EP1549667A1 - Agents de ciblage tumoral et leurs utilisations - Google Patents

Agents de ciblage tumoral et leurs utilisations

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Publication number
EP1549667A1
EP1549667A1 EP03748160A EP03748160A EP1549667A1 EP 1549667 A1 EP1549667 A1 EP 1549667A1 EP 03748160 A EP03748160 A EP 03748160A EP 03748160 A EP03748160 A EP 03748160A EP 1549667 A1 EP1549667 A1 EP 1549667A1
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EP
European Patent Office
Prior art keywords
targeting
acid
fmoc
units
amino
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP03748160A
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German (de)
English (en)
Inventor
Mathias Bergman
Merja Auvinen
Hannu Elo
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KARYON Oy
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KARYON Oy
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Publication of EP1549667A1 publication Critical patent/EP1549667A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to tumor targeting agents comprising at least one targeting unit and at least one effector unit, as well as to tumor targeting units and motifs. Further, the present invention concerns pharmaceutical and diagnostic compositions comprising such targeting agents or targeting units, and the use of such targeting agents and targeting units as pharmaceuticals or as diagnostic tools. The invention further relates to the use of such targeting agents and targeting units for the preparation of pharmaceutical or diagnostic compositions and for the preparation of reagents to be used in diagnosis or research. Furthermore, the invention relates to kits for diagnosing or treating cancer and metastases. Still further, the invention relates to methods of removing, selecting, sorting and enriching cells, and to materials and kits for use in such methods.
  • Malignant tumors are one of the greatest health problems of man as well as animals, being one of the most common causes of death, also among young individuals. Available methods of treatment of cancer are quite limited, in spite of intensive research efforts during several decades. Although curative treatment (usually surgery in combination with chemothreapy and/or radiotherapy) is sometimes possible, malignant tumors (cancer) still are one of the most feared diseases of civilization, requiring a huge number of lives every year. In fact, curative treatment is rarely accomplished if the disease is not diagnosed early. In addition, certain tumor types can rarely, if ever, be treated curatively. There are various reasons for this very undesirable situation but the most important one is clearly the fact that nearly all (if not all) treatment schedules (except surgery) lack sufficient selectivity.
  • Chemotherapeutic agents commonly used such as alkylating agents, platinum compounds (e.g. cisplatin), bleomycin-type agents, other alkaloids and other cytostatic agents in general, do not act on the malignant cells of the tumors alone but are highly toxic to other cells as well, being usually especially toxic to rapidly dividing cell types, such as hematopoietic and epithelial cells. The same applies to radiotherapy.
  • a specific field of cancer treatment namely neutron capture therapy, in which a non-radioactive nucleus (e.g. 10 B, 157 Gd or 6 Li) is converted into a radioactive nucleus in vivo in the patient with the aid of thermal (slow) neutrons from an external source.
  • a non-radioactive nucleus e.g. 10 B, 157 Gd or 6 Li
  • thermal neutrons from an external source.
  • some prior art agents are claimed to have some 2-3 fold selectivity for at least some types of tumors, but the results obtained have been mainly disappointing and negative.
  • Specific targeting agents would offer remarkable advantages also in this field. Also in the diagnosis of cancer and of metastases, including the follow-up of patients and the study of the effects of treatment on tumors and metastases, more reliable, more sensitive and more selective methods and agents would be a great advantage.
  • NMR nuclear magnetic resonance imaging
  • MRI magnetic resonance imaging
  • X-ray meth- ods histological staining methods (for light microscopy and electron microscopy and related methods, and in the future possibly also NMR, infrared, electron spin resonance and related methods) and in general any imaging as well as laboratory methods (histology, cytology, cell sorting, hematological studies, FACS and so on) known by specialists in the field.
  • agents capable of targeting an entity for detection a spin label, a radioactive substance, a paramagnetic contrast agent for NMR or a contrast agent for X-ray imaging or tomography, a boron atom for neutron capture and so on
  • Solid tumor growth is angiogenesis-dependent, and a tumor must continuously stimulate the growth of new microcapillaries for continued growth.
  • Tumor blood vessels are structurally and functionally different from their normal resting counterparts.
  • endothelial cells lining new blood vessels are abnormal in shape, they grow on top of each other and project into the lumen of the vessels. This neovascular heterogeneity depends on the tumor type and on the host organ in which the tumor is growing. Therefore vascular permeability and angiogenesisis are unique in every different organ and in tumor tissue derived from the organ.
  • v ⁇ 3 integrin When tumors switch to the angiogenic phenotype and recruit new blood vessels, endothelial cells in these vessels express proteins on the lu- minal surface that are not produced by normal quiescent vascular endothe- lium.
  • One such protein is v ⁇ 3 integrin.
  • US Patent publication, US 6,177,542 discloses a peptide that can bind specifically to ⁇ v ⁇ 3 integrin.
  • the tumor vessel specific targets described are adhesion molecules that mediate binding of endothelial cells to the vascular basement membrane. This peptide is a nine- residue cyclic peptide containing an ArgGlyAsp (RGD) sequence.
  • US Patent 5,628,979 describes oligopeptides for in vivo tumor imaging and therapy.
  • the oligopeptides contain 4 to 50 amino acids, which contain as a characteristic triplet the amino acid sequence Leu-Asp-Val (LDV). This triplet is reported to provide the oligopeptide with in vivo binding affinity for LDV binding sites on tumors and other tissues.
  • LDV Leu-Asp-Val
  • US Patent publication US 2002/0102265A1 describes a peptide, TSPLNIHNGQKL, that targets squamous cell cancer cell lines, and becomes internalized into cells in vitro. This peptide also targets experimental squamous carcinomas in nude mice.
  • US Patent Nos. 5,622,699 and 6,068,829 disclose a family of peptides comprising an SRL motif, which selectively home to brain.
  • International Patent publication WO 02/20769 discloses methods for identifying tissue specific peptides by phage display and biopanning. Some of the identified peptides are suggested to be tumor specific.
  • targeting peptides have been conju- gated to doxorubicin in an uncontrolled fashion, obviously resulting in mixtures of products or at least in an undefined structure and possibly also resulting in unefficient action and especially in difficulties in the identification, purification, quality control and quantitative analysis of the agent, even the amount of doxorubicin per peptide molecule remaining unknown (e.g. Arap et al., 1998).
  • the unspecific conjugation process might also impair the targeting functions of the peptide.
  • Another very serious disadvantage of the prior art is that most of the described targeting peptides appear to target to the tumor endothelium only and not to the tumor mass itself. For example, the targeting peptide used by
  • the targeting units according to the present invention offer an advantage over the prior art in that they seem to target to both the tumor endothelium and the tumor cell mass. This fact provides the possibility to target and destroy tumor endothelium supporting tumor growth as well as the tumor mass itself. A major advantage of this approach comes from the fact that the endothelium is a genetically stable tissue that will not acquire drug resistance but will be irreversibly eliminated.
  • sarcomas such as Kaposi ' s sarcoma, ornithine decarboxylase (ODC) overexpressing, highly angiogenic tumors, carcinomas, and to human primary and metastatic melanomas
  • the invention provides novel tumor and angiogenic tissue targeting agents that comprise at least one targeting unit and, optionally, at least one effector unit.
  • the invention provides targeting units comprising at least one motif that is capable of targeting both tumor endothelium and tumor cell mass.
  • Such targeting units, optionally coupled to at least one effector unit are therapeutically and diagnostically useful, especially in the treatment and diagnosis of cancer, including metastases.
  • the targeting agents according to the present invention are useful for cell removal, selection, sorting and enrichment.
  • pharmaceutical and diagnostic compositions comprising at least one targeting agent or at least one targeting unit comprising at least one motif capable of specifically targeting tumors, tumor cells and tumor endothelium.
  • it is a third object of the invention to provide novel diagnostic and therapeutic methods and kits for the treatment and/or diagnosis of cancer.
  • the present invention is based on the finding that a group of peptides having specific amino acid sequences or motifs are capable of selectively targeting tumors in vivo and tumor cells in vitro.
  • the peptides of this invention when administered to a human or animal subject, are capable of selectively binding to tumors but not to normal tissue in the body.
  • the present invention is also directed to the use of the targeting agents and analogues thereof for the manufacture of a pharmaceutical or diagnostic composition for treating or diagnosing cancer.
  • the targeting units of this invention may be used as such or coupled to at least one effector unit. Such substances can destroy the tumors or hinder their growth.
  • the targeting units and targeting agents of this invention can target also metastases and therefore they may be used to destroy or hinder the growth of metastases. As early diagnosis of metastases is very important for successful treatment of cancer, an important use of the targeting units and targeting agents of this invention is in early diagnosis of tumor metastases.
  • the present invention further encompasses salts, derivatives and analogues of the targeting units and targeting agents, as described herein, as well as uses thereof.
  • Especially preferred embodiments of the present invention relate to a group of small, cyclic tumor targeting peptides comprising a motif, LRS or SRL, optionally coupled to an effector unit and other additional units, as de- scribed in more detail herein.
  • Figure 1 is a graph showing the therapeutic effect of a targeting agent comprising doxorubicin.
  • cancer is used herein in its broadest sense, and includes any disease or condition involving transformed or malignant cells.
  • cancers are classified into five major categories, according to their tissue origin (histological type): carcinomas, sarcomas, myleomas, and lymphomas, which are solid tumor type cancers, and leu- kemias, which are "liquid cancers”.
  • tissue origin histological type
  • carcinomas sarcomas
  • myleomas myleomas
  • lymphomas which are solid tumor type cancers
  • leu- kemias which are "liquid cancers”.
  • cancer as used in the present invention, is intended to primarily include all types of diseases characterized by solid tumors, including disease states where there is no detectable solid tumor or where malignant or transformed cells, "cancer cells”, appear as diffuse infiltrates or
  • amino acid and “amino alcohol” are to be interpreted herein to include also diamino, triamino, oligoamino and polyamino acids and alcohols; dicarboxyl, tricarboxyl, oligocarboxyl and polycarboxyl amino acids; dihydroxyl, trihydroxyl, oligohydroxyl and polyhydroxyl amino alcohols; and analogous compounds comprising more than one carboxyl group or hydroxyl group and one or more amino groups.
