1. INTRODUCTION Analytical characterization of the BBB functionality of the opioid-peptides EM-1, EM-2 and CTAP. S. Van Dorpe 1, N. Pintelon 1, A. Adriaens 2, I. Polis 2, K. Peremans 2, W. Pan 3, C. Burvenich 2, B. De Spiegeleer 1 * 1 Drug Quality & Registration (DruQuaR) group, Faculty Pharmaceutical Sciences, and 2 Faculty of Veterinary Sciences, Ghent University, Belgium, 3 Blood– Brain Barrier Group, Pennington Biomedical Research Center, USA.*Corresponding author: bart.despiegeleer@ugent.be (O.Ref.: 2009 – 222c)bart.despiegeleer@ugent.be EXPERIMENTAL Peptides constitute a new group of promising drugs, with a diverse range of pharmacological activities. One of their most promising targets is the Central Nervous System (CNS), where the Blood-Brain Barrier (BBB) constitutes an important flux-regulating compartment. The development of selective and potent opioid peptide drugs for the major opioid receptor types (µ, δ and κ OR) continues to be a key objective in pharmacological research. We present here the BBB influx and efflux data of the 3 µ opioid peptides EM-1 (Endomorphin-1), EM-2 (Endomorphin-2) and CTAP (connective tissue activating peptide), as only limited literature data are available, indicating that both endomorphins are transported out of the brain [1], while CTAP can enter the brain from plasma [2]. RESULTS AND DISCUSSION CONCLUSIONS DruQuaR 1.Kastin, A.J., et al., Saturable brain-to-blood transport of endomorphins. Experimental Brain Research, 2001. 139(1): p. 70-75. 2. Abbruscato, T.J., et al., Blood-brain barrier permeability and bioavailability of a highly potent and mu-selective opioid receptor antagonist, CTAP: Comparison with morphine. Journal of Pharmacology and Experimental Therapeutics, 1997. 280(1): p. 402-409. REFERENCES Quality control (QC) The 3 peptides were analyzed using HPLC with UV-PDA and fluorescence detection. Impurities and peak purity of the peptide were quantified using a reporting threshold of 0.10% In-vitro metabolic stability Peptides are incubated at 37°C in mice brain, plasma, kidney and liver tissue homogenates using a mixing block heater. Aliquots are taken at suitable time points, and analyzed (after acidification, heating at 95°C and centrifugation) using HPLC. Results are expressed as half-life times, calculated from the regression curves. Typical HPLC conditions Everest C 18 238EV54 (250 × 4.6 mm, 300 Å, 5 µm) column (Grace Vydac) at 30°C, with (A) 0.1% formic acid in water, and (B) 0.1% formic acid in acetonitrile. A gradient program was used and the flow rate was set at 1.0 ml/min. BBB transport For the influx experiments, the 125 I-peptide was injected in the vena jugularis, blood and brain were collected at suitable time points. After brain collection, the brain is separated in capillaries and parenchyma. Both fractions are measured in the γ-counter. The distribution of the peptide in these fractions is evaluated. The efflux experiment is carried out by injection of the 125 I-peptide in the lateral ventricle of the brain. Residual radioactivity in brain is measured and from the regression curve, the disappearance half-life is calculated. CTAPEM-1 EM-2 Typical results are given here. For the BBB transport, only the results for EM-2 are given here, as the other peptides were successfully analyzed using the same methodology (data not given in this presentation). EM-1EM-2CTAP Purity peptide (%)97.6497.1299.44 Number impurities642 Sum impurties(%)2.362.880.56 Largest impurity (%)0.891.590.32 Table 1: Impurity profile (QC) Table 2: In-vitro metabolic stability half-lives Figure 1: Distribution between brain parenchyma and capillaries in the EM-2 brain influx experiment Figure 2: The efflux of EM-2 from brain. QC revealed that LC-peptide purities of more than 95% were obtained for the 3 opioid peptides. The in-vitro metabolic stability showed a relatively long half-life in brain, but very short half-lives in plasma. For EM-2, BBB transport analysis revealed a clear brain efflux with a disappearance half-life of 14.0 min, which is comparable to the literature data (12.5 min). The influx distribution experiment showed that EM-2 could cross the BBB, showing a slow influx of K in of 0.5 µl/g.min and with 96.36 ±0.86 % of the total brain activity in the brain parenchyma.