Link:  Pharm/Biotech Resources

Title:  PEG-LHRH analog conjugates

United States Patent:  6,914,121

Issued:  July 5, 2005

Inventors:  El-Tayar; Nabil (Milton, MA); Zhao; Xuan (Huntsville, AL); Bentley; Michael D. (Huntsville, AL)

Assignee:  Applied Research Systems Ars Holding N.V. (Curacao, NL)

Appl. No.:  184126

Filed:  June 28, 2002

PEG-LHRH analog conjugates, where a PEG moiety is covalently bound to a serine residue of a LHRH analog either directly or via a bifunctional linker molecule, such as an amino acid, and methods for producing these conjugates are provided in the present invention. Also provided are a pharmaceutical composition and a method for treating pathologies in which LHRH analog administration is beneficial.

SUMMARY OF THE INVENTION

This invention provides novel PEG-LHRH analogs conjugates wherein a PEG unit is covalently bound to Ser⁴ either directly or via a bifunctional linker molecule, such as an amino acid. PEG or PEG-linker molecule is bonded, specifically, to the alcohol function of the serine residue. The linkage between the LHRH analog and the polyethylene glycol or the PEG-linker molecule in these conjugates is subject to hydrolysis at physiological pH (7.2-7.4) and is preferably also subject to hydrolysis by esterases present in the blood.

DETAILED DESCRIPTION OF THE INVENTION

The conjugates of the present invention, preferably, show a solubility in water of at least 30 mg/ml at room temperature and physiological pH (7.2-7.4) and a solubility in a physiological saline solution of at least 10 mg/ml at the same conditions.

In the case in which the LHRH analog is antide, for example, such properties enable the use of antide as a drug whereas, previously, development of antide as a drug has been rendered difficult due to its poor water solubility.

The term "LHRH-analogs", as used herein, is meant to include any decapeptide which is an LHRH agonist or antagonist. Preferably the LHRH analog is an LHRH antagonist; more preferably it is antide.

The conjugates of the present invention can be prepared by any of the methods known in the art. According to an embodiment of the invention, the LHRH analog is reacted with a PEGylating agent in a suitable solvent and the desired conjugate is isolated and purified, for example, by applying one or more chromatographic methods.

"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.

The "PEGylating agent" as used in the present application means any PEG derivative, which is capable of reacting with the OH of a serine residue or a functional group of a bifunctional linker molecule, such as the amino group of an amino acid linker molecule. The other functional group of the linker molecule serves to form a covalent linkage to the serine residue of a LHRH analog, i.e., the carboxyl group of an amino acid linker molecule forms an ester linkage with serine. It can be an alkylating reagent, such as PEG aldehyde, PEG epoxide or PEG tresylate, or it can be an acylating reagent, such as PEG-O-(CH₂)_nCO₂-Z where n=1-3 and Z is N-succinimidyl or other suitable activating group.

The PEGylating agent is used in its mono-methoxylated form where only one terminus is available for conjugation, or in a bifunctional form where both termini are available for conjugation, such as for example in forming a conjugate with two LHRH analogs covalently attached to a single PEG moiety. It has a molecular weight between 500 and 100,000, preferably between about 5,000 and 40,000 (40 kDa) and more preferably between about 10 kDa and 40 kDa and most preferably between about 20 kDa and 40 kDa.

If the PEGylating agent is an acylating agent, it can contain either a norleucine or ornithine residue bound to the PEG unit 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).

A solvent for the PEGylation reaction is preferably a polar aprotic solvent, such as DMF, DMSO, pyridine, etc.

When the LHRH analog is reacted with the PEGylating agent, derivatization can occur on the OH of the Ser⁴ moiety, as well as on the amine nitrogen of other residues, such as, for example, on the ε-amino group of lysine (in case of antide, on N-Isopropyl-Lys⁸). In such reactions, high selectivity for amine PEGylation can occur. Products formed by PEGylation on amines are amides and while PEG amides can be water soluble, the amide linkage can be stable under physiological conditions, and thus the LHRH analog could not be substantially hydrolytically released in vivo. Therefore, using this method, the PEG-LHRH analog ester should be separated from the PEG-LHRH analog amide using chromatography. A limitation of this method is, therefore, low yield of the desired PEG-LHRH analog conjugate.

Therefore, in a preferred embodiment, the LHRH analog is protected on the amine groups which could either react with the PEGylating agent or with a bifunctional linker molecule prior to PEGylation.

