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Complexes of β-cylodextrin with five nucleotides of adenine (A), thymine (T) guanine (G), cytosine (C), and 5-methylcytosine have been investigated using Hatree-Fock (HF) and density functional theory (DFT) calculations of different quality.
This work was inspired by recent technological advances made in single-molecule sequencing of DNA. One molecule of single-stranded DNA can be sequenced by using nanopores made of α-hemolysin as the sequencing device. An exonuclease enzyme attached to the top of the pore cleaves bases from the DNA strand so they can traverse the pore one at a time. The different nucleotides then bind to cyclodextrin attached to the inside of the pore . The different bases are discriminated by the change they induce in the amplitude of the current carried by aqueous ions passing through the pore. Several variants of this method are currently developed by different companies and will be marketed soon.
We calculated low energy conformations of complexes of β-cyclodextrin with the five different nucleotides mentioned above, using different methods (HF, DFT). The interaction energy with β-cyclodextrin was estimated from the energy difference between a complex and the molecules it consists of. The presented results show, that it is possible to discriminate all five nucleotides very clearly. Because one can easily distinguish between cytosine and 5-methyl-cytosine, this method can be used to sequence methylated DNA directly in one step which might be an advantage compared to other similar sequencing methods.
Cationic lipids spontaneously bind, condense and coat DNA resulting in the formation of lipid/DNA complexes, so-called lipoplexes . These complexes transduce plasmids into cells causing expression of the genes (transfection). Low levels of transfection circumvent therapeutical efficacy of non-viral strategies. Nuclear accumulation of plasmid DNA is among the major obstacles of non-viral gene delivery .
This project aims to analyse nuclear transport of lipoplex-released pDNA as a potential transfection barrier in two cellular models, A10 and MDCK, which have been characterized regarding their endocytic profile and transfectability in previous work . To study nuclear import of pDNA, a novel strategy called “quantitative imaging” is applied, a combination of confocal laser scanning microscopy and image-based analysis using the open source software CellProfiler. This technique enables to track cy3-labeled complex DNA inside the cell and to quantify the amount of nuclear-accumulated complex released pDNA.
These studies reveal nuclear entry of the pDNA to represent a significant transfection barrier in MDCK cells. The investigated cellular models differ significantly in the amount of nuclear-accumulated complex DNA: nuclear transport is by far more efficient in A10 compared to MDCK cells.
Androgens and estrogens increase the number of cell division and the opportunity for random genetic errors and are thus involved in carcinogenesis of hormone related cancers. Pre-receptor regulatory enzymes interconvert the active forms of hormones with high affinities to corresponding receptors to their less active forms. They represent interesting targets for development of new drugs for prevention and treatment of conditions caused by disturbed hormone action. We have focused our attention to four hydroxysteroid dehydrogenases (HSDs). AKR1C1 converts potent progesterone to a weak 20α-hydroxyprogesterone; AKR1C2 inactivates potent androgen 5α-DHT, and AKR1C3 reduces a weak androgen androstenedione to a potent testosterone. Both AKR1C3 and 17β-HSD type 1 activate weak estrogen estrone to a potent estradiol [1, 2]. We have isolated the recombinant AKR1C1, AKR1C2, AKR1C3 and over-expressed 17β-HSD type 1 in bacterial and mammal cells. Different approaches were used for searching the inhibitors of these enzymes and structurally different compounds were shown to be potent inhibitors (e. g. [3, 4]). Inhibitors with IC50 values in micromolar range are good starting points for further design and synthesis of new and improved inhibitors of this important group of enzymes.
This work was supported by a J3-9448 grant to T.L.R. and a young researcher grant to P.B., both from the Slovenian Research Agency.
Deamidation of proteins is probably one of the most common chemical degradation pathways in protein pharmaceuticals. The reaction rate for deamidation of human growth hormone (hGH) is particularly pH-dependent and occurs typically faster at neutral and alkaline pH .
Protamine from salmon is an extremely basic peptide due to its high arginine content. It is isolated from sperm cells of salmon where it naturally builds a complex with DNA during spermatogenesis due to electrostatic interactions . We hypothesize a similar electrostatic effect between negatively charged hGH and positively charged protamine residues. Arising cationic complexes are stabilized by electrostatic repulsion. This stabilizing complexation allows solubilization of hGH down to a pH of 5.4 at high concentrations up to 6.8mg/ml. The minimal solubilizing molar ratio between hGH and protamine was found to be at least 1:13 (hGH:protamine). Deceleration of deamidation at pH 5.4 could be detected by reversed-phase high-performance liquid chromatography (RPHPLC). The presence of protamine did not alter the conformational structure of hGH which was determined by circular dichroism (CD) spectroscopy and fluorescence spectroscopy. Binding interactions between protamine and hGH were investigated by isothermal titration calorimetry (ITC) and Small-angle X-ray scattering (SAXS).
This work was supported by FFG, Land Steiermark and SFG.
Protein denaturation processes involving aggregation are among the prime factors impeding the development of stable protein drug formulations. Not only does aggregation limit the shelf-life of protein pharmaceuticals and potentially decreases the overall efficacy of therapeutic, it may also cause unwanted side effects such as immune reactions. Denaturation kinetically coupled to aggregation can occur in all stages of the production process and its prevention constitutes a major effort in biopharmaceutical technology. A problem in designing rational strategies counteracting the aggregation is that a sound molecular basis underlying the denaturation process is usually not available. Dissecting the overall denaturation pathway into discrete kinetic steps would allow one to evaluate stabilizing effects of certain process conditions more systematically. Generally, understanding of aggregation during or induced by the manufacturing process is an important goal to be achieved by the pharmaceutical industry.
For our studies we used human growth hormone (hGH), which is a common therapeutic protein, and examined its aggregation mechanism under a range of process conditions. hGH is a single domain, globular protein consisting of 191 amino acids and has a molecular mass 22 kDa. Its tertiary structure shows 4 antiparallel α-helices and two disulfide bridges (53 to 165 and 182 to 189). Experiments were carried out under accelerated aggregation conditions. We show that the gas/liquid interface generated by aeration is strongly promoting aggregation. Stirring likewise resulted in aggregation. Results from SEC measurements and native or SDS PAGE of time-course experiments should help understanding aggregation and its kinetic pathway. This may help in developing more stable drug formulations.
This work was supported by FFG, Land Steiermark and Steirische Wirtschaftsförderung (SFG)
Unsaturated Monoglycerides (or Phytantriol) dispersed in water form particles having a cubic nanostructure over a broad temperature range. The cubic structured phase has a high viscosity which requires high energy input in order to be dispersed in water.
By the addition of lipophilic substances the structure can be changed to hexagonal, micellar cubic and micellar, respectively [1, 2]. The low viscosity of the micellar phase allows the use of more gentle dispersion methods.
Those nanostructured dispersions have a high interfacial area, which allows them to be good carrier systems for amphiphilic substances. Due to their oil and water compartments they can also host hydrophilic as well as lipophilic molecules. Furthermore, if substances are incorporated in the phases their stability against degradation is enhanced. These properties make them interesting as possible drug delivery systems.
With the structural change not only the viscosity of the self assembled phase alters but also properties which can affect the release rate. The diffusion inside the phase is heavily dependent on the nanostructure.
The internal structure can further be expanded by the addition of lamellar phase forming amphiphiles such as Diglycerol Monooleate and Phosphatidylcholine . The symmetry of the structure is maintained even thought the hydrophilic compartments are enlarged. This enables the incorporation of larger molecules such as proteins.
In order to be able to use nanostructured dispersions as delivery vehicles it is important to gain information about the release mechanism and possible ways to control it. For this purpose the release from the self-assembled bulk as well as from the dispersed phases is studied.
In the present investigation, vancomycin was incorporated in liquid crystalline gel made of glyceryl monooleate and water, with the aim to achieve a sustained-release and to deliver locally sufficient concentrations of vancomycin to the eye, improving its efficacy against bacterial infections [1–3].
Technique for implants preparation was developed involving combining glyceryl monooleate and vancomycin solution by vortex mixing, centrifuging, preparing the shape of implants and lyophilization.
In vitro characterization of liquid crystalline gel/implants included structure analyses by light microscope with polariser and small angle X-ray scattering. Furthermore, the drug release from the implants was studied.
Structure analyses of samples with different percentage of water showed, that samples with 10% water show a lamellar phase, whereas samples with 23–30% of water have a cubic phase, Ia3d, and the samples with 40% of water show a mixture of two cubic phases, i. e. Ia3d and Pn3m.
Investigating release properties of implants with an initial water content of 30% slow release was found. Approx. 40% of vancomycin was released within 2 hours.
The in vitro characterization of the liquid crystalline gel formulation/implants containing vancomycin showed the possibility to obtain different phases, i. e. lamellar phase and cubic phase as well as the possibility to sustain the release of vancomycin from them. Therefore, the implants show a great potential for the local treatment of infections of the eye.
Semisolid products are 8–10% of dosage forms, but – in contrast with the solid dosage forms – we do not have any validated method for their drug release in any Pharmacopoeia. Kinetics of release process and its critical factors in case of 1% diclofenac sodium containing hydrogel, organogel, gelemulsion, o/w and w/o creams were observed under in vitro conditions. Comparison of results between Franz diffusion cell and paddle over disk method was made using synthetic cellulose acetate membrane soaked in buffer solution or in isopropyl myristate. In vivo studies were carried out on male Wistar rats; the carrageenan paw edema decreasing effect of 12 different formulations was measured in comparison with a control group. All products reduced paw edema in rats, although we found significant differences among them both in vitro and in vivo.
Gel emulsions represent special drug delivery systems. Up to present only some authors dealt with the formation and properties of these systems, although the gel emulsions assurance interesting and useful possibility as controlled drug delivers systems [1–4]. Several methods can be applied by their preparations: i) gelling of internal water phase with hydrophilic polymers, ii) gelling of oil-phase with hydrophobic colloids, iii) in situ polymerisation, iv) application of gel emulsifying agents. Their advantages are as follows: i) prominent mucoadhesion, ii) capable of floating on the gastric fluid [2, 3, 5].
In our experiments water-soluble (polar) and water-insoluble (unpolar) active agents were applied. Composition of gel emulsions, e. g. the ratio of water- and oil phase, the concentration of surface active copolymers, addition of coemulsifiers was changed systematically.
The drug release from emulsions was investigated by Hanson SR8 plus equipment and Franz vertical diffusion cell. The rheological characteristics was measured by Paar Physica oscillation rheometer Optical studies were accomplished with Leica image analyzer, and thermal-analytic investigations were carried out with DSC 281. Mettler Toledo equipment.
Relationships were established between following parameters: i) drug release vs. mucoadhesion, ii) concentration of polymer emulsifying agents vs. drug release iii) presence of microgel layer surrounding oil droplets vs drug release, iv) concentration of oil phase vs drug release.
The topic of this article was to monitor the impact of excipients on viscosity of gels with local anaesthetic. In previous studies the optimal excipients from the group of polymers, enhancers and humectants were examinated during the formulation of chlorhexidine, trimecain and terbinafine to the medical form – hydrogel [1, 2]. The choice of gel creating compound is influencing the reological properties of drug as directly affects its consistency. Reological properties – viscosity – are very important at dermal semisolid drugs, mainly at gels from the aspect of their use. The manufacturing process is significantly influencing the gels consistency as well.
The reological properties were evaluated in the study of hydrogels prepared from various gel creatig compounds (Natrosol 250 HX, Natrosol 250 HHX, Natrosol 250 HR and Chitosan) with active ingredient of trimecaini hydrochloridum and gels without the active ingredient. Then the influence of propylenglycol and glycerol humectants in various concentrations on Mesocain gel were examinated. Solitary influence of excipients was evaluated using the analyses of reological measurement results.
This work was supported by the Grant VEGA No. 1/ 0320/ 08
Quercetin is a flavonol with antioxidant properties, exhibiting poor water solubility and transdermal permeation ability. The objective of this study was to formulate a new microemulsion for the transdermal delivery of quercetin based on the use of Transcutol® P as a solubilizing agent and absorption promoter.
Quercetin hydrated (>95%) was purchased from Sigma-Aldrich (Germany). All the excipients were kindly supplied by Gattefossé (France). The microemulsion was prepared by mixing the water and the lipidic phase under gently and continuous stirring at room temperature. Formulations were characterized for physical stability (centrifugation), pH, viscosity (viscosimeter Rheomat RM 180), internal phase dimensions (optical microscope, Leitz), in vitro transdermal diffusion. For this last experiment, 6 vertical Franz’s cells were used. A polypropilen membrane (Celgard® k-381) soaked up with Labrafac® lipophile WL1349/CHCl3 (1:3) was used. The quercetin diffusion was determined by VIS-spectrophotometry.
The solubility of quercetin was spectrophotometrically determined in different excipients and whose in which it was more soluble were selected.
Through a ternary diagram, the percentages of the following phases were determined: lipidic phase, water phase, ratio surfactant/co-surfactant. The best formulation was: 1% of quercetin, 16.67% of water phase with 40% of Transcutol® P, 16.67% of lipidic phase (Labrafil®), 66.67% S/coS (Labraso®l, Capryol® 90). The most stable microemulsion was an O/W microemulsion, appearing as a yellow transparent liquid, with an internal phase size of 10–50 nm and a pH of 5.0. The dynamic viscosity was 0.068±0.003 Pa*s. The quercetin diffusion, compared to other different formulation, was good. From this study it was possible to highlight the fundamental role of the Transcutol® P in increasing the quercetin solubility in the microemulsion and as absortion promoter through the membranes.
Due to its topical irritant effect on gastric mucosa naproxen is an ideal candidate for encapsulation in pH sensitive drug delivery systems such as alginate– chitosan microparticles. In this study we aimed to investigate the feasibility of these carriers for oral delivery of naproxen, and to establish the kinetic mechanisms of naproxen release from microparticles under conditions simulating gastrointestinal environment. Sixteen different microparticles formulations in range between 262.3±14.9 and 358.4±21.7 μm have been prepared by well-known two-stage procedure using custom made air-jet device. Differences among formulations were hardening time in chitosan solution, drug/polymer ratio, chitosan molecular weight and chitosan concentration. After 2 h in acidic media (pH 1.2), naproxen release was less then 6.18% for all investigated formulations due to non-porous surface and low swelling ability of chitosan–alginate gel network in acidic environment. When the pH of the surrounding media was raised to 6.8, swelling of microparticles occurred, leading to the rapid release of encapsulated drug. ANOVA analysis revealed that all investigated factors have statistically significant influence on drug release from microparticles (p<0.05). Furthermore, ANOVA analysis showed existence of statistically significant negative interaction between chitosan molecular weight and its concentration in hardening medium. This observation may be explained by influence of the hardening medium viscosity on the formation of chitosan-alginate gel network. Naproxen release data obtained during second phase of experiment (pH 6.8) showed best fit to the Korsmeyer-Peppas equation, which can be confirmed by comparing the values of the correlation coefficient with those of other investigated models. The values of the Korsmeyer–Peppas release exponent (n) determined for all investigated formulations varied in range from 1.62 to 3.73 suggesting Super Case II transport mechanism for the release of naproxen from microparticles. Super Case II transport occurred due to a large increase in osmotic pressure driving forces, followed by rapid relaxation and swelling of alginate chitosan gel network.
The results indicate that chitosan-alginate microparticles represent convenient gastroresistance delivery system for oral administration of naproxen due to release controlled by pH dependent swelling ability.
Alginate-based microparticles have been widely investigated for applications like enzyme immobilization, immunoisolation in cell transplantation and drug release systems. Using the custom made air-jet device, by varying processing as well as several formulation factors, we aimed to prepare and investigate alginate-chitosan microparticles loaded with phenytoin, a standard antiepileptic agent. The extended-release formulations of antiepileptics simplify treatment of this chronic condition. Such commercial formulations with phenytoin appear to be well designed, with one possible shortcoming being the potential for irregular absorption that appears to occur particularly in elderly patients . The final aim of the project was to investigate microparticles loaded with phenytoin with possibly improved liberation profile. In order to evaluate the influence of formulation factors and hardening time in a chitosan solution on drug release rate, 24 full factorial design was used. The independent variables (inputs) investigated were the calcium chloride concentration (X1), chitosan molecular weight (X2), chitosan concentration in coating solution (X3) and hardening time (X4). The times for 50% and 90% of the drug to be released – t50% and t90% for each formulation were calculated and used as response parameters (outputs). ANOVA analysis revealed that among investigated factors X2, X3, X4 as well as interactions X2–X3 and X3–X4 have statistically significant influence on drug release from microparticles (p<0.05). Since no significant influence of X1 on drug release rate was observed, it was excluded from further statistical analysis. Calculated factor effects of investigated variables indicate that lower level of X2 (combination of high and low molecular weight chitosan), higher levels of X3 (0.2% w/w chitosan concentration in coating solution) and X4 (24h hardening time) favour sustained release of phenytoin. The present study suggests that alginate-chitosan microparticles may be used as a delivery system for sustained release of phenytoin with key factor affecting the release rate being the combination of hardening time, chitosan concentration and molecular weight.
Development of drug delivery systems is an indispensable strategy for successful transport of drug to its therapeutic site by the appropriate choice of carrier and route. Solid lipid nanoparticles (SLN) include the advantages of conventional carriers and are additionally utilized for protection of labile compounds, as well as in controlling of drug release, targeting and stability.
In general, physicochemical characteristics of SLN surface are those, which affect their behaviour both in vivo and in vitro [1, 2]. Consequently, development of appropriate design technique is crucial to effectively estimate the potential offered by SLN. The main objects of current research were to evaluate nanoparticles morphology (size, distribution, shape, structure, integrity), and their nano-mechanical properties such as adhesion forces and local hardness. Atomic force microscopy (AFM) has been used for these studies. More precisely, phase imaging as an extension of the tapping mode AFM, provided the mapping of the surface properties variations, such as composition, adhesion, friction and visco-elasticity.
SLN unlabelled and particles labelled with different coumarin based fluorescent dyes such as SPP 189 (SLN SPP-189) and 6-coumarine (SLN C), produced by melt-emulsification process were circular in shape. Photon correlation spectroscopy and AFM provided approximately the same particle size of investigated samples. Surface roughness of SLN SPP-189 was significantly elevated in comparison to unlabelled SLN, what should be the consequence of into surface incorporated fluorescent dye. Phase imaging and force spectroscopy measurement estimated the surface heterogeneity and local surface hardness. Phase contrast confirmed two different regions of SLN SPP- 189, which could be associated with surface heterogeneity caused by diverse nature of main constituents as well as fluorescent dye. Only these nanoparticles demonstrated light hallow in phase image, likely related to inhomogeneous distribution of lipophilic ingredients that also reflects differences in local surface hardness.
Current research will be additionally complemented with analysis of particles-cells interaction in order to improve the understanding of SLN composition involved in the cell uptake and in cell drug delivery.
The enhancement of dissolution rate is one of the most commonly used approaches to improve the bioavailability of drugs, since for a great extent of new drug substances their absorption is limited with their dissolution rate. The aim of this work was to prepare and optimize microparticles containing ketoprofen using spray-drying technology, which would enhance the dissolution rate of ketoprofen, which is poorly soluble at lower pH values. Different hydrophilic polymers with relatively low melting point (polyethylene glycol 4000 and 6000, poloxamer 188, Gelucire 44/14 and 50/13) were used to prepare microparticles by spray drying. Due to the low melting point of the polymers, spray drying parameters were carefully optimized to ensure reasonable yields. Of the process parameters, atomizing pressure was shown to be the most important factor influencing product yield. Product yield was also greatly impacted by the adhesion of the particles to the inside wall of the spray dryer due to the sticking of the carrier material. A positive correlation between the melting point of the polymers and the product yield was established - the lower the melting point of the polymer, the lower was the yield. Carriers with promising yield (polyethylene glycol 4000 and poloxamer 188 with the average yield of 34,4% and 43,0%, respectively) were chosen for the preparation of the drug loaded microparticles. A reference sample was prepared using lactose as a carrier. In all cases ketoprofen was not dissolved in the final mixture and ketoprofen suspensions in carrier polymer solutions were spray dried. During the preparation of drug loaded microparticles, the drug concentration in the suspension was shown to have a great impact on the overall yield. Due to extremely poor yield when polyethylene glycol 4000 was used as a carrier, only samples prepared with poloxamer 188 and lactose were further studied. Image analysis was used to determine the size and shape of microparticles, which were recorded by scanning electron microscope (SEM). The dissolution rate of ketoprofen from the microparticles prepared with poloxamer 188 was enhanced compared to microparticles prepared with lactose as a carrier. All prepared microparticles exhibited markedly increased dissolution rate compared to ketoprofen alone. The differential scanning calorimetry (DSC) confirmed a similar thermal behavior of ketoprofen microparticles and corresponding physical mixtures which indicates the absence of chemical interaction between ketoprofen and water soluble carrier. It was also shown that there was no significant conversion of crystalline ketoprofen to the amorphous form during the spray drying process.
In recent years there has been increasing attention given to the utility of self-microemulsifying systems (SMES) for improving the gastrointestinal absorption of drugs with inadequate biopharmaceutical properties. Microencapsulation of those systems into polymeric matrix is one of the solidification techniques that allow formulation of solid self-microemulsifing formulations.
Microcapsules with furosemide-loaded self-microemulsifying core that merge the advantages of SMES with those of solid dosage forms where produced by co-extrusion of liquid jet by vibrating nozzle device. The composition of self-microemulsifying core was optimized previously ; the aim of this study was therefore to modify the shell-forming phase in order to gain repeatable production of microcapsules with high encapsulation efficiency. The influence of lactose content in microcapsules` shell on drug release profile from dried micro-capsules and their swelling and erosion behaviour was examined additionally.
Best shaped microcapsules with highest encapsulation efficiency were obtained from the shell-forming phase with the Ca-alginate/pectinate ratio of 1/3 containing 5–10% lactose . Incorporation of furosemide in self-emulsifying core of microcapsules resulted in considerably enhanced drug release profile when compared to reference microspheres, which did not contain SMES. Drug release from microcapsules containing different content of lactose was further affected not only by pore inducing effect of lactose but also by the formation of hydrated visocous layer around the capsules, which acted as a barrier to drug release. The presented results constitute a step cloaser to a repeatable production of microcapsules with self-microemulsifying core by using method of liquid jet co-extrusion by vibrating nozzle device. Better solubility and permeability properties obtained with self-microemulsifying core also provide a promising alternative to ensure succesfull oral delivery of drugs with poor biopharmaceutical properties.
Egg lecithin is a complex mixture of phosphatides that consists mainly of phosphatidylcholine and phosphatidylethanolamine, combined with various amounts of other substances such as triglycerides and fatty acids . As pharmaceutical excipient lecithin is used in parenteral emulsions, liposomes and mixed micellar solutions. It has been proved, that aqueous dispersions of lecithin (Water-Lecithin Dispersion, WLD) may be used as safe and biocompatible solubilizing carriers for parenterally administered drugs with low solubility, such as paclitaxel [2, 3].
The aim of the study was to evaluate the solubilizing properties of WLD (containing 5% or 10% of lecithin) using 9 different active substances, demonstrating low water solubility: cyclosporin A, sulfamethoxazole, metronidazole, dexamethasone, hydrocortisone, carbamazepine, prednisolone, theophylline and testosterone. The solubility in water and WLDs was studied for untreated and lyophilized substances.
For solubility determination, active substance was suspended in 10 ml of water, 5% or 10% WLD and the mixture was stirred for 24 h at room temperature. Then the dispersion was centrifuged for 15 min and the supernatant was filtered and analysed by means of spectrophotometric or HPLC method.
Results indicated, that the solubility of the tested drugs in WLD was at least few times higher than in water and the amount of dissolved substance increased with the increase of the lecithin concentration in WLD. The only exceptions were drugs with relatively high water solubility (metronidazole, theophylline), for which WLD did not increase solubility. The highest increase in solubility was observed for cyclosporin A, for which measured solubilities were as follows [mg/ml]: 0.03 (water), 3.9 (5% WLD) and 5.7 (10% WLD). Microscopic analysis indicated, that in freeze-drying process reduction of the drug particles size was achieved and this resulted in much faster dissolution of the compounds in WLD. Due to biocompatibility, WLD may be considered as a carrier for poorly soluble parenterally administered drugs.
In modern pharmaceutical technology a number of methods offer themselves to enhancement of dissolution and rate of dissolution. The insert to a micelle or an inclusion complex and conjugatum between active agent and a polymer molecule can increase both solubility and the rate of dissolution, but then the microencapsulation increases only the rate of dissolution [1–3].
