Title:  Methods and compositions for delivery of therapeutic agents to bone tissue employing conjugates of negatively charged peptide oligomers with therapeutic agents

United States Patent:  6,455,495

Issued:  September 24, 2002

Inventors:  Orgel; Leslie (La Jolla, CA); Chu; Barbara Chen Fei (Del Mar, CA)

Assignee:  The Salk Institute for Biological Studies (La Jolla, CA)

Appl. No.:  367516

Filed:  December 14, 1999

PCT Filed:  February 13, 1998

PCT NO:  PCT/US98/02811

371 Date:  December 14, 1999

102(e) Date:  December 14, 1999

PCT PUB.NO.:  WO98/35703

PCT PUB. Date: August 20, 1998

The invention provides conjugates of negatively charged peptide oligomers with therapeutic agents, moieties capable of recruiting endogenous bone-affecting agents, or imaging agents, useful for delivering the agents to bone tissue or calcified masses, and the methods of use thereof. The negatively charged peptide oligomers bind strongly but reversibly to bone tissue and calcified masses with a controllable affinity and retention time on the tissue or mass.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

The one- and three-letter abbreviations used herein for the various common amino acids are as recommended in Pure Appl. Chem. 31, 639-645 (1972) and 40, 277-290 (1974) and comply with 37 CFR .sctn.1.822 (55 FR 18245, May 1, 1990). The abbreviations represent L-amino acids unless otherwise designated as D- or D,L-. Certain amino acids, both natural and non-natural, are achiral, e.g. glycine. All peptide sequences are presented with the N-terminal amino acid on the left and the C-terminal amino acid on the right.

The term "peptide oligomer" refers to a segment of at least about 3 amino acids, up to about 20 to about 50 amino acids, and does not include any natural proteins.

The term "negatively charged peptide oligomer" refers to a peptide oligomer of amino acid residues, wherein one or more amino acids, typically at least three amino acids, are negatively charged amino acids.

The term "negatively charged amino acid" refers to a natural or non-natural amino acid, regardless of chirality, containing, in addition to the C-terminal carboxyl group, at least one additional negatively charged group such as carboxyl, phosphate, phosphonate, sulfonate, or the like.

The term "conjugate" refers to and embraces a negatively charged peptide oligomer linked via a covalent bond to a therapeutic agent, moiety, or imaging agent, such as those described below, wherein such linkage is formed directly or indirectly via a linking agent.

Preferred Embodiments

The invention provides conjugates of negatively charged peptide oligomers with (a) therapeutic agents, (b) moieties capable of recruiting endogenous bone-affecting agents, and (c) imaging agents, useful for delivering such agents to bone tissue or calcified masses, and methods of use thereof. The negatively charged peptide oligomers bind strongly but reversibly to bone tissue and calcified masses. Both the binding affinity and the retention time can be controlled by use of the appropriate amino acids in the peptide oligomer. The term bone tissue as used herein, includes various calcified tissues such as bone and teeth; the term calcified masses includes such materials as calcium oxalate stones, and calcified implants.

One aspect of the invention provides a method of delivering a therapeutic agent to bone tissue of a mammalian subject comprising administering to the subject an effective amount of a composition comprising a conjugate of a negatively charged peptide oligomer with a therapeutic agent, and a pharmaceutically acceptable carrier. The negatively charged peptide oligomer, also referred to herein a peptide oligomer having negatively charged amino acids, has an affinity for the hydroxyapatite component of bone tissue and thereby binds to the bone tissue and brings the therapeutic agent with which it is conjugated into close contact with the bone tissue. In another aspect, the aforementioned composition may be used to deliver the therapeutic agent directly to cell surface receptors associated with bone tissue of the subject.

As described above, the negatively charged peptide oligomers can effectively deliver a therapeutic agent to bone tissue by being conjugated with the therapeutic agent. In another aspect of the invention, the peptide oligomers may be conjugated with moieties which are themselves capable of binding to, and thereby recruiting to bone tissue, endogenous bone affecting agents. Thus, one aspect of the invention provides a method of recruiting an endogenous bone affecting agent to bone tissue of a mammalian subject, comprising administering an effective amount of composition comprising a conjugate of a negatively charged peptide oligomer with a moiety capable of binding the bone affecting agent, and a pharmaceutically acceptable carrier. In a similar manner to that described above, such compositions may be used in a method of recruiting the endogenous bone affecting agent directly to cell surface receptors associated with bone tissue of a mammalian subject.

