Title:  Site-specific preparation of polyethylene glycol-grf conjugates

United States Patent:  6,528,485

Issued:  March 4, 2003

Inventors:  Veronese; Francesco Maria (Padova, IT); Caliceti; Paolo (Padova, IT); Schiavon; Oddone (Padova, IT)

Assignee:  Applied Research Systems ARS Holding N.V. (Curacao, AN)

Appl. No.:  587460

Filed:  June 5, 2000

Described are various human growth hormone releasing factor-PEG conjugates as well as their pharmaceutical use.

DESCRIPTION OF THE INVENTION

In contrast to the solid-phase preparation of mono-pegylated hGRF mentioned above, the present invention relates to site-specific pegylation of hGRF in solution phase.

hGRF was found to have a low solubility in a neutral/alkaline buffer solution, a chemical condition whereby most efficient pegylation reaction occurs. In a diluted hGRF solution, the hydrolysis of the activated PEG (such as the PEG ester) tends to decrease the yield of the pegylation reaction.

It was discovered by the Applicant that, in a suitable solvent whereby hGRF has a high solubility, it is possible to carry out a site-specific pegylation reaction in solution phase. In this way, even if the starting hGRF peptide is non-protected, the PEG chains will bind with high yields and almost exclusively to the primary amino groups (.epsilon.-amino groups) of Lys¹², Lys²¹ and/or N^.alpha.. depending upon the reaction conditions. The following four conjugates, which are also covered by the present invention, were obtained, the hGRF:PEG stoichiometric ratio in the conjugates mainly depending on the molar ratio of PEG to hGRF:

hGRF-PEG conjugate, in which 1 PEG molecule is covalently bound to Lys¹²,

hGRF-PEG conjugate, in which 1 PEG molecule is covalently bound to Lys₂₁,

hGRF-2PEG conjugate, in which 2 PEG molecules are covalently bound to both Lys¹² and Lys²¹ ; and

hGRF-3PEG conjugate, in which 3 PEG molecules are covalently bound to both Lys¹² and Lys²¹ and also to N^.alpha..

"N^.alpha. " through out the present invention means the amino group at the N-terminal position of the peptide (Tyr).

Further to this step, it is possible to carry out a simple chromatographic fractionation of the conjugates obtained in the reaction either by gel filtration or by direct application to a C18 HPLC column eluted by water/acetonitrile gradient. The second method is preferred, since large scale preparation and purification of the products could be obtained.

Therefore, the main embodiment of the present invention is a method for the site-specific preparation of different hGRF-PEG conjugates containing one or more than one PEG units (per hGRF) covalently bound to Lys¹² and/or Lys²¹ and/or N^.alpha., characterized in that the pegylation reaction is carried out in solution and the desired hGRF-PEG conjugate is purified, for example, by chromatographic methods.

hGRF-PEG conjugates containing one or more PEG units (per mole of hGRF) covalently bound to Lys¹² and/or Lys²¹ and/or N^.alpha. are also covered by the present invention. The hGRF-PEG conjugates, in which 1 PEG molecule is covalently bound to Lys¹² or to Lys²¹, are the preferred products of the present invention.

According to another embodiment of the present invention, if one or more of these three amino groups to which PEG chains bind, are reversibly protected by certain chemical groups from pegylation, the pegylation reaction will give directly the desired conjugate with specific pegylation sites, which can then be isolated from the reaction mixture, for example, by ultrafiltration or other chromatographic methods. In this case, the preparation method can further, optionally, comprise a de-protection reaction.

The de-protection reaction is preferably carried out according to known methods and depending on the chemical protective group to be removed.

According to this invention the term "hGRF", unless otherwise specified, is intended to cover any human GRF peptides, with particular reference to the 1-44, 1-40. 1-29 peptides and the corresponding amides thereof (containing an amide group at the N-terminus or C-terminus). The preferred hGRF peptide is hGRF(1-29)-NH₂ whose amino acid sequence is reported in SEQ ID NO:1.

The "activated PEG" (or "pegylating agent") is any PEG derivative, which can be used as protein modifier, because it contains a functional group capable of reacting with some functional croup in the protein/peptide to produce the PEG-protein/peptide conjugates. A review of PEG derivatives useful as protein modifiers can be found in Harris (1985). The activated PEG can be an alkylating reagent, such as PEG aldehyde, PEG epoxide or PEG tresylate, or it can be an acylating reagent, such as PEG ester.

The activated PEG is preferably used in its mono-methoxylated form. It has preferably a molecular weight between 2,000 and 20,000. Mono-methoxylated PEG_5,000 is particularly preferred for the preparation of the activated PEG according to the present invention.

If activated PEG is an acylating agent, it preferably contains either a norleucine or ornithine residue bound to the PEG moiety via an amide linkage. These residues allow a precise determination of the linked PEG units per mole of peptide (see for example Sartore et al., 1991). Therefore, more in particular, the preferred activated PEG is mono-methoxylated PEG_5,000 linked by means of an amide bond to the alpha amino group of norleucine, that is activated at the carboxy group as succinimidyl ester.

Branched PEGs are also in common use. The branched PEGs can be represented as R(-PEG-OH)_m in which R represents a central core moiety such as pentaerythritol or glycerol, and m represents the number of branching arms. The number of branching arms (m) can range from three to a hundred or more. The hydroxyl groups are subject to chemical modification.

