Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-12-20
2002-01-01
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S018700, C530S326000, C530S327000, C530S328000, C530S330000
Reexamination Certificate
active
06335320
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
PEPTIDIC PRODUCT, PROCESS and COMPOSITION The present invention relates to a peptidic product comprising an active tetrapeptide Acetyl-glutamyl-glutamyl-glutamyl-aspartyl (SEQ ID No. 5) associated with a vector which allows the introduction of said tetrapeptide into a cell.
The present invention is also related to pharmaceutical compositions comprising said peptidic product and to its use for the prevention and/or the treatment of wound contractions, hypertrophic scars, fibromatosis, in particular the so-called Dupuytren disease and fibrotic conditions (in particular lung fibrosis).
2. Description of the Related Art
Skalli et al. (J. of Cell Biology Vol. 103 No. 6, pp. 2787-2796 (1986)) describe that an antibody (anti-alpha SM-1) recognising exclusively the alpha smooth muscle (&agr;-SM) actin could be selected and characterised after immunisation of BALB/c mice with the amino terminal synthetic decapeptide of &agr;-SM actin coupled to a carrier molecule.
It is also known that the &agr;-SM actin is transiently expressed in myofibroblast during experimental wound healing (Darby et al., Laboratory Investigation Vol. 63, 21-29 (1990)). Chaponnier et al. (J. of Cell Biology Vol. 130 No. 4, 887-895 (1995)) describes the blocking effects of an amino terminal decapeptide of a-SM actin (AcEEEDSTALVC) (SEQ ID No: 6) on the binding of specific monoclonal antibody anti-alpha SM-1. This document indicates that the portion AcEEED (SEQ ID No: 5) is the epitope for the anti-alpha SM-1.
The anti-alpha SM-1 is the first monoclonal antibody described to be specific for a single actin isoform and is a reliable tool for the study of vascular SM cells and myofibroblast phenotypic modulation in physiological and pathological processes.
This document shows that said antibody (especially its Fab fragment) increases in vitro &agr;-SM actin polymerisation. Said document also suggests that the tetrapeptide AcEEED (SEQ ID No: 5) may be used for the study of the functions of the &agr;-SM actin in cells such as SM cells, fibroblasts, myofibroblasts or myoepithelial cells in physiological and pathological situation during which &agr;-SM actin expression is modulated. Said document also suggests that the tetrapeptide AcEEED (SEQ ID No: 6) inhibits the incorporation of &agr;-SM actin into stress fibers in the culture of SM cells when using microinjection technology.
It is also shown that an active turnover of &agr;-SM actin into stress fibers exists and that AcEEED (SEQ ID No: 5) traps a protein interacting with the amino-terminal sequence of &agr;-SM actin and activating polymerization (Chaponnier, C. et al., Experientia, 1995, 51, A62).
Järlebark, L. et al. (Biochemical and Biophysical Research Communications, 1996, 229 (2), 363-369), disclose that some peptidyl derivatives of adenosine 5′-carboxylic acid have inhibitory effects in certain P2 purinoceptor-carrying biological systems (gliome and smooth muscle cell lines and isolated smooth muscle tissue preparations).
The present invention aims to provide a peptidic product comprising the specific tetrapeptide AcEEED (SEQ ID No: 5) which allows its introduction into a cell, in order to interfere, in vitro and in vivo, with &agr;-SM-actin organisation in stress fibers.
Another aim of the present invention is to provide a pharmaceutical composition comprising said peptidic product in order to treat and/or to prevent various diseases related to &agr;-SM actin expression such as wound contraction, hypertrophic scars, fibromatosis, in particular Dupuytren diseases, and fibrotic condition (in particular lung fibrosis).