Aim of our research was to increase of solubility and rate of dissolution of poorly water-soluble active agents. Seven active agents poorly, slightly and very slightly soluble in water were investigated. In the course of preformulation the wettability of these active agents with different polymer solutions was studied. The contact angle of wetting was measured by OCA 20 equipment (produced Dataphysics). On the grounds of contact angle values the polymers with best wettability was chosen, which – presumaby – is most suitable to prepare microcapsules. The microcapsules was produced with Büchi’s spray drying equipment.
The microcapsules was produced with Büchi’s spray drying equipment. Relationships were appointed between the technical parameters of spraying (e. g. rate of spraying, the temperature of incoming and outgoing air) and the properties of microcapsules (e. g. size distribution, morphology and surfaces, and dissolution rate).
Diazepam is one of most frequently prescribed benzodiazepines. It is classified as a class I substance according to the Biopharmaceutics Classification Scheme (BCS). Diazepam tablets were prepared using different API raw-materials. Before the formulation, we measured particle size distribution on Malvern Instruments Ltd Mastersizer 2000. The tablets were prepared by employing conventional wet granulation. The formulations were evaluated for hardness, friability, content uniformity, in vitro disintegration time (DT), release profiles. The dissolution profile testing is performed on the two formulations with different particles size distribution.
Test parameters were: medium 0.1 mol / l hydrochloric acid, 900 ml, temperature 37°C ± 0.5°C, Mixing Speed 100 rpm, Number of tested tablets 12, Testing Cycle 10, 15, 20, 25 and 30 min. Instrument: Varian Van Kel VK7025, Varian Cary 50. We used the spectrophotometric method with absorbance measuring on 242 nm for quantification.
Results for particle size distribution for first Diazepam API particle distribution were d (0.10): 88.743 μm; d (0.50): 305.231 μm; d (0.90): 655.911 μm, and for the second raw-material of API was d (0.10):1.998 μm; d (0.50): 4.419 μm; d (0.90): 8.845 μm. The results of diazepam dissolution for two formulations and f2 value with reference drug are presented in Table 1:
It was concluded on the basis of formulation tests that Formulation B, for which micronized API was used, has a similar dissolution profile for API as compared to the reference medicine (f2=99.94) in comparison to Formulation A (f2=68.15)
Many drug substances exhibit low solubility in water, which results in their poor bioavailability. Among ophthalmic preparations, oil solutions, suspensions and ointments are formulations prepared for such compounds. Promising modern drug carries are self-emulsifying drug delivery systems (SEDDS) which are isotropic mixtures of oils and surfactants. Due to presence of surfactants improved solubility and transcorneal passage of the active substances can be expected .
The aim of this study was to compare the in vitro release of indomethacin (IND) and hydrocortisone (HC) from SEDDS and aqueous or oily suspensions. The release experiments were carried out for 6 h at 37°C using dialysis cellulose membrane and acceptor fluid imitating composition of lacrimal fluid.
Amount (mg) of the drug and the percentage of the dose delivered to the acceptor medium was measured and diffusion coefficients were calculated. After 6 h about 7 mg (40%) of IND was released from SEDDS and about 5 mg (30%) from 0,6% aqueous and oily suspensions. Only small percentage of HC was released (below 20%) from the aqueous and oily suspensions. However percentage of hydrocortisone released from SEDDS (containing 1% or 5% Cremophor EL) came to over 60%. The results suggest that increased solubility in SEDDS  does not necessarily increase diffusion through the dialysis membrane and in vivo effect on bioavailability can not be easily predicted.
In vitro dissolution testing serves as an important tool for drug manufacturing process control and quality assurance. In certain cases it can serve also as an indicator of how the formulation will perform in vivo .
Cefaclor, cephalosporin antibacterial drug, was chosen as a model drug, classified in BCS class III (high soluble and low permeable) . It is effective against many different bacterial organisms such as Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, E. coli, and many others .
The aim of this study was to investigate the impact of experimental conditions on drug release from capsules having the same composition, but containing 250 mg (product A) and 500 mg (product B) of cefaclor. Solubility, the drug intrinsic dissolution rate and in vitro drug release tests were performed in water as well as in three EP buffer media: pH 1.2, pH 4.5 and pH 6.8.
It has been shown that the solubility of cefaclor was two times lower comparing to that observed in other tested media. The drug intrinsic dissolution decreased in the following order: pH 1.2>pH 6.8>water>pH 4.5. The dissolution media composition strongly, and to variable extent, influence cefaclor release from the investigated drug products. At pH 1.2 the drug dissolution was very fast with more than 85% of cefaclor dissolved within 15 and 30 minutes for product A and B, respectively. In the case of EP pH 4.5 buffer medium, the drug dissolution was notably slower and, consequently, more than 75% of the drug dissolution was accomplished after 60 minutes. The employment of EP pH 6.8 buffer medium resulted in slow and incomplete drug release. The differences between the release profiles obtained for the investigated drug products A and B in each tested dissolution medium were found to be statistically insignificant. In addition to the drug characteristics, it is evident that the formulation factors strongly affected cefaclor dissolution rate and must be taken into account in selecting a test method that will be sensitive enough to reflect its bioavailability.
Due to complexity and interplay of variable gastrointestinal conditions with matrix tablets which are predominantly erosion controlled it is often difficult to predict in vivo release profile . Nascent gel layer of matrix tablets is prone to mechanical stress which is difficult to simulate in vitro.
Present study describes approaches to establish a biorelevant in vitro test for evaluation of two different matrix tablets containing a BCS class 2 model drug. Mechanism of release was also evaluated. Obtained results were compared with in vivo pharmacokinetic data (Cmax) and possibilities for IVIVC or IVIVR were explored.
The tablets were first tested with conventional dissolution tests. Secondly, a test that simulates mechanical stress potentially occuring during transition of tablets through GI tract was performed . This test incorporates glass beads manipulation. Additionally, a new approach using dissolution apparatus USP3 with plastic beads was also explored.
Obtained results were used to elaborate the mechanism of release from the tablets. One formulation was accelerated after applying the mechanical stress, while the other formulation exhibited no changes and followed a 0. order release. This was proven by calculating the Korsmeyer-Peppas release rate constants.
The release mechanism was further examined by inspecting the rates of dissolution ΔQ/Δt before and after mechanical stress manipulation. IVIVR with Cmax results was also shown, the dissolution profile when approximately 70% of the drug is released being relevant for in vivo.
The results from dissolution apparatus USP3 and plastic beads test were used to develop an IVIVC model. Based on this model the maximal plasma concentration (Cmax) was predicted and compared with the observed values from the in vivo BE study. The average absolute percent prediction value for each tested matrix tablet was less than 15%, which demonstrates a good internal predictability of the IVIVC model.
The holt-melt extrusion technique has proven to be an advantageous method for preparation of solid dosage forms. The aim of this work was to evaluate the release profiles of sustained release cylinders produced by hot-melt extrusion. The cylinders were composed of inner and outer part, both containing theophylline . Cylinders were 0.5cm or 1cm long. The two parts were extruded separately, cut into pieces and joined manually. Dissolution testing was performed on USP 2 apparatus at rotation speed 50, 100 and 150 rpm. Artificial gastric and intestinal media were used consecutively.
Due to their hydrophilic / lipophilic properties inner part of cylinder releases the drug fast while outer part represents extended release component. No influence of stirring rate on theophyline release was observed for 1cm cylinders while for 0.5cm cylinders the amount of released drug slightly increased with increase of stirring rate (Fig. 1). However, due to high variability of release results significant differences could not be proven. This high variability of release results was beside other factors (poor wettability due to lipophilic components etc.) at least partly ascribed to manual preparation of the dosage forms. Manual joining of the inner and outer part of the cylinder caused void spaces between both parts as shown on the stereomicrographs (Olympus SZX12) (Fig. 2). The consequence of void spaces is additional free surface available for dissolution which can not be controlled and thus increases the variability of release results. Thus, the automatic simultaneous co-extrusion would be better choice when preparing co-extrudates as void spaces between two parts of cylinders could be avoided and variability at least partly reduced.
The aim of this research is to prepare Moxifloxacin hydrochloride immediate release tablets by wet granulation using experimental design technique. Moxifloxacin is a fourth-generation fluoroquinolone that has been shown to be effective against Gram-positive, Gram-negative, and atypical strains, as well as multi-drug resistant Streptococcus pneumoniae.  Optimization has proven as an effective tool in product development. The purpose of carrying out optimization is to select the best possible formulation from pharmaceutical as well as consumer point of view. High dose drug that experience poor flow and poor compactibility was granulated to obtain suitable flow and cohesion for compaction. The selection of suitable filler and binder in the formulation of immediate release tablets and granulate residual moisture are very important because they affect physical parameters and in-vitro release profile.  The experimental design 23-1 was applied with following independent variables: filler type (X1), binder type (X2), granulate residual moisture (X3). Weight variation, thickness, hardness, friability, disintegration time and drug release after 15 minutes were examined as dependent variables.  Different fillers examined were Mannitol and Cellulose microcrystalline. Different binders examined were Maltodextrin and Povidone. The tablets prepared using Cellulose microcrystalline as a filler and Povidone as a binder in optimized formulation showed the best comparable results for physical parameters. In-vitro release profiles were tested in 900ml 0.1 M HCl, at 37 ± 0.5°C, in USP paddle apparatus with stirring rate of 50 rpm. Percent of drug released was determined by UV/VIS spectrophotometric method. Taking into consideration the fact that more than 85 % of the drug is released from product and optimized formulation within 15 minutes, in-vitro release profiles are considered similar without further mathematical evaluation. Analysis of the obtained results showed that the binder type (X2) and granulate residual moisture (X3) have significant influence on response parameters. The other independent variable, filler type (X1) does not have significant influence.
Administration of pellets in fed stomach state can prolong the gastric emptying of pellets in comparison to the administration under fasting conditions . On the basis of literature data we aimed to investigate the influence of caloric value of food on gastric emptying of pellets.
A systematic literature search on evaluation of human gastric emptying of pellets based on the technique of gamma scintigraphy was performed in the MEDLINE database. Studies estimating the gastric emptying of pellets under fed conditions were focused. In addition to the caloric value of the meal, a special attention was made on other factors such as, pellets size and density, pellets formulation (filled into capsules or compressed into tablets), a time delay for pellets administration in relation to the time of meal completion, and refreshments provision. The majority of pellets gastric emptying data was presented in terms of pellets t50 values – a time when 50% of the administered pellets emptied the stomach. In order to evaluate the influence of the caloric value of the meal on pellets t50 values a linear regression model was built. In this model the caloric value was regarded as a categorical variable, which was recorded into dummy variables with the lowest caloric value set as a reference.
In total 17 studies were recovered, however, only 6 of them met the selection criteria: pellets density between 1.2 and 1.5 g/cm3, administration of the pellets at the time of meal completion, pellets filled into capsules, and pellets size in the range from 0.6 to 1.4 mm. In these studies 48 individual values of pellets t50 were collected (6 or 8 subjects per study) and the obtained caloric values were 1200, 1500, 2300, 2800, and 3600 kJ. Mean value of t50 of pellets administered right after the 1200 kJ meal was 136 min. In comparison to the 1200 kJ meal, the mean values of t50 of pellets administered right after 2800 and 3600 kJ meal were significantly longer; 212 min (p=0.031) and 263 min (p=0.003), respectively. No significant difference in terms of mean values of pellets t50 could be noted between the 1200 kJ meal and 1500 or 2300 kJ meal.
Our results suggest that gastric emptying of pellets can be more prolonged if administered along with the food with higher caloric value.
Pellets are an attractive dosage form in the gender specific and pediatric therapy, since they offer the possibility of patient adapted dosing. Additionally, they are easily swallowed and unpleasant taste masking by coating is facilitated due to their smooth and homogenous surface. The development of disintegrating pellets is a new and innovative technology. The main advantage of this type of dosage form is the circumvention of the hepatic and the first pass metabolism . In the present study Ludiflash®/Kollidon® CL-SF (crospovidone) mixtures were used to prepare disintegrating micropellets via a wet extrusion/spheronization process.
Powders were blended and wetted with different water/ethanol mixtures (i. e., 10% 20%, 30%, 40% and 50% ethanol). The wet mass was subsequently extruded through a 0.8 mm multihole die plate and spheronized at different speeds for 2–4 minutes. Finally pellets were dried using three different methods (i.e, desiccation, tray drying and fluidized bed technology). The disintegration behavior in water at 37 ± 0.5 °C was evaluated according to Pharm. Eu. 6.0 2.9.1. In addition, the mechanical characteristics (tensile strength, friability) and the pellet shape were investigated.
For pellet formulations containing 5% Kollidon® CL-SF a minimum ethanol fraction of 30% in the granulation fluid was required for successful extrusion/spheronization. At lower alcohol contents extensive liquid movement occurred during extrusion resulting in non-reproducible process conditions. Pellets prepared with 30% and 40% ethanol disintegrated within 1 minute. Using 50% ethanol, however, yielded in increased disintegration times (>1min). Pellets containing 10% crospovidone were successfully prepared with all granulation liquids under investigation. Disintegration times exceeded 1 minute when the ethanol fraction was higher than 20%. All pellet formulations exhibited sufficient mechanical stability and an aspect ratio below 1.2.
Ludiflash® in combination with either 5% or 10% Kollidon® CL-SF is a promising extrusion and spheronization aid for the preparation of pellets showing disintegration times below 1 minute.
The objective of this study was to develop solid self-emulsifying pellets to deliver the poorly bioavailable Milk Thistle extract (Silybum Marianum) . These pellets were prepared via extrusion/spheronization procedure, using a self-emulsifying system or SES (composed of Akoline MCM®, Miglyol®, Tween 80®, Soy Lecithin and Propylene Glycol), microcrystalline cellulose and lactose monohydrate. To select the most suitable formulations for extrusion and spheronization, an experimental design of experiences was adopted. The screening amongst formulations (13 different blends) was carried out preparing pellets and evaluating extrusion profiles and quality of the spheronized extrudates. The pellets were characterized for sizing and shape, density and hardness. Although more than one type of pellets demonstrated adequate morphological and technological characteristics, pellets having best properties were selected for further biopharmaceutical investigations, including in vitro dissolution, determination of released droplet size and in vivo trials on rats to study serum and lymph levels after oral administration of the pellets. These preliminary technological and pharmacokinetic data demonstrated that extrusion/spheronization is a viable technology to produce self-emulsifying pellets of good quality and able to improve in vivo oral bioavailability of main components of a phytotherapic extract, also enhancing its lymphatic absorption.
The authors thank Indena for the kind gift of Sylibum Marianum dry extract.
In biomedical applications, it should be useful to have a system easy to administrate (the best would be a liquid injectable form), with structural properties in physiological conditions (the best would be a soft gel), able to resist to external stimulus (such us the erosion from body fluids, in this case the surface should be a hard gel). Such a system could be used as a drug delivery system, as well as a temporary scaffold.
From previous investigation, we found out that the acqueous solution of Pluronic F127 and alginate (18% F127 + 2% alginate), is liquid at low temperature (around 5°C), it gives a soft gel due to the Pluronic thermogelation at body temperature (around 37°C), and it gives a hard gel after the exposition at solutions of bivalent cations (reticulation, using Ca2+, Cu2+).
Prior of any other application, the system has to be thouroughly characterized. Therefore, 1) the mechanical properties of the solution were investigated by using rotational rheometry (monitoring the increase of solution moduli during heating); 2) the compressive behavior was investigated with impermeable and porous parallel plates (underlining the rule of the water to build up the properties of the gel); 3) the kinetics of cuprum diffusion and of hard gel formation were quantified (obtaining the thickness of reticulated layer – see the Figure – as function of cuprum solution concentrations and of exposure time. In the Figure, the symbols are experimental data and the surface curve is the model prediction). Further investigations are still ongoing. The data gatered will be used for the proper design of future biomedical applications.
The scope of the present work was to establish the role of the polymer charge on its ability to enhance the permeability of the urinary bladder wall.
Cationic poly-L-arginine and chitosan, anionic sodium carboxymethyl cellulose (NaCMC) and alginate as well as nonionic hydroxypropyl cellulose (HPC) and hydroxypropyl methylcellulose (HPMC) were tested. The polymers were tested in concentrations from 0.00005 to 1% (w/v) and a concentration of a model drug pipemidic acid (PPA) was 0.014% (w/v) in all experiments. All solutions were prepared in phosphate buffer and their pH was adjusted to 4.5. In diffusion cells the luminal side of isolated pig urinary bladder wall was exposed to tested solutions. In some experiments the tissue was exposed for 60 min to a polymer dispersion with PPA, while in other experiments the tissue was first exposed to a polymer dispersion for 45 min, rinsed and then the solution of PPA was applied for additional 45 min. At the end of the experiment the tissue was frozen and sectioned parallel to luminal surface. PPA was extracted from the tissue segments and its concentration was determined by HPLC.
We showed that the charge of the polymer affects its ability to enhance permeation of PPA into the urinary bladder wall, but to a limited extent. Positively charged chitosan and poly-L-arginine were established as the most promising absorption enhancer into the bladder wall. Compared to chitosan, higher concentrations of poly-L-arginine were needed to significantly increase the tissue permeability. Moreover, poly-L-arginine increased the permeability within 90 min constantly, while chitosan reached the plateau of its absorption enhancement activity after 60 min. In our previous study anionic polymer polycarbophil significantly increased permeation of PPA into the bladder wall . However, in this study anionic NaCMC and alginate did not significantly influence the permeation of PPA into the tissue. Interactions between NaCMC or alginate and PPA might prevail over the potential effect of NaCMC and alginate on the tissue permeability. Moreover, nonionic polymers HPMC and HPC did not significantly influence the bladder wall permeability.
We can conclude that positive charge has an important influence, but the selection of absorption enhancers for intravesical drug delivery systems can not be done exclusively on the basis of a polymer charge.
General anesthetic drugs were introduced more than 160 years ago and are indispensable in daily surgery at hospitals. Interestingly, their mode of action remains largely unresolved. At present, there are two schools, one favoring specific (direct) interactions of the drugs with proteins of the central nervous system and a second adhering to nonspecific mechanisms through a modulation of biophysical membrane properties. We have focused on the latter mechanism and studied the effects of R-(−), S-(+)-ketamine and propofol on the biophysical properties of lipid model membranes composed of palmitoyl oleoyl phosphatidylcholine by a combination of X-ray diffraction and all-atom molecular dynamics simulations. In agreement with several previous studies, we do not find significant changes to the overall membrane structure up to 8 mol % drug content. However, we observed that the insertion of drugs within the lipid/water interface caused significant changes of lateral pressures within the membrane . The changes are predicted to be stereospecific and large enough to affect the opening probability of ion channels at concentrations that compare remarkably well to clinically applied concentrations. Our results thus provide a novel basis for rational anesthetic drug design.
Cartilage lesions observed in osteoarthritis (OA) are related to an important release of cytokines. Current treatments though oral or systemic administration are suboptimal and not curative. Intra-articular administration of drugs is often proposed for localized forms of OA. MAPK inhibitors target important cytokines pathways and show promise for the treatment of OA .
The aim of the present work was to formulate nano- and microparticles loaded with a p38 MAPK inhibitor (VX-745) and to test their in vitro activity on human synoviocytes. We anticipated that this novel formulation will provide a high but local concentration of the active ingredient with a prolonged retention time.
Particles of different sizes were produced by a solvent evaporation method using a solution of PLGA and of VX-745 in dichloromethane and a solution of PVAL. The particles were characterized by laser diffraction, dynamic light scattering, scanning electron microscopy and reverse-phase HPLC.
In vitro studies were conducted by incubating nano- and microparticles with subconfluent human synoviocytes culture obtained from OA synovial samples. The ability of the particles to release the drug and consequently to inhibit the IL-6 biosynthesis was quantified by ELISA (eBioscience, San Diego, CA).
Spherical particles had a smooth surface and mean diameter of 300 nm, 2.5 μm and 25 μm. After 24 h of incubation with synoviocytes, IL-6 production was inhibited by VX-745-loaded nanoparticles in a dose-dependent and size-dependent pattern. For instance, the release of VX-745 from nanoparticles loaded at 100 nM inhibited IL-6 release to 52% compare to control and to 71% of control for a drug concentration of 800 nM.
To conclude, VX-745-loaded particles display extended release properties and inhibit significantly the production of IL-6 from human synoviocytes.
This work was supported by the De Reuter Fondation, Geneva, Switzerland.
Both the active pharmaceutical ingredient and the vehicle physicochemical characteristics are retained to be the main features responsible for favorable topical bioavailability. Emulsion systems based on natural-origin alkyl polyglucoside (APG) emulsifier (cetearyl glucoside & cetearyl alcohol) present promising vehicles in contemporary pharmaceutical compounding , but require further characterization using various model drugs. To this aim, APG test vehicles (with and without isopropyl alcohol addition) were loaded with ketoprofen as a model NSAID known for its non-optimal skin delivery characteristics , and compared to corresponding reference vehicles (Nonionic hydrophilic cream, DAB 2006). Physicochemical characterization included polarization microscopy, rheology and thermal analysis (DSC, TGA). In vitro release screening, in vitro skin permeation (Franz-type diffusion cells) and in vivo tape stripping were performed in order to evaluate ketoprofen biopharmaceutical profile. Additionally, safety considerations were assessed by in vitro skin irritation test (citotoxicity assay), alongside in vivo skin bioengineering measurements. Results obtained by tape stripping technique, showed significantly higher ketoprofen penetration profiles for test samples in comparison to reference ones. Although IPA addition tends to enhance ketoprofen penetration (probably through solubility enhancement – assessed by polarization microscopy) its influence cannot be considered significant (ANOVA, post hoc Tukey test). Ketoprofen demonstrated satisfactory but similar permeation and release profiles in investigated samples. Results of biopharmaceutical characterization comply with conducted physicochemical investigation, emphasizing overall difference in test and reference vehicles’ microstructure. APG vehicles showed satisfactory safety profile and skin performance, as well as suitable characteristics when loaded with ketoprofen.
In recent years, there has been an increasing interest in cocrystallization of active pharmaceutical ingredients. For example the physical stability can be enhanced by cocrystallizing . Thus, using cocrystals as solid-state forms is an attractive alternative. Theophylline and nicotinamide are known to form cocrystals for example via solid-state grinding . However, in appropriate conditions cocrystals can also form spontaneously .
The purpose of this work was to study whether teophylline and nicotinamide form cocrystals spontaneously. Theophylline and nicotinamide powders were gently mixed manually at 1:1 molar ratio and stored at different relative humidity conditions. The solid state of the samples was analysed by differential scanning calorimetry (DSC, Mettler Toledo DSC823) and Raman spectroscopy (Kaiser Rxn1 equipped with a PhAT probe). Three different variations of theophylline were used as starting materials, e. g., two size fractions of theophylline anhydrate (small: sieved through a 355 μm sieve; large: the fraction remaining on the sieve), and monohydrate (recrystallized from water). For reference, pure TP-NCT cocrystals were prepared by solid-state grinding with a Retsch MM400 mixer mill.
TP-NCT cocrystals can form spontaneously from manually mixed physical mixtures of TP and NCT during storage without any mechanical activation. For anhydrous samples, increasing storage humidity has an accelerating effect on the cocrystal formation. Particle size of the starting material was found to affect the cocrystal formation kinetics, especially at high relative humidity conditions. Polymorphism of the starting material (i. e. TP anhydrate vs. monohydrate) leads to complicated solid-state behavaviour during storage, particularly when TP anhydrate containing samples are stored at high humidity and vice versa.
Pharmaceutical powders intended to target the deeper regions of the lung have to consist of particles with aerodynamic diameters in the range of 0.5 μm - 5 μm. In such fine powders several forces, mainly Van der Waals and electrostatic forces, have strong influence on particle interactions. Particle interactions significantly determine material-loss during powder handling for example in mixing containers, flow-behavior of the powder and the amount of drug released from the inhaler. In addition to this, electrostatic charge affects the deposition of the powder in the lung. Since most pharmaceutical powders are insulators, electrostatic charge is acquired during every contact of the particles among themselves or with other surfaces. The phenomenon of the emergence of electrostatic charge in common powder handling processes has been investigated by several scientists such as Murtomaa , Rowley  and Zhu . Particularly Zhu examined the charging behavior and influencing variables on the arising of charge of an interactive mixture of salbutamol sulphate and lactose monohydrate in the mixing process.
The aim of this work is to extend these mixing studies with respect to powder blend composition and mixing conditions. The charging behavior is investigated in a T2F Turbula® mixer using a Faraday cup connected to a high resistance electrometer for charge measurement. When a powder is mixed the charge accumulates until a saturated level is reached. The sign and magnitude of the charge and the mixing time required to reach saturation is an important information in the evaluation of the charging behavior of the powder blend. The acquired charge is determined by several factors such as energy of mixing, mixing time, mixing ratio of the blend, relative humidity, particle size, material and wall-roughness of the mixing container. Mixing studies are carried out by systematically altering these factors in order to be able to perform statistical analysis. The results will be highly useful to improve the understanding of the emergence and the impact of electrostatic charge on the performance of pharmaceutical powders especially for pulmonary application.
