Transcript DruQuaR Functional quality of new peptide drugs: receptor-binding and tissue-interactions M. Verbeken1, V.

DruQuaR
Functional quality of new peptide drugs:
receptor-binding and tissue-interactions
M. Verbeken1, V. Vergote1, C. Burvenich1, C. Van de Wiele1, A. Ronai2, K. Gyires2, W. Luyten3 and B. De Spiegeleer1*
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: [email protected] (Ref.: 2009-238f)
INTRODUCTION
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.
EXPERIMENTAL
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.
Phenylephrine
Radio-ligand binding studies
Homogenates (equivalent to 30 µg of protein) of tissue, e.g. rat lung, were incubated with different concentrations of 125I-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 125I-peptide was determined experimentally from the difference between counts in the absence and presence
of excess unlabeled peptide. Adsorption was investigated on 125I-peptide samples containing no tissue homogenate.
RESULTS AND DISCUSSION
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)
Exp_Setting
Mean result (g)
% Specific binding
% Non-specific
binding
Non-specific / total
binding ratio
70.7
0.28
68.0
0.996
No additive
68.2
1.39
58.7
0.977
BSA
No additive
52.8
-2.38
61.2
1.040
-
No additive
BSA
68.4
-1.86
61.3
1.031
5
BSA
No additive
BSA
66.4
0.04
57.0
0.999
6
-
BSA
BSA
69.6
-0.55
58.0
1.010
7
PEI
No additive
No additive
0.6
2.43
1.4
0.368
0,8
8
-
PEI
No additive
0.6
2.97
1.1
0.263
0,6
9
PEI
No additive
BSA
0.5
3.91
1.0
0.197
10
-
PEI
BSA
0.5
2.21
1.3
0.373
11
PEI
BSA
No additive
0.6
9.13
1.6
0.147
12
BSA
PEI
No additive
0.8
5.75
0.9
0.135
13
BSA
BSA
No additive
64.5
-1.90
58.4
1.034
14
PEI
PEI
No additive
0.4
2.75
1.0
0.271
1. PE control contraction mean
0.8501
2. Ach control relaxation mean
0.5789
3. PE contraction mean after administration pept159
4. ACH relaxation mean after administration pept159
0.8312
0.4182
Figure 1: TB experiment pept159
1,0
Contraction (g)
Table 3: Influence of filtration in RLB studies [1]
0,4
0,2
Exp
#
Presoaking
(150 µl)
Prerinsing
(2 ml)
Washing
(4 × 2 ml)
% Adsorbed on
filter
1
-
No additive
No additive
2
BSA
No additive
3
-
4
0,0
PE_C
Ach_C
PE_SBO Ach_SBO
CONCLUSIONS
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.
REFERENCES
[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).