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WO2005120588A2 - Peptides delivered to cell nuclei - Google Patents

Peptides delivered to cell nuclei Download PDF

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Publication number
WO2005120588A2
WO2005120588A2 PCT/US2005/018700 US2005018700W WO2005120588A2 WO 2005120588 A2 WO2005120588 A2 WO 2005120588A2 US 2005018700 W US2005018700 W US 2005018700W WO 2005120588 A2 WO2005120588 A2 WO 2005120588A2
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WO
WIPO (PCT)
Prior art keywords
lys
arg
glu
gly
pro
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PCT/US2005/018700
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French (fr)
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WO2005120588A3 (en
Inventor
Thomas P. Quinn
Miao Yubin
Fabio Gallazzi
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The Curators Of The University Of Missouri
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Publication of WO2005120588A2 publication Critical patent/WO2005120588A2/en
Publication of WO2005120588A3 publication Critical patent/WO2005120588A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/68—Melanocyte-stimulating hormone [MSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/08—Ethers or acetals acyclic, e.g. paraformaldehyde
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the field of nuclear delivery of peptides and cell imaging.
  • the present invention also relates generally to the fields of cancer biology and cancer therapeutics.
  • G-protein coupled receptors are cell surface receptors that indirectly transduce extracellular signals to downstream effectors, which can be intracellular signaling proteins, enzymes, or channels, and changes in the activity of these effectors then mediate subsequent cellular events.
  • the interaction between the receptor and the downstream effector is mediated by a G-protein, a heterotrimeric protein that binds GTP.
  • G-protein coupled receptors (“GPCRs") typically have seven transmembrane regions, along with an extracellular domain and a cytoplasmic tail at the C-terminus. These receptors form a large superfamily of related receptors molecules that play a key role in many signaling processes, such as sensory and hormonal signal transduction.
  • GPCR G-protein coupled receptors
  • melanocortin receptors bind and are activated by peptides such as ⁇ -, ⁇ , or ⁇ -melanocyte stimulating hormones ( ⁇ -, ⁇ -, ⁇ -MSH) derived from the pro-opiomelanocortin (POMC) gene.
  • ⁇ -, ⁇ -, ⁇ -MSH ⁇ -melanocyte stimulating hormones
  • POMC pro-opiomelanocortin
  • U.S. Pat. No. 5,532,347 discloses human and mouse DNA molecules which encode MC-1R (also known in the art as ⁇ -MSH-R).
  • the expressed human protein contains 317 amino acids.
  • Alpha melanocyte stimulating hormone ( ⁇ -MSH) is a tridecapeptide [Ac-Ser- Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH 2 ] that regulates skin pigmentation in most vertebrates (Hruby, V. J., 1993).
  • the core ⁇ -MSH sequence His-Phe-Arg-Trp conserved in a number of species, has been found to be sufficient for receptor recognition (Hruby, V.
  • ⁇ -melanotropin is a naturally-occurring tridecapeptide that specifically recognizes melanotropin receptors. Since mouse melanoma cells and human melanocytes possess ⁇ - melanotropin ( ⁇ -MSH) receptors, the use of radiolabeled ⁇ -MSH analogs for diagnosis and treatment of ⁇ -MSH positive cancers (i.e., melanoma) was hypothesized.
  • ⁇ -MSH ⁇ - melanotropin
  • Various synthetic ⁇ - melanotropin analogs have been prepared and characterized for ⁇ -melanotropin activity by V. J. Hruby, M. E. Hadley et al. They have reported that cyclic analogs of ⁇ -MSH (see U.S. Pat. No. 4,485,039) display properties which increase their potency toward the ⁇ -MSH receptor, prolong their activity and increase their resistance to in vivo enzymatic degradation.
  • An embodiment of the invention is a method of delivering a peptide to a cell nucleus comprising contacting a cell expressing a G-protein coupled receptor with a peptide comprising a G-protein coupled receptor binding sequence and a nuclear localization signal.
  • the G-protein coupled receptor is an opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P, substance-R, or glutamate receptor.
  • the G- protein coupled receptor is a melanocortin- 1 receptor.
  • the peptide is linked to a radioisotope, a pharmaceutical compound, an MRI contrast reagent, or a fluorescent label.
  • the radioisotope is an alpha-emitter, a beta-emitter, a gamma-emitter, or an Auger emitter.
  • the radioisotope is In-111, Bi-214, At-211, or Bi-212.
  • the peptide is linked to an imaging agent.
  • the imaging agent is a DNA intercalator in one embodiment ofthe invention.
  • the cell expressing a G-protein coupled receptor is selected from the group consisting of a melanoma cell, liver cell, a brain cell, a pituitary cell, a keratinocyte cell, or an immune system cell.
  • the G-protein coupled receptor binding sequence comprises a melanocortin- 1 receptor binding sequence.
  • the melanocortin- 1 receptor binding sequence comprises His-Phe-Arg-Trp (SEQ ID NO:l).
  • the melanocortin- 1 receptor binding sequence comprises His-DPhe-Arg-Trp.
  • the peptide comprises D amino acids. In other embodiment ofthe invention, the peptide comprises a mix of D and L amino acids.
  • An embodiment ofthe invention is a peptide comprising the sequence: Lys-Gly- Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2), wherein Nle is norleucine.
  • An embodiment ofthe invention is a peptide comprising the sequence: Lys-Gly- Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-Trp-Cys-Arg-Pro-Val (SEQ ID NO:3).
  • An embodiment ofthe invention is a peptide comprising the sequence: Gly-Pro- Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val wherein Nle is norleucine.
  • An embodiment of the invention is a peptide comprising D amino acids comprising the sequence: Val-Pro-Lys-Gly-Trp-Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg- Lys-Lys-Lys-Pro-Gly-Lys wherein Nle is norleucine.
  • An embodiment of the invention is a peptide comprising D amino acids comprising the sequence: Val-Pro-Lys-Gly-Trp-Arg-Phe-His-Glu-Nle-Ala-Glu-Met-Ser-Lys-Ser wherein Nle is norleucine.
  • the nuclear localization signal comprises K(K/R)x(K R) (SEQ ID NO:4), where X is any amino acid.
  • the nuclear localization signal comprises KRXR.
  • An embodiment of the invention is a method of cell imaging comprising (a) contacting a cell expressing a G-protein coupled receptor with a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the peptide is linked to an imaging agent; and (b) obtaining an image ofthe cell.
  • An embodiment of the invention is a method of nuclear delivering of a pharmaceutical compound to a cell nucleus comprising contacting a cell expressing a melanocortin- 1 receptor with a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the peptide is linked to the pharmaceutical compound.
  • An embodiment ofthe invention is a method in vivo delivering of a peptide to a tumor comprising administering to a patient with a melanocortin- 1 receptor-expressing tumor a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the nuclear localization signal forms a positively charged helix.
  • An embodiment of the invention is a host cell expressing any of the peptides described herein.
  • FIG. 1 is a helical wheel projection of the NLS sequence (SEQ ID NO:5) with membrane translocation peptides.
  • the bracket highlights the positively charged face.
  • FIG. 2 shows the peptide MSH peptide containing the NLS sequence has significantly higher accumulation peptide in the nucleus.
  • the NLS sequence was successfully incorporated into the MSH sequence without affecting the biological activity of MCI receptor targeting.
  • the NLS signal is also active and allows a significant amount of the NLS-
  • NDP peptide to be transported to the cell nucleus.
  • FIG. 3 contains images of another cell from a well of cells incubated with (TMR)-NLS-NDP and counter stained with Cyto-5 (Green).
  • the left image is a combination overlay of the microscopic picture of the cell, the Cyto-5 image and the (TMR)-NLS-NDP image.
  • FIG. 4 shows a colony growth assay.
  • B16-F1 melanoma cells alone or treated with '"in, [ l u In]-DOTA-NDP or [ n ⁇ In]-DOTA-NLS-NDP were plated and examined for colony formation in 24-well tissue culture plates. Growth assays were performed in triplicate. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides peptides comprising G-protein coupled receptors binding sequences and nuclear localization signals that are delivered to cell nuclei.
  • the peptides bind G-protein coupled receptors, such as the melanocortin- 1 receptor, on the surface of he cell, and are internalized and targeted to the cell nuclei by virtue of cellular recognition of the nuclear localization signal.
  • the nuclear targeted peptides of the present invention escape lysosomal degradation by virtue of comprising a nuclear localization signal.
  • Also contemplated in the present invention are methods of delivering these peptides to cell nuclei, and delivering imaging compounds or therapeutic compounds to cell nuclei through the use of peptides as described herein.
  • Nuclear localization signals are present in the peptides described in the present invention.
  • Nuclear localization signal are peptide sequences that direct peptides to the nuclear transportation machinery.
  • Examples of nuclear localization signals (nuclear localization sequences) known in the art are the SV40 large T antigen nuclear localization signal, PKKKRKV (amino acids 3-9 of SEQ ID NO:5), and the c-myc proto-oncogene nuclear localization signal, with a sequence of PAAKRVKLD (SEQ ID NO:6).
  • Nuclear localization signals contemplated by the present invention include both monopartite and bipartite sequences.
  • Bipartite signals are characterized as a small cluster of basic residues positioned 10-12 residues N-terminal to a monopartite-like sequence.
  • An example of a bipartite nuclear localization signal is KRPAATKKAGQAKKKKL (SEQ ID NO:7).
  • the nuclear localization signal comprises the motif K(K R)x(K/R) (SEQ ID NO:4).
  • the nuclear localization signal is KRXR, wherein X is any amino acid.
  • G protein coupled receptors include all subtypes of the opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P and substance-R receptors, melanocortin, metabotropic glutamate, or any other GPCR known to couple via G proteins.
  • Polypeptide ligands of GPCRs include vasopressin, oxytocin, somatostatin, neuropeptide Y, GnRH, leutinizing hormone, follicle stimulating hormone, parathyroid hormone, orexins, urotensin II, endorphins, and enkephalins.
  • GPCRs respond not only to peptide ligands but also small molecule neurotransmitters (acetylcholine, dopamine, serotonin and adrenaline), light, odorants, taste, lipids, nucleotides, and ions. It is contempalted that GPCRs that respond to peptide ligands, including peptide hormones, are particularly useful for the present invention.
  • G-protein-coupled receptors are integral membrane proteins that possess seven membrane-spanning elements or transmembrane helices. The extracellular parts of the receptor can by glycosylated. These extracellular loops also contain highly conserved cysteine residues which build disulfide bonds to stabilize the receptor structure.
  • Signalling in G-protein coupled receptors causes interaction between the G- protein-coupled receptor and the G protein. Their interaction results in the dissociation between ⁇ and ⁇ subunits of the G protein.
  • the separated ⁇ and/or ⁇ subunits may then interact with effectors and cause downstream signaling events. These downstream signaling events includeintracellular calcium release and cAMP production.
  • the intracellular signaling systems used by peptide GPCRs are similar to those used by all GPCRs, and are typically classified according to the G-protein they interact with and the second messenger system that is activated.
  • Gs-coupled GPCRs activation of the G-protein Gs by receptor stimulates the downstream activation of adenylate cyclase and the production of cyclic AMP, while Gi-coupled receptors inhibit cAMP production.
  • cAMP production One of the key results of cAMP production is activation of protein kinase A.
  • Gq-coupled receptors stimulate phospholipase C, releasing IP3 and diacylglycerol. IP3 binds to a receptor in the ER to cause the release of intracellular calcium, and the subsequent activation of protein kinase C, calmodulin-dependent pathways.
  • GPCR pathways exhibit crosstalk with other signaling pathways including tyrosine kinase growth factor receptors and map kinase pathways. Transactivation of either receptor tyrosine kinases like the EGF receptor or focal adhesion complexes can stimulate ras activation through the adaptor proteins She, Grb2 and Sos, and downstream Map kinases activating Erkl and Erk2. Src kinases may also play an essential intermediary role in the activation of ras and map kinase pathways by GPCRs.
  • G-protein coupled receptors include alpha adrenergic ⁇ eceptors(ADRAlA OR ADRAIC; Accession No. P35348 (SEQ ID NO:8)), adreneocortpicotropic hormone receptors (MC2R OR ACTHR; Accession No. Q01718 (SEQ ID NO:9)), angiotensin type 1 receptors (AGTRl OR AGTRIA OR AT2R1; Accession No. P30556 (SEQ ID NO: 10)), melanocortin- 1 receptor (MC1R OR MSHR; Accession No. Q01726 (SEQ ID NO:l 1)), and Substance K receptor (TACR2 OR TAC2R OR NK2R; Accession No. P21452 (SEQ ID NO: 12)).
  • ADRAlA OR ADRAIC alpha adrenergic ⁇ eceptors
  • M2R OR ACTHR Accession No. Q01718 (SEQ ID NO:9)
  • angiotensin type 1 receptors
  • Linking/Coupling Agents can be used to combine to components or agents of the present invention, such as, for example, polyethylene glycol bifunctional linkages, antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, amino acid or modified amino acid linkages, or combinations thereof.
  • An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).
  • primary amine group e.g., N-hydroxy succinimide
  • a thiol group e.g., pyridyl disulfide, maleimides, halogens, etc.
  • the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
  • a cross-linker having reasonable stability in blood can be employed.
  • Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
  • Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups.
  • steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
  • the SMPT cross-linking reagent lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine).
  • cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-l,3'-dithiopropionate.
  • non-hindered linkers also can be employed in accordance herewith.
  • Other useful cross-linkers include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
  • Patent 4,680,3308 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like.
  • U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent.
  • Preferred uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
  • U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies.
  • the linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation.
  • U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.
  • V. Synthetic Peptides [0048] The present invention describes peptides for use in various embodiments of the present invention.
  • the peptides ofthe invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference..
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • peptides ma be isolated from natural sources using standard techniques, it will be advantageous to produce peptides using the solid-phase synthetic techniques (Merrifield, 1963).
  • Other peptide synthesis techniques are well known to those of skill in the art (Bodanszky et al, 1976;) Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984); .
  • Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973.
  • These synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group ofthe first amino acid residue is protected by a suitable, selectively removable protecting group.
  • a different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.
  • the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
  • the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support.
  • the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide.
  • the peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids.
  • Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
  • peptides are synthesized which comprises D amino acids. It is contemplated that peptides comprising L, D, or a mix of L and D amino acids are appropriate.
  • Imaging Agents there are provided diagnostic methods for detecting cells.
  • Many appropriate imaging agents for use in the present invention are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference).
  • the agent may comprise an imaging agent or region thereof, by which term is meant an agent which may be detected, whether in vitro in the context of a tissue, organ or organism in which the agent is located.
  • the imaging agent may emit a detectable signal, such as light or other electromagnetic radiation.
  • the imaging agent may be a radio-isotope as known in the art, for example 32 P, 35 S, 99 Tc, n ⁇ In, 86 Y, 18 F, and 99m Tc or a molecule such as a nucleic acid, polypeptide, or other molecule as explained below conjugated with such a radio-isotope
  • the imaging agent may be opaque to radiation, such as X-ray radiation.
  • the imaging agent may also comprise a targeting means by which it is directed to a particular cell, tissue, organ or other compartment within the body of an animal.
  • the agent may comprise a radiolabelled antibody specific for defined molecules, tissues or cells in an organism.
  • the imaging agent may be combined with, conjugated to, mixed with or combined with, any of the agents disclosed herein. Combinations of agents with multiple or overlapping specificities or utilities are clearly contemplated by this invention.
  • Radioisotopes [0055] h the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine , chromium, chlorine, cobalt, cobalt, copper , europium, gallium , iodine , iodine , iodine , indium , iron, phosphorus, rhenium , rhenium , 75 selenium, 35 sulphur, technicium m and/or yttrium 90 . Of particular interest are lutetium 177 , samarium , holmium and actinium" . Also, see Table 2, below.
  • Radioactively labeled peptides of the present invention may be produced according to well-known methods in the art.
  • peptides can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Peptides according to the invention may be labeled with technetium 99 TM by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
  • direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).
  • Fluorescent labels include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red. 3.
  • Paramagnetic contrasting agents There is a rapidly growing body of literature demonstrating the clinical effectiveness of paramagnetic contrast agents. The capacity to differentiate regions/tissues that may be magnetically similar but histologically distinct is a major impetus for the preparation of these agents. In the design of MRI agents, strict attention must be given to a variety of properties that will ultimately effect the physiological outcome apart from the ability to provide contrast enhancement. Two fundamental properties that must be considered are biocompatability and proton relaxation enhancement. Biocompatability is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Proton relaxation enhancement (or relaxivity) is chiefly governed by the choice of metal and rotational correlation times.
  • the first feature to be considered during the design stage is the selection of the metal atom, which will dominate the measured relaxivity of the complex.
  • Paramagnetic metal ions act as potent relaxation enhancement agents. They decrease the T ⁇ and T 2 relaxation times of nearby (r 6 dependence) spins. Some paramagnetic ions decrease the T] without causing substantial linebroadening (e.g., gadolinium (III), (Gd )), while others induce drastic linebroadening (e.g. superparamagnetic iron oxide).
  • the mechanism of Tj relaxation is generally a through space dipole-dipole interaction between the unpaired electrons of the paramagnet (the metal atom with an unpaired electron) and bulk water molecules (water molecules that are not "bound" to the metal atom) that are in fast exchange with water molecules in the metal's inner coordination sphere (are bound to the metal atom).
  • regions associated with a Gd 3+ ion appear bright in an MR image where the normal aqueous solution appears as dark background if the time between successive scans if the experiment is short (i.e., Ti weighted image).
  • Localized T 2 shortening caused by superparamagnetic particles is believed to be due to the local magnetic field inhomogeneities associated with the large magnetic moments of these particles.
  • Regions associated with a superparamagnetic iron oxide particle appear dark in an MR image where the normal aqueous solution appears as high intensity background if the echo time (TE) in the spin- echo pulse sequence experiment is long (i.e., T -weighted image).
  • chelator is derived from the Greek word chele which means a "crabs claw", an appropriate description for a material that uses its many “arms” to grab and hold on to a metal atom (see DTPA below).
  • chelator complexes include enthalpy and entropy effects (e.g. number, charge and basicity of coordinating groups, ligand field and conformational effects).
  • Various molecular design features of the ligand can be directly correlated with physiological results.
  • DTP A Diethylenetriaminepentaacetic
  • thermodynamic parameter indicates the fraction of Gd ions that are in the unbound state will be quite small and should not be confused with the rate (kinetic stability) at which the loss of metal occurs (k p /k d ).
  • the water soluble Gd(DTPA) 2 - chelate is stable, nontoxic, and one of the most widely used contrast enhancement agents in experimental and clinical imaging research. It is an extracellular agent that accumulates in tissue by perfusion dominated processes.
  • DTP A diethylenetriaminepentaacetic
  • DOTA diethylenetriaminepentaacetic
  • DTA l,4,7,10-tetraazacyclododecane'-N,N'N",N'"-tetracetic acid
  • DTP A diethylenetriaminepentaacetic
  • DAA l,4,7,10-tetraazacyclododecane'-N,N'N",N'"-tetracetic acid
  • DOTA diethylenetriaminepentaacetic
  • DOTA l,4,7,10-tetraazacyclododecane'-N,N'N",N'"-tetracetic acid
  • Gd contrast agents Another chelator used in Gd contrast agents is the macrocyclic ligand 1,4,7,10- tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA).
  • the Gd-DOTA complex has been thoroughly studied in laboratory tests involving animals and humans.
  • the GdDOTA complex was approved as an MRI contrast agent for use in adults and infants in France and has been administered to over 4500 patients.
  • paramagnetic Ions In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred.
  • Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
  • Therapeutic Agents include cardiovascular drugs, including cardioactive and vasoactive drugs, blood pressure increasing agents antihypertensive agents, antiarrhythmic drugs, beta blockers, cardiac glycosides and synthetic cardiotonic drugs, calcium antagonists, drugs affecting circulation, and diuretics.
  • cardiovascular drugs including cardioactive and vasoactive drugs, blood pressure increasing agents antihypertensive agents, antiarrhythmic drugs, beta blockers, cardiac glycosides and synthetic cardiotonic drugs, calcium antagonists, drugs affecting circulation, and diuretics.
  • Therapeutic agents also contemplated by the present invention include neuropharmaceuticals, gastrointestinal drugs, and respiratory tract agents such as analeptics, analgesics and antipyretics, anesthetics, appetite suppressants, antiepileptic drugs, sedatives, local anesthetics, Parkinsonism treatment, antipsychotics/neuroleptics, skeletal muscle relaxants, spasmolytics, anti-ulcer drugs, antiemetics, gallbladder and liver therapeutics, laxatives, gastroprokinetic agents, motilin, cough remedies, antiasthmatics, and antiallergics
  • Therapeutic agents also contemplated by the present invention include antiinfectives, endocrine and metabolic drugs, such as antibiotics, antimycotics, anthelmintics, HIV and AIDS therapeutics, steroids, protein hormones, chemical contraception, thyrotherapeutic agents, hormones, and oral antidiabetic drugs.
  • Other therapeutics contemplated for use in the present invention include interferons, monoclonal antibodies
  • chemopharmaceuticals refers to the use of drugs to treat cancer.
  • a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • One subtype of chemotherapy known as biochemotherapy involves the combination of a chemotherapy with a biological therapy.
  • Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing.
  • CDDP chlorambucil
  • cyclophosphamide cyclophosphamide
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.
  • Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. Alkylating agents can be implemented to treat, for example, chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary.
  • An alkylating agent may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines.
  • They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.
  • troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents, some of which are discussed below. i.
  • a nitrogen mustard may be, but is not limited to, mechlorethamine (HN ), which is used for Hodgkin's disease and non-Hodgkin's lymphomas; cyclophosphamide and/or ifosfamide, which are used in treating such cancers as acute or chronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilm's tumor, cervix testis and soft tissue sarcomas; melphalan (L-sarcolysin), which has been used to treat such cancers as multiple myeloma, breast and ovary; and chlorambucil, which has been used to treat diseases such as, for example, chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma,
  • HN mechlore
  • Chlorambucil (also known as leukeran) is a bifunctional alkylating agent of the nitrogen mustard type that has been found active against selected human neoplastic diseases. Chlorambucil is known chemically as 4-[bis(2-chlorethyl)amino] benzenebutanoic acid.
  • Chlorambucil is available in tablet form for oral administration. It is rapidly and completely absorbed from the gastrointestinal tract. For example, after a single oral doses of about 0.6 mg/kg to about 1.2 mg/kg, peak plasma chlorambucil levels are reached within one hour and the terminal half-life of the parent drug is estimated at about 1.5 hours. About 0.1 mg/kg/day to about 0.2 mg/kg/day or about 3 6 mg/m 2 /day to about 6 mg/m 2 /day or alternatively about 0.4 mg/kg may be used for antineoplastic treatment. Chlorambucil is not curative by itself but may produce clinically useful palliation. [0078] Cyclophosphamide. Cyclophosphamide is
  • Cyclophosphamide is prepared by condensing 3-amino-l-propanol with NN-bis(2-chlorethyl) phosphoramidic dichloride [(ClC ⁇ C ⁇ 2 ) 2 ⁇ POCl 2 ] in dioxane solution under the catalytic influence of triethylamine. The condensation is double, involving both the hydroxyl and the amino groups, thus effecting the cyclization.
  • Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day (usually in combination), depending upon gastrointestinal tolerance; or about
  • intravenous doses include, for example, initially about
  • a dose of about 250 mg/kg/day may be administered as an antineoplastic. Because of gastrointestinal adverse effects, the intravenous route is preferred for loading. During maintenance, a leukocyte count of about 3000/mm to 4000/mm usually is desired. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities. It is available in dosage forms for injection of about 100 mg, about 200 mg and about 500 mg, and tablets of about 25 mg and about 50 mg.
  • Melphalan also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard.
  • Melphalan is a bifunctional alkylating agent which is active against selective human neoplastic diseases. It is known chemically as 4-[bis(2-chloroethyl)amino]-L-phenylalanine.
  • Melphalan is the active L-isomer of the compound and was first synthesized in 1953 by Bergel and Stock; the D-isomer, known as medphalan, is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with the L-isomer.
  • the racemic (DL-) form is known as merphalan or sarcolysin.
  • Melphalan is insoluble in water and has a pKa ! of about 2.1.
  • Melphalan is available in tablet form for oral administration and has been used to treat multiple myeloma. Available evidence suggests that about one third to one half of the patients with multiple myeloma show a favorable response to oral administration ofthe drug.
  • Melphalan has been used in the treatment of epithelial ovarian carcinoma.
  • melphalan One commonly employed regimen for the treatment of ovarian carcinoma has been to administer melphalan at a dose of about 0.2 mg/kg daily for five days as a single course. Courses are repeated about every four to five weeks depending upon hematologic tolerance (Smith and Rutledge, 1975; Young et al, 1978). Alternatively in certain embodiments, the dose of melphalan used could be as low as about 0.05 mg/kg/day or as high as about 3 mg/kg/day or greater. ii.
  • An ethylenimene and/or a methylmelamine include, but are not limited to, hexamethylmelamine, used to treat ovary cancer; and thiotepa, which has been used to treat bladder, breast and ovary cancer.
  • Alkyl Sulfonates [0085] An alkyl sulfonate includes but is not limited to such drugs as busulfan, which has been used to treat chronic granulocytic leukemia.
  • Busulfan (also known as myleran) is a bifunctional alkylating agent. Busulfan is known chemically as 1,4-butanediol dimethanesulfonate. Busulfan is available in tablet form for oral administration, wherein for example, each scored tablet contains about 2 mg busulfan and the inactive ingredients magnesium stearate and sodium chloride.
  • Busulfan is indicated for the palliative treatment of chronic myelogenous
  • busulfan reduces the total granulocyte mass, relieves symptoms of the disease, and improves the clinical state of the patient. Approximately 90% of adults with previously untreated chronic myelogenous leukemia will obtain hematologic remission with regression or stabilization of organomegaly following the use of busulfan. Busulfan has been shown to be superior to splenic irradiation with respect to survival times and maintenance of hemoglobin levels, and to be equivalent to irradiation at controlling splenomegaly. iv. Nitrosourea [0088] Nitrosureas, like alkylating agents, inhibit DNA repair proteins.
  • a nitrosourea include but is not limited to a carmustine (BCNU), a lomustine (CCNU), a semustine (methyl-CCNU) or a streptozocin.
  • BCNU carmustine
  • CCNU lomustine
  • methyl-CCNU semustine
  • streptozocin has been used to treat diseases such as a malignant pancreatic insulinoma or a malignalnt carcinoid.
  • Carmustine (sterile carmustine) is one of the nitrosoureas used in the treatment of certain neoplastic diseases. It is 1,3 bis (2-chloroethyl)-l -nitrosourea. It is lyophilized pale yellow flakes or congealed mass with a molecular weight of 214.06. It is highly soluble in alcohol and lipids, and poorly soluble in water.
  • Carmustine is administered by intravenous infusion after reconstitution as recommended [0090] Although it is generally agreed that carmustine alkylates DNA and RNA, it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
  • Carmustine is indicated as palliative therapy as a single agent or in established combination therapy with other approved chemotherapeutic agents in brain tumors such as glioblastoma, brainstem glioma, medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors. Also it has been used in combination with prednisone to treat multiple myeloma. Carmustine has been used in treating such cancers as a multiple myeloma or a malignant melanoma. Carmustine has proved useful, in the treatment of Hodgkin's Disease and in non-Hodgkin's lymphomas, as secondary therapy in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
  • Sterile carmustine is commonly available in 100 mg single dose vials of lyophilized material.
  • the recommended dose of carmustine as a single agent in previously untreated patients is about 150 mg/rn 2 to about 200 g/m 2 intravenously every 6 weeks. This may be given as a single dose or divided into daily injections such as about 75 mg/m 2 to about 100 mg/m 2 on 2 successive days.
  • the doses should be adjusted accordingly. Doses subsequent to the initial dose should be adjusted according to the hematologic response of the patient to the preceding dose.
  • doses may be used in the present invention, for example about 10 mg/m 2 , O O 0 about 20 mg/m , about 30 mg/m , about 40 mg/m , about 50 mg/m , about 60 mg/m , about 70 mg/m 2 , about 80 mg/m 2 , about 90 mg/m 2 to about 100 mg/m 2 .
  • Lomustine is one ofthe nitrosoureas used in the treatment of certain neoplastic diseases. It is l-(2-chloro-ethyl)-3-cyclohexyl-l nitrosourea. It is a yellow powder with the empirical formula of C H 16 ClN 3 O 2 and a molecular weight of 233.71. Lomustine is soluble in 10% ethanol (about 0.05 mg/mL) and in absolute alcohol (about 70 mg/mL). Lomustine is relatively insoluble in water (less than about 0.05 mg/mL). It is relatively unionized at a physiological pH. Inactive ingredients in lomustine capsules are: magnesium stearate and mannitol.
  • lomustine alkylates DNA and RNA it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
  • Lomustine may be given orally. Following oral administration of 9 radioactive lomustine at doses ranging from about 30 mg/m to 100 mg/m , about half of the radioactivity given was excreted in the form of degradation products within 24 hours. The serum half-life of the metabolites ranges from about 16 hours to about 2 days. Tissue levels are comparable to plasma levels at 15 minutes after intravenous administration.
  • Lomustine has been shown to be useful as a single agent in addition to other treatment modalities, or in established combination therapy with other approved chemotherapeutic agents in both primary and metastatic brain tumors, in patients who have already received appropriate surgical and/or radiotherapeutic procedures. Lomustine has been used to treat such cancers as small-cell lung cancer. It has also proved effective in secondary therapy against Hodgkin's Disease in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
  • the recommended dose of lomustine in adults and children as a single agent in previously untreated patients is about 130 mg/m 2 as a single oral dose every 6 weeks. In individuals with compromised bone marrow function, the dose should be reduced to about 100 mg/m 2 every 6 weeks. When lomustine is used in combination with other myelosuppressive drugs, the doses should be adjusted accordingly. It is understood that other doses may be used for example, about 20 mg/m 2 , about 30mg/m 2 , about 40 mg/m 2 , about 50 mg/m 2 , about
  • Triazine include but is not limited to such drugs as a dacabazine (DTIC; dimethyltriazenoimidaz olecarboxamide), used in the treatment of such cancers as a malignant melanoma, Hodgkin's disease and a soft-tissue sarcoma.
  • DTIC dacabazine
  • Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds.
  • Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.
  • 5-fluorouracil 5-FU
  • cytarabine Ara-C
  • fludarabine gemcitabine
  • gemcitabine and methotrexate.
  • Folic Acid Analogs include but are not limited to compounds such as methotrexate (amethopterin), which has been used in the treatment of cancers such as acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, head and neck, lung and osteogenic sarcoma.
  • methotrexate amethopterin
  • Pyrimidine analogs include such compounds as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode-oxyuridine; FudR). Cytarabine has been used in the treatment of cancers such as acute granulocytic leukemia and acute lymphocytic leukemias. Floxuridine and 5-fluorouracil have been used in the treatment of cancers such as breast, colon, stomach, pancreas, ovary, head and neck, urinary bladder and topical premalignant skin lesions.
  • 5-Fluorouracil has the chemical name of
  • 5-fluoro-2,4(lH,3H)-pyrimidinedione Its mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid.
  • 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers. iii.
  • Purine analogs and related compounds include, but are not limited to, mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2-deoxycoformycin).
  • Mercaptopurine has been used in acute lymphocytic, acute granulocytic and chronic granulocytic leukemias.
  • Thrioguanine has been used in the treatment of such cancers as acute granulocytic leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia.
  • Natural products generally refer to compounds originally isolated from a natural source, and identified has having a pharmacological activity. Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.
  • Mitotic Inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis.
  • Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
  • Epipodophyllotoxins include such compounds as teniposide and VP16.
  • VP16 is also known as etoposide and is used primarily for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin for small-cell carcinoma of the lung.
  • Teniposide and VP16 are also active against cancers such as testis, other lung cancer, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia, acute nonlymphocytic leukemia, carcinoma of the breast, and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • VP16 is available as a solution (e.g., 20 mg/ml) for intravenous administration and as 50 mg, liquid-filled capsules for oral use.
  • the intravenous dose in combination therapy
  • the dose is can be as much as about 100 mg/m 2 or as little as about 2 mg/ m 2 , routinely about 35 mg/m 2 , daily for about 4 days, to about 50 mg/m 2 , daily for about 5 days have also been used.
  • the dose should be doubled.
  • the doses for small cell lung carcinoma may be as high as about 200 mg/m 2 to about 250 mg/m .
  • the intravenous dose for testicular cancer is about 50 mg/m 2 to about 100 mg/m 2 daily for about 5 days, or about 100 mg/m 2 on alternate days, for three doses. Cycles of therapy are usually repeated about every 3 to 4 weeks.
  • the drug should be administered slowly (e.g., about 30 minutes to about 60 minutes) as an infusion in order to avoid hypotension and bronchospasm, which are probably due to the solvents used in the formulation.
  • Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include but are not limited to compounds such as docetaxel and paclitaxel.
  • Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Paclitaxel is being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary. In certain aspects, maximal doses are about 30 mg/m 2 per day for about 5 days or about 210 mg/m 2 to about
  • Vinca Alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. They include such compounds as vinblastine (VLB) and vincristine. Vinblastine is an example of a plant alkaloid that can be used for the treatment of cancer and precancer. When cells are incubated with vinblastine, dissolution ofthe microtubules occurs.
  • VLB vinblastine
  • vincristine vinblastine
  • Vinblastine is an example of a plant alkaloid that can be used for the treatment of cancer and precancer. When cells are incubated with vinblastine, dissolution ofthe microtubules occurs.
  • vinblastine After intravenous injection, vinblastine has a multiphasic pattern of clearance from the plasma; after distribution, drug disappears from plasma with half-lives of approximately 1 and 20 hours. Vinblastine is metabolized in the liver to biologically activate derivative desacetylvinblastine. Approximately 15% of an administered dose is detected intact in the urine, and about 10% is recovered in the feces after biliary excretion. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 mM). [0113] Vinblastine sulfate is available in preparations for injection.
  • the drug When the drug is given intravenously; special precautions must be taken against subcutaneous extravasation, since this may cause painful irritation and ulceration.
  • the drug should not be injected into an extremity with impaired circulation. After a single dose of 0.3 mg/kg of body weight, myelosuppression reaches its maximum in about 7 days to about 10 days. If a moderate level of leukopenia (approximately 3000 cells/mm 3 ) is not attained, the weekly dose may be increased gradually by increments of about 0.05 mg/kg of body weight. In regimens designed to cure testicular cancer, vinblastine is used in doses of about 0.3 mg/kg about every 3 weeks irrespective of blood cell counts or toxicity.
  • vinblastine An important clinical use of vinblastine is with bleomycin and cisplatin in the curative therapy of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas, particularly Hodgkin's disease, where significant improvement may be noted in 50 to 90% of cases. The effectiveness of vinblastine in a high proportion of lymphomas is not diminished when the disease is refractory to alkylating agents. It is also active in Kaposi's sarcoma, testis cancer, neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well as in carcinoma ofthe breast and choriocarcinoma in women.
  • Doses of about 0.1 mg/kg to about 0.3 mg/kg can be administered or about 1.5 mg/m 2 to about 2 mg/m 2 can also be administered. Alternatively, about 0.1 mg/m 2 , about 0 0 0 o
  • Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes. Vincristine has a multiphasic pattern of clearance from the plasma; the terminal half-life is about 24 hours. The drug is metabolized in the liver, but no biologically active derivatives have been identified. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than about 3 mg/dl (about 50 mM).
  • Vincristine sulfate is available as a solution (e.g., 1 mg/ml) for intravenous injection.
  • Vincristine used together with corticosteroids is presently the treatment of choice to induce remissions in childhood leukemia; the optimal dosages for these drugs appear to be vincristine, intravenously, about 2 mg/m 2 of body-surface area, weekly; and prednisone, orally, about 40 mg/m 2 , daily.
  • Adult patients with Hodgkin's disease or non-Hodgkin's lymphomas usually receive vincristine as a part of a complex protocol. When used in the MOPP regimen, the recommended dose of vincristine is about 1.4 mg/m 2 .
  • Vincristine (and vinblastine) can be infused into the arterial blood supply of tumors in doses several times larger than those that can be administered intravenously with comparable toxicity. [0121] Vincristine has been effective in Hodgkin's disease and other lymphomas.
  • vincristine is an important agent, particularly when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone. Vincristine is more useful than vinblastine in lymphocytic leukemia.
  • Doses of vincristine include about 0.01 mg/kg to about 0.03 mg/kg or about 0.4 mg/m 2 to about 1.4 mg/m 2 can be administered or about 1.5 mg/m 2 to about 2 mg/m 2 can also be administered. Alternatively, in certain embodiments, about 0.02 mg/m 2 , about 0 0 0 o
  • Antitumor Antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers.
  • antitumor antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin. Widely used in clinical setting for the treatment of neoplasms these compounds generally are administered through intravenous bolus injections or orally.
  • Doxorubicin is absorbed poorly and is preferably administered intravenously.
  • the pharmacokinetics are multicompartmental. Distribution phases have half-lives of 12 minutes and 3.3 hours. The elimination half-life is about 30 hours, with about 40% to about 50% secreted into the bile. Most ofthe remainder is metabolized in the liver, partly to an active metabolite (doxorubicinol), but a few percent is excreted into the urine. In the presence of liver impairment, the dose should be reduced.
  • appropriate intravenous doses are, adult, about 60 mg/m 2 to about 75 mg/m 2 at about 21-day intervals or about 25 mg/m 2 to about 30 mg/m 2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg/m 2 once a week.
  • the lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
  • the dose should be reduced by about 50% if the serum bilirubin lies between about 1.2 mg/dL and about 3 mg/dL and by about 75% if above about 3 mg/dL.
  • the lifetime total dose should not exceed about 550 mg/m 2 in patients with normal heart function and about 400 mg/m in persons having received mediastinal irradiation.
  • and alternative dose regiment may comprise about 30 mg/m on each of 3 consecutive days, repeated about every 4 week.
  • Exemplary doses may be O O 0 o o about 10 mg/m , about 20 mg/m , about 30 mg/m , about 50 mg/m , about 100 mg/m , about 0 o o o o o
  • Daunorubicin (daunomycin; rubidomycin) intercalates into DNA, blocks DAN-directed RNA polymerase and inhibits DNA synthesis. It can prevent cell division in doses that do not interfere with nucleic acid synthesis.
  • Daunorubicin (daunomycin; rubidomycin) intercalates into DNA, blocks DAN-directed RNA polymerase and inhibits DNA synthesis. It can prevent cell division in doses that do not interfere with nucleic acid synthesis.
  • diseases such as, for example, acute granulocytic leukemia, acute myelocytic leukemia in adults (for induction of remission), acute lymphocytic leukemia and the acute phase of chronic myelocytic leukemia.
  • Oral absorption is poor, and it preferably given by other methods (e.g., intravenously).
  • the half-life of distribution is 45 minutes and of elimination, about 19 hours.
