JP2006518702A - Tumor targeting agents and uses thereof - Google Patents

Abstract

The present invention relates to novel tumor targeting motifs, units and substances, as well as tumor targeting peptides and analogs thereof. A targeting agent typically comprises at least one targeting motif, Aa-Bb-Cc, and at least one effector unit. The invention further relates to specific tumor targeting peptides, pharmaceutical compositions or diagnostic compositions comprising such peptides. Also disclosed are methods for diagnosing or treating cancer.

Description

  The present invention relates to tumor targeting agents comprising at least one targeting unit and at least one effector unit, as well as tumor targeting units and motifs. The invention further relates to pharmaceutical and diagnostic compositions comprising such targeting agents or targeting units, and the use of such targeting agents and targeting units as drugs or diagnostic tools. The invention further relates to the use of such targeting agents and targeting units for preparing pharmaceutical compositions or diagnostic compositions and for preparing reagents for use in diagnosis or research. Furthermore, the present invention relates to a kit for diagnosing or treating cancer and metastasis. Yet another aspect of the invention relates to cell removal, selection, sorting and expansion methods, and materials and kits for use in such methods.

  Malignant tumors are one of the greatest health problems in humans and animals and are one of the most common causes of death among young individuals. Despite decades of intensive research efforts, the available cancer treatments are very limited. While curative treatments (usually surgery combined with chemotherapy and / or radiation therapy) are sometimes possible, malignant tumors (cancers) are still one of the most threatening diseases of mankind and a vast number Life is lost every year. In fact, there is little curative treatment if the disease is not diagnosed early. Furthermore, some tumor types, when present, can hardly be cured curatively.

  There are various reasons for this highly undesirable situation, but the most important reason is the fact that almost all (if not all) treatment schedules (other than surgery) are not selective enough. It is clear. Commonly used chemotherapeutic agents, such as alkylating agents, platinum compounds (eg cisplatin), beromycins, other alkaloids and other cytostatics, are commonly used for tumor malignant cells alone. Although it does not work, it is also very toxic to other cells and is usually particularly toxic to rapidly dividing cell types such as hematopoietic cells and epithelial cells. The same applies to radiation therapy.

  In addition to the complexity described above, two other significant problems are plagued by non-surgical treatment of malignant solid tumors. First, physiological barriers within the tumor impair the delivery of therapeutic agents at effective concentrations to all cancer cells. Second, the acquired drug resistance resulting from genetic and epigenetic mechanisms reduces the effectiveness of available drugs.

  Treatment of cancer patients with currently available, mainly non-selective, chemotherapeutic agents or radiation therapy often results in undesirable side effects. Target specific organs or tissues or tumor tissue to specifically deliver the desired cytotoxicity or other drugs to these organs or tissues to improve the effects of chemotherapeutic agents and reduce side effects It seems very important to identify substances that can

The same applies to a specific area of cancer treatment, namely neutron capture therapy, in which non-radioactive nuclides (eg ¹⁰ B, ¹⁵⁷ Gd or ⁶ Li) are converted by thermal (slow) neutrons from an external source, Converted to radionuclide in the patient in vivo. In this case, some prior art substances are claimed to have about 2-3 fold selectivity for at least some types of tumors, but the results obtained are mainly disappointing. It is negative. Specific targeting agents would also provide significant advantages in this area.

  More reliable, more sensitive and more selective methods and substances in the diagnosis of cancer and metastases, including patient follow-up and study of the impact of treatment on tumors and metastases, It seems to be a big advantage. This includes nuclear magnetic resonance imaging (NMR, MRI), X-ray methods, tissue staining methods (optical and electron microscopy and related methods, and possibly NMR, infrared, electron spin resonance and related methods in the future), and This applies to all currently used methods known by experts in the field, including generally any image and experimental methods (histology, cytology, cell sorting, hematopoietic studies, FACS, etc.). Here, tumor tissue, metastasis or tumor cells and / or tumor endothelium is specifically or selectively detected as an entity to be detected (spin label, radioactive substance, paramagnetic phase substance for NMR, X-ray image or tomography) For example, materials that can target phase materials for neutrons, boron atoms for neutron capture, etc., would be a major advantage.

  Solid tumor growth is angiogenesis-dependent, and the tumor should constantly stimulate the growth of new capillaries for continuous growth. Tumor blood vessels are structurally and functionally different from their normal quiescent counterparts. In particular, the endothelial cells lining new blood vessels are abnormally shaped and they grow on top of each other and protrude into the lumen of the blood vessel. This heterogeneity of angiogenesis depends on the tumor type and the host organ in which the tumor is growing. Thus, vascular permeability and angiogenesis are unique in each different organ and tumor tissue derived from that organ.

  There are numerous publications that disclose peptides that host different cell and tissue types. Some of these are claimed to be useful as cancer targeting peptides. Among the early identified host peptides that have been described are integrin and NGR-receptor targeting peptides, described by Ruoslahti et al., For example, in US Pat. No. 6,180,084. These peptides host the angiogenic vasculature and bind to the NGR-receptor.

  As tumors change to an angiogenic phenotype and recruit new blood vessels, endothelial cells in these vessels express proteins on the luminal surface that are not produced by normal quiescent vascular endothelium. One such protein is αvβ3 integrin. US Patent Publication, US Pat. No. 6,177,542 discloses peptides that can specifically bind to αvβ3 integrin. The described tumor blood vessel specific targets are adhesion molecules that mediate the binding of endothelial cells to the vascular basement membrane. This peptide is a 9-residue cyclic peptide containing the ArgGlyAsp (RGD) sequence. Pasqualini et al. (1997) showed that, when injected intravenously, this peptide could be hosted in murine and human tumor blood vessels in mice 40-80 times more efficiently than control organ blood vessels. It has been suggested that RGD peptides may be suitable tools for primary targets for diagnostic and therapeutic purposes. However, integrin-binding peptides can generally impair cell adhesion and are therefore not suitable for clinical use to selectively target tumors.

  International Patent Publication WO 00/67771 provides endostatin peptides comprising the amino acid sequences RLQD, RAD, DGK / R. Other examples of peptides hosting the angiogenic vasculature are described in US Pat. Nos. 5,817,750 and 5,955,572. These peptides recognize RGD.

  US Pat. No. 5,628,979 describes oligopeptides for in vivo tumor imaging and therapy. This oligopeptide contains 4 to 50 amino acids, which contain the amino acid sequence Leu-Asp-Val (LDV) as a characteristic triplet. This triplet has been reported to give oligopeptides with in vivo binding affinity for LDV binding sites in tumors and other tissues.

  International patent publication WO 99/47550 describes cyclic peptides containing the HWGF motif, which are specific inhibitors of MMP-2 and MMP-9. The cyclic decapeptide CTTHWGFTLC specifically inhibits the activity of these enzymes, suppresses tumor and endothelial cell migration in vitro, hosts the tumor vasculature in vivo, and promotes tumor growth and in mice. It has also been found to prevent intrusion. However, peptides that act as inhibitors of MMP show basic binding with non-tumor tissue. The fact that MMPs are also expressed in normal tissues throughout the body makes the administration of such peptides to humans or animals dangerous and even lethal. Because the activity of these enzymes is required for normal tissue function (Hidalgo and Eckhardt, 2001).

  US Patent Publication US2002 / 0102265A1 describes a peptide, TSPLNIHNGQKL, that targets a squamous cell carcinoma cell line and internalizes to cells in vitro. This peptide also targets experimental squamous cell carcinoma in nude mice.

  US Pat. Nos. 5,622,699 and 6,068,829 disclose a family of peptides containing SRL motifs that are selectively hosted in the brain.

  International Patent Publication WO 02/20769 discloses a method for identifying tissue-specific peptides by phage display and biopanning. Some of the identified peptides have been suggested to be tumor specific.

  Although there are known host peptides that bind to tumor vasculature, there are still few reports on targeting agents that actually target tumor cells and tissues in vivo. Most of the previously described targeting peptides are vasculature specific. Thus, there remains a firm need for new substances that selectively target tumor tissue, tumor vasculature, or both.

  For therapeutic applications, the targeting peptide is conjugated to doxorubicin in an uncontrollable manner, apparently resulting in a mixture product, or at least an unidentified structure, possibly inefficient action, especially substance identification Also present difficulties in purification, quality control and quantitative analysis, and the amount of doxorubicin per peptide molecule is still unknown (eg, Arap et al., 1998). Non-specific binding processes can compromise the targeting function of the peptide.

  Another very significant disadvantage of the prior art is that most of the described targeting peptides appear to target only the tumor endothelium and not the tumor mass itself. For example, the targeting peptide used by Nicklin et al. (2000) rested endothelial cells in vitro, targeting adenoviral DNA transfection under conditions that seemed to be rarely applicable in vivo.

  The targeting units of the present invention offer an advantage over the prior art in that they appear to target both tumor endothelium and tumor cell mass. This fact gives the possibility of targeting and destroying the tumor growth bodies supported by the tumor endothelium and the tumor mass itself. The main advantage of this approach stems from the fact that the endothelium is a genetically stable tissue that does not acquire drug resistance but is irreversibly removed.

  It is not known whether prior art targeting peptides are universal in the sense that they can target any malignant tumor type. Thus, their use as targeted therapeutics for some specific tumors may not be useful at all and do not provide a therapeutic advantage or effect over the free state therapeutics themselves. An even more serious drawback is that by using such targeting agents in diagnostic procedures, not all existing tumors can be revealed and malignant processes may still not be recognized. It is.

  The present invention provides significant improvements in view of the prior art. This is because the targeting agents described here have been found to target all of the various tumor types tested. In particular, targeting agents target sarcomas such as Kaposi's sarcoma, ornithine decarboxylase (ODC) overexpression, highly angiogenic tumors, carcinomas, and human primary and metastatic melanoma.

(Brief description of the invention)
It is an object of the present invention to provide a novel tumor and angiogenic tissue targeting agent comprising at least one targeting unit and optionally at least one effector unit. In particular, the present invention provides a targeting unit comprising at least one motif that can target tumor endothelium and tumor cell mass. Such targeting units, optionally combined with at least one effector unit, are particularly therapeutically and diagnostically useful in the treatment and diagnosis of cancer, including metastases. Furthermore, the targeting agents of the present invention are useful for cell removal, selection, sorting and expansion.

  A second object of the present invention is to provide 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 to provide a pharmaceutical composition and a diagnostic composition comprising.

  Furthermore, a third object of the present invention is to provide a novel diagnostic method and therapeutic method and kit for treating and / or diagnosing cancer.

  The present invention is based on the discovery that peptides having specific amino acid sequences or motifs can selectively target in vivo tumors and in vitro tumor cells. Thus, the peptides of the present invention can selectively bind to tumors rather than normal tissues in the body when administered to human or animal subjects.

  The present invention is also directed to the use of targeting agents and analogs thereof for the manufacture of a pharmaceutical composition or diagnostic composition for treating or diagnosing cancer.

  The targeting units of the invention can be used in this way or can be combined with at least one effector unit. Such substances can destroy the tumor or inhibit its growth. The targeting units and targeting agents of the present invention can also target metastases and can therefore be used to destroy or inhibit metastatic tumor growth. Since the early diagnosis of metastases is very important for the successful treatment of cancer, the use of the targeting units and targeting agents of the present invention in the early diagnosis of tumor metastases is important.

  The invention further includes salts, derivatives and analogs of the targeting units and targeting agents described herein, and uses thereof.

  Other objects of the invention provide diagnostic and pharmaceutical compositions comprising the targeting agent of the invention, and therapeutic and diagnostic methods for treating and diagnosing cancer, use of the targeting agent of the invention. It is to be. Kits are also provided for use in such methods or research purposes, as well as for cell sorting or removal.

  A particularly preferred embodiment of the present invention is a group of small, circular tumor targeting comprising a motif, LRS or SRL, optionally combined with effector units and other additional units as described in more detail herein. Relates to peptides.

  For the purposes of the present invention, the term “cancer” is used herein in its broadest sense and includes any disease or condition associated with transformed or malignant cells. Cancers in the field are classified into five main categories according to their tissue source (histological type): solid tumor type cancers, carcinoma, sarcoma, melanoma, and lymphoma, and “liquid cancer”. Leukemia. The term cancer as used in the present invention is intended primarily to include all types of diseases characterized by solid tumors, including disease states, and in the absence of detectable solid tumors or malignant or transformed cells, “cancer cells” "Appears as diffuse infiltrating cells or sporadically in other cells in healthy tissue.

  The terms “amino acid” and “amino alcohol” refer to diamino, triamino, oligoamino, and polyamino acids and alcohols; dicarboxyl, tricarboxyl, oligocarboxyl and polycarboxyl amino acids; dihydroxyl, trihydroxyl, oligohydroxyl and polyhydroxylaminoalcohol. As well as similar compounds containing two or more carboxyl or hydroxyl groups and one or more amino groups.

According to established predicate, the term “peptide” means a single chain of amino acids (peptide units) linked together by peptide bonds to form an amino acid chain. As described below, the peptide may be cyclic. For the purposes of the present invention, compounds comprising one or more D-amino acids, β-amino acids and / or other unnatural amino acids (eg, amino acids having unnatural side chains) are also included in the term “peptide”. It is. For the purposes of the present invention, the term “peptide” is intended to include peptidyl analogs containing modified amino acids. Such modifications include the introduction or presence of substituents in the ring or chain; the introduction or presence of “excess” functional groups such as amino, hydrazide, carboxyl, formyl (aldehyde) or keto groups, or other moieties; The absence or removal of groups or other moieties can be included. The term includes amino- and / or carboxy-terminal modified analogs such as peptide amides and N-substituted amides, peptide hydrazides, N-substituted hydrazides, peptide esters, etc., and peptides that do not contain an amino-terminal -NH ₂ group Or, for example, a peptide containing a modified amino terminal amino group or imino or hydrazino group instead of the amino terminal amino group, a peptide not containing the carboxy terminal carboxyl group, or a peptide containing a modifying group instead.

  Some examples of possible reaction types that can be used to form “peptidyl analogs” that modify peptides include, for example, cycloaddition, condensation and nucleophilic addition reactions, and esterification, amide formation. , Substituted amide formation, N-alkylation, hydrazide formation, salt formation. Salt formation may be the formation of any type of salt, such as alkali or other metal salts, ammonium salts, salts and organic bases, acid addition salts and the like. Peptidyl analogs can be synthesized from the corresponding peptide or directly (by other routes).

  The compound that is a structural analog or functional analog of the peptide of the present invention may be a compound that does not consist of an amino acid or an amino acid alone, or a compound in which some or all of its basic components are modified amino acids. Different types of basic components can be used for this purpose, as is well understood by those skilled in the art. The function of these compounds in biological systems is essentially the same as that of peptides. The similarity between these compounds and the original peptide is therefore based on structural and functional similarities. Such compounds are referred to as peptidomimetic analogs because they mimic the function, conformation and / or structure of the original peptide, and for purposes of the present invention, they are referred to in the term “peptide”. include.

  Functional analogs of the peptides of the invention are characterized by a binding capacity for binding to a tumor, tumor tissue, tumor cell or tumor endothelium that is essentially the same as the binding capacity of similar peptides.

  For example, compounds such as benzolactam or piperazine can be used, including analogs based on the primary sequence of the original peptide (Adams et al., 1999; Nakanishi and Kahn, 1996a, 1996b; Houghten et al., 1999; Nargund et al. 1998). A wide variety of types of peptidomimetics have been reported in the scientific and patent literature and are well known to those skilled in the art. Peptidomimetics (analogues) can include, for example, one or more of the following structural components: reduced amides, hydroxyethylene and / or hydroxyethylamine isoesters, N-methyl amino acids, urea derivatives, thiourea derivatives. Cyclic urea and / or thiourea derivatives, poly (ester imides), polyesters, esters, guanidine derivatives, cyclic guanidines, imidazolyl compounds, imidazolinyl compounds, imidazolidinyl compounds, lactams, lactones, aromatic rings, bicyclic systems, hydantoins, and / Or thiohydantoin, as well as various other structures. Many types of compounds for synthesizing peptidomimetics are available from several commercial sources (e.g., Peptide and Peptidomic Synthesis, Reagents for Drug Discovery, Fluka Chemi GmbH, Books, Switzerland, 2000 and Novagem Chem. , Lauferfingen, Switzerland, 2000). The similarity between the peptidomimetic compound and the original peptide is based on structural and functional similarities. Thus, a peptidomimetic compound mimics the properties of the original peptide and for the purposes of this application its binding ability is similar to similar peptides. A peptidomimetic compound can be composed of non-natural amino acids (such as D-amino acids or amino acids containing non-natural side chains, or β-amino acids) that do not appear in the original peptide, for example, It is believed that they consist of or can be made from other compounds or structural units. Examples of synthetic peptidomimetic compounds include N-alkylamino cyclic ureas, thioureas, polyesters, poly (ester imides), bicyclic guanidines, hydantoins, thiohydantoins, and imidazole-pyridino-inols (Houghten et al., 1999 and Nargund). Et al., 1998). Such peptidomimetic compounds can be characterized as “structural or functional analogs” of the peptides of the invention.

  For the purposes of the present invention, the term “targeting unit” also selectively targets and binds to tumors, and preferably also the tumor stroma, tumor parenchyma, and / or tumor extracellular matrix. It represents a compound, a peptide that can. Another term used in the art for this specific binding is “host”. Tumor targeting means that the targeting unit specifically binds to the tumor when administered to the human or animal body. More specifically, the targeting unit can bind to a specific molecule or structure on the cell surface, on or within the cell, or the targeting unit can bind to an extracellular matrix that exists between the cells. Can do. The targeting unit can also bind to the endothelial cells or extracellular matrix of the tumor vasculature. Targeting units can also bind to metastatic tumor masses, tumor cells and extracellular matrix.

