Martin Kratzel

Synthesis and in vitro multidrug resistance modulating activity of a series of dihydrobenzopyrans and tetrahydroquinolines.

A series of dihydrobenzopyrans and tetrahydroquinolines was synthesized and pharmacologically tested for their ability to inhibit P-glycoprotein mediated daunomycin efflux in multidrug resistant CCRF-CEM vcr1000 cells. Several compounds exhibit activities in the range of the reference compounds verapamil and propafenone. Preliminary structure-activity relationship studies propose the importance of high molar refractivity values of the compounds and the presence of an additional basic nitrogen atom.

Two-dimensional electrophoresis of recombinant human erythropoietin: A future method for the European Pharmacopoeia?

Quality assurance of recombinant protein drugs concerning identity and purity represents a difficult task, in particular, when post‐translational modifications lead to a heterogeneous mixture of biomolecules. We chose Neorecormon® (rh‐EPO, Roche) for our studies to demonstrate the efficiency of two‐dimensional electrophoresis (2‐DE) to analyse post‐translationally modified recombinant drugs. More than 40 protein spots in the range from isoelectric point (pI) 3.5–4.5 and 32–45 kDa could be separated. Enzymatic deglycosylation revealed that the heterogeneity of the protein pattern is mainly caused by variations in glycosylation. In comparison to the separately performed isoelectric focusing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, as requested by the European Pharmacopoeia, we see a great synergy to use 2‐DE for the analysis of rh‐EPO. A by far higher resolution can be achieved, allowing an improved differentiation of the various rh‐EPO glycoforms. Sequential deglycosylation of sialic acids, N‐glycosides and the O‐glycoside lead to significant shifts both in apparent relative molecular mass and pI. Comparing the 2‐DE patterns of rh‐EPO before and after deglycosylation allows on the one hand valuable information to be gained on the glycosylation of the recombinant protein and shows on the other hand how significantly the 2‐DE protein pattern can be influenced by the glycosylation. As the equipment for the performance of 2‐DE has improved significantly over the last decade, we see 2‐DE as a reliable method, which should be approved for the routine quality assurance of recombinant drugs and also recommended for the European Pharmacopoeia.

Decrease of liposomal size and retarding effect on fluconazole skin permeation by lysine derivatives.

Liposomes are ideal dermal drug delivery systems because of their ability to alter the biodistribution profile of incorporated drugs. In a novel approach to optimize the liposomal microstructure, lysine derivatives were employed. The effect of the oligopeptides Lys-5 and Lys-7 on the structure as well as on the skin permeation of the antimycotic drug fluconazole in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles was studied using a variety of techniques. It was demonstrated by addition of the shift reagent praseodymium(III)chloride and subsequent (31)P NMR measurements that the liposomes produced consisted mainly of unilamellar vesicles. This was confirmed by cryo-transmission electron microscopy. The addition of Lys-5 and Lys-7 induced a structural change resulting in a decrease in particle size between 10% and 40% and a retarding effect on fluconazole skin permeation.

Modified mucoadhesive polymers for the peroral administration of mainly elastase degradable therapeutic (poly)peptides

Abstract A number of elastatinal–polymer conjugates, having the inhibitor linked to sodium carboxymethyl cellulose (Na-CMC), poly(acrylic acid) (PAA) and poly(acrylic acid-divinyl glycol) via a 1,8-diaminooctane spacer, were synthesized and their protective effect from enzymatic degradation caused by elastase as well as their mucoadhesive properties were evaluated. Unmodified polymers did not show any inhibitory effect under our enzyme assay conditions. However, 50 μ g of modified Na-CMC, PAA and poly(acrylic acid-divinyl glycol) inhibited the proteolytic activity of elastase (6 μ g/290 μ l 50 mM Tris–HCl, pH 7.8) at 20±0.5°C up to 77%, 41% and 44.5%, respectively. Whereas 1 mg of elastatinal–Na-CMC conjugates, resulting from reaction mixtures with a weight ratio of inhibitor to polymer of 1:10, 1:5 and 1:1, exhibited a protective effect, which was equivalent to 2.8±0.8 up to 9.2±1.2 μ g of unbound inhibitor, corresponding conjugates of elastatinal with PAA and poly(acrylic acid-divinyl glycol) were in the range between 0.8±0.4–3.2±0.4 and 1.6±0.4–4.2±0.8 μ g ( n =3; ±S.D.), respectively. Moreover, the mucoadhesive force of the polymers was not influenced by the slight modification. According to these results, the novel mucoadhesive polymers shielding from luminal enzymatic attack may be a useful tool for the peroral administration of mainly elastase degradable therapeutic (poly)peptides.

