Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

The role of ACE2 in the renin-angiotensin-system: Etiology and therapy of COVID-19 from a pharmaceutical perspective.

Angiotensin-2 converting enzyme (ACE2), a key element of the renin-angiotensin-system (RAS), is not only the direct target of infection by the human SARS-Cov-2 virus but is at the same the root for the complex pathogenetic events of COVID-19. From a pharmaceutical perspective, several established classes of medicines are involved in different phases of the disease. From their known mechanisms of action, a comprehensive understanding of COVID-19 will be hopefully soon delineated. A set of proven medicines is available to cope at least with some of the pathologies involved. To arrive back to normal life, vaccinations and broad consideration of hygienic measures are to be complemented by effective medicines to treat airborne viral infections. Therapeutic schemes based on a comprehensive understanding of the disease will include drug combinations made up from both established drugs as well as novel drugs presently under development.

The "sugar dilemma".

Type 2 diabetes mellitus is characterized by insulin resistance and elevated blood glucose levels. Treatment protocols generally include dietary restriction of sugar, as well as drugs aiming at a reduction of blood glucose, mainly by activating the insulin system or supplementing insulin. This established approach does not take into account the outstanding physiological role of glucose as a key molecule in metabolism. Glucose is crucial to meet the high energy demand of the brain, which depends on it as an exclusive nutrient. Insulin independent glucose transporters GLUT1 import glucose into the brain. Reduction of blood glucose, as in current treatment concepts, may lead to energy deficiency in the brain and consecutively to worsening of - possibly already impaired - neurocognitive function. Reduced cell membrane fluidity of the vascular endothelium of the bloodbrain-barrier (BBB) - due to malnutrition and/or aging - is considered a major factor in pathogenesis of the cerebral metabolic syndrome, which is a key step in neurodegeneration. Under this aspect we suggest a novel approach to prophylaxis and treatment focusing on a sufficient supply of glucose to the brain.

Charge heterogeneity study of a Fc-fusion protein, abatacept, using two-dimensional gel electrophoresis.

Medicinal products obtained by recombinant DNA technology are complex molecules and demonstrate a high degree of molecular heterogeneity. Charge heterogeneity and isoform pattern of this class of medicines, are parameters important for their quality, safety, and efficacy. In this study we report the application of two-dimensional gel electrophoresis (2-D electrophoresis) for the quality assessment, identification, charge heterogeneity and isoform pattern study of recombinant protein, CTLA4-Ig (abatacept), which has been selected as an example of the drug class, known as Fc-fusion proteins. In order to achieve an efficient separation of this complex analyte,2-D electrophoresis was optimized employing different experimental conditions regarding the selection of an immobilized pH gradient (IPG), sample pretreatment, presentation and detection procedure. Experimental datadocumented that 2-D electrophoresis is a suitable method for the assessment of identity, purity, structural integrity, isoform pattern and to monitor charge heterogeneity and post-translational glycosylation of the Fc-fusion protein, abatacept.

Determination of acarbose by capillary zone electrophoresis.

Acarbose (Glucobay, Bayer AG) acts as a potent alpha-glucosidase-inhibitor, which delays the intestinal starch digestion resulting in a reduction of postprandial blood glucose and insulin levels. Acarbose is a pseudo-tetrasaccharide, with two D-glucose units linked via an alpha 1-->4 glycosidic bond to acarviosin, which is a N-glycoside composed of an unsaturated cyclitol and 4-amino-4,6-dideoxy-alpha-D-glucopyranose. Several methods for the determination of acarbose by capillary electrophoresis can be found in literature. They are based either on the derivatisation with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) or on the detection of the unsaturated cyclitol at wavelengths below 200 nm. The aim of our work was the determination of acarbose making use of a previously developed method based on reductive amination with S-phenylethylamine. The aminoalditols generated in the reaction formed differently charged borate-complexes depending on the configuration of the sugar. After successful method optimisation we were able to separate two potential impurities of acarbose, D-maltose und D-glucose. For the quantitation of acarbose in Glucobay tablets an additional borate-buffer system was established, reducing the total time of analysis to less than 10 min.

Lactic acid oligomers (OLAs) as prodrug moieties.