Onychomycoses, fungal nail infections, are responsible for 50% of all nail disorders affecting up to 18% of general population in some countries . Nail lacquers (also transungual therapeutic systems) represent new drug forms specifically designed to enable longer release and sustained action of active principle into the infected nail plate .
We developed six formulations of nail lacquer containing 0,9% (w/v) fluconazole, Eudragit RS 100 and acetone. The formulations contain di-butyl phthalate, polyethylene glycol 400 or propylene glycol as plasticizers in two different concentrations. We characterized the developed formulations with regard to the drying time, fineness of formed film, fluconazole assay and viscosity.
The drying time for the formulations with lower plasticizer content was 25 s, and for those with higher plasticizer content was 30 s. All developed formulations gave homogenous, smooth, glossy and transparent films.
We developed simple derivative spectrophotometric method for fluconazole assay in all six formulations and the obtained results were in range of 97,5– 103,9% of the declared content.
Viscosity was measured using rotating viscometer in accordance to Ph.Eur.6.0 . The viscosity values were in range of 0,0055–0,0079 Pa×s, which are 50 times lower viscosity values in comparison to the cosmetic nail lacquers. Lower viscosities are preferred since they ensure better coverage and intimate contact with the nail plate leading to better therapeutic response.
The obtained results comply with the standards for this type of preparations with regard to the drying time, film appearance, as well as good flow characteristics. The content of fluconazole is within usually prescribed limits of 100±5%. The results indicate good compatibility of the used ingredients in all six developed formulations.
The drug release from solid matrices systems, made of polymes and drugs, is a basic concept for studies on controlled drug release. The most interesting class of polymers in this application is given by hydrogels. Matrices based on hydrogels, once swallowed (during the in-vitro tests, once immersed in the solvent mimicking body fluids), start to absorb water from the surrounding (water up-take) and the water causes a number of phenomena: the hydrogel swelling, the polymer plasticization (the lowering of the glass transition temperature), the increase of the diffusion coefficients, the polymer erosion (due to polymer chains disentanglement). As a result, the drug previously embedded in the matrix can be released (by diffusion into the matrix and then by dissolution in the outer solvent)
In this work, a model for the drug release kinetics was pointed out. Basically, it consists in differential balances of drug and water in the swelling matrices. It takes into account all the relevant phenomena: the diffusion of drug and water in polymer (with diffusion coefficients that vary with water concentration in the solid phase), the swelling and the dissolution of the polymer (which causes the boundary to be moving surfaces). The resulting code reveals itself able to correctly quantify all the observed phenomena, in terms of water, drug and polymer residual masses in the device, mass fractions of the three species, dimensions and shape of the matrix. To our knowledge, such a complete ability in description was never attained before. The model, therefore, could be a powerful tool in designing novel controlled drug release systems.
Drug liberation-release from the dosage form is a primary cause of the movement of a drug in the body. Pharmaceutical availability of the drug depends not only on the type of aditives but also on the selected concentration. In this study there are compared influence of the concentration of selected cationic surfactants on the liberation of antiseptic chlorhexidine from the hydrogels prepared on the chitosan basis.
The drug released was determined by using semipermeable membrane on the permeating apparatus. the released amounts were determined by spectrophotomentric method at 254 nm from 15 min to 3 hours
This work was supported by the Grant VEGA No. 1/ 0320/ 08
The drugs in a dosage form is rarely alone. Usually the dosage form is a system of several adjuvances and these more or less influence physico-chemical properties and the drug effect.
From the reason of increasing drug solubility many drugs and adjuvances have amphiphilic structure and are able to create associates. Since the drug is effective exclusively in monomeric form for formulation of a dosage form we need to know concentration at which the associates are created – so called critical micellar concentration (CMC).
The objective of this study is to observe the effect of adjuvances on hydrogel – surfactant
1-ethoxy-N,N,N-trimethyl-1-oxohexadecan-2-aminium bromide (Septonex – concentration 0.01% w/w) and polymer – Chitosan (2.5% w/w) on the release of chlorhexidine – member of group of antiseptics (CHH – 0.01% w/w).
Both substances – the drug and surfactant have amphiphilic structures, thus create micelles. CMC of the both was evaluated from conductivity and spectrophotometric dependences [Septonex at the temperature 25–50 °C, CMC increases non linearly in interval (8.07–9.59)·10−4 mol dm−3 and CHH at 25 °C – CMC = 7.25·10−5 mol dm−3].
The partition coefficients in system octanol / water were determined for both substances at the temperature 25 °C. Release profiles were evaluated at the temperature 25 °C. The released amount of drug was determined spectrophotometrically at λ = 281 nm.
The presence the Septonex as an adjuvance in the prepared hydrogel increased the amount of released drug (after 180 minutes 98.79 %) as compared with hydrogel containing polymer and drug only (51.9 %).
Surfactant used in prepared hydrogels positive effected the drug release.
This work was supported by the Grant VEGA No. 1/ 0320/ 08
The pharmaceutical industry currently experiences a trend towards continuous manufacturing as there are many advantages, including better control of process conditions, effectively eliminating batch-to-batch variability, and the lack of scale-up problems, since even bench-top continuous systems can produce the desired quantities required in the pharmaceutical or fine-chemicals industry. We present the development of continuous processes for the synthesis of active pharmaceutical ingredients (APIs). The set-ups include heterogeneous organometallic catalysts (titanocenes and Pd-complexes) that are ideally suited for these continuous processes. The covalent bond between the catalysts and the solid support effectively prevents metal leaching into the product. For this purpose we developed novel methods for catalyst immobilization [1, 2] including photolithographic techniques that allow a precise molecular control of the location and dispersion of the catalytic sites on the surface of the materials.
The application of the heterogeneous catalysts for the synthesis of chiral amines and substituted biphenyls  and the successful implementation in different lab-scale continuous flow systems, such as packed bed reactors, monolithic structures and microfluidic devices, showed that the continuous flow setups lead to improved practicability and flexibility of the processes. Thus, these novel reaction systems constitute promising alternatives to existing batch applications.
This work was supported by the Austrian Science Fund (Project No. 19410)
The broad application of the tetrazole scaffold in different fields, such as general and coordination chemistry, material science, microbiological and medicinal chemistry in recent years demands robust and safe methods for their preparation on production scale. From the many developed methods for the synthesis of the tetrazole nucleus, the [3+2] cycloaddition of an azide partner to a nitrile is by far the most simple, atom efficient and inexpensive strategy. However, hydrazoic acid (HN3) and its salts are extremely toxic and many metal azides and hydrazoic acid itself are extremely explosive.
Great advances in the synthesis of substituted tetrazoles were achieved since the first reports dating back to the mid 1950’s. Today, a variety of protocols are available that allow the synthesis of tetrazoles from azides and nitriles. However, none of these routes are suitable for a genuinely scalable production route, as these batch protocols often involve toxic/explosive intermediates or explosive sublimates, the use of toxic and/or expensive reagents or stoichiometric amounts of inorganic salts, in addition to long reaction times.
Here we present a simple, safe, fast and inexpensive protocol for the synthesis of 5-substituted tetrazoles under continuous flow conditions. We have discovered that the reaction can be cleanly performed using in-situ formed hydrazoic acid at very high temperatures and comparatively short reaction times using a process intensification regime to yield tetrazole products in excellent yields. The details of our microreactor strategy will be discussed.
Organic chemistry in microreactors, being generally characterized as chemistry in miniaturized reaction devices, comprising channel diameters below 1000 μm have many benefits. Mixing and heat transfer are exceptionally fast on the microscale, taking advantage of very short diffusion paths and a very high surface-to-volume ratio, which enables a very efficient heat transfer in both directions, flash heating, as well as excellent process control. Strongly exothermic reactions as well as transformations involving hazardous reactants or products can be handled safely on a large scale.
Very notably, microreactor techniques can be used to overcome the scale-up limitations inherent to high-speed microwave batch processing in sealed vessels. Vice versa, microwave reactors can be used for rapid reaction optimization for a given transformation conducted under continuous flow conditions, since the reaction time at a certain temperature obtained in a microwave reactor can be directly translated to a residence time in a flow reactor. An established flow process is readily scaleable (in contrast to a batch process) by either extending product collection time, increasing the length of microchannel (thereby allowing for a higher flow rate), parallel operation of many identical channels (“numbering up principle”), or by a combination of these principles .
We will present how continuous flow processing can be realized in meso-fluidic flow devices featuring stainless steel tubes, allowing for an exceptionally high operating window of up to 350 °C and 180 bar working pressure. Microwave reactors were advantageously used for rapid modification and optimization of the reactions conducted in flow reactors and directly transferred to continuous flow conditions.
Catalytic heterogeneous hydrogenation processes arguably belong to the most valuable synthetic transformations known. Current batch reactor technology is not only time consuming and difficult to set up but also catalyst addition and filtration is hazardous. Because of the aromatic character of many substrates, hydrogenation often requires the use of significant hydrogen pressures in combination with elevated temperatures and extended reaction times.
In this context, continuous flow hydrogenation technology presents an attractive alternative to batch processing and the recent introduction of the H-Cube™ – a continuous flow hydrogenation device incorporating in-situ hydrogen generation and pre-packed catalyst cartridges – has provided a safe and reliable method for performing hydrogenation reactions [1, 2].
In this poster we investigate the hydrogenations of olefins and heterocyclic aromatic rings. In general, the hydrogenations proceeded smoothly independent from the choice of the supported precious metal catalyst (Pd/C, Pt/C or Rh/C). Using 30–80 bar of hydrogen pressure at 60–80 °C full conversion was typically achieved in all cases at a flow rate of 0.5 mL min−1 providing the corresponding piperidines in high yields. For disubstituted pyridines variations in stereoselectivity were observed depending on both the metal catalyst and on the temperature/pressure in the reaction. For ethyl nicotinate the selectivity between partial and full hydrogenation could be tuned depending on hydrogen pressure and the choice of the supported metal catalyst. Changing the hydrogen source from H2O to D2O allowed the preparation of deuterated derivatives .
Efficient and effective powder mixing is an essential part of pharmaceutical manufacture. Positron emitting isotopes enable particles to be followed singly or in groups during mixing processes, using Positron Emission Particle Tracking (PEPT) or Positron Emission Tomography (PET) respectively. This paper shows how the two techniques can be adapted to pharmaceutical mixing operations and describes their application to mixing in V-mixers and binblenders. PEPT enables particle trajectories to be determined and methods of using trajectory information to quantify mixing are described.
Solid particle mixing is an important unit operation in process industry. In pharmaceutical industry it is of particular importance because the Active Pharmaceutical Ingredient (API) has to be properly mixed which is normally in the quantity of few milligrams in the final dosage form, and if not mixed properly can have no or severe therapeutic effects depending on API quantity.
Particle mixing is usually perceived as a simple unit operation because it does not involve any high-tech and complicated equipment but this operation is still under spotlight of latest research to understand complexity of the underlying physical phenomena. Mixing of solid particles depends on several parameters like nature of the material of the particles, their size, shape, number, and loading/unloading profile. It also depends on the size, shape, and material of the mixer container and likewise the size, shape, and material of the blades, fill level, position of the blade from bottom of the mixer, its speed, acceleration, any external surfaces/obstacles e. g. baffles present, phases involved, nature of the fluid (Newtonian or non-Newtonian) and its physical conditions like viscosity, surface tension, temperature, and pressure etc. Scale-up of powder flows and mixing is also a big issue because even small changes e. g. in equipment material, may change the powder flow and hence the mixing. Because of the number of parameters involved, mixing processes become complicated and expensive to understand experimentally.
In these mixing simulations of solid particles, DEM (Discrete Element Method) is used which treats every particle discretely and solves for each particle’s position, velocity, and all the forces acting on it including contact forces, body forces, hydrodynamic forces and cohesive forces. In this presentation, some physical parameters affecting mixing like blade rake angles, size of particles, and their loading profile will be studied by setting up a number of simulation cases for dry particles. First, velocity profiles for these mixing simulations will be analyzed and then the effects of blade rake angles, size of particles and their loading profile on mixing will be studied by calculating mixing index for each case.
Diffuse reflectance near-infrared (NIR) spectroscopy is a non-invasive and widely used technology with the potential for a wide range of applications in the pharmaceutical industry, ranging from raw material identification to final product qualification. Especially powder blending processes, as one of the key unit operations for solid dosage forms can be monitored in real-time for the blend quality and blending time until uniformity. Common NIR systems enable single point measurements at dedicated positions of the blender, based on the assumption, that the sampled position represents the whole blend. This is a problematic task as the mixing characteristics of powders significantly change (e. g., segregation, agglomeration, etc.) with the process parameters.
Here we introduce, a new process analytical technology (PAT) setup for a spatially resolved quasi-simultaneous process monitoring for batch and continuous manufacturing processes. Therefore, an FT-NIR spectrometer was combined with an optical multiplexer, simultaneously switching the excitation and detection path of the bifurcated fibers, which can be connected to a blender via sapphire sampling ports at different positions. Different multi-component powder mixing processes were investigated and quantitatively analyzed with multivariate data analysis (MVDA) using software packages of CAMO and Umetrics, which also support real-time process monitoring.
The new flexible PAT setup presented here, enables non-invasive in-line and real-time monitoring of dynamic processes, especially where spatial deviations can be expected e. g., segregation in solids (i. e., powder blends) and liquids (i. e., sedimentation).
The application of process analytical technology (PAT) has opened the possibility for continuous process monitoring and quality control in the pharmaceutical industry.
Transporting and mixing of pharmaceutical powders is a challenging task and changes significantly with the mixer type and with the process scale up. Therefore knowledge about the critical process parameters (CPPs) with respect to the powder and their influence on the mixing behavior are essential.
Traditional powder tests (e. g., shear cell, flow through a funnel, etc.) often lack reproducibility and depend strongly on the operator. In contrast to these powder characterization methods, here, an FT4 powder rheometer (Freeman Technology) was applied to determine standardized and repeatible results for powder properties, e. g., shear properties, permeability, cohesion, compressibility, etc.
Based on design of experiment (DoE), an experimental design was developed, testing a recent amount of parameters, which were evaluated for their responses on forming a uniform blend in a mixing process. Continuous process monitoring of the blending processes with near-infrared (NIR) spectroscopy allowed a non-destructive investigation of blend quality and mixing dynamics. Evaluation of the spectral data was performed with qualitative and quantitative multivariate data analysis (MVDA) methods, in order to look for correlations between FT4 parameters and blend quality.
Fluid mixing and dissolution processes are still an area of ongoing research. Numerical simulations have proven to be a valuable tool in understanding and optimizing industrial mixing problems . Computational Fluid Dynamics (CFD) is a well-developed and validated method that is frequently applied in many different fields of applications. Nevertheless, for more complex applications (e. g. industrial mixing) there is still much work left to be done until CFD can be effectively applied as engineering tool providing reliable results in reasonably short calculation times. In this contribution we focus on mixing and dissolution of a bulk powder in a solution inside a stirred tank reactor.
In our work the dissolution kinetics of solids in water were measured in a stirred tank reactor utilising inline Near Infrared (NIR) spectroscopy. The dissolution rate can thus be determined as a function of the powder and process properties such as particle size distribution, solution temperature, impeller speed, and concentrations of the solution.
A dissolution model was derived from the analytical investigations and implemented in the CFD code AVL FIRE. The dissolution test mentioned above was simulated with the CFD method in order to validate the dissolution model.
Due to the fact that real-time CFD simulations of mixing processes are very time–consuming, the dissolution rate of the particles was accelerated in the simulation process and the calculation time was thus reduced. The concentration of a passive scalar is used as a measure for the mass fraction of the dissolving solid particles. This approach allows an analysis of the variation of mixing parameters, and the respective effect on the mixing performance, respectively.
Finally industrial mixing and dissolution processes can be investigated and optimized by applying CFD simulations. In addition, NIR inline measurement techniques enable real-time analysis of the mixing status. Industrial mixing process can thus be controlled by Process Analytical Technology (PAT).
In the pharmaceutical sciences, interfacial phenomena play an important role in the processing of a wide variety of formulations, such as in the blending of solid materials. The surface energies can be utilized in the formulation of wet granulation processes in order to select a suitable device and to predict the properties of the resulting granules. The success of granulation depends on the spreading of the liquid and/or solid over the solid. In the method of Wu , the surface free energy is taken as the sum of dispersive and polar components. The surface free energies of solid materials can be determined by means of contact angle measurements on two liquids with known polarities. If the surface free energies of the solid materials are known, the spreading coefficient may be computed and the interactions between the two materials may be predicted. The spreading coefficient is calculated as the difference between the work of adhesion and the work of cohesion. The spreading coefficient (S12) of a material (1) over the surface of another material (2) can be determined as :
The purpose of this study was to investigate a device suitable for wet granulation via determination of the spreading coefficient. Contact angle measurements were carried out with an optical contact angle measuring instrument (OCA 20, DataPhysics Instruments GmbH, Filderstadt, Germany). The results were applied to calculate the surface free energy and the spreading coefficient according to the above-mentioned Wu method.
Granules were produced in a high-shear granulator (Pro-C-epT 4M8, Zelzate, Belgium). A lower work of adhesion was found in a steel vessel as compared with a glass process vessel.
This work was supported by Evonik Industries AG.
Hot-melt granulation techniques have received increasing attention due to the significant advantages compared to the conventional granulation methods. Fluidized hot melt granulation (FHMG) is recognized as promising technique, but there is still lack of information related to this topic . The aim of the present study was to investigate the influence of process and formulation parameters on granule characteristics and paracetamol dissolution from tablets.
Granulation process was performed in Mycrolab fluid bed processor, OYSTAR Hüttlin. Precirol ATO 5 (Gattefossé, France) and PEG 2000 (Fluka AG, Switzerland) were used as meltable binders that were initially present as discrete particles within fluidized bed. The effects of binder content, particle size of the binder, granulation time, and the inlet air speed on the granule flow properties (Carr index) and drug dissolution parameters (T40, T80) were investigated using factorial design.
Formulations with Precirol ATO 5 showed considerably slower drug release due to the forming of the lipid matrix. The binder content had significant influence on granule properties, as well as on drug release rate in the case of both formulations with Precirol ATO 5 and formulations with PEG 2000. Concerning the paracetamol dissolution from tablets prepared with Precirol ATO 5, particle size and granulation were also found to be significant factors. Furthermore, two-factor interaction between the binder content and the particle size was identified.
FHMG is simple and rapid granulation technique that enables a formulator to manipulate drug release rate depending on the nature and concentration of the meltable binder.
The Leistritz Micro Pelletizer (LMP) is a tool to cut extrudates from a twin-screw extruder to a defined shape and size. The purpose of this study was to evaluate the influence of the parameters to the granual properties in a wet granulation process. 33 experimental design was applied.
A formulation consisting of 40% microcrystalline cellulose (MCC M101, Pharmatrans Sanaq, Switzerland) and 60% lactose (Granulac 200, Meggle, Germany) was used. A preblend was transferred to the gravimetric powder feeder of the twin-screw extruder (18GL-40D, Leistritz, Germany). The extrusion took place at a constant powder feed rate of 25g/min and different liquid feed rates. Deionized water was used as granulation liquid. The Pelletizer (Leistritz Micro Pelletizer, Leistritz, Germany) cut the extrudates when emerging from the orifices of the die-head, using variable cutting blade speed. Image analysis was conducted by using the Camsizer (Retsch Technology, Germany).
The study dealt with the effect of 3 different process parameters – screw speed, liquid feed rate and cutting blade speed – on the pellet shape and size. These parameters were varied systematically in a 33 full factorial design. In addition, 3 experiments were performed at the center point in order to approximate the reproducibility.
The regression model was simplified by a backward regression. The pellet size and the pellet shape were characterized by equivalent diameter and aspect ratio, respectively. The most spherical pellets were obtained by using a liquid feed rate of 0.95kg/h and a cutting blade speed of 2500rpm. In the variance analysis, the cutting blade speed and the screw speed were insignificant, whereas the liquid feed rate had an effect to the pellet shape and size.
One of 3 investigated process parameters affected the pellet properties. Neither the screw speed nor the cutting blade speed showed an influence on the pellet shape and size. The liquid feed rate was crucial in the used experimental setup. The continuous granulation process using the Leistritz Micro Pelletizer (LMP) in combination with the twin-screw Extruder (Leistritz 18GL-40D) was robust and reliable.
Pellet manufacturing by extrusion/spheronisation is quite common in the pharmaceutical field because the obtained product is characterized by a high spericity as well as a narrow particle size distribution. After extrusion of the bulk material, the cylindrically shaped extrudates are transferred to the spheronizer, where they are plastically deformed into spherical pellets. The established mechanisms [1, 2] only consider deformation of the initial particles but do not account for mass transfer between the particles as a factor in achieving spherical particles.
This study investigates methods for visualizing and understanding the interparticle interactions and a possible mass transfer during the spheronisation step. Therefore, two common pelletization aids (Microcrystalline cellulose (MCC), kappa – carrageen) were used in combination with lactose as a filler. To visualize the interparticle exchanges of mass during the spheronization process, different colored extrudates were spheronized simultaneously, and the color change of the different particles was monitored.
The data obtained indicated that mass transfer between particles must be considered in addition to plastic deformation in order to capture the spheronization mechanism. A material transfer between pellet particles was observed in all investigations (fig. 1). Moreover, the images show regional distinctions in the amount of mass transfer. There seems to be an equatorial zone with a higher probability for agglomeration than at the former edges of the extrudate.
In conclusion, the commonly espoused pelletization mechanisms need to be extended to account for material transfer between pellet particles, which has not been considered before.
The problem emerging from particle coating is how to manufacture particles with just enough material deposited to achive the desired result . Especially in case of functional coatings, the uniformity of coating thickness over the entire particle surface plays an important role. In our study the performance of two bottom spray coaters and the effect of the particle motion on the coating uniformity were evaluated for the pellets with wide size distribution. In the first case the traditional Wurster coater was employed, while in the second one the coater with a swirl flow generator was used. Pellets (Cellets®, Syntapharm, Germany) of four different sieved fractions (300–400, 500–600, 700–800 and 900–1000 μm) were used in each coating experiment. Fractions of pellets comprising the bed were separated after dye dispersion coating, using the same sieves as used in the preparation of starting cores. Spectrofotometric evaluation of the dye concentration on the population of pellets from each fraction of every experiment was done. Results confirmed the fact that pellets having smaller diameter receive significantly less coating material compared to the larger ones when using conventional bottom spray coater with Wurster insert . The ratio of mass of coating deposited on the pellet to the pellet surface indicates the coating thickness to be much more evenly distributed over the pellets coated with the use of swirl flow generator equipped coating chamber. For example the values of dye per particle surface varied from 0.88 for smallest fraction to 2.12 μg/mm2 for largest fraction in case of conventional Wurster coating chamber and only from 1.21 to 1.79 μg/mm2 in case of application of swirling airflow generator. These results also indicate that the amount of dye deposited per particle is in a strong correlation with the particle diameter to the power of 2.36 in case of coater with swirl airfow, compared to the value of 2.85 obtained by a conventional bottom spray coater. The amount of dye per pellet would ideally be in correlation with pellet surface (d2,00), which makes swirl flow coater more suitable for coating particles with wide size distribution. The improvement in the yield of the process was also observed in the case of swirling airflow coater.
Coated multiparticulate systems are increasingly popular on the market. Their manufacture in a fluid bed often requires polymer binders. Polymers have been widely studied as inhibitors of crystallization . Migration of drugs into the coating layer has also been described  but the influence of the coating polymers on the crystallization of the drug is yet to be studied in more depth.
In our experiments we studied the thermal behaviour of diltiazem hydrochloride-layered pellets before and after coating. The multiparticulate pellet samples were prepared in a Strea-1 (Niro Aeromatic, Bubendorf, Switzerland) fluid bed Wurster chamber. Acryl-EZE dispersion (a fully formulated USP copolymer type C coating system) was prepared and applied following the guidelines provided by the manufacturer as a 20% aquous dispersion. The thermoanalytical examinations were carried out with a Mettler-Toledo 821e instrument with a dynamic method in the interval of 0–400°C, at a heating rate of 10°C/minute.
We found that the coated samples exhibited an exothermic peak between 90–110°C that wasn’t characteristic of any of the used materials. Further examinations suggested that the exothermic peak is the result of recrystallization; this theory was tested with XRD. Results showed that the uncoated drug-containing pellets contained only a relatively small amount of the crystalline drug. Coated samples behaved similarly. XRD examinations after heating the samples to 120°C (above the range of the exothermic peak) showed a definite increase in the crystallinity of the drug. Thermal examinations showed a correlation between the thickness of the coating and the extent of recrystallization.