In the case of antide, it is therefore preferable to reversibly protect the N-Isopropyl-Lys⁸ residue with a group that can be removed using photochemical, mild hydrolytic, or hydrogenation methods. With the nitrogen thus protected, the hydroxyl group on the serine residue is reacted with a PEGylating reagent to form a PEG ester and the protecting group on the N-Isopropyl-Lys⁸ is then removed to yield antide selectively PEGylated on the hydroxyl of the serine residue by an ester linkage. The conditions for removal of the amine protecting group must be sufficiently mild to avoid cleavage of the PEG-antide ester linkage. In another embodiment where a bifunctional linker or spacer molecule is used to link a PEG moiety to an LHRH analog such as antide, the protecting group is preferably removed after a bifunctional linker molecule is covalently bound to the serine residue of an LHRH analog or can be removed after PEGylation of the bifunctional linker molecule covalently bound to the LHRH analog.

Preferred reagents for protection include benzyloxycarbonyl chloride or ring-substituted derivatives of this compound, N-hydroxysuccinimidyl or 1-benzotriazolyl esters of benzyloxycarbonic acid or ring substituted derivatives of t-butoxycarbonyl chloride or the N-hydroxysuccinimidyl or 1-benzotriazolyl esters of t-butoxycarbonic acid.

In another embodiment of the invention, the conjugates of the invention can be prepared by using an appropriate PEGylated serine such as Fmoc-Ser(PEG)-OH or tBoc-Ser(PEG)-OH instead of serine during the solid-phase synthesis of the LHRH analogs.

A regulation of the rate of release in vivo of the LHRH analog can be accomplished by varying n and R in the PEG linkage. In general, as n increases, the rate of release of the LHRH analog decreases and if R is alkyl, the rate of release of the LHRH analog is lower than the rate of release if R is H. In general, as the size of R increases, the rate of release of the LHRH analog, or antide in particular, decreases. Variation of n and R thus provides substantially precise control of the delivery rate in vivo of antide when used as a drug.

According to recent studies, such as U.S. Pat. No. 5,840,900, higher molecular weight PEG appear to be important for obtaining therapeutic efficacy in certain cases. For antide, three PEG-antide conjugates with PEGylating agents having molecular weights of 20 kDa or 40 kDa have been prepared by modifying the protection-deprotection procedure. In a preferred embodiment, the PEGylation rate of antide, particularly with higher molecular weight PEG moieties, is increased by first attaching a linker molecule, such as glycine, to the antide and then PEGylating the linker molecule covalently attached to the antide. The reaction scheme for preparing a PEGylated antide with a glycine linker can be used for preparing, for example, conjugates such as PEG2-glycine-antide 20 k (a branched 20 kDa PEG can be used), mPEG-glycine-antide (a linear 20 kDa mPEG can be used), and PEG2-glycine-antide 40 k (a branched 40 kDa PEG can be used).

The linker molecule is preferably a small bifunctional molecule, which can rapidly react with the OH group on a serine residue of a LHRH analog. This linker molecule is preferably a heterobifunctional linker molecule, such as an amino acid, which forms an ester with a serine residue of a LHRH analog. The second functional group of the linker molecule serve as the site for PEGylation by the PEGylating agent. The amino acid, glycine, is a preferred heterobifunctional linker molecule according to the present invention. Other suitable linker molecules can be readily recognized or determined by those of skill in the art.

Another object of the present invention is to provide the conjugates in substantially purified form in order for them to be suitable for use in pharmaceutical compositions, as active ingredient for the treatment, diagnosis or prognosis of pathologies in which LHRH analogs administration is advisable. Such pharmaceutical compositions represent a further object of the present invention.

If the LHRH analog is antide, the above-mentioned pathologies include endometriosis, uterine fibroids, hormonal-dependent cancers (prostate, breast), uterine myoma, LH surge in women undergoing in-vitro fertilization and all the other pathological states reported in EP 377,665.

Further embodiments and advantages of the invention will be evident in the following description.

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 one of the diseases reported above or to subjects already showing such pathologies.

Any route of administration compatible with the active principle can be used. The preferred is 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 daily non-weighted dosage for the patient can be between 0.2 to 20 mg, and the preferable daily dose is between 0.2 to 10 mg.

The pharmaceutical composition for parenteral administration can be prepared in an injectable form comprising the active principle and a suitable vehicle. Vehicles for the parenteral administration are well known in the art and comprise, for example, water, saline solution, Ringer solution and/or dextrose.

The vehicle can contain small amounts of excipients in order to maintain the stability and isotonicity of the pharmaceutical preparation.

The preparation of the cited solutions can be carried out according to the ordinary modalities.

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 13 Claims

1. A method for treating a pathology selected from the group consisting of a hormonal dependent cancer, endometriosis, uterine fibroids, uterine myoma, and luteinizing hormone (LH) surge in woman undergoing in vitro fertilization, comprising administering to a subject in need thereof an effective amount of a conjugate having a polyethylene glycol (PEG) moiety covalently bound to the serine residue of antide to treat said pathology, wherein said conjugate is capable of hydrolysis to release antide.

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