In this paper we propose the use of lactic acid oligomers (OLAs) as prodrug moieties. Two synthetic approaches are presented, on the one hand a non selective oligomerisation of lactic acid and on the other hand a block synthesis to tetramers of lactic acid. Dimers of lactic acid were investigated with respect to their plasma stability and their adsorption to albumine. Ibuprofen was chosen as the first drug for OLAylation. The ester 19 of LA(1)-ibuprofen was evaluated with respect to the degradation to human plasma and the adsorption to albumine. All results indicate that lactic acid oligomers are promising prodrug moieties.

Blood-brain barrier in vitro models as tools in drug discovery: assessment of the transport ranking of antihistaminic drugs.

In the course of our validation program testing blood-brain barrier (BBB) in vitro models for their usability as tools in drug discovery it was evaluated whether an established Transwell model based on porcine cell line PBMEC/C1-2 was able to differentiate between the transport properties of first and second generation antihistaminic drugs. First generation antihistamines can permeate the BBB and act in the central nervous system (CNS), whereas entry to the CNS of second generation antihistamines is restricted by efflux pumps such as P-glycoprotein (P-gP) located in brain endothelial cells. P-gP functionality of PBMEC/C1-2 cells grown on Transwell filter inserts was proven by transport studies with P-gP substrate rhodamine 123 and P-gP blocker verapamil. Subsequent drug transport studies with the first generation antihistamines promethazine, diphenhydramine and pheniramine and the second generation antihistamines astemizole, ceterizine, fexofenadine and loratadine were accomplished in single substance as well as in group studies. Results were normalised to diazepam, an internal standard for the transcellular transport route. Moreover, effects after addition of P-gP inhibitor verapamil were investigated. First generation antihistamine pheniramine permeated as fastest followed by diphenhydramine, diazepam, promethazine and second generation antihistaminic drugs ceterizine, fexofenadine, astemizole and loratadine reflecting the BBB in vivo permeability ranking well. Verapamil increased the transport rates of all second generation antihistamines, which suggested involvement of P-gP during their permeation across the BBB model. The ranking after addition of verapamil was significantly changed, only fexofenadine and ceterizine penetrated slower than internal standard diazepam in the presence of verapamil. In summary, permeability data showed that the BBB model based on porcine cell line PBMEC/C1-2 was able to reflect the BBB in vivo situation for the transport of antihistaminc drugs and to distinguish between first and second generation antihistamines.

Serum-derived immunoglobulins alter amyloid beta transport across a blood-brain barrier in vitro model.

Since passive immunization with serum-derived immunoglobulins (intravenous immunoglobulins) showed several positive effects in some patients with Alzheimer's disease (AD), intravenous immunoglobulins (IVIG) are discussed as a possible treatment option. IVIG, an antibody product derived from human plasma, contains natural antibodies against amyloid beta(Abeta) peptide. Until now it is not known, how IVIG interferes with pathogenesis in AD, but several proposed mechanisms are in discussion. Receptor types which are involved in transport processes at the BBB are LRP, RAGE and hFcRn. We were looking for an in vitro BBB model expressing these receptors and studied the alteration of transport of Abeta peptides across this model under the influence of immunoglobulins. Cell line ECV304 was found to be suitable for our experiments. We found evidence for involvement of an improved clearance of Abeta across the BBB as well as a decreased Abeta influx from blood to the brain probably following complex formation of immunoglobulins with free Abeta in the periphery. Furthermore, we were able to confirm the activity of IVIG preparations which acted the same way but showed slightly less efficacy in comparison to monoclonal anti-Abeta antibodies. Based on these results we suggest multiple mechanisms responsible for the efficacy of immunotherapy in Alzheimer's disease.

Applying blood-brain barrier in vitro models to study the influence of drugs on endothelial cells--effects of selected COX-inhibitors.

