Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-02-29
2002-04-02
Russel, Jeffrey E. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C436S087000, C436S501000, C514S018700, C530S329000, C530S330000, C530S331000
Reexamination Certificate
active
06365572
ABSTRACT:
This application claims the benefit of foreign priority to European Application No. 99103869.6, filed on Mar. 1, 1999. This European priority document is incorporated by reference herein.
Objects of the present invention are low molecular weight peptide derivatives which are able to act as inhibitors of the interaction between laminin and nidogen (laminin
idogen interaction), a process for their preparation, pharmaceutical compositions prepared therefrom and their use for preparing pharmaceuticals and for identifying inhibitors of the laminin
idogen interaction.
The association of laminin (an 800 kDa glycoprotein) and nidogen (a 160 kDa glycoprotein) is regarded as a crucial biomolecular mechanism in the synthesis and stabilization of basement membranes (Mayer, U. and Timpl, R. (1994) in: Extracellular Matrix Assembly and Structure (P. D. Yurchenco, D. Birk and R. P. Mecham, Ed.) S. 389-416, Academic Press, Orlando, Fla.). The ability of nidogen to form ternary complexes with all main constituents of the basement membrane such as, for example, &ggr;1-containing laminin isoforms (for nomenclature see: Burgeson, R. E.; Chiquet, M.; Deutzmann, R.; Ekblom, P.; Engel, J.; Kleinmann, H.; Martin, G. R.; Meneguzzi, G.; Paulsson M.; Sanes, J.; Timpl, R.; Tryggvasson, K.; Yamada, Y.; Yurchenco, P. D. (1994) Matrix Biology 14; 209-211), collagen IV, perlecan and fibulin, and the association structures of each of them, means that it assumes the function of a linker which connects together, spatially organizes and stabilizes the independent macrostructures (Fox, J. W.; Mayer, U.; Nischt, R.; Aumailley, M.; Reinhardt, D.; Wiedemann, H.; Mann, K.; Timpl, R.; Krieg, T., Engel, J.; and Chu, M.-L. (1991) EMBO J. 10, 3137-3146).
Basement membranes are highly specialized extracellular structures which are attributed with important functions in the control of cell and tissue functions, tissue architecture, tissue interactions, cell growth, cell transformation, cell migration and in tissue-specific gene expression (Adams, J. C. and Waft, F. M. (1993) Development 117, 1183-1198).
Experiments with polyclonal antilaminin antibodies have provided clear evidence of the central function of the laminin
idogen interaction in the synthesis of a functional basement membrane. The described antibodies were obtained by immunizing rabbits with laminin P1 or with the recombinantly produced nidogen-binding domain of laminin (&ggr;1 III 3-5). The antibodies concentrated by affinity chromatography on laminin P1 or laminin &ggr;1 III 3-5 matrices showed complete inhibition of the laminin
idogen association in inhibition assays. However, this is based on steric blockade of the access of nidogen to laminin by the antibodies, whose binding regions are located in the vicinity of the nidogen-binding sequences of laminin (Mayer, U.; Nischt, R.; Pöschl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885).
In embryonic organ cultures, the described antibodies inhibited both the development of renal tubules, the formation of pulmonary alveoli and the morphogenesis of the embryonic salivary gland. These three models are representative of ontogenesis programs which depend on unimpeded synthesis of new basement membrane (Ekblom, P.; Ekblom, M.; Fecker, L.; Klein, G.; Zhang, H.-Y.; Kadoya, Y.; Chu, M.-L.; Mayer, U.; Timpl, R. (1994) Development 120; 2003-2014).
Antibodies directed against the laminin &ggr;1 chain sequence region which is essential for nidogen binding are likewise able to inhibit the laminin
idogen association. The inhibition is, however, competitive, in contrast to the antilaminin antibodies described above, because they compete directly with the nidogen for the binding site on laminin (WO 98/31709).
A monoclonal antibody of the IgM subclass (antilaminin P1 A6/2/4-DSM ACC2327; see WO 98/31709) inhibits the laminin
idogen interaction in vitro with an IC50 of 30 nM. Like the polyclonal antilaminin antibody preparation described above, it prevents the morphogenesis of the embryonic salivary gland in organ culture. This underlines the specificity of the laminin
idogen interaction, and the importance of the LE-4 module and of the identified sequence region in the laminin &ggr;1 III 4 domain in this interaction.
