the population of aggregation intermediates. We hypothesize that certain cytokines have increased stability in acid to enable receptor-mediated clearance, and that reengineering local endocytic trafficking can result in dramatic improvements in global serum half-life and therapeutic efficacy. The common feature of folding and aggregation intermediates has implications on kinetic folding pathways, membrane permeability, solubility, and precipitation properties that are critical for commercial production, formulation, and delivery. Understanding the structural stability properties of this class of cytokines may help elucidate new approaches to improving therapeutic efficacy.

Animals↗

PubMed2002

Screening drugs for metabolic stability using pulsed ultrafiltration mass spectrometry.

A pulsed ultrafiltration-mass spectrometric screening method has been developed to evaluate the metabolic stability of drugs. Pooled human liver microsomes containing cytochrome P450 enzymes were trapped by an ultrafiltration membrane in a stirred flow-through chamber, and eight beta-blocker drugs including acebutolol, alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol, and timolol were flow-injected through the chamber along with the cofactor NADPH. The ultrafiltrate was collected, concentrated and analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS-MS) in order to quantitate the unmetabolized fraction of each drug. The metabolic stability of each beta-blocker was determined based on the difference between the corresponding LC-MS-MS peak areas of an experimental incubation and a control without NADPH. A flow-through incubation method, pulsed ultrafiltration metabolic screening minimizes the potential for product feed back inhibition of cytochrome P450 enzymes. The importance of this phenomenon was illustrated by the observation that the metabolic stability of the set of beta-blocker drugs measured using pulsed ultrafiltration more closely resembled the in vivo stability than that determined using a conventional batch incubation with microsomes or an incubation with human hepatocytes. Since a mixture of compounds was analyzed, the relative metabolic stability of each compound could be assessed by comparison to the other compounds in the incubation. This approach might be particularly useful for the ranking of a directed library of drug leads with respect to metabolic stability and then the selection of lead compounds for further drug development.

Adrenergic beta-Antagonists↗

PubMedDate not supplied

Chemical, dissolution stability and microscopic evaluation of suspensions of ibuprofen and sustained release ibuprofen-wax microspheres.

Chemical stability studies of suspensions of ibuprofen powder and ibuprofen-wax microspheres were performed using an accelerated stability protocol with a modified high performance liquid chromatography (HPLC) procedure. The variables considered were pH, suspending agents and temperature. The study showed little or no chemical degradation in the different suspending agents after storage for three months. Dissolution stability was examined in suspensions of ibuprofen microspheres made from an optimized formulation with 17% drug loading. The storage temperature were 23, 37 and 45 degrees C. Other variables for the dissolution stability studies were suspending agents, wax types, suspending medium pH and microsphere size. Suspensions of ceresine wax microspheres stored at 37 degrees C showed faster drug release than room temperature storage, but suspensions stored at 45 degrees C showed an opposite effect. Microspheres suspended in syrup and stored at 37 degrees C had faster dissolution rates than microspheres suspended in methylcellulose at the same temperature, possibly as a result of an interaction between the syrup and the microsphere constituents. Suspensions of microcrystalline wax microspheres had better dissolution stability than microspheres made from ceresine wax. Higher suspending medium pH resulted in faster release of drug from the suspended microspheres, but particle size did not significantly affect the dissolution stability.

Anti-Inflammatory Agents, Non-Steroidal↗