Inactivation and Aggregation of β-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Purpose. To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized -galactosidase (-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation—time constant () and its distribution () is discussed. Methods. Protein aggregation in lyophilized -GA formulations at 10-70°C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl--D-galactopyranoside as a substrate. Results. Inactivation and aggregation of -GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (T_mc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in the formulation. The estimated parameter for protein aggregation decreased rapidly as temperature increased beyond T_mc because the mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t₉₀) calculated from the and parameters exhibited a change in temperature dependence gradient near T_mc. In contrast, t₉₀ for protein inactivation exhibited temperature dependence patterns varying with the excipients. Conclusions. The t₉₀ calculated from the estimated and parameters was found to be a useful parameter for evaluating the stability of lyophilized -GA formulations. The prediction of t₉₀ by extrapolation was possible in the temperature range in which did not rapidly vary with temperature.     

β-Relaxation of Insulin Molecule in Lyophilized Formulations Containing Trehalose or Dextran as a Determinant of Chemical Reactivity

Purpose The purpose of this study was to elucidate whether the degradation rate of insulin in lyophilized formulations is determined by matrix mobility, as reflected in glass transition temperature (T_g), or by β-relaxation, as reflected in rotating-frame spin-lattice relaxation time . Methods The storage stability of insulin lyophilized with dextran was investigated at various relative humidities (RH; 12–60%) and temperatures (40–90°C) and was compared with previously reported data for insulin lyophilized with trehalose. Insulin degradation was monitored by reverse-phase high-performance liquid chromatography. Furthermore, the of the insulin carbonyl carbon in the lyophilized insulin–dextran and insulin–trehalose systems was measured at 25°C by ¹³C solid-state NMR, and the effect of trehalose and dextran on was compared at various humidities. Results The degradation rate of insulin lyophilized with dextran was not significantly affected by the T_g of the matrix, even at low humidity (12% RH), in contrast to that of insulin lyophilized with trehalose. The insulin–dextran system exhibited a substantially greater degradation rate than the insulin–trehalose system at a given temperature below the T_g. The difference in degradation rate between the insulin–dextran and insulin–trehalose systems observed at 12% RH was eliminated at 43% RH. In addition, the of the insulin carbonyl carbon at low humidity (12% RH) was prolonged by the addition of trehalose, but not by the addition of dextran. This difference was eliminated at 23% RH, at which point the solid remained in the glassy state. These findings suggest that the β-relaxation of insulin is inhibited by trehalose at low humidity, presumably as a result of insulin–trehalose interaction, and thus becomes a rate determinant. In contrast, dextran, whose ability to interact with insulin is thought to be less than that of trehalose, did not inhibit the β-relaxation of insulin, and thus, the chemical activational barrier (activation energy) rather than β-relaxation becomes the major rate determinant. Conclusions β-Relaxation rather than matrix mobility seems to be more important in determining the stability of insulin in the glassy state in lyophilized formulations containing trehalose and dextran.     

The Effect of Salts on the Stability of β-Galactosidase in Aqueous Solution,as Related to the Water Mobility

The effect of salts (KI, KBr, NaCl, KC1, KF, phosphate, and Na₂SO₄) on the stability of -galactosidase in aqueous solution was studied from the aspect of changes in water mobility. At salt concentrations up to 200 mM, the inactivation rate of -galactosidase in all the salt solutions studied increased with increasing salt concentration. At higher concentrations, those salts which had little effect on the spin-lattice relaxation time, T ₁, of water (KI, KBr, and KC1) continued to increase the inactivation rate of -galactosidase with increasing concentration, while those salts which decreased the T _l of water (KF, phosphate, and Na₂SO₄) decreased the inactivation rate. It appeared that the decrease in water mobility caused by KF, phosphate, and Na₂SO₄ resulted in stabilization of -galactosidase. The results indicate that water mobility is an important factor in the denaturation rate of proteins.     