The nidogen binding domain of laminin has been unambiguously identified and characterized in terms of its location, sequence and its spatial structure (X-ray crystal structure and NMR structure) (Gerl, M.; Mann, K.; Aumailley, M.; Timpl, R. (1991) Eur. J. Biochem. 202; 167-174. Mayer, U.; Nischt, R.; Pöschl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885. Baumgartner, R.; Czisch, M.; Mayer, U.; Pöschl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668. Stetefeld, J.; Mayer, U.; Timpl, R.; Huber, R. (1996) J. Mol. Biol. 257; 644-657). It is located in an “LE module” (laminin type epidermal growth factor-like) of the short arm of the &ggr;1 chain of laminin, in the domain &ggr;1 III 4. “LE modules” are structural motifs of 50-60 amino acids which have a complex folding pattern, analogous to EGF, with 4 disulfide bridges (Bairoch, A.; (1995) Nomenclature of extracellular domains. The SWISS-PROT Protein sequence data bank. release 310. Engel, J. (1989) FEBS Letters 251; 1-7).
High-affinity binding of nidogen to the complementary laminin domain has been detected for laminin P1 from the EHS tumor of mice, laminin 2 and laminin 4 from human placenta and laminin from drosophila. The cause of this species-overlapping binding specificity is the extremely large identity of sequences present in the &ggr;1 III 4 domain for the species investigated. It is 97% between human and mouse, 61% between mouse and drosophila and, astonishingly, 51% between mouse and Caenorhabditis elegans when the whole domain is taken into account (Pikkarinen, T.; Kallunki, T.; Tryggvasson, K. (1987) J. Biol. Chem. 263; 6751-6758. Chi, H.-C.; Hui, C.-F. (1989) J. Biol. Chem. 264; 1543-1550. Wilson, R. et al.(1994) Nature 368: 32-38. Pöschl, E.; Mayer, U.; Stetefeld, J.; Baumgartner, R.; Holak, T. A.; Huber, R.; Timpl, R. (1996) EMBO J. 15: 5154-5159).
Besides the dependency of nidogen binding on an intact three-dimensional structure, unambiguous sequence regions located in the S—S stabilized loops a and c of the domain &ggr;1 III 4 have been identified. Five essential amino acids have been identified, four located inside a section of 7 amino acids in loop a, and a tyrosine side-chain in loop c (Mann, K.; Deutzmann, R.; Timpl, R. (1988) Eur. J. Biochem. 178; 71-80).
Synthetic peptides which can be derived from the appropriate regions of the &ggr;1 III 4 domain and are able to inhibit completely the laminin
idogen binding in specific binding assays have been disclosed by J. W. Fox and R. Timpl (U.S. Pat. No. 5,493,008).
The high-affinity binding to the laminin binding site of nidogen is thought to require an interaction with a tyrosine or histidine from a loop (loop c) adjacent to the actual binding sequence. This aromatic interaction was postulated as a precondition for inhibition in the IC50 range <500 nM on the basis of the 3D structure of the laminin &ggr;1 III 3-5 and as a result of the structure/function relations described in the U.S. Pat. No. 5,493,008. The question of whether loop c interacts directly with the nidogen, or whether it makes a contribution to stabilizing the suitable spatial structure of the NIDPNAV (SEQ ID NO:1) sequence region remained unclarified, however (Pöschl, E.; Fox, J. W.; Block, D.; Mayer, U.; Timpl, R, (1994) EMBO J. 13; 3741-3747. Baumgartner, R.; Czisch, M.; Mayer, U.; Pöschl, E.; Huber, R.; Timpl, R.; Holak, T. A. (1996) J. Mol. Biol. 257; 658-668. Stetefeld, J.; Mayer, U.; Timpl, R.; Huber, R. (1996) J. Mol. Biol. 257; 644-657).
The laminin
idogen interaction is influenced by a strong conformational component (Mayer, U.; Nischt, R.; Pöschl, E.; Mann, K.; Fukuda, K.; Gerl, M.; Yamada, Y.; Timpl, R. (1993) EMBO J. 12; 1879-1885. Mann, K.; Deutzmann, R.; Timpl, R. (1988) Eur. J. Biochem. 178; 71-80). The s
Binnie Robert A.
Flynn Gary A.
Gerl Martin
Stankova Magda
Stilz Hans Ulrich
Aventis Pharma Deutschland GmbH
Finnegan Henderson Farabow Garrett & Dunner LLP
Russel Jeffrey E.
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