Microemulsion-made gadolinium carbonate hollow nanospheres showing magnetothermal heating and drug release.

Gadolinium carbonate (Gd2(CO3)3) hollow nanospheres and their suitability for drug transport and magnetothermally-induced drug release are presented. The hollow nanospheres are prepared via a microemulsion-based synthesis using tris(tetramethylcyclopentadienyl)gadolinium(iii) and CO2 as the starting materials. Size, structure and composition of the as-prepared Gd2(CO3)3 hollow nanospheres are comprehensively validated by several independent analytical methods (HRTEM, HAADF-STEM, DLS, EDXS, XRD, FT-IR, DTA-TG). Accordingly, they exhibit an outer diameter of 26 ± 4 nm, an inner cavity of 7 ± 2 nm, and a wall thickness of 9 ± 3 nm. As a conceptual study, the nanocontainer-functionality of the Gd2(CO3)3 hollow nanospheres is validated upon filling with the anti-cancerogenic agent doxorubicin (DOX), which is straightforward via the microemulsion (ME) strategy. The resulting DOX@Gd2(CO3)3 nanocontainers provide the option of multimodal imaging including optical and magnetic resonance imaging (OI, MRI) as well as magnetothermal heating and drug release. As a proof-of-concept, we could already prove the intrinsic DOX-based fluorescence, a low systemic toxicity according to in vitro studies as well as the magnetothermal effect and a magnetothermally-induced DOX release. In particular, the latter is new for Gd-containing nanoparticles and highly promising in view of theranostic nanocontainers and synergistic physical and chemical tumor treatment.

Novel three-dimensional Boyden chamber system for studying transendothelial transport.

The rapid development in combinatorial chemistry of millions of novel potential drug candidates requires in vitro devices for reliable testing of their transendothelial transport and the uptake in specific cells. To date, this is often achieved in vitro by the use of regular planar Boyden chambers, which are not reflecting the three dimensionality of the blood vessel. This technical note describes the fabrication and biological validation of a novel three-dimensional Boyden chamber system for studying transendothelial transport. The key element of this new system is a porous thin-walled microchannel produced by a SMART (substrate modification and replication by thermoforming) process comprising a combination of microthermoforming and ion track technology. The membrane-like microstructure offers the opportunity to grow endothelial cells on the inner side of the channel resembling a more natural curved organization of vessels. After establishment of a confluent HUVECs layer in the porous microchannel this novel Boyden chamber was successfully applied to study the transendothelial transport of a polycationic cell penetrating peptoid through the 3D- or curved endothelial cell layer. Thus, this system will enable the investigation of such synthetic compounds as drug delivery systems with regard to their bioavailability and functionality under organotypic conditions.

Analytical method transfer using equivalence tests with reasonable acceptance criteria and appropriate effort: extension of the ISPE concept.

A method development process is commonly finalized by a method transfer from the developing to the routine laboratory. Statistical tests are performed in order to survey if a transfer succeeded or failed. However, using the classic two-sample t-test can lead to misjudgments and unsatisfying transfer results due to its test characteristics. Therefore the International Society of Pharmaceutical Engineering (ISPE) employed a fixed method transfer design using equivalence tests in their Guide for Technology Transfer. Although it was well received by analytical laboratories worldwide this fixed design can easily bring about high beta-errors (rejection of successful transfers) or high workload (many analysts employed during transfer) if sigma(AN) (error due to different analysts) exceeds 0.6%. Hence this work introduces an extended concept which will help to circumvent this disadvantage by providing guidance to select a personalized and more appropriate experimental design. First of all it demonstrates that former t-test related acceptance criteria can be scaled by a factor of 1.15, which allows for a broader tolerance without a loss of decision certainty. Furthermore a decision guidance to choose the proper number of analysts or series at given percentage acceptance limits (%AL) is presented.

Comparison of the recovery spread in analytical development and routine quality control--based on the ICH quality guideline Q2B.

In the present study, a simulation was performed for the ICH Q2B guideline for assessing the accuracy. By means of an experimental data set a permutation has been performed to investigate in which interval experimental mean recovery can be expected to scatter just by random effects. A good agreement has been found between the experimental intervals obtained by means of a permutation and the statistically derived confidence intervals. These findings could be confirmed with additionally generated virtual data sets with a true mean of 100% and a true standard deviation of 0.7%.

Evaluation of monolithic C18 HPLC columns for the fast analysis of pilocarpine hydrochloride in the presence of its degradation products.