  In general, the term "targeting" or "binding" refers to, for example, in vivo or ex vivo, in vitro tumor biopsy peptide competition experiments, or by in situ immunostaining, or other known to those skilled in the art. The method represents the adhesion, attachment, affinity or binding of the targeting unit of the present invention to a tumor, tumor cell and / or tumor tissue to the extent that binding can be objectively measured and determined. The exact mechanism of binding of the targeting unit of the present invention is not known. A target peptide of the invention is sufficiently strong to withstand normal sample processing, such as washing and rinsing with physiological saline or other physiologically acceptable salt or buffer solution at physiological pH, or An effector unit is considered “bound” to an in vitro tumor target when it binds to tumor targeting in vivo long enough to exhibit its function on the tumor.

  The binding of the targeting agent or targeting unit of the present invention to the tumor is “selective” and they do not bind to normal cells and organs, or bind to a very low extent compared to tumor cells and organs. Means.

  Pharmaceutically and diagnostically acceptable salts of the targeting units and targeting agents of the present invention include salts, esters, amides, hydrazides, N-substituted amides, N-substituted hydrazides, hydroxamic acid derivatives, their decarboxylations and There are N-substituted derivatives. Suitable pharmaceutically acceptable salts are readily understood by those skilled in the art.

The targeting motif of the present invention 3 amino acid motif Dd-Ee-Ff,
Wherein Dd-Ee-Ff is Aa-Bb-Cc or Cc-Bb-Aa and Aa is isoleucine, leucine or tert-leucine, or a structural or functional analog thereof;
A 3 amino acid motif Dd-Ee-Ff, wherein Bb is arginine, homoarginine or canavanine, or a structural analog or functional analog thereof; and Cc is serine or homoserine, or a structural analog or functional analog thereof, It has now been surprisingly found to target and exhibit selective binding to cancer and cancer cells and cancer cells.

  Aa of the present invention has in its side chain at least two same or different atoms selected from the group consisting of carbon, silicon, halogen bonded to carbon, ether-oxygen and thioether-sulfur, Unbranched or alicyclic structures can be included. The analogs are branched, unbranched or cyclic non-aromatic, lipophilic and hydrophobic having one or more lipophilic carborane-type or other lipophilic boron-containing side chains, or other lipophilic cage structures. Amino acid or amino acid analog or derivative, or a structural analog and / or functional analog thereof; amino acid or carboxylic acid, or amino acid analog or derivative, or carboxylic acid analog or derivative.

Aa can be selected from the group consisting of:
1) α-amino acid whose side chain is one of the following:
-Ethyl-propyl- 1-methylpropyl (side chain of isoleucine)
-2-methylpropyl (Leucine side chain)
-2,2-dimethylpropyl-1-ethylpropyl-tert-butyl-tert-pentyl-3-methylbutyl-2-methylbutyl-methylbutyl-ethylbutyl-2-ethylbutyl-cyclohexyl-2-methylcyclohexyl-cyclopentyl-2-methylcyclopentyl -3-methylcyclohexyl-cyclobutyl-cyclopropyl-2-methylcyclopropyl-methoxyethyl-methoxyethyl-methoxymethyl-ethoxymethyl-2-ethoxyethyl-1-ethoxyethyl-2-methoxypropyl-2,2-dimethoxypropyl -1-methylpropyl-1-methylbutyl-1-methylpentyl-1,1-dimethylpropyl-1,1-dimethylbutyl-1,1-dimethylpentyl-1 2-Dimethylpropyl-1-cyclopropylethyl-2-cyclopropylethyl-cyclopropylmethyl-1-cyclopropylethyl-1-cyclopropylpropyl-2-cyclopropylpropyl-3-cyclopropylpropyl-any cyclobutylalkyl -1-ethylpropyl-1-methylethyl-other mono-, di-, tri- or oligoalkyl-alkyl-other cyclic alkyl, or substituted cyclic alkyl, or one or more substituted or unsubstituted cycloalkyl groups And optionally substituted with one or more alkyl groups alkyl-allyl-vinyl-1-methylallyl-1-ethylallyl-1-ethylvinyl-1-propenyl-1-methyl-1-propenyl-methyl-1- Propenyl-methyl-1-pro Nyl-1-ethyl-1-propenyl-ethyl-1-propenyl-ethyl-1-propenyl-1-methyl-1-butenyl-methyl-1-butenyl-methyl-1-butenyl-1-ethyl-1-butenyl 2-ethyl-1-butenyl-ethyl-2-butenyl-ethyl-2-butenyl-ethyl-3-butenyl-ethyl-3-butenyl-ethyl-3-butenyl 2) any of the following carboxylic acids, any of which Including optical isomers:
-4-methylpentanoic acid-3-methylpentanoic acid-4,4-dimethylpentanoic acid-3,4-dimethylpentanoic acid-3,3-dimethylpentanoic acid-3-methylhexanoic acid-4-methylhexanoic acid-5 -Methylhexanoic acid-2-ethylpentanoic acid-3-ethylpentanoic acid-4-ethylpentanoic acid-2-cyclopropylpentanoic acid-3-cyclopropylpentanoic acid-4-cyclopropylpentanoic acid-2-methylbutanoic acid-3 -Methylbutanoic acid-4-methylbutanoic acid-2-cyclopropylbutanoic acid-3-cyclopropylbutanoic acid-4-cyclopropylbutanoic acid 3) optional optical and geometric isomers of any of the following compounds:
-2-amino-4-methyl-3-pentenoic acid-2-amino-4-methyl-4-pentenoic acid-2-amino-5-methyl-3-hexenoic acid-2-amino-5-methyl-4- Hexenoic acid-2-amino-5-methyl-5-hexenoic acid and 4)-one methyl, ethyl, propyl, isopropyl, or other alkyl group-one cycloalkyl group-one 9-fluore Nylmethyloxycarbonyl (FMOC) group-one benzyloxycarbonyl (Cbz) group-one tert-butoxycarbonyl (BOC) group-two selected from the aforementioned groups at this position (position 4) Amino-substituted (N-substituted) analogs of amino-containing compounds at positions 1 and 3 having the same, similar and / or different groups in the amino group.

Aa is an α-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 in the previous formula is the side chain R ² is hydrogen, methyl, ethyl, is selected from the group consisting of propyl, it may be α- amino acids.

  Bb of the present invention is an amino acid comprising one or more guanyl groups, aminozino groups or analogs and derivatives thereof, and structural or functional equivalents; one or more groups each containing at least two nitrogen atoms Alternatively, it can be selected from the group consisting of its structural analogs or functional analogs and has or can have a delocalized positive charge.

上式でＲ１〜Ｒ５が水素又はメチルであり、Ｒ２及びＲ３が−ＣＨ２−ＣＨ２−を形成することができ、且つｎが１〜６である化合物の群から選択することができる。 Bb is a compound of the following formula:

In the above formula, R1 to R5 are hydrogen or methyl, R2 and R3 can form -CH2-CH2-, and n can be selected from the group of compounds of 1-6.

Bb is preferably in the following L- or D-form.
Arginine,
Homoarginine,
Canavanine,
2-amino-8-guanidino-octanoic acid,
2-amino-7-guanidino-octanoic acid,
2-amino-6-guanidino-octanoic acid,
2-amino-5-guanidino-octanoic acid,
2-amino-7-guanidino-heptanoic acid,
2-amino-6-guanidino-heptanoic acid,
2-amino-5-guanidino-heptanoic acid,
2-amino-4-guanidino-heptanoic acid,
2-amino-5-guanidino-hexanoic acid,
2-amino-4-guanidino-hexanoic acid,
2-amino-3-guanidino-hexanoic acid,
2-amino-4-guanidino-pentanoic acid,
2-Amino-3-guanidino-pentanoic acid.

  Cc of the present invention includes amino acids, amino alcohols, diamino alcohols, tri-, oligos and polyaminos containing one or more hydroxyl groups, esterified hydroxyl groups, methoxyl groups and / or other esterified hydroxyl (ether) groups. It can be selected from the group consisting of alcohols and amino acid analogs, derivatives and structural or functional analogs thereof.

Cc as defined above may be serine or homoserine, or a structural or functional analog thereof containing at least one hydroxyl group; or any other mono-containing containing at least one alcohol hydroxyl group. Aminocarboxylic acid Any carboxylic acid containing at least one alcohol hydroxyl group Any containing aliphatic or other side chains containing one or more alcohol hydroxyl (OH) functional groups and / or esterified hydroxyl functional groups It can be selected from the group consisting of other aminocarboxylic acids.

Cc is preferably in the following L- or D-form.
Serine,
Homoserine,
2-amino-7-hydroxyheptanoic acid,
2-amino-5-hydroxypentanoic acid,
2-amino-6-hydroxyhexanoic acid,
2-amino-8-hydroxyoctanoic acid,
Or any other hydroxy-2-aminocarboxylic acid.

  Alternatively, the motif Aa-Bb-Cc of the present invention is generally a structural or functional analog of the structure where Aa, Bb and Cc are as defined above.

  Preferred embodiments of the present invention comprise the tumor targeting motif Aa-Bb-Cc, selected from those given in Table 1, and their structural and functional analogs.

  Thus, typical and preferred properties of Aa include lipophilicity, hydrophobicity and aliphaticity in at least one side chain, while Bb contains a delocalized positive charge and Cc is an OH-bond and Has the ability to get involved.

  Particularly preferred motifs Dd-Ee-Ff of the present invention are leucine-arginine-serine (LRS) and serine-arginine-leucine (SRL).

  The motif Dd-Ee-Ff of the present invention can form part of a larger structure, such as a peptide or some other structure. When the compound or structure in question contains more than one motif Dd-Ee-Ff, the orientation and orientation of the motif may change.

Targeting Units of the Invention Peptides and structural or functional analogs thereof comprising the tumor targeting motif of the invention have also been found to target and exhibit selective binding to tumor cells and tissues. Yes. Peptides containing the tumor targeting motif of the present invention and up to four optionally added amino acid residues, or analogs thereof, also exhibit such targeting and selective binding, This is a particularly preferred embodiment.

  Such peptides are very useful for use as targeting units in the present invention, for example because of their small size and their ease of synthesis, purification, analysis and quality control, more certainty and cheaper. Is advantageous.

  Particularly preferred targeting units of the present invention are peptides cyclized by amide bonds such as lactam bridges or by ester bonds such as lactone bridges.

  Such circular targeting units have now been shown to selectively target tumors in vivo.

A preferred tumor targeting unit of the present invention comprises a tumor targeting motif Dd-Ee-Ff as defined above and an additional residue selected from the group consisting of:
Natural amino acids,
Unnatural amino acids,
Amino acid analogs containing up to 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 up to 270,
And structural units and residues wherein the number of said additional residues is 0-4, preferably 0-3, more preferably 0-2, most preferably 0 or 2.

  As is known in the art, cyclic peptides are usually more stable in vivo and many other biological systems than their acyclic counterparts. However, it has now surprisingly been found that the targeting properties of the small peptides of the invention are even more pronounced when the targeting unit is cyclic or included in a cyclic structure.

上式で、Ｄｄ−Ｅｅ−Ｆｆが上記の本発明の腫瘍標的化モチーフであり；
Ｒｒが任意のアミノ酸残基であり；
ｎ及びｍが０〜４、好ましくは０〜３、より好ましくは０〜２の整数であり、ｎとｍの合計は４を超えず；且つ、
Ｃｙ及びＣｙｙが、ラクタム環によって環状構造を形成することができる実体である、構造を含むことができる。 Preferred targeting units of the invention are
Construction

Where Dd-Ee-Ff is the above-described tumor targeting motif of the present invention;
Rr is any amino acid residue;
n and m are integers of 0-4, preferably 0-3, more preferably 0-2, the sum of n and m does not exceed 4, and
Cy and Cyy can include structures that are entities capable of forming a cyclic structure with a lactam ring.

  Lactams are “head to tail” (carboxy terminus and amino terminus), “head to side chain” and “side chain to head” (carboxy or amino terminus, and one side chain amino or carboxyl group), And "subchain to sidechain" (one sidechain amino group, and the other sidechain carboxyl group).

  Preferred targeting units are targeting units in which Rr is any amino acid residue other than histidine, lysine or tryptophan. Particularly preferred are targeting units in which Rr is R or G.

Accordingly, a preferred structure is a compound of the general formula:
_{Cy-Rr n -Dd-Ee-} Ff-Rr m -Cyy
Wherein Cy and Cyy form a lactam bond, such as aspartic acid (D), glutamic acid (E), lysine (K), ornithine (O) or analogs thereof containing no more than 12 carbon atoms It is a compound that is a residue that can.

Particularly preferred targeting units of the invention having a cyclic structure with 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), DLRSGRRGK (SEQ ID NO: 7), OLRSE (SEQ ID NO: 8), OLRSGRGE (SEQ ID NO: 9) and KLRSD (SEQ ID NO: 10),
And their salts, esters, derivatives and analogs as defined above.

Targeting Agents of the Invention A targeting agent comprising at least one tumor targeting unit of the invention and at least one effector unit targets and selectively binds cancer cells and tissues and endothelial cells. It has also been found to show.

  The tumor targeting agent of the present invention can be a linking group, solubility modifier, stabilizer, charge regulator, spacer, dissolution or reaction or reactivity modifier, intrinsic unit or intrinsic enhancer, or membrane interaction unit, or other topical Units such as pathways, attachments, bonds and distribution units may optionally be included. Such additional units of the tumor targeting agents of the present invention can be linked together by any means suitable for that purpose.

上式でＥＵは「エフェクター単位」を示し、ＴＵは「標的化単位」を示し、ｎ、ｍ及びｋは独立に、０以外の任意の整数である。 Many possibilities are known to those skilled in the art for linking structures of interest or related types, molecules, groups, etc. to one another. The various units can be linked directly or linked by one or more identical, similar and / or different linking units. The tumor targeting agents of the present invention can have different structures, such as any of the non-limiting types of structures schematically shown below:
1. EU-TU
2. (EU) _n- (TU) _m
3. (EU) _n − (TU) _m − (EU) _k

In the above formula, EU represents “effector unit”, TU represents “targeting unit”, and n, m, and k are independently any integer other than 0.

  In the targeting agent of the present invention, as in many other pharmaceuticals and other substances, a long chain omega amino acid such as a spacer or a linking group, an amino acid and an analog thereof, and the like are included. May be wise to prevent it from being sterically and electrically “disturbed” or otherwise obstructed or “hidden” by other units of the effector unit or targeting agent. is there.

  In the targeting agents of the present invention, it may be useful for high activity to use dendritic or cyclic structures to give the possibility of incorporating multiple effector units or additional units per targeting unit. There is.

A preferred targeting agent of the present invention has the following structure:
Ef-TU-Eff
Where TU is the targeting unit of the present invention as defined above;
And Ef and Eff are
Effector unit, linking unit, solubility regulator unit, stabilizer unit, charge regulator unit, spacer unit, dissolution and / or reaction and / or reactivity regulator unit, intrinsic and / or intrinsic enhancer and / or membrane interaction unit And / or other local pathways and / or local attachment / local binding and / or distribution action units, adsorption promoting units, and other related units; and peptide sequences and other structures comprising at least one such unit; And no more than 20, preferably no more than 12, more preferably no more than 6, natural and / or non-natural amino acids; and natural containing no more than 25 non-hydrogen atoms and an unlimited number of hydrogen atoms And a peptide sequence comprising unnatural amino acids;
And a structure selected from the group consisting of these salts, esters, derivatives and analogs.

Effector Unit For the purposes of the present invention, the term “effector unit” means a molecule or radical or other chemical, as well as large particles such as colloidal particles; liposomes or microgranules. Suitable effector units may also form chemical structures, such as nanodevices or nanochips, or any combination thereof, and in some cases, to couple each or a portion of the effector unit components and the targeting agent. it can. The effector unit can also include components that affect the stabilization or increased solubility of the effector unit.

  Preferred effects provided by the effector units of the present invention include therapeutic (biological, chemical or physical) effects on the target tumor; properties that allow detection or imaging of tumors or tumor cells for diagnostic purposes; or different Binding capacity related to the use of targeting agents in the application.

  A preferred (biological) activity of the effector unit of the present invention is a therapeutic effect. Examples of such therapeutic activities include, for example, cytotoxicity, cytostatic effects, the ability to cause cell differentiation, or the ability to increase the degree of differentiation, or the ability to cause phenotypic or metabolic changes, run Activity, immunomodulatory activity, pain remission activity, radioactivity, ability to affect the cell cycle, ability to induce apoptosis, hormone activity, enzyme activity, ability to transfect cells, gene transfer activity, one or more Ability to mediate “knock-out” of genes, ability to cause gene substitution or “knock-in”, anti-angiogenic activity, ability to collect heat or other energy from external radiation or electric or magnetic fields, genetic information of cells or external Ability to affect transcription, translation or replication of relevant information; and ability to affect post-transcriptional and / or post-translational events.

  Other preferred therapeutic approaches made possible by the effector units of the present invention include the use of thermal (slow) neutrons (to make the nuclear radioactivity appropriate by neutron capture), or hydrolyzing ester bonds or other bonds, for example. It may be based on administration of an enzyme capable of degrading or administration of a targeted enzyme of the invention.

