A method is described for the site-specific preparation of hGRF-PEG conjugates containing one or more PEG units (per mole of hGRF) covalently bound to Lys.sup.12 and/or Lys.sup.21 and/or N.sub..alpha., characterized in that the conjugation reaction between the hGRF peptide and activated PEG is carried out in solution and the desired hGRF-PEG conjugate can be purified by chromatography. The conjugates prepared by this method, as well as their use in the treatment, prevention of diagnosis of growth hormone deficiency, are also an object of the present invention.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to a method for the site-specific preparation of hGRF-PEG conjugates containing one or more than one PEG units (per hGRF) covalently bound to Lys.sup.12 and/or Lys.sup.21 and/or N.sup..alpha., characterized in that the conjugation reaction between the hGRF peptide and activated PEG is carried out in solution and the desired hGRF-PEG conjugate is purified by chromatographic methods.

The conjugates prepared by this method, as well as their use in the treatment, prevention or diagnosis of growth-hormone related disorders, are also an object of the present invention.

BACKGROUND OF THE INVENTION

In the early 1980's several groups isolated and characterized growth hormone releasing factor (GRF).

GRF (also called Somatorelin) is a peptide secreted by the hypothalamus which acts on its receptor and can promote the release of growth hormone (GH) from the anterior pituitary. It exists as 44-, 40-, or 37-amino acid peptide; the 44-amino acids form may be converted physiologically into shorter forms. All three forms are reported to be active, the activity residing mainly in the first 29 amino acid residues. A synthetic peptide corresponding to the 1-29 amino acid sequence of human GRF [hGRF(1-29)], also called Sermorelin, has been prepared by recombinant DNA technology as described in European Patent EP 105 759.

Sermorelin has been used in the form of acetate for the diagnosis and treatment of growth hormone deficiency.

GRF has indeed a therapeutic value for the treatment of certain growth-hormone related disorders. The use of GRF to stimulate the release of GH is a physiological method in promoting long bone growth or protein anabolism.

One problem associated with the use of GRF relates to its short biological half-life (about 12 to 30 minutes). The hGRF(1-29)--NH.sub.2 is subject to enzymatic degradation and is rapidly degraded in the plasma via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala.sup.2 and Asp.sup.3.

It is therefore advantageous to develop biologically stable, long-acting GRF analogues using specific chemical modification of GRF, in order to prevent or slow down enzymatic degradation.

Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic polymer of general formula H(OCH.sub.2CH.sub.2).sub.nOH, wherein n.gtoreq.4. Its molecular weight could vary from 200 to 20,000 daltons.

It has been demonstrated that the chemical conjugation of PEG in its mono-methoxylated form to proteins and/or peptides significantly increases their duration of biological action. Like carbohydrate moieties in a glycoprotein, PEG provides a protective coating and increases the size of the molecule, thus reducing its metabolic degradation and its renal clearance rate.

PEG conjugation is an already established methodology for peptide and protein delivery pioneered by the fundamental studies of Davis and Abuchowski (Abuchowski et al., 1977a and 1977b). PEG conjugation to peptides or proteins generally resulted in non-specific chemical attachment of PEG to more than one amino acid residue. One of the key issues with this technology is therefore finding appropriate chemical methods to covalently conjugate PEG molecule(s) to specific amino acid residues.

For example, the trichiorotriazine-activated PEG, which was found to be toxic and reacted in a non-specific way, was later on replaced by various PEG reagents with chemical linkers that could react specifically to amino groups (Beauchamp et al., 1983; Veronese et al., 1985; Zalipsky et al., 1983; Zalipski et al., 1990; and Delgado et al., 1990), to suiphydryl groups (Sartore et al., 1991; and Morpurgo et al., 1996) or to guanidine residues (Pande et al., 1980).

Various PEG-protein conjugates were found to be protected from proteolysis and/or to have a reduced immunogenicity (Monfardini et al., 1995; and Yamsuki et al., 1988).