  • peptide is meant, according to established terminology, a chain of amino acids (peptide units) linked together by peptide bonds to form an amino acid chain. Peptides may be cyclic as described below. For the purposes of the present invention, also compounds comprising one or more D- amino acids, ⁇ -amino acids and/or other unnatural amino acids (e.g. amino acids with unnatural side chains) are included in the term “peptide”. For the purposes of the present invention, the term “peptide” is intended to include pepti- dyl analogues comprising modified amino acids.
  • Such modifications may comprise the introduction or presence of a substituent in a ring or chain; the intro- duction or presence of an "extra" functional group such as an amino, hy- drazino, carboxyl, formyl (aldehyde) or keto group, or another moiety; and the absence or removal of a functional group or other moiety.
  • the term also includes analogues modified in the amino- and/or carboxy termini, such as peptide amides and V-substituted amides, peptide hydrazides, ⁇ /-substituted hy- drazides, peptide esters, and their like, and peptides that do not comprise the amino-terminal -NH 2 group or that comprise e.g.
  • peptides that do not comprise the carboxy-terminal carboxyl group or comprise a modified group instead of it, and so on.
  • reaction types that can be used to modify peptides, forming "peptidyl analogues", are e.g., cycloaddition, condensation and nucleophilic addition reactions as well as esterification, amide formation, formation of substituted amides, ⁇ -alkylation, formation of hydrazides, salt formation.
  • Salt formation may be the formation of any type of salt, such as al- kali or other metal salt, ammonium salt, salts with organic bases, acid addition salts etc.
  • Peptidyl analogues may be synthesized either from the corresponding peptides or directly (via other routes).
  • Compounds that are structural or functional analogues of the peptides of the invention may be compounds that do not consist of amino acids or not of amino acids alone, or some or all of whose building blocks are modified amino acids. Different types of building blocks can be used for this purpose, as is well appreciated by those skilled in the art.
  • the function of these compounds in biological systems is essentially similar to the function of the peptides. The resemblance between these compounds and the original peptides is thus based on structural and functional similarities.
  • Such compounds are called peptidomimetic analogues, as they mimic the function, conformation and/or structure of the original peptides and, for the purposes of the present invention, they are included in the term "peptide".
  • a functional analog of a peptide according to the present invention is characterized by a binding ability with respect to the binding to tumors, tumor tissue, tumor cells or tumor endothelium which is essentially similar to that of the peptides they resemble.
  • Peptidomimetic substances may comprise for example one or more of the following structural components: re- prised amides, hydroxyethylene and/or hydroxyethylamine isosteres, ⁇ /-methyl amino acids, urea derivatives, thiourea derivatives, cyclic urea and/or thiourea derivatives, poly(ester imide)s, polyesters, esters, guanidine derivatives, cyclic guanidines, imidazoyl compounds, imidazolinyl compounds, imidazolidinyl compounds, lactams, lactones, aromatic rings, bicyclic systems, hydantoins and/or thiohydantoins as well as various other structures.
  • peptidomimetic compounds Many types of com- pounds for the synthesis of peptidomimetic substances are available from a number of commercial sources (e.g. Peptide and Peptidomimetic Synthesis, Reagents for Drug Discovery, Fluka ChemieGmbH, Buchs, Switzerland, 2000 and Novabiochem 2000 Catalog, Calbiochem-Novabiochem AG, Laufelfingen, Switzerland, 2000).
  • the resemblance between the peptidomimetic compounds and the original peptides is based on structural and/or functional similarities.
  • the peptidomimetic compounds mimic the properties of the original peptides and, for the purpose of the present application, their binding ability is similar to the peptides that they resemble.
  • Peptidomimetic compounds can be made up, for example, of unnatural amino acids (such as D-amino acids or amino acids comprising unnatural side chains, or of ⁇ -amino acids etc.), which do not appear in the original peptides, or they can be considered to consist of or can be made from other compounds or structural units.
  • Examples of synthetic peptidomimetic compounds comprise N-alkylamino cyclic urea, thiourea, polyesters, poly(ester imide)s, bicyclic guanidines, hydantoins, thiohydantoins, and imidazol-pyridino-inoles (Houghten et al. 1999 and Nargund et al., 1998).
  • Such peptidomimetic compounds can be characterized as being "structural or functional analogues" of the peptides of this invention.
  • the term "targeting unit” stands for a compound, a peptide, capable of selectively targeting and selec- tively binding to tumors, and, preferably, also to tumor stroma, tumor parenchyma and/or extracellular matrix of tumors. Another term used in the art for this specific association is "homing”.
  • Tumor targeting means that the targeting units specifically bind to tumors when administered to a human or animal body. More specifically, the targeting units may bind to a cell surface, to a specific molecule or structure on a cell surface or within the cells, or they may associate with the extracellular matrix present between the cells. The targeting units may also bind to the endothelial cells or the extracellular matrix of tumor vasculature.
  • the targeting units may bind also to the tumor mass, tumor cells and extracellular matrix of metastases.
  • targeting or “binding” stand for adhesion, attachment, affinity or binding of the targeting units of this invention to tumors, tumor cells and/or tumor tissue to the extent that the binding can be objectively measured and determined e.g., by peptide competition experiments in vivo or ex vivo, on tumor biopsies in vitro or by immunological stainings in situ, or by other methods known by those skilled in the art.
  • the exact mechanism of the binding of targeting units according to the present invention is not known.
  • Tageting peptides according to the present invention are considered to be "bound" to the tumor target in vitro, when the binding is strong enough to withstand normal sample treatment, such as washes and rinses with physiological saline or other physiologically acceptable salt or buffer solutions at physiologi- cal pH, or when bound to a tumor target in vivo long enough for the effector unit to exhibit its function on the target.
  • the binding of the present targeting agents or targeting units to tumors is "selective" meaning that they do not bind to normal cells and organs, or bind to such to a significantly lower degree as compared to tumor cells and organs.
  • compositions of the targeting units and agents of the present invention include salts, esters, amides, hy- drazides, N-substituted amides, N-substituted hydrazides, hydroxamic acid derivatives, decarboxylated and N-substituted derivatives thereof.
  • Suitable phar- maceutically acceptable salts are readily acknowledged by those skilled in the art.
  • Dd-Ee-Ff is either Aa-Bb-Cc or Cc-Bb-Aa, and Aa is isoleucine, leucine or tert-leucine, or a structural or functional analogue thereof;
  • Bb is arginine, homoarginine or canavanine, or a structural or functional analogue thereof; and Cc is serine or homoserine, or a structural or functional analogue thereof, targets and exhibits selective binding to tumors and cancers and tumor cells and cancer cells.
  • Aa according to the present invention may comprise in its sidechain a branched, non-branched or alicyclic structure with at least two siminal or different atoms selected from the group consisting of carbon, silicon, halogen bonded to carbon, ether-oxygens and thioether-sulphur.
  • the analogue may be selected from the group consisting of branched, non-branched or cyclic non-aromatic, lipophilic and hydrophobic amino acids or amino acid analogues or derivatives or structural and/or functional analogues thereof; amino acids or carboxylic acids or amino acid analogues or derivatives or carboxylic acid analogues or derivatives having one or more lipophilic carborane-type or other lipophilic boron-containing side chains or other lipophilic cage-type structures.
  • Aa may be selected from the group consisting of: 1) ⁇ -amino acids whose side chain is one of the following:
  • Aa may also be an ⁇ -amino acid (either L- or D- amino acid) of the formula R 1 - CR 2 (NH 2 ) - COOH wherein the side chain R 1 is selected from the side chains listed above, and the side chains R 2 is selected from the group consisting of: hydrogen, methyl, ethyl, propyl.
  • Bb may be selected from the group consisting of amino acids or structural or functional analogues thereof containing one or more guanyl groups, aminodino groups or their analogues and derivatives and structural or functional equivalents; one or more groups containing at least two nitrogen atoms each and have or can gain a delocal- ized positive charge.
  • Bb may be selected from the group of compounds of the following formula:
  • R1 - R5 is hydrogen or methyl
  • R2 and R3 may form -CH2-CH2- and n is 1-6.
  • Bb is the L- or Q- form of arginine, homoarginine, canavanine, 2-amino-8-guanidino-octanoic acid,
  • Cc may be selected from the group consisting of amino acids, amino alcohols, diamino alcohols, tri-, oligo- and polyamino alcohols and amino acid analogues, derivatives and structural or functional analogues thereof, comprising one or more hydroxyl group(s), es- terified hydroxyl group(s), methoxyl group(s) and/or other etherified hydroxyl
  • Cc as defined above may be serine or homoserine or a structural or functional analogue thereof, comprising at least one hydroxyl group; or may be selected from the group consisting of: any other monoaminocarboxylic acid comprising at least one alcoholic hydroxyl group any carboxylic acid comprising at least one alcoholic hydroxyl group any other aminocarboxylic acid comprising an aliphatic or other side chain that comprises one or more alcoholic hydroxyl (OH) function(s) and/or esterified hydroxyl fu notion (s).
  • OH alcoholic hydroxyl
  • Cc is the L- or D- form of serine, homoserine,
  • the motif Aa-Bb-Cc as a whole, according to the present invention is a structural or functional analogue of a structure where Aa, Bb and Cc are as defined above.
  • Preferred embodiments of the present invention include tumor targeting motifs Aa-Bb-Cc selected from those given in Table 1 as well as structural and functional analogues thereof.
  • typical and preferred characteristics of Aa include lipofilicity, hydrophobicity and aliphatic character in at least one side chain, wheras Bb includes a delocalized positive charge and Cc has the ability of participating in OH-binding.