One explanation of the above is that the water-soluable drug dissolves into the aqueous coating dispersion on the surface of the pellets during the coating and crystallizes only in a small extent as the film is dryed; most of the drug forms a molecular dispersion in the film. Crystallization is completed only on heating, as can also be proven by microscopic evidence.
This work was supported by “Stiftung Aktion Österreich-Ungarn“.
Flows encountered in processes involving turbulent bubbly flow are highly complex, especially when the gas holdup and gas-liquid interface mobility are such that coalescence and breakup of bubbles take place. These phenomena have an enormous impact on the performance and productivity of the processes. Attempts have been made by researchers to gain insight in the hydrodynamics of bubbly flows by detailed numerical simulation. However, resolving coalescence and breakup has often been ignored due to their large computational requirements.
The goal of this presentation is to assess the feasibility of using computational fluid dynamics (CFD) modeling as an engineering tool for analyzing, designing and scale-up of processes involving bubbly flow with coalescence and breakup of bubbles. The parallel and highly efficient modeling technique for bubbly flows described in the work of Sungkorn et al.  was extended to incorporate coalescence and breakup phenomena. The continuous liquid phase is simulated using the lattice Boltzmann (LB) scheme originally developed by Derksen and Van den Akker . The trajectory of the individual bubbles is tracked by solving their equations of motion including the effect of the fluid fluctuation along the bubble trajectory. Bubble-bubble collisions as well as coalescences are described based on the stochastic inter-particle collision model according to kinetic theory. The breakup of bubbles is considered using a model based on the local size of turbulent eddies.
Simulations of gas-liquid flow in a bubble column have been performed. The simulations provide detailed insight for the liquid flow field as well as the gas dispersion pattern. The accuracy of the simulations is demonstrated by comparing the predictions with experimental data. Additionally, excellent speedup and scalability on parallel computing platforms demonstrate the capability of the present modeling technique for simulations of large-scale reactors.
Granular flows are extremely important for the pharmaceutical and chemical industry, as well as for other scientific branches dealing with granular media. Thus, the understanding of the impact of particle size and related effects on the mean as well as on the fluctuating flow field in granular flows is critical for design and optimization of powder processing.
We use a specialized simulation tool written in C and CUDA, a massive parallelization technique which runs on the Graphics Processing Unit (GPU). It was chosen to be able to simulate significantly higher amounts of particles in acceptable runtime.
We focus on flow fields and wet-mixing properties obtained in the lower million particle range. In our talk, we present simulation results of a pitched blade mixer on a laboratory scale. Particles with a medium particle size of 365 micrometers are blended at 30–60 rpm for several turns (> 10 revolutions). The GPU implementation enable the DEM simulation of quartz powders in the real geometric resolution. In order to investigate the mixing process during the continuous spraying of a liquid, the DEM code was extended suitable. Liquid films and bridges implement the spread of the fluid in the aggregates. Depending on the wetness level on particle surfaces, capillary forces account for cohesion and agglomeration. The simulations will shed light on the optimum location and the type of fluid entry, and allow predictions about the time until a homogeneous moisture distribution in a blended bulk can be achieved.
Complex process engineering like drying of fluidized particles in hot air streams is a huge challenge for numerical simulations. Highly resolved two-phase flow of one million particles will be investigated. Here we focus on both, a gas-phase simulated using the Lattice Boltzmann Method (LBM) and a coupled solid-phase of discrete particles, simulated by using Discrete Element method (DEM). Subgrid scale two-way coupling of liquid and solid flow allows the investigation of residence time as well as other important process parameters and the prediction of an optimum operation regime.
Particle coating using fluid bed technology such as Wurster chamber is a common process in pharmaceutical technology. Coatings are used to achieve variety of functions, such as: taste masking, controlled release, increase of stability and others. Coating uniformity is one of the most important parameters of coated particles and can play an important role in the release of the active ingredient and is a result of number of process settings and material properties [1, 2]. One of the material parameter that effects particle movement and consequent coating is coefficient of restitution, which is defined as the ratio of particle velocity before and after the impact .
We have analysed the effect of coefficient of restitution on particle distribution and their velocity in the Wurster process chamber. Simulations were performed on a 2D axisymetric model using Ansys Fluent. Euler-Euler approach was used for simulation of gas and solid phase. The particle size was set at 1 mm and the coefficient of restitution was set from 0,1 to 1,0 in 0,1 steps. All other process parameters such as fluidizing and atomizing air flow were kept constant.10 s of two-phase flow was calculated in each simulation using timestep of 2.5*10−5 s, starting from the same initial condition.
The analysis of the simulation has established that in most cases higher values of coefficient of restitution increase the particle volume fraction in the coating region of the chamber. The lowest value of volume fraction of particles in the coating region was 6.82% and the highest was 9.41%. It was established that coefficient of restitution has effect on particle velocity and flow rate through the coating region, however far less pronounced as in the case of volume fraction, which is one of the main factors that affects uniformity of coating. Therefore, according to our findings elastic properties of the particles can affect the outcome of the coating process i. e. coating functionality, even when using the same process chamber and process parameters.
The aim of this study was to investigate the possibility of using Polyox® as the binding and/or coating agent for paracetamol sustained release formulation development.
Paracetamol granules were prepared in fluidized-bed apparatus, using different ratios of Polyox® as binding agent. Granules were coated with Polyox® solution by applying the bottom spray fluidized-bed technique. Tablets were prepared by compressing coated granulates with an excenter tablet press. For comparison purposes, sample tablets were also prepared by direct compression of the homogeneous mixture of paracetamol, Polyox® and microcrystalline cellulose. In vitro release studies were performed in the paddle dissolution apparatus, using USP phosphate buffer pH 5.8.
The rate of drug release from Polyox® based formulations was determined by swelling and diffusion through the gel layer formed. Dissolution study results of coated granules show that an increased Polyox® level in the formulation resulted in a markedly decreased drug release rate. Similarly, tablets made from coated granules exhibited slower and additionally decreased drug release compared to uncompressed granules [1, 2]. The in vitro dissolution rate of all the investigated granules and tablets was slower than drug release from the corresponding physical mixtures of the drug and polymer. Also, the results of dissolution studies show certain difference among the tablets made from coated and/or uncoated granulates.
The results obtained indicate the potential use of Polyox® polymer as the binding and coating agent in the sustained release tablet formulation.
This work was supported by the Ministry of Science and Technological Development, Republic of Serbia project TR23015.
In recent years, the interest in melt granulation has increased due to the advantages of this technique respect to traditional wet granulation; utilizable equipments for melt granulation are high-shear mixer and fluidised bed .
The aim of the study was to evaluate, by asymmetrical factorial design , the contribution of some variables on the in situ melt granulation process performed in fluidised bed in order to identify the critical parameters that influence the granule properties and the process.
The experiments have been performed in a lab scale apparatus (Mini-Glatt), employing spray dried lactose monohydrate as filler. Six factors were studied at different levels: the binder type was evaluated at 2 levels (Lutrol F68 and Lutrol F127), while the percentage of the binder, the size of the binder particles, the inlet air temperature, the inlet air rate and the granulation time were studied at 3 levels. The 16 experiments were run randomly in duplicate. Five variables were selected as experimental responses: the total process time, the yield, the particle size (d50), the friability and the flowability of the produced granules.
The analysis of the results showed that the percentage of the binder, the inlet air temperature and the inlet air rate significantly influenced all the experimental responses, while the binder type, the size of the binder and the granulation time had minor “weight” on the entire process. Five additional tests allowed to assess the validity of the screening.
In conclusion the factorial design results permitted to identify the most important variables of the in situ melt granulation process in fluidised bed. These findings result crucial for the ongoing further optimisation phase of the process.
One of several different ways to quantify the compactibility of pharmaceutical powders is the calculation of Pr , where tensile strength (σx) is normalized with the specific work (Wspec) of tablet compression (Equation 1):
in which Wspec is expressed as effective work (E2) invested in the compression of the unit mass of substance (m). E2 represents the area of hysteresis between compression and decompression curves in force-displacement measurements during tablet compression cycle.
In our previous experiments  we have noticed that the Pr is dependent on the maximum compression pressure used to produce the tablet, where Pr increases with compression pressure until a plateau is reached. This phenomenon is unwanted from a practical point of view, because it makes the comparison of results very difficult. It has been established, that both tensile strength and specific work yield a linear relationship with compression pressure, so Pr can be described as a quotient between two linear functions and is expected to be a constant value. However, the two linear functions do not go through origin of the co-ordinate system. Both functions have a negative y-intercept, which is the mathematical reason for apparent Pr dependence on compression pressure.
We propose a modification, indexed as Pr’ (Equation 2), based on the limit value of Pr:
where SLOPEs are the slopes of the two linear functions; P1 and P2 are two different compression pressures. In the compression pressure range from 68 to 211 MPa the Pr’ represents asymptotic value of Pr in plateau region, validating the equation. This modification enables the comparison of compactibility independently from the compression pressure.
The formulation of solid oral dosage forms significantly determines the release rate of active pharmaceutical ingredients (APIs). The release characteristics of the API are controled by the excipients in the tablet and the tablet microstructure. Computational simulation of tablet dissolution enables tablet designs and formulations to be tested quickly without resorting to many experiments. Crucially, the model offers a method of mapping the available design space with respect to both tablet composition and tablet geometry enabling parametric sensitivity studies to be carried out. This also enables the inverse problem to be solved i. e. for a desired API release rate, the model calculates the tablet composition and/or tablet geometry which achieves this.
The presented model discretises tablets using a grid of volume elements (for non swelling polymers) or discrete element method (DEM) (for swelling polymers), enabling the tablet microstructure to be explicitly defined. Mass transfer between volume elements is carried out using Fick's law with per-component porosity dependent diffusion coefficients. A concentration boundary layer is situated around the tablet whose thickness is defined by the Sherwood number based on flow conditions. Material diffusing across the boundary is considered released and plotted over time to form component release curves.
Optimisation minimises the difference between the simulated API release curve and the target release curve using the mean squared error and the downhill simplex method. This method can be used to find pure component parameters from dissolution experiments or can be used to optimise tablet formulations by adjusting one or more parameters. In this work, the method was applied to optimise a tablet formulation such that it meets a specified API release time. The tablet formulation contained a disintegrant whose fraction was varied in order to change this time. Two release times were specified (fast and slow) and the resulting formulations were then tested with dissolution experiments to validate the simulation.
The model demonstrates that optimisation can be applied to tablet formulation, leading to better Quality by Design (QbD) and faster realisation of custom formulations.
The “Drill&Fill”-Method is a suitable method to radiolabel compact dosage forms, such as tablets, and further to investigate their comportment in the human organism. Therefore, a hole is drilled into the tablet and filled with radioactive liquid. The hole is sealed with antiabsorbable glue. Hence, the radioactivity labels the tablet and allows its visualisation in the body using scintigraphy. The aim of the study was the evaluation and validation of a “Drill&Fill”-method for enteric coated tablets for a clinical study of dosage modification. Thereby the mandatory conditions were:
The tablet’s dimension was 18x 9x 5mm and it had a weight of approximately 1 gram. Drill: The hole was drilled using an electric drill (Proxxon®). Fill: The drill hole was filled with 99m-Tc-DTPA using a Hamilton syringe. Subsequently the drill hole was sealed with cyanacrylate glue (Pattex® Blitz Sekundenkleber) and air-dried. Thereafter, the activity of the tablet was assessed in a PTW Curiementor 2. The method was validated on the basis of 80 tablets.
For ideal filling conditions the drill hole had to be 12mm deep with a diameter of 1.5mm. The drill loss was 28.78mg ±0.4 and 2.64% ±0.04, respectively. The volume of 99m-Tc-DTPA in the hole was 21.92μl ±2.1. The glue was air dried for 90 minutes in order to assure a complete drying-out of the glue. The activity after drying was 2.77MBq ±0.4. The dissolution test showed no difference between drilled and intact tablets.
All required conditions could be achieved. The “Drill&Fill”-Method is a fast and easy method for labelling tablets. It is suitable for showing the residence and disintegration of the tablet.
Coated dosage forms in which the active pharmaceutical ingredient (API) is incorporated in the film layer in order to achieve rapid release have become very common in recent years. Our present aim was to develop a matrix pellet containing an API in the coated layer. The presence of insoluble particles in the coating liquid is a critical parameter in the preparation of films as such particles can modify the properties of the film formed [1, 2]. The film surface is required to be smooth, resistant against mechanical effects, heat and light. The excipients and APIs incorporated in a film layer influence the structure of the film significantly.
In this work, films were prepared on a teflon surface. The coating liquid was prepared as reported previously . Eudragit® L 30 D-55 dispersion was used as polymer, and Diclofenac sodium as API. The samples contained 0, 1 and 5% of the API. Measurements were made on the mechanical properties, surface properties and glass transitions of the free films, and on the free volume holes in the films.
The mechanical strength and the degree of deformation of the films were determined by the recording of deformation curves. Contact angles were measured to evaluate the surface free energy, solid surface free energy, liquid surface tension and polarity. Glass transition temperatures were detected by means of DSC. Positron annihilation spectroscopy was applied to study the free volume and track interactions between the polymer and the active ingredient. The size distributions of free volume holes were calculated from positron lifetime data for films of different compositions. The long-term stability of films was also studied.
An essential step for the development of probiotic products is the stabilisation (drying) of the microorganisms. The aim of this study was to compare the efficiency of drying cells by lyophilisation, a classical method for cell stabilisation , with fluid bed technology, which is an approved method for gentle drying as well as for mixing, granulation and coating processes . For this purpose the probiotic microorganism Enterococcus faecium M74 was dried by lyophilisation as well as by fluid bed drying. A further aim was to investigate the protective impact of excipients in different concentrations on microorganisms during the drying processes.
Both methods were studied regarding their effect on the culturability and the cellular activity of E. faecium M74. The culturability was determined by spreading the rehydrated cells onto kanamycin esculin azide agar plates and calculating the number of colony forming units (CFU). In order to investigate the cell membrane damage, the esterase activity, and the change in superoxide production of cells, the rehydrated cells were stained with the fluorescent dyes propidium iodide (PI), fluorescein diacetate (FDA), and dihydrorhodamine 123 (DHR 123), respectively, and measured with fluorimetry.
The addition of 50% skim milk, based on the wet mass of cells, had the best protective impact on E. faecium M74 during lyophilisation, whereas the best results for fluid bed drying were obtained by adding 100% skim milk. Remarkably, adding 50% or 100% skim milk were the only settings which significantly protected the cells during lyophilisation, contrary to the results of fluid bed drying, where nearly all of the used excipients had a significant protective impact on the cell culturability and the cellular activity of E. faecium M74. Furthermore the investigations showed that fluid bed drying is a capable method for the stabilisation of microorganisms. However, the selection of suitable protectants is important for the reliable application of both methods for drying probiotic microorganisms.
Ballistic powder injection is an emerging needle-free technology. Powder particles are accelerated towards the skin and penetrate the superficial layers. Biopharmaceuticals like vaccines can be placed into the epidermal layer of the skin, widely known to be an attractive site for immunization . Beneath avoiding hazards associated with needle-stick injuries using conventional needles and syringes, the use of dried sugar powders provides an enhanced stability for biopharmaceuticals . In the literature the use of spray freeze drying to generate sugar particles for ballistic powder injection has been evaluated . A ternary mixture of trehalose, mannitol and dextran was found to be appropriate to generate high density particles attributing appropriate characteristics for ballistic injection . Dextran has an antiplasticizing effect on the formulation and leads to a partially collapsed, highly wrinkled morphology of the spray freeze dried particles. Nevertheless, the stabilising effect of this matrix for proteins still needs to be improved. A polymer like dextran is vital for the partial collapse of the spray freeze dried particles, but it is also responsible for the reduced stabilizing effect on the protein . Our approach is to apply an aggressive freeze drying cycle as performed by Schersch et al. to evoke a collapse of the sugar matrix and generate a high density structure of the freeze dried cake . Characteristics like high density and low specific surface area of the lyophilizates can provide protein stability and appropriate properties for ballistic powder injection. We investigated different sugar formulations with and without dextran and evaluated the effect of collapse freeze drying on particle morphology and protein stability. With this more in-depth information about particle morphology and protein stability we can introduce an improved formulation strategy for biopharmaceutics entrapped in a high density sugar matrix intended for ballistic powder injection.
We considered poly(esteramide) hyperbranched polymers (trade names: Hybrane S1200 and Hybrane HA1690) as enhancers of glimepiride solubility and therewith associated dissolution rate. We prepared solid dispersions (SD) of glimepiride with hyperbranched polymers (HB) and compared their solubility with those of crystalline and amorphous glimepiride as well as with the solubility of SD of glimepridie with conventional linear polymers, i.ie. Poly(ethylene glycol) and Gelucire (stearoyl macroglyceride)s.
Since the solubility and dissolution rate depend also on glimepiride crystallinity we characterized the morphology of solid dispersions by X-ray powder diffraction (XRD) analysis. The nature of molecular interactions involved in complexation of glimepiride with HB was studied by infrared spectroscopy (FTIR).
The comparison of the results of in-vitro dissolution studies show that SD based on poly(ester amide) HB polymers showed significantly enhanced aqueous solubility of glimepiride and its dissolution rate as compared to glimepiride in crystalline or amorphous form as well as its SD with conventional linear polymers. We also calculated the amount of glimepiride complexed with particular HB polymer (loading capacity) in SD using dissolution results. The loading capacity for both HB polymers was estimated to be around 5 %, w/w of glimepiride. SD containing higher amounts of glimepiride appear to be oversaturated, so that non-complexed glimepiride crystallizes as a separate solid phase during the solvent evaporation. X-ray diffraction studies confirmed calculated loading capacity.
IR results indicated that glimepiride forms complexes with HB polymers through hydrogen bonds between the NH groups of glimepiride and carbonyls of ester (O)-C=O and amide (N)-C=O groups of HB polymers. Therefore, the improved glimepiride solubility was ascribed to complex formation between glimepiride and HB polymers.
The study demonstrates two approaches in formulation of solid self-emulsifying drug delivery systems (SSEDDS) of gliclazide (BCS class II drug). Self-emulsifying (SE) formulations can improve oral bioavaiability of poorly water-soluble and lipophilic drugs. Traditional SE formulations are liquids, having some disadvantages such as high production cost, low stability, low drug loading and few choices of dosage forms. To address these problems solid SEDDS are being investigated .
The aim of the study was formulation of solid oral self-emulsifying gliclazide preparations - hard capsules and tablets.
Twelve formulations have been prepared with varying type of solubilizer (Labrafil® M2130CS, Cremophor® RH40, Labrafil® M 1944CSCG, Labrafil® M2125CS), ratio gliclazide:solubilizer (1:1 to 1:20), presence of adsorbent (Neusilin® UFL2), as well as the ratio of gliclazide solution:adsorbent (1:1 and 2:1) and presence of disintegrator (Ludiflash®) in concentration of 10–50%. Formulations have been filled in hard capsules or compressed into tablets using excenter tablet press. Determination of gliclazide release from prepared capsules or tablets has been conducted in rotating basket apparatus (Erweka DT600, pH=7.5, 100 rpm, 900 ml, 45 minutes).
It has been demonstrated that gliclazide has the highest solubility in Cremophor® RH40, at ratio 1:20. Gliclazide release studies have demonstrated that capsules filled with gliclazide solution adsorbed on adsorbent, with ratio solution:adsorbent 1:1, show the fastest gliclazide release rate. Addition of disintegrator in formulations having ratio of solution:adsorbent 1:1, slows down gliclazide release rate. On the other hand, disintegrator enhances gliclazide release in formulations having ratio of solution:adsorbent 2:1. Gliclazide release studies from tablet formulations containing adsorbed solution (in ratios 1:1 and 2:1) and Ludiflash® demonstrate faster onset of drug release in first minutes followed by more sustained gliclazide release, in comparison to capsules. Obtained results demonstrate the possibility to formulate solid self-emulsifyng gliclazide formulations, once appropriate emulsifier, adsorbent and disintigator, in adequate ratio, are selected.
The study demonstrates two approaches in formulation of solid oral lipid-based diazepam preparations (BCS class II drug). The aim of the study was to formulate tablets and hard capsules, using emulsifier and adsorbent. Lipid-based preparations improve drug solubility and permeability, i. e. bioavailability. Solid silicate carriers are able to adsorb liquid lipid formulations, resulting in freely flowable and compressible powders .
Twelve formulations have been prepared, using emulsifier Labrafil®M2125CS (linoleoyl macrogolglycerides) to solubilize diazepam, Neusilin®UFL2 (magnesium aluminium silicate) as adsorbent, with the varying ratio of diazepam solution:adsorbent (1:1 to 4:1), and disintegrators Ac-Di-Sol® and Ludiflash® (with the ratio varying from 0–90 %). Formulations have been filled in hard capsules or compressed into tablets using excenter tablet press. Diazepam release study from prepared capsules or tablets has been conducted in rotating basket apparatus (ErwekaDT600, 0.1 M HCl, 100 rpm, 500 ml, 45 minutes).
Diazepam release studies have demonstrated that it is possible to formulate lipid-based solid oral preparations with immediate diazepam release. Capsules filled with diazepam solution adsorbed on adsorbent, with the ratio of solution:adsorbent 1:1, show the fastest diazepam release rate. Addition of 10% of disintegrator in capsule formulation enhanced diazepam release rate, but when the ratio of disintegrator is increased furthermore, diazepam release is sustained. It was observed that compression of powders in tablets also led to decrease in diazepam release rate. Addition of disintegrators further sustained diazepam release leading to conclusion that a physical interaction occurs between adsorbent and disintegrators. When adsorbent was excluded from the tablet formulations diazepam release rate was enhanced, but tableting properties of such powders were compromised.
Obtained results demonstrate the possibility to formulate diazepam immediate release formulations, with the necessity of careful selection of excipients and dosage forms.
Adsorption onto silica-based high surface area carriers is a long known method of improvement drug dissolution rate, which has been described in early 1970s . Porous silica has many silanol groups on its surface and can be used as pharmaceutical excipient, since it is regarded nontoxic after oral application. Due to its porous structure and large specific surface area, porous silica has a great capacity to adsorb organic compounds into its nano-sized pores and can improve physical stability of amorphous drug .
Solid dispersions of carvedilol in porous silica (Sylysia 350) were prepared by solvent evaporation in a vacuum evaporator which ensures efficient pore filling procedure . Two sets with different concentrations of carvedilol in solid dispersions were prepared, which differ in pore filling process. Set a) was prepared by one-step filling and set b) by multiple filling of smaller portions of dissolved carvedilol into porous silica. Solid dispersions were characterized by thermal analysis (DSC), X-ray diffraction and nitrogen adsoprtion experiments. Specific surface area and porosity parameters of solid dispersion samples confirm different mechanism of drug loading within silica pores in each experimental set. Results showed that carvedilol is more efficiently “packed” inside porous matrix when multiple drug filling procedure of smaller amounts is used as suggested by absence of crystalline state of a drug and greatly reduced porosity, while at the same time physical stability of amorphous carvedilol was most improved. Due to space restriction carvedilol can not crystallize when it is entrapped in a region of pores of smaller diameters than the minimum size of crystalline nucleus. Once the amorphous form of drug is stabilized inside porous silica it remains physically stable against crystallization and water sorption, while pure amorphous drug partially crystallized and adsorbed a detectable amount of moisture.
The authors acknowledge to KRKA, d.d., Novo mesto for support in the study. Present work was partly financed by the European Union, European Social Fund.
Taste-masking technologies are important for obtaining high-patient compliance and drug therapy efficiency, since many oral-delivery drugs have unpleasant qualities such as bitterness, sourness or saltiness.
Taste-masking technology includes two aspects: selection of appropriate taste-masking substances (polymers, flavourings, sweeteners, amino acids, etc.) and selection of appropriate taste-masking technique. Such taste-masked multiparticulate systems (granules, powders, dry syrups, or suspensions) represent further base for preparation of peroral formulations, such as dispersible tablets and oral liquids.
Selection of appropriate taste masking technique can strongly impact both, quality of taste masking and the process efficiency. There are many available techniques for taste masking developed today, including conventional granulation and polymer coating, but also more sophisticated is taste masking by encapsulation with lipids or cyclodextrins or by making multiple emulsions .
The aim of this study was to reduce or completely inhibit bitterness of model drug of defined physical-chemical characteristics. Fluid bed technology was used as the suitable method for taste masking. Drug particles were coated with the polymeric material, which ensured masking the bitter taste of the drug substance. Two different polymers, amino methacrylate copolymer (Eudragit® E PO) and ethylcellulose (Aquacoat ECD) were used. Influence of spray position and process parametrs on quality of coating of drug particles was studied. Physico-chemical properties of formed granules were characterised and compared in terms to ensure proper masking of drug bitterness and process efficiency .