The influence of three cyclooxygenase (COX) inhibitors (indometacin, lornoxicam and celecoxib) with different COX-1/COX-2 profiles on endothelial cells using in vitro blood-brain barrier (BBB) models was investigated. For the experiments two BBB mimicking cell lines (PBMEC/C1-2 and ECV304) and primary human umbilical vein endothelial cells (HUVEC) were used. In preliminary tests the two cell lines and HUVEC were characterized by cell ELISA in respect of the presence of the tight junction proteins occludin and zonula occludens protein-1 (ZO-1), the adhesion molecules ICAM-1 and VCAM-1 and the endothelial marker von Willebrand factor (vWF). Then, the influence of indometacin, lornoxicam and celecoxib on the expression of occludin, ZO-1, ICAM-1 and vWF of the two cell lines and HUVEC was analysed by cell ELISA. The COX inhibitors caused an effect on PBMEC/C1-2 and HUVEC but no influence was observed on ECV304. The results of PBMEC/C1-2 and HUVEC indicated that in comparable therapeutical concentrations celecoxib had a higher potential to impair endothelial cells and to decrease the expression of occludin, ZO-1 and ICAM-1 than indometacin and lornoxicam.

Determination of (R,R)-glycopyrronium bromide and its related impurities by ion-pair HPLC.

A simple, rapid and specific ion-pair HPLC method for the determination of (R,R)-glycopyrronium bromide and its related impurities is presented, and parameters affecting the chromatographic properties of these compounds are discussed. Optimal analyte separation was achieved on base deactivated Nucleosil at 40 degrees C, using phosphate buffer pH 2.30 with sodium-1-decanesulfonate (0.01 M)/methanol (35/65; v/v) as eluent for isocratic elution at a flow rate 1 ml x min(-1). The analytical assay was validated according to international guidelines. The methodis suitable for in-process control and as stability indicating assay.

Analogues and derivatives of tenoxicam. 1. Synthesis and antiinflammatory activity of analogues with different residues on the ring nitrogen and the amide nitrogen.

The synthesis of tenoxicam, 4-hydroxy-2-methyl-N-2-pyridyl-2H-thieno[2,3-e]-1,2-thiazine-3-carboxami de 1,1-dioxide (1e), and of the analogues with various residues on the ring nitrogen and the amide nitrogen is described. This new class of "oxicams" has pronounced antiinflammatory and analgesic properties. The very specific structure-activity relationship of isomeric and isosteric groups at the amide nitrogen has been evaluated. The substituent in position 2 also has a great influence on the pharmacological properties. Tenoxicam is presently undergoing clinical trials.

Interaction of long chain n-alkyl diamines with the NMDA receptor complex.

Long chain 1, omega-diamines with 8, 10, 12, 14 and 16 carbon atoms reduced the binding of [3H]MK 801 to rat hippocampal membranes with IC50 values of 333, 72, 16.5, 4.7 and 4.3 microM, respectively. In the presence of medium-effective concentrations of the diamines, the concentration response curve of the stimulation by the polyamine spermine was shifted to higher spermine concentrations, reaching similar maximal stimulation as in the absence of the diamines. In the case of 1,12-diaminododecane (N-12-N) and 1,14-diaminotetradecane (N-14-N), the extent of this shift was compatible with competitive antagonism of spermine. Inhibition of [3H]MK 801 binding by long chain diamines was sensitive to spermine, inhibition by N-12-N and N-14-N exhibited the highest sensitivities. Of all diamines tested, N-12-N and N-14-N seem to be the most likely candidates for inverse polyamine agonists.

Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides.

The paper describes a novel synthesis of cholest-5-en-3 beta-yl-6-aminohexyl ether (AH-Chol). AH-Chol was used to prepare positively charged liposomes. The liposomes consisted of phospholipon 90H and the cationic cholesterol derivative in an equimolar ratio. Liposome preparation was achieved by membrane homogenization after rehydration of a dry lipid film. Oligonucleotides (ODN) were adsorbed to the cationic liposomes very efficiently. At an ODN/liposome ratio of 1:5 (10:50 micrograms/ml) 84.2 +/- 5.4% of the ODNs were bound to the liposomal membrane. Within the range of 1:40 and 1:100 charge neutralization occurred and the liposome dispersion showed an increase in particle size due to aggregation. Below or above this range of charge neutralization the ODN loaded liposome preparation was physically stable, no sedimentation, increase of vesicle size or vesicle aggregation occurred.

Evaluation of aminoalkylmethacrylate nanoparticles as colloidal drug carrier systems. Part I: Synthesis of monomers, dependence of the physical properties on the polymerization methods.