  Examples of preferred functions of the effector units of the present invention suitable for detection are radioactivity, paramagnetic, ferromagnetic, weak magnetic, ferrimagnetic, or any type of magnetic, or the ability to be detected by NMR spectroscopy Or the ability to be detected by EPR (ESR) spectroscopy, the suitability of PET and / or SPECT images, or the presence of immunogenic structures, or the presence of antibodies or antibody fragments or antibody-type structures, or the presence of gold particles, or biotin Or the presence of avidin or other proteins, and / or luminescence and / or fluorescence and / or phosphorescence activity, or electron microscope, optical microscope (UV and / or visible light), infrared microscope, atomic force microscope or tunneling microscope, etc. The ability to enhance the detection of tumors, tumor cells, endothelial cells and metastases.

Preferred binding capacities of the effector units of the present invention include, for example:
a) the ability to bind to substances or structures such as histidine or other tags;
b) the ability to bind biotin or an analogue thereof,
c) the ability to bind to avidin or an analog thereof;
d) the ability to bind to an enzyme or a modified enzyme;
e) the ability to bind metal ions, for example by chelation,
f) the ability to bind to substances that affect cytotoxicity, apoptosis or metabolism, or substances that can be converted into such substances in situ;
g) the ability to bind integrins and other substances associated with cell adhesion, migration, or intracellular signals;
h) ability to bind to phage,
i) the ability to bind lymphocytes or other blood cells;
j) the ability to bind to any preselected substance by the presence of an antibody or structure selected by biopanning;
k) the ability to bind to a substance used for signal generation or amplification;
l) Ability to bind therapeutic agents.

  Such binding can be, for example, chelation, covalent bond formation, antibody-antigen type affinity, ion pair or ionic bond formation, specific interaction of the avidin-biotin type, or any type or form of binding or affinity. May be the result of sex.

  The one or more effector units or parts thereof may be part of the targeting unit itself. Thus, the effector unit is a target, such as a radioactive atom, or an atom that can become a radioactive atom, or a paramagnetic atom, or an atom (such as carbon-13) that is easily detected by MRI or NMR spectroscopy. It may be one or more atoms or nuclei of the chemical unit. Other examples are boron-containing structures such as carborane-type lipophilic side chains.

  The effector unit can be used for most, preferably all, or almost all of the targeting agent during an essential (required) targeting process, eg in a human or animal subject or biological sample under test or treatment. The effector unit can be linked to the targeting unit by any type of bond or structure, or any combination thereof, that is strong enough to remain linked to the targeting unit.

  The effector unit or part thereof may be in association with the targeting unit, or they may be partially or fully hydrolyzed, or otherwise by spontaneous chemical reaction or equilibrium. Or may be degraded from the targeting unit by spontaneous enzymatic processes or other biological processes, or as a result of intentional manipulations or procedures, such as administration of hydrolytic enzymes or other chemicals. It is also possible to cause or increase enzymatic processes or other reactions by administering a targeting agent such as an enzyme according to the present invention.

  One possibility is that the effector unit or part thereof is influenced by the influence of one or more different hydrolases present in or near the tumor (eg in the cell, in the cell membrane or in the extracellular matrix). Hydrolysis from targeting agents and / or hydrolysis to smaller units.

  Given that the targeting of the present invention can be very rapid, even non-specific hydrolysis that occurs throughout the body intentionally hydrolyzes one or more effector units May be acceptable and usable. Because such hydrolysis is very slow when appropriate (e.g., without steric hindrance, or any such hindering effect), as well understood by those skilled in the art, and therefore the body's hydrolysis. This is because the target substance is safely targeted despite the presence of the degrading enzyme. The formation of insoluble products and / or products that are rapidly absorbed into cells and / or products that bind to the cell surface after hydrolysis may result in targeted effector units and / or fragments thereof in or near the tumor. It may be useful to stay.

  In one preferred embodiment of the invention, the effector unit allows for “amplification” of therapeutic or other effects, signal detection, binding of preselected substances, including biological substances, molecules, ions, microorganisms or cells. It can include structures, features, fragments, molecules, etc. that directly or indirectly cause.

Such “amplification” may be based, for example, on one or more of the following non-limiting types:
Binding of other substances that can further bind to other substances by effector units (eg antibodies, fluorescent antibodies, other “label” substances, substances such as avidin, and preferably several molecules of other substances) Or "units" can be bound per each effector unit;
An effector unit comprising two or more substances capable of binding eg a protein, which allows direct amplification;
Amplification in two or more steps.

Preferred effector units of the present invention can be selected from the following group:
-Cytostatics or cytotoxic substances-Substances that cause or increase apoptosis-Enzymes or enzyme inhibitors-Antimetabolites-Substances that can impair membrane function-Radioactive substances or paramagnetic substances-One or Substances containing multiple metal ions-Substances containing boron, gadolinium and lithium-Substances suitable for neutron capture therapy-Labeling substances-Intercalators and substances containing them-Oxidizing and reducing agents-Nucleotides and their analogs-Metal chelates Or chelating agent.

  In a highly preferred embodiment of the invention, the effector unit comprises an alpha emitter.

  In other preferred embodiments of the present invention, the effector unit may comprise a copper chelate such as trans-bis (salisilaldoximalo) copper (II) and its analogs, or a platinum compound such as cisplatin, carboplatin. .

  Different types of structures, substances that can be used to cause or increase internalization into cells, including, for example, RQKIWFQNRRMKWKK; Penetratin (Prochiantz, 1996) and stearyl derivatives (Promega Notes Magazine, 2000) , And groups are known.

  Apoptosis-inducing structures can include, for example, the peptide sequence KLAKLAK that interacts with intracellular mitochondrial membranes. Ellerby et al. (1999).

For use in embodiments of the present invention, including cell sorting and any related applications, using the targeting units and substances of the present invention, for example,
a) coupled to or associated with magnetic particles;
b) adsorb, bind, couple or tie with plastic, glass or other solid, porous, fibrous material types or other substances, etc.
c) adsorbed, covalently bound, or otherwise linked, bound or associated in or on one or more substances or materials that can be used in the column and related systems;
d) adsorbed, covalently bound or otherwise linked in or onto one or more substances or materials that can be precipitated, centrifuged, or otherwise separated or removed; Can be combined or tied together.

Optional Units of the Targeting Agents of the Invention The targeting agents and targeting units of the invention can optionally include other units, such as the following units:
A linking unit linked to each other to a targeting unit, an effector unit, or other optional unit of the invention;
A solubility modifier unit that adjusts the solubility of the targeting agent or its hydrolysis product;
a stabilizer unit that stabilizes the targeting unit or the structure of the substance during synthesis, modulation, processing, storage or use in vivo or in vitro;
A charge control unit that modulates the electrical charge of the targeting unit or substance or their starting materials;
Spacer units to increase the distance between specific units of the targeting agents or their starting materials to reduce or reduce steric hindrance or structural burden of the product;
Reactivity modifier unit;
An endogenous unit or enhancer unit to increase targeting and uptage of the targeting agent;
Adsorption promoting units such as lipids or water soluble structures that facilitate the absorption of targeting agents in vivo; or other related units.

A number of suitable linking units are known in the art. Examples of suitable linking groups are:
1. Linking group for linking units containing amino groups: cyclic anhydride, dicarboxy or polyvalent, optionally activated or derivatized, carboxylic acid, compound having two or more reactive halogens, or at least one A compound having a reactive halogen atom and at least one carboxyl group;
2. Linking group for linking units containing carboxyl groups or derivatives thereof: at least two identical, such as amino, substituted amino, hydroxyl, —NHNH ₂ or substituted forms thereof, other known groups for this purpose, etc. Compounds with groups or different groups (activators can be used);
3. A linking group for linking an amino group and a carboxyl group: for example, amino acids and their active or protected forms or derivatives;
4). Formyl or keto group and other groups concatenated to a linking group for: example, compounds including at least one -N-NH ₂ or -O-NH ₂ or = N-NH _2;
5. Linking groups for linking several amino-containing units: polycarboxylic materials such as EDTA, DTPA and polycarboxylic acids, anhydrides, halides, and acyl halides;
6). A linking group for linking a substance containing an amino group and a substance containing a formyl group or a carboxyl group: hydrazino carboxylic acid, etc., preferably hydrazino carboxylic acid having a protected or activated hydrazino moiety or carboxyl group For example, 4- (FMOC-hydrazino) benzoic acid;
7). Linking group for linking the organic structure to the metal ion: can be bonded to the organic structure (eg, by their COOH group or their NH ₂ group) or is an essential part of the organic structure, a polycarboxyl moiety, For example, to link a substance further comprising an EDTA- or DTPA-like structure, a peptide comprising several histidines, etc., a peptide comprising several other components each containing a cysteine or -SH group, and an organic structure therewith Other chelating agents containing functional groups that can be used.

  A wide variety of the aforementioned materials, and other types of suitable linking materials, are known in the art.

A number of suitable solubility modifier units are known in the art. Suitable solubility modifier units include, for example:
To increase aqueous solubility: SO ₃ ⁻ , O—SO ₃ ⁻ , COOH, COO ⁻ , NH ₂ , NH ₃ ⁺ , OH groups, guanidino or amidino groups, or other ion or ionizable groups, and glycoform structures A molecule comprising:
To increase lipid solubility or solubility in organic solvents: (long) units comprising cyclic non-aromatic groups such as aliphatic branched or unbranched alkyl and alkenyl groups, cyclohexyl groups, aromatic rings and steric structures.

  A number of units known in the art can be used as stabilizer units. Bulk structures (eg tert-butyl groups, naphthyl and adamantyl and related groups), for example to increase steric hindrance, and D-amino acids and other unnatural amino acids (for example to prevent or prevent enzymatic hydrolysis) β-amino acids, ω-amino acids, amino acids having very large side chains, etc.).

  Units containing positive charge, negative charge, or both types of charge can be used as charge control unit units, or converted to units having positive charge, negative charge, or both types of charge. Structures that can be used can also be used.

  Spacer units can be very important, and the need to use such units depends on other components of the structure (eg, the type of biologically active substance used and their effects) Mechanism) and the synthesis procedure used.

  Suitable spacer units can 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 is a omega-amino acid with a long chain. Such compounds can also be used (simultaneously) as linking units between amino-containing units and carboxyl-containing units. Many such compounds are commercially available in this manner and in the form of various protected derivatives.

  Units susceptible to hydrolysis (spontaneous chemical hydrolysis or enzymatic hydrolysis by the body's own enzymes or enzymes administered to the patient) are, for example, internalization, intracellular or extracellular DNA or receptors It can be very advantageous if it is desired to separate the effector unit from the targeting agent for binding. Suitable units for this purpose include, for example, structures containing one or more ester or acetal functional groups, and various proteases can be used for reference purposes. Many groups used to make prodrugs may be suitable for purposes of increasing or causing hydrolysis, dissolution reactions, or other degradation processes.

  The effector unit, targeting unit, and optional unit of the present invention can perform more than one function simultaneously. Thus, for example, a targeting unit can be an effector unit at the same time or can include several effector units; a spacer unit can be a linking unit or a charge control unit unit, or both at the same time, The stabilizer unit can be an effector unit that has different properties than other effector units and the like. The effector unit can have, for example, several identical functions or even completely different functions.

  In one preferred embodiment of the invention, the tumor targeting agent comprises two or more different effector units. In that case, the effector units may be, for example, diagnostic and therapeutic units. Thus, for example, for boron neutron capture therapy, a substance is used that can be detected or quantified in vivo in the patient after administration of the substance, in addition to the boron atom containing the effector unit. It is preferable to be able to confirm proper accumulation in the tumor to be treated or to optimize the timing of neutron treatment. This goal can be achieved, for example, by using the targeting agents of the invention, such as those containing an effector unit containing a boron atom (preferably an isotope rich boron), and a group detectable by eg NMRI. Similarly, the presence of more than one type of therapeutically useful effector unit may be preferred. In addition, the targeting unit and targeting agent may comprise one or more “classical” or other tumor therapeutic agents, for example, surgery, chemotherapy, other targeted therapeutic agents, radiotherapy agents, if desired. It can be used in combination with an immunotherapeutic agent and the like.

Preparation of targeting unit and substance of the present invention The targeting unit of the present invention is preferably a synthetic peptide. Peptides can be synthesized in a wide 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 It can be synthesized by a combination of these. Even the automated equipment / devices of interest are commercially available, as are normal synthesis and purification services. All of these techniques are very well known to those skilled in the art. Some methods and materials are described, for example, in the following references.

  Bachem AG, SASRIN ™ (1999), The BACHEM Practice of SPPS (2000), Bachem 2001 catalog (2001), Novabiochem 2000 catalog (2000), “Synthesis of peptides and peptidomimetics (Peptide and Peptidomimetics”) , And “The Combinatorial Chemistry & Solid Phase Organic Chemistry (SPOC) Handbook” 98/99, “Combinatorial Chemistry Catalog and Solid State Organic Chemistry (SPOC) Handbook”. Peptide synthesis is also illustrated in the examples.

  As is known in the art, one or more protecting groups, most of which are known in the art, such as FMOC, BOC, and trityl groups, as well as other protecting groups described in the Examples, etc. Often it is sensible, important and / or necessary to use. Protecting groups are often used to protect amino, carboxyl, hydroxyl, guanyl and -SH groups, and any reactive groups / functional groups.

  As is well known to those skilled in the art, activation is often related to activation of carboxyl functional groups and / or activation of amino groups.

  The protection may be ortho and / or semi / quasi / quasi-ortho. Protecting and active groups, materials, and their use are exemplified in the examples and described in the references cited herein, and are numerous books and others commonly known in the art. (For example, “Protective Groups in Organic Synthesis”, 1999).

  Solid phase synthesis resins are also well known in the art and are described in the Examples and in the references cited above.

  The cyclic structures of the present invention can be synthesized by methods, for example based on the use of amino acids protected at the ortho position. Thus, for example, one amino acid containing an “excess” COOH functional group protected at the ortho position (eg, (N-(-FMOC-L-glutamic acid-allyl ester, ie, “FMOC-Glu-Oall”, Or (N-(-FMOC-L-glutamic acid -tert-butyl ester ("FMOC-Glu-OtBu", or N-(-FMOC-L-glutamic acid (-4 {N- [1- (4,4 -Dimethyl-2,6-dioxocyclohexylidene) -3-methylbutyl] -amino} benzyl ester ("FMOC-Glu-Odmab") or (N-(-FMOC-L-glutamic acid-2-phenylisopropyl) Esters ("FMOC-Glu (O-2-PhiPr) -OH") or other dicarboxyamino acids such as aspartic acid Related derivatives; or any of the aforementioned resin-bound forms) and one amino acid (eg, N-(-FMOC-N-(-4-methyltrityl) having an “excess” amino group protected at the ortho position. -L-lysine ("FMOC-Lys (Mtt) -OH"), or the corresponding derivative of ornithine or some other dicarboxylic acid, or one of these resin-bound forms; resin-bound form, but "excess" COOH A resin-bound form of an amino acid protected at the ortho position with a resin bond form that does not exist at the same time) can be incorporated into the structure, and after deprotection, the carboxyl group and amino group are usually activated. This type of method is well known and is described, for example, in the following reference, Novabiochem catalog (2000), pp. 19-21 and 33, and in particular B9-B15, and references therein, Bachem 2001 catalog (2001), pp. 31-32, Chan et al. (1995), Yue et al. (1993) and Hirschmann et al. (1998). It is described in.

  Suitable starting materials are commercially available and other starting materials can be made by methods known in the art. D-amino acid derivatives can also be used in this method. As those skilled in the art understand, quasi-ortho / semi-ortho / pseudo-ortho protecting groups can also be used in place of groups protected at the “true” ortho position.

Cyclic products made according to the methods described above are usually very stable in biological environments and are therefore preferred. This type of structure can be generated by any method (chemical, enzymatic or biological) to generate such a structure. Many such methods are well known to those skilled in the art. This type of cyclic structure can be synthesized chemically by solid phase synthesis, but can also be synthesized using solution methods or a combination of both, as is well known to those skilled in the art. it can. Amino acids with “excess” carboxyl or amino functionality suitable for cyclization purposes (when appropriately protected) include (as non-limiting candidates), for example, those having the structure shown below:

  As is well known by those skilled in the art, dilute solutions are usually advantageous for cyclization in any type of solution.

  The targeting units and targeting agents of the present invention can also be prepared as fusion proteins or by other suitable recombinant DNA methods known in the art. Such an approach for preparing the peptides of the present invention is particularly preferred when the effector unit and / or other optional units are peptides or proteins. One example of a useful protein effector unit is glutathione-S-transferase (GST).

Advantages of targeting units and targeting agents of the present invention There are several recognized problems with peptides for diagnostic or therapeutic use. One of these problems stems from the length of the sequence: there are other synthetic problems such as the presence of troublesome residues that require protection-deprotection and / or cause side reactions etc. In particular, the longer the sequence, the more difficult or even impossible to synthesize the desired product. Side reactions, as well as possible termination of the synthesis (not only completely reducing the yield of the desired product when it is formed, but also resulting in a product with an inappropriate length of peptide chain), and a large amount The tendency for the formation of harmful by-products depends on side chain protection (eg, basic side chains such as lysine, histidine and tryptophan side chains) and (naturally) the desired sequence of any amino acid that requires deprotection. Increases significantly in presence. Also, as is well known to those skilled in the art, all of these problems can make it more difficult to purify the desired peptide, making it impossible to produce properly purified material.

  Compared to known products that have problematic amino acid residues and contain sequences that are long and difficult to make, the peptides of the invention are clearly superior, as described in more detail below. ing.

  Accordingly, the products and methods of the present invention, and their use, provide very significant and very important advantages over the prior art.

