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Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis

This application is Continuation of U.S. Utility application Ser. No. 11/007,772 filed Dec. 7, 2004, which is a Continuation of U.S. Utility application Ser. No. 09/341,590, filed Jul. 12, 1999, which claims priority under 35 U.S.C. § 371 from international application PCT/DK99/00118, filed Mar. 9, 1999, which claims priority from Danish Patent Application No. 0317/98, filed Mar. 9, 1998.

There exist a large number of pharmacologically active peptides, e.g., naturally occurring in man or in animals, or synthetic analogues of such peptides. An illustrative example of such a peptide is the analgetically active peptide enkephalin that has given rise to a vast number of synthetic analogues. However, due to precisely their peptic nature, the routes of administration thereof have been rather limited. Thus, peptides are rapidly and very effectively degraded by enzymes, generally with half-lives in the range of minutes. Proteases and other proteolytic enzymes are ubiquitous, particularly in the gastro-intestinal tract, and therefore peptides are usually susceptible to degradation in multiple sites upon oral administration, and to some extent in the blood, the liver, the kidney, and the vascular endothelia. Furthermore, a given peptide is usually susceptible to degradation at more than one linkage within the backbone; each locus of hydrolysis is mediated by a certain protease. Even if such obstacles are overcome, for neuropeptides in particular, difficulties have been encountered in their transport across the blood-brain barrier.

There have been a number of attempts to protect peptides against premature degradation (reviewed in Prokai, 1997, Exp. Opin. Ther. Patent 7:233-245, Tamai et al., 1996, Adv. Drug Delivery Rev. 19:401-424 and Zhou et al., 1991, Int. J. Pharm. 75:97-115). One approach includes osmotically altering the blood-brain barrier by infusion of hypertonic solutions of mannitol, arabinose, lactamide, saline, urea, glycerol and radiographic contrast agents. However, there could be toxic side effects.

Another approach involves the use of protease inhibitors (reviewed in Zhou et al., 1991, Int. J. Pharm. 75:97-115). This approach has yielded mixed results.

A third approach has involved the use of absorption enhancers in peptide formulations (reviewed in Zhou et al., 1991, Int. J. Pharm. 75:97-115). Examples include fatty acids and bile salts. However, varying results have been obtained regarding efficacies, and the value of a particular enhancer is dependent on the route of administration used.

Another approach for enhancing the absorption of peptides involves chemically modifying the peptide by, for example, attaching a lipophilic moiety. It has also been found that attaching a pyroglutamyl residue at the N-terminal end can render a compound relatively resistant to hydrolysis. Tamai et al., 1996, Adv. Drug Delivery Rev. 19:401-404, discloses that E2078, a dynorphin analog, was chemically modified to make it more stable to enzyme degradation by adding an N-methyl group at the amino-terminus of Arg and replacing D-Leu with L-Leu and adding ethylamine at the carboxy-terminal.

A different approach involves the formation of chimeric peptides. This approach involves coupling the peptide that is not normally transported through the blood-brain barrier to peptide or protein “vectors” that undergo receptor-mediated or adsorptive-mediated transcytosis.

WO 98/22577 discloses a method for increasing the resistance of a “core protein” to proteolytic degradation by linking or inserting a “stabilizing polypeptide” having the formula [(Glya)X(Glyb)Y(Glyc)Z]n. X, Y, and Z may be alanine, serine, valine, isoleucine, leucine, methionine, phenylalanine, proline, and threonine.

U.S. Pat. No. 5,545,719 discloses molecules comprising protein fragments homologous to an active region of protein fragments capable of stimulating nerve growth (neuronotrophic proteins such as epidermal growth factor, tubulin, nerve growth factor, laminin, fibronectin, NCAM and ependymin) no greater than 80 amino acids long connected to a secondary molecule which can be a second protein fragment derived from the original protein, from another protein or from a non-proteinaceous moiety. This secondary molecule facilitates the transport of the peptide across the blood-brain barrier. It is stated in column 3, lines 3-7, “Upon entering the central nervous system, prodrug can remain intact or the chemical linkage between the carrier and the protein fragment may be hydrolyzed thereby separating the carrier from the fragment to release the nerve growth-stimulating fragment.” A preferred method for facilitating the coupling of the secondary molecule to the protein fragment is via one or more basic amino acids, preferably a pair of Lys residues, an Arg residue, or Arg-Lys.