Conventional nanoparticles based on acrylic compounds are lipophilic and possess a negative surface charge. This is due to their manufacturing process and to the chemical structure of the polymer. Hence, these particles are not suitable for the adsorption of hydrophilic anionic drugs. In the present investigation, positively charged copolymer nanoparticles prepared from aminoalkyl- and methylmethacrylates were evaluated, with regard to their physical properties. This report provides a detailed description of the synthesis of the non-commercially available monomers and their polymerization procedure. Various parameters were investigated, such as comonomer content, total amount of monomer, concentration of the radical initiator, and the composition of the polymerization medium. The resulting particle diameter and the surface charge were found to be strongly dependent on the polymerization conditions and on the pH. Optimization of the polymerization procedure yielded nanoparticles of about 200 nm exhibiting a positive surface charge. The charges of the different copolymer particles were then compared at different pH values. N-trimethylaminoethylmethacrylate (TMAEMC) nanoparticles with quaternary ammonium groups located at their surfaces, possessed a nearly constant positive zeta potential at various pH values and, consequently, pH-independent particle diameters. The physical characteristics of the other aminoalkyl copolymers correlated with the basicity of the monomers employed and were found to be strongly dependent on the pH of the dispersion medium. Aminoethylmethacrylate (AEMC), methylaminoethylmethacrylate (MMAEMC), and aminohexylmethacrylate (AHMC) as well as aminoethylmethacrylamide (AHMAC) copolymer nanoparticles exhibited a strong positively charged surface even at physiological pH and, therefore, are useful candidates for the adsorption of anionic drugs.

Evaluation of aminoalkylmethacrylate nanoparticles as colloidal drug carrier systems. Part II: characterization of antisense oligonucleotides loaded copolymer nanoparticles.

Aminoalkylmethacrylate methylmethacrylate copolymer nanoparticles were evaluated for their use as potential drug carrier systems. Their cytotoxicity, as well as the loading of antisense oligonucleotides that were employed as anionic model drugs depended on the substitution of the basic aminoalkyl copolymer. Toxic influences on the integrity of cell membranes depended on aminoalkyl groups located on the particle surfaces. Toxicity was observed either by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays using African green monkey kidney (AGMK) cells or by a hemolysis test, where the efflux of haemoglobin from disrupted erythrocytes was measured. The cytotoxic effects were increased by the elongation of the N-alkyl chain by four additional methylene groups. Lipophilic polymethylmethacrylate (PMMA) homopolymer nanoparticles showed a negative surface charge and, therefore, were not suitable for the adsorption of anionic drugs. The surface charge was changed to positive values by the incorporation of basic monomers. Consequently, the loading efficacy was increased by raising the basic copolymer portion. Additionally, a pH-dependent loading behaviour of oligonucleotides was observed. Substitution of the amino nitrogen protons by methyl groups led to a decreased oligonucleotide loading and to a reduced cytotoxicity. Nanoparticles with permanent positively charged quarternary ammonium groups showed a high pH-independent loading efficacy, but also possessed a high cytotoxic potential. In this study, cationic copolymer nanoparticles containing 30% (w/w) methylaminoethyl-methacrylate (MMAEMC) were found to be optimal with regard to biocompatibility and carrier properties for hydrophilic anionic antisense oligonucleotides. A significant portion of adsorbed oligonucleotides were protected from enzymatic degradation. The cellular uptake of oligonucleotides into Vero cells was significantly enhanced by this methylaminoethyl-methacrylate derivative.

Characterization of two blood-brain barrier mimicking cell lines: distribution of lectin-binding sites and perspectives for drug delivery.

In the present study plant lectins with distinct sugar specificities were applied to two blood-brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1-2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1-2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1-2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

Alteration of the glycocalyx of two blood-brain barrier mimicking cell lines is inducible by glioma conditioned media.