  The targeting unit of the present invention can be easily and reliably synthesized. One advantage compared to many prior art peptides is that the targeting units and motifs of the present invention need not include the problematic basic amino acids lysine and histidine, or tryptophan, both of which are It can cause significant side reactions in peptide synthesis, which can greatly reduce the yield of the desired product, or make it impossible to obtain the proper quantity or quality there is a possibility.

  When present, histidine, lysine and tryptophan must be properly protected using appropriate protecting groups that are intact during the synthetic procedure. This can be very difficult and at least increases cost and technology issues. In addition, reagents and workload can significantly increase costs, which can increase other costs of the deprotection step and the cost per unit of desired product.

  Due to their small size and thus significantly fewer steps in the synthesis, the peptides of the invention are much easier and cheaper to produce than prior art targeted peptides.

  Since histidine is not required in the products of the present invention, its risk of racemization is completely unrelated.

  It is a great advantage not to consider any racemization of histidine, not only for the economic synthesis of the products of the invention, but also for purification and analysis and quality control. This makes any administration to humans and animals safe and simpler.

  Because of its small size, the peptides of the invention can also be more reliably and easily purified with less effort and equipment-time, thus clearly reducing costs. The overall cost is thus greatly reduced and better products can be obtained in larger quantities. In addition, the reliability of purification is much better, and less toxic residues and lethal or otherwise serious side effects problems in therapeutic and diagnostic applications.

  Short synthetic protocols with relatively few steps generate small amounts of impurities, making the peptides of the invention very advantageous. Toxicity and even the risks of lethal impurities, allergens, etc. are dramatically reduced and further purification is facilitated.

  Analysis of the product of the present invention, and thus quality control, is easier and less expensive than that of long and more “troublesome” peptide sequences. This increases the reliability of analysis and quality control.

  Since residues such as lysine are not present in the targeting unit, there is no risk that the effector unit is inappropriately associated with such a residue. This is a significant advantage.

  The effector unit can be readily linked to the peptides and peptidyl analogs and peptidomimetics of the invention using, for example, protected lysine or ornithine (outside the targeting motif). This is because any lysine residue in the targeting motif is not at risk of reacting simultaneously.

  Protected lysine or ornithine can be used to cyclize the peptides of the present invention. This is because the targeting unit does not contain such amino acids. This is a huge advantage.

Lactam bridges are clearly superior to commonly used disulfide bridges. this is:
1. Lactams against reducing agents such as thiols that can easily break disulfide bridges and cause various undesirable reactions such as dimerization, polymerization, and formation of disulfide bridges with other thiols. Very stable;
2. Lactams can be synthesized in a well-controlled manner, for example by using protection at the ortho position;
3. Lactam is more stable than disulfide in the biological environment;
4). Purification of lactams is easier than disulfides;
5. Lactones are probably less prone to cause allergic reactions because they do not react with macromolecules having SH-groups;
For several reasons, such as:

  In the solid phase synthesis of the targeting agent of the present invention, the effector unit and optionally other units are targeted once they are still associated with the resin without the risk of removal of the protecting group causing destruction of the other unit. It can be linked to a peptide. Similar advantages apply to solution synthesis.

  Another important advantage of the present invention and the products of the present invention, the method and use according to the present invention is the targeting of highly selective and powerful products.

  Compared to targeted therapies using antibodies or antibody fragments, the products and methods of the invention are highly advantageous for several reasons. The potential immunological and related risks are also apparent for large biomolecules. In contrast to small synthetic molecules such as targeting agents, units and motifs of the present invention, allergic reactions are of great concern for such products.

  Compared to targeted antibodies or antibody fragments, the products and methods described in the present invention are highly advantageous. This is because their structures can be easily modified if needed or desired. Certain amino acids such as histidine, tryptophan, tyrosine, threonine can be omitted if desired and very few functional groups are required. On the other hand, a variety of different structural units can include certain desirable properties that are of great value in a particular application without compromising the targeting effect.

Use of the Targeting Agents of the Invention The targeting units and targeting agents of the invention are useful in cancer diagnosis and therapy. Because they selectively target tumors in vivo, as shown in the Examples. The effector unit can be selected according to the desired effect, detection or therapy. A desired effect can also be obtained by including an effector in a targeting unit or the like. For use in radiation therapy, the targeting unit itself can be, for example, radioactively labeled.

  The invention also relates to a diagnostic composition comprising an effective amount of at least one targeting agent of the invention. In addition to targeting agents, the diagnostic compositions of the present invention contain carriers, solvents, excipients, suspending agents, labeling substances, and other additives commonly used in diagnostic compositions. Can be included. Such diagnostic compositions are useful in diagnosing tumors, tumor cells and metastases.

The diagnostic composition of the present invention is an aqueous liquid comprising the targeting agent of the present invention as a liquid, gel or solid formulation, preferably in a concentration ranging from about 0.00001 μg / l to 25 × 10 ⁷ μg / l. Can be blended. The composition can further include stabilizers, surfactants such as polysorbate and Tween, and other additives. The concentration of these components can vary significantly depending on the formulation used. The diagnostic composition can be used in vivo or in vitro.

  The present invention also includes the use of targeting agents and targeting units to produce a pharmaceutical composition for the treatment of cancer.

  The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one targeting agent of the present invention. By using the pharmaceutical composition of the present invention to administer a therapeutically effective amount of a pharmaceutical composition comprising the targeting agent or targeting unit of the present invention, or a therapeutically acceptable salt, ester or other derivative thereof. Can treat, prevent or alleviate cancerous diseases. The compositions of the present invention can also include different combinations of targeting agents and targeting units, as well as labeling materials, imaging materials, agents and other additives.

  The therapeutically effective amount of the targeting agent of the present invention can vary depending on the formulation of the pharmaceutical composition. Preferably, the compositions of the present invention have concentrations varying from about 0.00001 μg / l to 250 g / l, more preferably from about 0.001 μg / l to 50 g / l, most preferably from 0.01 μg / l to 20 g / l. And can include a targeting agent.

  The pharmaceutical composition of the present invention is useful for administering the targeting agent of the present invention. Particularly preferred are pharmaceutical compositions suitable for oral use, intravenous or topical injection, or infusion. The pharmaceutical composition of the present invention can be used in vivo or ex vivo.

  The preparation can be lyophilized and reduced prior to administration, or as a solution, solution (s), suspension, suspension-solution, etc., ready for administration, or as a powder, concentrate, frozen It can be stored in any general shape or shape, including liquids and any other molds. The preparation may consist of separate entities that are mixed prior to use, possibly treated and / or otherwise treated. Liquid formulations offer the advantage that they can be administered without reduction. The pH of the solution product is in the range of about 1 to about 12, preferably around physiological pH. The osmolality of the solution can be adjusted to a preferred value using, for example, sodium chloride and / or saccharides, polyols and / or amino acids and / or similar components. The composition further comprises pharmaceutically acceptable excipients and / or stabilizers, such as albumin, sugars and various polyols, and any other active ingredient such as any acceptable additive or chemotherapeutic agent. be able to.

  The invention also relates to a method for treating cancer, in particular a solid tumor, by administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention.

  The therapeutic dose can be determined empirically by testing targeting agents and targeting units in available in vitro or in vivo test systems. Examples of such tests are given in the examples. Accordingly, an appropriate therapeutically effective dose can be estimated from these experiments.

  For oral administration, it is important that the targeting unit and the targeting agent are stable and properly absorbed from the intestinal tract.

  The pharmaceutical composition of the present invention may be selected from systemic, non-systemic, topical or local, parenteral and oral, for example, by subcutaneous, intravenous, intramuscular, oral, intranasal, pulmonary aerosol. It can be administered to the cheek, intracranial, or intraperitoneally by injection or infusion into the organ or area.

  The amount and formulation for administration of the tumor targeting agent of the present invention can be readily determined by one skilled in the clinical arts of cancer therapy. In general, the dose will depend on the type of targeting agent used; age; health condition; medical condition to be treated; type of combination therapy, if present, frequency of treatment, and nature of desired effect; gender; duration of symptoms; and If present, it will vary depending on considerations such as anti-indications and other variables adjusted by the individual physician. The preferred dose of administration of the targeting unit or targeting agent of the present invention to a human patient may vary from about 0.000001 μg to about 40 mg per kg body weight as a bolus or, for example, repeated as a daily dose.

  The targeting unit and targeting agent and pharmaceutical composition of the present invention comprise DNA or RNA, or a structural or functional analog thereof, such as phosphorothioate, or peptide nucleic acid (PNA), in a tumor and its metastasis, Alternatively, it can be used as a targeting device for delivery to isolated cells and organs in vitro, ie as a tool for both in vivo and in vitro gene therapy. In such cases, the targeting agent or targeting unit may be part of a viral capsid or envelope, liposome or other “container”, DNA / RNA or related substance, or DNA / RNA, or other such It may be bound directly to the molecule.

  The present invention also includes kits and kit components for diagnosing, detecting or analyzing cancer or cancer cells in vivo and in vitro. Such a kit comprises at least a targeting agent or targeting unit of the invention and a diagnostic entity that allows detection. The kit of the present invention comprises, for example, a targeting agent and / or a targeting unit coupled to a unit for detection by, for example, immunological methods, radiation or enzymatic methods, or other methods known in the art. Can be included.

  Furthermore, the targeting units and targeting agents, and targeting motifs and sequences of the present invention can be used as lead compounds to design peptidomimetics for any of the purposes described above.

  Still further, the targeting units and targeting agents of the present invention and targeting motifs and sequences, etc. and / or their binding to other substances may be combined into single cells, molecules, and related biological targets. Can be used for separation, purification and identification.

  The invention is further illustrated by the following non-limiting examples.

(Example)
The list of reagents used in the following examples, and the supplier of reagents, are included after the last numbered example.

Synthesis of Targeting Unit (Peptide) LRS A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS (Leucylarginylserine) was manually described in Example 2 below. Synthesized by synthesis.

The following reagents were used as starting materials (in this order):
Fmoc-Ser (tBu) resin Fmoc-L-Arg (Pbf) -OH
Fmoc-L-Leu-OH

  After the last cycle of the coupling process, a small sample of resin (including peptides that are still fully protected) is subjected to the treatment described in Example 2 for Fmoc removal (steps 1-10 of the same example). The peptide sample was then cleaved from the resin by treatment with the cleavage mixture described in Example 2 for 3 hours and isolated as described in the same Example.

The product (LRS) was then identified by its positive ion mode MALDI-TOF mass spectrum, in which it was clear that the M + 1 ion of LRS was dominant.
MALDI-TOF data (LRS):
Calculated molecular mass = 374.44
Observed signal:
375.30M + H
397.22M + Na

General procedure for peptide synthesis: manual solid-phase synthesis and mass spectrometric measurement All synthesis procedures consisted of a porous grade sintered glass filter disc between 2 and 4 and a screw kit made of polypropylene or phenolic plastic (sealed). And two PTFE key stopcocks: 1 stopcock under the filter disc (for discharge), 1 stopcock on the shoulder of the screw-kipped neck with an inclined angle (argon gas inlet) Carried out in a hermetic glass funnel.

  Fill the funnel with the appropriate solid phase synthetic resin and solution for each treatment and shake vigorously with the “Piston Pin Movement” bottle shaker (Gallenkamp) for the appropriate time, then filter with the appropriate argon gas pressure Went.

  The general procedure for one cycle of synthesis (= 1 amino acid unit addition) was as follows.

  About 1 mmol of Fmoc-peptide consisting of two or more amino acid units (= a peptide whose amino terminal amino group is protected with a 9-fluorenylmethyloxycarbonyl group), or about 1 mmol of a suitable Fmoc-amino acid (ie A suitable Wang resin (Applied Biosystems) charged with an amino acid having the above protecting group; about 2 g resin, 0.5 mmol / g) was treated in the manner as described below, each treatment step comprising: Shake for 2.5 minutes with 30 ml of the indicated solution or solvent, and filter unless otherwise stated.

  “DCM” means shaking with dichloromethane and “DMF” means shaking with N, N-dimethylformamide (DMF can be replaced by NMP, ie, N-methylpyrrolidinone).

The processing steps were as follows:
1. Shake DCM for 10-20 minutes. DMF
3. 5. 20% (volume) piperidine in DMF for 5 minutes 20% (volume) piperidine in DMF for 10 minutes 5-7. DMF
8-10. DCM
11. DMF
12 13. 3 mmol of active amino acid in DMF solution (prepared as described below), shake for 2 hours 13-15. DMF
16-18. DCM

  After the last treatment (18), argon gas was passed through the resin for about 15 minutes and the resin was stored under argon (in a sealed reaction funnel if the synthesis was continued with other units).

  Activation of 9-fluorenylmethyloxycarbonyl-N-protected amino acid (Fmoc-amino acid), which is added to amino acids or peptide chains on the resin, is processed in step no. In a separate container prior to 12, using the reagents listed below. In this way, Fmoc-amino acid (3 mmol) was dissolved in about 10 ml of DMF, treated with a solution of 3 mmol of HBTU in 6 ml of 0.5 M solution of HOBt in DMF for 1 minute, and then 2.0 M of 5 M for 5 minutes. Treated immediately with 3 ml of DIPEA solution.

The activation reagents used to activate the Fmoc-amino acids were as follows:
HBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphoric acid, CAS No. [94790-37-1], Applied Biosystems catalog number 401091, molecular weight: 379.3 g / mol
HOBt = 1-hydroxybenzotriazole, 0.5 M solution in DMF, Applied Biosystems catalog number 400934
DIPEA = N, N-diisopropylethylamine, 2.0 M solution in N-methylpyrrolidone, Applied Biosystems catalog number 401517

  The procedure described above was repeated several cycles using the appropriate different Fmoc-amino acids with appropriate protecting groups to generate the appropriate peptide (ie, “resin-bound” peptide) for the resin binding source. This procedure also provides a practical way of associating several effectors and / or spacers and / or linking units, such as biotin or Fmoc-Ahx (= 6- (Fmoc-amino) -hexanoyl) moieties, with resin-bound peptides. Given.

Cleavage from the resin was performed using the following reagent mixture:
Trifluoroacetic acid (TFA) 92.5 vol-%
Water 5.0 vol-%
Ethanedithiol 2.5 vol-%.

  After removal of the protected Fmoc group by steps 1-10 (similar to the general procedure described above), the resin was treated with each of the three component reagent mixtures (approximately 15 ml each for 1 g resin) for 1 hour each. These treatments were performed in an argon atmosphere by the method described above. The TFA solution obtained by filtration was then concentrated under reduced pressure using a rotary evaporator and refilled with argon. Some diethyl ether was added and the concentration was repeated. The concentrated residue was precipitated overnight under argon in diethyl ether in the refrigerator. The supernatant ether was removed and the precipitate was washed with diethyl ether. For mass spectral (MALDI-TOF +) measurements, the precipitated sample was dissolved in a solvent suitable for the spectroscopic method and then filtered, and the filtered solution was diluted as necessary. Further purification was performed using a reverse phase high performance liquid chromatography (HPLC) method with a “Waters 600” pump device, which included a particle size of 10 micrometers and a composition of 99.9% water / 30 in 30 minutes. A C-18 type column with a linear elution gradient changed from 0.1% TFA to 99.9% acetonitrile / 0.1% TFA was used. The dimensions of the HPLC column were 25 cm x 21.2 mm (Supelco catalog number 567212-U) and 15 cm x 10 mm (Supelco catalog number 567208-U). Detection was based on absorbance at 218 nm using a “Waters 2487” instrument.

  The cleavage mixture described above consists of the following protecting groups: Trityl (Trt) used for protecting cysteine-SH; 2,2,4,6,7-pentamethyl used for protecting the side chain of arginine Dihydrobenzofuran-5-sulfonyl (Pbf); tert-butyl group (ester group on carboxyl functional group; OtBu) used for protecting the side chain carboxyl group of glutamic acid and / or aspartic acid was also removed at the same time. It can also be used usually for the removal of these protecting groups on structures (thiol, guanyl, carboxyl). It did not cause Fmoc removal.

  The cleavage procedure described above can also be performed without removal of the Fmoc group to produce a peptide linked to the amino terminal N-Fmoc-derivative or effector unit (not including Fmoc) of the peptide.

Mass Spectrometry Used Matrix Assisted Laser Desorption / Ionization Method-Time of Flight (MALDI-TOF)
Instrument type: Bruker Biflex MALDI TOF mass spectrometer Instrument supplier: Bruker Daltonik GmbH, Fahrenheitstrasse 4, D-28359, Bremen, Germany

MALDI-TOF positive ion reflector mode External standard: Angiotensin II and ACTH (18-39)
Matrix: α-Cyano-4-hydroxycinnamic acid (saturated solution in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid).
The sample and matrix were dried on the target plate under a gentle warm air flow.

MALDI-TOF negative ion reflector mode:
External standard: cholecystokinin and glucagon Matrix: 2,4,6-trihydroxyacetophenone (3 mg / ml, 10 mM ammonium citrate in 50% acetonitrile).
The sample mixed with the matrix was immediately dried on the target plate under vacuum.

Sample Preparation Samples were mixed with the matrix solution described above at a concentration of 10-100 pmol / microliter.
“Shooting” with a nitrogen laser at a wavelength of 337 nm. The probe plate voltage was 19 kV in positive ion reflector mode and −19 kV in negative ion reflector mode.

General observations about the spectrum (only for positive ion mode)
In all cases, the M + 1 (ie, 1 protonated M + H +) signal, with its typical microstructure based on isotope association, was clearly dominant. In almost all cases, the pattern of M + 1 signal was accompanied by a similar but markedly weak band of the peak at M + 23 (Na + addition). In addition to the bands at M + 1 and M + 23, bands at M + 39 (K + addition) or M + 56 (Fe + addition) could often be observed.