Another technical difficulty in protein pegylation arises from the fact that PEG-protein conjugates usually have various number of PEG molecules attached and result in a mixture of conjugates with different PEG:protein stoichiometries. Site-specific pegylation remains a chemical challenge. The conjugation of PEG to GH represents a typical example of such problem (Clark et al., 1996). It was demonstrated that Lys-residues of GH were pegylated at random positions.

To avoid or reduce the loss of enzyme activity, the active site could be protected in advance, thus allowing enzyme pegylation to occur at non-active site(s) (Caliceti et al., 1993).

Another approach was recently proposed for the site-specific conjugation of PEG to low molecular weight peptides, such as GRF, which was prepared by solid-phase peptide synthesis. In these conjugates a pegylated amino acid, prepared in advance, was introduced into the peptide sequence during the solid-phase synthesis. This procedure, however, dramatically complicates product purification that is known to be the critical step in solid phase synthesis. The presence of PEG, for its high molecular weight and its polydispersivity, is likely to yield final products with unacceptable impurities and/or products with missing amino acids, the latter being considered to occur commonly in the Merrifield procedure.

Mono-pegylation, meaning that only one PEG molecule is attached, using solid-phase synthesis to specific amino acid residues of [Ala.sup.15]-hGRF(1-29)--NH.sub.2 has been recently reported in the literature (Felix et al., 1995). This study shows that [Ala.sup.15]-hGRF(1-29)--NH.sub.2 pegylated at residues 21 or 25 retains the full in-vitro potency of the parent [Ala.sup.15]-hGRF(1-29)--NH.sub.2. There is however no in-vivo data to show whether these pegylated conjugates exhibit a longer duration of action with respect to the non-pegylated counterpart.

More recently, it has been demonstrated (Campbell et al., 1997) that the attachment of PEG with different molecular weights to the C-terminus of several analogs of hGRF, again using solid-phase synthesis, had enhanced duration of action in both pig and mouse models as compared to the non-pegylated counterpart.

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 Lys.sup.12, Lys.sup.21 and/or N.sup..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 Lys.sup.12,

hGRF-PEG conjugate, in which 1 PEG molecule is covalently bound to Lys.sup.21,

hGRF-2PEG conjugate, in which 2 PEG molecules are covalently bound to both Lys.sup.12 and Lys.sup.21; and

hGRF-3PEG conjugate, in which 3 PEG molecules are covalently bound to both Lys.sup.12 and Lys.sup.21 and also to N.sup..alpha..

"N.sup..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 Lys.sup.12 and/or Lys.sup.21 and/or N.sup..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 Lys.sup.12 and/or Lys.sup.21 and/or N.sup..alpha. are also covered by the present invention. The hGRF-PEG conjugates, in which 1 PEG molecule is covalently bound to Lys.sup.12 or to Lys.sup.21, 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)--NH.sub.2 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 group 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.sub.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.sub.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).sub.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 (CH.sub.3O--PEG--).sub.pR--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 Lys.sup.12 and Lys.sup.21 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.sup..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.sup..alpha.-isopropyl-Tyr.sup.1,Lys(Alloc).sup.12]-hGRF and [Lys(Alloc).sup.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 invention, 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.

Claim 1 of 9 Claims

1. In a method for treating or diagnosing a growth hormone deficiency in which an effective amount of a growth hormone material is administered, the improvement wherein said growth hormone material administered to treat or diagnose a growth hormone deficiency is a human growth hormone releasing factor-polyethylene glycol (hGRF-PEG) conjugate having at least one PEG unit per hGRF covalently bound to at least one amino group of Lys.sup.12 of SEQ ID NO: 1, Lys.sup.21 of SEQ ID NO: 1 and the amino terminal group (N.sup..alpha.) of hGRF and wherein said hGRF-PEG conjugate consists of an hGRF comprising the amino acid sequence of SEQ ID NO: 1 and does not contain a triazine group.

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