  • the half-life of its active metabolite, daunorubicinol, is about 27 hours. Daunorubicin is metabolized mostly in the liver and also secreted into the bile (about 40%). Dosage must be reduced in liver or renal insufficiencies.
  • suitable intravenous doses are (base equivalent): adult, younger than 60 years, about 45 mg/m 2 /day (about 30 mg/m 2 for patients older than 60 year.) for about
  • injectable dosage forms base equivalent of about 20 mg (as the base equivalent to about 21.4 mg of the hydrochloride).
  • Exemplary doses may be about 10 mg/m 2 , 0 0 0 0 0 about 20 mg/m , about 30 mg/m , about 50 mg/m , about 100 mg/m , about 150 mg/m , about 0 0 0 0 0 0
  • Mitomycin also known as mutamycin and/or mitomycin-C
  • Mitomycin-C is an antibiotic isolated from the broth of Streptomyces caespitosus which has been shown to have antitumor activity.
  • the compound is heat stable, has a high melting point, and is freely soluble in organic solvents.
  • Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA).
  • the guanine and cytosine content correlates with the degree of mitomycin-induced cross-linking.
  • cellular RNA and protein synthesis are also suppressed.
  • Mitomycin has been used in tumors such as stomach, cervix, colon, breast, pancreas, bladder and head and neck.
  • mitomycin is rapidly cleared from the serum after intravenous administration. Time required to reduce the serum concentration by about 50% after a 30 mg. bolus injection is 17 minutes. After injection of 30 mg, 20 mg, or 10 mg I.V., the maximal serum concentrations were 2.4 mg/mL, 1.7 mg/mL, and 0.52 mg/mL, respectively. Clearance is effected primarily by metabolism in the liver, but metabolism occurs in other tissues as well. The rate of clearance is inversely proportional to the maximal serum concentration because, it is thought, of saturation ofthe degradative pathways. Approximately 10% of a dose of mitomycin is excreted unchanged in the urine. Since metabolic pathways are saturated at relatively low doses, the percent of a dose excreted in urine increases with increasing dose. In children, excretion of intravenously administered mitomycin is similar.
  • Actinomycin D (Dactinomycin) [50-76-0]; C 62 H 86 N 12 O 16 (1255.43) is an antineoplastic drug that inhibits DNA-dependent RNA polymerase. It is often a component of first-choice combinations for treatment of diseases such as, for example, choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor, Kaposi's sarcoma and Wilms' tumor. Tumors that fail to respond to systemic treatment sometimes respond to local perfusion. Dactinomycin potentiates radiotherapy. It is a secondary (efferent) immunosuppressive.
  • actinomycin D is used in combination with agents such as, for example, primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide.
  • agents such as, for example, primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide.
  • Antineoplastic activity has also been noted in Ewing' s tumor, Kaposi's sarcoma, and soft-tissue sarcomas.
  • Dactinomycin can be effective in women with advanced cases of choriocarcinoma. It also produces consistent responses in combination with chlorambucil and methotrexate in patients with metastatic testicular carcinomas. A response may sometimes be observed in patients with Hodgkin's disease and non-Hodgkin's lymphomas. Dactinomycin has also been used to inhibit immunological responses, particularly the rejection of renal transplants.
  • daily injections of about 100 mg to about 400 mg have been given to children for about 10 days to about 14 days; in other regimens, about 3 mg/kg to about 6 mg/kg, for a total of about 125 mg/kg, and weekly maintenance doses of about 7.5 mg/kg have been used.
  • direct intravenous injections have been given, with the precaution of discarding the needle used to withdraw the drug from the vial in order to avoid subcutaneous reaction.
  • Exemplary doses may be about 0 0 0 0 0 0 0
  • Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. Although the exact mechanism of action of bleomycin is unknown, available evidence would seem to indicate that the main mode of action is the inhibition of DNA synthesis with some evidence of lesser inhibition of RNA and protein synthesis.
  • mice high concentrations of bleomycin are found in the skin, lungs, kidneys, peritoneum, and lymphatics. Tumor cells ofthe skin and lungs have been found to have high concentrations of bleomycin in contrast to the low concentrations found in hematopoietic tissue.
  • the low concentrations of bleomycin found in bone marrow may be related to high levels of bleomycin degradative enzymes found in that tissue.
  • bleomycin In patients with a creatinine clearance of greater than about 35 mL per minute, the serum or plasma terminal elimination half-life of bleomycin is approximately 115 minutes. In patients with a creatinine clearance of less than about 35 mL per minute, the plasma or serum terminal elimination half-life increases exponentially as the creatinine clearance decreases. In humans, about 60% to about 70% of an administered dose is recovered in the urine as active bleomycin. In specific embodiments, bleomycin may be given by the intramuscular, intravenous, or subcutaneous routes. It is freely soluble in water. Because of the possibility of an anaphylactoid reaction, lymphoma patients should be treated with two units or less for the first two doses. If no acute reaction occurs, then the regular dosage schedule may be followed. [0140] In certain aspects, bleomycin should be considered a palliative treatment.
  • neoplasms either as a single agent or in proven combinations with other approved chemotherapeutic agents in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), esophagus, lung and genitourinary tract, Hodgkin's disease, non-Hodgkin's lymphoma, skin, penis, cervix, and vulva. It has also been used in the treatment of lymphomas and testicular carcinoma.
  • Platinum coordination Complexes include such compounds as carboplatin and cisplatin (cis-DDP).
  • Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors.
  • Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection.
  • Cisplatin can be used alone or in combination with other agents, with 0 efficacious doses used in clinical applications of about 15 mg/m to about 20 mg/m for 5 days every three weeks for a total of three courses being contemplated in certain embodiments.
  • Doses may be, for example, about 0.50 mg/m 2 , about 1.0 mg/m 2 , about 1.50 mg/m 2 , about 1.75 mg/m 2 , about 2.0 mg/m , about 3.0 mg/m , about 4.0 mg/m , about 5.0 mg/m , to about 10 mg/m .
  • An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer.
  • a substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis and malignant melanoma.
  • a methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease.
  • An adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease.
  • Toxins [0145] Various toxins are also useful in the treatment of cancers. As part of the present invention, toxins such as ricin A-chain (Burbage, 1997), diphtheria toxin A (Massuda et al, 1997; Lidor, 1997), pertussis toxin A subunit, E.
  • coli enterotoxin toxin A subunit, cholera toxin A subunit and Pseudomonas toxin c-terminal are suitable. It has demonstrated that transfection of a plasmid containing the fusion protein regulatable diphtheria toxin A chain gene was cytotoxic for cancer cells.
  • the present invention provides methods for the treatment of cancer, wherein the cancer is characterized by cells having an oxidative or hypoxic cellular environment.
  • the present invention relates to a peptide linked to a therapeutic agent that shows unexpected, potent anti-cancer activity in vitro.
  • the present invention provides a peptide linked to a therapeutic agent as an agent for treating a cancer in a subject.
  • Treatment methods will involve treating an individual with an effective amount of a composition containing a peptide linked to a therapeutic agent.
  • An effective amount is described, generally, as that amount sufficient to detectably and repeatedly to ameliorate, reduce, minimize or limit the extent of a disease or its symptoms. More specifically, it is envisioned that the treatment with the peptide linked to a therapeutic agent will kill cells, inhibit cell growth, cleave DNA, inhibit DNA synthesis, inhibit metastasis, decrease tumor size and otherwise reverse or reduce the malignant phenotype of tumor cells.
  • the peptide linked to a therapeutic agent is administered to a cell.
  • Cells that are encompassed by the present invention include, but are not limited to melanoma cells.
  • the cell may be a cancer cell, a non-cancerous cell or a benign hyperplastic cell.
  • a cancer cell may include, cells that are drug-resistant, primary cancer cells and/or metastatic cancer cells.
  • Other cells as contemplated in the present invention may be a cancer cell such as, but not limited to, a breast cancer cell, lung cancer cell, head and neck cancer cell, bladder cancer cell, bone cancer cell, bone marrow cancer cell, brain cancer cell, colon cancer cell, esophageal cancer cell, gastrointestinal cancer cell, gum cancer cell, kidney cancer cell, liver cancer cell, nasopharynx cancer cell, ovarian cancer cell, prostate cancer cell, skin cancer cell, stomach cancer cell, testis cancer cell, tongue cancer cell, or uterine cancer cell.
  • a cancer cell such as, but not limited to, a breast cancer cell, lung cancer cell, head and neck cancer cell, bladder cancer cell, bone cancer cell, bone marrow cancer cell, brain cancer cell, colon cancer cell, esophageal cancer cell, gastrointestinal cancer cell, gum cancer cell, kidney cancer cell, liver cancer cell, nasopharynx cancer cell, ovarian cancer cell, prostate cancer cell, skin cancer cell, stomach cancer cell, testis cancer cell, tongue cancer cell, or uter
  • An effective amount of the peptide linked to a therapeutic agent that may be administered to a cell includes a dose of about - 0.1 ⁇ M to about 100 ⁇ M. More specifically, doses ofthe peptide linked to a therapeutic agent to be administered are from about - 0.1 ⁇ M to about 1 ⁇ M; about 1 ⁇ M to about 5 ⁇ M; about 5 ⁇ M to about 10 ⁇ M; about 10 ⁇ M to about 15 ⁇ M; about 15 ⁇ M to about 20 ⁇ M; about 20 ⁇ M to about 30 ⁇ M; about 30 ⁇ M to about 40 ⁇ M; about 40 ⁇ M to about 50 ⁇ M; about 50 ⁇ M to about 60 ⁇ M; about 60 ⁇ M to about 70 ⁇ M; about 70 ⁇ M to about 80 ⁇ M; about 80 ⁇ M to about 90 ⁇ M; and about 90 ⁇ M to about 100 ⁇ M .
  • all of these amounts are exemplary, and any amount in-between these points is also expected to be of use in
  • an effective amount of the peptide linked to a therapeutic agent may be administered to a subject suffering from melanoma.
  • the effectiveness of the therapy according to the present invention can be determined in the treatment of melanoma by diagnostic methods that are known and used in the art, for example, but not limited to, analysis of a biopsy.
  • Other embodiments include methods for inhibiting development of melanoma in a subject at risk, inhibiting melanoma metastasis in a subject with primary melanoma, and/or inhibiting melanoma progression in subjects having Stage 1 or Stage 2 melanoma.
  • the effective amount or "therapeutically effective amounts" of the peptide linked to a therapeutic agent to be used are those amounts effective to produce beneficial results, particularly with respect to cancer treatment, in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated.
  • Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.
  • a specific dose level of active compounds such as the peptide linked to a therapeutic agent for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The person responsible for administration will determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • a therapeutically effective amount ofthe peptide linked to a therapeutic agent as a treatment varies depending upon the host treated and the particular mode of administration.
  • the dose range of the peptide linked to a therapeutic agent will be about 0.5 mg/kg body weight to about 500 mg/kg body weight.
  • body weight is applicable when an animal is being treated. When isolated cells are being treated, “body weight” as used herein should read to mean “total cell weight”. The term “total weight may be used to apply to both isolated cell and animal treatment. All concentrations and treatment levels are expressed as “body weight” or simply “kg” in this application are also considered to cover the analogous "total cell weight” and “total weight” concentrations.
  • Administration of the therapeutic the peptide linked to a therapeutic agent compositions ofthe present invention to a patient or subject will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, ofthe composition. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
  • the treatments may include various "unit doses.”
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition (the peptide linked to a therapeutic agent) calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.
  • Also of import is the subject to be treated, in particular, the state of the subject and the protection desired.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • the tumor may be infused or perfused with the composition using any suitable delivery vehicle.
  • Continuous administration also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease. Delivery via syringe or catherization is preferred. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment.
  • the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.
  • the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of ⁇ 4 cm, a volume of about 1-3 ml will be used (preferably 3 ml).
  • Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes. IX.
  • Combined Therapy with the Peptide linked to a Therapeutic Agent and/or Other Anticancer Agents may be used in combination with an additional therapeutic agent to more effectively treat cancer or other diseases.
  • Anticancer agents may include but are not limited to, radiotherapy, chemotherapy, gene therapy, hormonal therapy or immunotherapy that targets cancer/tumor cells.
  • the effective amounts of the additional therapeutic agent may simply be defined as that amount effective to inhibit and/or reduce the cancer growth when administered to an animal in combination with the peptide linked to a therapeutic agent. This may be easily determined by monitoring the animal or patient and measuring those physical and biochemical parameters of health and disease that are indicative of the success of a given treatment. Such methods are routine in animal testing and clinical practice.
  • compositions ofthe present invention To kill cells, induce cell-cycle arrest, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of cancer cells, using the methods and compositions ofthe present invention, one would generally contact a cell with the peptide linked to a therapeutic agent in combination with an additional therapeutic agent. These compositions would be provided in a combined amount effective to inhibit cell growth and or induce apoptosis in the cell. This process may involve contacting the cells with the peptide linked to a therapeutic agent in combination with an additional therapeutic agent or factor(s) at the same time.
  • treatment with the peptide linked to a therapeutic agent may precede or follow the additional agent treatment by intervals ranging from minutes to weeks.
  • the additional agent is applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent would still be able to exert an advantageously combined effect on the cell.
  • chemotherapy may be administered, as is typical, in regular cycles.
  • a cycle may involve one dose, after which several days or weeks without treatment ensues for normal tissues to recover from the drug's side effects. Doses may be given several days in a row, or every other day for several days, followed by a period of rest. If more than one drug is used, the treatment plan will specify how often and exactly when each drug should be given. The number of cycles a person receives may be determined before treatment starts (based on the type and stage of cancer) or may be flexible, in order to take into account how quickly the tumor is shrinking. Certain serious side effects may also require doctors to adjust chemotherapy plans to allow the patient time to recover.
  • Chemotherapeutic agents that may be used in combination with the peptide linked to a therapeutic agent in the treatment of cancer, include, but are not limited to cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil and methotrexate, or any analog or derivative variant of the foregoing.
  • CDDP cisplatin
  • carboplatin carboplatin
  • procarbazine mechloreth
  • Radiotherapeutic agents may also be use in combination with the compounds of the present invention in treating a cancer.
  • factors that cause DNA damage and have been used extensively include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • C. Immunotherapeutic Agents may also be employed in the present invention in combination with the peptide linked to a therapeutic agent thereof in treating cancer. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • the tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation.
  • the tumor suppressors p53, pl6 and C-CAM are described below.
  • mutant p53 has been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses.
  • the p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al, 1991) and in a wide spectrum of other tumors.
  • the p53 gene encodes a 393-amino acid phosphoprotein that can form complexes with host proteins such as large-T antigen and E1B.
  • the protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue
  • Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene. A single genetic change prompted by point mutations can create carcinogenic p53. Unlike other oncogenes, however, p53 point mutations are known to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity. Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line. Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991).
  • Another inhibitor of cellular proliferation is pi 6.
  • the major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's.
  • CDK cyclin-dependent kinase 4
  • the activity of this enzyme may be to phosphorylate Rb at late Gl .
  • the activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al, 1995). Since the pl6INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6.
  • pl6INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes ⁇ l6B, pi 9, p21WAFl, and p27KIPl.
  • the pl6INK4 gene maps to 9 ⁇ 21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6INK4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6LNK4 gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency ofthe pl6INK4 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al, 1994; Cheng et al, 1994; Arap et al, 1995). Restoration of wild-type pl6INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).
  • genes that may be employed according to the present invention include Rb, mda-7, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fins, trk, ret, gsp, hst, abl, El A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
  • angiogenesis e.g., VEGF, FGF, thrombospondin
  • Apoptosis or programmed cell death, is an essential process in cancer therapy (Kerr et al, 1972).
  • the Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems.
  • the Bcl-2 protein discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986).
  • Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists. [0177] Members of the Bcl-2 that function to promote cell death such as, Bax, Bak, Bik, Bim, Bid, Bad and Harakiri, are contemplated for use in combination with the peptide linked to a therapeutic agent in treating cancer.
  • a surgical procedure may be employed in the present invention. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4,
  • Hormonal therapy may also be used in conjunction with the peptide linked to a therapeutic agent as in the present invention, or in combination with any other cancer therapy previously described.
  • the use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
  • agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
  • additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers.
  • Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MlP-lbeta, MCP-1, RANTES, and other chemokines.
  • cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increased intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
  • cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
  • FAKs focal adhesion kinase
  • Lovastatin Lovastatin
  • compositions of the peptide linked to a therapeutic agent or any additional therapeutic agent disclosed herein in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention in an effective amount may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • composition(s) of the present invention may be delivered orally, nasally, intramuscularly, intraperitoneally, or intratumorally.
  • local or regional delivery of the peptide linked to a therapeutic agent, alone or in combination with an additional therapeutic agent, to a patient with cancer or pre-cancer conditions will be a very efficient method of delivery to counteract the clinical disease.
  • chemo- or radiotherapy may be directed to a particular, affected region of the subject's body.
  • Regional chemotherapy typically involves targeting anticancer agents to the region ofthe body where the cancer cells or tumor are located.
  • Other examples of delivery of the compounds of the present invention that may be employed include intra-arterial, intracavity, intravesical, intrathecal, intrapleural, and intraperitoneal routes.
  • Intra-arterial administration is achieved using a catheter that is inserted into an artery to an organ or to an extremity. Typically, a pump is attached to the catheter. Intracavity administration describes when chemotherapeutic drugs are introduced directly into a body cavity such as intravesical (into the bladder), peritoneal (abdominal) cavity, or pleural (chest) cavity. Agents can be given directly via catheter. Intravesical chemotherapy involves a urinary catheter to provide drugs to the bladder, and is thus useful for the treatment of bladder cancer.
  • Intrapleural administration is accomplished using large and small chest catheters, while a Tenkhoff catheter (a catheter specially designed for removing or adding large amounts of fluid from or into the peritoneum) or a catheter with an implanted port is used for intraperitoneal chemotherapy.
  • Abdomen cancer may be treated this way. Because most drugs do not penetrate the blood/brain barrier, intrathecal chemotherapy is used to reach cancer cells in the central nervous system. To do this, drugs are administered directly into the cerebrospinal fluid. This method is useful to treat leukemia or cancers that have spread to the spinal cord or brain. [0186] Alternatively, systemic delivery of the chemotherapeutic drugs may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
  • Intravenous therapy can be implemented in a number of ways, such as by peripheral access or through a vascular access device (VAD).
  • a VAD is a device that includes a catheter, which is placed into a large vein in the arm, chest, or neck. It can be used to administer several drugs simultaneously, for long-term treatment, for continuous infusion, and for drugs that are vesicants, which may produce serious injury to skin or muscle.
  • vascular access devices are available.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes but is not limited to, oral, nasal, or buccal routes. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. The drugs and agents also may be administered parenterally or intraperitoneally. The term "parenteral" is generally used to refer to drugs given intravenously, intramuscularly, or subcutaneously.
  • Solutions ofthe active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions of the present invention may be administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti- oxidants, chelating agents and inert gases. The pH, exact concentration of the various components, and the pharmaceutical composition are adjusted according to well known parameters.
  • Suitable excipients for formulation with the peptide linked to a therapeutic agent include croscarmellose sodium, hydroxypropyl methylcellulose, iron oxides synthetic), magnesium stearate, macrocrystalline cellulose, polyethylene glycol 400, polysorbate 80, povidone, silicon dioxide, titanium dioxide, and water (purified).
  • Additional formulations are suitable for oral administration.
  • Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.
