Transcript DruQuaR Analytical characterization of the BBB functionality of the opioid-peptides EM-1, EM-2 and CTAP. S.

DruQuaR
Analytical characterization of the BBB functionality of
the opioid-peptides EM-1, EM-2 and CTAP.
S. Van Dorpe1, N. Pintelon1, A. Adriaens2, I. Polis2, K. Peremans2, W. Pan3, C. Burvenich2, B. De Spiegeleer1*
1Drug
Quality & Registration (DruQuaR) group, Faculty Pharmaceutical Sciences, and 2Faculty of Veterinary Sciences, Ghent University, Belgium, 3Blood–
Brain Barrier Group, Pennington Biomedical Research Center, USA.*Corresponding author: [email protected] (O.Ref.: 2009 – 222c)
INTRODUCTION
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].
EXPERIMENTAL
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%
HN
O
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 C18 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.
O
O
O
H2N
HO
O
N
O
NH2
N
H
O
O
O
S
H2N
H3C
EM-2
O
CH3
CH3
HN
O
NH
C
H2
NH
CH3
EM-1
O
S
OH
H2N
O
O
HN
OH
HN
OH
N
H
NH2
O
O
H
N
H
N
N
H
N
NH
NH2
HN
H2N
N
NH2
OH
CTAP
BBB transport
For the influx experiments, the 125I-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 125I-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.
RESULTS AND DISCUSSION
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).
Table 2: In-vitro metabolic stability half-lives
Table 1: Impurity profile (QC)
EM-1
97.64
6
2.36
0.89
Purity peptide (%)
Number impurities
Sum impurties(%)
Largest impurity (%)
30
95.30
A tissue/A
serum (µl/g) 25
20
15
EM-2
97.12
4
2.88
1.59
96.03
CTAP
99.44
2
0.56
0.32
97.11
A capillaries/ A serum (µl/g)
97.02
A parenchyma/ A serum (µl/g)
10
5
0
2.98
3.97
4.70
2.89
3.900
4.600
8.200
14.717 Time (min)
Figure 1: Distribution between brain parenchyma and
capillaries in the EM-2 brain influx experiment
T ½ (min)
Liver
Brain
Kidney
Plasma
5.25
5.20
5.15
5.10
5.05
5.00
4.95
4.90
4.85
4.80
EM-1
13
205
8
7
EM-2
18
88
14
4
CTAP
225
119
101
60
Ln activity
(Brain)
y = -0.0496x + 5.3403
R² = 0.9739
0
2
4
6
8
10
Time (min)
12
Figure 2: The efflux of EM-2 from brain.
CONCLUSIONS
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 Kin of 0.5
µl/g.min and with 96.36 ±0.86 % of the total brain activity in the brain parenchyma.
REFERENCES
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.