Another branched form, such as that described in PCT patent application WO 96/21469, has a single terminus that is subject to chemical modification. This type of PEG can be represented as (CH₃ O-PEG-)_p R-X, whereby p equals 2 or 3. R represents a central core such as lysine or glycerol, and X represents a functional group such as carboxyl that is subject to chemical activation. Yet another branched form, the "pendant PEG", has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.

All these branched PEGs can be "activated" as indicated above.

"Chromatographic methods" means any technique that is used to separate the components of a mixture by their application on a support (stationary phase) through which a solvent (mobile phase) flows. The separation principles of the chromatography are based on the different physical nature of stationary and mobile phase.

Some particular types of chromatographic methods, which are well-known in the literature, include: liquid, high pressure liquid, ion exchange, absorption, affinity, partition, hydrophobic, reversed phase, gel filtration, ultrafiltration or thin-layer chromatography.

"Pegylation" is the reaction by which a PEG-protein/peptide conjugate is obtained starting from the activated PEG and the corresponding protein/peptide.

The molar ratio PEG:hGRF can be 1:1, 2:1 or 3:1, depending on which conjugate is sought at high yields.

The solvent of the pegylation reaction is selected from the group consisting of a highly concentrated nicotinamide aqueous solution. a buffered aqueous solution of a defolding agent (such as urea) or a polar organic solvent selected among dimethyl sulfoxide, dimethyl formamide/buffer or acetonitrile/buffer.

The pH of the solution is usually kept between 7 and 9.

A non-limitative list of protective chemical groups for Lys¹² and Lys²¹ includes: Alloc (allyloxycarbonyl), Dde (1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl), Adpoc (1-(1'-Adamantyl)-1-methyl-ethoxycarbonyl) or 2-Cl-Z (2-Chlorobenzyloxycarbonyl). Alloc is the preferred protective group for the lysine group.

After pegylation Alloc can be removed according to one of the methods described in Greene T. W. et al., 1991). Dde can be removed with 2% hydrazine in DMF (see W. C. Chan et al., 1995). Adpoc can be removed similarly to Alloc (see also D. Bourgin et al., 1997). 2-Cl-Z can be requires a stronger acid deprotection (HF, TFMSA, HBr) or hydrogenation (see also Tam et al., 1987).

The protective groups for N^.alpha. can be an alkyl group. such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, benzyl or cyclohexyl. Isopropyl is the preferred one. These alkyl groups can be introduced by reductive alkylation (see Murphy et al., 1988 or Hocart et al., 1987).

(N^.alpha. -isopropyl-Tyr¹,Lys(Alloc)¹²)-hGRF and (Lys(Alloc)¹².21)-hGRF are also covered by the present invention, as useful and new intermediates of the pegylation reaction.

It has also been discovered that the pegylation of the present invention:

1. does not modify the conformation of the peptide,

2. increases the resistance to the proteolytic degradation,

3. does not affect, or only slightly decreases, the biological activity, depending upon the extent of pegylation and

4. allows to obtain products (the conjugates), which are more soluble in aqueous buffered solutions.

Another object of the present invention is to provide the hGRF-PEG conjugates in substantially purified form in order for them to be suitable for use in pharmaceutical compositions as active ingredients.

In a further aspect, the present invention provides the use of the conjugates of the invention in the manufacture of a medicament for treatment, prevention or diagnosis of growth hormone-related disorders, such as for example growth hormone deficiency(GHD), in particular pediatric growth hormone deficiency.

The medicament is preferably presented in the form of a pharmaceutical composition comprising the conjugates of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical compositions form yet a further aspect of the present invention.

An embodiment of the invention is the administration of a pharmacologically active amount of the conjugates of the invention to subjects at risk of developing a growth hormone-related disease or to subjects already showing such pathology.

A further object of this invention is a method of treatment. prevention or diagnosis of growth hormone-related disorders, comprising administering an effective amount of the conjugates of the inven.ion, in the presence of one or more pharmaceutically acceptable excipients.

An "effective amount" refers to an amount of the active ingredients that is sufficient to affect the course and the severity of the disorders described above, leading to the reduction or remission of such pathology. The effective amount will depend on the route of administration and the condition of the patient.

"Pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered. For example, for parenteral administration, the above active ingredients may be formulated in unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.

Besides the pharmaceutically acceptable carrier, the compositions of the invention can also comprise minor amounts of additives, such as stabilizers, excipients, buffers and preservatives.

Any route of administration compatible with the active principle can be used. The preferred is the parenteral administration, such as subcutaneous, intramuscular or intravenous injection. The dose of the active ingredient to be administered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.

The dosage of the active ingredient for the human therapy can be between 5 and 6,000 .mu.g/Kg body weight and the preferable dose is between 10 and 300 .mu.g/Kg body weight.

The present invention has been described with reference to the specific embodiments, but the content of the description comprises all modifications and substitutions which can be brought by a person skilled in the art without extending beyond the meaning and purpose of the claims.

Claim 1 of 9 Claims

What is claimed is:

1. A human growth hormone releasing factor-polyethylene glycol (hGRF-PEG) conjugate consisting of a human growth hormone releasing factor (hGRF) comprising the amino acid sequence of SEQ ID NO:1, wherein said hGRF has at least one polyethylene glycol (PEG) unit per hGRF covalently bound to only the amino acids selected from the group consisting of at least one amino group of Lys¹² of SEQ ID NO:1, Lys²¹ of SEQ ID NO:1, and the amino terminal group (N^.alpha.) of said hGRF, and wherein said hGRF-PEG conjugate does not contain a triazine group.

 

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