  • XL Melanoma begins in melanocytes, which are cells that make the skin pigment melanin. Although melanoma accounts for only about 4% of all skin cancer cases, it is the cause of most skin cancer-related deaths. Melanoma is often curable if it is detected and treated in its early stages. In men, melanoma is found most often on the area between the shoulders and hips or on the head and neck. In women, melanoma often develops on the lower legs. It may also appear under the fingernails or toenails or on the palms or soles. The chance of developing melanoma increases with age, but it affects all age groups and is one of the most common cancers in young adults. [0192] The number of new melanomas diagnosed in the United States is increasing.
  • melanoma When melanoma starts in the skin, it is called cutaneous melanoma. Melanoma may also occur in the eye (ocular melanoma or intraocular melanoma) and, rarely, in other areas where melanocytes are found, such as the digestive tract, meninges, or lymph nodes.
  • cancer cells are also found in the lymph nodes and possibly also other parts of the body, such as the liver, lungs, or brain. In these cases, the cancer cells are still melanoma cells, and the disease is called metastatic melanoma.
  • the nuclear localization sequence is recognized as an extended peptide structure to our NLS peptide would have to be able to adopt a helix conformation with positive charges to escape the membrane and the relax into an extended form in the cytoplasm to be recognized by the nuclear localization machinery. This was possible since short peptides have dynamic structures in solution.
  • the prototype NLS peptide with translocation properties had the sequence KGPKKKRK (SEQ ID NO:5). This sequence was consistent with the nuclear localization consensus sequence and if projected as an ⁇ -helix it would have positive charges long one face.
  • a proline in the first three positions of a helix acted to promote it, not destroy it.
  • the NLS sequence did not contain a completely optimal translocation sequence in order to prevent uncontrolled diffusion throughout and out of the cell. This was the basis for incorporating only one Arg residue since the translocation properties have been linked to Arg residues displayed on one face of a helix
  • Example 2 Peptide Synthesis [0196] The MSH peptide analogs were synthesized using standard Fmoc/HBTU chemistry on amide resin with an Advanced ChemTech 396 Omega solid phase peptide synthesizer (Advanced ChemTech Inc., Louisville, KY). The peptides were acetylated with glacial acetic acid at the N-terminus. Amino acids and resin were purchased from Advanced ChemTech Inc (Louisville, KY). The peptides were deprotected and cleaved from the resin by using a cleavage mixture of trifluoroacetic acid (TFA), thioanisol, ethandiol, and water at room temperature for 3 hrs.
  • TFA trifluoroacetic acid
  • the peptides were purified by HPLC (Isco, Inc., Lincoln, NE) on a C- 18 reverse phase column (218TP54, Vydac, Hesperia, CA) and lyophilized. The identities of peptides were confirmed by electrospray ionization mass specfrometry (ESI-MS) on a Finnigan TSQ7000 mass spectrometer (Thermo Finnigan, San Jose, CA).
  • Example 3 In Vitro Cell Binding [0197] The IC 50 values for the ⁇ -MSH peptide analogs were determined by competitive binding assays with 125 I-Tyr 2 -NDP (Amersham Pharmacia Biotech, UK). B16/F1 murine melanoma cells (5xl0 5 /well) were seeded into a 24-well cell culture plate and incubated at 37°C overnight.
  • binding media MEM with 25 mM HEPES, pH 7.4, 0.2% BSA, 0.3 mM 1,10-phenathroline
  • the cells were incubated at 25°C for 2 hours with approximately 50,000 cpm of 125 I-Tyr 2 -NDP and different concentrations ofthe ⁇ -MSH peptides in 0.3 ml of binding media.
  • Cells were rinsed with 0.5 ml of ice-cold pH 7.4, 0.2% BSA / 0.01 M PBS two times and lysed in 0.5 ml of 1 N NaOH for 5 minutes. The activity in cells was measured in a Nal well counter.
  • IC 50 values for the peptides were calculated by using the Grafit software (Erithacus Software Limited, UK). All of the ⁇ -MSH peptide analogs bound the murine melanoma cells and exhibited IC 50 values ranging from the low nanomolar to low micromolar.
  • DF D-Phenylalanine
  • Nle Norleucine
  • DOTA l ,4,7, 10-tetraazacyclododecane-N,N' ,N" ,N" ' - tetracetic acid. Description notes chirality ofthe amino acids except where differences are explicitly designated.
  • TMR yielding the peptide analogs (TMR)-NLS-NDP and (TMR)-NDP.
  • the fluorescent peptides were added to live murine melanoma cells and observed under an Olympus 1X70 microscope/BioRad Radiance 2000 scanning confocal fluorescent system. Briefly, B16/F1 murine melanoma cells were seeded into 24 well tissue culture plates that contained glass coverslips which had been coated with poly-D-lysine. The cells were plated at a final density of 1 x 10 5 cells per well and allowed to grow over night at 37°C in a humidified incubator with 5% CO 2 .
  • the cells were washed with Opti-MEM media without fetal calf serum and then the peptide, in Opti-MEM was added and incubated with the cells for 2 hr at 25°C. The final peptide concentration was 1 micromolar. Prior to examination, the Opti-MEM media containing the peptide was removed and the cells were gently rinsed with 200 microliters of Opti-MEM 25 ° C. The glass cover slip containing the cells was mounted and sealed on the confocal microscope's stage.
  • Example 5 Melanoma Cell Killing Assay
  • the radionuclide targeting and melanoma cell killing ability of DOTA-NLS- NDP was compared to DOTA-NDP. Both peptides were synthesized with a N-terminal DOTA chelator for radiolabeling with n i In. Indium-Ill is an Auger emitter. The therapeutic properties of Auger emitters are limited to approximately 8-10 micrometers or less than or equal to one cell diameter. The closer an Auger emitting radionuclide is located to the cell nuclear the greater its therapeutic potential.
  • Radiolabeling DOTA-NLS-NDP and DOTA-NDP were radiolabeled in an acetate buffered solution at 80°C.
  • the radiolabeld peptides were purified via HPLC on a C-18 column using a 16 to 26% gradient acetonitrile/water (of 0.1% triflouroacetic acid (TFA)).
  • the purified radiolabeled peptides were radiochemically stable in phosphate buffered saline.
  • Colony growth assay The colony growth test was performed to determine if nuclear targeting of an Auger emitter ( U1 ln) was more effective in killing cells than cytoplasmic targeting or extra-cellular localization.
  • the colony growth assay was performed with B16-F1 melanoma cells. Briefly, twenty-five thousand B16-F1 cells were divided evenly into 4 vials.
  • NDP was more effective in killing melanoma cells than [ m In]-DOTA-NDP or ⁇ ⁇ In (FIG. 4).
  • Nuclear localization of m In ([ ni In]-DOTA-NLS-NDP) versus cytoplasmic localization ([ m In]- DOTA-NDP) or extracellular localization ( ⁇ n In chloride) resulted in significantly fewer melanoma colonies.

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Abstract

The present invention provides peptides that selectively target the nuclei of cells. The peptides are useful for nuclear localization of radioisotopes, therapeutics, and imaging agents. The peptides are useful for in vitro as well as in vivo applications. The present invention also provides methods of targeting and imaging cell nuclei, and methods to treat diseases using such peptides.

Description

DESCRIPTION
PEPTIDES DELIVERED TO CELL NUCLEI
[0001] The government owns rights in the present invention pursuant to grant number DOE FG02 93ER 61661 from the Department of Energy. The present invention claims priority to U.S. Provisional Application Serial No. 60/574,558, filed May 26, 2004, the entire content of which is hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates generally to the field of nuclear delivery of peptides and cell imaging. The present invention also relates generally to the fields of cancer biology and cancer therapeutics.
BACKGROUND OF THE INVENTION
[0003] G-protein coupled receptors are cell surface receptors that indirectly transduce extracellular signals to downstream effectors, which can be intracellular signaling proteins, enzymes, or channels, and changes in the activity of these effectors then mediate subsequent cellular events. The interaction between the receptor and the downstream effector is mediated by a G-protein, a heterotrimeric protein that binds GTP. G-protein coupled receptors ("GPCRs") typically have seven transmembrane regions, along with an extracellular domain and a cytoplasmic tail at the C-terminus. These receptors form a large superfamily of related receptors molecules that play a key role in many signaling processes, such as sensory and hormonal signal transduction.
[0004] Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR's). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as α-, β, or γ-melanocyte stimulating hormones (α-, β-, γ-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
[0005] U.S. Pat. No. 5,532,347, discloses human and mouse DNA molecules which encode MC-1R (also known in the art as α-MSH-R). The expressed human protein contains 317 amino acids. [0006] Alpha melanocyte stimulating hormone (α-MSH) is a tridecapeptide [Ac-Ser- Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 ] that regulates skin pigmentation in most vertebrates (Hruby, V. J., 1993). The core α-MSH sequence His-Phe-Arg-Trp, conserved in a number of species, has been found to be sufficient for receptor recognition (Hruby, V. J., 1993). α-melanotropin is a naturally-occurring tridecapeptide that specifically recognizes melanotropin receptors. Since mouse melanoma cells and human melanocytes possess α- melanotropin (α-MSH) receptors, the use of radiolabeled α-MSH analogs for diagnosis and treatment of α-MSH positive cancers (i.e., melanoma) was hypothesized. Various synthetic α- melanotropin analogs have been prepared and characterized for α-melanotropin activity by V. J. Hruby, M. E. Hadley et al. They have reported that cyclic analogs of α-MSH (see U.S. Pat. No. 4,485,039) display properties which increase their potency toward the α-MSH receptor, prolong their activity and increase their resistance to in vivo enzymatic degradation.
[0007] Bard et al. described targeting malignant melanoma with multiple α-MSH analogs chemically attached to a chelating molecule (e.g., DTP A) which can subsequently be linked to a cytotoxic agent or radionuclide (e.g., In-111 or 1-131). These authors also describe the use of In- 111 -labeled analogs of the patented moieties for use as diagnostic agents for malignant melanoma. The peptide analog used as the starting material for Re and Tc labeling reactions was first described by Cody et al. as a superpotent α-MSH analog with prolonged biological activity. One radiolabeled α-MSH analog has been reported as a targeting agent of the radionuclide Indium-I l l. This analog consists of two des-acetyl-MSH molecules crosslinked through the chelating group DPTA. In vivo work on this analog was reported in Bard, D. R. et al. U.S. patent No. 6,338,834 describes α-MSH analogs with integrally located radionuclides.
SUMMARY OF THE INVENTION
[0008] An embodiment of the invention is a method of delivering a peptide to a cell nucleus comprising contacting a cell expressing a G-protein coupled receptor with a peptide comprising a G-protein coupled receptor binding sequence and a nuclear localization signal.
[0009] In one embodiment, the G-protein coupled receptor is an opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P, substance-R, or glutamate receptor. In a specific embodiment, the G- protein coupled receptor is a melanocortin- 1 receptor. [0010] In another embodiment, the peptide is linked to a radioisotope, a pharmaceutical compound, an MRI contrast reagent, or a fluorescent label. In certain embodiments of the invention, the radioisotope is an alpha-emitter, a beta-emitter, a gamma-emitter, or an Auger emitter. In certain embodiments of the invention, the radioisotope is In-111, Bi-214, At-211, or Bi-212. In certain embodiments of the invention, the peptide is linked to an imaging agent. The imaging agent is a DNA intercalator in one embodiment ofthe invention.
[0011] hi another embodiment, the cell expressing a G-protein coupled receptor is selected from the group consisting of a melanoma cell, liver cell, a brain cell, a pituitary cell, a keratinocyte cell, or an immune system cell. [0012] In one embodiment, the G-protein coupled receptor binding sequence comprises a melanocortin- 1 receptor binding sequence. In another embodiment, the melanocortin- 1 receptor binding sequence comprises His-Phe-Arg-Trp (SEQ ID NO:l). In a specific embodiment, the melanocortin- 1 receptor binding sequence comprises His-DPhe-Arg-Trp. In certain embodiments of the invention, the peptide comprises D amino acids. In other embodiment ofthe invention, the peptide comprises a mix of D and L amino acids.
[0013] An embodiment ofthe invention is a peptide comprising the sequence: Lys-Gly- Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2), wherein Nle is norleucine.
[0014] An embodiment ofthe invention is a peptide comprising the sequence: Lys-Gly- Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-Trp-Cys-Arg-Pro-Val (SEQ ID NO:3).
[0015] An embodiment ofthe invention is a peptide comprising the sequence: Gly-Pro- Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val wherein Nle is norleucine. [0016] An embodiment of the invention is a peptide comprising D amino acids comprising the sequence: Val-Pro-Lys-Gly-Trp-Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg- Lys-Lys-Lys-Pro-Gly-Lys wherein Nle is norleucine. [0017] An embodiment of the invention is a peptide comprising D amino acids comprising the sequence: Val-Pro-Lys-Gly-Trp-Arg-Phe-His-Glu-Nle-Ala-Glu-Met-Ser-Lys-Ser wherein Nle is norleucine.
[0018] In one embodiment of the invention, the nuclear localization signal comprises K(K/R)x(K R) (SEQ ID NO:4), where X is any amino acid. In a specific embodiment, the nuclear localization signal comprises KRXR.
[0019] An embodiment of the invention is a method of cell imaging comprising (a) contacting a cell expressing a G-protein coupled receptor with a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the peptide is linked to an imaging agent; and (b) obtaining an image ofthe cell.
[0020] An embodiment of the invention is a method of nuclear delivering of a pharmaceutical compound to a cell nucleus comprising contacting a cell expressing a melanocortin- 1 receptor with a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the peptide is linked to the pharmaceutical compound. [0021] An embodiment ofthe invention is a method in vivo delivering of a peptide to a tumor comprising administering to a patient with a melanocortin- 1 receptor-expressing tumor a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the nuclear localization signal forms a positively charged helix.
[0022] An embodiment of the invention is a host cell expressing any of the peptides described herein.
[0023] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."
[0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0026] FIG. 1 is a helical wheel projection of the NLS sequence (SEQ ID NO:5) with membrane translocation peptides. The bracket highlights the positively charged face.
[0027] FIG. 2 shows the peptide MSH peptide containing the NLS sequence has significantly higher accumulation peptide in the nucleus. The NLS sequence was successfully incorporated into the MSH sequence without affecting the biological activity of MCI receptor targeting. Likewise, the NLS signal is also active and allows a significant amount of the NLS-
NDP peptide to be transported to the cell nucleus.
[0028] FIG. 3 contains images of another cell from a well of cells incubated with (TMR)-NLS-NDP and counter stained with Cyto-5 (Green). The left image is a combination overlay of the microscopic picture of the cell, the Cyto-5 image and the (TMR)-NLS-NDP image.
[0029] FIG. 4 shows a colony growth assay. B16-F1 melanoma cells alone or treated with '"in, [l uIn]-DOTA-NDP or [n ιIn]-DOTA-NLS-NDP were plated and examined for colony formation in 24-well tissue culture plates. Growth assays were performed in triplicate. DETAILED DESCRIPTION OF THE INVENTION
[0030] It is readily apparent to one skilled in the art that various embodiments and modifications can be made to the invention disclosed in this Application without departing from the scope and spirit ofthe invention.
I. Present Invention [0031] The present invention provides peptides comprising G-protein coupled receptors binding sequences and nuclear localization signals that are delivered to cell nuclei. The peptides bind G-protein coupled receptors, such as the melanocortin- 1 receptor, on the surface of he cell, and are internalized and targeted to the cell nuclei by virtue of cellular recognition of the nuclear localization signal. The nuclear targeted peptides of the present invention escape lysosomal degradation by virtue of comprising a nuclear localization signal.
[0032] Also contemplated in the present invention are methods of delivering these peptides to cell nuclei, and delivering imaging compounds or therapeutic compounds to cell nuclei through the use of peptides as described herein.
II. Nuclear Localization Signals [0033] Nuclear localization signals are present in the peptides described in the present invention. Nuclear localization signal are peptide sequences that direct peptides to the nuclear transportation machinery. Examples of nuclear localization signals (nuclear localization sequences) known in the art are the SV40 large T antigen nuclear localization signal, PKKKRKV (amino acids 3-9 of SEQ ID NO:5), and the c-myc proto-oncogene nuclear localization signal, with a sequence of PAAKRVKLD (SEQ ID NO:6).
[0034] Nuclear localization signals contemplated by the present invention include both monopartite and bipartite sequences. Bipartite signals are characterized as a small cluster of basic residues positioned 10-12 residues N-terminal to a monopartite-like sequence. An example of a bipartite nuclear localization signal is KRPAATKKAGQAKKKKL (SEQ ID NO:7).
[0035] In one embodiment of the invention, the nuclear localization signal comprises the motif K(K R)x(K/R) (SEQ ID NO:4). In a specific embodiment, the nuclear localization signal is KRXR, wherein X is any amino acid.
III. G protein coupled receptors [0036] The terms "G protein coupled receptors" and "GPCRs" as used interchangeably herein include all subtypes of the opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P and substance-R receptors, melanocortin, metabotropic glutamate, or any other GPCR known to couple via G proteins. Polypeptide ligands of GPCRs include vasopressin, oxytocin, somatostatin, neuropeptide Y, GnRH, leutinizing hormone, follicle stimulating hormone, parathyroid hormone, orexins, urotensin II, endorphins, and enkephalins. GPCRs respond not only to peptide ligands but also small molecule neurotransmitters (acetylcholine, dopamine, serotonin and adrenaline), light, odorants, taste, lipids, nucleotides, and ions. It is contempalted that GPCRs that respond to peptide ligands, including peptide hormones, are particularly useful for the present invention.
[0037] Pathways involving GPCRs are the targets of hundreds of drugs, including antihistamines, neuroleptics, antidepressants, and antihypertensives. Like all so-called 7TM receptors, G-protein-coupled receptors are integral membrane proteins that possess seven membrane-spanning elements or transmembrane helices. The extracellular parts of the receptor can by glycosylated. These extracellular loops also contain highly conserved cysteine residues which build disulfide bonds to stabilize the receptor structure. [0038] Signalling in G-protein coupled receptors causes interaction between the G- protein-coupled receptor and the G protein. Their interaction results in the dissociation between α and βγ subunits of the G protein. The separated α and/or βγ subunits may then interact with effectors and cause downstream signaling events. These downstream signaling events includeintracellular calcium release and cAMP production. The intracellular signaling systems used by peptide GPCRs are similar to those used by all GPCRs, and are typically classified according to the G-protein they interact with and the second messenger system that is activated. For Gs-coupled GPCRs, activation of the G-protein Gs by receptor stimulates the downstream activation of adenylate cyclase and the production of cyclic AMP, while Gi-coupled receptors inhibit cAMP production. One of the key results of cAMP production is activation of protein kinase A. Gq-coupled receptors stimulate phospholipase C, releasing IP3 and diacylglycerol. IP3 binds to a receptor in the ER to cause the release of intracellular calcium, and the subsequent activation of protein kinase C, calmodulin-dependent pathways. In addition to these second messenger signaling systems for GPCRs, GPCR pathways exhibit crosstalk with other signaling pathways including tyrosine kinase growth factor receptors and map kinase pathways. Transactivation of either receptor tyrosine kinases like the EGF receptor or focal adhesion complexes can stimulate ras activation through the adaptor proteins She, Grb2 and Sos, and downstream Map kinases activating Erkl and Erk2. Src kinases may also play an essential intermediary role in the activation of ras and map kinase pathways by GPCRs.