Significance of hydrogen sulfide production in the pancreatic β-cell

Heart failure is a major cause of death in developed countries, and the development of an epoch-making cure is desired from the viewpoint for improving the quality of life and reducing the medical cost of the patient. The importance of neurohumoral factors, such as angiotensin (Ang) II and catecholamine, for the progression of heart failure has been supported by a variety of evidence. These agonists stimulate seven transmembrane-spanning receptors that are coupled to heterotrimeric GTP-binding proteins (G proteins). Using specific pharmacological tools to assess the involvement of G protein signaling pathways, we have revealed that α subunit of G(q) (Gα(q)) activates Ca(2+)-dependent hypertrophic signaling through diacylglycerol-activated transient receptor potential canonical (TRPC) channels (TRPC3 and TRPC6: TRPC3/6). In contrast, activation of Gα(12) family proteins in cardiomyocytes confers pressure overload-induced cardiac fibrosis via stimulation of purinergic P2Y(6) receptors induced by extracellular nucleotides released from cardiomyocytes. In fact, direct or indirect inhibition of TRPC3/6 or P2Y(6) receptors attenuates pressure overload-induced cardiac dysfunction. These findings will provide a new insight into the molecular mechanisms underlying pathogenesis of heart failure.     

Aggregates Formed During Storage of β-Galactosidase in Solution and in the Freeze-Dried State

Aggregates formed during storage of freeze-dried -galactosidase were compared with those formed in solutions. Freeze-dried -galactosidase aggregated during storage in the presence of moisture, producing a protein precipitate which was soluble in guanidine hydrochloride solution but not in buffer solution. The protein precipitate dissolved in guanidine solution exhibited a large molecular size by high-performance size exclusion chromatography and converted to proteins of original size in the presence of dithiothreitol. It is suggested that the aggregation involves chemical interaction via covalent disulfide bonding. In contrast, -galactosidase in aqueous solution aggregated without formation of protein precipitates. Soluble aggregates were converted to proteins of original size in guanidine solution without dithiothreitol, suggesting noncovalent bonding. The difference in aggregation behavior may be ascribed to the difference in the water:protein ratio. We propose that inactivation of -galactosidase is due to formation of thermally denatured (unfolded) protein, which aggregates dependent on the water:protein ratio, either via noncovalent interactions at a high water:protein ratio in solution or via covalent interaction at a low water:protein ratio in the freeze-dried state.     

Stability of β-Galactosidase,a Model Protein Drug,Is Related to Water Mobility as Measured by 17O Nuclear Magnetic Resonance (NMR)

The inactivation of freeze-dried -galactosidase during storage was studied, focusing on the effect of water mobility as measured by the spin-lattice relaxation time, T ₁, of water using ¹⁷O NMR. Inactivation of -galactosidase lyophilized from phosphate buffer solution was studied as a function of water content, which in turn affected the T _l of water. An increase in the water content of freeze-dried -galactosidase brought about an increase in the T _l of water, as well as a rise in pH. For the freeze-dried enzyme with sufficient water content to be dissolved, the inactivation rate was related to the T _l of water rather than to the pH change. It is suggested that as the water content increases, the mobility of water around the enzyme increases, resulting in enhanced enzyme inactivation. The freeze-dried samples with limited moisture showed inactivation rates faster than those expected from the pH and water mobility, suggesting that the inactivation mechanism is different from that for the freeze-dried enzyme with a larger amount of water. Inactivation of -galactosidase in solutions was also studied as a function of phosphate buffer and sodium chloride concentrations, which in turn affected the T _l of water. Because the inactivation rate increased with increasing salt concentrations and the rate extrapolated to zero concentration was negligible, inactivation of the freeze-dried enzyme was apparently induced by the salts used as additives for lyophilization. The enhancing effect of phosphate buffer components, however, was reduced at higher concentrations, an effect related to the decrease in the T _l of water. This result may be ascribed to the decrease in water mobility caused by phosphate buffer components and is consistent with the observation that the inactivation rate of the freeze-dried enzyme with a relatively large amount of water decreased with decreasing T ₁ of water.     

Trehalose Preserves the Integrity of Lyophilized Phycoerythrin–AntiHuman CD8 Antibody Conjugates and Enhances their Thermal Stability in Flow Cytometric Assays

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment–protein complex widely used as fluorescent marker, from thermal denaturation. Once embedded into a dried trehalose matrix, R-PE retains its optical absorption–emission characteristics even when exposed to 70°C for h or when subjected to freeze-drying. We subsequently examined the protection exerted by trehalose on freeze-dried antihuman CD8–RPE (CD8–RPE) conjugated antibodies. Flow cytometric analysis showed that colyophilized trehalose–CD8–RPE preparations can be exposed for 4 weeks at 45°C without significant loss of functionality. Remarkably, even following 4 weeks incubation at 70°C, the preparations are still able to specifically recognize CD8⁺ lymphocyte populations. These results show that colyophilization with trehalose makes possible the preparation of antibody-based diagnostic kits which can withstand breaks in the “cold chain” distribution, particularly suited for use in less-developed countries of the tropical areas.     