  • Especially preferred motifs Dd-Ee-Ff according to the present invention are leucine-arginine-serine (LRS) and serine-arginine-leucine (SRL).
  • motifs Dd-Ee-Ff may form part of a larger structure, such as a peptide or some other structure.
  • the orientation and direction of the motifs may vary.
  • peptides and structural or functional analogues thereof comprising a tumor targeting motif according to the present in- vention target to and exhibit selective binding to tumor cells and tissues.
  • Peptides comprising a tumor targeting motif according to the present invention and, optionally, up to four additional amino acid residues or analogues thereof likewise exhibit such targeting and selective binding and are especially preferred embodiments of the present invention.
  • Such peptides are highly advantageous for use as targeting units according to the present invention, e.g., because of their small size and their easier and more reliable and much cheaper synthesis, purification, analysis and quality control.
  • Especially preferred targeting units according to the present invention are peptides cyclized by an amide bond, such as a lactam bridge or by an ester bond, such as a lacotone bridge.
  • Such cyclic targeting units have now been shown to selectively target to tumors in vivo.
  • Preferred tumor targeting units according to the present invention comprise a tumor targeting motif Dd-Ee-Ff as defined above, and additional residues selected from the group consisting of: natural amino acids; unnatural amino acids; amino acid analogues comprising maximally 30 non-hydrogen atoms and an unlimited number of hydrogen atoms,; and other structural units and residues whose molecular weight and/or formula weight is maximally 270; wherein the number of said additional residues ranges from 0 to 4, preferably from 0 to 3, more preferably from 0 to 2, and most preferably is either 0 or 2.
  • Cyclic peptides are usually more stable in vivo and in many other biological systems than are their non-cyclic counterparts, as is known in the art. It has now, however, surprisingly been found that the targeting property of the small peptides according to the present invention is more pronounced when the targeting unit is cyclic or contained in a cyclic structure.
  • Preferred targeting units according to the present invention may comprise a structure
  • Dd-Ee-Ff is a tumor targeting motif according to the present invention, as described above,
  • Rr is any amino acid residue
  • n and m are integers 0-4, preferably 0-3, more preferably 0-2, whereby the sum of n and m does not exceed four
  • Cy and Cyy are entities capable of forming a cyclic structure by means of a lactam ring.
  • Lactams can be of several subtypes, such as “head to tail” (carboxy terminus plus amino terminus), “head to side chain” and “side chain to head” (carboxy or amino terminus plus one side chain amino or carboxyl group) and
  • side chain to side chain (amino groug of one side chain and carboxys group of another side chaine).
  • Preferred targeting units are such, where Rr is any amino acid residue, except histidine, lysine or tryptophane. Especially preferred are targeting units wherein Rr is R or G.
  • Cy and Cyy are residues capable of forming a lactam bond, such as aspartic acid (D), glutamic acid (E), lysin (K), omithine (O) or analogues thereof comprising no more than 12 carbon atoms.
  • Especially preferred targeting units according to the present invention having a cyclic structure by virtue of a lactam bridge are : DLRSK (SEQ ID NO: 1), DGRGLRSK (SEQ ID NO: 2), DRGLRSK (SEQ ID NO: 3), DRYYNLRSK (SEQ ID NO: 4), DSRYNLRSK (SEQ ID NO: 5), DLRSGRK (SEQ ID NO: 6), DLRSGRGK (SEQ ID NO: 7), OLRSE (SEQ ID NO. 8), OLRSGRGE (SEQ ID NO. 9) and KLRSD (SEQ ID NO. 10), as well as salts, esters, derivatives and analogues thereof, as defined above.
  • DLRSK SEQ ID NO: 1
  • DGRGLRSK SEQ ID NO: 2
  • DRGLRSK SEQ ID NO: 3
  • DRYYNLRSK SEQ ID NO: 4
  • DSRYNLRSK SEQ ID NO: 5
  • DLRSGRK SEQ ID NO: 6
  • targeting agents comprising at least one tumor targeting unit according to the present invention, and at least one effector unit, target to and exhibit selective binding to cancer cells and tissues as well as endothelial cells.
  • the tumor targeting agents according to the present invention may optionally comprise unit(s) such as linkers, solubility modifiers, stabilizers, charge modifiers, spacers, lysis or reaction or reactivity modifiers, internalizing units or intemalization enhancers or membrane interaction units or other local route, attachment, binding and distribution affecting units.
  • Such additional units of the tumor targeting agents according to the present invention may be coupled to each other by any means suitable for that purpose. Many possibilities are known to those skilled in the art for linking structures, molecules, groups etc. of the types in question or of related types, to each other. The various units may be linked either directly or with the aid of one or more identical, similar and/or different linker units.
  • the tumor targeting agents of the invention may have different structures such as any of the non- limiting types schematically shown below:
  • EU indicates "effector unit” and TU indicates “targeting unit” and n, m and k are independently any integers except 0.
  • targeting agents as in many other medicinal and other substances, it may be wise to include spacers or linkers, such as amino acids and their analogues, such as long-chain omega-amino acids, to prevent the targeting units from being 'disturbed', steri- cally, electronically or otherwise hindered or 'hidden' by effector units or other unit of the targeting agent.
  • spacers or linkers such as amino acids and their analogues, such as long-chain omega-amino acids
  • targeting agents according to the present invention it may be useful for increased activity to use dendrimeric or cyclic structures to provide a possiblility to incorporate multiple effector units or additional units per targeting unit.
  • Preferred targeting agents according to the present invention com- prise a structure Ef-TU-Eff, wherein
  • TU is a targeting unit according to the present invention as defined abover; and Ef and Eff are selected from the group consisting of: effector units, linker units, solubility modifier units, stabilizer units, charge modifier units, spacer units, lysis and/or reaction and/or reactivity modifier units, internalizing and/or intemalization enhancer and/or membrane interaction units and/or other local route and/or local attachment local binding and/or distribu- tion affecting units, adsorption enhancer units, and other related units; and peptide sequences and other structures comprising at least one such unit; and peptide sequences comprising no more than 20, preferably no more than 12, more preferably no more than 6, natural and/or unnatural amino acids; and natural and unnatural amino acids comprising no more than 25 non-hydrogen atoms and an unlimited number of hydrogen atoms; as well as salts, esters, derivatives and analogues thereof.
  • effector unit means a molecule or radical or other chemical entity as well as large particles such as colloidal particles and their like; liposomes or microgranules. Suitable effector units may also consitute nanodevices or nanochips or their like; or a combination of any of these, and optionally chemical structures for the attachment of the constituents of the effector unit to each or to parts of the targeting agents. Effector units may also contain moieties that effect stabilization or solubility enhancement of the effector unit.
  • Preferred effects provided by the effector units according to the present invention are therapeutical (biological, chemical or physical) effects on the targeted tumor; properties that enable the detection or imaging of tumors or tumor cells for diagnostic purposes; or binding abilities that relate to the use of the targeting agents in different applications.
  • a preferred (biological) activity of the effector units according to the present invention is a therapeutic effect.
  • therapeutic activities are for example, cytotoxicity, cytostatic effect, ability to cause differentiation of cells or to increase their degree of differentiation or to cause phenotypic changes or metabolic changes, chemotactic activities, immunomodulating activities, pain relieving activities, radioactivity, ability to affect the cell cycle, abil- ity to cause apoptosis, hormonal activities, enzymatic activities, ability to trans- feet cells, gene transferring activities, ability to mediate "knock-out" of one or more genes, ability to cause gene replacements or "knock-in", antiangiogenic activities, ability to collect heat or other energy from external radiation or elec- trie or magnetic fields, ability to affect transcription, translation or replication of the cell ' s genetic information or external related information; and to affect post-transcriptional and/or post-translational events.
  • Other preferred therapeutic approaches enabled by the effector units according to the present invention may be based on the use of thermal (slow) neutrons (to make suitable nuclei radioactive by neutron capture), or the administration of an enzyme capable of hydrolyzing for example an ester bond or other bonds or the administration of a targeted enzyme according to the present invention.
  • Examples of preferred functions of the effector units according to the present invention suitable for detection are radioactivity, paramagnetism, ferromagnetism, ferrimagnetism, or any type of magnetism, or ability to be detected by NMR spectroscopy, or ability to be detected by EPR (ESR) spectros- copy, or suitability for PET and/or SPECT imaging, or the presence of an immunogenic structure, or the presence of an antibody or antibody fragment or antibody-type structure, or the presence of a gold particle, or the presence of biotin or avidin or other protein, and/or luminescent and/or fluorescent and/or phosphorescent activity or the ability to enhance detection of tumors, tumor cells, endothelial cells and metastases in electron microscopy, light microscopy (UV and/or visible light), infrared microscopy, atomic force microscopy or tun- neling microscopy, and so on.
  • EPR EPR
  • Preferred binding abilities of an effector unit according to the present invention include, for example: a) ability to bind to a substance or structure such as a histidine or other tag, b) ability to bind to biotin or analogues thereof, c) ability to bind to avidin or analogues thereof, d) ability to bind to an enzyme or a modified enzyme, e) ability to bind metal ion(s) e.g.
  • f ability to bind a cytotoxic, apoptotic or metobolism affectin substance or a substance capable of being converted in situ into such a substance, g) ability to bind to integrins and other substances involved in cell adhesion, migration, or intracellular signaling, h) ability to bind to phages, i) ability to bind to lymphocytes or other blood cells, j) ability to bind to any preselected material by virtue of the presence of antibodies or structures selected by biopanning, k) ability to bind to material used for signal production or amplification, I) ability to bind to therapeutic substance.
  • Such binding may be the result of e.g. chelation, formation of covalent bonds, antibody-antigen-type affinity, ion pair or ion associate formation, specific interactions of the avidin-biotin-type, or the result of any type or mode of binding or affinity.
  • the effector unit may also be a part of the targeting units themselves.