[0039] Examples of G-protein coupled receptors include alpha adrenergic τeceptors(ADRAlA OR ADRAIC; Accession No. P35348 (SEQ ID NO:8)), adreneocortpicotropic hormone receptors (MC2R OR ACTHR; Accession No. Q01718 (SEQ ID NO:9)), angiotensin type 1 receptors (AGTRl OR AGTRIA OR AT2R1; Accession No. P30556 (SEQ ID NO: 10)), melanocortin- 1 receptor (MC1R OR MSHR; Accession No. Q01726 (SEQ ID NO:l 1)), and Substance K receptor (TACR2 OR TAC2R OR NK2R; Accession No. P21452 (SEQ ID NO: 12)).
IV. Linkers/Coupling Agents [0040] Linking/coupling agents and/or mechanisms known to those of skill in the art can be used to combine to components or agents of the present invention, such as, for example, polyethylene glycol bifunctional linkages, antibody-antigen interaction, avidin biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, amino acid or modified amino acid linkages, or combinations thereof.
TABLE 1 HETERO-BIFUNCTIONAL CROSS-LINKERS
Figure imgf000009_0001
Figure imgf000010_0001
[0041] An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
[0042] It is contemplated that a cross-linker having reasonable stability in blood can be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents. [0043] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
[0044] The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-l,3'-dithiopropionate. The N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue. [0045] In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers. [0046] U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Preferred uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
[0047] U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques. V. Synthetic Peptides [0048] The present invention describes peptides for use in various embodiments of the present invention. Because of their relatively small size, the peptides ofthe invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference.. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
[0049] While peptides ma be isolated from natural sources using standard techniques, it will be advantageous to produce peptides using the solid-phase synthetic techniques (Merrifield, 1963). Other peptide synthesis techniques are well known to those of skill in the art (Bodanszky et al, 1976;) Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984); . Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973. These synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group ofthe first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.
[0050] Using solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide. The peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids. Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
[0051] In certain embodiments ofthe present invention, peptides are synthesized which comprises D amino acids. It is contemplated that peptides comprising L, D, or a mix of L and D amino acids are appropriate.
VI. Imaging Agents [0052] In accordance with the present invention, there are provided diagnostic methods for detecting cells. Many appropriate imaging agents for use in the present invention are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference).
[0053] The agent may comprise an imaging agent or region thereof, by which term is meant an agent which may be detected, whether in vitro in the context of a tissue, organ or organism in which the agent is located. The imaging agent may emit a detectable signal, such as light or other electromagnetic radiation. The imaging agent may be a radio-isotope as known in the art, for example 32P, 35S, 99Tc, n ιIn, 86Y, 18F, and 99mTc or a molecule such as a nucleic acid, polypeptide, or other molecule as explained below conjugated with such a radio-isotope The imaging agent may be opaque to radiation, such as X-ray radiation. The imaging agent may also comprise a targeting means by which it is directed to a particular cell, tissue, organ or other compartment within the body of an animal. For example, the agent may comprise a radiolabelled antibody specific for defined molecules, tissues or cells in an organism.
[0054] The imaging agent may be combined with, conjugated to, mixed with or combined with, any of the agents disclosed herein. Combinations of agents with multiple or overlapping specificities or utilities are clearly contemplated by this invention.
1. Radioisotopes [0055] h the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine , chromium, chlorine, cobalt, cobalt, copper , europium, gallium , iodine , iodine , iodine , indium , iron, phosphorus, rhenium , rhenium , 75selenium, 35sulphur, technicium m and/or yttrium90. Of particular interest are lutetium177, samarium , holmium and actinium" . Also, see Table 2, below. Radioactively labeled peptides of the present invention may be produced according to well-known methods in the art. For instance, peptides can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Peptides according to the invention may be labeled with technetium99™ by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).
2. Fluorescent labels [0056] Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red. 3. Paramagnetic contrasting agents [0057] There is a rapidly growing body of literature demonstrating the clinical effectiveness of paramagnetic contrast agents. The capacity to differentiate regions/tissues that may be magnetically similar but histologically distinct is a major impetus for the preparation of these agents. In the design of MRI agents, strict attention must be given to a variety of properties that will ultimately effect the physiological outcome apart from the ability to provide contrast enhancement. Two fundamental properties that must be considered are biocompatability and proton relaxation enhancement. Biocompatability is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Proton relaxation enhancement (or relaxivity) is chiefly governed by the choice of metal and rotational correlation times. [0058] The first feature to be considered during the design stage is the selection of the metal atom, which will dominate the measured relaxivity of the complex. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation enhancement agents. They decrease the Tγ and T2 relaxation times of nearby (r6 dependence) spins. Some paramagnetic ions decrease the T] without causing substantial linebroadening (e.g., gadolinium (III), (Gd )), while others induce drastic linebroadening (e.g. superparamagnetic iron oxide). The mechanism of Tj relaxation is generally a through space dipole-dipole interaction between the unpaired electrons of the paramagnet (the metal atom with an unpaired electron) and bulk water molecules (water molecules that are not "bound" to the metal atom) that are in fast exchange with water molecules in the metal's inner coordination sphere (are bound to the metal atom).
[0059] For example, regions associated with a Gd3+ ion (near-by water molecules) appear bright in an MR image where the normal aqueous solution appears as dark background if the time between successive scans if the experiment is short (i.e., Ti weighted image). Localized T2 shortening caused by superparamagnetic particles is believed to be due to the local magnetic field inhomogeneities associated with the large magnetic moments of these particles. Regions associated with a superparamagnetic iron oxide particle appear dark in an MR image where the normal aqueous solution appears as high intensity background if the echo time (TE) in the spin- echo pulse sequence experiment is long (i.e., T -weighted image). The lanthanide atom Gd3+ is by the far the most frequently chosen metal atom for MRI contrast agents because it has a very 0 0 Jϊ high magnetic moment (u =63BM ), and a symmetric electronic ground state (S ). Transition metals such as high spin Mn(II) and Fe(III) are also candidates due to their high magnetic moments.
[0060] Once the appropriate metal has been selected, a suitable ligand or chelate must be found to render the complex nontoxic. The term chelator is derived from the Greek word chele which means a "crabs claw", an appropriate description for a material that uses its many "arms" to grab and hold on to a metal atom (see DTPA below). Several factors influence the stability of chelate complexes include enthalpy and entropy effects (e.g. number, charge and basicity of coordinating groups, ligand field and conformational effects). Various molecular design features of the ligand can be directly correlated with physiological results. For example, the presence of a single methyl group on a given ligand structure can have a pronounced effect on clearance rate. While the addition of a bromine group can force a given complex from a purely extracellular role to an effective agent that collects in hepatocytes. [0061] Diethylenetriaminepentaacetic (DTP A) chelates and thus acts to detoxify lanthanide ions. The stability constant (K) for Gd(DTPA)2- is very high (logK=22.4) and is more commonly known as the formation constant (the higher the logK, the more stable the complex). This thermodynamic parameter indicates the fraction of Gd ions that are in the unbound state will be quite small and should not be confused with the rate (kinetic stability) at which the loss of metal occurs (kp /kd). The water soluble Gd(DTPA)2- chelate is stable, nontoxic, and one of the most widely used contrast enhancement agents in experimental and clinical imaging research. It is an extracellular agent that accumulates in tissue by perfusion dominated processes.
[0062] To date, a number of chelators have been used, including diethylenetriaminepentaacetic (DTP A), l,4,7,10-tetraazacyclododecane'-N,N'N",N'"-tetracetic acid (DOTA), and derivatives thereof. See U.S. Pat. Nos. 5,155,215, 5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et al, Invest. Radiol. 25: S53 (1990).
[0063] Image enhancement improvements using Gd(DTPA) are well documented in a number of applications (Runge et al, Magn, Reson. Imag. 3:85 (1991); Russell et al, AJR 152:813 (1989); Meyer et al, Invest. Radiol. 25:S53 (1990)) including visualizing blood-brain barrier disruptions caused by space occupying lesions and detection of abnormal vascularity. It has recently been applied to the functional mapping of the human visual cortex by defining regional cerebral hemodynamics (Belliveau et al. (1991) 254:719).
[0064] Another chelator used in Gd contrast agents is the macrocyclic ligand 1,4,7,10- tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA). The Gd-DOTA complex has been thoroughly studied in laboratory tests involving animals and humans. The complex is conformationally rigid, has an extremely high formation constant (logK=28.5), and at physiological pH possess very slow dissociation kinetics. Recently, the GdDOTA complex was approved as an MRI contrast agent for use in adults and infants in France and has been administered to over 4500 patients.
[0065] Previous work has resulted in MRI contrast agents that report on physiologic or metabolic processes within a biological or other type of sample. As described in U.S. Pat. No. 5,707,605, PCT US96/08549, and U.S. Ser. No. 09/134,072, MRI contrast agents have been constructed that allow an increase in contrast as a result of the interaction of a blocking moiety present on the agent with a target substance. That is, in the presence of the target substance, the exchange of water in one or more inner sphere coordination sites of the contrast agent is increased, leading to a brighter signal; in the absence of the target substance, the exchange of water is hindered and the image remains dark. Thus, the previous work enables imaging of physiological events rather than just structure.
4. Paramagnetic Ions [0066] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
VII. V. Therapeutic Agents [0067] The present invention also provides for the delivery of therapeutic agents to cells using peptides to target such agents. Some examples of therapeutic agents are discussed in the following pages. A. General Therapeutic Agents [0068] Therapeutic agents contemplated by the present invention include cardiovascular drugs, including cardioactive and vasoactive drugs, blood pressure increasing agents antihypertensive agents, antiarrhythmic drugs, beta blockers, cardiac glycosides and synthetic cardiotonic drugs, calcium antagonists, drugs affecting circulation, and diuretics. Therapeutic agents also contemplated by the present invention include neuropharmaceuticals, gastrointestinal drugs, and respiratory tract agents such as analeptics, analgesics and antipyretics, anesthetics, appetite suppressants, antiepileptic drugs, sedatives, local anesthetics, Parkinsonism treatment, antipsychotics/neuroleptics, skeletal muscle relaxants, spasmolytics, anti-ulcer drugs, antiemetics, gallbladder and liver therapeutics, laxatives, gastroprokinetic agents, motilin, cough remedies, antiasthmatics, and antiallergics Therapeutic agents also contemplated by the present invention include antiinfectives, endocrine and metabolic drugs, such as antibiotics, antimycotics, anthelmintics, HIV and AIDS therapeutics, steroids, protein hormones, chemical contraception, thyrotherapeutic agents, hormones, and oral antidiabetic drugs. Other therapeutics contemplated for use in the present invention include interferons, monoclonal antibodies, dermatologic drugs, and opthalmological agents.
B. Anticancer Therapeutics
1. Radiopharmaceuticals [0069] A number of different radioactive substances can be used in cancer therapy. Examples of radioactive isotopes for therapeutic applications include astatine211, 51chromium, chlorine, cobalt, cobalt, copper , europium, gallium , iodine , iodine , iodine , indium 111 , 59 i-ron, 32„ phosphorus, rhenium .186 , rhenium 188 , 75 s,elenium, 35 s„ulphur, technicium ,99m yttrium , lutetium , samarium , holmium , and actinium . Also, see Table 2, below. TABLE 2. THERAPEUTIC AND DIAGNOSTIC RADIOACTIVE ISOTOPES
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
A = June 1996 SNM Abstracts B = Holmes 91 C = Herac 89 D = Fairchild 87 E = Everyone's Guide to Cancer Therapy (Dollinger, Rosenbaum, Cable), 1991 F = SNM (Society of Nuclear Medicine)
2. Chemopharmaceuticals [0070] The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. One subtype of chemotherapy known as biochemotherapy involves the combination of a chemotherapy with a biological therapy.
[0071] Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof. [0072] Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the Goodman & Gilman's 'The Pharmacological Basis of Therapeutics" and in "Remington's Pharmaceutical Sciences", incorporated herein by reference in relevant parts), and may be combined with the invention in light ofthe disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Examples of specific chemotherapeutic agents and dose regimes are also described herein. Of course, all of these dosages and agents described herein are exemplary rather than limiting, and other doses or agents may be used by a skilled artisan for a specific patient or application. Any dosage in-between these points, or range derivable therein is also expected to be of use in the invention. a. Alkylating agents [0073] Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. Alkylating agents can be implemented to treat, for example, chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary. An alkylating agent, may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. [0074] They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan. In specific aspects, troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents, some of which are discussed below. i. Nitrogen Mustards [0075] A nitrogen mustard may be, but is not limited to, mechlorethamine (HN ), which is used for Hodgkin's disease and non-Hodgkin's lymphomas; cyclophosphamide and/or ifosfamide, which are used in treating such cancers as acute or chronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilm's tumor, cervix testis and soft tissue sarcomas; melphalan (L-sarcolysin), which has been used to treat such cancers as multiple myeloma, breast and ovary; and chlorambucil, which has been used to treat diseases such as, for example, chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma, Hodgkin's disease and non-Hodgkin's lymphomas.
[0076] Chlorambucil. Chlorambucil (also known as leukeran) is a bifunctional alkylating agent of the nitrogen mustard type that has been found active against selected human neoplastic diseases. Chlorambucil is known chemically as 4-[bis(2-chlorethyl)amino] benzenebutanoic acid.
[0077] Chlorambucil is available in tablet form for oral administration. It is rapidly and completely absorbed from the gastrointestinal tract. For example, after a single oral doses of about 0.6 mg/kg to about 1.2 mg/kg, peak plasma chlorambucil levels are reached within one hour and the terminal half-life of the parent drug is estimated at about 1.5 hours. About 0.1 mg/kg/day to about 0.2 mg/kg/day or about 3 6 mg/m2/day to about 6 mg/m2/day or alternatively about 0.4 mg/kg may be used for antineoplastic treatment. Chlorambucil is not curative by itself but may produce clinically useful palliation. [0078] Cyclophosphamide. Cyclophosphamide is
2H-l,3,2-Oxazaphosphorin-2-arnine, N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide, monohydrate; termed Cytoxan available from Mead Johnson; and Νeosar available from Adria. Cyclophosphamide is prepared by condensing 3-amino-l-propanol with NN-bis(2-chlorethyl) phosphoramidic dichloride [(ClCΗ CΗ2)2Ν~POCl2] in dioxane solution under the catalytic influence of triethylamine. The condensation is double, involving both the hydroxyl and the amino groups, thus effecting the cyclization.
[0079] Unlike other β-chloroethylamino alkylators, it does not cyclize readily to the active ethyleneimonium form until activated by hepatic enzymes. Thus, the substance is stable in the gastrointestinal tract, tolerated well and effective by the oral and parental routes and does not cause local vesication, necrosis, phlebitis or even pain.
[0080] Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day (usually in combination), depending upon gastrointestinal tolerance; or about
1 mg/kg/day to about 2 mg/kg/day; intravenous doses include, for example, initially about
40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day. In some aspects, a dose of about 250 mg/kg/day may be administered as an antineoplastic. Because of gastrointestinal adverse effects, the intravenous route is preferred for loading. During maintenance, a leukocyte count of about 3000/mm to 4000/mm usually is desired. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities. It is available in dosage forms for injection of about 100 mg, about 200 mg and about 500 mg, and tablets of about 25 mg and about 50 mg.
[0081] Melphalan. Melphalan, also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard. Melphalan is a bifunctional alkylating agent which is active against selective human neoplastic diseases. It is known chemically as 4-[bis(2-chloroethyl)amino]-L-phenylalanine.
[0082] Melphalan is the active L-isomer of the compound and was first synthesized in 1953 by Bergel and Stock; the D-isomer, known as medphalan, is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with the L-isomer. The racemic (DL-) form is known as merphalan or sarcolysin. Melphalan is insoluble in water and has a pKa! of about 2.1. Melphalan is available in tablet form for oral administration and has been used to treat multiple myeloma. Available evidence suggests that about one third to one half of the patients with multiple myeloma show a favorable response to oral administration ofthe drug. [0083] Melphalan has been used in the treatment of epithelial ovarian carcinoma.
One commonly employed regimen for the treatment of ovarian carcinoma has been to administer melphalan at a dose of about 0.2 mg/kg daily for five days as a single course. Courses are repeated about every four to five weeks depending upon hematologic tolerance (Smith and Rutledge, 1975; Young et al, 1978). Alternatively in certain embodiments, the dose of melphalan used could be as low as about 0.05 mg/kg/day or as high as about 3 mg/kg/day or greater. ii. Ethylenimenes and Methymelanunes [0084] An ethylenimene and/or a methylmelamine include, but are not limited to, hexamethylmelamine, used to treat ovary cancer; and thiotepa, which has been used to treat bladder, breast and ovary cancer. iii. Alkyl Sulfonates [0085] An alkyl sulfonate includes but is not limited to such drugs as busulfan, which has been used to treat chronic granulocytic leukemia.
[0086] Busulfan (also known as myleran) is a bifunctional alkylating agent. Busulfan is known chemically as 1,4-butanediol dimethanesulfonate. Busulfan is available in tablet form for oral administration, wherein for example, each scored tablet contains about 2 mg busulfan and the inactive ingredients magnesium stearate and sodium chloride.
[0087] Busulfan is indicated for the palliative treatment of chronic myelogenous
(myeloid, myelocytic, granulocytic) leukemia. Although not curative, busulfan reduces the total granulocyte mass, relieves symptoms of the disease, and improves the clinical state of the patient. Approximately 90% of adults with previously untreated chronic myelogenous leukemia will obtain hematologic remission with regression or stabilization of organomegaly following the use of busulfan. Busulfan has been shown to be superior to splenic irradiation with respect to survival times and maintenance of hemoglobin levels, and to be equivalent to irradiation at controlling splenomegaly. iv. Nitrosourea [0088] Nitrosureas, like alkylating agents, inhibit DNA repair proteins. They are used to treat non-Hodgkin's lymphomas, multiple myeloma, malignant melanoma, in addition to brain tumors. A nitrosourea include but is not limited to a carmustine (BCNU), a lomustine (CCNU), a semustine (methyl-CCNU) or a streptozocin. Semustine has been used in such cancers as a primary brain tumor, a stomach or a colon cancer. Stroptozocin has been used to treat diseases such as a malignant pancreatic insulinoma or a malignalnt carcinoid. Streptozocin has beeen used to treat such cancers as a malignant melanoma, Hodgkin's disease and soft tissue sarcomas. [0089] Carmustine. Carmustine (sterile carmustine) is one of the nitrosoureas used in the treatment of certain neoplastic diseases. It is 1,3 bis (2-chloroethyl)-l -nitrosourea. It is lyophilized pale yellow flakes or congealed mass with a molecular weight of 214.06. It is highly soluble in alcohol and lipids, and poorly soluble in water. Carmustine is administered by intravenous infusion after reconstitution as recommended [0090] Although it is generally agreed that carmustine alkylates DNA and RNA, it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
[0091] Carmustine is indicated as palliative therapy as a single agent or in established combination therapy with other approved chemotherapeutic agents in brain tumors such as glioblastoma, brainstem glioma, medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors. Also it has been used in combination with prednisone to treat multiple myeloma. Carmustine has been used in treating such cancers as a multiple myeloma or a malignant melanoma. Carmustine has proved useful, in the treatment of Hodgkin's Disease and in non-Hodgkin's lymphomas, as secondary therapy in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
[0092] Sterile carmustine is commonly available in 100 mg single dose vials of lyophilized material. The recommended dose of carmustine as a single agent in previously untreated patients is about 150 mg/rn2 to about 200 g/m2 intravenously every 6 weeks. This may be given as a single dose or divided into daily injections such as about 75 mg/m2 to about 100 mg/m2 on 2 successive days. When carmustine is used in combination with other myelosuppressive drugs or in patients in whom bone marrow reserve is depleted, the doses should be adjusted accordingly. Doses subsequent to the initial dose should be adjusted according to the hematologic response of the patient to the preceding dose. It is of course understood that other doses may be used in the present invention, for example about 10 mg/m2, O O 0 about 20 mg/m , about 30 mg/m , about 40 mg/m , about 50 mg/m , about 60 mg/m , about 70 mg/m2, about 80 mg/m2, about 90 mg/m2 to about 100 mg/m2.
