JP2007503382A - Possible activation of high molecular weight prodrugs - Google Patents

Abstract

The present invention eliminates steric hindrance while maintaining high action specificity, low toxicity, and stability in blood and / or serum, preferably mammals, when masking groups and / or stabilizing groups are large. Prodrug structures that allow or facilitate the cleavage of oligopeptides are provided.
A “molecular arm” or “molecular spacer” is inserted between a masking group and / or a stabilizing group (eg, PEG).
[Selection] Figure 1

Description

  The present invention relates to the field of prodrugs, and more particularly to the field of prodrugs intended for the treatment and / or diagnosis of cancerous tumors and / or inflammatory responses.

  Prodrugs are pharmacologically inactive molecules that can be converted in vivo to pharmaceuticals (active therapeutic agents) after chemical or enzymatic modification of their structure. A prodrug is capable of releasing a pharmaceutical product, i.e., a pharmaceutical product resulting from the conversion of the prodrug, to a site of action or target tissue rather than to circulating tissue or non-target tissue. Prodrugs also improve the in vivo therapeutic index (ratio of activity to toxicity) of anticancer drugs such as anthracyclines (eg doxorubicin) and vinca alkaloids or anti-cancer drugs such as methotrexate that have anti-inflammatory effects. Can do. Therefore, several types of prodrugs have been developed so far in order to obtain high action specificity in blood and / or serum, low toxicity and improved stability.

  Conventionally, prodrugs having the basic structure shown below for therapeutic agents, oligopeptides that can be cleaved by enzymes present in the extracellular environment of target cells, stabilizing groups or masking groups have been described.

  The PCT patent specification of WO 96/05863 specifically describes β-alanyl-leucyl-alanyl-leucyl-doxorubicin (also referred to as βALA-LEU-ALA-LEU-Dox or βALAL-Dox). This prodrug is stable in blood, that is, it is relatively difficult to cleave with peptidases contained in blood, and is reactivated in vivo with peptidases secreted from the periphery of various tumor cells. This prodrug is continuously hydrolyzed in the extracellular environment around the tumor to become Ala-Leu-Dox, and further to Leu-Dox. Leu-Dox enters the cell by diffusion, where it is activated to the doxorubicin form (Trouet et al., 2001). Examination of the in vivo toxicity and activity of the prodrug reveals that toxicity is reduced and tumor growth is significantly suppressed as compared to doxorubicin itself. However, pharmacokinetic studies show that the half-life for renal excretion is short. This prodrug is thought to be rapidly excreted via the urinary tract (Dubois et al., 2002).

  The PCT patent specification of WO 00/33888 is a proposal to increase its effectiveness by adding a group that masks the positive charge of β-alanine to a prodrug represented by βAla-Leu-Ala-Leu-Dox. I am doing. This masking group may be, for example, polyethylene glycol (PEG).

  The PCT patent specification of WO 01/91798 describes prodrugs that have improved stability in the circulating tissue. For example, the prodrug may be PEGylated, in which case PEG is used as a stabilizing group and / or masking group. This polymer (PEG) linkage improves the pharmacokinetic and pharmacodynamic properties of the prodrug and reduces the renal excretion due to the larger molecule. In fact, the larger the molecule, the slower its excretion (Harris & Chess, 2003).

Within the framework of research leading to the present invention, Applicants have used PEGs of different sizes to produce PEGylated prodrugs from doxorubicin prodrugs (described in WO 96/05863). Attempts were made to maintain and maintain the properties of the prodrug (reactivated by enzymes secreted from tumor cells) while reducing ultrafiltration in the kidney by increasing the compound. Applicants have conjugated PEGs of various sizes (molecular weights 350-20,000, with 750, 2000 and 5000 in between) to the prodrug βAla-Leu-Ala-Leu-Dox. To examine the reactivation of the PEGylated derivative of this prodrug, an in vitro cleavage test was performed. The purpose of this study was to re-activate PEGylated derivatives by enzymes secreted from tumor cells (LS-174T and MCF-7 / 6) into Leu-Dox in the presence of conditioned medium containing cancer cells. It is to evaluate in comparison with βAla-Leu-Ala-Leu-Dox which is decomposed. From these test results, 1) Regardless of the size of PEG, reactivation of the drug by cleavage of the peptide sequence (Ala-Leu-Ala-Leu) is not as effective as a prodrug without a PEG group, 2) Correlation between PEG size and cleavage of PEGylated prodrug: It was found that the larger the PEG attached, the less prodrug cleavage by the enzyme in the extracellular environment of the target cell. Applicants hypothesized that the reduction in peptide bond cleavage of the prodrug is probably due to the steric hindrance phenomenon of PEG. In other words, prodrugs containing higher molecular weight stabilizing groups or masking groups are less reactivated, which contradicts the challenge of providing prodrugs with longer half-lives.
International Publication No. 96/05863 International Publication No. 00/33888 International Publication No. 01/91798

  In particular, the present invention provides steric hindrance for large masking and / or stabilizing groups, while maintaining high action specificity, low toxicity, and stability in blood and / or serum, preferably mammals. The object is to provide a prodrug structure that allows or facilitates the cleavage of oligopeptides.

