Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2001-06-05
2004-09-14
Kemmerer, Elizabeth (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S402000, C435S181000, C424S009350, C424S198100, C536S024200
Reexamination Certificate
active
06790942
ABSTRACT:
The present invention concerns a conjugate derivative, in particular a polymer conjugate derivative, of a biological active molecule such as a protein, a peptide, a polypeptide. The present invention concerns also a method of chemical identification of said conjugate derivative.
Although a great number of new peptides and proteins with potentially useful pharmacological activities are now synthesised thanks to genetic engineering, their therapeutic potential is often drastically limited by negative properties that are intrinsic to their chemical structure. For example, polypeptides, once administered, may often be easily cleaved by endo- or exo-proteases, be rapidly excreted by renal filtration, and may often provoke immunological reactions, in spite of possessing human sequences. Furthermore, the size and nature of the macromolecule can dictate a targeting inside the body that is not always the one desired for their therapeutic action.
Conjugation at the protein surface of biocompatible, non toxic, non immunogenic, water soluble polymers, has been found to be a procedure that may reduce these problems and allows for, in few cases, useful therapeutic applications. A polymer often used for such conjugation is poly(ethylene-glycol) (PEG) due in part to its very favourable biological properties, (see “Poly(ethylene glycol) chemistry and biological application” M. J. Harris Ed. Plenum Press, (1994), (incorporated herein by reference) as well as dextran, albumin, poly(N-vinylpyrrolidone), and poly(N-acryloylmorpholine, among others. “New synthetic polymers for enzyme and liposome modification Poly(ethylene glycol), p.182, ACS Symp Series 680, 1997, also discloses some of the later polymers.
Proteins and peptides have also being conjugated to antibodies and other high affinity ligands in order to target to a specific site inside the body.
Various amino acid residues in proteins, have been found suitable for polymer conjugation, including, for example, the amino groups of lysine and the alpha amino terminal groups. The thiol group of cysteine, guanidino group of arginine and the terminal carboxylic group, as well as the carboxylic group of aspartic or glutamic acids have been considered. Reported studies and methodologies for modification of protein amino groups include those of Davies and Abuchowski (Abuchowski et al. 1977a and 1977b), those of Benchamp et al., 1983, Veronese et al., 1985, Zalipsky et al., 1983, Delgrado et al., 1990. Protein SH group modification is described in the work of Morpurgo et al., 1996, while guanidino modification is described by Pande et al. Polysaccarides residues, when present in proteins, have been exploited more rarely, and principally, for binding. However, among these residues the conjugation to the amino groups is by far the most important one.
In spite of the current state of knowledge related to polymer conjugation in prodrugs preparations, it remains true that since in polypeptides and proteins all of these groups are present in a number that may be very high, (and also the groups themselves present different exposure at the macromolecule surface, or possess different nucleophylicity), an heterogeneous and complex pattern of products is generally obtained. Up to the present time it has remained very difficult to state the position of polymer conjugation in the primary sequence of the protein with much success.
This difficulty is compounded by the fact that it is extremely difficult, if not impossible, in the majority of the cases, to fractionate the different conjugates from the conjugation mixture, even when the most sophisticated methods now available are employed. The hindrance of the protein molecule may mask the charges at the protein surface, thus reducing the binding to ion exchange resins, as well as the selectivity of binding to affinity ligands, and reduces the potentials of various methods, including gel filtration chromatography, in accomplishing this task.
Various approaches have been reported in the literature on the identification of the conjugation site, especially when an high molecular polymer is the conjugation species.
One approach is based on the comparison of the finger printings, obtained by HPLC, in the case of the PEG-conjugated product with that obtained in the case of the native unconjugated polypeptide. Though some information on binding sites was obtained on the basis of the identification of the missing PEG peptides in the tryptic digest of the conjugate, this information relied on the assumption that trypsin does not act where PEG is bound or in its close surroundings, and that there is no resultant release of the corresponding peptides.
Though this approach was found useful in the study of the peptide growth hormone (Ross Clark et al. J. Biol. Chem. 271, 21969-21977, 1996), it cannot be always considered conclusive because it is based on indirect evidence, and relies on a method that can give information on the site of binding of PEG only in the cases of relatively short polypeptides that give rise to simple patterns of proteolitic digestion.
Another approach was based on the protein “PEGilation” with a polymer that carries a succinic acid arm between PEG and protein. Succinic acid was linked to PEG by a labile ester function and to protein by a stable amide. The PEG chains were removed from the protein by mild basic hydrolysis of the labile ester bond that links PEG to succinic acid, while leaving succinic acid moiety, as a reporter group, linked to the residues where PEG was bound, with labelled peptides then recognised by mass spectrometry (M. M. Vestling et al. Drug Metab. Disp. 21, 911-917). There are at least two severe limitations of this method: the first resides in the linking chemistry of PEG to protein, whereby the chemistry is not generally considered appropriate or convenient for products of human use, because the ester linkage between PEG and succinic acid may be easily cleaved in physiologic circulation, giving rise to new products; the second limitation resides in the fact that the identification of the succinic acid labelled peptide, in the peptide map, may be carried out by mass spectrometry only. Standard amino acid analysis, which is the usual method for sequence studies, cannot be applied in such a case since, during the strong acid hydrolysis that has to be done before the amino acid identification (AAA), the succinic acid moiety is removed from the amino acid where it is bound. For this reason one cannot identify by the most accurate AAA procedure the peptides where succinic acid was bound. Furthermore, the weakness of the same bond hampers the use of other important procedures of sequence analysis determinations.
U.S. Pat. No. 5,286,637 (Veronese et al.), issued Feb. 15, 1994, also describes an approach and a method using M-PEG. The method of their invention is based on the linkage of art amino acid or peptide spacer arm of various structures and properties to the hydroxyl function of monoalkoxypolyethylene glycol through a carbonate linkage which involves the NH
2
group of the amino acid or peptide. This reaction is followed by the activation of the COOH function of the amino acid or peptide spacer arm as succinimidyl ester which, thus, becomes reactive towards the amino group of the biologically active peptide, protein or drug. Use of this method has the disadvantage, common to all methods of conjugation of PEG chains, to hamper the use of most, if not all, of the fractionation procedures of the conjugate product, as well as a suitable fractionation of the PEGylated digestion mixture.
For this reason, the exact identification of the site of polymer binding into the protein remains a still problem, when preparing the corresponding prodrug, although it is an essential prerequisite for a rational drawing of correlation between structure and activity of the prodrug conjugates. The knowledge of this correlation may be in fact one of the most useful guides to design new conjugates with more convenient pharmacokinetic, pharmacological and therapeutic properties.
The exact identification of t
Caliceti Paolo
Orsolini Piero
Schiavon Oddone
Veronese Francesco
Debio Recherche Pharmaceutique
Kemmerer Elizabeth
Nixon & Vanderhye P.C.
Wegert Sandra
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