The Use of Disaccharides in Inhibiting Enzymatic Activity Loss and Secondary Structure Changes in Freeze-Dried β-Galactosidase during Storage

Purpose  

The purpose of this study is to show how disaccharides differ in their ability to protect lyophilized β-galactosidase from enzymatic activity loss and secondary structure changes during storage.     

Expression of β-catenin in lung tissues of mice with pulmonary fibrosis

目的 观察β-连环蛋白在肺纤维化模型小鼠肺组织中的表达.方法 昆明小鼠40只随机均分成实验组和对照组,气管内分别一次性注入博来霉素5 mg/kg(0.04 ml)和生理盐水0.04 ml.给药后第3、7、14、28天,每组处死小鼠5只,取肺组织做病理切片和HE染色;用碱水解法检测肺组织羟脯氨酸含量,免疫组化法及Western blot检测肺组织中β-连环蛋白表达.结果 实验组肺泡炎和肺纤维化程度均明显高于对照组(P＜0.05),羟脯氨酸的含量在第7-28天明显高于对照组(P＜0.01).实验组在各时间点肺组织中的β-连环蛋白表达高于对照组(P＜0.05);其表达高峰在第14天.结论 β-连环蛋白在模型小鼠肺纤维化发生过程中存在高表达.     

Effects of intrathecal or intracerebroventricular pretreatment with pertussis toxin on antinociception induced by β-endorphin or morphine administered intracerebroventricularly in mice

We have previously demonstrated that -endorphin and morphine, when administered supraspinally, produce antinociception by activating different descending pain inhibitory systems in both rats and mice. However, the signal transduction mechanisms involved in the descending pain-inhibitory systems that are activated by -endorphin and morphine administered intracerebroventricularly (i.c.v.) have not been characterized. Therefore, in the present study, the effects of intrathecal (i.t.) and i.c.v. pretreatments with pertussis toxin (PTX) on antinociception induced by -endorphin or by morphine administered i.c.v. were studied in ICR mice. Antinociception was assessed by the tail-flick assay and by the hotplate assay. Intrathecal pretreatment with PTX (0.5 g) for 6 days effectively reduced the inhibition of the tail-flick response induced by -endorphin (1 g) or by morphine (1 g) administered i.c.v. However, i.t. pretreatment with PTX was not effective in reducing the inhibition of the hot-plate response induced by -endorphin or by morphine administered i.c.v. Intracerebroventricular pretreatment with PTX (0.5 g) for 6 days effectively reduced the inhibition of the tail-flick and hot-plate responses induced by morphine (1 g), but not that induced by -endorphin (1 g), administered i.cv. Our results suggest that there are PTX-sensitive G proteins coupled to the spinal descending pain inhibitory systems that are activated by -endorphin and morphine administered i.c.v. At a supraspinal level, i.cv. morphine- but not -endorphin-induced antinociception is mediated by PTX-sensitive G proteins. Correspondence to: Hong W. Suh at the above address     

Effects of long-term active immunization with the second extracellular loop of human β1- or β3-adrenoceptors in thoracic aorta and mesenteric arteries in Lewis rats

ObjectiveTo evaluate whether active immunization producing β₁- or β₃-antibodies (β₁-ABs and β₃-ABs) detected in sera of patients with dilated cardiomyopathies has deleterious effects on vascular reactivity in Lewis rat thoracic aorta (TA) and small mesenteric arteries (SMA).Design and methodLewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of β₁- and β₃-adrenoceptors (ARs). During the immunization, systolic blood pressure (SBP) was monitored using the tail cuff method. The vascular reactivity of immunized rats was assessed by ex vivo studies on SMA and TA using various β-AR agonists, phenylephrine and KCl.ResultsThe immunizations producing functional β₁-ABs and β₃-ABs did not affect the SBP. However, in TA from β₁-AR-immunized rats, the relaxations mediated by dobutamine and salbutamol were significantly impaired in comparison with adjuvant rats whereas nebivolol-induced relaxation was not modified. Moreover, phenylephrine and KCl-mediated contractions were enhanced in these rats. In contrast, immunization with β₃-AR peptide led to the increase of relaxations induced by dobutamine in TA but did not change those induced by salbutamol and nebivolol. Surprisingly, in SMA from both rats immunized with β₁- or β₃-peptides, relaxations induced by the various β-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired.ConclusionsOur study shows that β₁- and β₃-ABs can affect vascular reactivity. β₁-ABs would have a pathogenic action whereas β₃-ABs would have a beneficial effect on aorta reactivity.     