  • the effector unit may for example be one or more atoms or nuclei of the targeting unit, such as radioactive atoms or atoms that can be made radioactive, or paramagnetic atoms or atoms that are easily detected by MRI or NMR spectroscopy (such as carbon-13).
  • boron-comprising structures such as carborane-type lipophilic side chains.
  • the effector units may be linked to the targeting units by any type of bond or structure or any combinations of them that are strong enough so that most, or preferably all or essentially all of the effector units of the targeting agents remain linked to the targeting units during the essential (necessary) targeting process, e.g. in a human or animal subject or in a biological sample under study or treatment.
  • the effector units or parts of them may remain linked to the target- ing units, or they may be partly or completely hydrolyzed or otherwise disintegrated from the latter, either by a spontaneous chemical reaction or equilibrium or by a spontaneous enzymatic process or other biological process, or as a result of an intentional operation or procedure such as the administration of hy- drolytic enzymes or other chemical substances. It is also possible that the en- zymatic process or other reaction is caused or enhanced by the administration of a targeted substance such as an enzyme in accordance with the present invention.
  • effector units or parts thereof are hydrolyzed from the targeting agent and/or hydrolyzed into smaller units by the ef- feet of one or more of the various hydrolytic enzymes present in tumors (e.g., intracellularly, in the cell membrane or in the extracellular matrix) or in their near vicinity.
  • the targeting according to the present invention may be very rapid, even non-specific hydrolysis that occurs every- where in the body may be acceptable and usable for hydrolysing one or more effector unit(s) intentionally, since such hydrolysis may in suitable cases (e.g., steric hindrance, or even without any such hindering effects) be so slow that the targeting agents are safely targeted in spite of the presence of hydrolytic enzymes of the body, as those skilled in the art very well understand.
  • the for- mation of insoluble products and/or products rapidly absorbed into cells and/or bound to their surfaces after hydrolysis may also be beneficial for the targeted effector units and/or their fragments etc. to remain in the tumors or their closest vicinity.
  • the effector units may comprise structures, features, fragments, molecules or the like that make possible, cause directly or indirectly, an "amplification" of the therapeutic or other effect, of signal detection, of the binding of preselected substances, including biological material, molecules, ions, microbes or cells.
  • Such “amplification” may, for example, be based on one or more of the following non-limiting types:
  • the effector units comprise more than one entity capable of binding e.g. a protein, thus making direct amplification possible;
  • radioactive or paramagnetic substances ._ substances comprising one or more metal ions
  • the effector unit comprises alpha emittors.
  • the effector units may comprise copper chelates such as frar?s-bis(salicylaldoximaro) copper(ll) and its analogues, or platinum compounds such cisplatin, carboplatin.
  • the peptide sequence KLAKLAK that interacts with mitochondrial membranes inside cells can be included Ellerby et al. (1999).
  • the targeting units and agents of the invention can, for example, be used a) coupled or connected to magnetic particles, b) adsorbed, coupled, linked or connected to plastic, glass or other solid, porous, fibrous material-type or other surface(s) and the like, c) adsorbed, covalently bonded or otherwise linked, coupled or connected into or onto one or more substance(s) or material(s) that can be used in columns and related systems d) adsorbed, covalently bonded or otherwise linked, coupled or connected into or onto one or more substance(s) or material(s) that can be precipitated, centri- fuged or otherwise separated or removed.
  • the targeting agents and targeting units of the present invention may optionally comprise further units, such as: linker units coupling targeting units, effector units or other optional units of the present invention to each other; solubility modifying units for modifying the solubility of the targeting agents or their hydrolysis product; stabilizer units stabilizing the structure of the targeting units or agents during synthesis, modification, processing, storage or use in vivo or in vitro; charge modifying units modifying the electrical charges of the targeting units or agents or their starting materials; spacer units for increasing the distance between specific units of the targeting agents or their starting materials, to release or decrease steric hindrance or structural strain of the products; reactivity modifyer units; internalizing units or enhancer units for enhancing targeting and uptage of the targeting agents; adsorption enhancer units, such as fat or water soluble structures enhancing absorption of the targeting agents in vivo; or other related units.
  • linker units coupling targeting units, effector units or other optional units of the present invention to each other
  • solubility modifying units for modifying the solubility
  • linker units are known in the art.
  • suitable linkers are: 1. for linking units comprising amino groups: cyclic anhydrides, dicarboxylic or multivalent, optinally activated or derivatized, carboxylic acids, compounds with two or more reactive halogens or compounds with at least one reactive halogen atom and at least one carboxyl group;
  • linking units comprising carboxyl groups or derivatives thereof: com- pounds with at least two similar or different groups such as amino, substituted amino, hydroxyl, -NHNH 2 or substituted forms thereof, other known groups for the purpose (activators may be used);
  • hydrazinocarboxylic acids or their like for linking a substance comprising an amino group to a substance comprising either a formyl group or a carboxyl group: hydrazinocarboxylic acids or their like, preferably so that the hydrazino moiety or the carboxyl group is protected or activated, such as 4-(FMOC-hydrazino)benzoic acid;
  • substances that can be coupled to the organic structure e.g. by virtue of their COOH groups or their NH 2 groups) or that are integral parts of it, and that in addition comprise a polycarboxylic part for example an EDTA- or DTPA-like structure, peptides comprising several histidines or their like, peptides comprising several cysteines or other moieties comprising an -SH group each, and other chelating agents that comprise functional groups that can be used to link them to the organic structure.
  • substances that can be coupled to the organic structure e.g. by virtue of their COOH groups or their NH 2 groups
  • a polycarboxylic part for example an EDTA- or DTPA-like structure
  • peptides comprising several histidines or their like peptides comprising several cysteines or other moieties comprising an -SH group each
  • other chelating agents that comprise functional groups that can be used to link them to the organic structure.
  • Suitable solubility modifier units comprise, for example: - for increasing aqeous solubility: molecules comprising SO 3 " , O-SO 3 " , COOH, COO " , NH 2 , NH 3 + , OH groups, guanidino or amidino groups or other ionic and ionizable groups and sugar-type structures;
  • units comprising (long) aliphatic branched or non-branched alkyl and alkenyl groups, cyclic non- aromatic groups such as the cyclohexyl group, aromatic rings and steroidal structures.
  • a large number of units known in the art can be used as stabilizer units, e.g. bulky structures (such as ferf-butyl groups, naphthyl and adamantyl and related radicals etc.) for increasing steric hindrance, and D-amino acids and other unnatural amino acids (including ⁇ -amino acids, ⁇ -amino acids, amino acids with very large side chains etc.) for preventing or hindering enzymatic hydrolysis.
  • bulky structures such as ferf-butyl groups, naphthyl and adamantyl and related radicals etc.
  • D-amino acids and other unnatural amino acids including ⁇ -amino acids, ⁇ -amino acids, amino acids with very large side chains etc.
  • Units comprising positive, negative or both types of charges can be used as charge modifier units, as can also structures that are converted or can be converted into units with positive, negative or both types of charges.
  • Spacer units may be very important, and the need to use such units depends on the other components of the structure (e.g. the type of biologically active agents used, and their mechanisms of action) and the synthetic procedures used Suitable spacer units may include for example long aliphatic chains or sugar-type structures (to avoid too high lipophilicity), or large rings. Suitable compounds are available in the art. One preferred group of spacer units are ⁇ - amino acids with long chains. Such compounds can also be used (simultaneously) as linker units between an amino-comprising unit and a carboxyl- comprising unit. Many such compounds are commercially available, both as such and in the forms of various protected derivatives.
  • Units that are susceptible to hydrolysis may be very advantageous in cases where it is desired that the effector units are liberated from the targeting agents e.g. for internalization, intra- or extracellularl DNA or receptor binding.
  • Suitable units for this purpose include, for example, structures comprising one or more ester or acetal functionality, Various proteases may be used for the purposes mentioned.
  • Many groups used for making pro-drugs may be suitable for the purpose of increasing or causing hydrolysis, lytic reactions or other decomposi- tion processes.
  • the effector units, the targeting units and the optional units according to the present invention may simultaneously serve more than one function.
  • a targeting unit may simultaneously be an effector unit or comprise several effector units;
  • a spacer unit may simultaneously be a linker unit or a charge modifier unit or both;
  • a stabilizer unit may be an effector unit with properties different from those of another effector unit, and so on.
  • An effector unit may, for example, have several similar or even completely different functions.
  • the tumor targeting agents comprise more than one different effector units.
  • the effector units may be, for example, diagnostic and therapeutic units.
  • targeting agents by using such a targeting agents according to the invention that comprise an effector unit comprising boron atoms (preferably isotope-enriched boron) and groups detectable e.g. by NMRI.
  • an effector unit comprising boron atoms (preferably isotope-enriched boron) and groups detectable e.g. by NMRI.
  • the presence of more than one type of therapeutically useful effector units may also be preferred.
  • the targeting units and targeting agents may, if desired, be used in combination with one or more "classical" or other tumor therapeutic modalities such as surgery, chemotherapy, other targeting modalities, radiotherapy, immunotherapy etc.
  • the targeting units according to the present invention are preferably synthetic peptides.
  • Peptides can be synthesized by a large variety of well- known techniques, such as solid-phase methods (FMOC-, BOC-, and other protection schemes, various resin types), solution methods (FMOC, BOC and other variants) and combinations of these. Even automated apparatuses/devices for the purpose are available commercially, as are also routine synthesis and purification services. All of these approaches are very well known to those skilled in the art. Some methods and materials are described, for example, in the following references: Bachem AG, SASRINTM (1999), The BACHEM Practise of SPPS
  • Peptide synthesis is exemplified also in the Examples.
  • protecting groups a large variety of which are known in the art, such as FMOC, BOC, and trityl groups and other protecting groups mentioned in the Examples.
  • protecting groups are often used for protecting amino, carboxyl, hydroxyl, guanyl and -SH groups, and for any reactive groups/functions.
  • activation often involves carboxyl function activation and/or activation of amino groups.
