Pharmaceutical process optimisation of bulk lyophilisates:implications of powder handling

Lyophilisation or freeze drying is the preferred dehydrating method for pharmaceuticals liable to thermal degradation. Most biologics are unstable in aqueous solution and may use freeze drying to prolong their shelf life. Lyophilisation is however expensive and has seen lots of work aimed at reducing cost. This thesis is motivated by the potential cost savings foreseen with the adoption of a cost efficient bulk drying approach for large and small molecules. Initial studies identified ideal formulations that adapted well to bulk drying and further powder handling requirements downstream in production. Low cost techniques were used to disrupt large dried cakes into powder while the effects of carrier agent concentration were investigated for powder flowability using standard pharmacopoeia methods. This revealed superiority of crystalline mannitol over amorphous sucrose matrices and established that the cohesive and very poor flow nature of freeze dried powders were potential barriers to success. Studies from powder characterisation showed increased powder densification was mainly responsible for significant improvements in flow behaviour and an initial bulking agent concentration of 10-15 %w/v was recommended. Further optimisation studies evaluated the effects of freezing rates and thermal treatment on powder flow behaviour. Slow cooling (0.2 °C/min) with a -25°C annealing hold (2hrs) provided adequate mechanical strength and densification at 0.5-1 M mannitol concentrations. Stable bulk powders require powder transfer into either final vials or intermediate storage closures. The targeted dosing of powder formulations using volumetric and gravimetric powder dispensing systems where evaluated using Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta Galactosidase models. Final protein content uniformity in dosed vials was assessed using activity and protein recovery assays to draw conclusions from deviations and pharmacopeia acceptance values. A correlation between very poor flowability (p<0.05), solute concentration, dosing time and accuracy was revealed. LDH and IgG lyophilised in 0.5 M and 1 M mannitol passed Pharmacopeia acceptance values criteria with 0.1-4 while formulations with micro collapse showed the best dose accuracy (0.32-0.4% deviation). Bulk mannitol content above 0.5 M provided no additional benefits to dosing accuracy or content uniformity of dosed units. This study identified considerations which included the type of protein, annealing, cake disruption process, physical form of the phases present, humidity control and recommended gravimetric transfer as optimal for dispensing powder. Dosing lyophilised powders from bulk was demonstrated as practical, time efficient, economical and met regulatory requirements in cases. Finally the use of a new non-destructive technique, X-ray microcomputer tomography (MCT), was explored for cake and particle characterisation. Studies demonstrated good correlation with traditional gas porosimetry (R2 = 0.93) and morphology studies using microscopy. Flow characterisation from sample sizes of less than 1 mL was demonstrated using three dimensional X-ray quantitative image analyses. A platinum-mannitol dispersion model used revealed a relationship between freezing rate, ice nucleation sites and variations in homogeneity within the top to bottom segments of a formulation.

The effect of the generation and handling in the acquired electrostatic charge in airborne particles

The measurement of the charge distribution in laboratory generated aerosols particles was carried out. Four cases of electrostatic charge acquisition by aerosol particles were evaluated. In two of these cases. the charges acquired by the particles were naturally derived from the aerosol generation procedure itself, without using any additional charging method. Ill the other two cases, a corona charger and an impact charger were utilized as Supplementary methods for charge generation. Two types of aerosol generators were used in the dispersion of particles in the gas Stream: the vibrating orifice generator TSI model 3450 and the rotating plate generator TSI model 3433. In the vibrating orifice generator. a Solution of methylene blue Was used and the generated particles were mono-dispersed. Different mono-aerosols were generated with particle diameters varying from 6.0 x 10(-6) m to 1.4 x 10(-5) m. In the rotating plate generator, a poly-dispersed phosphate rock concentrate with Stokes mean diameter of 1.30 x 10(-6) m and size range between 1.5 x 10(-7) m and 8.0 x 10(-6) m Was utilized as powder material in all tests. In the tests performed with the mono-dispersed particles. the median charges of the particles varied between -3.0 x 10-(16) C and -5.0 x 10(-18) degrees C and a weak dependence between particle size and charge was observed. The particles were predominantly negatively charged. In the tests with the poly-dispersed particles the median charges varied fairly linearly with the particle diameter and were negative. The order of magnitude of the results obtained is in accordance with data reported in the literature. The charge distribution, in this case, was wider, so that an appreciable amount of particles were positively charged. The relative spread of the distribution varied with the charging method. It was also noticed that the corona charger acted very effectively in charging the particles. (C) 2008 Elsevier BY. All rights reserved.

