Ehrlich II –2nd World Conference on Magic Bullets

Application of Electrochemical Detection to the Determination of (A) Trace Amounts Minoxidil in Hamster Ear Skin Follicles (Magic Bullet-like Delivery) and (B) Residual Hydrogen Peroxide Present in the Minoxidil Formulation Excipients HUANG T^1, GARCEAU ME^2, RAMSTAD T^3, STEHLE RG^4, GAO P^5 ^1Bausch & Lomb, Rochester, USA; ^2 Washington Univ. St. Louis, USA ; ^3Pfizer, Kalamazoo, USA; ^4Covance, Madison, USA ; ^5Abbott Laboratories, Chicago, USA. Background: A highly sensitive electrochemical method was needed for the determining (A), the low concentrations of the hair growth drug, Minoxidil, within hamster ear hair follicles (Magic Bullet-like Delivery), as well as, (B), the low levels of hydrogen peroxide in the formulation excipients used in the Topical Minoxidil formulations. Methods: The Minoxidil located specifically in the sebaceous glands of the hamster ear was isolated from the skin and hair follicles in different skin layers by treatment with aqueous trichloroacetic acid followed by acetonitrile. The amount of Minoxidil in the drug containing supernatant layer was determined by liquid chromatography with detection by electrochemical oxidation (LCEC). Quantitation of the low residual levels of hydrogen peroxide in the formulation excipients was also achieved with LCEC by oxidation using the platinum electrode or by reduction using a wired enzyme electrode with dual channel electrochemical detection. Results: The lower detection limit for Minoxidil was found to be 1 ng/ml using LCEC. The analytical recoveries ranged between 94.4-103.1% and linearity was excellent up to 250 µg/ml with a regression coefficient (r^2) of 0.9988. The LCEC method is more cost effective and the detection limit is similar to that of the widely used radiolabeled scintillation method. The analysis time for determination of the residual hydrogen peroxide present in the formulation excipients was 1 min and the detection limit was 10 ng/mL using the platinum electrode, whereas, the detection limit was 1 ng/mL using the wired enzyme electrode. Peak purity was assured by showing that the peak ratios were constant at different potentials on the dual electrodes. Conclusions: The LCEC method allows rapid determination of the Magic Bullet-like delivery of Minoxidil to the hair follicles with levels as low 1 ng/mL, as well as, the hydrogen peroxide levels as low as 1 ng/mL in various excipients in the Minoxidil Formulations.

Characterised Nanomaterials for Biomarker Profiling Based Cancer Detection HUCK CW, RAINER M, HEIGL N, BONN GK Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020 Innsbruck, Austria Background: Proteomics pattern analysis based biomarker discovery has recently emerged as a novel tool for early diagnostics of diseases including various cancers. In this presentation, the development of derivatized nano-structured affinity materials such as carbon nano-tubes, nano crystalline diamond, fullerene and carbon nanofiber derivatives etc. for efficient material enhanced laser desorption ionization time of flight mass spectrometry (MELDI-TOF MS) based protein profiling and biomarker detection is reported (1). Methods: The utilization of nano-structured affinity materials with various surface properties (hydrophilic, hydrophobic, etc.) enabled rapid and reliable protein profiling to obtain characteristic mass fingerprints for rapid and reliable biomarker discovery. The applicability of MELDI-TOF-MS for rapid protein profiling was evaluated with human serum samples. Results: First, derivatised carbon nano-tubes (2) were explored to reveal protein profile differences in human body fluids e.g., serum, by MELDI-TOF-MS (Figure 1) that can be related to disease and subsequently find relevant biomarkers. Figure 1. Principle of material enhanced laser desoprtion ionisation mass spectrometry (MELDI-MS) The versatility of this technology is meant to increase the amount of information from biological samples at the protein level, which will have a major impact to serve the cause of diagnostic markers. The unique properties of carbon nano-tubes were exploited for manipulating this nano-structured material to construct a novel tool for efficient protein profiling and biomarker discovery by MELDI-TOF-MS. Another nanotechnology approach utilized attachment of specific parts of biomolecules to nano-crystalline diamond surface that has been selectively prepared to contain hydrophobic and hydrophilic regions (3). Nano-crystalline diamond surfaces were selected because of their unique characteristic of enormous bio-compatibility and chemical inertness, i.e. lack of cross-reactivity with such analytes as serum peptides and proteins. Finally, derivatisation of fullerenes (4, 5) and carbon nano-fibers (6) were found to alter its adsorption characteristics toward proteins, thus differences in protein profiling mass patterns were expected when employing the fullerene derivatives as MELDI carrier materials. The success of individual derivatisation steps of these and other materials surface as well as physical properties, e.g. surface area, porosity were measured by a novel near-infrared spectroscopic (NIRS) method, enabling an extreme fast and presice physico-chemical characterization (7-9). Conclusions: The novel technology allows to distinguish cancer, non-cancer, benign in case of prostate cancer with a sensitivity and selectivity > 85%. 1) M. Najam-ul-Haq, M. Rainer, Z. Szabó, R. Vallant, C. W. Huck*, G. K. Bonn, J. Biochem. Biophys. Meth., (2007), 70, 319-328 2) M. Najam-ul- Haq , M. Rainer, T. Schwarzenauer, C.W. Huck* , G.K. Bonn , Anal. Chim. Acta, 561, 2006, 32-39 3) M. Najam-ul-Haq, M. Rainer, C.W. Huck, G. Stecher, I. Feuerstein, D. Steinmüller, G.K. Bonn, Current Nanoscience, 2006, 2, 1-7 4) R.M. Vallant, Z. Szabo, S. Bachmann, R. Bakry, M. Najam-Ul-Haq, M. Rainer, N. Heigl, C. Petter, C.W. Huck, G.K. Bonn, Anal. Chem., 2007, 79(21), 8144-8153 5) M. Najam-ul-Haq, M. Rainer, N. Heigl, Z. Szabo, R. Vallant, C.W. Huck*, H. Engelhardt, R. Bischoff, G.K. Bonn, Amino Acids, 2008, 34, 279-286, DOI 10.1007/s00726-007-0492-5 6) A. Greiderer, M. Rainer, M. Najam-ul-Haq, R.M. Vallant, C.W. Huck*, Günther K. Bonn, , Amino Acids, 2008, in press 7) N. Heigl, C.H. Petter, M. Najam-Ul-Haq, M. Rainer, R.M. Vallant, G.K. Bonn, C.W. Huck*, J. Near Infrared Spectrosc., 2008, in press 8) C.W. Huck*, R. Ohmacht, Z. Szabo, G.K. Bonn, J. Near Infrared Spectrosc., 14, 2006, 51-57 9) R.M. Vallant, Z. Szabo, L. Trojer, M. Najam-Ul Haq, M. Rainer, C.W. Huck, R. Bakry, G.K. Bonn, J. Proteom. Res., (2007), 6(1), 44-53.