Recent studies showed that glioma conditioned medium is able to induce blood-brain barrier properties in in vitro models. In this regard, it was investigated whether glioma conditioned medium can also influence the lectin-binding capacity of blood-brain barrier in vitro models. For the presented study cell lines PBMEC/C1-2 and ECV304 were chosen because it was previously shown that glioma conditioned medium was able to induce specific blood-brain barrier properties in these cell lines. Six different plant lectins (WGA, STL, LCA, UEA-I, DBA, PNA) with distinct sugar specificities were applied in order to elucidate the glycosylation patterns of cell line PBMEC/C1-2 and ECV304. Lectin-binding studies were carried out with monolayers as well as with single cells. In the case of PBMEC/C1-2 monolayers, results showed a significant increase of the binding of lectins WGA, STL, UEA-I, DBA and PNA after application of 25 pmol lectin when cultured in media containing soluble factors derived from glioma cell line C6, whereas the binding capacity for LCA remained similar. For ECV304 monolayers, a significant decrease of WGA, STL and LCA was observable, whereas UEA-I binding increased in comparison to cells grown in the corresponding basal growth medium without soluble C6 factors. Single cell studies showed less significant, but similar changes in the lectin-interactions with the cell surfaces. In conclusion, it was shown that soluble factors derived from glioma cell line C6 can modulate the "glycocalyx" of blood-brain barrier mimicking cell lines.

[Thiophene as a structural element in physiologically active substances. 18. A thiophene analog of propafenone]. / Thiophen als Strukturelement physiologisch aktiver Substanzen, 18. Mitt.: Ein Thiophenanalogon des Propafenons.

The preparation of compounds 1, which are thiophene analogues of propafenon (A) is described. Compound 1a shows antiarrhythmic activity in various in vivo test-systems.

Functional expression of recombinant N-methyl-D-aspartate receptors in the yeast Saccharomyces cerevisiae--localization and pharmacological characterization.

The yeast Saccharomyces cerevisiae was used for expressing the genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits from rats (NR1a, NR2A, NR2C) and mice (NR2B). Four plasmids were constructed by cloning the different NMDA receptor genes in the two multi-copy yeast-Escherichia coli shuttle vectors pMB01 (--> NR1a gene) and pMB02 (--> NR2A-2C genes). The protease-deficient S. cerevisiae strain cI3-ABYS-86 (leu-, ura-) was transformed or co-transformed with the resulting plasmids pMBNR1a (leu+) or pMBNR1a/pMBNR2A-C (ura+) respectively. Western blotting analysis with antibodies raised against amino acid sequences at the C-termini of the respective subunits revealed that the recombinant receptor proteins were differently expressed and only partially glycosylated in the cell membranes of the recombinant yeast strains. The expression and localization of the recombinant NMDA receptor proteins were also proved by immunofluorescence microscopy which indicated a distinct expression of the different NMDA receptor subunits in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells showed saturable binding of the glycine antagonist [3H]MDL105,519 with Kd values of 56.9 +/- 6.19 nM (NR1a/NR2A), 26.72 +/- 2.13 nM (NR1a/NR2B), and 21.22 +/- 1.64 nM (NR1a/NR2C), and bound capacities of 17.94 +/- 1.24 pmol/mg membrane protein (NR1a/NR2A), 11.45 +/- 0.67 pmol/mg (NR1a/NR2B), and 16.15 +/- 0.86 (NR1a/NR2C) pmol/mg. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate, ethyl-2-carboxy-4,6-dichloro-3-indoleacetate, and itself, but not by glycine, D-serine or 1-amino-cyclopropanecarboxylic acid. Specific binding of [3H]glycine or the NMDA channel blocker [3H]dizolcipine were not observed.

Benzimidazole-type glycine antagonists: the role of the ring nitrogen atoms.

Several derivatives of 1H-benzimidazole-2-carboxylic acid (BICA, 2a) were tested in vitro in comparison to 1H-indole-2-carboxylic acid (ICA, 1e) for their ability to displace [3H]glycine from rat hippocampal membranes. Compound 2a was 8 times more potent than 1e (Ki 5.3 microM, as compared to 42 microM). However, introduction of a carboxymethyl group or a corresponding ester at position 3 had no positive effect on the potency of 2a, while this type of structural modification increased the potency of 1e significantly. Nevertheless, 1-carboxymethyl-BICA (2b) displaced [3H]glycine with similar potency as the corresponding 3-carboxymethyl-ICA 1c, indicating that also a nitrogen atom lacking a hydrogen atom can be engaged in glycine receptor interaction. N-Methylation strongly reduced the potencies of both BICA and ICA derivatives.