Link:  Pharm/Biotech Resources

Title:  Method for increasing the serum half-life of a biologically active molecule

United States Patent:  6,884,780

Issued:  April 26, 2005

Inventors:  Drummond; Robert J. (Richmond, CA); Rosenberg; Steve (Oakland, CA)

Assignee:  Chiron Corporation (Emeryville, CA)

Appl. No.:  123092

Filed:  April 11, 2002

Abstract

A method is provided for preparing a biologically active molecule having an increased serum half-life. The method involves conjugating a polymer such as polyethylene glycol to the biologically active molecule. Also provided are polypeptide drugs having an increased serum half-life, e.g., human urokinase plasminogen activator (human "uPA" or "hUPA") or a fragment or derivative thereof. Pharmaceutical compositions containing such molecules and methods of using them to treat uPA-mediated and uPA receptor-mediated disorders are also provided.

Description of the Invention

TECHNICAL FIELD

This invention relates generally to the chemical modification of biologically active molecules, and more particularly relates to a method for modifying biologically active molecules to increase their serum half-life.

BACKGROUND

Unfavorable pharmacokinetics, such as a short serum half-life, can prevent the pharmaceutical development of many otherwise promising drug candidates. Serum half-life is an empirical characteristic of a molecule, and must be determined experimentally for each new potential drug. For example, with lower molecular weight polypeptide drugs, physiological clearance mechanisms such as renal filtration can make the maintenance of therapeutic levels of a drug unfeasible because of cost or frequency of the required dosing regimen. Conversely, a long serum half-life is undesirable where a drug or its metabolites have toxic side effects.

A possible solution to an undesirably short serum half-life of a pharmaceutical agent is to covalently attach to the agent molecules which may increase the half-life. Previously, it has been shown that attachment of polymers to polypeptides may increase their serum half-lives. See, for example, European Patent Publication No. 0 442 724 A2, which describes "PEGylated" interleukin-6 derivatives (i.e., interleukin derivatives bound to polyethylene glycol, or "PEG") having an extended serum half-life. Attachment of drugs to polymers has also been reported to increase their water solubility, stability during storage and reduce their immunogenicity (published patent applications EP 0 539 167 A2, WO 94/13322). Conjugates of IL-2 or muteins thereof with polymers have also been reported to have reduced immunogenicity, increased solubility and increased half-lives (U.S. Pat. Nos. 5,362,852, 5,089,261, 5,281,698 and published patent application WO 90/07938).

However, the attachment of polymers can lead to decreases in drug activity. Incomplete or nonuniform attachment leads to a mixed population of compounds having differing properties. Additionally, the changes in half-lives resulting from such modifications are unpredictable. For example, conjugation of different polyethylene glycols to IL-8, G-CSF and IL-1ra produced molecules having a variety of activities and half-lives (Gaertner and Offord, (1996), Bioconjugate Chem. 7:38-44). Conjugation of IL-8 to PEG_{20 kD} produced no change in its half-life, while conjugation of PEG_{20 kD} to IL-1ra gave an almost seven-fold increase in half-life. Additionally, the IL-8/PEG_{20 kD} conjugate was ten- to twenty-fold less effective than the native protein.

Accordingly, a method which is capable of increasing the serum half-life of a biologically active molecule, without seriously diminishing the biological function of the molecule, would be highly desirable.

SUMMARY OF THE INVENTION

Thus there is a need in the art for a method for modifying a biologically active molecule without abolishing its biological activity. There is a further need in the art to provide a method for increasing the serum half-life of such a molecule. There is yet a further need for a method of increasing the serum half-life of a biologically active molecule which produces a single species of product having uniform biological and pharmacokinetic properties.

Accordingly, it is a primary object of the invention to address the above-described needs by providing a method for increasing the serum half-life of a biologically active molecule.

It is another object of the invention to provide such a method which avoids modification of sites necessary for biological activity present within the molecule.

It is still another object of the invention to provide such a method wherein the molecule is a polypeptide.

It is an additional object of the invention to provide such a method wherein the polypeptide is "PEGylated," i.e., coupled to PEG, for example by reaction of a PEG hydrazide with an aldehyde moiety present at the N-terminus of the polypeptide.

It is yet another object of the invention to provide such a method wherein the serum half-life of the polypeptide is increased by site-specific attachment of a polymer such as polyethylene glycol to the N-terminus of the polypeptide chain.

It is a further object of the invention to provide a modified molecule having a longer serum half-life than the native molecule.

It is still another object of the invention to provide a pharmaceutical composition comprising a conjugate of a polymer and a biologically active molecule in combination with a pharmaceutically acceptable carrier or excipient.

It is yet a further object of the invention to provide a method of treating a urokinase plasminogen activator-("uPA-") mediated disorder by administering a pharmaceutically acceptable composition comprising a conjugate of uPA_1-48 and a polymer.

Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

In one embodiment, the present invention relates to a method for chemically modifying a molecule to increase the serum half-life thereof, preferably in a site-specific manner without modification of any sites necessary for biological activity, such as receptor binding or enzymatic activity. The molecule is preferably, although not necessarily, a polypeptide. The method involves binding the molecule to a polymer such as polyethylene glycol, e.g. by reacting a hydrazide-activated form of polyethylene glycol (referred to herein as "polyethylene glycol hydrazide" or "PEG hydrazide") with an aldehyde moiety present on the molecule. When the molecule to be modified is a polypeptide, the aldehyde moiety is introduced at the N-terminus, and can be generated by oxidative cleavage at adjacent hydroxyl and amino groups found in N-terminal serine or threonine residues.

In another embodiment of the invention, a modified molecule is provided having an increased serum half-life relative to the molecule per se, i.e., the "unmodified" molecule. Preferably, the modified molecule is a polypeptide conjugated to polyethylene glycol through a hydrazone or semicarbazone linkage. In a particularly preferred embodiment, the polypeptide is human urokinase or a fragment thereof, e.g., uPA_1-48.

In another embodiment of the invention, a method is provided for producing a conjugate of the uPA epidermal growth factor-("EGF-") like domain, particularly uPA_1-48.

In still another embodiment of the invention, a conjugate of uPA_1-48 is provided which is useful for inhibiting the mitogenic activity of uPA in cancer cells.

In another embodiment of the invention, a method is provided for treating a uPA-mediated or uPA receptor-mediated disorder by administering a conjugate of uPA_1-48 and a polymer to a patient in need thereof.

In yet another embodiment of the invention, a method is provided for treating cancer and metastasis by administering an effective amount of a conjugated uPA EGF-like domain, particularly uPA_1-48.

DETAILED DESCRIPTION OF THE INVENTION

Overview and Definitions:

By "increase in serum half-life" is meant the positive change in circulating half-life of a modified biologically active molecule relative to its non-modified form. Serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. The increase is desirably at least about two-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids a toxic effect. Preferably the increase is at least about three-fold, more preferably at least about five-fold, and most preferably at least about ten-fold, and most preferably at least about fifteen-fold. Increases of up to 28.8-fold in serum half-life are demonstrated herein.

The increase in serum half-life preferably occurs through a method that at least preserves biological activity, measured, for example, in a binding assay. In some instances, the method may even increase biological activity. However, where the method does provide a decrease in biological activity, it is preferable that the proportionate increase in serum half-life is at least as great as the proportionate decrease in biological activity. More preferably, the increase in serum half-life is greater than the decrease in biological activity, proportionately. This is not an absolute requirement, and depends, for example, on the pharmacokinetics and toxicity of the specific derivative. The percentage of biological activity which is retained is preferably about 10 to about 100%, more preferably about 15 to about 100%, and most preferably about 20 to about 100%. In an especially preferred embodiment, about 25 to about 100% of the biological activity is retained.

In a preferred embodiment, the biologically active molecule is a polypeptide. A particularly preferred polypeptide is uPA_1-48. uPA_1-48 is herein demonstrated to have a short serum half-life. Increasing the serum half-life of rapidly cleared compounds is desirable, particularly where the compounds are recombinant molecules which are difficult and costly to produce. Such an increase in half-life can reduce treatment costs, decrease the amount of agent administered, decrease the duration of administration, and lessen the exposure of patients to large volumes of pharmaceutical preparations. Conjugation of PEG to uPA_1-48 is shown herein to dramatically increase its serum half-life by as much as 28.8 fold.

The polypeptide can be produced by any suitable means, such as expression in a recombinant host cell or by chemical synthesis. The polypeptide is then purified through standard methods. Where the polypeptide is uPA_1-48, production in a yeast host cell, as described in published PCT patent application WO 94/28145, is suitable. For example, DNA encoding residues 1-48 of mature human uPA are cloned into a yeast expression vector as a fusion with the yeast alpha-factor leader (αF1), under transcriptional control of a hybrid ADH2-GAP promoter. The PCR fragment of the gene encoding huPA primer and a template plasmid, and the alkaline phosphatase treated pCBR subcloning vector containing the yeast expression cassette are digested with BgIII, followed by ligation. The subclone thus obtained (pCBRuPAα13) is subjected to BamHI digestion and the isolated expression cassette is ligated into the yeast shuttle vector. The expression plasmid is then transformed into the yeast host under conditions to promote the expression of the polypeptide. uPA_1-48 can then be purified as described therein, or by suitable techniques known in the art, such as centrifugation, column chromatography, anion exchange chromatography, cation exchange chromatography, or combinations thereof. Diafiltration can be used to bring the polypeptide solution to a desired concentration and/or to change the composition of the solution.

The biologically active molecule can be linked to a polymer through any available functionality using standard methods well known in the art. It is preferred that the biologically active molecule be linked at only one position in order to minimize any disruption of its activity and to produce a pharmacologically uniform product. Nonlimiting examples of functional groups on either the polymer or biologically active molecule which can be used to form such linkages include amine and carboxy groups, thiol groups such as in cysteine resides, aldehydes and ketones, and hydroxy groups as can be found in serine, threonine, tyrosine, hydroxyproline and hydroxylysine residues.