[0093] Lomustine. Lomustine is one ofthe nitrosoureas used in the treatment of certain neoplastic diseases. It is l-(2-chloro-ethyl)-3-cyclohexyl-l nitrosourea. It is a yellow powder with the empirical formula of C H16ClN3O2 and a molecular weight of 233.71. Lomustine is soluble in 10% ethanol (about 0.05 mg/mL) and in absolute alcohol (about 70 mg/mL). Lomustine is relatively insoluble in water (less than about 0.05 mg/mL). It is relatively unionized at a physiological pH. Inactive ingredients in lomustine capsules are: magnesium stearate and mannitol. [0094] Although it is generally agreed that lomustine alkylates DNA and RNA, it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
[0095] Lomustine may be given orally. Following oral administration of 9 radioactive lomustine at doses ranging from about 30 mg/m to 100 mg/m , about half of the radioactivity given was excreted in the form of degradation products within 24 hours. The serum half-life of the metabolites ranges from about 16 hours to about 2 days. Tissue levels are comparable to plasma levels at 15 minutes after intravenous administration.
[0096] Lomustine has been shown to be useful as a single agent in addition to other treatment modalities, or in established combination therapy with other approved chemotherapeutic agents in both primary and metastatic brain tumors, in patients who have already received appropriate surgical and/or radiotherapeutic procedures. Lomustine has been used to treat such cancers as small-cell lung cancer. It has also proved effective in secondary therapy against Hodgkin's Disease in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
[0097] The recommended dose of lomustine in adults and children as a single agent in previously untreated patients is about 130 mg/m2 as a single oral dose every 6 weeks. In individuals with compromised bone marrow function, the dose should be reduced to about 100 mg/m2 every 6 weeks. When lomustine is used in combination with other myelosuppressive drugs, the doses should be adjusted accordingly. It is understood that other doses may be used for example, about 20 mg/m2, about 30mg/m2, about 40 mg/m2, about 50 mg/m2, about
60 mg/m , about 70 mg/m , about 80 mg/m , about 90 mg/m , about 100 mg/m to about 120 mg/m2.
[0098] Triazine. A triazine include but is not limited to such drugs as a dacabazine (DTIC; dimethyltriazenoimidaz olecarboxamide), used in the treatment of such cancers as a malignant melanoma, Hodgkin's disease and a soft-tissue sarcoma. b. Antimetabolites [0099] Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate. i. Folic Acid Analogs [0100] Folic acid analogs include but are not limited to compounds such as methotrexate (amethopterin), which has been used in the treatment of cancers such as acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, head and neck, lung and osteogenic sarcoma. ii. Pyrimidine Analogs [0101] Pyrimidine analogs include such compounds as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode-oxyuridine; FudR). Cytarabine has been used in the treatment of cancers such as acute granulocytic leukemia and acute lymphocytic leukemias. Floxuridine and 5-fluorouracil have been used in the treatment of cancers such as breast, colon, stomach, pancreas, ovary, head and neck, urinary bladder and topical premalignant skin lesions.
[0102] 5-Fluorouracil (5-FU) has the chemical name of
5-fluoro-2,4(lH,3H)-pyrimidinedione. Its mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid. Thus, 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers. iii. Purine Analogs and Related Inhibitors [0103] Purine analogs and related compounds include, but are not limited to, mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2-deoxycoformycin). Mercaptopurine has been used in acute lymphocytic, acute granulocytic and chronic granulocytic leukemias. Thrioguanine has been used in the treatment of such cancers as acute granulocytic leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. Pentostatin has been used in such cancers as hairy cell leukemias, mycosis fungoides and chronic lymphocytic leukemia. c. Natural Products [0104] Natural products generally refer to compounds originally isolated from a natural source, and identified has having a pharmacological activity. Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers. i. Mitotic Inhibitors [0105] Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
[0106] Epipodophyllotoxins. Epipodophyllotoxins include such compounds as teniposide and VP16. VP16 is also known as etoposide and is used primarily for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin for small-cell carcinoma of the lung. Teniposide and VP16 are also active against cancers such as testis, other lung cancer, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia, acute nonlymphocytic leukemia, carcinoma of the breast, and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS).
[0107] VP16 is available as a solution (e.g., 20 mg/ml) for intravenous administration and as 50 mg, liquid-filled capsules for oral use. For small-cell carcinoma ofthe lung, the intravenous dose (in combination therapy) is can be as much as about 100 mg/m2 or as little as about 2 mg/ m2, routinely about 35 mg/m2, daily for about 4 days, to about 50 mg/m2, daily for about 5 days have also been used. When given orally, the dose should be doubled. Hence the doses for small cell lung carcinoma may be as high as about 200 mg/m2 to about 250 mg/m . The intravenous dose for testicular cancer (in combination therapy) is about 50 mg/m2 to about 100 mg/m2 daily for about 5 days, or about 100 mg/m2 on alternate days, for three doses. Cycles of therapy are usually repeated about every 3 to 4 weeks. The drug should be administered slowly (e.g., about 30 minutes to about 60 minutes) as an infusion in order to avoid hypotension and bronchospasm, which are probably due to the solvents used in the formulation.
[0108] Taxoids. Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include but are not limited to compounds such as docetaxel and paclitaxel.
[0109] Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Paclitaxel is being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary. In certain aspects, maximal doses are about 30 mg/m2 per day for about 5 days or about 210 mg/m2 to about
250 mg/m2 given once about every 3 weeks.
[0110] Vinca Alkaloids. Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. They include such compounds as vinblastine (VLB) and vincristine. Vinblastine is an example of a plant alkaloid that can be used for the treatment of cancer and precancer. When cells are incubated with vinblastine, dissolution ofthe microtubules occurs.
[0111] Unpredictable absorption has been reported after oral administration of vinblastine or vincristine. At the usual clinical doses the peak concentration of each drug in plasma is approximately 0.4 mM. Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes.
[0112] After intravenous injection, vinblastine has a multiphasic pattern of clearance from the plasma; after distribution, drug disappears from plasma with half-lives of approximately 1 and 20 hours. Vinblastine is metabolized in the liver to biologically activate derivative desacetylvinblastine. Approximately 15% of an administered dose is detected intact in the urine, and about 10% is recovered in the feces after biliary excretion. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 mM). [0113] Vinblastine sulfate is available in preparations for injection. When the drug is given intravenously; special precautions must be taken against subcutaneous extravasation, since this may cause painful irritation and ulceration. The drug should not be injected into an extremity with impaired circulation. After a single dose of 0.3 mg/kg of body weight, myelosuppression reaches its maximum in about 7 days to about 10 days. If a moderate level of leukopenia (approximately 3000 cells/mm3) is not attained, the weekly dose may be increased gradually by increments of about 0.05 mg/kg of body weight. In regimens designed to cure testicular cancer, vinblastine is used in doses of about 0.3 mg/kg about every 3 weeks irrespective of blood cell counts or toxicity. [0114] An important clinical use of vinblastine is with bleomycin and cisplatin in the curative therapy of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas, particularly Hodgkin's disease, where significant improvement may be noted in 50 to 90% of cases. The effectiveness of vinblastine in a high proportion of lymphomas is not diminished when the disease is refractory to alkylating agents. It is also active in Kaposi's sarcoma, testis cancer, neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well as in carcinoma ofthe breast and choriocarcinoma in women.
[0115] Doses of about 0.1 mg/kg to about 0.3 mg/kg can be administered or about 1.5 mg/m2 to about 2 mg/m2 can also be administered. Alternatively, about 0.1 mg/m2, about 0 0 0 o
0.12 mg/m , about 0.14 mg/m , about 0.15 mg/m , about 0.2 mg/m , about 0.25 mg/m , about 0 0 0 0 0 0.5 mg/m , about 1.0 mg/m , about 1.2 mg/m , about 1.4 mg/m , about 1.5 mg/m , about 0 0 0 0 0 0
2.0 mg/m , about 2.5 mg/m , about 5.0 mg/m , about 6 mg/m , about 8 mg/m , about 9 mg/m , 0 about 10 mg/m , to about 20 mg/m , can be given.
[0116] Vincristine blocks mitosis and produces metaphase arrest. It seems likely that most of the biological activities of this drug can be explained by its ability to bind specifically to tubulin and to block the ability of protein to polymerize into microtubules. Through disruption of the microtubules of the mitotic apparatus, cell division is arrested in metaphase. The inability to segregate chromosomes correctly during mitosis presumably leads to cell death.
[0117] The relatively low toxicity of vincristine for normal marrow cells and epithelial cells make this agent unusual among anti-neoplastic drugs, and it is often included in combination with other myelosuppressive agents. [0118] Unpredictable absorption has been reported after oral administration of vinblastine or vincristine. At the usual clinical doses the peak concentration of each drug in plasma is about 0.4 mM.
[0119] Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes. Vincristine has a multiphasic pattern of clearance from the plasma; the terminal half-life is about 24 hours. The drug is metabolized in the liver, but no biologically active derivatives have been identified. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than about 3 mg/dl (about 50 mM).
[0120] Vincristine sulfate is available as a solution (e.g., 1 mg/ml) for intravenous injection. Vincristine used together with corticosteroids is presently the treatment of choice to induce remissions in childhood leukemia; the optimal dosages for these drugs appear to be vincristine, intravenously, about 2 mg/m2 of body-surface area, weekly; and prednisone, orally, about 40 mg/m2, daily. Adult patients with Hodgkin's disease or non-Hodgkin's lymphomas usually receive vincristine as a part of a complex protocol. When used in the MOPP regimen, the recommended dose of vincristine is about 1.4 mg/m2. High doses of vincristine seem to be tolerated better by children with leukemia than by adults, who may experience sever neurological toxicity. Administration ofthe drug more frequently than every 7 days or at higher doses seems to increase the toxic manifestations without proportional improvement in the response rate. Precautions should also be used to avoid extravasation during intravenous administration of vincristine. Vincristine (and vinblastine) can be infused into the arterial blood supply of tumors in doses several times larger than those that can be administered intravenously with comparable toxicity. [0121] Vincristine has been effective in Hodgkin's disease and other lymphomas.
Although it appears to be somewhat less beneficial than vinblastine when used alone in Hodgkin's disease, when used with mechlorethamine, prednisone, and procarbazine (the so-called MOPP regimen), it is the preferred treatment for the advanced stages (III and IV) of this disease. In non-Hodgkin's lymphomas, vincristine is an important agent, particularly when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone. Vincristine is more useful than vinblastine in lymphocytic leukemia. Beneficial response have been reported in patients with a variety of other neoplasms, particularly Wilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, small cell lung, and carcinomas of the breast, bladder, and the male and female reproductive systems.
[0122] Doses of vincristine include about 0.01 mg/kg to about 0.03 mg/kg or about 0.4 mg/m2 to about 1.4 mg/m2 can be administered or about 1.5 mg/m2 to about 2 mg/m2 can also be administered. Alternatively, in certain embodiments, about 0.02 mg/m2, about 0 0 0 o
0.05 mg/nT, about 0.06 mg/m , about 0.07 mg/m , about 0.08 mg/m , about 0.1 mg/m , about O 0 0 0 o
0.12 mg/m , about 0.14 mg/m , about 0.15 mg/m , about 0.2 mg/m , about 0.25 mg/m can be given as a constant intravenous infusion. [0123] Antitumor Antibiotics. Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers. Examples of antitumor antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin. Widely used in clinical setting for the treatment of neoplasms these compounds generally are administered through intravenous bolus injections or orally.
[0124] Doxorubicin hydrochloride, 5,12-Naphthacenedione,
(8s-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,8,1 l-trihydroxy-8-(hydroxyacetyl)-l-methoxy-hydrochloride (hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wide antineoplastic spectrum. It binds to DNA and inhibits nucleic acid synthesis, inhibits mitosis and promotes chromosomal aberrations.
[0125] Administered alone, it is the drug of first choice for the treatment of thyroid adenoma and primary hepatocellular carcinoma. It is a component of 31 first-choice combinations for the treatment of diseases including ovarian, endometrial and breast tumors, bronchogenic oat-cell carcinoma, non-small cell lung carcinoma, stomach, genitourinary, thyroid, gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostatic carcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' tumor, Hodgkin's disease, adrenal tumors, osteogenic sarcoma, soft tissue sarcoma, Ewing' s sarcoma, rhabdomyosarcoma and acute lymphocytic leukemia. It is an alternative drug for the treatment of other diseases such as islet cell, cervical, testicular and adrenocortical cancers. It is also an immunosuppressant.
[0126] Doxorubicin is absorbed poorly and is preferably administered intravenously. The pharmacokinetics are multicompartmental. Distribution phases have half-lives of 12 minutes and 3.3 hours. The elimination half-life is about 30 hours, with about 40% to about 50% secreted into the bile. Most ofthe remainder is metabolized in the liver, partly to an active metabolite (doxorubicinol), but a few percent is excreted into the urine. In the presence of liver impairment, the dose should be reduced.
[0127] In certain embodiments, appropriate intravenous doses are, adult, about 60 mg/m2 to about 75 mg/m2 at about 21-day intervals or about 25 mg/m2 to about 30 mg/m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg/m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs. The dose should be reduced by about 50% if the serum bilirubin lies between about 1.2 mg/dL and about 3 mg/dL and by about 75% if above about 3 mg/dL. The lifetime total dose should not exceed about 550 mg/m2 in patients with normal heart function and about 400 mg/m in persons having received mediastinal irradiation. In certain embodiments, and alternative dose regiment may comprise about 30 mg/m on each of 3 consecutive days, repeated about every 4 week. Exemplary doses may be O O 0 o o about 10 mg/m , about 20 mg/m , about 30 mg/m , about 50 mg/m , about 100 mg/m , about 0 o o o o
150 mg/m , about 175 mg/m , about 200 mg/m , about 225 mg/m , about 250 mg/m , about
275 mg/m , about 300 mg/m , about 350 mg/m , about 400 mg/m , about 425 mg/m , about 450 mg/m2, about 475 mg/m2, to about 500 mg/m2.
[0128] Daunorubicin hydrochloride, 5,12-Naphthacenedione, (8S-cw)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-
7,8,9, 10-tetrahydro-6,8,l l-trihydroxy-10-methoxy-, hydrochloride; also termed cerubidine and available from Wyeth. Daunorubicin (daunomycin; rubidomycin) intercalates into DNA, blocks DAN-directed RNA polymerase and inhibits DNA synthesis. It can prevent cell division in doses that do not interfere with nucleic acid synthesis. [0129] In combination with other drugs it is often included in the first-choice chemotherapy of diseases such as, for example, acute granulocytic leukemia, acute myelocytic leukemia in adults (for induction of remission), acute lymphocytic leukemia and the acute phase of chronic myelocytic leukemia. Oral absorption is poor, and it preferably given by other methods (e.g., intravenously). The half-life of distribution is 45 minutes and of elimination, about 19 hours. The half-life of its active metabolite, daunorubicinol, is about 27 hours. Daunorubicin is metabolized mostly in the liver and also secreted into the bile (about 40%). Dosage must be reduced in liver or renal insufficiencies.
[0130] Generally, suitable intravenous doses are (base equivalent): adult, younger than 60 years, about 45 mg/m2/day (about 30 mg/m2 for patients older than 60 year.) for about
1 day, about 2 days or about 3 days about every 3 weeks or 4 weeks or about 0.8 mg/kg/day for about 3 days, about 4 days, about 5 days to about 6 days about every 3 weeks or about 4 weeks; no more than about 550 mg/m2 should be given in a lifetime, except only about 450 mg/m2 if there has been chest irradiation; children, about 25 mg/m once a week unless the age is less than
2 years, or the body surface less than about 0.5 m, in which case the weight-based adult schedule is used. It is available in injectable dosage forms (base equivalent) of about 20 mg (as the base equivalent to about 21.4 mg of the hydrochloride). Exemplary doses may be about 10 mg/m2, 0 0 0 0 0 about 20 mg/m , about 30 mg/m , about 50 mg/m , about 100 mg/m , about 150 mg/m , about 0 0 0 0 0
175 mg/m , about 200 mg/m , about 225 mg/m , about 250 mg/m , about 275 mg/m , about 0 0 0 0 0
300 mg/m , about 350 mg/m , about 400 mg/m , about 425 mg/m , about 450 mg/m , about 0 0
475 mg/m , to about 500 mg/m . [0131] Mitomycin (also known as mutamycin and/or mitomycin-C) is an antibiotic isolated from the broth of Streptomyces caespitosus which has been shown to have antitumor activity. The compound is heat stable, has a high melting point, and is freely soluble in organic solvents.
[0132] Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA). The guanine and cytosine content correlates with the degree of mitomycin-induced cross-linking. At high concentrations of the drug, cellular RNA and protein synthesis are also suppressed. Mitomycin has been used in tumors such as stomach, cervix, colon, breast, pancreas, bladder and head and neck.
[0133] In humans, mitomycin is rapidly cleared from the serum after intravenous administration. Time required to reduce the serum concentration by about 50% after a 30 mg. bolus injection is 17 minutes. After injection of 30 mg, 20 mg, or 10 mg I.V., the maximal serum concentrations were 2.4 mg/mL, 1.7 mg/mL, and 0.52 mg/mL, respectively. Clearance is effected primarily by metabolism in the liver, but metabolism occurs in other tissues as well. The rate of clearance is inversely proportional to the maximal serum concentration because, it is thought, of saturation ofthe degradative pathways. Approximately 10% of a dose of mitomycin is excreted unchanged in the urine. Since metabolic pathways are saturated at relatively low doses, the percent of a dose excreted in urine increases with increasing dose. In children, excretion of intravenously administered mitomycin is similar.
[0134] Actinomycin D (Dactinomycin) [50-76-0]; C62H86N12O16 (1255.43) is an antineoplastic drug that inhibits DNA-dependent RNA polymerase. It is often a component of first-choice combinations for treatment of diseases such as, for example, choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor, Kaposi's sarcoma and Wilms' tumor. Tumors that fail to respond to systemic treatment sometimes respond to local perfusion. Dactinomycin potentiates radiotherapy. It is a secondary (efferent) immunosuppressive.
[0135] In certain specific aspects, actinomycin D is used in combination with agents such as, for example, primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide. Antineoplastic activity has also been noted in Ewing' s tumor, Kaposi's sarcoma, and soft-tissue sarcomas. Dactinomycin can be effective in women with advanced cases of choriocarcinoma. It also produces consistent responses in combination with chlorambucil and methotrexate in patients with metastatic testicular carcinomas. A response may sometimes be observed in patients with Hodgkin's disease and non-Hodgkin's lymphomas. Dactinomycin has also been used to inhibit immunological responses, particularly the rejection of renal transplants.
[0136] Half of the dose is excreted intact into the bile and 10% into the urine; the half-life is about 36 hours. The drug does not pass the blood-brain barrier. Actinomycin D is supplied as a lyophilized powder (0/5 mg in each vial). The usual daily dose is about 10 mg/kg to about 15 mg/kg; this is given intravenously for about 5 days; if no manifestations of toxicity are encountered, additional courses maybe given at intervals of about 3 weeks to about 4 weeks. Daily injections of about 100 mg to about 400 mg have been given to children for about 10 days to about 14 days; in other regimens, about 3 mg/kg to about 6 mg/kg, for a total of about 125 mg/kg, and weekly maintenance doses of about 7.5 mg/kg have been used. Although it is safer to administer the drug into the tubing of an intravenous infusion, direct intravenous injections have been given, with the precaution of discarding the needle used to withdraw the drug from the vial in order to avoid subcutaneous reaction. Exemplary doses may be about 0 0 0 0 0
100 mg/m , about 150 mg/m , about 175 mg/m , about 200 mg/m , about 225 mg/m , about o o
250 mg/m , about 275 mg/m , about 300 mg/πT, about 350 g/m", about 400 mg/m , about 425 mg/m2, about 450 mg/m2, about 475 mg/m2, to about 500 mg/m2.