  • Protection may also be orthogonal and/or semi/quasi/pseudo- orthogonal.
  • Protecting and activating groups, substances and their uses are exemplified in the Examples and are described in the references cited herein, and are also described in a large number of books and other sources of information commonly known in the art (e.g. Protective Groups in Organic Synthesis, 1999).
  • Cyclic structures according to the present invention may be synthesized, for example, by methods based on the use of orthogonally protected amino acids.
  • one amino acid containing an orthogonally protected "extra" COOH function e.g the (-allyl ester of N-(-FMOC-L-glutamic-acid, i.e., "FMOC-Glu-Oall", or the (-tert-butyl ester of N-(-FMOC-L-glutamic acid (“FMOC-Glu-OtBu), or the
  • Suitable starting materials are available commercially, and further ones can be made by methods known in the art. D-amino acid derivatives can also be used in this methodology. Instead of "truly" orthogonal protective groups, also quasiorthogonal/semi-orthogonal/pseudoorthogonal protecting groups can be employed, as those skilled in the art understand.
  • Cyclic products made according to the above described methods are usually especially stable in biological milieu, and are thus preferred.
  • This type of structures may be produced by any of the methods for the production of such structures (chemical, enzymatic or biological). Many such methods are well known for those skilled in the art. Cyclic structures of this type can be syntesized chemically with the aid of solid-phase synthesis but they can likewise be synthesized using solution methods or a combination of both, as those skilled in the art well know.
  • Amino acids with an "extra" carboxyl or amino func- tion suitable for cyclization purposes (when adequately protected) include (as non-limiting possibilities), for example, those with the structures shown below:
  • the targeting units and agents according to the present invention may also be prepared as fusion proteins or by other suitable recombinant DNA methods known in the art. Such an approach for preparing the peptides according to the present invention is preferred especially when the effector units and/or other optional units are peptides or proteins.
  • One example of a useful protein effector unit is glutathion-S-transferase (GTS).
  • the targeting units of this invention can be synthesized easily and reliably.
  • An advantage as compared to many prior art peptides is that the targeting units and motifs of this invention do not need to comprise the problematic basic amino acids lysine and histidine, nor tryptophan, all of which may cause serious side-reactions in peptide synthesis, and, due to which the yield of the desired product might be lowered radically or even be impossible to obtain in adequate amounts or with adequate quality.
  • histidine, lysine and tryptophan When present, histidine, lysine and tryptophan must be adequately protected using suitable protecting groups that remain intact during the synthesis prodecures. This may be very difficult and at least increases the costs and technical problems. Also costs are remarkably increased by the reagents and work-load and other costs of the deprotection steps and the costs per unit of desired product may be increased.
  • the peptides of the present invention are much easier and cheaper to produce than targeting peptides of the prior art.
  • the peptides of the present invention can also be purified much more reliably and easily and with much less labor and apparatus-time, and thus with clearly lower costs. Overall costs are thus drastically reduced and better products can be obtained and in greater amounts. Furthermore, the reliability of the purification is much better, giving less concern of toxic remainders and of fatal or otherwise serious side-effects in therapeutic and diagnostic applications. Shorter synthesis protocols with relatively few steps produce less impurities, making the peptides of the present invention highly advantageous. The risks of toxic and even fatal impurities, allergens etc. are dramatically lowered and, in addition, purification is easier. The analysis and thus the quality control of the products of the present invention is easier and less costly, than that of the longer and more 'difficult' peptide sequences. This increases the reliability of the analyses and of quality control.
  • the effector unit can easily be linked to the peptides and peptidyl analogues and peptidomimetic substances of the present invention using (outside the targeting motif) for example protected lysine or ornithine as there is no risk of simultaneous reaction of any lysine residue in the targeting motif.
  • protected lysine or ornithine can be used, as the targeting units do not contain such amino acids. This is an enormous advantage
  • lactam bridge is clearly superior to the commonly used disulphide bridge. This is due to a number of reasons, such as:
  • Lactams are highly stable against reducing agents such as thiols that easily destroy disulphide bridges and can cause various undesired reactions, for example dimerization, polymerization and formation of disulphide bridges to other thiols; 2. Lactams can be synthesized in a highly controlled fashion for example by using orthogonal protection;
  • Lactams are more stable than disulphides in biological milieu.
  • lactones Because of lack of reaction with macromolecules having SH- groups lactones probable are less prone to cause allergic reactions.
  • the effector units and optional additional units may be linked to the targeting peptide when still connected to the resin without the risk that the removal of the protecting groups will cause destruction of additional unit. Similar advantage applies to solution syntheses. Another important advantage of the present invention and its products, methods and uses according to it is the highly selective and potent targeting of the products.
  • the products and methods of in the present invention are highly advantageous because of several reasons. Potential immunological and related risks are also obvious in the case of large biomolecules. Allergic reactions are of great concern with such products, in contrats to small synthetic molecules such as the targeting agents, units and motifs of the present invention.
  • the products and methods described in the present invention are highly advantageous because their structure can be easily modified if needed or desired. Specific amino acids such as histidine, tryptophan, tyrosine, threonine can be omitted if dersired, and very few functional groups are necessary.
  • the targeting units and targeting agents according to the present invention are useful in cancer diagnostics and therapy, as they selectively target to tumors in vivo, as shown in the examples.
  • the effector unit may be chosen according to the desired effect, detection or therapy. The desired effect may also be achieved by including the effector in the targeting unit as such.
  • the targeting unit itself may be e.g., radioactively labelled.
  • the present invention also relates to diagnostic compositions comprising an effective amount of at least one targeting agent according to the present invention.
  • a diagnostic composition according to the present invention may, optionally, comprise carriers, solvents, vehicles, suspending agents, labelling agents and other additives commonly used in diagnostic compositions. Such diagnostic compositions are useful in diagnosing tumors, tumor cells and metastasis.
  • a diagnostic composition according to the present invention may be formulated as a liquid, gel or solid formulation, preferably as an aqueous liquid, containing a targeting agent according to the present invention in a concentra- tion ranging from about 0.00001 ⁇ g/l to 25 x 10 7 ⁇ g/l.
  • the compositions may further comprise stabilizing agents, detergents, such as polysorbates and Tween, as well as other additives. The concentrations of these components may vary significantly depending on the formulation used.
  • the diagnostic compositions may be used in vivo or in vitro.
  • the present invention also includes the use of the targeting agents and targeting units for the manufacture of pharmaceutical compositions for the treatment of cancer.
  • the present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one targeting agent according to the present invention.
  • the pharmaceutical compositions may be used to treat, prevent or ameliorate cancerdiseases, by administering an therapeutically effective dose of the pharmaceutical composisiton comprising targeting agents or targeting units according to the present invention or thera- Railically acceptable salts, esters or other derivatives thereof.
  • the compositions may also include different combinations of targeting agents and targeting units together with labelling agents, imaging agents, drugs and other additives.
  • a therapeutically effective amount of a targeting agent according to the present invention may vary depending on the formulation of the pharma- ceuticakl composition.
  • a composition according to the present invention may comprise a targeting agent in a concentration varying from about 0.00001 ⁇ g/l to 250 g/l, more preferably about 0,001 ⁇ g/l to 50 g/l, most preferably 0,01 ⁇ g/l to 20 g/l.
  • a pharmaceutical composition according to the present invention is useful for administration of a targeting agent according to the present invention.
  • Pharmaceutical compositions suitable for peroral use, for intravenous or local injection, or infusion are particularly preferred.
  • the pharmaceutical compositions may be used in vivo or ex vivo.
  • the preparations may be lyophilized and reconstituted before ad- ministration or may be stored for example as a solution, solutions, suspensions, suspension-solutions etc. ready for administration or in any form or shape in general, including powders, concentrates, frozen liquids, and any other types. They may also consist of separate entities to be mixed and, possibly, otherwise handled and/or treated etc. before use. Liquid formulations provide the advantage that they can be administered without reconstitution.
  • the pH of the solution product is in the range of about 1 to about 12, prefera- bly close to physiological pH.
  • the osmolality of the solution can be adjusted to a preferred value using for example sodium chloride and/or sugars, polyols and/or amino acids and/or similar components.
  • the compositions may further comprise pharmaceutically acceptable excipients and/or stabilizers, such as albumin, sugars and various polyols, as well as any acceptable additives, or other active ingredients such as chemotherapeutic agents.
  • the present invention also relates to methods for treating cancer, especially solid tumors by administering to a patient in need of such treatment a therapeutically efficient amount of a pharmaceutical composition according to the present invention.
  • Therapeutic doses may be determined empirically by testing the targeting agents and targeting units in available in vitro or in vivo test systems. Examples of such tests are given in the examples. Suitable therpeutically effective dosage may then be estimated from these experiments. For oral administration it is important that the targeting units and targeting agent are stable and adequately absorbed from the intestinal tract.
  • compositions according to the present invention may be administered systemically, non-systemically, locally or topically, par- enterally as well as non-parenterally, e.g. subcutaneously, intravenously, in- tramuscularly, perorally, intranasally, by pulmonary aerosol, by injection or infusion into a specific organ or region, buccally, intracranically or intraperitoneally.
  • Amounts and regimens for the administration of the tumor targeting agents according to the present invention can be determined readily by those with ordinary skill in the clinical art of treating cancer. Generally, the dosage will vary depending upon considerations such as: type of targeting agent employed; age; health; medical conditions being treated; kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired; gender; duration of the symptoms; and, counterindications, if any, and other variables to be adjusted by the individual physician.
  • Preferred doses for administration to human patients targeting targeting units or agents according to the present invention may vary from about 0.000001 ⁇ g to about 40 mg per kg of body weight as a bolus or repeatedly, e.g., as daily doses.
  • the targeting units and targeting agents and pharmaceutical com- positions of the present invention may also be used as targeting devices for delivery of DNA or RNA or structural and functional analogues thereof, such as phosphorothioates, or peptide nucleic acids (PNA) into tumors and their metastases or to isolated cells and organs in vitro; i.e. as tools for gene therapy both in vivo and in vitro.