This work was supported by scientifically assistance and knowledge share of Gordana Matijašević and Ivan Simčić in performing analytical tests.
The dispersion and emulsification in micro channels by high pressure have several characteristics which recommend these methods especially for pharmaceutical applications, e. g. the possibility to use very small educt batches, a narrow residence time distribution and a relatively accurate adjustment of the induced stresses with a good reproducibility. Due to the small dimensions “Cleaning In Place” (CIP) is easy to realize without a large loss of product or time and for high hygienic requirements even the possibility of disposable devices exists. Nevertheless deposits, which can lead to blockages, and the abrasion of the micro systems with the associated product contamination pose big challenges.
In order to determine the dispersion efficiency of different micro channel geometries, experiments with nanoparticle agglomerates were carried out. The results show that the dimensions as well as the geometries of the micro channels influence the dispersion efficiency, the appearance of blockages and the amount of abrasion. Especially areas with low velocities as behind rough edges or oversized in- and outlets run the risk of depositions. With computational fluid dynamics calculations (CFD) and flow measurements (μPIV) areas of low velocities or backflow which run the risk of depositions and the occurence of cavitation are identified and subsequently eliminated or minimized in optimized channel designs.
The CFD calculations are also conducted to get a better understanding of the stress field which leads to the dispersion or emulsification. The turbulent flow fields of the different micro channels were solved and used for a stationary particle tracking. Based on these particle pathes the impressed elongential, shear and turbulent stresses are calculated. The agglomerate size distribution measured after the dispersion experiments are compared to the calculated stresses to determine the mechanisms involved in the dispersion or emulsification.
The authors gratefully acknowledge the DFG for financial support within the DFG research group 856 “Mikrosysteme für partikuläre Life-Science-Produkte“ (mikroPART).
The group of poly(2-oxazoline)s has scarcely been considered as scaffold for hydrogels [1, 2], despite of the FDA approval for two prominent congeners, namely poly(2-ethyl-2-oxazoline) and poly(2-phenyl-2-oxazoline) (21 CFR 175.105). The polymerization of 2-oxazolines has significantly benefited from the advent of microwave reactors specially designed for chemical syntheses by a remarkable decrease of reaction times with a factor of 60, maintaining the livingness of the polymerization [3, 4]. These accelerations have paved the way to the synthesis of hydro-, amphi-, and lipogel libraries.
In this presentation, the influence of the ratio of poly(2-ethyl-2-oxazoline) vs. poly(2-phenyl-2-oxazoline), the degree of cross-linking and the type of cross-linker on the swelling degree and the proton-mediated degradation of the gels will be shown, and the potential of 2-oxazoline-based gels as toolbox for tailor-made hydro-, lipo- and amphigels will be discussed .
Shrinkage cracking during the deliquoring of compressible filter cakes by means of differential gas pressure is an undesired phenomenon of great practical importance for the pharmaceutical industry amongst others. Cracks are detrimental to the deliquoring process as they can lead to a channelling effect whereby the gas flows preferentially through the network of cracks rather than displacing the interstitial liquid, reducing the effectiveness of the deliquoring process. This has economic consequences of increased gas consumption, as well as higher residual moisture content, which would lead to an increased energy input in the later thermal drying stages.
The precise mechanism of crack formation is not fully understood at present. Crack formation is usually preceded by shrinkage of the filter cake, the driving potential for this shrinkage being the action of the capillary forces on particles forming the cake. When the stresses generated by the capillary forces exceed the tensile strength of the filter cake, the formation and propagation of cracks can be observed . The tensile strength can be significantly reduced by local cake inhomogeneities.
In this contribution we will present the results of an experimental parametric study on cake cracking. The cracking probability was investigated for a model system of calcium carbonate-water on a laboratory-scale pressure filtration rig. The effects of the following parameters on crack formation were studied in detail: particle size distribution, filtration and deliquoring pressure, cake height, concentration of slurry and filter medium. The results will be presented in the form of graphs and correlations allowing the estimation of both the probability and severity (expressed by an increase in cake permeability) of cracking. For selected cases the crack network structure will be characterised by digital image analysis and three-dimensional techniques such as x-ray microtomography.
This work was supported by the UK Engineering and Physical Sciences Research Council (CASE grant number 0900052) and by GlaxoSmithKline Ltd.
Metoprolol is a cardioselective β-blocker that is classified as a class I substance according to the Biopharmaceutics Classification Scheme BCS, meaning that it is highly soluble and highly permeable. Dissolution test was carried out according to EP 2.9.3. in medium 0.1N HCl, 900 ml, 100 r/min, sampling time 5 minutes. Quantification was spectrophotometric measurement on 275 nm.
The results of dissolution analysis are summarized in Table 1, sample 1 hardness 123–137 N, disintegration 5.21–9.17 min, sample 2 hardness 157–168 N, disintegration 8.38–14.10 min and sample 3 hardness 132–151 N, disintegration 12.23–14.23 min.
Sample 3 has the highest f2 compared to reference drug, Samples 2 and 1 have values significantly different from the reference drug results. This study helps in revealing the effect of formulation processing variables on tablet properties.
The application of a coating layer on a tablet is a commonly used technique to selectively control tablet characteristics. Among the most important functions of the coating is the regulation of the release of active ingredient.
In the course of a typical coating process, the tablet passes a spray zone multiple times. Each time, a partial coating layer is applied. For each tablet, this is repeated numerous times until a film of desired thickness is achieved. The number of repetitions needed as well as the quality of the final coating depends strongly on the quality of the partial coating. Therefore, it is of great interest to study the outcome of a single coating event in detail.
In this work, we show the application of numerical simulations to the investigation of film formation on a tablet (see fig. 1). External parameters entering the simulation were taken from measurements of an industrial coating process. Adapted computational fluid dynamics (CFD) models are applied to study the influence of central process characteristics (e. g. temperature, residence time, tablet rotation or droplet diameter distribution) on the quality of partial coating. As a result, a deeper understanding on the influence of each parameter is gained, helping to optimize the tablet coating process.
Particle shape and size have an influence on the solubility of an API-particle and hence on the bioavailability of the substance. Thus, bulk properties such as the crystal size distribution (CSD) are important quality attributes of powders. Furthermore, downstream processes (e. g., filtering, washing, drying etc.) and the handling abilities (e. g., flowability, tabletability) of the particles are positively affected by narrow CSDs. In order to obtain product crystals with desired features it is important to control numerous parameters during a crystallization process.
We present a versatile, continuously operated tubular crystallizer system, which is based on the growth of seeds to product particles in a tube. Due to the tubular appearance and the small inner dimensions of the crystallizer in the few millimeter range, it is possible to adjust the temperatures along the tubing according to the needs of the crystallization. Thus, the product can be manipulated under controlled conditions [1–3]. Parameters that can be altered in the crystallization process include seed loadings, temperatures, solution concentrations, and flow rates of the suspension in the tubing. As a model system we have chosen the crystallization of acetylsalicylic acid (ASA) in ethanol (EtOH).
The effects of various process parameters on product characteristics have been investigated. All experiments resulted in significant crystal growth despite short residence times in the few minute range. Moreover, crystal masses increased by a few g/min and steady-state conditions were obtained in less than 5 minutes. Suppression of nucleation events by avoiding rapid cooling, thus high levels of supersaturation allowed successful achievement of narrow CSDs.
Additionally, simulations regarding the temperature gradients and the crystal growth have been performed and compared to the experimental results.
The aim of this work is to investigate image processing and process monitoring technologies which can provide a systematic, robust and sensitive solution for nucleation detection using bulk video imaging (BVI). The external BVI method is based on a video camera and a capture hardware, which captures 25 frames of the crystallization bulk per second . Another set of experimental data is generated using in-situ endoscopy and a small scale crystallization calorimeter . In this work the images are converted into time series and these are monitored using Shewhart and exponentially moving average (EWMA) statistical process control (SPC) charts; nucleation is detected when the signals exceed the control limits. A second approach to nucleation detection is histogram matching. Histograms of the images are generated and similarity measures are applied to detect significant changes indicating the beginning of nucleation; the current study evaluates the use of Kolmogorov-Smirnof and Chi2 statistics to determine the statistically significant change between the observed and the reference histograms for a 95% confidence interval.
The third approach relies on the advantages offered by the multivariate image analysis . According to this, multiway principal component analysis (PCA) is performed on the color images to obtain the first score, which is also an image. Furthermore, the PCA model residual monitoring performance was investigated. The image analysis techniques are applied for the crystallization of caffeine as a model for active pharmaceutical ingredients, both externally and in-situ.
The compression during tabletting is a complex and irreversible dynamic process and the study of it with instrumented tablet presses is beneficial for process and formulation optimization. The compaction and ejection cycle provides us suitable signals about the tabletting process. Instrumented presses used in tabletting research and development are normally equipped to measure punch force and displacement . By measuring the compression force both of the upper and the lower punch and the displacement of the upper punch force as a function of time and as a function of displacement can be analyzed. Moreover, several energy parameters, such as the lubrication of the tablets and the occurred friction during the compaction can be calculated from these data concerning for example to the binding characteristics of the applied excipients [2, 3].
The aim of our work was to investigate the effect of pharmaceutical excipients and process parameters on the tabletting properties of poorly compactable drugs analyzing the force-displacement profile of drug-excipient systems recorded by tablet press instrumentation. Furthermore with matching the forces measured on the upper and lower punches various parameters of the compaction – lubrication, friction coming up during tabletting – were calculated as well. A 32-type face centered full factorial design was applied to study the energy parameters as response variables (Y) and surface plot was made which is to describe with the following equation:
where according to the polynomial terms the effect of each independent variables (b, c), their linearity (d, e) and the interaction between them (f) are described. The force-time curves are useful indicators of the compaction behavior regarding elasticity and plasticity. In addition, studies on compression profiles may serve as fingerprints for tablet compositions.
This paper presents results of the simulation of unsteady mixing processes using a unique, mesh-less particle approach. Similar simulations, performed by Hörmann et.al  using common Computational Fluid Dynamics (CFD) have already been published in the past. The new method applied offers computational possibilities for problems involving transient dynamics, free surface analysis, moving boundaries or fluid-structure interaction. The simulations performed within the framework of this project are based on the Navier Stokes Equations using Large Eddy or Detached Eddy approaches. Turbulent energy dissipation models are used to resolve scales which are not captured by the particle resolution.
The investigations presented focus on the reproduction flow phenomena in a mixing tank, including viscosity adaption due to particle dissolution and analysis of particle concentration using a Discrete Phase Model (DPM). Applying CFD packages, problems including moving boundaries require time consuming work-arounds, which often lead to errors or divergence of the simulation. Due to the used mesh-less method, only the physical and mechanical properties of the problem, like number of revolutions, filling height or initial fluid viscosity need to be defined.
The results of the simulation are compared with Particle Image Velocimetry (PIV) measurements, showing good agreement.
Process performance in the synthesis of API and its intermediates can be reached through new technologies and methods. Significant development time reduction, operating cost reduction by yield improvement, energy savings and a fundamental improvement of plant safety are some of the main results of process intensification concepts. The activities can be divided into 4 phases: 1. evaluation, 2. verification, 3. development and 4. realization. A riskbased approach according to ICH Q9 is used during the whole project.
First of all the identification of reaction performance potential of existing chemical plants needs to be analyzed ending with the formulation of a target for the verification (basic feasibility). The evaluation starts with a process capability check, followed by the generation of a cost flow analysis and ends with a technology evaluation and comparison. Microinnova compares the needs for a chemical reaction with technology profiles to find out the most suitable solution for a process.
In the second phase the intensification potential of the reaction needs to be verified. Typically a continuous plant setup is developed and modified according to the needs of the chemical process. An operating procedure will be generated and optimized. Finally, a rough parameter scan of a few key parameters will deliver a relevant overview for the verification.
Once the potential is proven the process development needs to be done, where the basic feasibility delivers a good basis for the work (phase 3). The reaction process will be optimized. New process options are used as well as new technology concepts like microreactors and flow chemistry to find the best possible process. A knowledge based approach according to ICH Q8 will be used for the development by applying methods like the critical parameter concept and cause effect chains. Engineering data necessary for the scale up will be generated in this part of the work.
Finally in the fourth phase the observed performance needs to be realized. The deep specific knowledge in process intensification equipment needs to be combined with process experience and competence. The best possible equipment solution needs to be selected and calculated under considerations regarding the common standards like ATEX or cGMP. The gap between continuous processing and multi-purpose flexibility can be bridged by modularization of continuous plants.
Freeze dried products form a special group of sterile medications because of the unique manufacturing process. Their importance is ever growing due to the appearance of a number of unstable active pharmaceutical ingredients and nanosystems. This instability can be overcome with the formulation of a freeze dried product in most cases, thus ensuring a pharmaceutically acceptable shelf-life. On the other hand, in a number of compositions which comprise low concentrations of active ingredients the addition of a bulking agent is necessary to produce an optimal product .
Isomalt is a disaccharide sugar alcohol, which offers several important health benefits. The most important is its suitability for diabetics as it does not raise blood glucose or insulin levels. Its glycaemic index is very low .
The aim of our work was to assess isomalt as a new bulking agent. Furthermore, our purpose was to determine and optimize the parameters for freeze drying and to compare important properties (e. g. water content, water uptake) of freeze dried isomalt to conventionally used excipients (lactose).
Our experiment led us to the elaboration of the optimal parameters for freeze drying. We determined the acceptable rate of freezing and the time of primary sublimation during lyophilisation using statistical analysis of data. Latter was performed using the TableCurve®3D v4.0 (Systat Software Inc., London, UK). The effect of the independent variables on response y was modelled by the following polynomial equation:
where x1 and x2 are the factors (x1: time, x2: temperature) and b parameters mark the coefficients characterizing the main (b1, b2), the quadratic (b11, b22), and the interaction effects (b12). These equations were used in a two factorial, three level study to determine the water content of the product.
Furthermore we determined the water uptake of freeze dried isomalt over time and compared it to lactose lyophilised under the same process parameters.
Nowadays, a considerable range of industrial applications (pharmaceutics, cosmetics, foods agriculture products) relies on microencapsulation of solids or liquids by polymer coating and entrapment into polymer matrices.
Production of these micro-systems doubtless represents one of the most important improvements in the development of functional and therapeutic products belonging to the industrial fields above reported.
Generally, microencapsulation is widely used in pharmaceutical and nutraceutical fields, mainly to improve stability or to modify active principles release. Smart micro-systems release active molecules in fuction of external stimuli (pH, temperature). Preparation of the microparticles is achieved by both physico-chemical and mechanical processes. Feasibility studies about a process of microencapsulation are achieved by manual procedures in a lab-scale. The path toward commercialization of micro-particles needs plants or automated equipments on lab- or pilot-scale first, eventually on industrial scale. However, the state-of-art about microencapsulation techniques displays several processes based on manual operations that often don’t find an applicative realization in industrial field. Moreover, equipments reported in literature, especially in lab-scale systems, are not versatile devices and are penalized by a high consumption of resources. This work is focused on the development of techniques able to overcome such limitations. A promising new technique with low energy consumption consists in the coupling between ultrasonic atomization and microwave drying to produce micro-systems through an intensified process. The variation of energy/frequency of ultrasounds gives the dimensional distribution more suitable for the final applications. The microwave drying modifies materials structure giving unique properties to the micro-particles.
The propylene glycol Liquorice, Marigold and Yarrow extracts (LE, ME, YE-respectively) were obtained by the repercolation method, in the plant:extract ratio 1:2. Its appearance, relative density, index of refraction, pH values and dry materials content were evaluated. The extracts were encapsulated into the instant liposome (Natipide® II, Nattermann Phospholipids, Germany). Liposomal encapsulation of LE, ME and YE extracts was done by mixing of instant liposomes and the extract (2:1) in an ultrasound bath for 10 min. The obtained dispersions were diluted with purified water, in the ratio 1:1 and homogenised during 15 min to get final dispersion LD, MD and YD, respectively. Average radius of the liposoms size in dispersions was determined spectrofotometrically. For obtaining the O/W emulsion-type creams-vehicle, applying the cold-cold method, we used 5%w/w Emulgin®VL75 emulsifier [Lauryl Glycoside (and) Polyglyceryl-2 Dipolyhydroxystearate (and) Glycerine] (Cognis, Germany). The composition of vehicle is the same as described previously . The extracts LE, ME and YE (5% w/w) were incorporated in the vehicle (LC, MC and YC creams obtained). The liposomal dispersions LD, MD and YD were incorporated (30%) in the vehicle to obtain LLC, MLC and YLC creams, respectively. Liposomal creams (LLC, MLC, YLC) and nonliposomal creams (LC, MC, YC) were inspected simultanously: stability under storage: at room temperature (22°C±2), in hot air at 45°C±2 and at 5°C±2; centrifuge test: centrifuged in two 15-minutes runs, each at 3000 rpm; organoleptic properties inspected both visually and by smear tests on glass slabs: appearance, colour, odour and homogeneity of the samples; pH values of the creams were obtained by direct potentiometric method; rheological characterization of creams: Rotovisko RV 12 with coaxial sensor cylinder systems SVst, Svdin, was used (measurements carried out at 20±0,1°C with shearing rates continuously changing within the ratio from 0 to 110s−1). The results indicate that emulsifier mentioned in the text above is suitable excipient for preparation of stable o/w creams-vehicles for incorporation of LE, ME and YE as well as liposomes filled with extracts.
This work was supported by Serbian Ministry of Science and Technological Development, project number TR 20137.
In order to reach the deep lung API (active pharmaceutical ingredient) particles must have an aerodynamic diameter of 1 μm to 5 μm. Powders of this size are rather cohesive and exhibit poor flowing properties. Due to the fact that dosing is done volumetrically sufficient flowability is crucial. One solution to cope with cohesivity is to mix the API with coarser carrier particles of sufficient flowability. On the one hand interparticular forces of such interactive mixtures must be high enough to guarantee powder uniformity during mixing and powder handling and on the other hand low enough to allow API detachement form the carrier upon inhalation. Since interparticular forces depend on the contact area between API and carrier particles and consequently surface morphology the variation of the carrier surface may lead to optimized dry powder inhaler (DPI) formulations. Experiments of Maas  showed that spray drying aqueous mannitol samples at different outlet temperatures lead to varying morphologies. However these particles which had been prepared at a lab scale spray dryer were too small (d50,3=12 μm) as to be used as carriers in DPIs.
The aim of this work was to prepare spray dried mannitol carrier particles of variable surface roughness. Intended particle size was between 50 μm and 100 μm. Interactive mixtures of carrier particles with salbutamol were prepared and the respirable fraction was determined according to the European Pharmacopoeia using the next generation impactor (NGI).
The lipophilicity of a drug plays a key role for its distribution and accumulation in the human organism. The analysis of the distribution behaviour between buffer and a model membrane is most suitable to ascertain the partition coefficient between hydrophilic and biological membranes. Liposomes are common membrane models and ideal systems to characterize drug-membrane interactions .
We verified an indirect screening method to investigate the liposome-buffer partition coefficient using the fluorescent dye 6-Lauryl-2-dimethylaminonaphtalene (Laurdan). Laurdan offers a distinctive emission spectrum which is characterized by a shoulder at 434 nm and a maximum at 486 nm. The ratio of the fluorescence intensities at these two wavelengths is termed as the membrane state parameter (MSP). MSP changes depend on the polarity of the dye’s enviroment. After insertion of drug molecules into the liposomal membrane, the MSP value decreases as a function of the amount of inserted drug. The calculation of the MSP for different drug concentrations and various lipid amounts enables the determination of the drug’s partition coefficient [2, 3].
This indirect method offers the possibility to analyze the partition behaviour of a broad range of different drug classes without requiring further operations like separation steps or specific drug detection.
In an initial study, we determined the membran partition behavior of bile salts as amphiphilic model drugs.
Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Especially the alveolar rhabdomyosarcoma (ARMS) shows poor prognosis when metastases have developed.
The aim of this project is to downregulate the expression of several genes that are involved in the aggressive behaviour of ARMS by means of siRNA. Therefore, a liposomal delivery system, which is able to transport siRNA selectively to the ARMS cells, is being developed.
For enhancing the interaction of the delivery systems with the target cells and thus the effectivity, active targeting is intended. Therefore, the surfaces of the liposomes were modified with ligands binding to receptors expressed on the ARMS. Two different kinds of targeting devices were utilized: a RGD-peptide (arginine-glycine-aspartic acid tripeptide) recognizing integrins on ARMS as well as an antibody binding to the IGF1-receptor (insulin-like growth factor 1 receptor).
siRNA was encapsulated into the liposomes during liposome preparation by speedmixing (dual asymmetric centrifugation). This method is well suitable for efficient siRNA entrapment .
To modify liposomes for active targeting the ligands were coupled to the liposomal surface using the sterol-based post-insertion technique (SPIT) .
For optimization of the delivery systems, cellular interaction of fluorescently labeled liposomes with the ARMS cell line RH-30 and gene silencing of siRNA-loaded (anti-GFP siRNA) liposomes in stably GFP-transfected RH-30 cells were determined by flow cytometry.
RGD-peptide as well as anti-IGF1-receptor antibody modified liposomes showed significant interaction with RH-30 cells. Furthermore, specificity and extend of gene silencing was analyzed.
Blood vessel diseases like antherosclerosis lead to formation of blood clots with severe consequences like acute ischemic stroke or myocardial infarction .
A well-directed treatment of thrombotic events implies fast localization of the blood clot as well as fast and effective destruction with low doses of lytic agents to prevent the patient from severe damage. Standard procedure is application of high doses of thrombolytic agents with bleeding complications as negative side effects. Daffertshofer et al. investigated clot lysis efficiency of thrombolytic drugs alone, in combination with ultrasosund and ultrasound alone and proofed that even ultrasound alone is able to burst clots . The well known fact that blood clots are fibrin rich areas can be used for the development of a site specific drug vehicle for diagnostic and therapeutic applications.
The aim of this study was to proof the concept that ultrasound active liposomes, surface modified with antifibrin antibodies, can act as drug carriers. We focused our work on the development of novel ultrasound active liposomes which are stable under similar conditions as in human body like blood pressure, physiological pH and body temperature. They are composed of DPPC/PEG40-stearate or DSPC/PEG40-stearate and show a small diameter between 100-200 nm with a sharp polydispersity. In order to develop long-circulating immunoliposomes, which combine sterical stabilization with a superior targetability, we attached fibrin antibodies directly onto the distal ends of s synthesized DPPE-PEG derivative, which had been endgroup-functionalized with cyanuric chloride . The binding efficiency of the antibody, the ultrasound activity and the binding ability to fibrin under streaming conditions was investigated.
The authors would like to thank the DFG “Forschergruppe Nanohale 695” for support.
Recently, increasing attention has been paid to lecithin/chitosan nanoparticles as colloidal drug delivery system showing the great potential to improve the permeation of encapsulated drug across various biological barriers [1, 2]. However, the overall poor stability of colloidal nanoparticles in an aqueous medium is a significant drawback in their development perspective. Therefore, the aim of this study was to set up a freeze-drying process of melatonin-loaded lecithin/chitosan nanoparticles in order to improve their physico-chemical stability. Suspensions of nanoparticles were freeze-dried in the presence of glucose or trehalose as cryoprotectants. Nanoparticles were characterised in terms of size, polydispersity, zeta-potential and melatonin content before and after the lyophilisation process.
Before the lyophilisation process, lecithin/chitosan nanoparticles obtained were characterised by mean diameters ranging between 117.4 ± 3.9 and 328.5 ± 3.1 nm, and were significantly larger than comparable lecithin nanoparticles. The zeta potential was inverted from negative values for lecithin nanoparticles to positive values for lecithin/chitosan nanoparticles (up to 30.2 mV).The size and the surface charge of the nanoparticles increased with the increase in the chitosan content and the negative charge of lecithin used in the preparation. Compared to lecithin nanoparticles, lecithin/chitosan nanoparticles were characterised by significantly higher melatonin loadings (up to 7.1 %, w/w).
In the lyophilisation process it was shown that a minimal concentration of 2.5 % (w/v) of trehalose or glucose was needed for a suitable reconstitution of nanoparticles lyophilised at particle concentration of 2 mg/ml. No significant difference in the mean particle size, size distribution or zeta-potential of nanoparticles in suspension before and after freeze-drying and reconstitution were observed. No significant melatonin leakage caused by the stress of freezing and dehydration in the presence of cryoprotectants occurred.
This work was supported by Grant 006-0061117-1244 of the Ministry of Science, Education and Sports of the Republic of Croatia.