Crystal structure of β-hexosaminidase B in complex with pyrimethamine, a potential pharmacological chaperone

β-Hexosaminidases (β-hex) are a group of glycosyl hydrolase isozymes that break down neutral and sialylated glycosphingolipids in the lysosomes, thereby preventing their buildup in neuronal cells. Some mutants of β-hex have decreased folding stability that results in adult-onset forms of lysosomal storage diseases. However, prevention of the harmful accumulation of glycolipids only requires 10% of wild-type activity. Pyrimethamine (PYR) is a potential pharmacological chaperone that works by stabilizing these mutant enzymes sufficiently to allow more β-hex to arrive in the lysosome, where it can carry out its function. An X-ray structure of the complex between human β-hexosaminidase B (HexB) and PYR has been determined to 2.8 ?. PYR binds to the active site of HexB where several favorable van der Waals contacts and hydrogen bonds are introduced. Small adjustments of the enzyme structure are required to accommodate the ligand, and details of the inhibition and stabilization properties of PYR are discussed.     

The aggregative stability of β-lactoglobulin in glassy mixtures with sucrose, trehalose and dextran

The aim of this study was to investigate the effect of the addition of different carbohydrates on the thermally induced aggregation of a model globular protein, β-lactoglobulin (BLG), in the glass state. Amorphous mixtures of BLG with trehalose, sucrose and dextran were prepared by freeze-drying, their glass behaviour was characterised using calorimetry and thermally induced aggregation was measured using size exclusion chromatography. Pure BLG shows increasing levels of aggregation when heated in the temperature range 70-100 °C for 48-144 h. The addition of the disaccharides sucrose and trehalose both resulted in a decrease in aggregation rate which approached negligible rates at 50 wt.% carbohydrate. The effect of dextran addition was similar to that of the disaccharides when preparations containing 9 wt.% carbohydrate were heated at 70 °C for 2 days. However, when the concentration exceeded 23 wt.%, the reaction temperature was 70 °C or above or the reaction time was longer than 48 h, the addition of the polysaccharide did not protect the protein from thermally induced aggregation, suggesting that protein-polymer phase separation could have occurred during freeze-drying. Overall the results support the proposal that one aspect of carbohydrate additive functionality is as a diluent with the added condition that the carbohydrate remains miscible with the protein during processing.     

Synthesis,Characterization, and Evaluation of the Local Irritant Action of an Ibuprofen - β-Cyclodextrin Inclusion Complex

Pharmaceutical Chemistry Journal -     

No role of homologous recombination in dealing with β-lapachone cytotoxicity in yeast

β-Lapachone (β-lap) is a promising antitumoral agent. DNA base oxidation and alkylation are among the expected damages by β-lap. Herein, we have explored the role that the homologous recombination pathway (HR), a critical DNA repair process in Saccharomyces cerevisiae, has in the cytotoxic profile of β-lap. We have further compared β-lap to the closely related compound menadione and the well-known alkylating agent methyl methanesulfonate (MMS). Surprisingly, we found that β-lap does not trigger HR, as seen for (i) the mutant sensitivity profiles, (ii) concentration-dependent arrest profiles, (iii) absence of nuclear DNA repair factories, and (iv) frequency of recombination between direct repeats.     

Effects of chronic treatment with the new ultra-long-acting β2-adrenoceptor agonist indacaterol alone or in combination with the β1-adrenoceptor blocker metoprolol on cardiac remodelling

Background and Purpose

The ability of a chronic treatment with indacaterol, a new ultra-long-acting β₂-adrenoceptor agonist, to reverse cardiac remodelling and its effects in combination with metoprolol, a selective β₁-adrenoceptor antagonist, were investigated on myocardial infarction in a rat model of heart failure (HF).

Experimental Approach

We investigated the effects of indacaterol and metoprolol, administered alone or in combination, on myocardial histology, β-adrenoceptor-mediated pathways, markers of remodelling and haemodynamic parameters in a rat model of HF. Five groups of rats were assessed: sham-operated rats; HF rats; HF + indacaterol 0.3 mg·kg⁻¹·day⁻¹; HF + metoprolol 100 mg·kg⁻¹·day⁻¹; HF + metoprolol + indacaterol. All pharmacological treatments continued for 15 weeks.

