1. INTRODUCTION Functional quality of new peptide drugs: receptor-binding and tissue-interactions M. Verbeken 1, V. Vergote 1, C. Burvenich 1, C. Van de Wiele 1, A. Ronai 2, K. Gyires 2, W. Luyten 3 and B. De Spiegeleer 1* 1 Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium 2 Semmelweis University, H-1085 Budapest, Hungary 3 Leuven University, Naamsestraat 59, B-3000 Leuven, Belgium * Corresponding author: bart.despiegeleer@UGent.be (Ref.: 2009-238f)bart.despiegeleer@UGent.be EXPERIMENTAL RESULTS AND DISCUSSION CONCLUSIONS DruQuaR [1] V. Vergote, S. Van Dorpe, M. Verbeken, C. Burvenich, C. Van de Wiele, W. Banks, B. De Spiegeleer. Development of peptide receptor binding assays: methods to avoid false negatives. Submitted for publication (2009). REFERENCES Tissue-bath experiments The effect on smooth muscle contraction of peptide-mixtures is tested covering 4 different tissues from different species: vas deferens (mouse, mVD), aortic rings (rat, rAOR), trachea and ileum longitudinal muscle strips (guinea pig, gpT and gpI resp). Different experimental conditions are used to extract maximal useful information. In mVD and gpI, the inhibitory effect of peptides is tested on electrical stimulated contraction, in rAOR and gpT on phenylephrine (PE) induced contractions. Inhibition of contraction is expressed in comparison to respectively maximum electrical or chemical (PE) stimulated contractions. Typical results are given here. Other peptides were successfully analyzed using the same methodology (data not given in this presentation). Table 1: TB experiment pept159 (chemical stimulation, rAOR)Table 3: Influence of filtration in RLB studies [1] Results of the present RLB study demonstrate that multi-factorial DOE is a powerful tool for investigating the influence of variables in the RLB development. DOE allows a reduction in the number of experiments with a complete exploration of the experimental domain to be studied. Furthermore, false negative results can be significantly reduced when applying this approach. Moreover, tissue bath experiments can give overall physiologically significant information, where a screening approach with different tissues under different operational conditions is considered the most efficient approach. Phenylephrine Figure 1: TB experiment pept159 Exp # Presoaking (150 µl) Prerinsing (2 ml) Washing (4 × 2 ml) % Adsorbed on filter % Specific binding % Non-specific binding Non-specific / total binding ratio 1-No additive 70.70.2868.00.996 2BSANo additive 68.21.3958.70.977 3-BSANo additive52.8-2.3861.21.040 4-No additiveBSA68.4-1.8661.31.031 5BSANo additiveBSA66.40.0457.00.999 6-BSA 69.6-0.5558.01.010 7PEINo additive 0.62.431.40.368 8-PEINo additive0.62.971.10.263 9PEINo additiveBSA0.53.911.00.197 10-PEIBSA0.52.211.30.373 11PEIBSANo additive0.69.131.60.147 12BSAPEINo additive0.85.750.90.135 13BSA No additive64.5-1.9058.41.034 14PEI No additive0.42.751.00.271 Exp_SettingMean result (g) 1. PE control contraction mean0.8501 2. Ach control relaxation mean0.5789 3. PE contraction mean after administration pept1590.8312 4. ACH relaxation mean after administration pept1590.4182 The study of biomedical effects of peptide pharmaceuticals, not only lead drug structures but their derived fragments, label-modified analogues, metabolites, related synthesis and degradation impurities for qualification evaluation as well, are studied using receptor-ligand binding (RLB) and tissue-bath (TB) experiments. The importance of such a biomedical, functional quality, screening approach is substantiated by the wide variety of physiological effects already seen with known peptides. However, errors of the type I (false positives or α) and type II (false negatives or β) need to be minimized and controlled. Radio-ligand binding studies Homogenates (equivalent to 30 µg of protein) of tissue, e.g. rat lung, were incubated with different concentrations of 125 I-peptide, e.g. VIP, in a total volume of 500 µl. The reaction conditions are screened by a very efficient, high content PB-DoE [1]. After incubation, the reaction was terminated by rapid filtration through (overnight) presoaked and (just before use) prerinsed glass fiber filterplates on a cell harvester and washing four times with 2 ml of ice-cold buffer. The radioactivity of the filters (after being transferred into polypropylene tubes) was determined using a gamma counter. The specific binding of 125 I-peptide was determined experimentally from the difference between counts in the absence and presence of excess unlabeled peptide. Adsorption was investigated on 125 I-peptide samples containing no tissue homogenate.