  In the case of substances with a small molecular mass, the “matrix signal” (the signal due to the matrix / “ionization environment” component) was omitted (ie the signals at 294 and 380 Da were omitted).

  The calculated molecular mass values reported in the synthesis examples correspond to the most abundant isotope or “exact mass” of each component. The interpretation given for the signal is only tentative.

General Procedure for I ₂ -Promoted Cyclization of Peptides / Targeting Units or Targeting Agents on Resin (For Peptides Containing Cysteine and Targeting Units and Targeting Agents) Resin (1 g) in CH ₂ Cl ₂ (15 ml) and stirred for 20 minutes. The solvent was removed by filtration and the resin was treated once with DMF (15 ml) for 3 minutes. After filtration, the resin-bound peptide (or targeting agent) was treated with iodine (5 molar equivalent) dissolved in DMF (10 ml) for 1 hour.

The iodine solution was removed by filtration DMF-, 3 times with DMF (15 ml), and _CH 2 _Cl 3 times with 2 (15 ml), each time for three minutes, the residue was washed.

When preparing a “plain” peptide (without the Fmoc group), 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 reverse phase HPLC. In the case of targeting agents that did not contain an Fmoc group, the product was released from the resin and purified similarly.
Substances used:
Iodine CAS No. 7553-56-2
Molecular weight: 253.81
Merck Art. No. 4760

Synthesis of targeting unit (peptide) DLRSK A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was synthesized by manual synthesis as described in Example 2 above.

The following reagents were used as starting materials (in this order):
Fmoc-Lys (Mtt) resin Fmoc-Ser (tBu) -OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-L-Leu-OH
Fmoc-Asp (2-phenylisopropyl ester) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. After the cyclization process (macrolactam formation), a small sample of the resin (which still contains the fully protected cyclic peptide) was described in Example 2 for Fmoc removal (Steps 1-10 of Example 2). A sample of the peptide was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product (DLRSK macrolactam) was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of the cyclic DLRSK was clearly predominant.
MALDI-TOF data (circular DLRSK):
Calculated molecular mass = 599.34
Observed signal:
600.42M + H
622.40M + Na
638.29M + K
Fmoc-DLRSK macrolactam

Cyclic Fmoc-DLRSK was prepared and identified in the same manner as cyclic DLRSK except for the last Fmoc removal which was omitted in this case.
MALDI-TOF data (annular Fmoc-DLRSK):
Calculated molecular mass = 821.41
Observed signal:
822.60M + H
844.62M + Na

Synthesis of targeting unit (peptide) DLRSGRK (cyclic by lactam bridge) A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was synthesized manually as described in Example 2 above. Was synthesized.

The following reagents were used 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-Gly-OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-Gly-OH
Fmoc-Asp (2-phenylisopropyl ester) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. After the cyclization process (macrolactam formation), a small sample of the resin (which still contains the fully protected cyclic peptide) was described in Example 2 for Fmoc removal (Steps 1-10 of Example 2). A sample of the peptide was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product (DGRGLRSK macrolactam) was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of cyclic DGRGLRSK was clearly dominant.
MALDI-TOF data (circular DLRSGRK):
Calculated molecular mass = 869.48
Observed signal:
870.48M + H

Synthesis of targeting unit (peptide) DRGLRSK (cyclic by lactam bridge) A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was manually synthesized as described in Example 2 above. Was synthesized.

The following reagents were used 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-Gly-OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-Asp (2-phenylisopropyl ester) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. After the cyclization process (macrolactam formation), a small sample of the resin (which still contains the fully protected cyclic peptide) was described in Example 2 for Fmoc removal (Steps 1-10 of Example 2). A sample of the peptide was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product (DRGLRSK macrolactam) was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of cyclic DRGLRSK was clearly dominant.
MALDI-TOF data (annular DRGLRSK):
Calculated molecular mass = 812.46
Observed signal:
813.34M + H

Synthesis of targeting unit (peptide) DRYYNLRSK (cyclic by lactam bridge)
A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was synthesized by manual synthesis as described in Example 2 above. The following reagents were used 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, in two subsequent cycles Fmoc-L-Arg (Pbf) -OH, again Fmoc-Asp (2-phenylisopropyl ester) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. After the cyclization process (macrolactam formation), a small sample of resin (including cyclic peptides that are still fully protected) is treated as described in Example 2 for Fmoc removal (steps 1-10 of the same example). The peptide sample was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product (DRYYNLRSK macrolactam) was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of cyclic DRYYNLRSK was clearly dominant.
Mass spectral data (annular DRYYNLRSK):
Calculated molecular mass = 1195.61
Observed signal:
1096.28M + H

Synthesis of targeting unit (peptide) DSRYNLLRSK (cyclic by lactam bridge)
A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was synthesized by manual synthesis as described in Example 2 above. The following reagents were used as starting materials (in this order):
Fmoc-Lys (Mtt) resin Fmoc-L-Ser (tBu) -OH
Fmoc-L-Arg (Pdf) -OH
Fmoc-L-Leu-OH
Fmoc-L-Asn-OH
Fmoc-L-Tyr (tBu) -OH
Fmoc-L-Arg (Pdf) -OH, again Fmoc-L-Ser (tBu) -OH, again Fmoc-Asp (2-phenylisopropyl ester) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. After the cyclization process (macrolactam formation), a small sample of resin (including cyclic peptides that are still fully protected) is treated as described in Example 2 for Fmoc removal (steps 1-10 of the same example). The peptide sample was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product (DSRYNLRSK macrolactam) was then identified by its positive ion mode MALDI-TOF mass spectrum where the M + 1 ion of the cyclic DSRYNLRSK was clearly predominant.
Mass spectral data (cyclic DSRYNLRSK):
Calculated molecular mass = 1119.58
Observed signal:
1120.24M + H

標的化モチーフＬＲＳを含む、機能的に保護された、樹脂結合標的化単位（保護ペプチド）を、上記の実施例２に記載した手動合成によって合成した。 Synthesis of targeting unit (peptide) AhxDLRSK The unit of interest has the following structure:

A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was synthesized by manual synthesis as described in Example 2 above.

The following reagents were used 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-Asp (2-phenylisopropyl ester) -OH
Fmoc-6-aminohexanoic acid

After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. After the cyclization process (macrolactam formation), a small sample of resin (which still contains a fully protected cyclic peptide) was treated for 3 hours with the cleavage mixture described in Example 2. In this way, a peptide sample was cleaved from the resin, except for the final Fmoc removal which was omitted in this case, removing the side chain protecting groups of the sample. Samples were isolated as described in Example 2. The product (DLRSK macrolactam) was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of the cyclic DLRSK was clearly predominant.
MALDI-TOF data (annular Fmoc-AhxDLRSK):
Calculated molecular mass = 938.50
Observed signal:
939.50M + 1

A targeting unit comprising the effector unit diaminopropionic acid, linked by an amide bond to the N-terminal amino group of the peptide DLRSK via its carboxyl group (directly linked without a specific linking unit) and cyclic by a lactam bridge Synthesis of targeting agent Fmoc2Dap-DLRSK (Dap = diaminopropionyl), also including DLRSK. This targeting agent was synthesized using the manual synthesis described in Example 2 above (including the cyclization described above). Similar to the synthesis of Example 4). The sequence DLRSK was then continued with DL-2,3-bis (Fmoc-amino) propionic acid by the general coupling method described in Example 2.

Preparation of DL-2,3-bis (Fmoc-amino) propionic acid:
DL-2,3-diaminopropionic acid monohydrochloride was dissolved in 15 mL of aqueous 10% Na2CO3 solution. 7 mL of dioxane was then added and the reaction mixture was cooled to + 4 ° C. Fmoc-chloride in 20 mL dioxane was added and the reaction mixture was stirred at + 4 ° C. for 1 hour. After stirring overnight at room temperature, the reaction mixture was extracted with ethyl acetate, which was then evaporated. The residue was triturated with n-hexane and washed with a small amount of hot ethyl acetate to give a white solid that was dried in vacuo overnight.

Reagents used:
DL-2,3-diaminopropionic acid monohydrochloride Fmoc-chloride; 9-fluorenylmethyl chloroformate 98%; C.I. A. S. no: 28920-43-6 Acros, catalog number 170940250
MALDI-TOF data (Fmoc2Dap-DLRSK, circular):
Calculated molecular mass = 1129.53
Observed signal:
1130.32M + H

General procedure used in the synthesis of biotinylated compounds [targeting agent containing one D-biotin (vitamin H) as effector unit] The appropriate protected peptide was immobilized according to the general procedure described in Example 2. Synthesized using phase synthesis. The peptide was not deprotected and was not removed from the resin. Resin bound peptide was added to the reaction flask. The resin was swelled using CH2Cl2 (15 ml) and stirred for 20 minutes. The solvent was removed by filtration and the resin was treated once with DMF for 3 minutes. The peptide was deprotected using a 20% piperidine solution (20 ml) in DMF and shaken with it for 5 minutes, and this process was repeated using (here 10 minutes shaking). The resin was washed 3 times with DMF (15 ml), 3 times with CH2Cl2 (15 ml) and once with DMF (15 ml) for 3 minutes each time.

  D-biotin (3 molar equivalent) was suspended in DMF (10 ml) (heterogeneous suspension) for 1 minute using a 0.5 M solution of HBTU / HOBT in DMF (3 molar equivalent). Processed in a separate container. To this container was added a 2M solution of di-isopropylethylamine in NMP (6 molar equivalents). After the addition, the reaction mixture became homogeneous. This mixture was added to the reactor and the apparatus was shaken for 2 hours.

The reaction mixture was then filtered and the residue was washed 3 times with DMF (15 ml) and 3 times with CH ₂ Cl ₂ (15 ml), 3 minutes each time.

Cyclization was performed after the biotinylation procedure when the peptide was biotinylated as described herein, and when it was cyclized by iodine treatment as described in Example 3.
Substances used:
D-biotin (vitamin H)
CAS No. 58-85-5
Molecular weight: 244.3 g
Sigma B-4501
99%

Targeting comprising an effector unit D-biotin linked to the N-terminal amino group of peptide LRS by an amide bond via its carboxyl group (directly linked without a specific linking unit) and also including a targeting unit LRS Synthesis of Agent Bio-LRS (Bio = D-Biotin = Vitamin H) This targeting agent was further synthesized using the manual synthesis described in Example 2 above (similar to the synthesis in Example 1 above). The biotinylation procedure described in Example 11 was synthesized as a final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used in this way. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-LRS):
Calculated molecular mass = 600.31
Observed signal:
601.34M + H
623.23M + Na
639.25M + K

An amide bond between the D and K side chains, containing the effector unit D-biotin linked to the N-terminal amino group of the peptide DLRSK by its amide bond via its carboxyl group (directly linked without a specific linking unit) Synthesis of targeting agent Bio-DLRSK (Bio = D-biotin = vitamin H), which also contains targeting unit DLRSK, which is cyclic by binding. This targeting agent is a manual synthesis (cyclization) described in Example 2 above. And the biotinylation procedure described in Example 11 above was further used as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used in this way. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-DLRSK, circular):
Calculated molecular mass = 825.42
Observed signal:
826.49M + H
848.35M + Na

It contains the effector unit D-biotin, which is linked to the N-terminal amino group of the peptide DGRGLRSK via its carboxyl group by an amide bond (directly linked without a specific linking unit), and between the side chains of aspartic acid and lysine. Synthesis of targeting agent Bio-DGRGLRSK (Bio = D-biotin = vitamin H), which also contains the targeting unit DGRGLRSK, which is cyclic by the amide bond of This targeting agent is a manual synthesis described in Example 2 above ( Was synthesized using the biotinylation procedure described in Example 11 above as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used as is. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-DGRGLRSK, circular):
Calculated molecular mass = 1095.56
Observed signal:
1096.51M + H

It contains the effector unit D-biotin, which is linked to the N-terminal amino group of the peptide DRGLRSK by its amide bond via its carboxyl group (directly linked without a specific linking unit), between the side chains of aspartic acid and lysine. Synthesis of targeting agent Bio-DRGLRSK (Bio = D-biotin = vitamin H), which also contains the targeting unit DRGLRSK, which is cyclic by an amide bond. This targeting agent is synthesized manually (cyclic) as described in Example 2 above. And the biotinylation procedure described in Example 11 above was further used as a final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used in this way. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-DRGLRSK, circular):
Calculated molecular mass = 1038.56
Observed signal:
1039.59M + H

It contains the effector unit D-biotin, which is linked to the N-terminal amino group of the peptide DLRSK by an amide bond via its carboxyl group (directly linked without a specific linking unit), between the side chains of aspartic acid and lysine. Synthesis of targeting agent Bio-AhxDLRSK (Bio = D-biotin = vitamin H), which also contains the targeting unit DRYYNLRSK, which is cyclic by an amide bond. Was synthesized using the biotinylation procedure described in Example 11 above as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used in this way. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
Mass spectral data (Bio-DRYYNLRSK, cyclic):
Calculated molecular mass = 1421.69
Observed signal:
142.34M + H

A peptide in the form of a lactam, and / or a targeting unit, and / or a targeting agent and / or a targeting motif, and / or part thereof (as a macrolactam; “mediated by a peptide bond between lysine and aspartic acid” “Included in the sequence at the end of the sequence”, a general method for cyclization. Acyclic, fully protected, resin-bound peptide was manually prepared by the general method described in Example 2 above. It was prepared with.

  Prior to cyclization, a selective, one-step, degradation of lysine and aspartic acid side chain protecting groups [the groups are: 4-methyltrityl on lysine units, and 2-phenylisopropyl (esters on aspartate units) Was carried out using diluted TFA (4% in dichloromethane). Cyclization involved condensation between the side chain carboxyl group of the aspartic acid unit and the 6-amino group (side chain amino group) of the lysine unit. Activation was by the PyAOP / HOAt / DIPEA reagent mixture (see below for details and abbreviations) or the HBTU / HOB + / DIPEA mixture described in Example 2. The equipment, common solvents, and actual techniques were similar to those described in Example 2.

The first fully protected and resin-bound peptide (0.3 mmol) was shaken at room temperature under an argon atmosphere with different solutions (about 10 mL) for the times indicated below and then filtered:
1. 1. Dichloromethane, 20 minutes 4. 4% (volume) trifluoroacetic acid in dichloromethane, 15 minutes 3. 3 minutes in a 1:10 mixture of 0.2M DIPEA, NMP and dichloromethane 4. Dichloromethane, 3 minutes Dichloromethane, 3 minutes 6. 6. Dichloromethane, 3 minutes DMF, 3 minutes 8. Activation, 4 hours, follow description below.

  None of the components were filtered with a mixture of PyAOP and HOAt, or a mixture of HBTU and HOBt, which was 3 moles relative to a resin-bound peptide (and thus both 0.9 mmol) in DMF (7 mL). Was shaken with the resin for 1 minute, then 6 molar equivalents of 2M DIPEA in NMP were added.

  After step 8 above, starting with step 13, the procedure was continued as described in Example 2.

The reagents for activation in this type of cyclization were:
PyAOP = 7-azabenzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate, CAS No. 156311-83-0, PE Biosystems catalog number GEN076531, molecular weight: 521.4 g / mol
HOAt = 1-hydroxy-7-azabenzotriazole, 0.5 M solution in DMF, Applied Biosystems catalog number 4330631
DIPEA = N, N-diisopropylethylamine, 2.0 M solution in N-methylpyrrolidone, Applied Biosystems catalog number 401517

  See materials shown in Example 2 for “HBTU and HOBt” alternative materials.

Starting material for “special” amino acid units (aspartic acid and lysine) during which an “excess” peptide bond is formed:
Fmoc-Lys (Mtt) resin, 0.68 mmol / g, Bachem catalog number D-2565.0005
Fmoc-Asp (2-phenylisopropyl ester) -OH, molecular weight: 473.53 g / mol, Bachem catalog number B-2475.0005

It contains an effector unit D-biotin linked to the N-terminal amino group of the peptide DLRSK by an amide bond via its carboxyl group (directly linked with a specific linking unit), and between the side chains of aspartic acid and lysine. Synthesis of the targeting agent Bio-DSRYNLRSK (Bio = D-biotin = vitamin H), which also contains the targeting unit DSRYNLRSK, which is cyclic by an amide bond. This targeting agent is synthesized manually (cyclic) as described in Example 2 above. And the biotinylation procedure described in Example 11 above was further used as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used as is. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
Mass spectral data (Bio-DSRYNLRSK, cyclic):
Calculated molecular mass = 1345.66
Observed signal:
1346.32M + H

It contains an effector unit D-biotin linked to the N-terminal amino group of the peptide AhxDLRSK via its carboxyl group (directly linked without a specific linking unit) by an amide bond, and between the side chains of D and K Synthesis of targeting agent Bio-AhxDLRSK (Bio = D-biotin = vitamin H), which also contains targeting unit AhxDLRSK, which is cyclic by an amide bond. And the biotinylation procedure described in Example 11 above was further used as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used as is. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-AhxDLRSK, circular):
Calculated molecular mass = 938.50
Observed signal:
939.50M + H

Via the N-terminal amino group of the lysine residue (unit) and its carboxyl group, this is then the amino group of Ahx (6-aminohexanoic acid) by an amide bond, which in turn is the amino terminus of DLRSK by an amide bond. Targeting unit comprising one effector unit D-biotin bound to (by one and one linking unit and / or spacer unit and / or as one large spacer and / or linking unit) Synthesis of targeting agent Bio-K-AhxDLRSK (cyclic by amide bond between aspartic acid and C-terminal lysine; Bio = D-biotin = vitamin H), also including DLRSK. This synthesis was performed as follows: Fully protected resin-bound cyclic targeting unit (peptide with spacer / linking unit) AhxDLRSK, Prepared similarly as described in Example 9 above. The sequence AhxDLRSK is then performed by the general coupling method described in Example 2 using one lysine unit (protected with an Fmoc group at the N-terminal amino group and a Boc group at the side chain amino group). To keep being maintained. The following reagents were used as starting materials:
Fmoc-L-Lys (tBoc) -OH

Finally, K-AhxDLRSK still bound to the resin and fully protected was biotinylated according to the general method described in Example 11. Purification by HPLC gave an overall yield of 30% theoretical. Product identification:
Positive ion mode MALDI-TOF mass spectrum: M + 1 ion was clearly dominant.
MALDI-TOF data (Bio-K-AhxDLRSK, circular):
Calculated molecular mass = 1066.60
Observed signal:
1067.5M + H

４倍ビオチン化した４個の分岐鎖連結／スペーサー単位を、Ｋ−ＡｈｘＤＬＲＳＫのアミノ−末端に含むことを示すことができる。 1 amino group of lysine residue (unit), N-terminal amino group or side chain amino group, and dendritic structure (2 lysines each have 2 effector biotin units, It is linked to one other lysine via the carboxyl function, which is then linked to the N-terminal amino group of one lysine (side chain unbound) by an amide bond. This is linked in the same manner as Ahx (6-aminohexanoic acid), which is linked to the amino terminus of the peptide DLRSK by an amide bond) and its carboxyl group, respectively (linkage unit) And / or a combination of spacer units and / or connected through a dendritic structure that can be considered as one large spacer and / or connecting unit). Includes a restrictor unit D- biotin, targeting units DLRSK including, targeting agent _Bio 4 _-K 3 _-K-AhxDLRSK (between aspartic acid and terminal lysine, cyclic by an amide bond; Bio = D- Biotin = Vitamin H The product has the formula shown below:

It can be shown that 4 branched biotinylated 4 branched chain linkage / spacer units are included at the amino-terminus of K-AhxDLRSK.