SUMMARY OF THE INVENTION
The present invention is related to a peptidic product comprising the active tetrapeptide Acetyl-glutamyl-glutamyl-glutamyl-aspartyl (SEQ ID No: 5) (identified hereafter by the formulation AcEEED) associated with a chemical entity which is able to introduce said tetrapeptide into a cell.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
According to the invention, in the active tetrapeptide Ac means Acetyl, E means glutamic acid residue, D means aspartic acid residue.
According to the invention, by a chemical entity we understand a vector.
According to the invention, by a cell we understand in vivo cells, such as cells composing the organs or tissues of living animals or humans, or microorganisms found in living animals or humans; or in vitro cells,such as cultured animal cells, human cells or microorganisms. Preferably it means human cells, and more preferably fibroblastic or smooth muscle cells.
According to a first embodiment of the present invention, the chemical entity can be a liposome, a lipidic vesicle made of lipids and/or fatty acids or a cationic vesicle such as the one described by Gao X. and Huang L. (Cationic Liposomes - Mediated Gene Transfer, Gene Therapy, pp. 710-722 (1995)) and/or a mixture thereof.
According to a second embodiment of the present invention, the chemical entity is a polypeptide having preferably at least about 8 amino acids, preferably between 8 and 40, more preferably between 10 and 30 amino acids.
According to another embodiment of the present invention, said polypeptide is the tat mediated protein, having advantageously a capacity of binding with membranous receptor(s) of specific cell(s).
The preferred linking of a chemical entity like the tat mediated protein to the tetrapeptide according to the invention for the delivery of heterologous peptides into a cell, is described in the International Patent Publication WO 94/04686 incorporated herein by reference. The best results have been obtained with the tat mediated protein of formula tat37-72, tat37-58, tat38-58GGC, tatCGG47-58, tat47-58GGC and tat&Dgr;C.
According to a preferred embodiment of the present invention, the chemical entity is a homeobox peptide, preferably the pAntp peptide described in the International Patent Publication WO 97/12912, also incorporated herein by reference. The best results have been obtained with the peptide pAntp[43-58], and the peptide pAntp[43-58]-Pro
50
. The peptide pAntp[43-58] means Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys, (SEQ ID No: 7) wherein, for this description, the first amino acid Arg is named amino acid in position 43 and the last amino acid Lys is named amino acid in position 58. The peptide pAntp[43-58]-Pro
50
(SEQ ID No: 7) represents the same sequence of pAntp[43-58]wherein Gln has been substituted by Pro in position 50.
The present invention relates also to the preparation method of such product, using known methods by the person skilled in the art such as preparation methods of lipidic vesicles (Martin F. G.,
Pharmaceutical Manufacturing of Liposomes
, Praveen Tyle Ed., Specialised Drug Delivery Systems, Marcel Decker, N.Y. (1993)), automatic peptides synthesis, cross-linking between olecules.
Possibly, the tetrapeptide is linked through a spacer, preferably a peptidic spacer, to the chemical entity. Said spacer is preferably an amino acid or a peptide comprising no more than 5 amino acids. Preferably, said amino acid used as a spacer is a Lysyl residue (“K”) to which the AcEEED (SEQ ID No: 7) tetrapeptide is linked through the &agr;-amino group, or through the &egr;-amino group, or a Cystinyl residue (meaning two Cysteinyl residues (“C”) linked by their &bgr; sulfide functions forming a disulfide link represented by “C-[S-S]-C”) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the &agr;-amino group, or &bgr;-Alanyl-&bgr;-Alanyl dipeptide (“&bgr;-A-&bgr;-A”) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the &egr;-amino group, or through a Lysyl residue (“K”) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the &egr;-amino group and a BiotynylGG residue (meaning a biotinyl functionnal group linked through its carboxylic function to two successive glycyl residues) to which the AcEEED (SEQ ID No: 5) tetrapeptide is linked through the &agr;-amino group.
The best results have been obtained with the AcEE
Gabbiani Giulio
Scarso Alain
Carlson Karen Cochrane
UCB S.A.
Wenderoth , Lind & Ponack, L.L.P.
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