The affinity of the negatively charged peptide oligomers for bone tissue and calcified masses is also useful in imaging such tissue or mass. Accordingly, one aspect of the invention provides a method of imaging bone tissue or a calcified mass of a mammalian subject, comprising administering to said subject an effective amount of a composition comprising a conjugate of a negatively charged peptide oligomer with an imaging agent, and a pharmaceutically acceptable carrier, and detecting the presence of said imaging agent bound to said bone tissue or calcified mass. A wide variety of imaging agents known in the art may be employed, such as radionuclides, (e.g., technetium 99), various particles (e.g., gold, ferritin, magnetic particles, red blood cells), fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, numerous moieties (particularly haptens), and chemiluminescers. Radionuclides are preferred and the preferred radionuclide is technetium-99 m. Chelating agents may be necessary to bind certain imaging agents, particularly radionuclides and certain particles, to the negatively charged peptide oligomer, and such chelating agents for purposes herein shall be considered to be part of the imaging agent conjugated to the oligomer. Suitable chelating agents are well known in the art and include, e.g., ethylenediaminetetraacetate (EDTA), diethylenetriaminepentaacetate, and the like. EDTA is a preferred chelating agent. As described in more detail below, controlling the affinity and rate of degradation of the oligomer conjugates enables one to precisely deliver the imaging agent for the desired period of time, thereby avoiding the shortcomings of procedures currently employed for such purpose.

Negatively charged peptide oligomers bound to hydroxyapatite are also useful in separating substances of interest from a solution by transferring the substance from a liquid phase to a solid phase. Another aspect of the invention, therefore, provides a method of separating a substance of interest from a solution containing the substance, comprising contacting hydroxyapatite with a conjugate of a negatively charged peptide oligomer with a moiety capable of binding the substance of interest to form an adsorption support and intimately contacting the adsorption support with the solution whereby the substance is bound to the adsorption support. The adsorption support and solution may be intimately contacted by a variety of methods including for example passing the solution through a column of the adsorption support, or by shaking the solution with the support. The bound substance is separated from the spent solution, as for example by filtration or by other gravitational means. The bound substance may then be separated from the adsorption support, as for example by contacting the substance bound to the support with an eluting agent that causes the substance to be eluted from the support, and collecting the eluate containing the substance of interest. It will be apparent to the skilled artisan that a number of suitable anion exchangers exist, in addition to hydroxyapatite, for binding to the negatively charged peptide oligomer. Therefore, the adsorption support could be formed from any suitable anion exchange material bound to a conjugate of a negatively charged peptide oligomer with a moiety capable of binding the substance of interest.

Another aspect of the invention provides compositions of matter and pharmaceutical compositions employed in the methods discussed above.

The binding affinity of the invention oligomer conjugates to bone tissue, calcified mass, or hydroxyapatite can be controlled by varying the total negative charge of the peptide oligomer and the ratio of total negative charge to number of amino acid residues in the peptide oligomer.

The negatively charged peptide oligomer binds to bone tissue, calcified mass, or hydroxyapatite with an affinity, and therefore a retention time, that increases with the total negative charge of the peptide oligomer. The total negative charge on the peptide oligomer must be sufficient to ensure that the peptide oligomer binds to the bone tissue, calcified mass, or hydroxyapatite. The total negative charge of the peptide oligomer is also dependent on the number of negatively charged amino acid residues in the oligomer. The presently preferred negatively charged peptide oligomer has between about 3 and about 20 negatively charged amino acids. As the negative charge is increased above the minimum charge needed for binding, the strength of the bond between the peptide oligomer and bone tissue, calcified tissue or hydroxyapatite increases. The minimum total negative charge of the peptide oligomer is preferably at least 6. A total negative charge of 6 is provided for example, by the hexamer of glutamic acid (Glu₆) or the trimer of 2-amino-5-phosphonovaleric acid.

Binding affinity of the peptide oligomer conjugate can also be controlled by varying the ratio of the total negative charge to the number of amino acid residues in the peptide oligomer. The presently preferred ratio of total negative charge to number of amino acid residues in the peptide oligomer is between about 0.5:1 to about 2:1. The specific ratio for a given oligomer will depend on the identity and charge density of the negatively charged groups, and the size of the oligomer.