  • the targeting agents or targeting units may be parts of viral capsids or envelopes, of liposomes or other "containers" of DNA/RNA or related substances, or may be directly coupled to the DNA/RNA or other molecules mentioned above.
  • kits and components for kits for diagnosing, detecting or analysing cancer or cancer cells in vivo and in vitro comprise at least a targeting agent or targeting unit of this invention together with diagnostic entities enabling detection.
  • kits comprise for example a targeting agent and/or a targeting unit coupled to a unit for detection by e.g. immunological methods, radiation or enzymatic methods or other methods known in the art.
  • targeting units and agents of this invention as well as the targeting motifs and sequences can be used as lead compounds to design peptidomimetics for any of the purposes described above.
  • the targeting units and agents as well as the targeting motifs and sequences of the present invention can be used for the isolation, purification and identification of the cells, molecules and related biological targets.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS (leucyl arginyl serine), was synthesized by means of manual synthesis as described in Example 2 below.
  • the following reagents were employed as starting materials (in this order):
  • the funnel was loaded with the appropriate solid phase synthesis resin and solutions for each treatment, shaken powerfully with the aid of a "wrist movement" bottle shaker (Gallenkamp) for an appropriate period of time, followed by filtration effected with a moderate argon gas pressure.
  • 'DCM' means shaking with dichloromethane
  • 'DMF' means shaking with ⁇ /, ⁇ /-dimethylformamide (DMF may be replaced by NMP, i.e. N- methylpyrrolidinone).
  • Fmoc-amino acid 9-fluorenylmethyloxycarbonyl- ⁇ /-protected amino acid
  • Activation of the 9-fluorenylmethyloxycarbonyl- ⁇ /-protected amino acid (Fmoc-amino acid) to be added to the amino acid or peptide chain on the resin was carried out, using the reagents listed below, in a separate vessel prior to treatment step no. 12.
  • the Fmoc-amino acid (3 mmol) was dis- solved in approximately 10 ml of DMF, treated for 1 min with a solution of 3 mmol of HBTU dissolved in 6 ml of a 0.5 M solution of HOBt in DMF, and then immediately treated with 3 ml of a 2.0 M DIPEA solution for 5 min.
  • HBTU 2-(1 H-benzotriazol-1 -yl)-1 ,1 ,3,3-tetramethyluronium hexafluoro- phosphate, CAS No. [94790-37-1], Applied Biosystems Cat. No. 401091 , molecular weight: 379.3 g/mol
  • HOBt 1-Hydroxybenzotriazole, 0.5 M solution in DMF, Applied Biosystems Cat. No. 400934
  • DIPEA N,N-Diisopropylethylamine, 2.0 M solution in N-methylpyrrolidone, Applied Biosystems Cat. No. 401517
  • the procedure described above was repeated in several cycles using the appropriate different Fmoc-amino acids, carrying suitable protecting group(s), to produce a resin-bound source of the appropriate peptide (i.e., a "resin-bound" peptide).
  • the resin was treated with three portions of the above reagent mixture (each about 15 ml for 1 g of the resin), each for one hour. The treatments were carried out under argon atmosphere in the way described above.
  • the TFA solutions obtained by filtration were then concentrated under reduced pressure using a rotary evaporator and were recharged with argon. Some diethyl ether was added and the concentration re- peated. The concentrated residue was allowed to precipitate overnight under argon in dietyl ether in a refrigerator. The supernatant ether was removed and the precipitate rinsed with diethyl ether.
  • the cleavage mixture described above also simultaneously re- moved the following protecting groups: trityl (Trt) as used for cysteine -SH protection; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) as used for protection of side chain of arginine; the t ⁇ rt-butyl group (as an ester group on the carboxyl function; OtBu) as used for protection of the side-chain carboxyl group of glutamic acid and/or aspartic acid, and can normally be used also for removal of these protecting groups on analogous structures (thiol, guanyl, car- boxyl). It did not cause Fmoc removal.
  • the cleavage procedure described above can be carried out also without the removal of the Fmoc group, to produce the amino terminal N- Fmoc-derivative of the peptide, or for a peptide linked to an effector unit (comprising no Fmoc).
  • MALDI -TOF Matrix Assisted Laser Desorption lonization - Time of Flight
  • Angiotensin II and ACTH(18-39) Matrix alpha-cyano-4-hydroxycinnamic acid (saturated solution in aqueous 50% ace- tonitrile containing 0.1% of trifluoroacetic acid). The sample, together with the matrix, was dried onto the target plate under a gentle stream of warm air.
  • MALDI-TOF NEGATIVE ION REFLECTOR MODE External standards: cholecystokinin and glucagon Matrix:
  • the specimen was mixed at a 10-100 picomol/microliter concentration with the matrix solution as described.
  • the M+1 ⁇ i.e. the one proton adduct M+H+) signal with its typical fine structure based on isotope satellites was clearly predominant.
  • the M+1 signal pattern was accompanied by a similar but markedly weaker band of peaks at M+23 (Na+ adduct).
  • bands at M+1 and M+23 also bands at M+39 (K+ adduct) or M+56 (Fe+ adduct) could be observed in many cases.
  • the 'matrix signals' (signals due to the constituents of the matrix/ 'the ionization environment') have been omitted (i.e., signals at 294 and 380 Da have been omitted).
  • the resin (1 g) was swelled on CH2CI2 (15 ml) and stirred for 20 minutes. The solvent was removed by filtration and the resin was treated once with DMF (15 ml) for three minutes. After filtration, the resin-bound peptide (or targeting agent) was treated with iodine (5 molar equivalents) in DMF (10 ml) for 1 hour.
  • the DMF-iodine solution was removed by filtration and the residue was washed three times with DMF (15 ml) and three times with CH2CI2 (15ml) for 3 minutes each time.
  • the Fmoc group was removed and the peptide was released from the resin according to the general procedure described in Example 2 and purified by reversed phase HPLC.
  • the product was released from the resin and purified analogously.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of man- ual synthesis as described in Example 2 above.
  • the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • a small sample of the resin (containing the still fully protected cyclized peptide) was subjected to the treatment described in Example 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture described in Example 2, and isolated as described in the same Example.
  • Cyclic Fmoc-DLRSK was prepared and identified in analogous manner to cyclic DLRSK with the exeption of the final Fmoc removal that was o om itted in th is case .
  • the functionally protected, resin bound targeting unit (protected 0 peptide), comprising targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above.
  • the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • cyclization process 5 (macrolactam formation)
  • a small sample of the resin (containing the still fully protected cyclized peptide) was subjected to the treatment described in Example 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture described in Example 2, and isolated as described in the same Exam- pie.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above.
  • the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • a small sample of the resin (containing the still fully protected cyclized peptide) was subjected to the treatment described in Example 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture described in Example 2, and isolated as described in the same Example.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of man- ual synthesis as described in Example 2 above.
  • the following reagents were employed as starting materials (in this order): Fmoc-Lys(Mtt) Resin Fmoc-L-Ser(tBu)-OH Fmoc-L-Arg(Pbf)-OH Fmoc-L-Leu-OH Fmoc-L-Asn-OH
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above.
  • the following reagents were employed as starting materials (in this order): Fmoc-Lys(Mtt) Resin Fmoc-L-Ser(tBu)-OH Fmoc-L-Arg(Pbf)-OH Fmoc-L-Leu-OH Fmoc-L-Asn-OH Fmoc-L-Tyr(tBu)-OH
  • the still resin-bound tar- geting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • a small sample of the resin (containing the still fully protected cyclized peptide) was subjected to the treatment described in Example 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture described in Example 2, and isolated as described in the same Example.
  • the targeting unit has the structure:
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above.
  • the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • a small sample of the resin (containing the still fully protected cyclized peptide) was subjected to three hours' treatment with the cleavage mixture described in Example 2.
  • a sample of peptide was cleaved from the resin and the protecting groups of side chains of that sample were removed with the exeption of the final Fmoc removal that was omitted in this case.
  • the sample was isolated as described in the same Example.
  • the product DLRSK macrolactam
  • DL-2,3-diaminopropionic acid monohydrochloride was dissolved in 15 mL of aqueous 10% Na2C03 solution. Then 7 mL of dioxane was added and the reaction mixture cooled to +4°C. Fmoc-chloride in 20 mL of dioxane was added and the reaction mixture stirred for one hour at +4°C. After contin- ued stirring at room temperature overnight the reaction mixture was extracted with ethyl acetate that was then evaporated. The residue was triturated with n- hexane and washed with small amount of hot ethyl acetate to afford white solid that was dried in vacuo overnight. Reagents used: DL-2,3-diaminopropionic acid monohydrochloride
  • the appropriate protected peptide was synthesized on using solid- phase synthesis according to the general procedure described in Example 2.
  • the peptide was not deprotected and also not removed from the resin.
  • the resin-bound peptide was added to the reaction flask.
  • the resin was swelled using CH2CI2 (15 ml) and stirred for 20 minutes.
  • the solvent was removed by filtration and the resin was treated once with DMF for three minutes.
  • the peptide was deprotected using 20% piperidine solution in DMF (20ml) and shaking therewith for 5, and the process was repeated using (now shaking for 10 minutes).
  • the resin was washed three times with DMF (15 ml) and three times with CH2CI2 (15ml) and once with DMF (15 ml) for three minutes each time.
  • reaction mixture was then filtered and the residue was washed three times with DMF (15 ml) and three times with CH2CI2 (15ml) for 3 minutes each time.
  • the targeting agent was synthesized using manual synthesis as de- scribed in Example 2 above (analogously to the synthesis in Example Example 1 above) and using the biotinylation procedure described in Example 11 above as the final coupling step. In this final coupling process, D-biotin was employed instead of a protected amino acid. D-biotin was not protected but was employed as such. The product was isolated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • the targeting agent was synthesized using manual synthesis as de- scribed in Example 2 above (analogously to the synthesis in Example Example 4 above, including cyclization) and using the biotinylation procedure described in Example 11 above as the final coupling step.