Atherosclerosis is the major cause of mortality in the western world today. The main objective for the use of nanotechnology is to address prospects of imaging atherosclerotic (AS) plaques, which lead to clinical endpoints. Today various biomarkers are known to be involved in the pathophysiologic scenario of AS-plaques. For the targeting the biomarker globular Adiponectin (gAd)  was coupled to two different nanoparticles (NP). The gAd-targeted NPs were investigated towards their potency to characterize critical scenarios within early and advanced AS-plaque lesions applying an AS-mouse model. Aortas of wt and ApoE-deficient mice, fed a high fat diet, were dissected and first stained with fluorescence-labelled gAd. Ex vivo imaging was performed using confocal laser-scanning microscopy (CLSM). Second, gAd was coupled to fluorescence-labelled protamine-oligonucleotide nanoparticles (proticles), respectively flu-labelled Stealth®-liposomes to enhance the imaging performance. The gAdproticle constructs were characterized using scanning electron microscopy. Modified native gel electrophoresis was used for the characterization of the gAd-targeted Stealth®-liposomes. Successful fluorescence labelling of gAd was achieved. According to WB analysis no critical structural changes occurred. Ex vivo CLSM imaging showed that flu-gAd binds to the AS-plaque but not to the lesser injured aortic surface. Both, successful coupling of gAd to proticles, respectively to the reactive PEGylated lipids exposed on the liposomal surface of Stealth®-liposomes, was achieved. Compared to the plaque-staining using only flu-labelled gAd, the gAd targeted, flu-labelled Stealth®-liposomes showed a strong signal enhancement in CLSM imaging, while the gAd targeted, flu-labelled proticles generated a different, more spotty-like staining on the surface of the AS-plaques. Results by now suggest a promising role of the applied gAd-targeted NPs for enhanced AS-imaging in vivo and their potential use for new targeted therapeutic strategies in cardiovascular medicine.
The eye provides a variety of defense mechanisms, which protect the organ against numerous different substances. These mechanisms include the bony orbit, the eyelids and cilia, the tear film and nasolacrimal drainage, corneal and conjunctival epithelium, and a submucosal secretory immune defense. In order to overcome the strong physiological barriers, new drug delivery systems have been established, including the development of nanocarriers. However, it is known that nanostructured materials are able to invade cellular structures and trigger toxic reactions.
For the establishment of an in vitro system, fluorescence-labelled carboxyl polystyrene particles (CPP 20 nm and 200 nm) were characterized in terms of their physico-chemical properties (size, distribution, agglomeration, surface charge) in different biological media (i. e. MQ-water, 0.9% NaCl and tear fluid) using a ZetaSizer Nano-ZS (Malvern). Ex vivo permeability studies were performed with static Franz diffusion cells. Donor and receiver compartment were separated by insertion of three weeks cultured excised human corneae. CPP (20 nm) were suspended in physiological NaCl, applied in concentrations of 100 μg/ml and 250 μg/ml and incubated for 6 hours. Samples were withdrawn from the sampling port of each cell and fluorimetrically analyzed. After 6 hours the tissue was fixed, frozen, sliced into 10 μm thin slides and observed with a fluorescence microscope.
Size and surface charge of the particles differed according to the medium. Particles suspended in tear fluid showed reduced surface charges and diameters three times higher, compared to those, suspended in MQ-water and 0.9% NaCl. This increase in particle size can not only be explained by partial aggregation of the particles but also by the fact that in biological fluids, proteins attach to the nanoparticle surface, which leads to the so called ‘protein corona‘. Histopathological Analysis showed that corneal epithelium and endothelium detached during prolonged storage and incubation. Fluorescent particles were seen in the entire corneal stroma.
20 nm CPP can penetrate through Bowman′s and Descement membrane and the entire corneal stroma. Corneae with shorter culturing times will be used for the assessment of the epithelial barrier function.
In studying in vitro drug release from nanoparticles (NPs) an important issue to consider is the method that can be used to measure drug release . The characterization of the in vitro drug release from a colloidal carrier is technically difficult to achive. This can be attributed to the inability of effective and rapid separation of the particles from the dissolved or released drug in the surrounding medium owing to the very small size of nanocarriers .
The aim of our study was to compare in vitro release profiles of budesonide from NPs, which were obtained using four different methods for the assessment of drug release: membrane diffusion technique (dialysis or reverse dialysis) and sample and separate technique (filtration or ultracentrifugation).
Budesonid as a hydrophobic drug was incorporated into poly(lactic acid) NPs, which were prepared by the nanoprecipitation method using vibrating nozzle device . The mean size of NPs was 260 nm, the entrapment efficiency of budesonid 70 % and drug content 5,6 %. Our results show that in vitro drug release profiles from NPs obtained by four methods were different. In vitro release profiles obtained by filtration, centrifugation and reverse dialysis exhibited a typical biphasic release phenomenon namely initial burst release and consequently sustained release. The critical points in filtration are the pore size and adsorption of dissolved drug on filter membrane. The problem with centrifugation is that smallest particles are very slow to sediment and further dissolution can occur over this time. The fastest burst release phase was obtained after centrifugation following by filtration and reverse dialysis. The release of budesonide from NPs determined by dialysis was much slower and showed no burst release. In this method the transport across the membrane was rate limiting, and therefore the true release rate was not measured.
No clear agreement exists on the suitability of the techniques reported to date for drug release determination from particles in the nanometer size range. Our results show that the comparison of in vitro release results obtaned by different methods is not reasonable.
Ketoprofen is a potent non-steroidal anti-inflammatory drug and an excellent candidate for transdermal/dermal delivery because of its adverse side effects and short elimination half-time after oral administration [1, 2]. It is previously reported that drug loaded nanoparticles may provide a number of advantages compared to free drug such as increasing of bioavailability and drug skin penetration, ensuring controlled drug delivery, delayed and prolonged drug action in the application site [3, 4]. Therefore, the aim of our work was to prepare poly-D,L-lactide nanospheres of ketoprofen by modified precipitation method and to characterize them [5, 6]. The size and morphology of the obtained particles have been determined by field-emission scanning electron microscopy (FESEM) and laser light diffraction. Span value and mean diameter were about 1,3 and 65 nm, respectively. The encapsulation of ketoprofen into the PDLLA nanospheres was confirmed by X-ray diffraction (XRD) and infrared spectroscopy (FT-IR). Nanoparticles of ketoprofen were obtained with high encapsulation efficiency.
Formulation development of nanostructured lipid carriers (NLC) aims physical stability in both particle size and crystalline state. Besides production parameters, the selection of appropriate stabilizers is the most crucial parameter in the development. Very often the influence of different stabilizers on the size and the physical stability is investigated. However, little is known about the influence of stabilizers on the crystalline state of lipid nanoparticles (LN). Thus, two surfactants of polyglycerol fatty acid ester type were screened for their ability to form physically stable NLC and for their influence on the crystalline state of the lipid matrix. The investigated surfactants are polyglycerol 6-distearate (PD) and polyglycerol 6-isostearate (Pl). Physical stability study was performed using light microscopy, dynamic and low angle static light scattering. DSC study was used to analyze the crystalline status of LN. Both surfactants in the concentration of 1.0 % (w/w) led to almost similar LN with a mean diameter between 180–200 nm. LN were stable over the period of 90 days. The absence of large particles was also confirmed by light microscopy and low angle static light scattering. In contrast to this significant differences were found within DSC thermograms. LN stabilized with PD show a melting event, whereas LN stabilized with PI show no melting event. Hence, PI inhibited the crystallization of the lipid matrix, leading to a non-crystalline LN instead of NLC. A possible explanation is that PD which has a solid lipid tail leads to a different interaction with lipid matrix, than the liquid lipid tail surfactant (PI). These interactions can lead either to an initiated crystallization or to the circumvention of crystallization. In conclusion: Stabilizers can influence the crystalline state of the lipid matrix of LN. Thus, for a successful development, this parameter needs to be investigated. Furthermore, it might be a helpful tool to develop NLC with a “tailor-made” crystalline state and release profile in the future.
The authors would like to acknowledge the financial support obtained from DAAD (Deutscher Akademischer Austauschdienst) for the present research.
Solid lipid nanoparticles (SLN) represent rapidly growing class of colloidal transport system, particularly interesting for pharmaceutical applications . Various physical methods can be used for characterization of nanoparticles (NPs), but evidently, their imaging and monitoring, especially in biological system, are of primary importance. Fluorescence microscopy may provide valuable information regarding interactions between nanoparticulate carriers and target cells as well as their intracellular fate. For this purpose, good fluorescent labels are needed. Since numerous different dyes with affinity to particular target object in the cell are available, for colloidal carriers only few are frequently used (e. g. derivatives of cyanine, fluorescein, rhodamine and coumarin), which are not design for particular nanosized system and often make comprehension difficult .
The advantages of the in vitro imaging of SLN by using novel fluorescent dye (SPP-189) was investigated in comparison with frequently used dye 6-coumarin (C). SPP-189 is derivative of coumarin with long lipophilic chain  which enables its stronger integration into lipid matrix of SLN than 6-coumarin, what was proved by release study. SPP-189 shows overall brightness in the pictures of long lasting monitoring of SLN-SPP-189 internalization and intracellular localization comparing to the dye 6-coumarin (Figure 1).
Accordingly, we present an extremely sensitive tool to monitor the location of fluorescently labelled NPs by SPP-189 within respective organelles that should find a wide application in drug study.
Nanoparticles composed of silica and its derivatives are currently in use both in basic scientific research and applied enginnering development. Precursor silica particles have been investigated for applications in microeletronics, chemical, biological sensors, as targeting devices and in drug delivery . The efficacy and quality of these products is highly dependent on the size and polydispersity of the silica nanoparticles . The aim of this work was to compare two mixing modes, i. e. ultrasonication vs magnetic stirring, in the synthesis of silica nanoparticles. These nanoparticles were produced applying the aqueous sol-gel method  at room temperature via hydrolysis and condensation of tetraethyl orthosilicate (TEOS) using HCl as catalytic agent. Particle size was measure by dynamic light scattering (DLS) and the morphology of nanoparticles was assessed by transmission electron microscopy (TEM). Ultrasonication and magnetic stirring allowed the production of particles with mean average size of 485.9 nm and 81.8 nm, respectively. TEM micrographs showed that particle aggregation is stronger under ultrasonication process in comparison to magnetic stirring. This result was attributed to the nanoparticles collision enhanced by the higher input of energy of sonication which is minimized under magnetic stirring. Furthermore, the latter provides a more uniform energy distribution in the dispersion contributing to better assembling of these particles. The formation of homogenous, monodispersed nanometer silica particles depends on the reaction conditions and parameters e. g. TEOS concentration, pH, temperature and H2O/TEOS ratio.
This work was supported by FCT, SFRH/BD/60640/2009 to T. Andreani and by Capes to A.L.R. Souza. Whe are thankyfull to Dr. L. Fernandes for TEM assistance.
Aspergillosis is a common fungal infection in falcons. Standard treatment, systemic application of Itraconazol, is limited due to hepatotoxicity. The aim of the present study was to develop Itraconazol-loaded Nanostructured Lipid Carriers (NLC) for pulmonary application. NLC are a nanoparticulate carrier system composed of a blend of solid lipid and liquid lipid, with a particle matrix being solid at body temperature. NLC have shown many advantages for pulmonary application such as deep lung deposition due to the small particle size, adhersive properties leading to an accumulation in the lung, prolonged release properties leading to a reduction of dosing frequency and low toxicity due to the use of well tolerated excipients.
NLC composed of 0.02% Itraconazol (Jai Rhades Sales, India), 4.5% Precirol ATO 5 (Gattefossé, Germany), 0.5% oleic acid (Croda, Germany), 2% Tween 20 (Cognis, Germany) and 92.81% Milli-Q water were prepared by hot-high pressure homogenization method (Panda K2, GEA Niro Soavi, Germany). The particle size of NLC was determined using a NanoSizer ZS (Malvern Instruments, UK). The melting point of NLC was assassed by differential scanning calorimetry using a DSC 204 F1 Phoenix (Netzsch, Germany). NLC dispersion was nebulized using a Nanonebulizer (Medic Activ, Germany). The particle size of the aerosol was analysed using a wide range aerosolspectrometer (Grimm Aerosol Technik, Germany).
Itraconazol-loaded NLC with an average particle size of 119 nm and a polydispersity index (PI) of 0.254 were obtained applying 5 homogenisation cycles at 500 bar. NLC showed a melting point of 45.1°C proving the solid state of the particle matrix of the carrier system. Nebulizing the NLC dispersion using a Nanonebulizer led to an aerosol with an average particle size of 265 ± 1.78 nm. The particle size of NLC in the aerosol was found to be of the same order of magnitude as before nebulization, which shows that after nebulization the favorable characteristics of the delivery system are maintained.
Nebulization of Itraconazol-loaded NLC using a Nanonebuizer by MedicActiv leads to an aerosol with favorable characteristics for pulmunar treatment of aspergilosis in falcons.
This work was supported by MedicActiv Vertriebs-GmbH (www.medicactiv.de).
In order to use API (active pharmaceutical ingredient) particles in a dry powder formulation, they have to exhibit an aerodynamic diameter of 1 μm – 5 μm. Only this order of magnitude guarantees a penetration to the deeper parts of the lungs. Particles of this scale are very cohesive and possess rather poor flow properties , which lead to difficulties concerning volumetrically dosing. To handle cohesivity, the drug particles may be coated with particles in the nanometer range. Nanoparticles will act as spacers between the contacting API particles and may cause a reduction of the interparticle forces. Already implemented experiments showed that Aerosil® particles can enormously alter the particles′ flow characteristics. [2–4]. Due to the fact that Aerosil® is not approved in DPI formulations, there is a rising demand for substances, which can be used as an alternative.
The aim of this work is to create nanoparticles via a novel spray drying technique. With the help of this apparatus, it is feasible to produce API particles in the nanometer range. The intended benefit is the absence of any excipient, because both, the drug itself (1 μm – 5 μm) and the nanoparticles, acting as flowability enhancers, are made of the same API substance. During a mixing process  the nanoparticles are attached on the microparticles` surface. This alternative DPI formulation should facilitate a reproducible dosing, which is a quite important requirement for using DPI’s.
The major limitations for the use of small interfering RNA (siRNA) in vivo and in vitro are the relatively high molecular weight, the negative charge, and the susceptibility to nuclease degradation . Therefore it is imperative to use suitable carrier systems to ensure internalization and intracellular separation of the components to subsequently induce RNA interference. The aim of this study was to silence a specific protein expression with a nanoparticulate system by RNA interference and analyze the consequences on tumor cell growth, proliferation and protein expression. Fluorescence microscopy was used for intracellular detection and localization of the components. This study was performed to determine differences between intracellular siRNA delivery after the application of different developed protamine-based nanoparticle systems and their effect on human glioma cells. Therefore two particulate systems composed of i) siRNA and protamine (binary particls) and ii) siRNA, protamine and human serum albumin (ternary particles) were assembled and analysed by size and surface charge before applying on cells. Our study has yielded the following results: fluorescence microscopy showed differences in intracellular particle separation. Binary particles could not be separated after internalization. Protamine-rhodamine B was colocalized with siRNA-FAM dye in the cytoplasm while in case of ternary particles no intracellular protamine signal could be detected. In this case siRNA-FAM was uniformly distributed in the cytoplasm. Furthermore PCR studies showed a protein expression of 76% for ternary particles and 112% for the binary system referring to control (100%). Proliferation of the cells after transfection was 85% for ternary particles and 106% for binary particles. This data verified the necessarity of intracellular separation of siRNA from the nanoparticle complex to induce RNA interference.
The authors would like to acknowledge the financial support of the study by FFG, Bridge-program: P810994.
Pharmaceutical drug formulations in the nanometer scale received significant interest during the last years as they provide the possibility to stabilize drugs and to transport them directly to their target. Biodegradable self-assembled nanoparticles consisting of protamine and oligonucleotides, so-called “proticles”, have already been successfully developed for different applications [1, 2]. However, the production could only be performed at the milliliter scale due to the sensibility with respect to parameter changes at larger volumes. For further investigations, such as in-vivo or clinical studies, it is essential to scale-up the production process to achieve larger amounts of proticles. As the introduction of a microreactor has been shown to have many advantages for the scale-up of nanoparticle formation processes, this approach is investigated for the production of proticles. The advantages of microreactors include the ability for continuous mixing of small volumes and the precise control of the operating parameters . Because of the high costs of oligonucleotides, alternative nanoparticles comprising protamine and polyacrylic acid were developed for the first scale-up experiments. The characterization of these nanoparticles produced at the milliliter scale, included size and zeta potential measurements which were performed by Dynamic Light Scattering and Laser Doppler Electrophoresis. Protamine binding efficiency was determined by fluorimetry. After optimization of the process, particles of about 150 nm and a zeta potential of around 30 mV were produced exhibiting a suspension stability of at least 5 days. First experiments with a microreactor can now be started. This presentation will highlight some details of the microreactor, its advantages in the scale-up and the latest results concerning the nanoparticle production.
The project is supported by one-A engineering austria, Prager Elektronik and the Austrian NANO Initiative (Nano-HEALTH).
Ischemic stroke results from a cerebral vessel occlusion by a blood clot. Treatments are intended to restore the cerebral blood flow as soon as possible to avoid major brain damage. Intravenous administration of recombinant tissue plasminogen activator (rtPA) is the only thrombolytic drug approved for treatment of acute ischemic stroke .
In vitro and in vivo studies have shown that rtPA in combination with ultrasound and microbubbles can improve clot lysis . However, the exact mechanisms involved in this sonothrombolysis process remain unknown. Additionnally, the optimal conditions for clot lysis are not mastered.
The aim of the present work was to set up a robust in vitro human clot model and to evaluate the thrombolytic effect of rtPA and microbubbles while varying various acoustic parameters.
Practically, fresh whole human blood clots were formed after three hours at 37°C in 200 μL glass micropipettes through which a silk suture had been threaded. The clots were characterized by their diameter, by scanning electron microscopy (SEM) and by immunostaining. The clots were placed in a cell mounted with transparent Mylar® films, and filled with human plasma. The cell was then placed in a thermostated water bath (37°C) and the clot was exposed to different experimental conditions of ultrasound (0.65MHz), rtPA and microbubble suspensions in a continuous infusion. Every five minutes during 60 minutes photographs were taken and analysed to determine the mean diameter decrease of the clot.
The clots had an initial diameter of 0.89 ± 0.06 mm (mean diameter ± sd). In the presence of rtPA at 3 μg/mL, microbubbles and ultrasound, the mean clot diameter dropped by more than 0.5 mm in 60 min. Under SEM and with the immunostaining, sonothrombolysis effects were clearly observed.
These preliminary experiments are encouraging and will enable a thorough evaluation of the synergetic effects of microbubbles, ultrasound and rtPA.
The majority of topical ocular preparations available today are in the form of aqueous eye drops. This might be due to existing problems related to ocular drug delivery systems such as cost, bulk manufacturing, and patient compliance. Commercial eye drops are commonly used by patients, due to their ease in usage and low interference with vision. A homogenous dosage solution form offers many industrial advantages including the simplicity of large-scale manufacturing. At the ame time, commercial eye drops are often ineffective and require frequent application. Only 1–5% of the applied drug penetrates the cornea and goes into intraocular tissues [1, 2]. Therefore, the aim of this study was to formulate a polyelectrolyte/surfactant mixture that can maintain the advantage of commercial eye drops while enhancing biopharmaceutical properties i.e., enhanced ocular bioavailability.
Micelle systems composed of the polyoxyethylated nonionic surfactant Pluronic® F127 (F127) and cationic polyelectrolyte chitosan (CH) were prepared with dexamethasone (DEX) as a hydrophobic model drug. The F127/CH micelles were characterised by their hydrodynamic diameter and a zeta-potential ranging between 25.4 and 28.9 nm and +9.3 and +17.6 mV, respectively. The DEX loading was between 0.48% and 0.56%, and no significant influence of CH on DEX loading was observed.
This colloidal carrier was well tolerated in rabbit eyes, and no clinically abnormal signs in various ocular structures were observed. The increase in intraocular pressure (IOP) in rabbits was used to evaluate DEX ocular bioavailability. The AUC values showed a 1.7- and 2.4-fold increase in bioavailability with F127 and F127/0.015 w/v % CH micelle systems, respectively, as compared to a standard DEX suspension. These data indicate improved intraocular DEX absorption from the micelle systems, which can be ascribed to both F127 and CH corneal permeability enhancement.
This work was supported by Grant 006-0061117-1244 of the Ministry of Science, Education and Sports of the Republic of Croatia
Different promising colloidal delivery systems including microemulsions and liquid crystals could be obtained with suitable combination of oil, surfactants/cosurfactants and water. However, the main problem from a formulation point of view has been to choose the surfactants capable of forming such systems and be physiologically acceptable in the same time. To overcome the later shortcoming the use of less toxic non ionic or biocompatible naturally obtained surfactant is proposed .
The aim of this study was to develop and characterize colloidal systems for dermal delivery based on nontoxic components, i. e. lecithin and Tween 80 as surfactant mixture, isopropyl myristate as oily phase and water.
Pseudoternary phase diagrams were constructed using conventional titration technique with 1:1 and 2:1 mass ratios of Tween 80 to lecithin, respectively, isopropyl myristate and water. Clear samples of high viscosity were deemed to be lyotropic liquid crystals. Representative samples were further evaluated for their physical stability and rheological characteristics, while the structure of the samples was examined with a polarization microscope.
The region of clear and highly viscous mixtures was detected at higher concentrations of Tween 80/lecithin (at both mass ratios) that were further on confirmed as lamellar lyotropic liquid crystals with polarization microscopy due to characteristic “Maltese cross” textures The samples were physically stable at room temperature during 2 months and after centrifuge test. Determination of viscosity and flow curves showed shear-thinning characteristics and thixotropic behaviour of samples containing more than 35% of water, while samples with less than 30% of water revealed rheopectic properties. Additionally, the viscosity was decreasing with increasing temperature, and was affected by composition of surfactant mixture (higher content of Tween 80 induced higher viscosity) and water content (up to 40% water content increased viscosity due to hydration of lecithin polar head groups).
Lamellar lyotropic liquid systems were identified from phase diagrams at higher surfactant concentration. They show great potential for dermal application due to nontoxicity of surfactants, physical stability and moreover, convenient rheological properties.
Solid dispersions of active pharmaceutical ingredients (APIs) in freely water soluble carriers are often used in order to improve dissolution rate and/or solubility of poorly water soluble drugs  in the aqueous gastro intestinal fluids. Depending on the dispersion of the drug in the carrier one may distinguish between solid solutions, in which the drug is molecularly dispersed within the carrier and solid suspensions, which is a dispersion of either amorphous or crystalline drug particles in the carrier. Whether a solid solution or a solid suspension is obtained, depends on the preparation conditions of the solid dispersion. Moreover, storage may lead to changes in the dispersion of the drug in the carrier, for example leading to the recrystallisation of initially dissolved API in the carrier. These changes may substantially alter the dissolution profile/solubility of the drug in the aqueous dissolution media.
There are several techniques that are used in order to either determine the state of dispersion of the drug in the carrier or to monitor storage induced changes. Conventional techniques include thermal analysis and wide angle X-ray diffraction, although the detection of very fine drug crystals in the carrier or the transition of an initial solid solution to a solid suspension at a very early stage is almost impossible.
We apply simultaneous small and wide-angle X-ray scattering, which is becoming more important for solid-state pharmaceutical analytics , in order to gain information of solid nano-structure and dispersions stability. The study presents examples consisting of nimodipine and polyethylene glycol and/or polyvinylpyrrolidone, characterized by SWAXS analytical parameters.
In order to fulfill the qualtity requirements introduced by regulatory authorities for pharmaceutical products, sure and adequate methods are needed for the product qualification. A considerable portion of process analytical technology (PAT) tools exists, which satisfy many expected requirements, as nondestructive and sensitive examination of material properties. Recently continuous manufacturing (e. g. mixing, extruding, etc.) became a big issue, which has the need for fast process monitoring systems with the capability for real-time and in-line measurements.
Here, a near-infrared (NIR) spectral imaging system (Helios Core from EVK DI KERSCHHAGGL) with an InGaAs detector (240x316 Pixel) was tested for its applicability in the pharmaceutical industry. The system characterization was performed in order to examine detection limits for low active pharmaceutical ingredient (API) content or powder blends, impurities in powder mixtures and the coating thickness of tablets. Multivariate data analysis (MVDA) was applied for qualitative and quantitative evaluation.
Spray coating is an important unit operation in the pharmaceutical industry. The ultimate goal is to produce uniformly coated products with the desired amount of coating material, to guarantee controlled active pharmaceutical ingredient (API) release. The coating thickness and homogeneity can be determined with spectroscopic process analytical technology (PAT) tools, like near-infrared (NIR) and Raman spectroscopy.
