Patent 6,790,942

Biologically active drug polymer derivatives, namely peptides or protein derivatives, are useful medicaments and are represented by the generic formula: RO--(CH₂ --CH₂ O)_n --(CO)--NH--X--(CO)--NH--Z wherein R represents a lower alkyl group, n is an integer between 25 and 250, X when combined with adjcacent NH and CO groups represents a dipeptide residue, and Z when combined with the adjacent group represents a biologically active peptide or protein.

Description of the Invention

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 NH₂ 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 the site of conjugation is also important for a more precise analytical characterisation of a biological active macromolecular product, particularly when the conjugate is intended for therapeutic use, and requires an accurate characterisation in order to meet health authority requirements.

The importance of finding a method for identifying the original location of the functional entity is demonstrated in the use of macromolecules in therapy: there, conjugates are necessary. In such conjugates, (which can use in therapeutical applications, for example, a PEG+protein combination), the PEG may be linked to many different sites on the macromolecule. It is important to have a method to discover at exactly which site or sites the PEG+protein linked to the macromolecule was bound. The present invention retains an attached amino acid reporter moiety, thereby helping identify and describe, through analysis, the conjugation product.

The aims of the present invention are to provide with biological active conjugate derivatives while overcoming the limitations reported in the aforementioned procedures for the polymer modifications of polypeptides and proteins.

To that effect, one of the objects of the present invention is a biological active conjugate derivative having the following general formula (I)

FE-L-M (1);

where:

M represents the corresponding radical of a biological active molecule selected from the group consisting of proteins, peptides and polypeptides;

FE represents a functionalizing entity; and

L represents a linking arm having a reporter moiety detectable by standard analytical methods, the linking arm being able to be cleaved by chemical or enzymatic in vitro treatment, the reporter moiety remaining attached to the biological active molecule once the linking arm has been cleaved.

According to the present invention, it has been found that specific spacer groups lead to the possibility to remove the functional entity FE from the conjugate with a diminished harshness to avoid destroying or otherwise denaturing the molecule, while retaining an attached amino acid reporter moiety to the molecule for identification.

The linking arm L is stable under physiological conditions but cleavable by specific and selective physical-chemical means, whereby a stable reporter moiety detectable by standard analytical methods remains attached to M and thereby its presence may optionally be identified by mass spectroscopy and may be cleaved by chemical or enzymatic treatment for evaluation by standard amino acid analysis.

An alternative identification procedure may include Maldi mass spectroscopy analysis of an uncleaved conjugate.

Preferably, in the biological active conjugate derivative according to the present invention, the functionalizing entity FE is selected from PEG, PVP, PacM, dextran, hormones, antibodies or antibody fragments. More preferably, the functionalizing entity FE is a polymer with a molecular weight in the range of 2 Kd to 50 Kd. The functionalizing entity FE can be either a linear polymer or a branched polymer.

Preferably, in the biological active conjugate derivative according to the present invention, the linking arm L comprises the following fragment Met-X in which X represents the reporter moiety and the reporter moiety is an amino acid. More preferably, the amino acid X is selected from Nle and beta Ala.

Also preferably, the linking arm L comprises either the dipeptide Gln-Gly or Asp-Pro.

Preferably, in the biological active conjugate derivative according to the present invention, the radical M corresponds to a biological active molecule and said molecule is a protein selected from insulin, lysozime, interferon, in particular interferon .alpha.-2b, erithropoietin, G-CSF, GH.

Another object of the present invention is a method for identifying, is on the above mentioned biological active drug conjugate derivative, sites of conjugation of the functionalizing entity FE along the biological active molecule M comprising a specific chemical or enzymatic in-vitro cleavage of the linking arm L, releasing, removing and separating FE by classical methods.

Another object of the present invention is an intermediate compound, for the preparation of the biological active conjugate of claim 1, having the following general formula (II)

FE-L (II);

where:

FE represents a functionalizing entity; and

L represents a lining arm having a reporter moiety detectable by standard analytical methods.