  • D-biotin was employed instead of a protected amino acid. D-biotin was not protected but was employed as such.
  • the product was isolated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • the targeting agent was synthesized using manual synthesis as described in Example 2 above (analogously to the synthesis in Example 6 above, including cyclization) and using the biotinylation procedure described in Exam- pie 11 above as the final coupling step. In this final coupling process, D-biotin was employed instead of a protected amino acid. D-biotin was not protected but was employed as such.
  • the product was isolated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • the targeting agent was synthesized using manual synthesis as described in Example 2 above (analogously to the synthesis in Example 7 above, including cyclization) and using the biotinylation procedure described in Example 11 above as the final coupling step.
  • D-biotin was employed instead of a protected amino acid. D-biotin was not protected but was employed as such.
  • the product was isolated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spec- troscopy (M+1 ion clearly predominant).
  • Activation was by a PyAOP/HOAt/DIPEA reagent mixture (for details and abbreviation explanation, see below) or, alternatively, by the HBTU/HOBt/DIPEA mixture described in Example 2.
  • the equipment, common solvents, and practical techniques were similar to those described in Example 2.
  • the initially fully protected resin-bound peptide (0.3 mmol) was shaken under argon atmosphere at room temperature with different solutions (about 10 mL) for the periods of time indicated below, followed by filtration: 1. dichloromethane, for 20 min. 2. 4 % (by volume) trifuoroacetic acid in dichloromethane, for 15 min.
  • D-biotin was employed instead of a protected amino acid. D-biotin was not protected but was employed as such.
  • the product was isolated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • the targeting agent was synthesized using manual synthesis as de- scribed in Example 2 above (analogously to the synthesis in Example 9 above, including cyclization) and using the biotinylation procedure described in Example 11 above as the final coupling step.
  • D-biotin was employed instead of a protected amino acid. D-biotin was not protected but was employed as such.
  • the product was isolated and purified in the man- ner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • the synthesis was carried out as follows: The fully protected resin- bound cyclized targeting unit (peptide with spacer/linker unit) AhxDLRSK was prepared as described in Example 9 above. Next, the sequence AhxDLRSK was continued with one lysine unit (protected with Fmoc-group on N-terminal amino group and with Boc-group on side branch amino group) by means of the general coupling methods described in Example 2.
  • the reagent used as start- ing material was carried out as follows:
  • the product has the formula shown below:
  • the synthesis was carried out as follows: The fully protected resin- bound, 'on resin' cyclized targeting unit (peptide with two spacer/linker units) K- AhxDLRSK was prepared as described in Example 20 above. The branched structure comprising the four biotins and the three lysines was conctructed by means of the general coupling methods described in Example 2, so that the sequence K-AhxDLRSK was continued first with one lysine unit (protected with one Fmoc-group on each of its two amino groups).
  • Biotinylation was done according to the general method described in Example 11 using 12 molecular equivalents of coupling reagents and biotin, employing the resin-bound branched peptide, to afford a stucture comprising four biotin units. Purification by HPLC gave 44% of the theoretical as overall yield.
  • the product has the formula shown below:
  • the synthesis was carried out as follows: The isolated and purified targeting agent Bio4K3-K-AhxDLRSK was prepared as described in Example 21 above. The product thus obtained was then treated with 10 molecular equivalents of diethylenetriaminepentaacetic dianhydride in DMF solution (0.01 M solution as calculated on the basis of the biotinylated peptide) for 18 hours. After this treatment, the volume was doubled by addition of water to the DMF solution, and the solution was put aside and allowed to stay still for 4 hours. Finally, the solvents were evaporated in vacuo and the residue was mixed in water containing 0.1% trifluoroacetic acid and was filtered and the filtrate was purified by reversed-phase HPLC. The product was identified by its M+1 peak in the MALDI-TOF mass spectrum. Identification of the product: positive mode MALDI-TOF mass spectrum: M+1 ion clearly predominant.
  • the targeting agent was synthesized using manual synthesis as de- scribed in Example 2 above (analogously to the synthesis in Example Example Example
  • the targeting agent was synthesized using manual synthesis as de- scribed in Example 2 above (analogously to the synthesis in Example Example Example
  • the targeting agent was synthesized using manual synthesis as de- scribed in Example 2 above (analogously to the synthesis in Example 4 above, including cyclization).
  • Reagent used 4-amino-10-methylfolic acid hydrate; (+)amethopterin; methotrexate CAS No. 59-05-2 Formula weight: 454.4 g/mol Sigma Cat. No. A-6770
  • the targeting agent was synthesized by stirring daunomycine with equimolar amount of Aoa-DLRSK described above in Example 23 in methanol solution (concentration 0.0025M) at room temperature in dark for three days. The product was isolated by evaporation of solvents and purified by HPLC. Reagent used: Daunomycin hydrochloride CAS No. 20830-81-3 Molecular weight: 564.0 g/mol ICN Biomedicals, Aurora, Ohio, USA Cat. No. 44583
  • the targeting agent was synthesized by stirring doxorubicine with equimolar amount of Aoa-DLRSK described above in Example 23 in methanol solution (concentration 0.0025M) at room temperature in dark for three days. The product was isolated by evaporation of solvents and purified by HPLC. Reagent used: Doxorubicin hydrochloride CAS No. 25316-40-9 Molecular weight: 580.0 g/mol Fluka Cat. No. 44583
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above.
  • the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • a small sample of the resin (containing the still fully protected cyclized peptide and being suitable starting material for further synthesis e.g. biotinylation) was subjected to the treatment described in Example 2 for Fmoc removal (steps 1-10 in that Example), after which the sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture described in Example 2, and isolated as described in the same Example.
  • the targeting agent was synthesized using manual synthesis as described in Example 2 above (analogously to the synthesis in Example 28 above, including cyclization) and using the biotinylation procedure described in Example 11 above as the final coupling step.
  • D- biotin was employed instead of a protected amino acid.
  • D-biotin was not protected but was employed as such.
  • the product was isolated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRSGRG, was synthesized by means of manual synthesis as described in Example 2 above.
  • the targeting agent was synthesized using the biotinylation proce- dure described in Example 11 above as the final coupling step.
  • D-biotin was employed instead of a protected amino acid.
  • D- biotin was not protected but was employed as such.
  • the biotinylated, still resin-bound targeting agent was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17.
  • the product was iso- lated and purified in the manner indicated in Example 2 and identified by positive-mode MALDI-TOF spectroscopy (M+1 ion clearly predominant).
  • ODC sarcoma cells originally derived from tumors that were formed in nude mice to which had been administered NIH3T3 mouse fibro- blasts transformed by virtue of ornithine decarboxylase (ODC) overexpression and have been described earlier (Auvinen et al., 1997);
  • the cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Bio-Whittaker) supplemented with 5-10% fetal calf serum (FCS; Bio- Whittaker), 1 % L-glutamine (Bio-Whittaker) and 1 % penicillin/streptomycin (Bio-Whittaker).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • Bio-Whittaker 1 % L-glutamine
  • penicillin/streptomycin Bio-Whittaker
  • the cells listed above (OS, KS and melanoma: 0.5 x 106 cells were injected subcutaneously into both flanks of nude mice of the strains Balb/c Ola Hsd-nude, NMRI/nu/nu or Athymic-nu (all mice of both strains were from Harian Laboratories). Tumors were harvested when they had reached a weight of about 0.4 g.
  • Metastases (mostly formed in the lungs) were produced by injection of melanoma cells i.v. into Balb/c Ola Hsd-nude mice. The mice were kept 4-6 weeks, and then targeting experiments were performed. Tumor-bearing or metastase-bearing mice were anesthesized by administering 0.02 ml/g body weight Avertin [10 g 2,2,2-tribromoethanol (Fluka) in 10 ml 2-methyl-2-butanol (Sigma Aldrich)] intraperitoneally (i.p.).
  • Avertin 10 g 2,2,2-tribromoethanol (Fluka) in 10 ml 2-methyl-2-butanol (Sigma Aldrich)
  • mice For localization of the targeting peptides OS and melanoma tumor- bearing NMRI nude mice were anesthesized and 1 or 2 mg of biotinylated synthetic targeting peptide (prepared in Example 12) was injected i.v. 5-10 min after the i.v. injections, the mice were perfused via the heart using a winged infusion 25G needle set (Terumo) with 50 ml DMEM. Then, their organs were dissected and frozen in liquid nitrogen. Tumors and control organs (liver, kidney, spleen, heart, brain) were dissected and frozen in liquid nitrogen.
  • Biotinylated peptides were detected on 10 micrometer cryosections using AB (avidin-biotin) -complex containing avidin, and biotinylated HRP (Vectastain ABC-kit, cat no. PK6100; Vector Laboratories) with diaminobenzidine (DAB substrate kit, cat no. 4100, Vector Laboratories). The results of the experiments are shown in Table 2
  • the targeting agent Dxrb-Aoa-DLRSK prepared in Example 24, comprising a cytotoxic effector unit, doxorubicin, linked by oxime ligation to the cyclic targeting unit DLRSK comprising the targeting motif
  • LRS was used to demonstrate in vivo targeting and therapeutic effect on melanoma tumors.
  • mice 1 million C8161 M/T1 melanoma cells were injected subcutaneously into flank of eight Athymic-nu mice and tumours were allowed to grow for one week. The mice were then divided into three groups those received the following treatments:
  • Tumours were measured with a calli- per in two perpendicular directions on each injection day and on the day the animals were sacrificed. Tumour volume was calculated by the formula for ellipsoid:
  • a selective, one-process, dismantling of par- ticular protecting groups of ornithine and gutamic acid [the said groups are: 2- N-Fmoc on the ornithine unit and 5-(2-trimethylsilylethyl ester) on the glutamic acid unit] is carried out with tetrabutylammonium fluoride solution in DMF.