Fawell et al., 1994, Proc. Natl. Acad. Sci. USA 91: 664-668 discloses chemically crosslinking various Tat peptide fragments to B-galactosidase, RNase A, and domain III of Pseudomonas exotoxin A. These included Tat-(37-72), Tat-(37-58) and Tat-(47-58). All of these peptides appeared to promote uptake of galactosidase, RNase A, and domain III into cells. It was stated that this is the basic region of Tat. Conjugates containing poly (L-lysine) or poly (L-arginine) were not taken up by the cells.

WO 97/24445 discloses fusion proteins of albumin and growth hormone or variants thereof. It is stated in the specification that variants of albumin should have the oncotic, ligand-binding, and non-immunogenic properties of full-length albumin and that variants of growth hormone should have its non-immunogenicity and ability to bind and activate the growth hormone receptor. Further, it is stated that the albumin variant will be at least 100 amino acids long and that the growth hormone variant should have growth hormone activity and will generally have at least 10 amino acids.

WO98/28427 discloses an Fc-OB fusion protein. Fc is an immunoglobulin fragment and OB is leptin. It has been found that such conjugates are more stable than OB alone. The Fc fragment is 378 amino acids in length. The Fc fragment can be conjugated directly or via a linker to OB or an OB fragment.

Another approach involves preparing peptide analogs with increased stability and/or activity by adding a peptide tail. Greene et al., J. Pharm. Exp. Therap. 277:1366-1375, discloses results of studies with various enkephalin analog prodrugs of [D-Pen2, D-Pen5] enkephalin (DPDPE) and [D-Pen2, L-Cys5] enkephalin (DPLCE)(SEQ ID NO: 1), specifically DPLCE-Arg-Pro-Ala (SEQ ID NO: 2), DPDPE-Phe (SEQ ID NO: 3), DPLCE-Phe (SEQ ID NO: 4), DPDPE-Arg-Gly (SEQ ID NO: 5), DPLCE-Arg-Gly (SEQ ID NO: 6), DPDPE-Phe-Ala-NH—C6H13 (SEQ ID NO: 7), DPDPE-Phe-Ala-CONH2 (SEQ ID NO: 7). The half-lives of most of the analogs, except for DPDPE-Arg-Gly are less than the parent compounds. It is stated on page 1372, column 2 that “the ideal CNS-targeted prodrug would have a long half-life in the serum and a short half-life in the brain.” U.S. Pat. No. 4,724,229 discloses vasopressin antagonists, which have a tripeptide side chain having three basic amino acids, such as arginine, lysine, or ornithine that have potent antagonistic activity. U.S. Pat. No. 4,542,124, discloses vasopressin antagonists, which have a dipeptide side chain having two amino acids, one of which has potent vasopressin antagonistic activity.

In the international patent application PCT/DK97/00376 (Bjarne Due Larsen and Arne Holm), prodrugs of pharmacologically active peptides are described, wherein the pharmacologically active peptide is coupled at its C terminus to a peptide pre-sequence via a linker, the linker typically being an α-hydroxy carboxylic acid. These special peptide derivatives were found to have a prolonged half-life in the presence of proteolytic enzymes such as carboxypeptidase A, leucine aminopeptidase, pepsin A and α-chymotrypsin. In addition, PCT/DK97/00376 discloses (as reference compounds) four different peptides equipped with a peptide pre-sequence but without a linker, namely DSIP-(Lys-Glu)3 (SEQ ID NO: 8), DSIP-(Glu)6 (SEQ ID NO: 9), Leu-enkephalin-(Glu)6 (SEQ ID NO: 10), and Leu-enkephalin-(Lys)6 (SEQ ID NO: 11).

It is evident that there is a need for a peptide conjugate which contains a pharmacologically active peptide and a stabilising protein that is relatively simple to synthesize, retains its activity even without removing the stabilising peptide, is stable in plasma or serum and is relatively resistant to enzyme degradation. Therefore, it is an object of the invention to provide a peptide conjugate comprising a pharmacologically active peptide and stabilising peptide that is relatively resistant to enzyme degradation.