Development of a Sustained Release Dosage Form for α‐Lipoic Acid. II. Evaluation in Human Volunteers

Within this study an oral sustained release dosage form of α‐lipoic acid (thioctic acid) has been generated and evaluated in healthy volunteers. A granulate comprising 56.8% α‐lipoic acid and 43.2% chitosan acetate was compressed to tablets (weight: 0.45 g; diameter: 10.0 mm; thickness: 4 mm). Three of these tablets were administered at once orally to each volunteer. Prior to administration and then every hour for 12 hours blood samples were taken from the antebrachial vein. α‐Lipoic acid concentrations in plasma were quantified via precolumn derivatization and reversed‐phase high‐performance liquid chromatography (HPLC). Results demonstrated that an increased plasma level of α‐lipoic acid can be achieved by this formulation for at least 12 hours. Within this time period at least two maximum plasma concentrations were reached. The first one is based on the release of α‐lipoic acid, which is not ionically and therefore only loosely bound to chitosan, whereas a second maximum is based on the release of the drug during the enzymatic degradation of the chitosan matrix in the colon. The AUC0→12 was determined to be 183.8 ± 101.4 µg × min/mL (mean ± SD; n = 8). Because of the pulsed sustained release of α‐lipoic acid, the dosage form described here seems to be highly beneficial in order to stimulate the glucose uptake in the case of diabetes type II.

Channelling of substrate promiscuity of the skeletal-muscle ADP-ribosyl cyclase isoform

The novel Ca2+-mobilizing second messengers cADPr (cyclic ADP-ribose) and NAADP (nicotinic acid-adenine dinucleotide phosphate) are both synthesized by ADP-ribosyl cyclases. Using HSR (heavy sarcoplasmic reticulum) fractions from rabbit skeletal muscle, NAADP-induced Ca2+ release was observed. In the present paper, we show in HSR membranes the formation of authentic cADPr, cGDPr (cyclic GDP-ribose) and NAADP. The cyclization reaction to form cADPr and cGDPr as well as the base-exchange reaction to form NAADP were strictly dependent on pH. Although the formation of cGDPr is optimized at pH 6, the synthesis of NAADP was most pronounced at a pH below 5. A novel regulation mechanism is provided for nicotinic acid, the co-substrate for NAADP synthesis. Nicotinic acid had virtually no influence on the cyclization reaction, but increased the affinity of NADP at an acidic pH and had the opposite effect at alkaline pH. Nicotinamide, the side product of cADPr synthesis, is an inhibitor of the cyclization reaction (IC50, 0.7+/-0.1 mM) and was 30-fold more potent at suppressing the base-exchange reaction. Although the synthesis of NAADP was highly sensitive to nicotinamide inhibition, this was not via a competition with the nicotinic-acid-binding site. In contrast with the ecto-ADP-ribosyl cyclase (CD38), the cyclization and base-exchange reaction of the skeletal muscle isoform was inhibited by Cu2+ and Zn2+, while other bivalent cations such as Ca2+, Mg2+ and Mn2+ had virtually no effect. These findings allow for the prediction of a novel ADP-ribosyl cyclase isoform in skeletal muscle HSR, other than CD38. Hence the enzymic prerequisite for cADPr- and NAADP-mediated Ca2+ signalling is present.

Synthesis, development and in vitro evaluation of drug delivery systems with protective effect against degradation by pepsin.