One aspect of the invention provides the negatively charged peptide oligomer contain negatively charged groups selected from the group consisting of carboxyl, phosphate, phosphonate, and sulfonate. The presently preferred negatively charged groups are carboxyl and phosphonate. The charge density of the negatively charged

groups varies depending upon the group selected, for example the carboxyl group has a negative charge of 1 whereas the phosphonate group has a negative charge of 2, so that the total negative charge of the peptide oligomer will depend upon the type of negatively charged groups that are present. The negatively charged groups may all be the same or a mixture of groups may be used in forming the negatively charged peptide oligomer.

A wide variety of amino acids may be employed in the negatively charged peptide oligomers, including naturally occurring amino acids such as glutamic acid and aspartic acid, and non naturally occurring amino acids such as 2-amino-5-phosphonovaleric acid, and the like. The peptide oligomer may consist of only one type of amino acid such as glutamic acid, or a combination of two or more different amino acids such as glutamic acid and aspartic acid. Presently preferred amino acids are glutamic acid and aspartic acid. One preferred peptide oligomer is glutamic acid decapeptide (glu₁₀). One aspect of the invention provides a negatively charged peptide oligomer wherein the negatively charged groups are carboxy groups and the peptide oligomer contains between about 6 to about 10 amino acids. Another aspect of the invention provides a negatively charged peptide oligomer wherein the negatively charged groups are phosphonate groups and the peptide oligomer contains between about 3 to about 5 amino acids.

The negatively charged peptide oligomers bind strongly but reversibly to bone tissue. By varying the total negative charge of the peptide oligomers as well as the ratio of total negative charge to number of amino acid residues in the peptide oligomer, a wide range of binding affinities of the peptide oligomer for bone tissue is provided. The binding affinity of the negatively charged peptide oligomers for bone tissue, calcified masses or hydroxyapatite is at least sufficient that a given size oligomer will bind thereto.

With respect to (a) the delivery of therapeutic agents, (b) recruitment of endogenous bone affecting agents, or (c) imaging of bone tissue or calcified masses, the rate of proteolytic degradation of the negatively charged peptide oligomer can be controlled by incorporating at least one D-amino acid. It will be apparent to the skilled artisan that the number and position of D-amino acids incorporated in the peptide oligomer will vary depending upon the agent to be delivered and the extent of resistance to proteolytic degradation desired. The presently preferred percentage of D-amino acid residues to total number of amino acid residues is between about 5% to about 100%. The N-terminal positions on the peptide oligomer are preferred for the D-amino acids.

The peptide oligomer may contain a random mixture of D and L amino acids. Alternatively, it may be preferable for the peptide oligomer to consist solely of D amino acids, or an ordered arrangement of D and L amino acids. It is preferred that a single diastereomeric conjugate be utilized in the methods and compositions of the present invention.

The conjugates of negatively charged peptide oligomers referred to above are useful for delivering a wide variety of therapeutic agents to bone tissue. Representative examples include antineoplastic agents such as methotrexate; bone formation stimulating agents such as insulin-like growth factors, bone morphogenic protein, fibrobast growth factor, and platelet derived growth factor; bone formation inhibiting agents such as glucocorticoids, and vitamin D derivatives such as 1, 25-dihydroxyvitamin D3; cathepsin K inhibitors, and agents which affect bone resorption such as macrophage colony stimulating factor, interleukins and other cytokines, bisphosphonate, calcitonin; and the like.

The negatively charged peptide oligomer may also be conjugated with a wide variety of moieties capable of binding endogenous bone affecting agents. This is particularly useful in making the delivery of such endogenous agents more effective than by relying solely upon their delivery from body fluids. Such moieties include, for example, antibodies capable of binding antigens, and portions of proteins, such as recognition sequences, that bind the agent of interest. The endogenous bone affecting agents include proteins, antigens, and the like, including endogenous therapeutic agents such as those mentioned above.

It is particularly useful when the therapeutic agent or the moiety to be conjugated with the peptide oligomer is itself a peptide, thereby allowing the conjugate to be prepared, for example, by a single solid-phase peptide synthesis. In addition, the conjugate could be prepared by genetic engineering, whereby recombinant DNA is used which encodes a conjugate of an endogenous or nonendogenous protein therapeutic agent or moiety with a negatively charged peptide oligomer. The recombinant DNA, which may be produced using known genetic engineering techniques, therefore encodes a conjugate of a protein with a terminal segment, or "tail", comprising the negatively charged peptide oligomer. Such conjugate would thus have a greater affinity for bone tissue than the protein itself. It should be noted that while the negatively charged peptide oligomers are no more than about 50 amino acids in length, the protein therapeutic agent or moiety which is conjugated with a negatively charged peptide oligomer is not limited as to the number of amino acids.