Here, both spectroscopic techniques were applied for the analysis and characterization of tablet coatings. Due to the relatively small coating thickness with respect to the penetration depth of the excitation, both laser light and Raman scattering penetrate through the coated layer. Hence, Raman chemical mapping can also be used to analyse qualitatively the chemical composition (i. e. the distribution of (active) components) in tablets. A quantitative multivariate data analysis (MVDA) based model for the tablet coating thickness was developed on basis of tablets sampled from different stages of a coating process [1, 2]. Calibration was carried out by monitoring weight gain, which is also used in industrial quality control. A comparison between calibration models for four different sampling modes (Mapping, Line, Super Macro and Single Point) was performed. The root mean squared error of prediction (RMSEP) of the different sampling methods showed 1.25, 1.33, 2.1 and 2.05 mg for Mapping, Line, Super Macro and Single Point measurement respectively. In accordance, NIR measurements with a fiber optical reflectance probe showed a RMSEP of 1.07 mg, representing a good correlation between the two methods. Although the RMSEP of the mapping mode is the smallest, it does not compensate for the much longer measurement time. Raman chemical mapping is conditionally applicable for the determination of coating thickness, but it is not a rapid standard method.
We investigate the compaction behavior of the most usual pharmaceutical excipients mixture (placebo) contained lactose monohydrate (LmH), microcrystalline cellulose (MCC), silicum dioxyd (SiO2) and magnesium stearate (MgSt) with combined small- and wide-angle X-ray scattering (SWAXS). In general, the method is becoming an increasingly important technique in pharmaceutical solid-state characterization [1, 2]. Highly relevant questions of polymorphism in crystalline materials, stability and nanostructure of amorphous states, total inner surface in controlled-release formulations, stability and ageing formulations can be addressed by this technique. The information to be gained by SAXS expands largely the scope of conventional powder diffraction techniques. A particular advantage lies in the simultaneous observation of nano-scale (SAXS) and atomic scale (WAXS). With the development of high-brilliance laboratory SWAXS systems (Hecus S3MICROpix) the times for analysis have been greatly reduced, and hence the method can be applied to quality screening and process analytical technology (PAT). Furthermore, examples will be presented for technologically relevant systems, such as polymorphic forms of active ingredient carbamazepine, compactness of granulate, amorphous formulations of their coating lack, and dissolution behavior of pentoxifylline after process granulation. The results show, that an analysis in terms of robust SAXS parameters, such as total inner surface, scattering power intensity, Porod exponent and average correlations length, can provide highly valuable technological information about granulate compactness and dissolution behavior in correlation with tablets hardness.
Protein aggregation represents probably the most common and troubling manifestation of protein instability. Shelf life of therapeutic proteins is particularly impaired by aggregation, which can occur almost in all phases of protein drug development. Administration of protein aggregates may lead to reduced pharmacological activity and adverse drug reactions. Thus, prediction of protein aggregation is of major interest for pharmaceutical industry. Existing sequence based bioinformatic tools are barely able to predict the aggregation behavior of therapeutic proteins. To implement new approaches for this, detailed analysis of protein aggregation, its underlying mechanisms and its kinetics is necessary.
In our study we simulate process conditions for induction of aggregation of a common therapeutic protein (granulocyte colony stimulating factor, G-CSF). Size exclusion HPLC is performed to quantify protein aggregates and to enable kinetic pathway analysis by identification of different aggregation intermediates under certain process and formulation conditions over time. Further characterization is accomplished using intrinsic fluorescence, polyacrylamid gel electrophoresis and mass spectrometry.
This work was supported by FFG, Land Steiermark and Steirische Wirktschaftsförderung (SFG).
To investigate the nonlinear kinetics of in vitro hepatic uptake the OATP substrate, Pitivastatin, was used as a probe. Experiments were conducted using freshly isolated rat hepatocytes, utilising the ‘oil spin’ methodology described by Hassen et al . Briefly, freshly isolated rat hepatocytes were incubated with Pitvastatin (5–300μM). At 10 s, 30 s, 50 s and 70 s aliquots were spun through a silicone oil layer to separate the hepatocytes from the media. [Pitivastatin]hepatocyte was detemined using LCMSMS.
Uptake to rat hepatocytes was saturable and progressed according to Michaelis-Menten kinetics. The Km and Vmax of Pitvastatin were 2050 pmols/min/106 cells and 33 μM respectively, which was in in good agreement with other literature reports .
A nonlinear pharmacokinetic model has been derived to characterise the uptake process. A structural identifiablity analysis was performed on the model to establish that all unknown parameters could be identified from the experimental observations available. The model was then subsequently used for parameter estimation and model validation using the data collected. Sensitivity analysis and model robustness analyses were also performed. Once fully validated the model has the potential to perform robust, predictive simulations to ascertain optimal levels of uptake and the effects of the use of appropriate inhibitors.
This work was supported by AZ & TG was supported by AZ and the MRC Capacity Building initiative.
Stressful life style of modern human societies leads to many diseases and one of the growing indispositions related to this fact is hypertension. Thus, there is a strong need for novel effective drugs. One of the new categories of antihypertensive drugs consists of AT1 antagonists (SARTANs), whose action is based on blocking of the active site of the AT1 receptor. The aim of the present study is to contribute to a basic understanding of the their molecular mode of action on membranes. We studied influences on the binary mixtures of DMPC/cholesterol and POPC/cholesterol. Losartan led to a complete loss of positional correlations between adjacent bilayers for all single component model membranes. This can be explained by the negative surface charged conferred to the bilayers upon losartan insertion. The effect was however, counter-balanced upon the addition of cholesterol. Both, POPC and DMPC bilayers exhibited no positional correlations up to 5 mol% cholesterol, respectively. However, only POPC remained uncorrelated at 20 mol% and above, while DMPC/cholesterol bilayers exhibited multilamellar vesicles, i. e. a reentrant transition of the positionally uncorrelated bilayers into correlated ones. Our results may be understood in view of the different hydrocarbon chain packing densities in saturated versus unsaturated bilayers and their respective affinities to interaction with cholesterol. This shows that the insertion of losartan into the membrane may be overridden by a tighter bilayer interface and emphasizes the role of hydrocarbon chain composition in its mode of action. Additionally, we also the influence of the SARTANs (e. g. losartan, candesartan-cilexiril and valsartan) on the global structure of phospholipid bilayers composed of pure dimyristoyl phosphatidylcholine (DMPC), palmitoyl oleoyl phosphatidylcholine (POPC) and dipalmitoil phosphatidylcholine (DPPC) applying synchrotron small-angle x-ray scattering. Valsartan is the only one which shows a disruption of the neutral DPPC bilayer, more visible at higher drug concentrations. This could be attributed to the valsartan chemical structure, which poses a carbonyl acid (carboxyl) group providing higher polarity compared to the other two drugs in the neutral environment.
The interaction of active pharmaceutical ingredients with various surfaces and solutions are of high interest in pharmaceutical industry in particular with respect to development of new formulations, new packaging material  and cleaning validation of production plants . Experimental adsorption/absorption studies as well as the performance of cleaning validations provide valuable data for these research fields; however appropiate analytical methods are needed for the drug of interest. In the case of diisopropylphenol – a small lipophilic drug which is typically formulated as lipid emulsion for intravenous injection – the main analytical challenge is a highly sensitive method (in the ng/ml range) suitable to be applied to a variety of different sample matrices (lipid emulsions, aqueous solutions, organic solvents as well as various surfaces). In particular, the analysis of diisopropylphenol in complex matrices such as lipid formulations requires efficient samplecleanup methods. We here report the development and evaluation of a new headspace-solid phase microextraction (HS-SPME) methodology for GC-MS as a cleanup procedure for the moderately volatile drug diisopropylphenol in various matrices. Optimization of the HS-SPME procedure with respect to temperature, incubation times and matrix was carried out and linear calibration curves in the range of 2–200 ng/ml could be achieved for various matrices including lipid emulsions.
This work was supported by the RCPE K1 Competence Center – Initiated by the Federal Ministry of Transport, Innovation & Technology (MMVIT) and the Federal Ministry of Economics & Labour (BMWA). Funded by FFG, Land Steiermark and Steirische Wirtschaftsförderung (SFG).
Amlodipine is a long-acting calcium channel blocker used in the treatment of hypertension and angina pectoris. S-(−)-amlodipine besylate is a safer and longer-acting alternative to the existing racemate . Although several products based on the active enantiomer have reached the market, information on its chemical, physical and thermal stability during storage is lacking [2, 3]. The objective of the present study was to evaluate the critical properties of the S-enantiomer and to obtain information on the degradation pathways during storage of the bulk drug. The degradation products formed upon subjecting S-(−)-amlodipine besylate to different conditions (hydrolysis, oxidation, high and low pH, dry heat and photolysis) were resolved on a Lichrospher RP-18 column using 237 nm as detection wavelength [4, 5]. A good understanding of the chemical and physical stability of the drug was gained based on the results of the forced degradation study.
This work was supported by ALIMS B&H.
Fundamental scientific knowledge is required to develop and optimize pharmaceutical manufacturing processes that yield constantly high quality products and provide most cost efficient manufacturing strategies. In our work we present combined Quality-by-Design and computer simulation approaches (Discrete Element Method, DEM, and Computational Fluid Dynamics, CFD) to characterize three key unit operations of solid and liquid dosage form manufacturing, namely powder blending , tablet coating  and fluid mixing. The aim is to evaluate the impact of formulation parameters and process variables on process and product quality.
Understanding the variability of both material attributes and process parameters, as well as their overall impact on the unit operations are critical elements for QbD. In a first step, the QbD-methodology is systematically used to (1) establish the critical quality attributes representative for the selected dosage forms, (2) identify potentially critical input factors that may affect process and product quality and (3) risk-rank these factors to define activities for process characterization. Subsequently, computer simulation-based characterizations of the three unit operations are performed. For the blending process the concentration of the active pharmaceutical ingredient, as well as the mixing time are related to the blending uniformity. The film homogeneity on a single tablet is then analyzed as critical quality attribute for the coating unit operation. Finally, a functional design space (engineering design space) for a mixing tank is established. The computational data are used to map out three-dimensional knowledge spaces, leading finally to the definition of design spaces as subset of them. Based on these results, appropriate strategies for process control are presented as well.
The aim of the study was to present an example of experimental design application to set up the dissolution test conditions for the two immediate-release products of levothyroxine sodium (L-Na) with proven bioequivalence: the generic product A and the reference product B [1, 2]. The description of the dissolution profiles by using model-independent methods included the calculation of mean dissolution time (MDT) from the in vitro data for both formulations. MDT for the products were compared one to each other as well as with mean absorption time (MAT), calculated from the in vivo data . The experimental factorial design 23 was applied with following independent variables: concentration of surfactant used (X1), volume of dissolution medium (X2), and paddle stirring speed (X3). Dependent variables were set up as a difference between the MDT observed under various experimental conditions for the investigated products (Y1), as well as the difference between MDT and MAT for each product (Y2 = MDTprod. A − MATprod. A; Y3 =MDTprod. B − MATprod. B). The obtained results showed that the paddle rotation speed was the most significant drug release factor. The medium volume had very small effect on responses Y1 and Y2 but its impact could not be regarded as negligible in the case of response Y3. The surfactant used for dissolution testing showed significant effect on the tested parameters, but the observed effects were contradictory and general conclusion could not be made. This study showed the limited applicability of experimental design in the optimization of the dissolution conditions for two different L-Na formulations. The significant differences among in vitro release profiles were obtained and there was not unique dissolution test model applicable to both investigated products.
The most commonly used drug physicochemical property to access its in vivo performance is in vitro dissolution of a drug product. It is, therefore, important to define drug release methodology that would be predictive of its bioperformance and to establish quantitative in vitro-in vivo correlation (IVIVC).
The objective of this study was to: i) to develop drug-specific absorption models for the selected BCS Class II model compounds (nimesulide, glimepiride, gliclazide) using gastrointestinal simulation technology (GST), ii) to use the generated absorption models to provide the target in vivo dissolution profiles for IVIVC, iii) to identify biorelevant dissolution specifications for IR tablet forms of the tested model drugs.
GST  was used for in silico prediction of oral drug absorption. The input parameters required for the simulation were experimentally determined, in silico predicted and/or taken from the literature. Parameter Sensitivity Analysis (PSA) was used to assess the sensitivity of the predicted rate and extent of drug absorption to the selected input parameters. A set of experimentally observed and virtual in vitro data were used for correlation purposes. Level A IVIVC was applied to assess the relationship between the in vitro and in vivo data.
The results obtained indicate that: i) PSA can aid to identify critical parameters affecting the rate and extent of drug absorption, ii) GST can be successfully used to predict drugs absorption profiles, iii) IVIVC in conjunction with GST can aid to identify biorelevant dissolution specifications for the in vitro assessment of the tested model compounds.
This work was done under the project Biopharmaceutical Characterization of the selected BCS Class II and III drugs: In Vitro and In Silico Methods Evaluation (TR-23015) supported by the Ministry of Science and Technological Development, Republic of Serbia.
Highly purified organic materials are essential in various fields, including the fine-chemical, pharmaceutical and food industry. A method widely used to purify, e. g., active pharmaceutical ingredients (APIs), is preparative chromatography. In this context, continuous processing is becoming increasingly important. The development of a novel prototype for continuous annular electro-chromatography (CAEC) is the goal of a project which was established by an international consortium of industrial and academic research partners. The CAEC process combines the principles of electrophoresis and chromatography, thus leading to an increase of throughput, whilst maintaining high separation efficiency. An online detection system enables advanced automation and quality control, so that the process accords to the PAT (Process Analytical Technology) guidelines specified by the FDA.
We present the development of stationary phases for this CAEC process. Our approaches include functionalized silica-based monolithic materials , which were implemented in different devices, such as capillaries for capillary electrochromatography (CEC) as well as planar test cells that can be continuously operated. The functional groups attached to the monolithic silica backbone can be easily adapted to different separation problems. Alternativly, the functionalization was achieved by implementation of functionalized silica particles in the precursor mixture of the monolithic material. Using these approaches, we succeeded in the preparation of reversed phase as well as amino-, cyano- and thiol-functionalized materials. In addition to these inorganic materials, organic monoliths  were successfully implemented in CEC capillaries and planar glass plates. Preliminary results show that the implementation of the stationary phases in the continuous operation set-ups leads to improved feasibility to gain high-value added products.
This work is supported by the 7th Research Framework Programme of the European Commission (Grant agreement number: NMP2-SL-2008-206707).
Pregabalin is a structural analogue of γ-aminobutyric acid approved for treatment of neuropathic pain, as adjunctive therapy for partial seizures, and in generalized anxiety disorder. It has no significant UV or visible absorption, therefore most often derivatization methods have to be employed for its determination. Previusly published methods for pregabalin determination in pharmaceutical dosage forms are either time-consuming  or use chromatographic separation . Herein, we propose a novel high-throughput method utilizing a simple derivatization with fluorescamine and fluorescence detection with a microplate reader. The derivatization process is fast (5 min), proceeds at a room temperature in borate buffer at pH 9.5. Using fluorescent detection (λex 395 nm, λem 476 nm), the method was linear (r2 > 0.997) over the concentration range (0.75–30 μg/ml). The intraday and interday precision of quality control samples was < 4.5%, and accuracy was 97.3–105.3%. Evaluation of the method robustness revealed that the emission wavelength and fluorescamine concentration are the most influential parameter. The method was applied to the analysis of pregabalin in 25 mg capsules. The content of pregabalin did not differ from the declared amount by more than 3.7%. Additionally, the method was applied to dissolution studies using USP 29 Apparatus 2 and FDA recommendation for dissolution testing of pregabalin capsules. It was demonstarted that more than 80% of pregabaline released in 10 min and more than 99.7% in 45 min. Finally, the universality of the method was proven by analyzing the content and dissolution profiles of pregabalin structural analogues vigabatrin, and gabapentin in pharmaceutical dosage forms.
Sodium naproxen (SN) exists in an anhydrous form and in four different hydrated forms: one monohydrate (MSN), two dehydrates (DSN and CSN) and one tetrahydrate (TSN). Mechanisms of dehydration have been previously determined by an isothermal method [1, 2]. In this study, their dehydration was followed by an isoconvertional analysis (ICA), which is based on a non-isothermal method.
SN (B.P.) was kindly supplied by A.C.R.A.F. (Italy).
Thermogravimetric analysis (TGA) was performed by a Simultaneous Thermal Analyzer (STA 6000, Perkin Elmer, Inc., Waltham, MA, USA), under nitrogen atmosphere (20 mL/min) in a 0.07 ml open aluminium oxide pans. After calibration, samples (approximately 10 mg) were tested in quadruplicate by heating from 293 to 413 K at four different heating rates (5, 10, 20, 40 K/min).
For each hydrate form, the degre of conversion (% dehydration) was plotted versus temperature at any heating rate. The temperature relative to each conversion degree was then determined and plotted versus temperature. The slope of the regression lines permitted the calculation of the activation energy (Eatt). The Eatt ranged approximately from 60 to 80 kJ*mol−1 for the MSN, from 70 to 90 kJ*mol−1 for the DSN, from 28 to 50 kJ*mol−1 for the CSN and from 30 to 70 kJ*mol−1 for the TSN. A dehydration occurring by one-step mechanism has been revealed for the MSN and DSN, while a multi-step mechanism explains the dehydration of CSN and TSN. The results of this study showed a good correlation with those previously determined by applying an isothermal method . In conclusion, the isoconversional analysis may be considered as a simple and fast method to predict the conversional behaviour of pharmaceutical substances and it may be applied to predict the phase transition of complex systems.
The poorly-soluble Vinpocetine was successfully coground with micronized crospovidone in a planetary mill, which allowed an enhancement of bioavailability through a solid-state activation. Drug-to-polymer weight ratio and milling time variables led to statistically significant effects on the activation/amorphysation of the product. An ad hoc software was then used to calulate the dimensions of the drug crystallites in the samples on the basis of the calorimetric data . The thermal analyses were then accompanied by a multisciplinary characterization of the samples, by means of X-ray diffraction, Raman imaging/spectroscopy, DRIFT and SS-NMR spectroscopy, laser light scattering and solubilization kinetics tests. All the analyses attested the progressive loosing of crystalline structure of the drug when increasing milling time and amount of polymer in the formulations. Further, the analyses revealed the perturbation of the VIN carbonyl environment as a result of the disruption of the crystalline lattice, the absence of hydrogen bonding between components and the insurgence of hydrophobic interaction between components. The activated status of the drug, that resulted to be homogeneously distributed on the coground sample and stable for at least 1 year, was reflected on favorable solubilization kinetics. Finally, the in vivo studies on rats revealed that coground systems promoted a five-fold higher oral bioavailability enhancement in comparison to a marketed oral formulation (Vimpocetin® 5 mg C, Pharma).
The authors thank Linnea for the kind gift of Vinpocetine and Fondazione CRTrieste for funding.
The use of enzyme-catalyzed reactions for the synthesis of sugar substrates represents a more desirable approach in terms of “green chemistry”. As the azidonitratization reaction of cellobial furnishes the 2-azido-2-deoxycellobiose stereospecifically, we have prepared 2-azido-2-deoxyglucose from 2-azido-2-deoxycellobiose using recombinant cellobiose phosphorylase (CeP) from Clostridium thermocellum NCIMB 10682 . This enzyme catalyzes the reversible phosphorolysis of cellobiose to form α-D-glucose and α-D-Glucose-1-phosphate with inversion of the anomeric configuration (Scheme 1) .
2-Azido-2-deoxycellobiose is cleaved quantitatively by the action of cellobiose phosphorylase and is separated from glucose 1-phosphate by filtration through DEAE-cellulose. In this manner, we have demonstrated that the substitution of the hydroxyl group by an azido group in position 2 of the reducing cellobiose moiety is tolerated by the recombinant CeP.
The knowledge of surface charge and the isoelectric point is important for many biomedical applications. The zeta potential, formally defined as the electrical potential at the electrokinetic plane of shear, is an important property of charged solid/liquid interfaces and a descriptor of the actual surface charge of a solid immersed in a dielectric. For macroscopic solid surfaces, the zeta potential is commonly determined by the measurement of streaming potential and streaming current. The versatility of the streaming potential method allows handling of planar surfaces, cylindrical capillaries, and packed beds of granular or fibrous materials.
Besides the evaluation of the charging behavior of solid surfaces, the zeta potential is a valuable indicator for surface stability in the presence of various liquids, and for the adsorption of proteins, polypeptides, and other biomacromolecules on biomedical and biomaterial surfaces.
In this paper the application of the streaming potential method in the field of biomedical surface characterization is presented with different case studies.
The aim of the presented study is to propose the utilization of dynamic neural networks and fuzzy algorithms as in silico tools for prediction of drug release rate from hydrophilic and lipid matrix tablets. Dynamic neural networks are neural networks used for analysis of nonlinear, time dependent processes, having the ability of multiple presentations of inputs to the network. Fuzzy algorithms, on the other hand, enable creation of set of rules defining desired formulation properties. Compositions of matrix tablets were as follows: (a) hydrophilic: polyethylene oxide (Polyox® WSR Coagulant), diclofenac sodium, microcrystalline cellulose (Avicel® PH 102); (b) lipid: glyceryl palmitostearate (Precirol® ATO 5), caffeine, mannitol; and magnesium stearate. Matrix tablets, prepared using compression simulator (Zwick 1478), were evaluated on porosity, tensile strength and drug release rate. Commercially available software Peltarion® has been used to construct dynamic neural networks and fuzzy algorithms. Modeling of drug release rate has been conducted taking into account formulation factors and manufacturing method (formulations composition, compression force used for tableting, porosity and tensile strength of manufactured tablets). Focused, Elman’s dynamic network was employed to model drug release rate from both hydrophilic and lipid matrix tablets, optimizing the number of neurons in hidden layers as well as signal time delay with genetic algorithms. Separate sets of rules were generated using fuzzy algorithms for hydrophilic and lipid matrix tablets. Testing of the developed dynamic networks and fuzzy algorithms has demonstrated that dissolution profiles for matrix tablets of known composition and mechanical properties can be predicted in silico with great accuracy. In the case of diclofenac sodium release modeling, obtained values for similarity and difference factors for test formulations release profiles were f2 = 79,08 and f1 = 4,36 i. e. f2 = 75,23 and f1 = 6,69. On the other hand, in the case of caffeine release modeling obtained values were f2 = 87,61 and f1 = 1,58 i. e. f2 = 72,30 and f1 = 5,24. For all predicted drug release profiles the correlation coefficient among predicted and true values was higher than 0,9950. This study demonstrates the feasibility of application of dynamic networks and fuzzy algorithms for modeling of drug release rate from hydrophilic and lipid matrix tablets.
Existing concepts for personnel locks in clean rooms are characterized by cost and time consuming procedures. Clean room personnel have to change their clean room garment in personnel locks when moving from one clean room class to another. The changing process of clothes is a source of contamination due to human imperfection.
It is a fact that the major source for airborne microbes is the human body. The results of confocal laser scanning microscopy show that the microstructure of synthetic fibers is very complex and offers many niches for the attachment of microbes to the fiber. These niches drastically reduce the point of action for example of UV sterilization.
Clean room clothing is intended to hinder particles from the wearer’s body to contaminate the environment. There is a demand of the industries for the design of new textiles with new features and new concepts for innovative personnel lock systems. Innovation will take place either in the modification of the textile as well in the design of new cleaning systems for personnel locks. To overcome this huge challenge an interdisciplinary team will completely reconsider common processes to create new standards in clean room technology. This will be achieved by the screening for new bio-based antimicrobials, the usage of light-activated disinfection and the modeling of the behavior of particles on the textile as well as in clean room environment. The great challenge is to create an innovative concept with the basic requirement of maximum safety for the wearer’s body as well for the subsequent processes.
This work was supported by the FFG, SFG, Das Land Steiermark, Ortner Clean Rooms Unlimited and Dastex.
Mechanical stress caused by gastrointestinal motility might have a great impact on drug release from oral dosage forms. In our experiments different mechanical influences on the tablet during dissolution experiments were tested using extended release tablets containing diclofenac. Dissolution experiments were performed on the flow-through apparatus, developed on Faculty of Pharmacy, University of Ljubljana . The novel peristaltic movement simulating stirring device contains a working vessel with a certain amount of small glass beads that are stirred by a magnetic stirrer. Tablets were kept in simulated gastric fluid (pH 1.2) for 2 hours and in simulated intestinal fluid (pH 6.8) afterwards. The intensity of mechanical stress on the tablet was modified by varying the amount of glass beads, stirring speed, and by using different barriers in the working beaker in a different position for 5 or 20 minutes.