Monolithic Performance C18 HPLC columns (Chromolith Performance RP-18e, Merck) were applied for the determination of pilocarpine hydrochloride in the presence of its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid. The method was validated using a set of six monolithic columns and compared to a conventional C18 column. The separation of pilocarpine from its degradation products was investigated on monolithic columns at different flow rates from 1 to 9 ml/min. Superior resolution was obtained using monolithic columns over the conventional C18 column at the same flow rate of 1 ml/min with a total run time of 9 min compared to 13.5 min for the conventional C18 column. Comparable resolution to that obtained in the C18 column (but with better peak symmetry) was obtained at a flow rate of 4 ml/min, although the total run time was reduced to 3.5 min. The precision for both retention time and peak area was investigated over a wide concentration range and found to be equal, or slightly better on Chromolith Performance compared to the conventional column. The overall RSDs% ranged from 0.5% to 1.16% for the conventional column, while for monolithic columns ranged from 0.38% to 0.87% at a flow rate of 1 ml/min and from 0.38% to 0.89% at a flow rate of 4 ml/min. Monolithic column to column reproducibility (n = 6) was measured. The RSDs% ranged from 0.34% to 0.68% for retention time and from 0.3% to 0.94% for peak areas. The detection and quantitation limits on monolithic columns at both flow rates (1 and 4 ml/min) were found to be 0.17 microg/ml and 0.5 microg/ml, compared to 0.31 microg/ml and 1 microg/ml on the conventional column. Monolithic silica rods have also shown the advantage of reducing the time to wash and to re-equilibrate the column. The method showed good linearity and recovery and was found to be suitable for the analysis of pilocarpine hydrochloride formulations.

Application of the equivalence test for analytical method transfers: testing precision using the United States Pharmacopoeia concept (1010).

In this work, the performance of the USP (1010) concept for comparing precision has been investigated. A diagram has been constructed to relate common variance ratios, sample sizes and the corresponding powers. The choice of the upper acceptable limit of variance ratios strongly influences the power. Small upper limits, such as 2.25, are not practical. The proposed upper limit of 4 requires sample sizes of 14 or higher to achieve a power of 80%. If the precision of a method is very good, higher ratios seem to be acceptable, with a significant reduction in measurements. For example, using n = 6 is sufficient to obtain a power of 90%, if the variances are in fact the same and the acceptable variance ratio is 16.

Application of the equivalence test according to a concept for analytical method transfers from the International Society for Pharmaceutical Engineering (ISPE).

The performance of the equivalence test in the context of analytical method transfers was investigated by means of a simulation study. An ISPE design proposal and typical error contributions for pharmaceutical routine control have been used for the testing of accuracy. Acceptable results (probability of a correct decision) have been obtained here. For total variations above 0.4% R.S.D. the basic design was not sufficient. An overview for the number of additional series needed corresponding to higher variations has been developed based on further simulations. An alternative approach may be the choice of wider acceptance criteria, which was also evaluated.

Wide concentration range investigation of recovery, precision and error structure in liquid chromatography.

Using a typical HPLC assay, the characteristics of recovery, system precision and repeatability were investigated over a wide concentration range. In the presence of a constant amount of typical tablet excipients, the antidiabetic drug glibenclamide was analyzed in the range from 0.24 to 0.005mg/mL (18 concentration levels, 6 independent sample preparations each). On the basis of a typical concentration for an HPLC glibenclamide assay of 0.2mg/mL, this corresponds to a relative amount of 120-0.025% label claim. In the range from 120 to 0.075%, the recovery was found to be quite constant and systematically heightened mainly due to the evaporation from vials during centrifuging and the displacement of solvent volume by the added matrix. Both system precision and repeatability remain almost constant in the interval from 120 to 10% at a R.S.D.% of 0.31 and 0.70%, respectively, indicating that the sample preparation is the major error source in this range (0.63%). Between 10 and 0.25%, a linear relationship between the logarithmized concentration and the repeatability was noted. However, for lower amounts close to the limit of quantitation, the R.S.D.% of measurements increases much more distinctly. This increase is caused by a strong rise of the system precision. At this concentration range, system precision and repeatability are not significantly different any longer. This leads to the conclusion that with the injection error being constant the peak integration error becomes the dominating error source at low concentrations, e.g. at concentrations below the five-fold of the LOQ. The results obtained here agree well with earlier published data. As the quantitation limit of 0.05% can be regarded as typical for a pharmaceutical impurity control test, generalizations of these findings from this extensive data set should be possible. In this context, peak integration and improvements of the signal-to-noise ratio are the most promising measures to improve an unsatisfactory precision in LC.

Long endogenous dsRNAs can induce complete gene silencing in mammalian cells and primary cultures.

Recently, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has rapidly developed to a powerful instrument for specific silencing of gene expression in several organisms, including Caenorhabditis elegans, Drosophila melanogaster, and plants. The finding that synthetic small interfering RNAs (siRNAs) of 21 nt as well as stable, endogenously expressed, large dsRNA are suited to specifically induce gene silencing in mammalian cells offered the possibility of expanding this technique to mammalian systems. In this work, we engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes. We used this technique to specifically silence genes coding for glucosylceramide synthase (GCS), the sphingolipid activator protein precursor (SAP), and glucocerebrosidase (GBA), all implicated in glycosphingolipid metabolism. From a 1600-bp inverted repeat DNA template, a dsRNA of 800 bp is expressed and predicted to mediate the specific suppression of the corresponding gene by RNAi. Remarkably, we were able to use this method to achieve complete inhibition of those genes we targeted in different cultured human cell lists. These findings testify to the generality of RNAi application in suppressing gene expression in mammalian cells.