The reporter moiety is useful to identify the original location of the functional entity. The present inventions described in detail below, takes advantage of the use of a special PEG amino acid sequences, in which PEG may be released in such a way to leave a reporter amino acid bound to the macromolecule that is exploited for the identification of the site of PEG binding.

Without limitation and in the spirit of the invention, several functionalizing entities can be foreseen--for example, FE is typically a polymer when M is intended for therapeutic applications. While many polymers may be included in the present invention, preferentially, the polymer may be selected from the group consisting of PEG, PVP, PAcM, dextran and others. Even more preferred in PEG, PVP and PacM. Even more preferred is PEG for its very favourable biological properties. Even more preferred is MPEG.

As described above, FE, as described in the present invention, may be a polymer or functional entity consisting of polymers. Preferably, the FE/Polymer has a molecular weight greater than 2 kd; more preferably, the molecular weight is in the range of 2 to 50 kd. When FE is a polymer, FE can be a linear or branched polymer.

Other functionalizing entities can be included in the present invention, for example high affinity ligands for targeting to specific sites. FE, in this case, may be any of a number of high affinity ligands. Preferably, FE can be an hormone, antibody, or an antibody fragment, or a compound selected with high output technique to a recognition site. More preferably, FE can be selected from the group consisting of antibody(ies) or antibody fragment(s).

The linking arm L may be preferably devised to react with amino groups from the M moiety such as the alpha amino or the epsilon amino group of lysine. Preferably the linking arm L may be represented by an activated carboxilic group of amino acid. Most preferably, an unnatural amino acid, nor-leucine or beta-alanine is preferred for its easier identification after acid hydrolysis.

When the derivative compound has the following structure:

FE-Met-X-M,

X is a suitable reporter group such as an either unnatural or natural amino acid. Preferably the unnatural amino acid is nor-leucine (Nleu) or the amino acid beta-alanine(beta-Ala), which is absent among the protein constituents. Also of preference are other amino acids which are non common constituents of proteins.

X may be bound to the protein M by, for example, stable amide linkage, through procedures known to the man of ordinary skill in the art. The advantage to use of these types of amino acids as reporters resides in the fact that, not being natural constituents of the proteins, they are very convenient in analysis--their presence indicates and quantifies the previous presence of PEG.

In the case of one or more of these more preferred embodiments of the present invention, FE can be removed from the protein by selective CNBr treatment, leaving X still covalently bound to M by a stable amide bond.

When the conjugate has the following structure:

FE-Gln-Gly-M,

specific removal of FE can be preferentially obtained by hydrazine treatment, leaving the amino acid Gly bound to M.

When the conjugate has the following structure:

FE-Asp-Pro-M,

specific removal of FE can be obtained by mild acid treatment, leaving Pro still bound to M.

In the above specified embodiments of the present invention, the M bound reporter moieties are very stable to acid hydrolysis and therefore easily identified by the standard amino acid analysis following acid hydrolysis, as well as by mass spectrometry if the non hydrolysed peptide containing them is analysed by this technique. Preferably, M bound reporter moieties are selected from the group of Nle, betaAla, Gly or Pro. More preferably, M bound reporter moieties are selected from unnatural amino acids, Nle or beta Ala. Even more preferably, the reporter moiety is norleucine.

It is particularly preferred in the present invention, and particularly using Met-X, that one uses Nle or betaAla as amino acid reporter moieties.

In general, in a main embodiment of the present invention, the method of the present invention consists of identifying or analysing linkage sites on macromolecules using a compound having the composition FE-L-M by identifying the binding site of FE on to the M moiety based on a specific physical--chemical cleavage of L and, thereafter, releasing, removing and separating FE by chromatographic methods.