[0137] Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. Although the exact mechanism of action of bleomycin is unknown, available evidence would seem to indicate that the main mode of action is the inhibition of DNA synthesis with some evidence of lesser inhibition of RNA and protein synthesis.
[0138] In mice, high concentrations of bleomycin are found in the skin, lungs, kidneys, peritoneum, and lymphatics. Tumor cells ofthe skin and lungs have been found to have high concentrations of bleomycin in contrast to the low concentrations found in hematopoietic tissue. The low concentrations of bleomycin found in bone marrow may be related to high levels of bleomycin degradative enzymes found in that tissue.
[0139] In patients with a creatinine clearance of greater than about 35 mL per minute, the serum or plasma terminal elimination half-life of bleomycin is approximately 115 minutes. In patients with a creatinine clearance of less than about 35 mL per minute, the plasma or serum terminal elimination half-life increases exponentially as the creatinine clearance decreases. In humans, about 60% to about 70% of an administered dose is recovered in the urine as active bleomycin. In specific embodiments, bleomycin may be given by the intramuscular, intravenous, or subcutaneous routes. It is freely soluble in water. Because of the possibility of an anaphylactoid reaction, lymphoma patients should be treated with two units or less for the first two doses. If no acute reaction occurs, then the regular dosage schedule may be followed. [0140] In certain aspects, bleomycin should be considered a palliative treatment.
It has been shown to be useful in the management of the following neoplasms either as a single agent or in proven combinations with other approved chemotherapeutic agents in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), esophagus, lung and genitourinary tract, Hodgkin's disease, non-Hodgkin's lymphoma, skin, penis, cervix, and vulva. It has also been used in the treatment of lymphomas and testicular carcinoma. [0141] Improvement of Hodgkin's Disease and testicular tumors is prompt and noted within 2 weeks. If no improvement is seen by this time, improvement is unlikely. Squamous cell cancers respond more slowly, sometimes requiring as long as 3 weeks before any improvement is noted. 3. Miscellaneous Agents [0142] Some chemotherapy agents do not qualify into the previous categories based on their activities. They include, but are not limited to, platinum coordination complexes, anthracenedione, substituted urea, methyl hydrazine derivative, adrenalcortical suppressant, amsacrine, L-asparaginase, and tretinoin. It is contemplated that they are included within the compositions and methods ofthe present invention for use in combination therapies. a. Platinum Coordination Complexes [0143] . Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis-DDP). Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with 0 efficacious doses used in clinical applications of about 15 mg/m to about 20 mg/m for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. Doses may be, for example, about 0.50 mg/m2, about 1.0 mg/m2, about 1.50 mg/m2, about 1.75 mg/m2, about 2.0 mg/m , about 3.0 mg/m , about 4.0 mg/m , about 5.0 mg/m , to about 10 mg/m . b. Other Agents [0144] An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer. A substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis and malignant melanoma. A methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease. An adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease. 4. Toxins [0145] Various toxins are also useful in the treatment of cancers. As part of the present invention, toxins such as ricin A-chain (Burbage, 1997), diphtheria toxin A (Massuda et al, 1997; Lidor, 1997), pertussis toxin A subunit, E. coli enterotoxin toxin A subunit, cholera toxin A subunit and Pseudomonas toxin c-terminal are suitable. It has demonstrated that transfection of a plasmid containing the fusion protein regulatable diphtheria toxin A chain gene was cytotoxic for cancer cells.
VIIL Methods of Treatment [0146] In a particular aspect, the present invention provides methods for the treatment of cancer, wherein the cancer is characterized by cells having an oxidative or hypoxic cellular environment. The present invention relates to a peptide linked to a therapeutic agent that shows unexpected, potent anti-cancer activity in vitro. Thus, the present invention provides a peptide linked to a therapeutic agent as an agent for treating a cancer in a subject.
[0147] Treatment methods will involve treating an individual with an effective amount of a composition containing a peptide linked to a therapeutic agent. An effective amount is described, generally, as that amount sufficient to detectably and repeatedly to ameliorate, reduce, minimize or limit the extent of a disease or its symptoms. More specifically, it is envisioned that the treatment with the peptide linked to a therapeutic agent will kill cells, inhibit cell growth, cleave DNA, inhibit DNA synthesis, inhibit metastasis, decrease tumor size and otherwise reverse or reduce the malignant phenotype of tumor cells.
[0148] In certain embodiments, the peptide linked to a therapeutic agent is administered to a cell. Cells that are encompassed by the present invention include, but are not limited to melanoma cells. The cell may be a cancer cell, a non-cancerous cell or a benign hyperplastic cell. A cancer cell may include, cells that are drug-resistant, primary cancer cells and/or metastatic cancer cells.
[0149] Other cells as contemplated in the present invention may be a cancer cell such as, but not limited to, a breast cancer cell, lung cancer cell, head and neck cancer cell, bladder cancer cell, bone cancer cell, bone marrow cancer cell, brain cancer cell, colon cancer cell, esophageal cancer cell, gastrointestinal cancer cell, gum cancer cell, kidney cancer cell, liver cancer cell, nasopharynx cancer cell, ovarian cancer cell, prostate cancer cell, skin cancer cell, stomach cancer cell, testis cancer cell, tongue cancer cell, or uterine cancer cell.
[0150] An effective amount of the peptide linked to a therapeutic agent that may be administered to a cell includes a dose of about - 0.1 μM to about 100 μM. More specifically, doses ofthe peptide linked to a therapeutic agent to be administered are from about - 0.1 μM to about 1 μM; about 1 μM to about 5 μM; about 5 μM to about 10 μM; about 10 μM to about 15 μM; about 15 μM to about 20 μM; about 20 μM to about 30 μM; about 30 μM to about 40 μM; about 40 μM to about 50 μM; about 50 μM to about 60 μM; about 60 μM to about 70 μM; about 70 μM to about 80 μM; about 80 μM to about 90 μM; and about 90 μM to about 100 μM . Of course, all of these amounts are exemplary, and any amount in-between these points is also expected to be of use in the invention.
[0151] In further aspects, an effective amount of the peptide linked to a therapeutic agent may be administered to a subject suffering from melanoma. The effectiveness of the therapy according to the present invention can be determined in the treatment of melanoma by diagnostic methods that are known and used in the art, for example, but not limited to, analysis of a biopsy.
[0152] Other embodiments include methods for inhibiting development of melanoma in a subject at risk, inhibiting melanoma metastasis in a subject with primary melanoma, and/or inhibiting melanoma progression in subjects having Stage 1 or Stage 2 melanoma. [0153] The effective amount or "therapeutically effective amounts" of the peptide linked to a therapeutic agent to be used are those amounts effective to produce beneficial results, particularly with respect to cancer treatment, in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value. [0154] As is well known in the art, a specific dose level of active compounds such as the peptide linked to a therapeutic agent for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The person responsible for administration will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
[0155] A therapeutically effective amount ofthe peptide linked to a therapeutic agent as a treatment varies depending upon the host treated and the particular mode of administration. In one embodiment ofthe invention the dose range of the peptide linked to a therapeutic agent will be about 0.5 mg/kg body weight to about 500 mg/kg body weight. The term "body weight" is applicable when an animal is being treated. When isolated cells are being treated, "body weight" as used herein should read to mean "total cell weight". The term "total weight may be used to apply to both isolated cell and animal treatment. All concentrations and treatment levels are expressed as "body weight" or simply "kg" in this application are also considered to cover the analogous "total cell weight" and "total weight" concentrations. However, those of skill will recognize the utility of a variety of dosage range, for example, 1 mg/kg body weight to 450 mg/kg body weight, 2 mg/kg body weight to 400 mg/kg body weighty, 3 mg/kg body weight to 350 mg/kg body weighty, 4 mg/kg body weight to 300 mg/kg body weight, 5 mg/kg body weight to 250 mg/kg body weighty, 6 mg/kg body weight to 200 mg/kg body weight, 7 mg/kg body weight to 150 mg/kg body weighty, 8 mg/kg body weight to 100 mg/kg body weight, or 9 mg/kg body weight to 50 mg/kg body weight. Further, those of skill will recognize that a variety of different dosage levels will be of use, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 120 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg, 200 mg/kg, 225 mg/kg, 250mg/kg, 275mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1250 mg/kg, 1500 mg/kg, 1750 mg/kg, 2000 mg/kg, 2500 mg/kg, and/or 3000 mg/kg. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention. Any of the above dosage ranges or dosage levels may be employed for the peptide linked to a therapeutic agent.
[0156] Administration of the therapeutic the peptide linked to a therapeutic agent compositions ofthe present invention to a patient or subject will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, ofthe composition. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
[0157] The treatments may include various "unit doses." Unit dose is defined as containing a predetermined-quantity of the therapeutic composition (the peptide linked to a therapeutic agent) calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. Also of import is the subject to be treated, in particular, the state of the subject and the protection desired. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
[0158] According to the present invention, one may treat the cancer by directly injection a tumor with the peptide linked to a therapeutic agent. Alternatively, the tumor may be infused or perfused with the composition using any suitable delivery vehicle. Local or regional administration, with respect to the tumor, also is contemplated. More preferably, systemic administration or oral administration may be performed. Continuous administration also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease. Delivery via syringe or catherization is preferred. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. For tumors of > 4 cm, the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of < 4 cm, a volume of about 1-3 ml will be used (preferably 3 ml). Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes. IX. Combined Therapy with the Peptide linked to a Therapeutic Agent and/or Other Anticancer Agents [0159] In the context ofthe present invention, it is contemplated that the peptide linked to a therapeutic agent may be used in combination with an additional therapeutic agent to more effectively treat cancer or other diseases. Anticancer agents may include but are not limited to, radiotherapy, chemotherapy, gene therapy, hormonal therapy or immunotherapy that targets cancer/tumor cells.
[0160] When an additional therapeutic agent is administered, as long as the dose of the additional therapeutic agent does not exceed previously quoted toxicity levels, the effective amounts of the additional therapeutic agent may simply be defined as that amount effective to inhibit and/or reduce the cancer growth when administered to an animal in combination with the peptide linked to a therapeutic agent. This may be easily determined by monitoring the animal or patient and measuring those physical and biochemical parameters of health and disease that are indicative of the success of a given treatment. Such methods are routine in animal testing and clinical practice.
[0161] To kill cells, induce cell-cycle arrest, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of cancer cells, using the methods and compositions ofthe present invention, one would generally contact a cell with the peptide linked to a therapeutic agent in combination with an additional therapeutic agent. These compositions would be provided in a combined amount effective to inhibit cell growth and or induce apoptosis in the cell. This process may involve contacting the cells with the peptide linked to a therapeutic agent in combination with an additional therapeutic agent or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the peptide linked to a therapeutic agent and the other includes the additional agent.
[0162] Alternatively, treatment with the peptide linked to a therapeutic agent may precede or follow the additional agent treatment by intervals ranging from minutes to weeks. In embodiments where the additional agent is applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hr of each other and, more preferably, within about 6-12 hr of each other, with a delay time of only about 12 hr being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
[0163] It also is conceivable that more than one administration of either the peptide linked to a therapeutic agent in combination with an additional therapeutic agent such as anticancer agent will be desired. Various combinations may be employed, where the peptide linked to a therapeutic agent is "A" and the additional therapeutic agent is "B", as exemplified below:
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
A. Chemotherapeutic Agents [0164] In some embodiments of the present invention chemotherapy may be administered, as is typical, in regular cycles. A cycle may involve one dose, after which several days or weeks without treatment ensues for normal tissues to recover from the drug's side effects. Doses may be given several days in a row, or every other day for several days, followed by a period of rest. If more than one drug is used, the treatment plan will specify how often and exactly when each drug should be given. The number of cycles a person receives may be determined before treatment starts (based on the type and stage of cancer) or may be flexible, in order to take into account how quickly the tumor is shrinking. Certain serious side effects may also require doctors to adjust chemotherapy plans to allow the patient time to recover.
[0165] Chemotherapeutic agents that may be used in combination with the peptide linked to a therapeutic agent in the treatment of cancer, include, but are not limited to cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil and methotrexate, or any analog or derivative variant of the foregoing.
B. Radiotherapeutic Agents [0166] Radiotherapeutic agents may also be use in combination with the compounds of the present invention in treating a cancer. Such factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. C. Immunotherapeutic Agents [0167] Immunotherapeutics may also be employed in the present invention in combination with the peptide linked to a therapeutic agent thereof in treating cancer. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.
[0168] Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context ofthe present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
D. Inhibitors of Cellular Proliferation [0169] The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors p53, pl6 and C-CAM are described below.
[0170] High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al, 1991) and in a wide spectrum of other tumors.
[0171] The p53 gene encodes a 393-amino acid phosphoprotein that can form complexes with host proteins such as large-T antigen and E1B. The protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue
[0172] Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene. A single genetic change prompted by point mutations can create carcinogenic p53. Unlike other oncogenes, however, p53 point mutations are known to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity. Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line. Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991). [0173] Another inhibitor of cellular proliferation is pi 6. The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin- dependent kinase 4 (CDK4), regulates progression through the Gl . The activity of this enzyme may be to phosphorylate Rb at late Gl . The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al, 1995). Since the pl6INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6.
[0174] pl6INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes ρl6B, pi 9, p21WAFl, and p27KIPl. The pl6INK4 gene maps to 9ρ21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6INK4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6LNK4 gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency ofthe pl6INK4 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al, 1994; Cheng et al, 1994; Arap et al, 1995). Restoration of wild-type pl6INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).
[0175] Other genes that may be employed according to the present invention include Rb, mda-7, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fins, trk, ret, gsp, hst, abl, El A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
E. Regulators of Programmed Cell Death [0176] Apoptosis, or programmed cell death, is an essential process in cancer therapy (Kerr et al, 1972). The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists. [0177] Members of the Bcl-2 that function to promote cell death such as, Bax, Bak, Bik, Bim, Bid, Bad and Harakiri, are contemplated for use in combination with the peptide linked to a therapeutic agent in treating cancer.
F. Surgery [0178] It is further contemplated that a surgical procedure may be employed in the present invention. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
[0179] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments maybe of varying dosages as well.
G. Hormonal Therapy [0180] Hormonal therapy may also be used in conjunction with the peptide linked to a therapeutic agent as in the present invention, or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
H. Other agents [0181] It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MlP-lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increased intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
X. Formulations and Routes for Administration of the Peptide Linked to a Therapeutic Agent [0182] Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions of the peptide linked to a therapeutic agent, or any additional therapeutic agent disclosed herein in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. [0183] One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention in an effective amount may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
[0184] The composition(s) of the present invention may be delivered orally, nasally, intramuscularly, intraperitoneally, or intratumorally. In some embodiments, local or regional delivery of the peptide linked to a therapeutic agent, alone or in combination with an additional therapeutic agent, to a patient with cancer or pre-cancer conditions will be a very efficient method of delivery to counteract the clinical disease. Similarly, chemo- or radiotherapy may be directed to a particular, affected region of the subject's body. Regional chemotherapy typically involves targeting anticancer agents to the region ofthe body where the cancer cells or tumor are located. Other examples of delivery of the compounds of the present invention that may be employed include intra-arterial, intracavity, intravesical, intrathecal, intrapleural, and intraperitoneal routes.
[0185] Intra-arterial administration is achieved using a catheter that is inserted into an artery to an organ or to an extremity. Typically, a pump is attached to the catheter. Intracavity administration describes when chemotherapeutic drugs are introduced directly into a body cavity such as intravesical (into the bladder), peritoneal (abdominal) cavity, or pleural (chest) cavity. Agents can be given directly via catheter. Intravesical chemotherapy involves a urinary catheter to provide drugs to the bladder, and is thus useful for the treatment of bladder cancer. Intrapleural administration is accomplished using large and small chest catheters, while a Tenkhoff catheter (a catheter specially designed for removing or adding large amounts of fluid from or into the peritoneum) or a catheter with an implanted port is used for intraperitoneal chemotherapy. Abdomen cancer may be treated this way. Because most drugs do not penetrate the blood/brain barrier, intrathecal chemotherapy is used to reach cancer cells in the central nervous system. To do this, drugs are administered directly into the cerebrospinal fluid. This method is useful to treat leukemia or cancers that have spread to the spinal cord or brain. [0186] Alternatively, systemic delivery of the chemotherapeutic drugs may be appropriate in certain circumstances, for example, where extensive metastasis has occurred. Intravenous therapy can be implemented in a number of ways, such as by peripheral access or through a vascular access device (VAD). A VAD is a device that includes a catheter, which is placed into a large vein in the arm, chest, or neck. It can be used to administer several drugs simultaneously, for long-term treatment, for continuous infusion, and for drugs that are vesicants, which may produce serious injury to skin or muscle. Various types of vascular access devices are available.
[0187] The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes but is not limited to, oral, nasal, or buccal routes. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. The drugs and agents also may be administered parenterally or intraperitoneally. The term "parenteral" is generally used to refer to drugs given intravenously, intramuscularly, or subcutaneously.
[0188] Solutions ofthe active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0189] The therapeutic compositions of the present invention may be administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti- oxidants, chelating agents and inert gases. The pH, exact concentration of the various components, and the pharmaceutical composition are adjusted according to well known parameters. Suitable excipients for formulation with the peptide linked to a therapeutic agent include croscarmellose sodium, hydroxypropyl methylcellulose, iron oxides synthetic), magnesium stearate, macrocrystalline cellulose, polyethylene glycol 400, polysorbate 80, povidone, silicon dioxide, titanium dioxide, and water (purified). [0190] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.
XL Melanoma [0191] Melanoma begins in melanocytes, which are cells that make the skin pigment melanin. Although melanoma accounts for only about 4% of all skin cancer cases, it is the cause of most skin cancer-related deaths. Melanoma is often curable if it is detected and treated in its early stages. In men, melanoma is found most often on the area between the shoulders and hips or on the head and neck. In women, melanoma often develops on the lower legs. It may also appear under the fingernails or toenails or on the palms or soles. The chance of developing melanoma increases with age, but it affects all age groups and is one of the most common cancers in young adults. [0192] The number of new melanomas diagnosed in the United States is increasing.
Since 1973, the rate of new melanomas diagnosed per year has more than doubled from 6 per 100,000 to 14 per 100,000. The American Cancer Society estimates that about 51,400 new melanomas will be diagnosed in the United States during 2001. About 7,800 cancer deaths will be attributed to malignant melanoma in 2001. [0193] When melanoma starts in the skin, it is called cutaneous melanoma. Melanoma may also occur in the eye (ocular melanoma or intraocular melanoma) and, rarely, in other areas where melanocytes are found, such as the digestive tract, meninges, or lymph nodes. When melanoma metastasizes, cancer cells are also found in the lymph nodes and possibly also other parts of the body, such as the liver, lungs, or brain. In these cases, the cancer cells are still melanoma cells, and the disease is called metastatic melanoma.