Characterization of regulatory elements in the 5'-flanking region of the GM2 activator gene.

Lysosomal degradation of the ganglioside GM2 by human beta-hexosaminidase A requires the presence of the GM2 activator protein as an essential cofactor. Here we demonstrate that GM2 activator mRNA is differentially expressed and mainly localized to the apical part of the epithelial cells of distal renal tubules and the collecting duct. In order to understand the mechanism underlying the regulation of the GM2 activator gene, we analyzed the genomic organization upstream exon 2 as well as the 5'-flanking region. The GM2 activator gene spans about 16.8 kb with a first intron of 6.5 kb, and the transcription start is located at position -96 upstream from the ATG. DNA elements responsible for GM2 activator expression were identified in a PCR-based method of long-distance DNA walking. Sequence analysis revealed a 2.9 kb region upstream of the ATG that contained regulatory elements like CAAT boxes, Sp1 binding sites as well as AP1, and AP2 sites. Transfection experiments in COS-1 cells with a series of chimeras of 5'-stepwise deletion mutants of the GM2 activator gene 5'-flanking region and the secretory alkaline phosphatase (SEAP)-reporter gene indicated that a genomic fragment encompassing -323 to +1 bp had significant promoter activity. EMSA experiments showed that Sp1 and other transcription factors like AP1, AP2 and CCAAT-Box binding proteins are involved in GM2 activator gene regulation.

GM2 gangliosidosis AB variant: clinical and biochemical studies of a Japanese patient.

OBJECTIVE: To determine the clinical features and biochemical basis of the first Japanese patient with the GM2 gangliosidosis AB variant. METHODS: The clinical manifestations and laboratory findings in the patient were investigated. Cultured fibroblasts from the patient were analyzed by means of immunofluorescence staining with an anti-GM2 ganglioside monoclonal antibody and thin-layer chromatography and immunostaining. GM1 ganglioside catabolism in cultured cells was analyzed by pulse labeling, and the amount of GM2 activator in cells was determined by Western blot analysis. Gene analysis was performed according to standard protocols. RESULTS: The patient showed progressive neurologic manifestations of quite early onset. Muscular weakness and hypotonia became evident by 1 month of age, and the patient then developed a startle reaction, severe psychomotor retardation, and myoclonic seizures. Immunocytochemical analysis clearly revealed the accumulation of GM2 ganglioside in cultured fibroblasts from the patient, and thin-layer chromatography confirmed it. Western blot and metabolic studies showed a complete deficiency of GM2 activator. Gene analysis did not reveal any mutations in the protein coding region of the GM2 activator gene. CONCLUSION: The clinical features and biochemical basis of this Japanese patient with GM2 gangliosidosis AB variant were determined. Immunocytochemical analysis using cultured fibroblasts as samples is available for the diagnosis of this disease.

Molecular analysis of a GM2-activator deficiency in two patients with GM2-gangliosidosis AB variant.

Lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (hex A) requires the presence of the GM2 activator protein (GM2AP) as an essential cofactor. A deficiency of the GM2 activator causes the AB variant of GM2 gangliosidosis, a recessively inherited disorder characterized by excessive neuronal accumulation of GM2 and related glycolipids. Two novel mutations in the GM2 activator gene (GM2A) have been identified by the reverse-transcriptase-PCR method--a three-base deletion, AAG262-264, resulting in a deletion of Lys88, and a single-base deletion, A410, that causes a frameshift. The latter results in substitution of 33 amino acids and the loss of another 24 amino acid residues. Both patients are homoallelic for their respective mutations inherited from their parents, who are heteroallelic at the GM2A locus. Although the cultured fibroblasts of both patients produce normal levels of activator mRNA, they lack a lysosomal form of GM2AP. Pulse/chase labeling of cultured fibroblasts of the patients, in presence and absence of brefeldin A, indicates a premature degradation of both--mutant and truncated--GM2APs in the endoplasmic reticulum or Golgi. These results were supported by in vitro translation experiments and expression of the mutated proteins. When the mutated GM2APs were expressed in Escherichia coli, both mature GM2AP forms turned proved to exhibit only residual activities in an in vitro assay.

Regional localization of the gene coding for the GM2 activator protein (GM2A) to chromosome 5q32-33 and confirmation of the assignment of GM2AP to chromosome 3.

The gene coding for the GM2 activator protein (GM2A) was previously mapped by us to chromosome 5 by an ELISA-based technique. Here we confirm this assignment using a PCR analysis of somatic cell hybrids and describe a regional localization to chromosome 5q32-33 by in situ hybridization. We also confirm the assignment of a pseudogene GM2AP to chromosome 3.