Flame Synthesis of Complex Fluoride-Based Nanoparticles as Upconversion Phosphors

Recent improvements in precursor chemistry, reactor geometry and run conditions extend the manufacturing capability of traditional flame aerosol synthesis of oxide nanoparticles to metals, alloys and inorganic complex salts. As an example of a demanding composition, we demonstrate here the one-step flame synthesis of nanoparticles of a 4-element non-oxide phosphor for upconversion applications. The phosphors are characterized in terms of emission capability, phase purity and thermal phase evolution. The preparation of flame-made beta-NaYF4 with dopants of Yb, Tm or Yb, Er furthermore illustrates the now available nanoparticle synthesis tool boxes based on modified flamespray synthesis from our laboratories at ETH Zurich. Since scaling concepts for flame synthesis, including large-scale filtration and powder handling, have become available commercially, the development of industrial applications of complex nanoparticles of metals, alloys or most other thermally stable, inorganic compounds can now be considered a feasible alternative to traditional top-down manufacturing or liquid-intense wet chemistry.

Fire protection precautions for handling uranium turnings, chips, borings, sawdust, and powder /

"4-10-50--825-A11578."

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

Age polyethism in Plebeia emerina (Friese) (Hymenoptera: Apidae) colonies related to propolis handling

Stingless bees collect plant resins and make it into propolis, although they have a wider range of use for this material than do honey bees (Apis spp.). Plebeia spp. workers employ propolis mixed with wax (cerumen) for constructing and sealing nest structures, while they use viscous (sticky) propolis for defense by applying it onto their enemies. Isolated viscous propolis deposits are permanently maintained at the interior of their colonies, as also seen in other Meliponini species. Newly-emerged Plebeia emerina (Friese) workers were observed stuck to and unable to escape these viscous propolis stores. We examined the division of labor involved in propolis manipulation, by observing marked bees of known age in four colonies of P. emerina from southern Brazil. Activities on brood combs, the nest involucrum and food pots were observed from the first day of life of the marked bees. However, work on viscous propolis deposits did not begin until the 13th day of age and continued until the 56th day (maximum lifespan in our sample). Although worker bees begin to manipulate cerumen early, they seem to be unable to handle viscous propolis till they become older.

Identification of Neoplasias of Breast Tissues Using a Powder Diffractometer

An investigation was carried out to study the potential use of the angular distribution of scattered photons by human breast samples for a rapid identification of neoplasias of breast tissues. This technique has possible applications as diagnostic aid for breast cancer. In this work, a commercial powder diffractometer was used to obtain the scattering profiles from breast tissues histopathologically classified as normal breast tissues, fibroadenomas (benign breast diseases) and carcinomas (malignant breast diseases), in the interval 0.02 angstrom(-1) < x < 0.62 angstrom(-1). The experimental methods and data corrections are discussed in detail, and they included background subtraction, polarization, self-attenuation and geometric effects. The validation of the experimental procedure was achieved through an analysis of water sample. The results showed that the scattering profile is a unique impression of each type of tissue, being correlated with their microscopic morphological features. Multivariate analysis was applied to these profiles in order to verify if the information carried by these scattering profiles allow the differentiation between normal, benign and malignant breast tissues. The statistical analysis results showed that a correct identification of 75% of the analyzed samples is accomplished. The values of sensibility and specificity of this method in correctly differentiating between normal and neoplastic samples were 95.6% and 82.3%, respectively, while the values for differentiation between benign and malignant neoplasias were 78.6% and 62.5%. These initial results indicate the feasible use of commercial powder diffractometer to provide a rapid diagnostic with a high sensitivity.

Synchrotron X-ray powder diffraction and extended X-ray absorption fine structure spectroscopy studies on nanocrystalline ZrO(2)-CaO solid solutions

The crystal structure and the local atomic order of a series of nanocrystalline ZrO(2)-CaO solid solutions with varying CaO content were studied by synchrotron radiation X-ray powder diffraction and extended X-ray absorption fine structure (EXAFS) spectroscopy. These samples were synthesized by a pH-controlled nitrate-glycine gel-combustion process. For CaO contents up to 8 mol%, the t' form of the tetragonal phase (c/a > 1) was identified, whereas for 10 and 12 mol% CaO, the t '' form (c/a=1; oxygen anions displaced from their ideal positions in the cubic phase) was detected. Finally, the cubic phase was observed for solid solutions with CaO content of 14 mol% CaO or higher. The t'/t '' and t ''/cubic compositional boundaries were determined to be at 9 (1) and 13 (1) mol% CaO, respectively. The EXAFS study demonstrated that this transition is related to a tetragonal-to-cubic symmetry change of the first oxygen coordination shell around the Zr atoms.