Drug-Peptide Conjugates with Antitumour or Anti Parasite Activity HUDECZ F^1,2, ORBÁN E^1, MIKLÁN ZS^1, SZABÓ R^1, BÁNÓCZI Z^1, REMÉNYI J^1, GAÁL D^2 ^1Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, Budapest, Hungary. ^2Department of Organic Chemistry, Eötvös L. University, Budapest, Hungary. ^2National Institute of Oncology, Budapest, Hungary Background: Peptide/protein carriers can be used as soluble drug conjugates for “passive targeting” resulting in altered pharmacokinetics (e.g. accumulation in tumour), increased intracellular uptake, decreased non–specific toxicity and immunogenicity. Methods: We have developed a new group of water-soluble oligo- or polypeptid-drug-conjugates in which daunomycin (Dau), methotrexate (MTX) or other anitumour agents are coupled to amphoteric or polycationic branched polypeptides or to oligoarginine. Toxicity, in vitro and in vivo antitumor activity of conjugates with daunomycin were investigated using sensitive and multidrug resistant, mouse (L1210 and P388 leukemia, S180 sarcoma, MXT breast carcinoma) and human (HL60 leukaemia) tumors. The antiparasitic activity of the MTX containing conjugates was analysed also in vitro and in vivo using Leishmania donovani infected macrophages and animals. Results: We found that attachment of peptide to the bioactive cargo significantly improved the antitumour or antiparasitic properties of the drug, respectively and also significant reduction of drug related side effects could be demonstrated. Conclusions: The covalent conjugation of drugs with antitumour or antiparasite activity to oligoarginine or macromolecular polypeptide typa carrier could be useful strategy to develop new compounds with improved therapeutic efficacy.

Glucose transporters in the blood-brain barrier HUSSAR P, BENNO I, HUSSAR Ü University of Tartu, Tartu, Estonia Backround: The central nervous system and peripheral nerves are guardedagainst free access from the outside by the blood-brain,blood-cerebrospinal fluid and blood-nerve barriers. The glucose transporterGLUT1 mediates the specific transfer of glucose across thesebarriers while GLUT3 is a high-affinity isoform of Type I glucose transporter expressed mostly in neurons where it is believed to be the main glucose transporter isoform. As for a long time it was an open question whether GLUT1 andGLUT3 are present in the olfactory system the aim of the present study was to give answers to these questions. Methods: In the study including 20 male Wistar rats (4 weeks old) mucous membranes of the olfactory region were studied by double immunofluorescence labeling. Rabbit anti-GLUT1, guinea pig anti-GLUT1, rabbit anti-chicken tubulin, rabbit anti-GLUT3 and mouse anti-PGP served as primary antibodies.Fluorescein isothiocyanate-labeled donkey antiguinea pig immunoglobulinG (IgG), dichlorotriazinyl amino fluorescein-labeled and rhodaminered X-labeled donkey anti-rabbit IgG, and Cy3-labeled donkey anti-mouseIgG were used as secondary antibodies. Results: The studies indicated the abundant presence of GLUT1 in the endothelial cells of olfactory mucosa while the upper cells of olfactory epithelium (cellulae neurosensoria olfactoriae) stained srongly positive for GLUT3. Anti-tubulin antibody strongly stained the apices of the olfactory epithelialcells as well as nervefiber bundles emanating from the epithelium. Anti-PGP antibody stained olfactory receptor neurons in theolfactory epithelium and the nerve fibers running underneath. Conclusions: The immunolocalization of GLUT1 in the endothelial cells of olfactory mucosa and GLUT3 expressed primarily in olfactory receptor neurons allow glucose to crossthe blood-brain barrier and enter neurons. Moreover, the results also showed that PGP serves as a marker forthe olfactory epithelium (nerve fibers emanating thereof)and that tubulin acts as a marker for the nerve fibers in olfactory mucosa.