De Spiegeleer, Bart

Lefebvre, Romain

Van Dorpe, Sylvia

Verbeken, Mathieu

Ghent University Academic Bibliography

INTRODUCTION Bioactive peptides as potential new drugs: from analytics to tissue bath functionality.M. Verbeken1, S. Van Dorpe1, R. Lefebvre2 and B. De Spiegeleer1*Ghent university: 1 DruQuaR, Harelbekestraat 72, B-9000 Ghent; 2 Departement of Pharmacology, De Pintelaan 185, B-9000 Ghent.*Corresponding author: bart.despiegeleer@UGent.be (Ref.: 2010-260c)RESULTS AND DISCUSSIONCONCLUSIONSDruQuaR[1] Vergote, V., et al., Quality specifications for peptide drugs: a regulatory-pharmaceutical approach. Journal of Peptide Science, 2009. 15(11): p. 697-710.REFERENCESTissue-bath experimentsIsolated smooth muscle strips, maintained in temperature controlled Krebs buffer, weremounted between two platina electrodes attached to the mounting hook in glass tissuebath containers and connected to a force transducer to record smooth musclecontractions (Fig. 1). Four smooth muscle preparations were used for the functionalscreening of candidate bioactive peptides (Table 1).Twenty-five candidate bioactive peptides were selected, based on a multi-criteria decision strategy, and screened in tissue organ bath experiments using fourisolated preparations. Three peptides induced significant alterations of smooth muscle contractions: SBO121 and SBO215/215_7 (gpI) and SBO 291(mVD).However, it was observed that SBO215_7 (>95% purity) did not influence smooth muscle contractions, contrary to “crude” SBO215_7 peptide quality.Therefore, analytical controls are crucial to establish the correct peptide structure responsible for the bioactivity and to control the peptide purity.Peptides show great pharmaceutical potential as active drugs and diagnostics in several clinical areas such as endocrinology, obstetrics or oncology [1]. Thesearch towards novel lead-peptides with a biological function has thus attracted renewed interest. Within a collaborative, multidisciplinary approach, severalnew peptides were identified with the potential of being new drugs or serving as a basis towards new drugs. Twenty-five peptides, selection based on a multi-criteria decision strategy, were tested in tissue baths using different smooth muscle preparations. A positive bioactivity was confirmed if a significant alterationof the smooth muscle contractility was induced by the addition of the investigated peptides. Analytical controls to establish the correct peptide structureresponsible for the bioactivity have been performed as well, stressing the importance of purity control.Analytical control of bioactive peptidesThe purity of peptides used for functionalitytesting ranged from crude to 98%. Stockand tissue bath samples of bioactivepeptides were analyzed using HPLC UV-MS to identify the correct peptide structureresponsible for the bioactivity. A C18reversed phase column with gradientprogram (acetonitril/water mixture) and UVdetection was used for quantification, whileHPLC-ESI/iontrap MSn was used foridentification purposes [1].EXPERIMENTAL Table 1: Isolated muscle preparations; experimental conditions.Isolated preparations Guinea pig ileum Guinea pig trachea Mouse vas deferens Rat aortic ringPre-stretch (g) 0.7 2.0 0.2 2.0Krebs buffer Mg2+ containing Mg2+ containing Mg2+ free Mg2+ containingTKrebs buffer (°C) 36 36 31 36Electrical stimulation Yes No Yes NoFig.1: Tissue bath model with isolated smooth muscle strip.Twenty-five peptides were screened in four isolated smooth muscle preparations, three peptides were able to induce significant alterations of smooth musclecontractions: SBO121 and SBO215/215_7 (both in guinea pig ileum, gpI) and SBO291 (in mouse vas deferens, mVD) (Fig. 2).SBO215_7 (>95% purity)SBO121SBO291Fig. 3: SBO121 inhibition of EFS induced contractions.1g 1minAddition of SBO121 inhibited electrical induced smoothmuscle contractions (Fig. 2-3, relative to EFS inducedcontractions before), while SBO215 induced baselinecontractions without significant alterations of the magnitudeof the EFS induced contractions in gpI. However, no baselinecontractions were observed with pure SBO215_7 peptide.SBO291 exerts similar actions as SBO215, but on mVD.7080901001101 5 10% EFS induced contractionsTime (Min)No peptideSBO121Fig. 2: Representative tracings showing the effect of SBO121 and SBO215/215_7 in gpI and SBO291 in mVD (left), and the apposite MS spectra (right). The repetitive contractions were induced by electrical field stimulation (EFS), the non-stimulated contractions by acetylcholine (Ach) and phenylephrine (PE). PEAchSBO215/215_7 (crude)SBO121MW: 1070.31SBO215 (crude)MW: 1378.53SBO215_7 (crude)MW: 1378.53SBO215_7 (>95% purity)MW: 1378.53Drug Quality & Registration (DruQuaR) format Man06.001.006-v01AchAch

Bioactive peptides as potential new drugs: from analytics to tissue bath functionality

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Bioactive peptides as potential new drugs: from analytics to tissue bath functionality

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