Our experiments showed that mechanical stress induced by increasing stirring rate or decreasing the amount of glass beads resulted in increased diclofenac release. The insertion of different types of barriers into the working vessel increased the release rate, however, some crushing of tablets appeared. Thus, no subsequent decrease of release was observed when the barrier was removed. Namely, such dissolution kinetics was desired as we wanted to develop a dissolution test to predict double peaks in plasma profiles after oral administration of diclofenac extended release tablets. It seems that only squeezing the tablet and not other types of mechanical influences, simulates well in vivo situation . Additionally, tablets were more sensitive to mechanical stress in intestinal than in gastric medium which is a consequence of different diclofenac solubility in both media. Thus, greater mechanical sensitivity of the tablet might also be expected in vivo when passing ileo-cecal sphincter than in pylorus. However, it is questionable if this influence could be observed in vivo as a large amount of drug has already been released when tablet reaches the end of ileum.
The purpose of this work is two fold: it describes the proof of concept of the newly introduced bulk video imaging (BVI) method and it presents the nucleation detection in comparison with existing process analytical technologies (PAT) such as focused beam reflectance measurement (FBRM) and ultra violet/visible (UV/Vis) spectroscopy. While the latter two sample the system closely to the probe, the BVI approach monitors the entire crystallizer volume.
The external BVI (eBVI) method is based on a video camera and a capture hardware, which captures 25 frames or pictures of the crystallization bulk per second . It can also be regarded as a matrix of external turbidity probes. Another set of experimental data is generated using in-situ endoscopy and a small scale crystallization calorimeter  (iBVI). The eBVI can be implemented as an external sensor providing significantly lower sensitivity to mixing conditions than other probes based on local measurements. Furthermore, the contamination related problems are circumvented. The advantage of the eBVI approach is that it does not require high speed video camera or advanced illumination technologies such as laser or xenon flash. The BVI method is proposed as a complementary PAT tool and it is shown that it is able to detect the nucleation onset with comparable or better performance to the FBRM and UV/VIS probes. Additionally, the endoscopy based BVI is a low cost sensor which can be easily integrated in the existing laboratory hardware and software environments. Recently, multivariate image analysis methods were evaluated for the purpose of nucleation detection .
One of the key steps in the manufacturing of solid oral dosage formulations is the blending of several ingredients in powdered form which are then processed further to achieve the final dosage form. Up until this blending process, it is relatively straightforward to measure critical quality attributes such as particle size and/or shape of the input materials using well established techniques such as laser light scattering or particle image analysis. A much more difficult challenge is to be able to characterise the size and shape of the individual ingredients once they have been through the blending process. This may be especially important when trying to understand any possible impact of the blending process on the size and shape of the API and how this might impact the bioavailability or bioequivalence of the drug. The technique of NIR chemical imaging has been used to get some information of the homogeneity of powder blends , however limitations in spatial resolution impact its applicability for smaller particle sizes.
In this paper we describe a novel approach using a hybrid technique of Morphologically directed Raman Spectroscopy to study the effects of processing on a laboratory scale of a model formulation consisting of ibuprofen, starch and lactose. The sample is first of all dispersed in its dry state, and then images are captured of the individual particles in the mixture in order to determine their size and shape. Each particle is then targeted in an automated manner with a Raman microprobe to obtain a chemical ID. Particle size and shape distributions can then be constructed for each of the ingredients and compared directly with measurements made before the blending process. Results for the model formulation are presented and implications for a number of different types of pharmaceutical formulations are discussed.
14N NQR is a new method in pharmacy that has a potential to establish itself as an additional and valuable analytical tool for characterizing solid state of a substance . 14N NQR is a method that can precisely and accurately determine a specific polymorphic state of a solid compound. It is non-destructive and there is usually no spectral interference from other polymorphs or other solids.
The objective of presented study was to use 14N NQR as a way to characterize qualitatively and quantitatively different solid state forms of sulfanilamide, nifedipine and piroxicam . Thermodynamically stable forms of said substances were obtained on the market. Subsequently, samples of unstable amorphous or polymorphic forms were prepared. DSC and ATR FT-IR were used additionally to identify differences between the samples. 14N NQR method was used with the same samples to (re)confirm the already established polymorphic states . 14N NQR method proved itself capable of accurately and precisely determining the solid-state forms of studied pure compounds.
The possibility of accurately determining the mixture composition of different forms was also demonstrated. Mixtures of piroxicam forms in different ratios were prepared and then measured with 14N NQR. Determined mixture ratios were in close agreement with the known values.
For many biomedical applications of proteins the knowledge of the three-dimensional structure of proteins is of great importance. It is the key to understand their function in biological processes. Small-Angle X-ray Scattering (SAXS), in contrast to other methods e. g. Protein Crystallography and Cryo-TEM, allows investigating proteins in solution, i. e. in their native state and under biological conditions. Furthermore SAXS does not need any specific sample preparation which might change the protein sample.
SAXS enables to study structural changes upon changing the external conditions and is a valuable tool for determining several important parameters : (1) protein size and shape, (2) aggregated structures, (3) folding state, (4) subunit structures.
By knowing these parameters of the proteins in native, valuable conclusions can be derived in order to explain the proteins’ functionalities. In this contribution the application of laboratory-based small- angle x-ray scattering studies performed with the SAXSess mc2 system in the field of biomedical applications is presented and discussed.
Overcoming biological barriers, including the gastrointestinal epithelial barriers, is a great challenge for pharmaceutical research. There is a need for new absorption enhancers with more favorable profile. Sucrose esters are widely used in the food and cosmetic industry, and there are reports on their potential use in pharmaceutical formulations as excipients [1, 2], however no data are available on their toxicity profile and their usefulness as absorption enhancers in epithelial models.
We tested three water soluble sugar esters having HLB value of 16, laurate (L-1695), myristate (M-1695), palmitate (P-1695) esters, with 12, 14 and 16 carbon fatty acid chains, respectively, on human Caco-2 epithelial monolayers, a widely used model for the human intestinal epithelial barrier. Cremophor RH40 and Tween 80 were used as reference absorption enhancers. For toxicity tests cells were cultured in 96-wells, and MTT dye conversion and lactate dehydrogenase relase were determined after treatments. For permeability tests cells were cultured on Millipore CM inserts (hydrophilic PTFE membranes, pore size: 0.4 μm, surface 4.2 cm2) in 6-well plates for 3 weeks to reach stationary phase and barrier properties. Fluorescein was selected as a marker of paracellular permeability. Untreated monolayers formed a good barrier as reflected by a transepithelial resistance of 722 ± 80 Ω cm2 and positive immunostaining for tight junction proteins claudin-4 and ZO-1. Sucrose esters showed 50 % cytotoxicity (1 h treatment) at 300 μg/ml, while cell death was measured at concentrations above 1 000 μg/ml. The reference molecules were only toxic at doses above 10 000 μg/ml. L-1695 in non-toxic concentrations reduced TEER and increased permeability for fluorescein in Caco-2 monolayers indicating an absorption enhancer property.
This work was supported by TÁMOP-Hungary research project: Development of teranostics in cardiovascular, metabolic, and inflammatory diseases (TÁMOP-4.2.2-08/1-2008-0013).
Delayed release dosage form development usually requires an effective intermediate layer for separation of acid liable drug and acidic gastric polymer. A case is presented where an effective intermediate layer was developed for duloxetine pellets. Duloxetine pellets were produced in Wurster process using suspension/solution layering technique. Neutral pellet cores were coated with duloxetine layer, intermediate layer and gastric layer. Intermediate layer usually comprises water soluble polymer (e. g. povidone, hypromellose); additionally talc may be added as filler and a processing aid (it reduces tackiness of coating dispersion). These kind of intermediate layer compositions provide simple separation of API and gastric layer and are usually sufficient for effective separation. In duloxetine pellets development, however, accelerated stability tests indicated poor long term stability. Diffusion of acidic species (e. g. phthalic acid is a hypromellose phthalate degradation product) across intermediate layer was suspected. Addition of small amounts of sodium chloride (4.8% wt./wt. relative to mass of intermediate layer, dissolved in aqueous coating dispersion) drastically improved stability of duloxetine pellets. Increase in coat thickness resulted in further decrease of duloxetine degradation products: this fact indicates that stabilization is induced by strengthening the diffusion barrier and thus minimizing diffusion of acidic species between the layers. Scanning electron microscope images were used to determine the structure of intermediate coat: talc particles are bonded with hypromellose: within hypromellose phase small crystals of sodium chloride (sub micrometer size range) were detected. Sodium chloride is dissolved in coating dispersion and once applied onto pellet surface it re-crystallizes in form of small crystals (due to fast water removal during coating). It is sugested that these finely dispersed sodium chloride crystals within hypromellose phase provide effective diffusional barrier which prevents migration of acidic species between layers and thus enabling stabilization of duloxetine. In order to assure long term stability of duloxetine pellets it was shown that an effective diffusional barrier is required.
Topotecan (TPT) is a semi-synthetic derivative of the natural extract camptothecin, which has been found to act as an inhibitor of the DNA enzyme topoisomerase I in a specific and reversible fashion. The drug undergoes reversible hydrolysis from the pharmacologically active parent lactone form (TPTL) to an inactive hydroxy acid form. In the cytoplasm the irreversible inactivation of TPTL is catalysed by the enzyme aldehyde dehydrogenase (ALDH). Over-expression of the human breast cancer resistance protein (BCRP/ABCG2) has been linked to high levels of resistance to the anti-cancer agent TPT by promoting an active efflux pump mechanism. The expressions of both ALDH and BCRP have been experimentally identified in a large number of solid tumours and thus play an important role in clinical drug resistance of cancers. To investigate the catalytic reaction and efflux pump mechanism, a compartmental model for the in vitro uptake kinetics of TPT has been extended to better describe the drug activity and delivery of TPTL to the DNA target as well as the catalysis by ALDH and the elimination of drug from the cytoplasm via the efflux pump. All unknown model parameters were uniquely estimated to a high level of confidence. Model simulations have been compared with live human breast cancer cells (MCF-7 cell line) data and found to give good qualitative agreement. The kinetic model has then been linked to a cell cycle model, based on , which facilitates analysis of the response of the growth of single cells in the presence and absence of TPT. Parameter estimation is performed using green fluorescent protein tagged cyclin B1 (cell cycle regulatory protein) data for the osteosarcoma cell line U-2 OS. Linking both models allows the study of drug perturbation to the cell cycle as well as in silico estimation and prediction of the relationship between the target binding and the dose, also permitting the effects of different expressions of the drug resistance protein and the ALDH enzyme. Such a coupled kinetic/dynamic model, once fully validated, has the potential for enhancing the design of optimal dosing regimens.
MIA is funded by the University of Warwick Vice Chancellor’s PhD Scholarship.
In the light of the current trend for natural surfactants to be used as emulsifiers , a physicochemical characterization of a new mixed alkyl polyglucoside (APG) emulsifier (Arahidyl&Behenyl alcohol&Arahidyl glucoside) was performed. The characterization was carried out through the polarization microscopy (PLM), x-ray diffraction studies (WAXD and SAXD), thermal analysis (DSC and TGA) and rheological measurements. In order to investigate the emulsifier potential to build the lyotropic liquid crystalline phase, binary systems (with increasing percentage of emulsifier) and series of cream samples with fixed emulsifier/water ratio and increasing percentage of oil phase were prepared. Additionally, 5 cream samples with different oil phases (Candelilla oil, Avocado oil, Isopropyl myristate, Decyl oleate and Liquid Paraffin oil) were evaluated.
Anisotropic structures defined as distorted Maltese crosses were seen in PLM micrographs, as well as birefringence at the emulsion oil droplets border, indicating the liquid lamellar phase . The DSC, WAXD and SAXD patterns additionally proved PLM findings, implying synergism of lamellar liquid and lamellar gel phases within the colloidal structure. Complex lamellar structures were preserved upon addition of various oils, with the exception of liquid paraffin which reduced the specific birefringence (observed in PLM micrographs and DSC thermograms). Percentage of water evaporation (TG results) indicated that more than one third of the water within the system is entrapped - interlamellarly fixed (“depot” water). In steady state rheological measurements all samples showed “shear-thinning” behavior with thixotropy. Oscillatory measurements pointed at more pronounced elastic over viscous component (typical for lamellar phase). Overall, investigated APG emulsifier demonstrated good potential in lamellar liquid and gel phases formation, a property desirable for mixed emulsifier useful in stabilization of vehicles anticipated for dermopharmaceutics and dermocosmetics incorporation.
At least three enzymes (i. e., trypsin, chymotrypsin and neutrophil elastase) are responsible for the degradation of salmon calcitonin (sCT) in the human body . Here, we investigated how these proteinases affect drug disposition of sCT in the lungs.
Stability of sCT was studied in human respiratory epithelial cells. The presence of trypsin, chymotrypsin and neutrophil elastase in cell supernatant and homogenate was studied by Western blot. Enzymes’ activities were studied in cell supernatant by measuring UV absorption, caused by interaction with model substrates. In vitro stability studies were carried out by adding sCT to cell supernatant, homogenate, as well as on intact cell monolayers, followed by incubation at 37°C for 2 h. Samples were serially withdrawn and analysed by HPLC for sCT content. Degradation of sCT by pure trypsin, chymotrypsin and neutrophil elastase was investigated after incubation for 2 h at 37°C.
We observed that sCT concentrations remained unchanged over the period of 2 h, when incubated in supernatant or on cell monolayers. When cell homogenates were studied, sCT concentrations were reduced to 82.5% (hBEpC), 45.9% (Calu-3), 45.1% (16HBE14o-), 4.3% (A549) and 18.5% (Caco-2), respectively. sCT was also degraded when incubated with pure enzymes. Western blot revealed strong signals for all proteinases in the cell homogenates and weaker, varying expression in supernatants.
Calcitonin is rapidly degraded in homogenate, indicating high intracellular enzymatic activity. Epithelial proteases hence, appear to play a role in interactions of sCT with lung epithelium. Although trypsin, chymotrypsin and neutrophil elastase are secreted in vitro, sCT remained intact in supernatant for 2 h. The concentration of the relevant enzymes (indicated by very low measured activities) might be too low to cause degradation.
This work was funded by a Strategic Research Cluster grant (07/SRC/B1154) under the National Development Plan co-funded by EU Structural Funds and Science Foundation Ireland and by an IRCSET Embark Postgraduate Scholarship.
Wound healing is a complex process with many potential factors that can delay healing. Imbalance between tissue deposition stimulated by growth factors and tissue destruction mediated by proteases causes nonhealing of chronic wounds . To overcome the destroyable effect of proteases, the application of growth factors or protease’s inhibitors has been shown as successful. The aim of our research work is to formulate and charaterize the biocompatible modern wound dressings with model protein for protease’s inhibitor, which would enable protein stability, delivery and controlled release.
Hydrogels of hydroxyethylcellulose (HEC) and polyvinyl alcohol (PVA) were made at different m/m ratios and their rheological behaviors were performed by rotational viscosimeter (Rheolab MC 100, Physica). For HEC effects of sterilization process and osmolarity of the medium and for PVA effects of freeze-thaw cycles were evaluated. Egg albumin as model protein was incorporated into the hydrogels and drug release studies were performed by developed chamber simulating conditions at wound place. To increase protein stability hydrogels were lyophilized and tested for rehydration ability, viscosity and protein release. Electrospinning, which is a nanofiber-forming process where polymer solution or melt is charged to high voltages, was used for producing nanofibers from the same polymers.
HEC hydrogel at 3w/w % has appropriate rheologic properties, regardless of the medium used (water, phosphate buffer) or sterilization process performed. Strenght and rigidity of PVA hydrogels increases with number of freeze/thaw cycles. Mild process conditions enable homogenous incorporation of protein compound into HEC and PVA hydrogel without deteoriation of its native structure. The release of protein is in accordance with Fick’s diffusion. Freeze-dryed HEC hydrogels were spontaneously rehydrated in 24 hours. Viscosity and thixothropy of rehydrated samples were reestablished and neither lyophilization nor freezing mode affects the protein structure or releases kinetics. Additionaly some data about nanofibers obtained by electro-spinning will be presented. The nanofibers potentially mimic many roles of extracellular matrix to promote tissue growth.
Hydrogels with incorporated drugs that actively interfere in healing process and nanofibers with their unique properties represent improvement for treatment of chronic wounds.
Multiple carbohydrate structures in the chondrocyte glycocalyx can serve as targets for biorecognitive lectins. Recently, we have outlined the applicability of selected plant lectins as mediators of bioadhesion in cartilage research . However, given the diversity of glycan structures at cell surfaces as well as the limited specificity of most lectins for certain tissues, in-depth knowledge about altered oligosaccharide structures under disease conditions is required. We therefore aimed at defining in detail the chondrocyte glycome following exposure to pro-inflammatory cytokines.
Primary human chondrocytes were isolated from donors (n=5) and cultured as high density monolayers. Cells were treated with 10 ng/ml IL-1ß or 40 ng/ml TNF-α. The transcription of 19 glycosyltransferases was quantified using RT-qPCR. N- and O-glycan analysis was performed using LC-ESI-MS.
We found that both IL-1ß and TNF-α increased overall sialylation of N- and O-glycans and induced a major shift from α2,6-linked sialic acid residues towards α2,3-linked sialic acids. These results were supported by RT-qPCR showing increased expression of α2,3 sialyltransferases in treated cells. In this context, ST6Gal1 mRNA levels were markedly reduced 4.7-fold in case of IL-1ß and 3.3-fold in case of TNF-α, whereas the expression of ST3Gal4 was enhanced 2.8-fold and 3.2-fold, respectively. Moreover, we found that both cytokines induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans, whereas core α1,6-fucosylation was found to be reduced particularly by TNF-α.
In conclusion, IL-1ß and TNF-α induce a range of specific alterations in chondrocyte glycoproteins, which might be of relevance for malfunctioning cell-matrix interactions in osteoarthritis. Interestingly, such specific changes in the chondrocyte glycome could also be exploited to target functionalized pharmaceutical delivery systems to osteoarthritic chondrocytes.
This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 and other SARTANS (losartan, valsartan and candesartan) with the model membrane system of dipalmitoylphosphatidylcholine (DPPC). SARTANS are designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT1). Small angle X-ray scattering (SAXS) experiments on the interaction of SARTANS with DPPC bilayers were carried out and results demonstrate that all studied SARTANS are well incorporated into the membrane leaflets and furthermore cause partial bilayer interdigitation. Further structural as well as dynamical effects will be discussed, and compared to their overall efficiency as antihypertensive drug.
Density functional theory studies at the B3LYP/6-31G(d,p) and M06-2X/6-31G(d,p) levels have been used to determine the geometries and the enthalpies of formation of several methylated β-cyclodextrins.
Various degrees of methylation can be performed at the hydroxyl groups of β-cyclodextrin at the 2-, 3-, and 6-positions. The number of methyl groups and the position influence the physico-chemical properties of the derivatives as well as their inclusion ability. Large changes of the association constants with different guest molecules are accompanied with increase or decreased solubilities, as well as by a modification of the inclusion mechanisms. In this study three different types of methylated β-CDs were investigated: Heptakis(2-O-methyl)-β-cyclodextrin, heptakis(6-O-methyl)-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin, and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin.
During building up the various β-CD derivatives C7-symmetry was used throughout. By applying systematic B3LYP/6-31G(d,p) and M06-2X/6-31G(d,p) calculations, the oxygen-oxygen distances were scanned and all remaining geometry parameters were optimized. Three conformational minima were obtained. In the lowest energy conformation two homodromic hydrogen bond rings were formed, one with very short hydrogen bonds at the primary hydroxyls and a second at the secondary hydroxyls of the CD. Four orientations of the homodromic hydrogen bond rings, which are different in energy, have been taken into consideration: both hydrogen bond rims orientated counterclockwise (cccc) or clockwise (cwcw) and the primary rim clockwise and the secondary rim counterclockwise (cwcc) and vice versa (cccw). These four conformations were used to construct the methyl-derivatives of the β-CD molecules. For the optimization with both DFT methods no constraints were imposed on the molecules. To compare the calculated energies with related values obtained experimentally, the respective energies of five crystallographic structures have also been calculated.
The essential manufacturing step of probiotic products involves dehydration of microorganisms, resulting in cellular stabilization and improved formulation and storage characteristics. Spray drying is one of the most common dehydration techniques used for the preparation of pharmaceutical formulations containing probiotics. This method is less time- and cost- consuming as compared to other drying techniques such as lyophilization [1, 2]. However, spray drying of probiotics involves the challenge of maintaining viable microorganisms in spite of high temperatures involved in the process.
The aim of the present study was investigation of the impact of heat on the cellular activity and culturability of probiotic strains Enterococcus faecium M 74 and Bifidobacterium bifidum 12. Alterations in membrane permeability, esterase activity and production of superoxide radicals were investigated after exposure of cells to heat at different time intervals. The measurements were obtained using fluorimetry and flow cytometry after staining of cells with fuorochromes. The results of heat stress showed deleterious alterations in the membrane integrity and esterase activity of both strains after exposure to temperatures of 60°C to 90°C. However, B. bifidum 12 cells were more affected than E. faecium M 74. The maximum damage of cell membrane and active metabolism occurred after shorter periods of heat exposure in the case of B. bifidum 12. In addition, increasing the temperature or extending the exposure time resulted in a remarkable impairment of the esterase activity of B. bifidum 12. The obtained results were thereafter used for the improvement of cell stabilization during spray drying of Enterococcus faecium M 74 and Bifidobacterium bifidum 12.
Oral drug administration is mainly regulated by the step of intestinal absorption. For various substances uptake is strongly superimposed by an active efflux back into the lumen via ATP-driven pumps like the P-glycoprotein (P-gp), significantly decreasing their bioavailability.
P-gp inhibitory effects can be determined via the calcein accumulation assay (CAA) or transport studies in cellular systems. Hereby it was previously shown that particular phospholipids (PL) reduce the efflux of a common P-gp substrate .
The aim of this study is to identify further potent PL derivatives and their molecular mechanism of action and to investigate the influence of different experimental parameters.
CaCo2 and MDCKII-mdr1 cells were routinely maintained in supplemented DMEM. Cells were grown for either 21 days in Transwell® plates in case of transport studies, or for 8, resp. 4 days (MDCKII-mdr1) in 96-well plates in case of CAA.
Lipid derivatives were applied as liposomal formulations in HBSS.
CaCo2 cell layers were pre-incubated with lipid and digoxin (3H-labeled) was added apically for absorptive, resp. basolaterally for secretory studies. Monolayer integrity was determined via transepithelial electrical resistance (TEER) measurements and the ratio of the apparent permeability coefficients (Papp) of both directions displayed P-gp effects.
After pre-incubation with lipid or Verapamil as a positive control the intra-cellular accumulation of the fluorescent dye calcein indicated P-gp inhibition.
In the transport studies one achieved a concentration-dependent enhancement of netto drug absorption with C12-PG, C6-PS and various unsaturated symmetric and asymmetric PC lipids.CAA confirmed these findings in both celllines for C12-PG and other middle-chained saturated PC derivatives.
This work was financially supported by Phospholipid e.V., Heidelberg, Germany
A mathematical model has been developed describing the pharmacokinetics of Hoechst 33342 following administration into a culture medium containing a population of transfected cells (HEK293 hBCRP) with a potent inhibitor of the BCRP, Fumitremorgin C (FTC), present. FTC is reported to almost completely annul resistance mediated by BCRP in vitro. The non-linear and multi-compartmental model describes the relationship between the concentration of Hoescht 33342 and FTC initially spiked in the medium and the observed change in fluorescence due to Hoescht 33342 binding to DNA. This model has been extended to consider multi-cell, multi-input responses.
Structural identifiability arises from the inverse problem of inferring from the known properties of a biomedical or biological system a suitable model structure and estimates for the corresponding rate constants and other parameters. Structural identifiability analysis considers the uniqueness of the unknown model parameters from the input-output structure corresponding to proposed experiments to collect data for parameter estimation. This is an important theoretical prerequisite to experiment design, system identification and parameter estimation, since numerical estimates for unidentifiable parameters are effectively meaningless. If parameter estimates are to be used to inform about intervention or inhibition strategies, or other critical decisions, then it is essential that the parameters be uniquely identifiable. Such analysis is highly relevant to large-scale, highly complex systems, typical in chemical kinetics and systems biology.
Structural identifiability analysis has been performed on the Hoechst 33342 pharmacokinetic models developed using a method based on the similarity transformation/exhaustive modelling approach. The analysis demonstrated that all models derived are uniquely identifiable for the experiments/observations available. This permitted subsequent numerical parameter estimation to be performed with greater confidence.
A kinetic modelling software package, FACSIMILE (MPCA Software, UK), was used to obtain numerical solutions for the system equations and for parameter fitting. Model fits gave very good agreement with in-vitro data provided by AstraZeneca across a variety of experimental scenarios. This should ultimately permit predictive analysis to be performed using the model in an attempt to optimise targeting of the compound to cancerous tumours.
Work supported by the UK MRC Capacity Building Intitiative & AstraZeneca.