The conjugation of the negatively charged peptide oligomer to a therapeutic agent or moiety as set forth herein, can be effected by chemical conjugation procedures well known in the art, such as by creating peptide linkages, use of condensation agents, and by employing well known bifunctional cross-linking reagents. The conjugation may be direct, which includes linkages not involving any intervening group, e.g., direct peptide linkages, or indirect, wherein the linkage contains an intervening moiety, such as a protein or peptide, e.g., plasma albumin, or other spacer molecule. For example, the linkage may be via a heterobifunctional or homobifunctional cross-linker, e.g., carbodiimide, glutaraldehyde, N-succinimidyl 3-(2-pyridydithio) propionate (SPDP) and derivatives, bis-maleimide, 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, and the like. Cross-linking may also be accomplished without exogenous cross-linkers by utilizing reactive groups on the molecules being conjugated. Methods for chemically cross-linking peptide molecules are generally known in the art, and a number of hetero- and homobifunctional agents are described in, e.g., U.S. Pat. Nos. 4,355,023, 4,657,853, 4,676,980, 4,925,921, and 4,970,156, and Immuno Technology Catalogue and Handbook, Pierce Chemical Co. (1989), each of which is incorporated herein by reference. Cleavable cross-linkers, particularly those that form cleavable disulfide bonds, may be employed to allow cleavage of the conjugate to free the therapeutic agent under physiological conditions. An example of such a cleavable cross-inker is 4-succinimidyloxycarbonyl-a-(2-pyridyldithio)-toluene. Such conjugation, including cross-linking, should be performed so as not to substantially affect the desired function of the peptide oligomer or entity conjugated thereto, including therapeutic agents, and moieties capable of binding substances of interest.

Conjugation of a negatively charged peptide oligomer to an imaging agent can be effected by well known procedures in the art, including having the negatively charged peptide oligomer complex a radionuclide directly, or by chemical conjugation of the negatively charged peptide oligomer with a coordination complex of a radionuclide and a chelating agent.

Conjugation of a negatively charged peptide oligomer can be effected by a linkage via the N-terminal or the C-terminal of the peptide oligomer, resulting in an N-linked peptide oligomer or a C-linked peptide oligomer, respectively.

Classical synthesis of invention peptide oligomers can be accomplished by suitable methods, such as exclusively solid phase techniques, partial solid-phase techniques, fragment condensation or classical solution couplings. For example, techniques of exclusively solid phase synthesis are set forth in the textbook "Solid-Phase Synthesis", Stewart & Young, Freemen & Company, San Francisco, 1969, and are exemplified by the disclosure of U.S. Pat. No. 4,105,603, issued Aug. 8, 1979. Classical solution synthesis is described in detail in "Methoden der Organischen Chemic (Houben-Weyl): Synthese von Peptiden", E. Wunsch (editor) (1974) Georg Thieme Verlag, Stuttgart West Germany. The fragment condensation method of synthesis is exemplified in U.S. Pat. No. 3,972,859, issued Aug. 3, 1976. Other available syntheses are exemplified in U.S., Pat. No. 3,842,067, issued Oct. 15, 1974 and U.S. Pat. No. 3,872,925, issued Jan. 28, 1975. The foregoing disclosures are incorporated herein by reference.

Alternatively, recombinant DNA synthesis may be employed to synthesize invention peptide oligomers containing natural amino acid residues. Recombinant techniques are well known to those skilled in the art. Representative methods are disclosed in Maniatis, et al., Molecular cloning, a Laboratory Manual, 2nd edition, Cold Springs Harbor Laboratory (1989), incorporated herein by reference. As mentioned above, recombinant DNA synthesis can be used to produce not only the negatively charged peptide oligomer, but also a conjugate of the peptide oligomer with an endogenous or nonendogenous protein therapeutic agent or moiety.

The invention conjugates of negatively charged peptide oligomers with therapeutic agents and moieties capable of binding endogenous bone affecting agents are useful for the prevention and treatment of a variety of conditions involving mammalian bone tissue. In particular, such conjugates are indicated for the prophylaxis and therapeutic treatment of mammalian bone conditions such as osteoporosis or osteosarcoma. Moreover, conjugates of negatively charged peptide oligomers with imaging agents are useful in bone imaging.