A (poly)peptide drug delivery system providing a protective effect against degradation by pepsin has been generated. A simplified pepstatin analogue was thereby synthesised displaying a terminally located primary amino group allowing an easy covalent attachment to anionogenic mucoadhesive polymers by the formation of amide bonds. The IC50 of this novel inhibitor was determined to be 6.65+/-1.05x10(-6) M. Mediated by a carbodiimide it was covalently bound to polycarbophil and sodium carboxy-methyl cellulose (NaCMC). In contrast to polycarbophil-inhibitor conjugates, NaCMC-inhibitor conjugates displayed a high inhibitory effect towards pepsin. The protective effect of tablets containing a NaCMC-pepsin inhibitor conjugate (10%), NaCMC (56.7%), insulin (3.3%), and mannitol (30%) was evaluated in vitro. Tablets were therefore incubated for 2 h at 37 degrees C with simulated gastric fluid according to the Pharmacopoeia Europea. Following analysis demonstrated that 50.8+/-8.6% (mean +/- SD; n = 3) of insulin were degraded within the swollen carrier matrix, whereas insulin was completely metabolised in tablets without the NaCMC-pepsin inhibitor conjugate. This protective effect against degradation by pepsin might make such dosage forms useful tools for a targeted (poly)peptide delivery to the stomach.

Rapid Determination of Diclofenac in Pharmaceutical Formulations by Capillary Zone Electrophoresis

Capillary electrophoresis is competitive to HPLC and other chromatographic methods, predominantly when charged analytes have to be separated. The time of analysis can be reduced by the use of very short capillaries applying a high voltage. In most instruments which are commercially available the so-called ‘short end’ of the capillary can be used for separation, leading to very rapid separations. In this contribution we want to demonstrate this approach by using Diclofenac Sodium as an analyte.

Binding and structure‐activity‐relation of benzo[f]isoquinoline‐ and norcodeinone‐derivatives at μ‐opioid receptors in the rat cerebral cortex

1 We have probed the ligand binding site of the μ‐opioid receptor using a series of isoquinoline‐ and norcodeinone‐derivatives; in these morphine‐ and codeine‐analogues, the position of the piperidine‐nitrogen as well as its mobility is altered relative to that found in morphine. 2 The μ‐receptor in rat cortical membranes was labelled with [3H]‐naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine‐nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity. 3 In contrast, we find that the piperidine‐nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl‐group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)‐10, 11‐epoxy‐1, 2, 3, 4, 5, 6‐hexahydro‐9‐hydroxy‐3‐methyl‐4a,10b‐butano‐benzo[f]isochinolin‐12‐on(10). This compound is an agonist with an affinity comparable to that of morphine. 4 We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the μ‐opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2Å distance that exists between the position of the nitrogen in morphine and WB4/PH.

BENZOANALOGOUS CONGENERS OF STREPTAZOLIN

Summary. Streptazolin, a unique natural compound with antibiotic and antifungal activities, is based on a hexahydro-1H-1-pyrindine system containing an internal urethane unit and an exocyclic ethylidene side chain. The diene system, which represents an important structural feature for biological activity, is also responsible for the propensity of streptazolin to polymerize upon concentration from organic solutions. The formal annulation of an aromatic ring under preservation (and prolongation) of the diene system leads to congeners with enhanced stability but reduced antibiotic activity.Zusammenfassung. Das antibiotisch und antifungal wirksame Streptazolin besitzt eine für einen Naturstoff außergewöhnliche Struktur. Diese basiert auf einem Hexahydro-1H-1-pyrindin-System, das als weitere Strukturmerkmale eine endocyclische Urethangruppierung und eine exocyclische Ethylidenfunktionalität aufweist. Das für die biologische Aktivität essentielle Diensystem zeichnet jedoch auch für die Polymerisationsneigung der Verbindung verantwortlich, die zu unwirksamen Polymeren führt. Die formale Anellierung eines Aromaten unter Erhalt (und Verlängerung) des Diensystem liefert Derivate mit erhöhter Stabilität, aber reduzierter antibiotischer Wirkung.