The polymer can be activated by coupling a reactive group such as trichloro-s-triazine (Abuchowski et al., (1977), J. Biol. Chem. 252:3582-3586), carbonylimidazole (Beauchamp et al., (1983), Anal. Biochem. 131:25-33), or succinimidyl succinate (Abuchowski et al., (1984), Cancer Biochem. Biophys. 7:175-186) in order to react with an amine functionality on the biologically active molecule. Another coupling method involves formation of a glyoxylyl group on one molecule and an aminooxy, hydrazide or semicarbazide group on the other molecule to be conjugated (Fields and Dixon, (1968), Biochem. J. 108:883-887; Gaertner et al., (1992), Bioconjugate Chem. 3:262-268; Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146; Gaertner et al., (1994), J. Biol. Chem. 269:7224-7230). Other methods involve formation of an active ester at a free alcohol group of the first molecule to be conjugated using chloroformate or disuccinimidylcarbonate, which can then be conjugated to an amine group on the other molecule to be coupled (Veronese et al., (1985), Biochem. and Biotech. 11:141-152; Nitecki et al., U.S. Pat. No. 5,089,261; Nitecki, U.S. Pat. No. 5,281,698). Other reactive groups which may be attached via free alcohol groups are set forth in Wright, published European patent application 0 539 167 A2, which also describes the use of imidates for coupling via free amine groups.

An aldehyde functionality useful for conjugating the biologically active molecule can be generated from a functionality having adjacent amino and alcohol groups. Where the biologically active molecule is a polypeptide, for example, an N-terminal serine, threonine or hydroxylysine can be used to generate an aldehyde functionality via oxidative cleavage under mild conditions using periodate. These residues, or their equivalents, can be normally present, for example at the N-terminus of a polypeptide, may be exposed via chemical or enzymatic digestion, or may be introduced via recombinant or chemical methods. The reaction conditions for generating the aldehyde typically involve addition of a molar excess of sodium meta periodate and under mild conditions to avoid oxidation at other positions in the protein. The pH is preferably about 7.0. A typical reaction involves the addition of a 1.5 fold molar excess of sodium meta periodate, followed by incubation for 10 minutes at room temperature in the dark.

The aldehyde functionality can then be coupled to an activated polymer containing a hydrazide or semicarbazide functionality to form a hydrazone or semicarbazone linkage. Hydrazide-containing polymers are commercially available, and can be synthesized, if necessary, using standard techniques. PEG hydrazides preferred for the invention can be obtained from Shearwater Polymers, Inc., 2307 Spring Branch Road, Huntsville, Ala. 35801. The aldehyde is then coupled to the polymer by mixing the solution of the two components together and heating to about 37° C. until the reaction is substantially complete. An excess of the polymer hydrazide is typically used to increase the amount of conjugate obtained. A typical reaction time is 26 hours. Depending on the thermal stability of the reactants, the reaction temperature and time can be altered to provide suitable results. Detailed determination of reaction conditions for both oxidation and coupling is set forth in Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146, and in Geoghegan, U.S. Pat. No.

Such a conjugate formed between uPA_1-48 and a polymer can be used therapeutically to treat uPA- and uPA receptor-mediated disorders. A pharmaceutically acceptable solution containing the conjugate is prepared, and a therapeutically effective dose of the conjugate is administered to an individual having a uPA-mediated or a uPA receptor-mediated disorder. The conjugate is preferably administered via injection either intravenously or, more preferably, subcutaneously. Administration is repeated as necessary in order to maintain therapeutically effective levels of the conjugate.

Pharmaceutical compositions comprising a conjugate of a biologically active molecule and a polymer can be prepared by mixing the conjugate with any pharmaceutically acceptable component, such as, for example, a carrier, a medicinal agent, an adjuvant, a diluent, and the like, as well as combinations of any two or more thereof. Suitable pharmaceutical carriers, medicinal agents, adjuvants, and diluents are described in "Remington's Pharmaceutical Sciences," 18^th edition, by E. W. Martin (Mack Publ. Co., Easton, Pa.).

The composition may be administered in a variety of ways, including, for example, orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, as suppositories, etc. The specific amount of active conjugate administered will, of course, depend on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician. Information concerning dosages of various pharmacological agents may be found in standard pharmaceutical reference books, e.g., "Remington's Pharmaceutical Sciences," supra. The pharmaceutical compositions may be in solid, semi-solid or liquid dosage forms, such as, for example, tablets, pills, capsules, powders liquids, suspensions, and the like.

It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Claim 1 of 16 Claims

1. A method for preparing a biologically active polypeptide having an increased serum half-life, comprising coupling the polypeptide to a polymer by reacting an aldehyde functionality on the polypeptide with a hydrazide or semicarbazide group on the polymer under reaction conditions effective to promote formation of a conjugated polypeptide, wherein the polypeptide is bound to the polymer through a hydrazone or semicarbazone linkage.

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