In general, such conjugates when used for therapeutic purposes will be administered in effective amounts for the desired purpose, with such effective amounts dependent on the disease. For example, for intravenous administration the amounts will range from between about 1.0 .mu.g/kg body weight per hour of administration and 1.0 mg/kg body weight per hour of administration, preferably from about 10 to about 100 .mu.g/kg body weight per hour of administration. For a 50 kg human female subject, the daily dose of active ingredient (conjugate) would be from about 50 .mu.g/hour to about 50 mg/hour, preferably from about 500 .mu.g/hour to about 5 mg/hour. Single or multiple administrations or a controlled release formulation of the compositions can be delivered in conventional pharmaceutical compositions as needed, to achieve the most effective results. The conjugates of negatively charged peptide oligomers with imaging agents would typically be administered in amounts between about .0.01 and about 1.0 mg/kg body weight, and the dosage would typically contain between about 5 and about 20 mCi of radioactivity when the peptide oligomer is conjugated with a radionuclide.

The selection of the exact dose and composition and the most appropriate delivery regimen will be influenced by, inter alia, the pharmacological properties of the selected therapeutic agent or endogenous agent, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient.

Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular and intravenous), topical, rectal, buccal (including sublingual), transdermal, and intranasal. The presently preferred mode of administration is parenteral, particularly intravenous.

A further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a conjugate of a negatively charged peptide oligomer with (a) a therapeutic agent, (b) a moiety capable of recruiting a bone affecting agent, or (c) an imaging agent, in admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned above, such compositions may be prepared for parenteral (subcutaneous, intramuscular or intravenous) administration, particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; for intranasal administration, particularly in the form of powders, nasal drops or aerosols; and for rectal or transdermal administration.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., (1985), incorporated herein by reference. Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. For oral administration, the formulation can be enhanced by the addition of bile salts or acylcarnitines. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered spray. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across the nasal mucous membrane may be enhanced by surfactant acids, such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrins and the like.

Delivery of the conjugates of the present invention to a subject over prolonged periods of time, for example, for periods of one week to one year, may be accomplished by a single administration of a controlled release system containing sufficient active ingredient for the desired release period. Various controlled release systems, such as monolithic or reservoir-type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms may be utilized for this purpose. Localization at the site to which delivery of the active ingredient is desired is an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders.

One form of controlled release formulation contains the invention conjugates dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as copoly(lactic/glycolic) acid, as described in the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No. 4,675,189, incorporated by reference herein. The conjugates may also be formulated in cholesterol or other lipid matrix pellets, or silastomer matrix implants. Additional slow release, depot implant or injectable formulations will be apparent to the skilled artisan. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, Controlled Release of Biologically Active Agents, John Wiley & Sons, New York, 1987, incorporated by reference herein.

As described above, conjugates of endogenous or non-endogenous proteins with negatively charged peptide oligomers are particularly useful inasmuch as they have a greater affinity for bone tissue than the proteins themselves. It has also been described that such conjugates may easily be produced by recombinant DNA synthesis. Accordingly, another valuable method of delivering a therapeutic protein to bone tissue of a mammalian subject comprises the in vivo production of a conjugate of such protein with a negatively charged peptide oligomer. Such in vivo production can be accomplished by gene therapy techniques known in the art whereby a gene encoding such conjugate is inserted into the subject and expressed.

The following specific Examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims. Glutamic acid decapeptide (Glu₁₀) and hexapeptide (Glu₆) was synthesized by standard solid phase peptide synthesis. D,L-2-amino-5-phosphonovaleric acid, methotrexate, chicken liver dihydofolate reductase and N-hydroxy-succinimide were obtained from Sigma Chemical Company. 1,1-carbonyldiimidazole and dicyclohexylcarbodiimde were obtained from Aldrich Chemical Company. Succinimidyl-6-(biotinamido) hexanoate was obtained from Pierce Chemical Company, and hydroxyapatite from BioRad Laboratories.

Claim 1 of 29 Claims

What is claimed is:

1. A method of delivering a therapeutic agent to bone tissue of a mammalian subject, comprising:

administering to said subject an effective amount of a composition comprising a conjugate of a negatively charged peptide oligomer directly bonded to said therapeutic agent, wherein said conjugate is administered in a pharmaceutically acceptable carrier.
 

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