This synthesis was performed as follows: a fully protected resin-bound “on-resin” cyclic targeting unit (a peptide with two spacers / linking units) K-AhxDLRSK, as described in Example 20 above. Prepared as described. A branched chain structure containing 4 biotins and 3 lysines was constructed by the general coupling method described in Example 2, resulting in 1 lysine unit (each of the 2 amino groups being 1 Was first used to keep the sequence K-AhxDLRSK maintained. The procedure (lysine addition) was then repeated using twice the amount of coupling reagent and two times the protected (Fmoc group) lysine, and two more lysine units, one of which was coupled first. The lysine unit was linked at the amino group at the amino terminal with one side chain amino group. Reagents used (other than those described in the reference examples):
Fmoc-L-Lys (Fmoc) -OH

  Using a resin-bound branched peptide, using 12 molar equivalents of a coupling reagent and biotin, biotinylation was performed according to the general method described in Example 11 to obtain a structure containing 4 biotin units. Obtained. Purification by HPLC gave an overall yield of 44% theoretical.

Product identification:
Positive ion mode MALDI-TOF mass spectrum: M + 1 ion was clearly dominant.
MALDI-TOF data (Bio ₄ -K ₃ -K-AhxDLRSK, cyclic):
Calculated molecular mass = 2129.12.
Observed signal:
2129.89 M + H (maximum isotopomer is 2130.9)

４倍ビオチン化した５個の分岐鎖連結／スペーサー単位を含み、ペプチドＡｈｘＤＬＲＳＫのＮ−末端の、１個の分岐鎖上にＤｔｐａ−部分を有することを示すことができる。 1 amino group of lysine residue (unit), N-terminal amino group or side chain amino group, and dendritic structure (2 lysines each have 2 effector biotin units, It is linked to one other lysine via the carboxyl function, which is then linked by an amide bond to the N-terminal amino acid of one lysine (the side chain is bound by an amide bond with Dtpa). Which is linked in the same way as Ahx (6-aminohexanoic acid), which is with the amino terminus of the peptide DLRSK by an amide bond) and its carboxyl group, respectively. A bond (through a dendritic structure which can be regarded as a combination of linking units and / or spacer units and / or as one large spacer and / or linking unit Binding) were two types of effector unit: includes four identical effector unit D- biotin, targeting units DLRSK including, targeting agent _{Bio 4 -K 3 -K (Dtpa)} -AhxDLRSK ( aspartic acid and Synthesis by cyclic amide bond between terminal lysines; Bio = D-biotin = vitamin H; DTPA = diethylenetriaminepentaacetic acid—one OH) The product has the formula:

It can be shown that it contains 5 branched chain linkage / spacer units that are 4-fold biotinylated and has a Dtpa- moiety on one branched chain at the N-terminus of the peptide AhxDLRSK.

This synthesis was performed as follows: The isolated and purified targeting agent Bio _4- K ₃ -K-AhxDLRSK was prepared as described in Example 21 above. The product thus obtained was treated with 10 molar equivalents of diethylenetriaminepentaacetic dianhydride in DMF solution (0.01 M solution, calculated based on biotinylated peptide) and then treated for 18 hours. did. After this treatment, the volume was doubled by adding water to the DMF solution and the solution was placed in another place and left for another 4 hours. Finally, the solvent was evaporated in vacuo, the residue was mixed with water containing 0.1% trifluoroacetic acid, filtered and the filtrate was purified by reverse phase HPLC. The product was identified by its M + 1 peak in the MALDI-TOF mass spectrum.
Product identification:
Positive ion mode MALDI-TOF mass spectrum: M + 1 ion was clearly dominant.
MALDI-TOF data [Bio4-K3-K (Dtpa) -AhxDLRSK, cyclic]:
Calculated molecular mass = 2504.24
Observed signal:
2505.29M + H

Targeting containing the effector unit amino-oxyacetic acid, linked by an amide bond to the N-terminal amino group of the peptide DLRSK via its carboxyl group (directly linked without a specific linking unit) and cyclic by a lactam bridge Synthesis of targeting agent Aoa-DLRSK (Aoa = aminooxyacetyl = NH 2 OCH 2 CO), which also contains the unit DLRSK. This targeting agent was synthesized using the manual synthesis described in Example 2 above (including cyclization). , Similar to the synthesis of Example 4 above). The sequence DLRSK was then continued with amino-oxyacetic acid by the general coupling method described in Example 2.

Reagents used:
Boc-amino-oxyacetic acid; Boc-NH-OCH2-COOH
MALDI-TOF data (Aoa-DLRSK, circular):
Calculated molecular mass = 674.37
Observed signal:
673.54M + H

An effector unit 5- (1-O-carboranyl) -pentanoic acid linked by an amide bond to the N-terminal amino group of the peptide DLRSK via its carboxyl group (directly linked without a specific linking unit), Synthesis of targeting agent Cbp-DLRSK [Cbp = 5- (1-O-carboranyl) -pentanoyl], which also contains the targeting unit DLRSK, which is cyclic by a lactam bridge This targeting agent is described in Example 2 above Was synthesized using the same manual synthesis (similar to the synthesis of Example 4 above, including cyclization). The sequence DLRSK was then maintained using 5- (1-O-carboranyl) -pentanoic acid by the general coupling technique described in Example 2. However, PyAoP (in place of HBTU) and HOAT (in place of HOBt), and the reaction time of the processing step 12 of Example 2 is 4 hours.
Reagents used:
5- (1-o-carboranyl) -pentanoic acid, Katchem, Prague, Czech Public, formula weight 244.34 g / mol
MALDI-TOF data (Cbp-DLRSK, circular):
Calculated molecular mass = 817.60 (basis B10, abund. 20%), 827.57 (basis B11 abund. 80%)
Average molecular weight = 826.01 g / mol
Observed signal:
Multiplet, highest peak at 826.55 and 827.55: M + H
Multiplet, highest peak at 848.45 and 849.50: M + Na

It contains the effector unit 4-amino-10 methylfolic acid linked directly to the N-terminal amino group of the peptide DLRSK by an amide bond via its carboxyl group (directly linked without a specific linking unit), and cyclic by a lactam bridge. Synthesis of the targeting agent Amf-DLRSK [Amf = 4-amino-10 methyl folate acyl], which also includes the targeting unit DLRSK. This targeting agent is a manual synthesis (including cyclization) described in Example 2 above. In the same manner as in Example 4 above). Next, the general coupling technique described in Example 2 was used to keep the sequence DLRSK using 4-amino-10 methylfolic acid. However, PyAoP (in place of HBTU) and HOAT (in place of HOBt), and a reaction time of 5 hours in the processing step 12 of Example 2, and a reagent (peptide / PyAOP / HOAT / DIPEA = 1: equimolar ratio with the resin-bound peptide) 1: 1: 2).
Reagents used:
4-amino-10 methyl folic acid; (+) amethopterin; methotrexate hydrate CAS No. 59-05-2
Formula weight: 454.4 g / mol
Sigma catalog number A-6770
MALDI-TOF data (Amf-DLRSK, ring):
Calculated molecular mass = 1035.50
Observed signal:
1036.35M + H

Aoa-DLRSK [targeting containing a linking (binding) unit aminooxyacetic acid, linked via an amide bond to the N-terminal amino group of the peptide DLRSK via its carboxyl group (directly linked without a specific linking unit) Agent (Derivative of Peptide)] Targeting agent Dnm-, comprising the effector unit daunomycin, linked via an oxime bond with the amino-oxy group of the agent (peptide derivative), and also the targeting unit DLRSK cyclic by a lactam bridge Synthesis of Aoa-DLRSK (Dnm = daunomycin, Aoa = amino-oxyacetyl = NH 2 OCH 2 CO) -Dissolved in DLRSK methanol (concentration 0.00 25M) was synthesized by stirring. The product was isolated by evaporating the solvent and purified by HPLC.
Reagents used:
Daunomycin hydrochloride CAS No. 20830-81-3
Molecular weight: 564.0 g / mol
ICN Biomedicals, Aurora, Ohio, USA Catalog No. 44583
MALDI-TOF data (Dnm-Aoa-DLRSK, circular):
Calculated molecular mass = 1181.52
Observed signal:
1182.41M + H

Aoa-DLRSK [target containing a linking (binding) unit amino-oxyacetic acid, linked via an amide bond to the N-terminal amino group of the peptide DLRSK via its carboxyl group (directly linked without a specific linking unit) Targeting agent Dxrb, which comprises the effector unit doxorubicin linked by an amino-oxy group and an oxime bond of the agent (derivative of the peptide) via its carboxyl group and also includes the targeting unit DLRSK that is cyclic by a lactam bridge -Synthesis of Aoa-DLRSK (Dxrb = doxorubicin, Aoa = amino-oxyacetyl = NH2OCH2CO) This targeting agent was described in Example 23 above in equimolar amounts with doxorubicin hydrochloride at room temperature in the dark for 3 days. Aoa-DLRSK dissolved in methanol (concentration 0.0025M) was synthesized by stirring. The product was isolated by evaporating the solvent and purified by HPLC.
Reagents used:
Doxorubicin hydrochloride CAS No. 25316-40-9
Molecular weight: 580.0 g / mol
Fluka catalog number 44583
MALDI-TOF data (Dxrb-Aoa-DLRSK, ring):
Calculated molecular mass = 1197.52
Observed signal:
1198.17M + H
1198.17M + H

Synthesis of targeting unit (peptide) DLRSGRK, cyclic by lactam bridges A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRS was prepared manually as described in Example 2 above. Synthesized by synthesis.

The following reagents were used as starting materials (in this order):
Fmoc-Lys (Mtt) -resin Fmoc-L-Arg (Pbf) -OH
Fmoc-Gly-OH
Fmoc-L-Ser (tBu) -OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-L-Leu-OH
Fmoc-Asp (2-phenylisopropyl ester) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 17. Example 2 on Fmoc removal after a cyclization process (macrolactam formation) for a small sample of resin (which still contains a fully protected cyclic peptide and is a suitable starting material for further synthesis eg biotinylation) The same treatment as described in (steps 1 to 10 of the same example) was applied, after which the peptide sample was cleaved from the resin by treatment for 3 hours with the cleavage mixture described in example 2 and carried out. Isolated as described in Example 2.

The product (cyclic DLRSGRK) was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of the cyclic DLRSK was clearly dominant.
MALDI-TOF data (circular DLRSGRK):
Calculated molecular mass = 812.46
Observed signal:
813.69M + H

It contains the effector unit D-biotin, which is linked to the N-terminal amino group of the peptide DLRSK by an amide bond via its carboxyl group (directly linked without a specific linking unit), between the side chains of aspartic acid and lysine. Synthesis of targeting agent Bio-DLRSGRK (Bio = D-biotin = vitamin H), which also contains the targeting unit DLRSGRK, which is cyclic by an amide bond. This targeting agent was synthesized manually (cyclic) as described in Example 2 above. Was synthesized using the biotinylation procedure described in Example 11 above as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used in this way. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-DLRSGRK, circular):
Calculated molecular mass = 1038.54
Observed signal:
1039.74M + H
1061.76M + Na
1077.60M + K

Synthesis of targeting unit (peptide) DLRSGRGK. A target comprising an effector unit D-biotin linked to the N-terminal amino group of the peptide DLRSGRRGK via its carboxyl group (directly linked without a specific linking unit) by an amide bond and also including a targeting unit DLRSGRGK Synthesis of the agent Bio-DLRSGRGK (Bio = D-biotin = vitamin H). Cyclization of the targeting agent Bio-DLRSGRGK (as a macrolactam) in lactam form.
A functionally protected, resin-bound targeting unit (protected peptide) containing the targeting motif LRSGRG was synthesized by manual synthesis as described in Example 2 above.

The following reagents were used as starting materials (in this order):
Fmoc-Lys (Mtt) -resin 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-Asp (2-phenylisopropyl ester) -OH

This targeting agent was synthesized by using the biotinylation procedure described in Example 11 above as the final coupling step. In this final coupling process, D-biotin was used in place of the protected amino acid. D-biotin was not protected but was used as is. The biotinylated, still resin-bound targeting agent was subjected to a cyclization process that forms excess amide bonds as described in Example 17. The product was isolated and purified by the method shown in Example 2 and identified by positive ion mode MALDI-TOF spectroscopy (M + 1 ion was clearly dominant).
MALDI-TOF data (Bio-DLRSGRGK, circular):
Calculated molecular mass = 1065.56
Observed signal:
1096.55M + H

In Vivo Targeting of Mouse Tumors In this example, in vivo targeting of the targeting units prepared in the above example was compared to three different types of primary tumors, fibrosarcoma, melanoma, and Shown for melanoma in the lung. The tested targeting units of the present invention have been shown to selectively target primary tumors and metastases in vivo but not normal tissues or organs.

Mice with cell lines and tumors The following tumor cell lines were used to generate experimental tumors in mice:
"ODC sarcoma cells", (OS), originally derived from tumors formed in nude mice administered NIH3T3 mouse fibroblasts transformed by overexpression of ornithine decarboxylase (ODC) Described (Auvinen et al., 1997);
The human melanoma cell line, C8161 (M), Welch et al. (1991).

  These cell lines were supplemented with 5-10% fetal calf serum (FCS; BioWhittaker), 1% L-glutamine (Bio-Whittaker) and 1% penicillin / streptomycin (Bio-Whittaker), Dulbecco's modified eagle The cells were cultured in a medium (DMEM; Bio-Whittaker).

Experimental Tumor Generation To generate experimental tumors, the cells listed above (OS, KS and melanoma: 0.5 × 10 6 cells) were transformed into the line Balb / c Ola Hsd-nude, NMRI / nu / nu. Alternatively, Athymic-nu (both strains of mice were from Harlan Laboratories) were injected subcutaneously on both flank of nude mice. Tumors were harvested when a weight of approximately 0.4 g was reached.

  Metastases (mostly formed in the lung) were generated by injecting melanoma cells intravenously into Balb / c Ola Hsd-nude mice. Mice were raised for 4-6 weeks and then targeted for experiments.

  For mice with tumors or mice with metastases, 0.02 ml Avertin [10 g 2,2,2-tribromoethanol (Fluka) 10 ml 2-methyl-2-butanol (Sigma Aldrich)] per gram body weight Anesthesia was performed by intraperitoneal (ip) administration of the solution in solution.

In vivo targeting and detection of targeting In order to localize the targeting peptide, NMRI nude mice with OS and melanoma tumors were anesthetized and 1 or 2 mg of biotinylated (prepared in Example 12). Synthetic targeting peptides were injected intravenously, and 5-10 minutes after the intravenous injection, the perfusate was delivered to the heart of the mice using a 25G needle set for wing perfusion (Terumo) and 50 ml DMEM. The organs were then dissected and frozen in liquid nitrogen. Tumors and control organs (liver, kidney, spleen, heart, brain) were dissected and frozen in liquid nitrogen.

  Biotinylated peptide (targeting agent), avidin, and biotinylated HRP (Vectorstein ABC-kit, catalog number PK6100; Vector Laboratories) AB (avidin-biotin) -complex, and diaminobenzidine (DAB substrate kit, catalog No. 4100, Vector Laboratories) and detected on 10 micrometer frozen sections.

  The results of these experiments are shown in Table 2.

In vivo effect of targeting agents comprising cytotoxic effector units In this experiment, prepared in Example 24, which contains a cytotoxic effector unit, doxorubicin, linked by oxime binding to a cyclic targeting unit DLRSK containing a targeting motif LRS. A targeting agent, Dxrb-Aoa-DLRSK, was used to demonstrate in vivo targeting and therapeutic effects on melanoma tumors.