  • the cyclization involves a condensation between the side-chain carboxyl group of the glutamic acid unit and the 2-amino group (N-terminal amino group) of the ornithine unit.
  • Activation is by a PyAOP/DIPEA reagent mixture (for details and abbreviation explanation, see below) instead of the HBTU/HOBt/DIPEA mixture described in Example 2.
  • PyAOP/DIPEA reagent mixture for details and abbreviation explanation, see below
  • HBTU/HOBt/DIPEA mixture described in Example 2.
  • the equipment, common solvents, and practical techniques are similar to those described in Example 2.
  • This method can be modified for lysine (instead of ornithine) and aspartic (instead of glutamic) acid unitst by empoying respective derivatives of those amino acids.
  • the reagent for deprotection prior to cyclization is: Tetrabutylammonium fluoride trihydrate, CAS No. 87749-50-6, molecular weight: 315.51 g/mol, Acros Organics Cat. No. 221080500.
  • Fmoc-L-Glu(OTMSEt)-ONa ⁇ /-2-Fmoc-glutamic acid 5-(2-trimethylsilylethyl) ester sodium salt, molecular weight: 468.60 g/mol, Novabiochem Cat. No. 04- 12-1231.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising targeting motif LRS, was synthesized by means of man- ual synthesis as described in Example 2 above, in which the the "empty" resin was deprotected prior to the first coupling in the same manner as described for the the pre-loaded resins (steps 1-11 in Example 2).
  • the functionally protected, resin bound targeting unit (protected peptide), was synthesized by means of manual synthesis as described in Example 2 above.
  • the following reagents were employed as starting materials (in this order):
  • Fmoc-Gly Resin Fmoc-L-Glu(OTMSEt)OH Fmoc-Gly-OH Fmoc-L-Arg(Pbf)-OH Fmoc-Gly-OH Fmoc-L-Ser(tBu)-OH Fmoc-L-Arg(Pbf)-OH Fmoc-L-Leu-OH Fmoc-D-Orn(Mtt)-OH
  • the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 33.
  • a sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture de- scribed in Example 2, and isolated as described in the same example. Then, the product was identified with the aid of its positive mode MALDI-TOF mass spectrum by means of the M+1 ion of cyclic D- OrnLRSGRGEG.
  • the functionally protected, resin-bound, and cyclized targeting unit comprising the targeting motif LRS, was synthesized by means of manual synthesis as described in Example 34 above.
  • the resin was treated with diluted TFA (4 % in dichloromethane) in the manner described in example 17 (steps 1 -7) to cleave the side chain protecting Mtt-group of ornithine.
  • the still resin-bound unit was then coupled with DOTA-tris-ferf-butyl ester by means of the general method described in Example 2 (steps 12-18) using HBTU/HOBt/DIPEA activation.
  • Reagent used DOTA-tris(tBu ester).
  • the product was cleaved and isolated as described in Example 2 and identified with the aid of its positive mode MALDI-TOF mass spectrum, in which the M+1 ion of cyclic D-Om(Dota)LRSE-amide was clearly predominant.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising the targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above, in which the the "empty" resin was deprotected prior to the first coupling in the same manner as de- scribed for the the pre-loaded resins (steps 1-11 in Example 2).
  • the still resin-bound tar- geting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 33 (as modification which replaces Glu with Asp and Lys with Orn).
  • a sample of peptide was cleaved from the resin by three hours' treatment with the cleavage mixture described in Example 2, and isolated as described in the same example. Then, the product was identified with the aid of its positive mode
  • the functionally protected, resin-bound, and cyclized targeting unit comprising the targeting motif LRS, was synthesized by means of manual synthesis as described in Example 37 above.
  • the resin was treated with di- luted TFA (4% in dichloromethane) in the manner described in example 17 (steps 1-7) to cleave the side chain protecting Mtt-group of lysine.
  • the still resin-bound unit was then coupled with DOTA-tris-terf-butyl ester by means of the general method described in Example 2 (steps 12-18) using HBTU/HOBt/DIPEA activation.
  • DMF N,N-dimethylformamide
  • the resin was filtered and washed sev- eral times with DMF and dichloromethane in the way described in Example 2 (steps 13 -18).
  • the resin was shaken for 2 hours with a mixture of acetic anhydride (2M solution, 94 equivalents) and /N/V-diisopropylethylamine (DIPEA, 1.6 M solution, 80 equivalents) in -methyl pyrrolidinone (NMP) solution, filtered and washed like earlier ending up in drying at argon gas flow.
  • DIPEA /N/V-diisopropylethylamine
  • NMP -methyl pyrrolidinone
  • Fmoc-6-aminohexanoic acid (Fmoc-Ahx-OH) , CAS No. 88574-06-5, Novabiochem Cat. No. 04-12-1111 , Molecular Weight: 353.4 g/mol.
  • Acetic anhydride Fuka Cat. No. 45830.
  • the still resin-bound product was next cyclized according to Example 1 . Finally the sequence was continued with acetic acid (i.e. end-capped at amino terminal) as follows: Amino protecting Fmoc-group was removed as de- scribed in Example 2 (steps 1-10). Then the still resin-bound product was treated with the mixture of acetic anhydride and DIPEA in NMP like was done after the initial binding of Ahx moiety to the resin. In the end the product was released from the resin and purified as described in Example 2. Identification was based on M+1 ion of MALDI mass spectrum.
  • Doxorubicin hydrochloride CAS No. 25316-40-9, molecular weight: 580.0 g/mol, Sigma Cat. No. D-1515.
  • Example 2 After isolation and purification, according to Example 2, the product was identified on the basis of M+1 ion in positive mode MALDI mass spectrum.
  • Paclitaxel succinate described in the end of this example, was dissolved as 0.012 M solution in DMF and equimolar amount of 0.05 M PyAOP in DMF was added, followed by double molar amount of 2.0 M DIPEA in NMP. After 2 minutes equimolar amount (per paclitaxel succinate) of side-chain-to- side-chain cyclic targeting compound AhxDLRSK, described in Example 9 above, was added as 0.015 M solution in DMF. After staying overnight the mixture was diluted with diethyl ether. The centrifuged solid precipitate was purified by reverse phase HPLC chromatography as described in Example2, including the identification of the product based on its M+1 ion in the positive mode MALDI-TOF mass spectrum.
  • Paclitaxel from Taxus yannesis
  • CAS No. 33069-62-4 molecular weight: 853.9 g/mol
  • Succinic anhydride CAS No. 108-30-5, molecular weight: 100.08 g/mol, Fuka product No. 14089.
  • Camptothecin p-nitrophenylcarbonate described in the end of this example, was dissolved as 0.02 M solution in DMF and combined with 0.04 M solution of equimolar amount of cyclic targeting compound AhxDLRSK, described in Example 9 above, in the same solvent. After staying overnight, 2 M DIPEA in NMP was added in 10% excess (i.e. equimolar amount multiplied by 1.1). After being stirred overnight the mixture was diluted with diethyl ether and the centrifuged solid precipitate was purified by reverse phase HPLC chromatography as described in Example2, including the identification of the product based on its M+1 ion in the positive mode MALDI-TOF mass spectrum.
  • the functionally protected, resin bound targeting unit (protected peptide), comprising the targeting motif LRS, was synthesized by means of manual synthesis as described in Example 2 above, in which the the "empty" resin was deprotected prior to the first coupling in the same manner as described for the the pre-loaded resins (steps 1-11 in Example 2).
  • Example 17 After these coupling cycles the still resin-bound targeting unit was subjected to the cyclization process in which an extra amide bond is formed as described in Example 17. Next, still two couping cycles were carried out as described in Example 2 above employing following starting materials: Fmoc-6-aminohexanoic acid DOTA-tris-(tBu ester). Finally the the resin was cleaved and the product purified as described in Example 2, and identified by means of its M+1 ion in positive mode MALDI-TOF mass spectrum.
  • MALDI-TOF data Calculated molecular mass: 1522.66 based on the most abundant isotopes Observed signal M+1 : 1523.66 as typical isotopic pattern
  • Boc-amino-oxyacetic acid Boc-NH-OCH2-COOH, Molecular weight: 191.2 g/mol, CAS No., Novabiochem Cat. No. 01-63-0060
  • Boc-Cys (Trt)-OH CAS No: 21947-98-8, Novabiochem, cat. no 04-12-0020

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Abstract

L'invention concerne de nouveaux motifs structuraux, unités et agents de ciblage tumoral, ainsi que des peptides de ciblage tumoral et des analogues de ceux-ci. Ces agents de ciblage comprennent généralement au moins un motif structural de ciblage Aa-Bb-Cc et au moins une unité effectrice. L'invention concerne en outre de nouveaux peptides de ciblage tumoral spécifiques, et des compositions pharmaceutiques et diagnostiques contenant de tels peptides. L'invention concerne également des méthodes de diagnostic ou de traitement des cancers.
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US20070231258A1 (en) * 2006-03-31 2007-10-04 Karyon Ctt Ltd. Peptide conjugates
US8636978B2 (en) 2008-06-23 2014-01-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Cyclized NGR peptide compounds, compositions, and methods of their use
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US5516889A (en) * 1993-06-21 1996-05-14 University Technologies International, Inc. Synthetic thrombin receptor peptides
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US5981478A (en) * 1993-11-24 1999-11-09 La Jolla Cancer Research Foundation Integrin-binding peptides
US5622699A (en) * 1995-09-11 1997-04-22 La Jolla Cancer Research Foundation Method of identifying molecules that home to a selected organ in vivo
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Publication number Priority date Publication date Assignee Title
US5516889A (en) * 1993-06-21 1996-05-14 University Technologies International, Inc. Synthetic thrombin receptor peptides
WO2002090503A2 (fr) * 2001-05-04 2002-11-14 The Scripps Research Institute Peptides antimicrobiens et compositions

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