Key Results

Treatment with either indacaterol or metoprolol significantly reduced the infarct size in HF rats. However, the combination of indacaterol and metoprolol reduced the infarct size even further, reduced both BP and heart rate, reversed the decrease in ejection fraction, normalized left ventricular systolic and diastolic internal diameters, normalized the decreased β₁ adrenoceptor mRNA expression as well as cardiac cAMP levels and reduced cardiac GPCR kinase 2 expression, compared with the untreated HF group.

Conclusion and Implications

The results of our study demonstrated an additive interaction between indacaterol and metoprolol in normalizing and reversing cardiac remodelling in our experimental model of HF. The translation of these findings to clinical practice might be of interest, as this combination of drugs could be safer and more effective in patients suffering from HF and COPD.Tables of Links

PK-PD modeling of β-lactam antibiotics: in vitro or in vivo models?

A modified E(max)-pharmacokinetic-pharmacodynamic (PK-PD) model was previously proposed in literature for describing the antimicrobial activity of β-lactam antibiotics based on in vitro experiments. However, bacteria behave differently in vitro and in vivo. Thus, the aims of this study were to model the killing effect of piperacillin (PIP) against Escherichia coli on immunocompromised infected rats using this model and to compare the parameters obtained in vitro and in vivo for the same bacteria/drug combination. The PK-PD parameters determined in vitro and in vivo were as follows: generation rate constant of 1.30 ± 0.10 and 0.76 ± 0.20?h(-1), maximum killing effect of 3.11 ± 0.27 and 1.38 ± 0.20?h(-1) and concentration to produce 50% of the maximum effect of 5.44 ± 0.03 and 1.31 ± 0.27?μg?ml(-1), respectively. The comparison between the in vitro and in vivo parameters was not straightforward and had to take into consideration the intrinsic differences of the models involved. So far, the main application of the PK-PD model evaluated is for the comparison of different antimicrobial agent's potency and efficacy, under equivalent conditions.     

Significance of toxic interactions in medicolegal evidence. Complex fatal poisoning with drugs of abuse in the material of the Chair of Forensic Medicine, Collegium Medicum, Jagiellonian University in Kraków

The subject of the study was fatal complex poisonings with drugs of abuse in two young men. In the first case, postmortem investigation revealed cardiotoxic death as the result of an interaction between opiates, amphetamine derivatives and oxazepam. In the second case, death followed the administration of amphetamine derivatives and cocaine (xenobiotics known on the illicit drug market as "UFO"). Based on the toxicological postmortem analysis the authors discuss the interpretation of the results in the light of general problems of interactions taking place in toxicokinetic and toxicodynamic phases of intoxication processes.     

Effects of valsartan with or without benazepril on blood pressure,angiotensin Ⅱ,and endoxin in patients with essential hypertension

AIM：To eveluate the effects of valsartan(Val)with or without benazepril(Ben)on blood pressure and plasma levels of angiotensin(Ang Ⅱ)and digoxin-immunoreactive factors(endoxin)in patients with essential hypertension. METHODS:Ninety patients with essential hypertension were randomly divided into 3 groups(n=30 per group):Ben group(Ben 10 mg/d,po);Val group(Val 80mg/d,po);combination drug therapy group(Val 80mg/d Ben 10mg/d,po);all patients were treated for 12 weeks.Age and sex-matched 20 normal subjects were served as control group.RESULTS:The levels of plasma endoxin and ang Ⅱ in patients with essential hypertension were remarkably higher than those in normal subjects.The levels of plasma ang Ⅱ and endoxin were all obvious positive correlation with systolic blood pressure(SBP)and diastolic blood pressure(DBP)(Ang Ⅱ:r=0.5151,0.7978;endoxin:r= 0.4706,0.7274,respectively),within 6 weeks of drug intervene,SBP and DBP were remarkably decreased in 3 groups.After 6 weeks,SBP and DBP were continuously decreased in Ben group and Val Ben group,but not in Val group.Level of plasma AngⅡ was remarkably decreased as SBP and DBP decreased in Ben group and Val Ben group;level of plasma AngⅡ was remarkably increased in Val group.CONCLUSION:Val with or without Ben remarkably decreased SBP and DBP in patients with essential hypertension within 6 weeks.Antihypertensive efficacy was weakened after long-term use of Val alone.The antihypertensive effect of Val Ben group was the most remarkable among 3 groups and could avoid the side effects of high plasma AngⅡ.