One million C8161M / T1 melanoma cells were injected subcutaneously into the flank of eight Athymic-nu mice and tumors were allowed to grow for one week. These mice were then divided into three groups, which were given the following treatments:
DMEM group: 2 mice, DMEM only Dox group: 2 mice, 1, 43 mg / kg doxorubicin dissolved in DMEM pept + dox group: 4 mice, 1, 43 mg / kg Dxrb-Aoa-DLRSK (Doxorubicin linked to targeting motif LRS) dissolved in DMEM (Dox group and equimolar dose)

The treatment was administered intravenously twice a week (Tuesday and Friday) for a total of 5 doses. Tumors were measured with calipers in the vertical direction twice on each injection day and the day the animals were killed. Tumor volume was calculated by the elliptical formula:
Volume = (length x width ² ) x 0.5

The results of this experiment are shown in FIG. 1, confirming that the targeting agent of the present invention selectively targets melanoma tumors in vivo and significantly increases the therapeutic effect of doxorubicin.
Reagents used:
Doxorubicin hydrochloride, CAS No. 25316-40-9, molecular weight: 580.0 g / mol, Fluka catalog number 44583

General method for cyclizing a peptide or related substance by an amide bond between D-ornithine and glutamic acid contained in the sequence at the end of the “intervening” sequence: “head-side chain macrolactam”, Formation of “Glu (D-Orn) -Ring” Acyclic, fully protected, resin-bound peptides are prepared manually by the general method described above.

  Prior to cyclization, a selective, one-step degradation of the individual protecting groups of ornithine and glutamic acid [the groups are: 2N-Fmoc on the ornithine unit, and 5- (2-trimethylsilylethyl ester on the glutamic acid unit )] Is performed using a solution of tetrabutylammonium chloride in DMF. Cyclization involves 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 the PyAOP / DIPEA reagent mixture instead of the HBTU / HOBt / DIPEA mixture described in Example 2 (see below for details and abbreviations). The equipment, common solvents, and actual techniques are similar to those described in Example 2.

  This method can be modified for lysine (instead of ornithine) and aspartic acid (instead of glutamic acid) units by using the respective derivatives of these amino acids.

Initially fully protected and resin-bound peptide (0.3 mmol) is shaken at room temperature under an argon atmosphere with different solutions (about 10 mL) for the time indicated below and then filtered:
1. 1. Dichloromethane, 20 minutes 1M tetrabutylammonium chloride dissolved in DMF, 20 minutes 3-5. DMF, 1 minute (3 treatments).
6-8. DCM, 1 minute (3 treatments).
9. DMF, 1 minute 10. A solution of 0.9 mmol of PyAOP (3 molar equivalents relative to the resin-bound peptide) in DMF (7 mL) is shaken with the resin for 1 minute without filtration.
11. Add 6 molar equivalents of 2M DIPEA (hence 1.8 mmol) in NMP and then shake for 4 hours.

  After the foregoing steps, the resin is washed, for example, as described in the general procedure for (manual) peptide synthesis (step after adding active amino acids).

The reagents for deprotection before cyclization are as follows:
Tetrabutylammonium chloride trihydrate, CAS No. 87749-50-6, molecular weight: 315.51 g / mol, Acros Organics catalog number 221080500

The reagents for activation in this type of cyclization are:
PyAOP = 7-azabenzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate, CAS No. 156311-83-0, PE Biosystems catalog number GEN076531, molecular weight: 521.4 g / mol
DIPEA = N, N-diisopropylethylamine, 2.0 M solution in N-methylpyrrolidone, Applied Biosystems catalog number 401517

Starting materials for “special” amino acid units (glutamate and ornithine) during which “excess” peptide bonds are formed:
Fmoc-D-Orn (Mtt) -OH; 2-N-Fmoc-5-N- (4-methyltrityl) -D-ornithine, molecular weight: 610.8 g / mol, Novabiochem catalog number 04-13-1012.
Fmoc-L-Glu (OTMSEt) -ONa; N-2-Fmoc-glutamic acid 5- (2-trimethylsilylethyl) ester sodium salt, molecular weight: 468.60 g / mol, Novabiochem catalog number 0412-1231.

Synthesis of targeting unit (peptide) D-OrnLRSE-amide, which is cyclic by an amide bond between the side chain of the glutamic acid unit and the α-amino group of D-ornithine, including a targeting motif LRS, functionally protected, Resin-bound targeting units (protected peptides) were synthesized by manual synthesis as described in Example 2 above, in which the “empty” resin was the same as described for the pre-filled resin (Example In steps 1-11) of 2, deprotection prior to the first coupling.

The following reagents were used as starting materials (in this order):
Rink amide MBHA resin Fmoc-L-Glu (OTMSEt) -OH
Fmoc-L-Ser (tBu) -OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-L-Leu-OH
Fmoc-D-Orn (Mtt) -OH

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 33. The peptide sample was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product was then identified by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of the cyclic D-OrnLRSE-amide was clearly predominant.
MALDI-TOF data (cyclic _D-OrnLRSE-NH 2):
Calculated molecular mass = 598.36
Observed signal:
599.42M + 1

Synthesis of targeting unit (peptide) D-OrnLRGSRGEG, which is cyclic by an amide bond between the side chain of the glutamic acid unit and the α-amino group of D-ornithine Functionally protected, resin-bound targeting unit (protected peptide) ) Was synthesized by the manual synthesis described in Example 2 above.

The following reagents were used as starting materials (in this order):
Fmoc-Gly resin Fmoc-L-Glu (OTMSEt) OH
Fmoc-GIy-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

  After the final cycle of the coupling process, the targeting unit that is still resin bonded was subjected to a cyclization process that forms excess amide bonds as described in Example 33. The peptide sample was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in the same example.

The product was then identified by its positive ion mode MALDI-TOF mass spectrum with M + 1 ion of cyclic D-OrnLRGSRGEGG.
MALDI-TOF data (circular D-OrnLRGSGRGEG):
Calculated molecular mass = 926,50
Observed signal:
927.45M + 1

標的化モチーフＬＲＳを含む、機能的に保護され、樹脂結合した、環状標的化単位を、上記の実施例３４に記載した手動合成によって合成した。次に、実施例１７（ステップ１〜７）に記載した方法で、希釈したＴＦＡ（４％、ジクロロメタン中）を用いて、樹脂を処理して、リジンの側鎖保護Ｍｔｔ基を切断した。次いで、依然として樹脂に結合した単位を、ＨＢＴＵ／ＨＯＢｔ／ＤＩＰＥＡ活性化を使用する、実施例２（ステップ１２〜１８）に記載した一般的方法によって、ＤＯＴＡ−トリス−ｔｅｒｔブチルエステルと結合させた。
使用した試薬：ＤＯＴＡ−トリス−（ｔＢｕエステル）。 Targeting agent D-Orn (DOTA) LRSE-amide (DOTA = linked by its one carboxyl, 1,4, cyclic by an amide bond between the side chain of the glutamic acid unit and the α-amino group of D-ortinin , 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)

A functionally protected, resin-bound, cyclic targeting unit containing the targeting motif LRS was synthesized by manual synthesis as described in Example 34 above. The resin was then treated with the diluted TFA (4% in dichloromethane) as described in Example 17 (Steps 1-7) to cleave the side chain protected Mtt group of lysine. The units still bound to the resin were then coupled with DOTA-Tris-tertbutyl ester by 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 by its positive ion mode MALDI-TOF mass spectrum, where the M + 1 ion of the cyclic D-Orn (Dota) LRSE-amide was clearly predominant. .
MALDI-TOF data [cyclic D-Orn (Dota) LRSE-NH ₂ ]:
Calculated molecular mass = 984.54
Observed signal:
985.52M + 1

Synthesis of a targeting unit (peptide) KLRSD-amide that is cyclic by an amide bond between the side chain of the aspartic acid unit and the α-amino group of lysine Functionally protected, resin-bound target containing the targeting motif LRS Embedded image (protected peptide) was synthesized by manual synthesis as described in Example 2 above, in which the “empty” resin was prepared in the same manner as described for the pre-filled resin (steps of Example 2). 1-11) was deprotected prior to the first coupling.

The following reagents were used as starting materials (in this order):
Rink amide MBHA resin Fmoc-L-Asp (OTMSEt) -OH
Fmoc-L-Ser (tBu) -OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-L-Leu-OH
Fmoc-L-Lys (Mtt) -OH

  After the final cycle of the coupling process, the targeting units that are still resin-bound were subjected to a cyclization process that forms excess amide bonds as described in Example 33 (alternatively, Glu and Asp, as well as Lys and Orn). The peptide sample was then cleaved from the resin by treatment for 3 hours with the cleavage mixture described in Example 2 and isolated as described in Example 2.

The product was then identified by its positive ion mode MALDI-TOF mass spectrum with M + 1 ions.
MALDI-TOF data (cyclic _KLRSD-NH 2):
Calculated molecular mass = 598.36
Observed signal:
599.21M + 1

Targeting agent K (DOTA) LRSD-amide (DOTA = linked by its one carboxyl, 1,4,7, cyclic by an amide bond between the side chain of the aspartic acid unit and the α-amino group of lysine Synthesis of 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) A functionally protected, resin-bound, cyclic targeting unit comprising the targeting motif LRS is described in Example 37 above. Synthesized by manual synthesis. The resin was then treated with the diluted TFA (4% in dichloromethane) as described in Example 17 (Steps 1-7) to cleave the side chain protected Mtt group of lysine. The units still bound to the resin were then coupled with DOTA-Tris-tertbutyl ester by the general method described in Example 2 (Steps 12-18) using HBTU / HOBt / DIPEA activation. Reagents used:
DOTA-Tris- (tBu ester).

The product was cleaved and isolated as described in Example 2 and identified with M + 1 ions by its positive ion mode MALDI-TOF mass spectrum.
MALDI-TOF data [annular K (Dota) LRSE-NH ₂ ]:
Calculated molecular mass = 984.54
Observed signal:
985.52M + 1

Synthesis of the targeting unit Ac-DLRSK-Ahx, which is cyclic by the side chains of aspartic acid and lysine. The preparation of Ac-DLRSK-Ahx was performed by the manual solid phase peptide synthesis technique described in detail in Example 2.

  A first structural component (component), 6-aminohexanoic acid (= Ahx), wherein the amino functional group was protected by a 9-fluorenylmethyloxycarbonyl group (= Fmoc group), and a hydroxyl functionalized peptide The binding of the synthetic resin was performed by the dichlorobenzoyl chloride method (the “equivalent” below is the amount of molecules or “mol” relative to the resin filling capacity).

  Unfilled (“empty”) resin was first washed by shaking with N, N-dimethylformamide (= DMF) for 20 minutes and filtered. 5 equivalents of Fmoc protected 6-aminohexanoic acid (Fmoc-Ahx-OH) in DMF (0.2 M solution) and 8 equivalents of pyridine were added to the resin and then it was shaken for 3 minutes. . Then 5 equivalents of 2,6-dichlorobenzoyl chloride were added and the mixture was shaken for 18 hours (overnight).

  After a long treatment, the resin was filtered and washed several times with DMF and dichloromethane as described in Example 2 (Steps 13-18). Next, a mixture of acetic anhydride (2M solution, 94 eq) and N, N-diisopropylethylamine (DIPEA, 1.6M solution, 80 eq) dissolved in N-methylpyrrolidone (NMP) solution for 2 hours. The resin was shaken, filtered and washed as above, and finally dried in a stream of argon gas.

The reagents used so far were the following:
HMP resin, packing capacity: 1.16 mmol / g, Applied Biosystems catalog number 400957
2,6-dichlorobenzoyl chloride, CAS No. 225-102-4, molecular weight: 209.46 g / mol, Lancaster (Morcam, England) catalog number 8922
Pyridine, Merck Art. No. 9728.
Fmoc-6-aminohexanoic acid (Fmoc-6-Ahx-OH), CAS No. 88574-06-5, Novabiochem catalog number 04-12-1111, molecular weight: 353.4 g / mol,
Acetic anhydride, Fuka catalog number 45830.

From this point on, the synthesis proceeds according to the general method described in Example 2. The next structural reagents used in this synthesis are in order:
Fmoc-Lys (Mtt) -OH
Fmoc-L-Ser (tBu) -OH
Fmoc-L-Arg (Pbf) -OH
Fmoc-L-Leu-OH
Fmoc-Asp (2-phenylisopropyl ester) -OH

The still resin bonded product was then cyclized according to Example 17. Finally, acetic acid was used to keep the sequence maintained (ie, the end was covered at the amino terminus) as follows: The amino protected Fmoc group is described in Example 2 (Steps 1-10). Removed as before. The still resin bonded product was then treated with a mixture of acetic anhydride and DIPEA in NMP, as was done after the initial bonding of the Ahx moiety and the resin. Finally, the product was cleaved from the resin and purified as described in Example 2. Identification was based on the M + 1 ion of the MALDI-TOF mass spectrum.
MALDI-TOF data (circular Ac-DLRSK-Ahx)
Calculated molecular mass = 754.43
Observed signal: 755.60

Includes doxorubicin linked by an amide bond to the carboxyl group of the C-terminal spacer moiety (Ahx = 6-aminohexanoyl) of the N-coated (Ac = acetyl) cyclic targeting unit Ac-DLRSK-Ahx containing the targeting motif LRS , Synthesis of Ac-DLRSK-Ahx-Dox (Dox = doxorubicin linked via its amino group) targeting agent The “targeting unit” compound (peptide derivative) Ac-DLRSK-Ahx was described in Example 39. Prepared in the same manner. Doxorubicin was ligated with purified Ac-DLRSK-Ahx in N, N-dimethylformamide (= DMF) solution by the following PyAOP / IDIPEA activation.

  Equimolar amounts of Ac-DLRSK-Ahx and PyAOP were combined as a 0.05M solution in DMF and 2 molar equivalents of DIPEA (2M solution in NMP) were mixed therein and after 5 minutes (Ac-DLRSK An equimolar amount of doxorubicin hydrochloride (0.05 M solution in DMF) was added (relative to Ahx). After the reaction was allowed to proceed for 1 hour in the dark (to protect from light), the mixture was diluted with diethyl ether. The centrifuged solid precipitate was purified by reverse phase HPLC chromatography as described in Example 2 and identified by positive ion mode MALDI mass spectrum.

使用した物質：
ドキソルビシン塩酸塩、ＣＡＳ Ｎｏ．２５３１６−４０−９、分子量：５８０．０ｇ／ｍｏｌ、Ｓｉｇｍａカタログ番号Ｄ−１５１５。
ＭＡＬＤＩ−ＴＯＦデータ（Ａｃ−ＤＬＲＳＫ−Ａｈｘ−Ｄｏｘ、−ＤＬＲＳＫ−部分環状）：
計算した分子質量＝１２７９．６０
観察したシグナル：
１２８０．２９Ｍ＋１ Ac-DLRSK-Ahx-Dox formula:

Substances used:
Doxorubicin hydrochloride, CAS No. 25316-40-9, molecular weight: 580.0 g / mol, Sigma catalog number D-1515.
MALDI-TOF data (Ac-DLRSK-Ahx-Dox, -DLRSK-partial ring):
Calculated molecular mass = 1279.60
Observed signal:
1280.29M + 1

It contains the effector unit 4-amino-10 methylfolic acid linked to the N-terminal amino group of the peptide AhxDLRSK via its carboxyl group by an amide bond, and is cyclic by a lactam bridge between the side chains of the outermost component of the sequence. Synthesis of a targeting agent Amf-AhxDLRSK [Amf = 4-amino-10 methyl folate acyl], which also contains a targeting unit DLRSK A resin-bound spacer (= Ahx) containing a targeting unit (AhxDLRSK) Prepared as described in Example 9 above, including cyclization (according to Example 21). Next, the general coupling technique described in Example 2 was used to keep the sequence AhxDLRSK on the resin using glutamic acid. Upon final coupling, the sequence (here E-AhxDLRSK, “E” becomes part of the “Amf” part) is converted to “Amf-E acid” by the general coupling technique described in Example 2. Ie, 4- [N- (2,4-diamino-6-pteridinyl-methyl) -N-methylamino] -benzoic acid, hemihydrate chloride dihydrate. However, in processing step 12 of Example 2, PyAOP (instead of HBTU and HOBt) as an activating reagent, a reaction time of 5 hours, and an approximately equimolar ratio of the reagent and the resin-bound peptide.

The ratio of stoichiometric reagents for this step was as follows:
“Resin-bound peptide” / “Amf-E acid” /PyAOP/DIPEA=1:1.2:1.2:2.4 (time 5 hours).

  After isolation and purification according to Example 2, the product was identified based on the M + 1 ion in the positive ion mode MALDI mass spectrum.

Reagents used:
Fmoc-L-Glu (OtBu) -OH, CAS No. 71989-18-9, Applied Biosystems catalog number GEN911036, molecular weight: 425.5 g / mol.
4- [N- (2,4-Diamino-6-pteridinyl-methyl) -N-methylamino] -benzoic acid, hemihydrate chloride dihydrate, CAS No. 1974-14-1, Aldrich catalog number 86, 155-3, molecular weight: 379.59 g / mol, indicated as “Amf-E acid”.