High-temperature X-ray powder diffraction study of the tetragonal-cubic phase transition in nanocrystalline, compositionally homogeneous ZrO2-CeO2 solid solutions

Crystal structure of compositionally homogeneous, nanocrystalline ZrO2-CeO2 solutions was investigated by X-ray powder diffraction as a function of temperature for compositions between 50 and 65 mol % CeO2 center dot ZrO2-50 and 60 mol % CeO2 solid solutions, which exhibit the t'-form of the tetragonal phase at room temperature, transform into the cubic phase in two steps: t'-to-t '' followed by t ''-to-cubic. But the ZrO2-65 mol % CeO2, which exhibits the t ''-form, transforms directly to the cubic phase. The results suggest that t'-to-t '' transition is of first order, but t ''-to-cubic seems to be of second order. (C) 2008 International Centre for Diffraction Data.

Synchrotron X-ray powder diffraction study of the tetragonal-cubic phase transition in nanostructured ZrO2-Sc2O3 solid solutions

The transition between tetragonal and cubic phases in nanostructured ZrO2-Sc2O3 solid solutions by high-temperature X-ray powder diffraction using synchrotron radiation is presented. ZrO2-8 and 11 mol% Sc2O3 nanopowders that exhibit the t'- and t ''-forms of the tetragonal phase, respectively, were synthesized by a stoichiometric nitrate-lysine gel-combustion route. The average crystallite size treated at 900 degrees C was about 25 nm for both compositions. Our results showed that t'-t '' and t ''-cubic transitions take place for the 8 and 11 mol% Sc2O3 samples, respectively. (C) 2008 International Centre for Diffraction Data.

Seed phytate and protein content in beans depending on the application of basalt powder

Seed phytate and protein content in beans depending on the application of basalt powder. The content of phytate in the grains is correlated with the supply of phosphorus to the plant, but there is a lack of knowledge as to possible effect of slower availability of nutrients in the soil. The objectives of this study were to assess the effect of rock powder, alone or combined with cattle manure, on the productivity, levels of phosphorus, protein and phytate content in beans. The experiment was carried out in a randomized blocks design, with four replications. The treatments were control (limestone, granite and natural phosphate); conventional fertilization; powder basalt (2.5, 5.0, 10.0 and 20.0 ton. ha(-1)); cattle manure, and doses of powder basalt with cattle manure. In the treatment with conventional fertilizer, the total phosphorus content in grain was higher than the control, but the application of powder of basalt did not show a difference significant. Increase in the doses of basalt powder increased the phosphorus content, but phytate content remained constant. Basalt powder proved to be an alternative to maintain low levels of phosphorus in the form of phytate in the grains.

Angiotensin receptor blockade improves the net balance of cardiac Ca(2+) handling-related proteins in sympathetic hyperactivity-induced heart failure

Aims: The clinical benefits of angiotensin II type 1 (AT1) receptor blockers (ARB) in heart failure (HF) include cardiac anti-remodeling and improved ventricular function. However, the cellular mechanisms underlying the benefits of ARB on ventricular function need to be better clarified. In the present manuscript, we evaluated the effects of AT1 receptor blockade on the net balance of Ca(2+) handling proteins in hearts of mice lacking alpha(2A) and alpha(2C) adrenoceptors (alpha(2A)/alpha(2C)ARKO), which develop sympathetic hyperactivity (SH) induced-HF. Main methods: A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)ARKO mice in a C57BL6/J genetic background (5-7 mo of age) was randomly assigned to receive either placebo or ARB (Losartan, 10 mg/kg for 8wks). Ventricular function (VF) was assessed by echocardiography, and cardiac myocyte width and ventricular fibrosis by a computer-assisted morphometric system. Sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), phospholamban (PLN), phospho-Ser(16)-PLN, phospho-Thr(17)-PLN, phosphatase 1 (PP1), Na(+)-Ca(2+) exchanger (NCX), Ca(2+)/calmodulin-dependent protein kinase 11 (CaMKII) and phospho-Thr(286)-CaMKII were analyzed by Western blot. Key findings: alpha(2A)/alpha(2C)ARKO mice displayed ventricular dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis paralleled by decreased SERCA2 and increased phospho-Thr(17)-PLN, CaMKII, phospho-Thr(286)-CaMKII and NCX levels. ARB induced anti-cardiac remodeling effect and improved VF in alpha(2A)/alpha(2C)ARKO associated with increased SERCA2 and phospho-Ser(16)-PLN levels, and SERCA2:NCX ratio. Additionally, ARB decreased phospho-Thr(17)-PLN levels as well as reestablished NCX, CaMKII and phospho-Thr(286)-CaMKII toward WT levels. Significance: Altogether, these data provide new insights on intracellular Ca(2+) regulatory mechanisms underlying improved ventricular function by ARB therapy in HF. (c) 2011 Elsevier Inc. All rights reserved.