The suitability of the general methods of the present invention is demonstrated further by the using as non-limiting examples (the localisation of the site of PEG conjugation in the primary sequence of a PEGilated nonapeptide and insulin, either when insulin was PEGilated at one or at the level of two amino groups).

In a specific embodiment of the present invention the polypeptides were modified by PEG-Met-Nle. The conjugation of PEG to the therapeutically relevant protein, lysozime, followed by its complete removal by CNBr, is thereby illustrated.

In several embodiments of the present invention, several PEG peptide derivatives compounds are illustrated as examples of the structure: FE-L.

Among these, the following are reported as illustrative and appear in several examples: PEG-Met-Nle, PEG-Met-Val, PEG-Met-.beta.-Ala, PEG-Gln-Gly, PEG-Asp-Pro. All of these selected PEG derivative compounds of the present invention were activated at the terminal COOH with one of the methods reported in the literature, namely hydroxysuccinimidylester (OSu), and were then coupled to the representative polypeptides and proteins.

As additional preferred embodiment of the present invention, lysozime was PEGilated with PEG-Met-Nle, activated as OSU, and in turn, the polymer moiety was removed by CNBr treatment.

In all of these above selected example embodiments the FE-L-M complexes, after conjugation, were purified by HPLC or FPLC treatment in order to remove the activated unreacted polymer as well as the unmodified M.

As a further step, the purified conjugates were examined and the composition quantified, in a preferred way, by amino acid analysis after acid hydrolysis in order to evaluate, on the basis of the ratio of the reporter amino acid group to the amino acid content of M, (as well as by mass spectrometry of the non hydrolysed derivative), the number of polymer chains that were bound to M.

Furthermore, by trinitrobenzenesulphonate (TNBS) calorimetric assay, the purified conjugates were analysed to evaluate the number of the amino groups that did not react, (and by iodine assay, specific for PEG) in a preferred embodiment, to reveal its presence.

For the compounds and using the method of the present invention, analyses demonstrate that all of the FE-L were linked to the polypeptides similarly to what occurs when PEG is activated according to other procedures. It was also found that the increase in molecular weight, as evaluated by the Maldi mass spectrometry, corresponds to the polypeptide molecular weight increased of the FE-L weight or of its multiples. Furthermore, the presence of the reporter amino acid groups in the polypeptide could surprisingly always be revealed and quantitatively evaluated by amino acid analysis, and the value obtained corresponded to the modification calculated on the basis of the amino groups calorimetric evaluation. Finally it was found that in all of the cases the conjugates are reactive to iodine and a decrease or disappearance of polypeptide amino groups takes place at an extent that corresponds to the number of bound FE-L.

In the method of the present invention, after removal of PEG by the aforementioned procedure, the PEG-free polypeptide is purified by gel filtration from where it is eluted at higher volume due to its reduction in weight. The new elution volume approximately corresponds to that of M before the PEG modification; in theory, the small difference being due to the increase in weightowing to the binding of the reporter group that remains bound to M after FE removal.

Steps of sequence structure evaluation, such as Edman degradation, reduction and carboxymethylation, or proteolitic degradation could also be used in the present invention to indicate the site of PEG attachment, with easier detection of the reporter amino acid by amino acid analysis after acid hydrolysis or, when possible, mass spectrometry of the non-hydrolysed peptide.

Claim 1 of 15 Claims

What is claimed is:

1. A biologically active conjugate derivative having the following general formula (I)

FE-L-M (I)

where:

M represents the corresponding radical of a biologically active molecule selected from the group consisting of proteins, peptides, and polypeptides;

FE represents a functionalizing entity selected from the group consisting of PEG, PVP, PacM, dextran, hormones, antibodies, and antibody fragments; and

L represents a linking arm comprising a dipeptide selected from the group consisting of Met-Nle, Met-.beta.Ala, Gln-Gly, and Asp-Pro,

which is capable of being cleaved by chemical treatment to leave Nle, .beta.Ala, Gly or Pro, respectively, as a reporter group linked to M.

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