MALDI-TOF data (Amf-AhxDLRSK, ring):
Calculated molecular mass = 1148.58
Observed signal:
1149.62M + H

この実施例の最後に記載する、パクリタクセルスクシネートを、０．０１２Ｍ溶液としてＤＭＦに溶かし、等モル量の０．０５ＭのＰｙＡＯＰをＤＭＦに溶かしたものを加え、次に２倍モル量の２．０ＭのＤＩＰＥＡをＮＭＰに溶かしたものを加えた。２分後に、（パクリタクセルスクシネート当たり）等モル量の、上記の実施例９に記載した、側鎖−側鎖環状標的化化合物ＡｈｘＤＬＲＳＫを、０．０１５Ｍ溶液としてＤＭＦに加えた。一晩放置した後、混合物をジエチルエーテルで希釈した。遠心分離にかけた固形沈殿を、正イオンモードＭＡＬＤＩ−ＴＯＦ質量スペクトルにおけるそのＭ＋１イオンに基づく生成物の同定を含めて、実施例２に記載したのと同様の逆相ＨＰＬＣクロマトグラフィーによって精製した。
ＭＡＬＤＩ−ＴＯＦデータ（ＰｔｘＳｕｃ−ＡｈｘＤＬＲＳＫ、環状）：
計算した分子質量＝１６４７．７６
観察したシグナル：
１６４８．５７Ｍ＋１ Containing the effector unit paclitaxel as monosuccinate, linked to the amino group via its succinyl (carbonyl succinate) group in the 6-aminohexanoyl (= Ahx) part of the peptide AhxDLRSK by an amide bond, and the outermost component of the sequence The targeting agent PtxSuc-AhxDLRSK (PtxSuc = paclitaxel monosuccinate), which also includes the targeting unit DLRSK, which is cyclic by an amide bond between side chains (or the effector unit paclitaxel is a spacer unit 6- (succinylamino) -Targeting agent linked to targeting unit DLRSK by hexanoyl)

The paclitaxel succinate described at the end of this example was dissolved in DMF as a 0.012M solution, equimolar amounts of 0.05M PyAOP dissolved in DMF were added, and then a 2-fold molar amount. 2.0M DIPEA dissolved in NMP was added. After 2 minutes, an equimolar amount (per paclitaxel succinate) of the side-chain cyclic targeting compound AhxDLRSK described in Example 9 above was added to DMF as a 0.015 M solution. After standing overnight, the mixture was diluted with diethyl ether. The centrifuged solid precipitate was purified by reverse phase HPLC chromatography similar to that described in Example 2, including identification of the product based on its M + 1 ion in the positive ion mode MALDI-TOF mass spectrum.
MALDI-TOF data (PtxSuc-AhxDLRSK, ring):
Calculated molecular mass = 1647.76
Observed signal:
1648.57M + 1

  Paclitaxel succinate is described in the following literature: Chun-Ming Huang, Ying-Ta Wu and Shui-Tein Chen (2000). "Targeting delivery of paclitaxel into tumor cells via somatostatin receptor endocytosis" by endocytosis of somatostatin receptors. "Targeting delivery of paclitaxel into tumor cells via somatostatin receptor endocytosis". Synthesized by following the procedure described in Chemistry & Biology 2000, Vol 7 No 7.453-461.

  Here, 0.05 M paclitaxel dissolved in pyridine was stirred with 12-fold excess of succinic anhydride for 3 hours. After evaporating the solvent under reduced pressure, the residue was dissolved in water and freeze-dried (lyophilized).

Materials used in the synthesis of paclitaxel succinate:
Paclitaxel (from Tacsus yarns), CAS No. 33069-62-4, molecular weight: 853.9 g / mol, Sigma product number T-1912.
Succinic anhydride, CAS No. 108-30-5, molecular weight: 100.08 g / mol Fuka product number 14089.

この実施例の最後に記載する、カンプトテシンｐ−ニトロフェニルカルボネートを、０．０２Ｍ溶液としてＤＭＦに溶かし、同じ溶媒の上記の実施例９に記載した等モル量の環状標的化化合物ＡｈｘＤＬＲＳＫの０．０４Ｍ溶液と組み合わせた。一晩放置した後、２ＭのＤＩＰＥＡをＮＭＰに溶かしたものを、１０％過剰に（即ち等モル量の１．１倍）加えた。一晩攪拌した後、混合物をジエチルエーテルで希釈し、遠心分離にかけた固形沈殿を、正イオンモードＭＡＬＤＩ−ＴＯＦ質量スペクトルにおけるそのＭ＋１イオンに基づく生成物の同定を含めて、実施例２に記載したのと同様の逆相ＨＰＬＣクロマトグラフィーによって精製した。
ＭＡＬＤＩ−ＴＯＦデータ（Ｃｐｔｃ−ＡｈｘＤＬＲＳＫ、環状）：
計算した分子質量＝１０８６．５１
観察したシグナル：
１０８７．２６Ｍ＋１ A target comprising an effector unit camptothecin carbonate, linked to an amino group in the 6-aminohexanoyl (= Ahx) portion of the peptide AhxDLRSK by an amide bond, cyclic by the amide bond between the side chains of the outermost components of the sequence The targeting agent Cptc-AhxDLRSK [Cptc = carbonyl acyl, ie (S)-(+)-linked as an ester at its hydroxyl group via the (S)-(+)-camptothecin carbonyl moiety) Camptothecin] (or targeting agent in which effector unit (S)-(+)-camptothecin is linked to targeting unit DLRSK by spacer unit 6- (carbonylamino) -hexanoyl)

Camptothecin p-nitrophenyl carbonate, described at the end of this example, was dissolved in DMF as a 0.02M solution and 0. 0 of equimolar amounts of the cyclic targeting compound AhxDLRSK described in Example 9 above in the same solvent. Combined with 04M solution. After standing overnight, 2M DIPEA in NMP was added in 10% excess (ie 1.1 times the equimolar amount). After stirring overnight, the mixture was diluted with diethyl ether and the solid precipitate centrifuged was described in Example 2, including identification of the product based on its M + 1 ion in the positive ion mode MALDI-TOF mass spectrum. Purified by reverse phase HPLC chromatography as in.
MALDI-TOF data (Cptc-AhxDLRSK, circular):
Calculated molecular mass = 1086.51
Observed signal:
1087.26M + 1

  Synthesis of camptothecin p-nitrophenyl carbonate: 0.29 mmol 4-nitrophenyl chloroformate and 0.10 mmol (S)-(+)-camptothecin were dissolved in 12 mL dichloromethane (DCM). Next, 1.71 mmol of 4- (dimethylamino) -pyridine (DMAP) was added to the DCM solution in the cold water bath. The mixture was then shaken for 2 hours and then diluted with 30 mL DCM. After washing twice with 0.1% hydrochloric acid and once with saturated aqueous sodium chloride solution: the DCM solution was dried with disodium sulfate, filtered and concentrated to a small volume. The product was precipitated by adding diethyl ether and collected after centrifugation.

Materials used in the synthesis of camptothecin p-nitrophenyl carbonate:
4-nitrophenyl chloroformate, GAS No. 4 7693-46-1, molecular weight: 201.57 g / mol, Fluka product number 23240
(S)-(+)-camptothecin, CAS No. 7689-03-4, molecular weight: 348.36 g / mol, Aldrich product number 36,563-7
DMAP; 4-dimethylaminopyridine, CAS No. 1122-58-3, molecular weight: 122.17 g / mol, Fluka product number 29224

It contains a targeting unit “DLRSGRGK” and a potential metal ligand group “DOTA” linked via a spacer unit, 6-aminohexanoyl (= Ahx) as an effector, and thus an aspartic acid moiety (“D”) And the lysine ("K") side chain, the targeting agent DOTA-AhxDLRSGRGK-amide (DOTA = 1,4,7,10-tetraazacyclododecane-1, linked by its one carboxyl, 4,7,10-tetraacetic acid). Any of the linkages required for the aforementioned combinations are functionally protected, resin-bound targeting units (protected peptides) containing an amide bond targeting motif LRS by manual synthesis as described in Example 2 above. The “empty” resin was synthesized and deprotected prior to the first coupling in the same manner as described for the pre-filled resin (steps 1-11 of Example 2).

The following reagents were used as starting materials (in this order):
Rink amide MBHA resin Fmoc-L-Lys (Mtt) -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-L-Asp (2-phenylisopropyl ester) -OH

After these coupling cycles, the targeting units that were still resin bonded were subjected to a cyclization process that formed excess amide bonds as described in Example 17. Then, two more coupling cycles were performed as described in Example 2 above, using the following starting materials:
Fmoc-6-aminohexanoic acid DOTA-tris- (tBu ester).

Finally, the resin was cleaved and the product purified as described in Example 2 and identified by its M + 1 ion in the positive ion mode MALDI-TOF mass spectrum.
MALDI-TOF data (Dota-AhxDLRSGRGK-amide):
Calculated molecular mass = 1367.76
Observed signal:
1368.70M + 1

The targeting unit “DLRSGRKK” contains a gadolinium atom as a detectable effector linked by a chelating agent “DOTA” via a spacer unit, 6-aminohexanoyl (= Ahx), and thus an aspartic acid moiety (“D”). ) And lysine ("K") side chains, the targeting agent Gd-DOTA-AhxDLRSGRGK-amide (Gd = chelated gadolinium ^III , DOTA = 1,4,7,10-tetraazacyclododecane-1) , 4,7,10-tetraacetic acid—3 hydrogens, linked by one acyl to form an amide bond) 14.9 mg of the chelator-peptide compound described in Example 44, and 24 mg Of gadolinium trichloride hydrate (5-fold excess) in 1 mL of aqueous 0.05 M ammonium acetate It was dissolved in Umm aqueous solution. The next day, the solution was subjected to reverse phase HPLC purification similar to that described in Example 2. The exception is 0.05M ammonium acetate as a buffered aqueous solution (instead of an aqueous TFA solution). Identification was by positive ion mode MALDI-TOF mass spectrum on a neutral substrate (without TFA). The purified yield was 9.4 mg.
MALDI-TOF data:
Calculated molecular mass = 1522.26 based on the most abundant isotope
Observed signal M + 1: 1523.66 as a typical isotope pattern

List of Reagents 4-Amino-10 methyl folic acid; (+) amethopterin; methotrexate hydrate; formula weight: 454.4 g / mol, CAS No. 59-05-2, Sigma A-6770
Boc-amino-oxyacetic acid; Boc-NH-OCH2-COON, molecular weight: 191.2 g / mol, CAS No. Novabiochem catalog number 01-63-0060
Boc-Cys (Trt) -OH, CAS No: 21947-98-8, Novabiochem, catalog number 04-12-0020
D-biotin (vitamin H), CAS No. 58-85-5, molecular weight: 244.3, Sigma B-4501, 99%
DL-2,3-diaminopropionic acid monohydrochloride, CAS No. 54897-59-5, C3H8N2O2. HCl, Acros Organics (New Jersey, USA; Hale, Belgium), catalog number 204670050
Diethylenetriaminepentaacetic acid dihydrate, CAS No. 23911-26-4, molecular weight: 357.32, Aldrich catalog number 28, 402-5
Fmoc-6-aminohexanoic acid (Fmoc-6-Ahx-OH), CAS No. 88574-06-6-5
Fmoc-Asp (2-phenylisopropyl ester) -OH, molecular weight: 473.53 g / mol, Bachem catalog number B-2475.0005
Fmoc-L-Asn-OH, CAS No. 71089-16-7, Applied Biosystems, catalog number: GEN9111018
Fmoc-Gly resin, Applied Biosystems catalog number 401421 0.65 mmol / g
Fmoc-Gly-OH, CAS No. 29022-11-5, Novabiochem catalog number 04-12-1001, molecular weight: 297.3 g / mol
Fmoc-L-Asn-OH, Applied Biosystems catalog number Gen 9111018, molecular weight: 354.40
Fmoc-L-Arg (Pbf) -OH, CAS No. 154445-77-9, Applied Biosystems catalog number GEN911097, molecular weight: 648.8 g / mol.
Fmoc-L-Cys (Trt) -OH, CAS No. 103213-32-7, Applied Biosystems catalog number GEN911027, molecular weight: 585.7 g / mol
Fmoc-L-Leu-OH, CAS No. 35661-60-0, Applied Biosystems catalog number GEN9111048, molecular weight: 353.4 g / mol
Fmoc-L-Lys (Fmoc) -OH, CAS No. 78081-87-5, molecular weight: 590.7 g / mol, PerSeptive Biosystems, catalog number GEN911095, Hamburg, Germany Fmoc-Lys (Mtt) resin, 0.68 mmol / g, Bachem catalog number D-2565.0005
Fmoc-L-Lys (tBoc) -OH, CAS No. 71989-26-9, molecular weight: 468.6 g / mol, Applied Biosystems catalog number GEN911051.
Fmoc-Ser (tBu) resin, Applied Biosystems catalog number 401429, 0.64 mmol / g
Fmoc-L-Ser (tBu) -OH, CAS No. 71989-33-8, Perseptive Biosystems catalog number GEN911062, molecular weight: 383.4 g / mol.
Fmoc-L-Tyr (tBu) -OH, Applied Biosystems catalog number GEN911068
CAS No. 71989-38-3, M.M. W. 459.5

List of suppliers Applied Biosystems, Warrington, WA14SR, UK Bachem AG, Hauptstrasse (Hautopstraße) 144, CH-4416, Bubendorf, Switzerland Calbiochem-Novabiochem, CH-4448, Laufelfingen Hb Switzerland Merck KGaA, Darmstadt, Germany PE Biosystems, Warrington, UK Perseptive Biosystems, Warrington, UK / Hamburg, Germany Sigma Aldrich Chemie, Steinheim, Germany (also Riedel-deHaen)
Sigma-Genosys LTD, Pampsford, Cambridgeshire, UK Bio-Whittaker, Berbie, Belgium Harlan Laboratories, Horst, Genset SA, Paris, France , Burlingame, usa

It is a graph which shows the therapeutic effect of the targeting agent containing doxorubicin.

[Sequence Listing]

Claims (26)

Peptide sequence:
_{Cy-Rr n -Dd-Ee-} Ff-Rr m -Cyy
A tumor targeting unit comprising, or a pharmaceutically or physiologically acceptable salt thereof,
Where Dd-Ee-Ff is Aa-Bb-Cc or Cc-Bb-Aa;
Aa is isoleucine, leucine or tert-leucine, or a structural or functional analog thereof;
Bb is arginine, homoarginine or canavanine, or a structural or functional analog thereof;
Cc is serine or homoserine, or a structural or functional analog thereof;
Each Rr is independently any amino acid residue, or a structural or functional analog thereof;
n and m are each independently an integer of 0 to 4, and the sum of n and m does not exceed 4, and
A tumor targeting unit, or a pharmaceutically or physiologically acceptable salt thereof, wherein Cy and Cyy are entities capable of forming a cyclic structure through an amide or ester bond.   The tumor targeting unit according to claim 1, wherein the peptide is cyclic or forms part of a cyclic structure.   The tumor targeting unit according to claim 2, wherein the cyclic structure is formed by a lactam or lactone bond.   The tumor target according to claim 3, wherein one of Cy and Cyy is aspartic acid, glutamic acid, or a structural analog or functional analog thereof, and the other is lysine, ornithine, or a structural analog or functional analog thereof. Unit.   The tumor targeting unit according to any one of claims 1 to 4, wherein the sum of n and m is 2 to 4.   The tumor targeting unit according to any one of claims 1 to 4, wherein n or m or both are 0.   The tumor targeting unit according to any one of claims 1 to 6, wherein Rr is any amino acid residue other than histidine or lysine.   The tumor targeting unit according to claim 7, wherein Rr is selected from the group consisting of glycine, arginine, and structural or functional analogs thereof.   The tumor targeting unit according to any one of claims 1 to 8, wherein Dd-Ee-Ff is LRS or SRL.   Formula 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) DLRSGRRGK (SEQ ID NO: 7), OLRSE The tumor targeting unit of claim 9, comprising (SEQ ID NO: 8), OLRSGRGE (SEQ ID NO: 9) or KLRSD (SEQ ID NO: 10).   The tumor targeting unit according to any one of claims 1 to 10, wherein the unit is derivatized, activated, protected, resin bound, or other carrier bound state.   A tumor targeting agent comprising at least one targeting unit according to any one of claims 1 to 11 bound to at least one effector unit.   The tumor targeting agent according to claim 12, wherein the effector unit is a drug or therapeutic agent that can be detected directly or indirectly.   15. The tumor targeting agent of claim 14, wherein the detectable agent comprises an affinity label, a fluorescent or luminescent label, a chelating agent, a metal complex, a concentrated isotope, a radioactive substance or a paramagnetic substance.   16. The tumor targeting agent of claim 15, wherein the detectable agent is a rare earth metal.   The tumor targeting agent according to claim 13, wherein the therapeutic agent is selected from the group consisting of cytotoxic and cytostatic substances and radiation-emitting substances.   21. The tumor targeting agent of claim 20, wherein the detectable agent is gadolinium.   18. The tumor targeting agent of claim 17, wherein the therapeutic agent comprises doxorubicin, daunorubicin, methotrexate or boron.   The tumor targeting agent according to any one of claims 12 to 18, further comprising an optional unit.   A diagnostic or pharmaceutical composition comprising at least one targeting unit according to any one of claims 1 to 11, or at least one targeting agent according to any one of claims 12 to 19. object.   The targeting unit according to any one of claims 1 to 11 or the targeting according to any one of claims 12 to 19 for the preparation of a medicament for treating cancer or cancer-related diseases. Agent use.   The use according to claim 21, wherein the cancer or cancer-related disease is a solid tumor.   23. Use according to claim 22, wherein the cancer is selected from the group consisting of carcinoma, sarcoma, melanoma or metastasis.   21. A method for treating cancer or cancer-related diseases comprising providing a patient in need with a therapeutically effective amount of the pharmaceutical composition of claim 20.   25. The method of claim 24, wherein the cancer or cancer-related disease is a solid tumor.   26. The method of claim 25, wherein the solid tumor is selected from the group consisting of carcinoma, sarcoma, melanoma or metastasis.

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