Exercise training and caloric restriction prevent reduction in cardiac Ca(2+)-handling protein profile in obese rats

Previous studies show that exercise training and caloric restriction improve cardiac function in obesity. However, the molecular mechanisms underlying this effect on cardiac function remain unknown. Thus, we studied the effect of exercise training and/or caloric restriction on cardiac function and Ca(2+) handling protein expression in obese rats. To accomplish this goal, male rats fed with a high-fat and sucrose diet for 25 weeks were randomly assigned into 4 groups: high-fat and sucrose diet, high-fat and sucrose diet and exercise training, caloric restriction, and exercise training and caloric restriction. An additional lean group was studied. The study was conducted for 10 weeks. Cardiac function was evaluated by echocardiography and Ca(2+) handling protein expression by Western blotting. Our results showed that visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels were higher in rats on the high-fat and sucrose diet compared with the lean rats. Cardiac nitrate levels, reduced/oxidized glutathione, left ventricular fractional shortening, and protein expression of phosphorylated Ser(2808)-ryanodine receptor and Thr(17-)phospholamban were lower in rats on the high-fat and sucrose diet compared with lean rats. Exercise training and/or caloric restriction prevented increases in visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels and prevented reduction in cardiac nitrate levels and reduced: oxidized glutathione ratio. Exercise training and/or caloric restriction prevented reduction in left ventricular fractional shortening and in phosphorylation of the Ser(2808)-ryanodine receptor and Thr(17)-phospholamban. These findings show that exercise training and/or caloric restriction prevent cardiac dysfunction in high-fat and sucrose diet rats, which seems to be attributed to decreased circulating neurohormone levels. In addition, this nonpharmacological paradigm prevents a reduction in the Ser(2808)-ryanodine receptor and Thr(17-)phospholamban phosphorylation and redox status. (Hypertension. 2010;56:629-635.)

Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure

Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum PC. Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure. J Appl Physiol 109: 702-709, 2010. First published July 1, 2010; doi: 10.1152/japplphysiol.00281.2010.-The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remain unclear. We investigated potential Ca(2+) abnormalities in skeletal muscles comprising different fiber compositions and investigated whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5-mo-old wild-type (WT) and congenic alpha(2A)/alpha(2C) adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of a 8-wk running session of 60 min, 5 days/wk (from 5 to 7 mo of age). After completion of the exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function test by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography, and Ca(2+) handling-related protein expression by Western blot. alpha(2A)/alpha(2C)ARKO mice displayed decreased ventricular function, exercise intolerance, and muscle weakness paralleled by decreased expression of sarcoplasmic Ca(2+) release-related proteins [alpha(1)-, alpha(2)-, and beta(1)-subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca(2+) reuptake-related proteins [sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA) 1/2 and Na(+)/Ca(2+) exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance and muscle function and reestablished the expression of proteins involved in sarcoplasmic Ca(2+) handling toward WT levels. We provide evidence that Ca(2+) handling-related protein expression is decreased in this HF model and that exercise training improves skeletal muscle function associated with changes in the net balance of skeletal muscle Ca(2+) handling proteins.

Aerobic exercise training improves Ca(2+) handling and redox status of skeletal muscle in mice

Exercise training is known to promote relevant changes in the properties of skeletal muscle contractility toward powerful fibers. However, there are few studies showing the effect of a well-established exercise training protocol on Ca(2+) handling and redox status in skeletal muscles with different fiber-type compositions. We have previously standardized a valid and reliable protocol to improve endurance exercise capacity in mice based on maximal lactate steady-state workload (MLSSw). The aim of this study was to investigate the effect of exercise training, performed at MLSSw, on the skeletal muscle Ca(2+) handling-related protein levels and cellular redox status in soleus and plantaris. Male C57BL/6J mice performed treadmill training at MLSSw over a period of eight weeks. Muscle fiber-typing was determined by myosin ATPase histochemistry, citrate synthase activity by spectrophotometric assay, Ca(2+) handling-related protein levels by Western blot and reduced to oxidized glutathione ratio (GSH:GSSG) by high-performance liquid chromatography. Trained mice displayed higher running performance and citrate synthase activity compared with untrained mice. Improved running performance in trained mice was paralleled by fast-to-slow fiber-type shift and increased capillary density in both plantaris and soleus. Exercise training increased dihydropyridine receptor (DHPR) alpha 2 subunit, ryanodine receptor and Na(+)/Ca(2+) exchanger levels in plantaris and soleus. Moreover, exercise training elevated DHPR beta 1 subunit and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 1 levels in plantaris and SERCA2 levels in soleus of trained mice. Skeletal muscle GSH content and GSH:GSSG ratio was increased in plantaris and soleus of trained mice. Taken together, our findings indicate that MLSSw exercise-induced better running performance is, in part, due to increased levels of proteins involved in skeletal muscle Ca(2+) handling, whereas this response is partially dependent on specificity of skeletal muscle fiber-type composition. Finally, we demonstrated an augmented cellular redox status and GSH antioxidant capacity in trained mice.