XII. Examples [0194] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope ofthe invention. Example 1 . Peptide Design [0195] The prototype nuclear targeting MSH peptide was based on the wild-type α- MSH peptide found in most vertebrates including humans. Its sequence is Ac- SYSMEHFRWGKPV-NH2 (SEQ ID NO: 13). The N-terminus of the wild-type α-MSH peptide is acetylated and the C-terminus is amidated. The consensus sequence for nuclear targeting is K(K/R)x(K R). Membrane translocation properties were achieved through the use of optimization of known translocation peptide sequences. One optimized translocation peptide, PTD-4 had the sequence YARAAARQARA (SEQ ID NO: 14). It has been shown that the translocation peptides are likely to form an ampliipathic α-helix with the positive charges on one face to the helix. Computer aided molecular design was used to incorporate the critical distribution of positively charged residues of the translocation peptide into the nuclear localization sequence. The nuclear localization sequence is recognized as an extended peptide structure to our NLS peptide would have to be able to adopt a helix conformation with positive charges to escape the membrane and the relax into an extended form in the cytoplasm to be recognized by the nuclear localization machinery. This was possible since short peptides have dynamic structures in solution. The prototype NLS peptide with translocation properties had the sequence KGPKKKRK (SEQ ID NO:5). This sequence was consistent with the nuclear localization consensus sequence and if projected as an α-helix it would have positive charges long one face. A proline in the first three positions of a helix acted to promote it, not destroy it. The NLS sequence did not contain a completely optimal translocation sequence in order to prevent uncontrolled diffusion throughout and out of the cell. This was the basis for incorporating only one Arg residue since the translocation properties have been linked to Arg residues displayed on one face of a helix
Example 2 Peptide Synthesis [0196] The MSH peptide analogs were synthesized using standard Fmoc/HBTU chemistry on amide resin with an Advanced ChemTech 396 Omega solid phase peptide synthesizer (Advanced ChemTech Inc., Louisville, KY). The peptides were acetylated with glacial acetic acid at the N-terminus. Amino acids and resin were purchased from Advanced ChemTech Inc (Louisville, KY). The peptides were deprotected and cleaved from the resin by using a cleavage mixture of trifluoroacetic acid (TFA), thioanisol, ethandiol, and water at room temperature for 3 hrs. The peptides were purified by HPLC (Isco, Inc., Lincoln, NE) on a C- 18 reverse phase column (218TP54, Vydac, Hesperia, CA) and lyophilized. The identities of peptides were confirmed by electrospray ionization mass specfrometry (ESI-MS) on a Finnigan TSQ7000 mass spectrometer (Thermo Finnigan, San Jose, CA).
Example 3 In Vitro Cell Binding [0197] The IC50 values for the α-MSH peptide analogs were determined by competitive binding assays with 125I-Tyr2-NDP (Amersham Pharmacia Biotech, UK). B16/F1 murine melanoma cells (5xl05/well) were seeded into a 24-well cell culture plate and incubated at 37°C overnight. After being washed once with binding media (MEM with 25 mM HEPES, pH 7.4, 0.2% BSA, 0.3 mM 1,10-phenathroline), the cells were incubated at 25°C for 2 hours with approximately 50,000 cpm of 125I-Tyr2-NDP and different concentrations ofthe α-MSH peptides in 0.3 ml of binding media. Cells were rinsed with 0.5 ml of ice-cold pH 7.4, 0.2% BSA / 0.01 M PBS two times and lysed in 0.5 ml of 1 N NaOH for 5 minutes. The activity in cells was measured in a Nal well counter. The IC50 values for the peptides were calculated by using the Grafit software (Erithacus Software Limited, UK). All of the α-MSH peptide analogs bound the murine melanoma cells and exhibited IC50 values ranging from the low nanomolar to low micromolar.
Table 3
Figure imgf000059_0001
DF=D-Phenylalanine; Nle=Norleucine; DOTA=l ,4,7, 10-tetraazacyclododecane-N,N' ,N" ,N" ' - tetracetic acid. Description notes chirality ofthe amino acids except where differences are explicitly designated. Example 4 Confocal Microscopy [0198] The infra-cellular targeting properties of the NLS-NDP peptide were compared to the NDP peptide alone. Both NLS-NDP and NDP sequences were synthesized with a N- terminal lysine residue which was conjugated to the fluorescent dye teframethylrhodamine. (TMR) yielding the peptide analogs (TMR)-NLS-NDP and (TMR)-NDP. The fluorescent peptides were added to live murine melanoma cells and observed under an Olympus 1X70 microscope/BioRad Radiance 2000 scanning confocal fluorescent system. Briefly, B16/F1 murine melanoma cells were seeded into 24 well tissue culture plates that contained glass coverslips which had been coated with poly-D-lysine. The cells were plated at a final density of 1 x 105 cells per well and allowed to grow over night at 37°C in a humidified incubator with 5% CO2. The next day the cells were washed with Opti-MEM media without fetal calf serum and then the peptide, in Opti-MEM was added and incubated with the cells for 2 hr at 25°C. The final peptide concentration was 1 micromolar. Prior to examination, the Opti-MEM media containing the peptide was removed and the cells were gently rinsed with 200 microliters of Opti-MEM 25°C. The glass cover slip containing the cells was mounted and sealed on the confocal microscope's stage.
[0199] This experiment confirmed the nuclear localization of the (TMR)-NLS-NDP peptide and the Cyto-5 staining confirms that the cell is alive and that its membranes are intact. These results demonstrated that the only way the peptide can get into the nucleus is to bind the MCI- receptor on the melanoma cell's surface and be translocated inside the cell. Once inside the cell, a significant portion of the peptide escaped the lysosomal vesicles and were transported and accumulated in the nucleus. The cells were alive and not fixed so their membranes were intact and the compound was not just diffusing into the cells non-specifically. [0200] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments ofthe process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one will readily appreciate from the disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
[0200] Example 5 Melanoma Cell Killing Assay [0201] The radionuclide targeting and melanoma cell killing ability of DOTA-NLS- NDP was compared to DOTA-NDP. Both peptides were synthesized with a N-terminal DOTA chelator for radiolabeling with n iIn. Indium-Ill is an Auger emitter. The therapeutic properties of Auger emitters are limited to approximately 8-10 micrometers or less than or equal to one cell diameter. The closer an Auger emitting radionuclide is located to the cell nuclear the greater its therapeutic potential. [0202] Radiolabeling. DOTA-NLS-NDP and DOTA-NDP were radiolabeled in an acetate buffered solution at 80°C. Briefly, 20 μg of DOTA-NLS-NDP (1 mg/ml in water) was mixed with 40 μl of 0.5 N NH4OAc (pH 5.4), and 10 μl of * ' 'in (10 mCi/ml). The mixture was heated at 80°F for 35 minutes and then cooled to room temperature. Sodium ethylenediaminetetraacetic acid (EDTA) was added to quench the reaction. An aliquot of the mixture was spotted onto thin-layer chromatography (TLC) strip and developed in 5% sodium EDTA in order to determine the complexation percentage. An identical procedure was carried out for the DOTA-NDP peptide except that the mixture was only heated for 25 minutes at 80°F. TLC tests deteraiined that approximately 95% of the mIn complexed to both the peptides. The radiolabeld peptides were purified via HPLC on a C-18 column using a 16 to 26% gradient acetonitrile/water (of 0.1% triflouroacetic acid (TFA)). The purified radiolabeled peptides were radiochemically stable in phosphate buffered saline.
[0203] Colony growth assay. The colony growth test was performed to determine if nuclear targeting of an Auger emitter (U1ln) was more effective in killing cells than cytoplasmic targeting or extra-cellular localization. The colony growth assay was performed with B16-F1 melanoma cells. Briefly, twenty-five thousand B16-F1 cells were divided evenly into 4 vials. Twenty-five microcuries of purified [π ιIn]-DOTA-NLS-NDP was added to one vial of cells, 25 μCi of purified [ι πIn]-DOTA-NDP was added to a second vial of cells, 25 μCi of ιnIn was added to a third vial of cells and a forth vial was used as a control. Before addition to the cells both purified peptides and the ι πIn were diluted to 50% with CCM in order to neutralize the pH to 7.4. The 4 vials were incubated in a humidified, 5% CO2 incubator at 37°C for 2 hours while gently shaking every 15 minutes. The vials were then centrifuged and the solution was decanted from the cells. Cell culture media was added to the cells, which were then plated at 500 cells per well in a 24 well tissue culture plates. The colonies were allowed to grow for 10 days upon which time they were fixed with 5% formaldehyde. The formaldehyde was removed and the cells were rinsed twice with 500 μl of PBS. An aliquot of 500 ml of crystal violet stain mixture was pipetted into each well and mixed. The crystal violet mixture consisted of 75 mg of crystal violet, 25.5 mg of sodium chloride (NaCl) and 3.345 ml of 22.3% ethanol in water producing a total volume of 15 ml. Once applied to the cells, the stain was allowed to sit for 10 minutes at room temperature. The cells were then rinsed 3 times with 500 μl of distilled water and the colonies were counted. [0204] The results of the colony growth assay demonstrated that [ulIn]-DOTA-NLS-
NDP was more effective in killing melanoma cells than [mIn]-DOTA-NDP or π ιIn (FIG. 4). Nuclear localization of mIn ([niIn]-DOTA-NLS-NDP) versus cytoplasmic localization ([mIn]- DOTA-NDP) or extracellular localization (ιnIn chloride) resulted in significantly fewer melanoma colonies. These results highlight the melanoma therapeutic potential of nuclear delivery of Auger emitters, as well as other high linear energy transfer emitters, using the NLS- NDP peptide.
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Claims

1. A method of delivering a peptide to a cell nucleus comprising contacting a cell expressing a G-protein coupled receptor with a peptide comprising a G-protein coupled receptor binding sequence and a nuclear localization signal.
2. The method of claim 1, wherein the G-protein coupled receptor is an opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P, substance-R, or glutamate receptor.
3. The method of claim 1 , wherein the G-protein coupled receptor is a melanocortin- 1- receptor.
4. The method of claim 1, wherein the peptide is linked to a radioisotope, a pharmaceutical compound, an MRI contrast reagent, or a fluorescent label.
5. The method of claim 4, wherein the radioisotope is an alpha-emitter, a beta-emitter, a gamma-emitter or an Auger emitter.
6. The method of claim 4, wherein the radioisotope is In-111, Bi-214, At-211, or Bi-212.
7. The method of claim 1 , wherein the peptide is linked to an imaging agent.
8. The method of claim 7, wherein the imaging agent is a DNA intercalator.
9. The method of claim 1, wherein the cell is a melanoma cell.
10. The method of claim 1, wherein the cell is selected from the group consisting of a liver cell, a brain cell, a pituitary cell, a keratinocyte cell, or an immune system cell.
11. The method of claim 1, wherein the G-protein coupled receptor binding sequence comprises a melanocortin- 1 receptor binding sequence.
12. The method of claim 11, wherein the melanocortin- 1 receptor binding sequence comprises His-Phe-Arg-Trp (SEQ ID NO:l).
13. The method of claim 11, wherein the melanocortin- 1 receptor binding sequence comprises His-DPhe-Arg-Trp.
14. The method of claim 1, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2).
15. The method of claim 1, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-Trp-Cys-Arg-Pro-Val (SEQ ID 5 NO:3).
16. The method of claim 1, wherein the peptide comprises the sequence Gly-Pro-Lys-Lys-Lys- Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val.
17. The method of claim 1, wherein the nuclear localization signal comprises K(K/R)x(K/R) (SEQ ID NO:4).
10 18. The method of claim 17, wherein the nuclear localization signal comprises KRXR.
19. The method of claim 1 , wherein the peptide comprises D amino acids.
20. The method of claim 19, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg-Lys-Lys-Lys-Pro-Gly-Lys.
21. The method of claim 19, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- 15 Arg-Phe-His-Glu-Nle-Ala-Glu-met-Ser-Lys-Ser.
22. A method of cell imaging comprising (a) contacting a cell expressing a G-protein coupled receptor with a peptide comprising a melanocortin- 1 receptor binding sequence and a nuclear localization signal, wherein the peptide is linked to an imaging agent; and (b) obtaining an image ofthe cell.
20 23. The method of claim 22, wherein the G-protein coupled receptor is an opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P, substance-R, or glutamate receptor.
24. The method of claim 22, wherein the G-protein coupled receptor is a melanocortin- 1 receptor.
25 25. The method of claim 22, wherein the imaging agent is a radioisotope, an MRI contrast reagent, or a fluorescent label.
26. The method of claim 22, wherein the radioisotope is an alpha-emitter, a beta-emitter, or an Auger emitter.
27. The method of claim 22, wherein the radioisotope is In- 111 , Bi-214, At-211 , or Bi-212.
28. The method of claim 22, wherein the imaging agent is a DNA intercalator. 5
29. The method of claim 22, wherein the cell is a melanoma cell.
30. The method of claim 22, wherein the cell is selected from the group consisting of a liver cell, a brain cell, a pituitary cell, a keratinocyte cell, or an immune system cell.
31. The method of claim 22, wherein the melanocortin-1 receptor binding sequence comprises His-Phe-Arg-Trp (SEQ ID NO:l).
10 32. The method of claim 22, wherein the melanocortin-1 receptor binding sequence comprises His-DPhe-Arg-Trp.
33. The method of claim 22, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2).
34. The method of claim 22, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- 15 Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-Trp-Cys-Arg-Pro-Val (SEQ ID NO:3).
35. The method of claim 22, wherein the peptide comprises the sequence Gly-Pro-Lys-Lys-Lys- Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val.
36. The method of claim 22, wherein the nuclear localization signal comprises K(K/R)x(K/R) 0 (SEQ ID NO:4).
37. The method of claim 36, wherein the nuclear localization signal comprises KRXR.
38. The method of claim 22, wherein the peptide comprises D amino acids.
39. The method of claim 38, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg-Lys-Lys-Lys-Pro-Gly-Lys.
40. The method of claim 38, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-met-Ser-Lys-Ser.
41. A method of delivering a phaπnaceutical compound to a cell nucleus comprising contacting a cell expressing a melanocortin-1 receptor with a peptide comprising a melanocortin-1 5 receptor binding sequence and a nuclear localization signal, wherein the peptide is linked to the pharmaceutical compound.
42. The method of claim 41, wherein the pharmaceutical compound is a chemotherapeutic agent.
43. The method of claim 42, wherein the chemotherapeutic agent is selected from the group consisting of immunotoxins, alkylating agents, anti-metabolites, enediynes, alkaloids, and
10 antitumor antibiotics.
44. The method of claim 41 , wherein the cell is a melanoma cell.
45. The method of claim 41 , wherein the cell is selected from the group consisting of a liver cell, a brain cell, a pituitary cell, a keratinocyte cell, or an immune system cell.
46. The method of claim 41, wherem the melanocortin-1 receptor binding sequence comprises 15 His-Phe-Arg-Trp (SEQ ID NO:l).
47. The method of claim 41, wherein the melanocortin-1 receptor binding sequence comprises His-DPhe-Arg-Trp.
48. The method of claim 41, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2).
20 49. The method of claim 41, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-T -Cys-Arg-Pro-Val (SEQ ID NO:3).
50. The method of claim 41, wherein the peptide comprises the sequence Gly-Pro-Lys-Lys-Lys- Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val.
25 51. The method of claim 41, wherein the nuclear localization signal comprises K(K/R)x(K/R) (SEQ ID NO:4).
52. The method of claim 51 , wherein the nuclear localization signal comprises KRXR.
53. The method of claim 41 , wherein the peptide comprises D amino acids.
54. The method of claim 53, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg-Lys-Lys-Lys-Pro-Gly-Lys.
5 55. The method of claim 53, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-met-Ser-Lys-Ser.
56. A method of in vivo delivering of a peptide to a tumor comprising administering to a patient with a melanocortin-1 receptor-expressing tumor a peptide comprising a melanocortin-1 receptor binding sequence and a nuclear localization signal, wherein the nuclear localization
10 signal forms a positively charged helix.
57. The method of claim 56, wherein the administration is intravenous, topical, or oral.
58. The method of claim 56, wherein the tumor is a melanoma tumor.
59. The method of claim 56, wherein the peptide is linked to a radioisotope, a pharmaceutical compound, an MRI contrast reagent, or a fluorescent label.
15 60. The method of claim 56, wherein the melanocortin-1 receptor binding sequence comprises His-Phe-Arg-Trp (SEQ ID NO:l).
61. The method of claim 56, wherein the melanocortin-1 receptor binding sequence comprises His-DPhe-Arg-Trp.
62. The method of claim 56, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- 0 Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2).
63. The method of claim 56, wherein the peptide comprises the sequence Lys-Gly-Pro-Lys-Lys- Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-Trp-Cys-Arg-Pro-Val (SEQ ID NO:3).
64. The method of claim 56, wherein the peptide comprises the sequence Gly-Pro-Lys-Lys-Lys- 5 Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val.
65. The method of claim 56, wherein the nuclear localization signal comprises K(K/R)x(K/R) (SEQ ID NO:4).
66. The method of claim 65, wherein the nuclear localization signal comprises KRXR.
67. The method of claim 56, wherein the peptide comprises D amino acids.
68. The method of claim 67, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg-Lys-Lys-Lys-Pro-Gly-Lys.
69. The method of claim 67, wherein the peptide comprises the sequence Val-Pro-Lys-Gly-Trp- Arg-Phe-His-Glu-Nle-Ala-Glu-met-Ser-Lys-Ser.
70. An isolated peptide comprising a melanocortin-1 receptor binding sequence and a nuclear localization signal.
71. The isolated peptide of claim 70, wherein the nuclear localization signal comprises K(K/R)x(K/R) (SEQ ID NO:4).
72. The isolated peptide of claim 71 , wherein the nuclear localization signal comprises KRXR.
73. The isolated peptide of claim 70, wherein the melanocortin-1 receptor binding sequence comprises His-Phe-Arg-Trp (SEQ ID NO: 1).
74. The isolated peptide of claim 70, wherein the melanocortin-1 receptor binding sequence comprises His-DPhe-Arg-Trp.
75. The isolated peptide of claim 70, wherein the peptide comprises the sequence Lys-Gly-Pro- Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala- le-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO:2).
76. The isolated peptide of claim 70, wherein the peptide comprises the sequence Lys-Gly-Pro- Lys-Lys-Lys-Arg-Lys-Val-Glu-Ala-Gly-Cys-Cys-Glu-His-Phe-Arg-Trp-Cys-Arg-Pro-Val (SEQ ID NO:3).
77. The isolated peptide of claim 70, wherein the peptide comprises the sequence Gly-Pro-Lys- Lys-Lys-Arg-Lys-Val-Glu-Ala-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val.
78. The isolated peptide of claim 70, wherein the peptide comprises D amino acids.
79. The isolated peptide of claim 78, wherein the peptide comprises the sequence Val-Pro-Lys- Gly-Trp-Arg-Phe-His-Glu-Nle-Ala-Glu-Val-Lys-Arg-Lys-Lys-Lys-Pro-Gly-Lys.
80. The isolated peptide of claim 78, wherein the peptide comprises the sequence Val-Pro-Lys- Gly-Trp-Arg-Phe-His-Glu-Nle-Ala-Glu-met-Ser-Lys-Ser.
81. The isolated peptide of claim 70, wherein the peptide is linked to a radioisotope, a pharmaceutical compound, an MRI contrast reagent, or a fluorescent label.
82. A host cell expressing the isolated peptide of claim 70.
83. An imaging composition comprising a peptide linked to at least one imaging agent, wherein the peptide comprises a melanocortin-1 receptor binding sequence and a nuclear localization signal.
84. The imaging composition of claim 83, wherein the imaging agent is a radioisotope, an MRI contrast reagent or a fluorescent label.
PCT/US2005/018700 2004-05-26 2005-05-26 Peptides delivered to cell nuclei WO2005120588A2 (en)

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