  We insert a “molecular arm” or “molecular spacer” between the masking group and / or stabilizing group (eg PEG) and this peptide sequence can be cleaved by an enzyme “specific” to the sequence. As a result, the present invention has been completed.

  The molecular spacer of the present invention (hereinafter also referred to as “spacer”) was selected in consideration of the hydrophilicity of the units constituting the spacer.

Therefore, the first subject of the present invention is the formula (A) _p- (EB) _n- (I) _m :
(Where
-I is an active substance that favors target cells,
-A is a group that extends the half-life of BI during blood circulation;
-EB is a group for linking A and I;
-B is a structure that can be selectively cleaved by an enzyme present alone or preferably by an enzyme present near or on the target cell;
-E is a hydrophilic group that is stable in the circulating tissue, which allows or facilitates cleavage of B near or on the target cell by pulling A away from B; As a result, release of I or release of I with the group B is possible or easy.
-N is an integer from 1 to the total number of reactive groups I to which the linking group EB can bind, or up to the total number of reactive groups A to which the linking group EB can bind,
-M is an integer from 1 to the total number of reactive groups A to which the linking group EB can bind,
-P is an integer from 1 to the total number of reactive groups I to which the linking group EB can bind,
(If p = 1, n = m; if m = 1, n = p)
It is a compound shown by these.

  Hereinafter, embodiments of the present invention will be described. The first preferred embodiment is a case where p, n and m are equal to 1. This compound is hereinafter referred to as formula A-E-B-I.

A second preferred form of the invention is when m is equal to 1 and n and p are equal and greater than 1. This compound is hereinafter referred to as formula (A-E-B) _{t> 1-} I, where t means an integer from 2 to the total number of reactive groups I to which the group A-E-B- can be bound. . Advantageously, I may be a polypeptide such as a TNF-α cytokine molecule in which several AEB groups are branched on the molecule.

A third preferred form of the invention is when p is equal to 1, and n and m are equal and greater than 1. This compound is hereinafter referred to as formula A- (E-B-I) _{k> 1} , where k is an integer from 2 to the total number of reactive groups A to which the group -E-B-I can be bound. . Advantageously, A may be a polymer such as a branched PEG molecule in which several -E-B-I groups are branched on the molecule.

  A fourth preferred form of the invention is when p and m are greater than 1 and n may be different from p and m. For example, when p = 2, n = 3, and m = 2, the compound can be represented by the following formula: (I)-(BE)-(A)-(EB)-(I)-(B -E)-(A).

  Advantageous active substances (I) may be directly linked to one or more structures B by covalent bonds or indirectly via a “link arm”. For example, if structure B is an amino acid sequence that is directly linked to the advantageous substance I, the N-terminal or C-terminal end of the amino acid sequence, or any part of the oligopeptide (eg one amino acid), depending on its orientation A covalent bond can be formed on the side chain of Furthermore, when the bond between B and I is indirect, the linking arm may have several functional groups that facilitate cleavage between BI and between BI. Suitable chemical coupling means are provided, resulting in an improvement in the synthesis process of the compound, improving the physical properties of the advantageous substance (I), additional to the intracellular or extracellular release of the advantageous substance (I) Mechanisms are provided. Such indirect coupling can be performed by any chemical, biochemical, enzymatic or genetic coupling method known to those skilled in the art. Such linking arms include, for example, cross-linking reagents having the same or different functionality, such as succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC); bifunctional having an alkyl, aryl, aralkyl or peptide group Or polyfunctional agents; esters, aldehydes or alkyls, aryl or arylalkyl acids; anhydrides, sulfhydryl groups or carboxyl groups such as maleyl benzoate, maleyl propionate and succinimidyl derivatives; bromocyan or chlorocyan derivatives; carbonyldiimidazole , Thiocarbonyldiimidazole of succinimide ester or sulfonic acid halide; phosgene, thiophosgene; self-relocating (or “self-sacrificing”) space Can be given over (Schmidt et al., 2001).

  The spacer (E) of the present invention links group A to structure B. The spacer is preferably hydrophilic and stable in the circulating tissue.

  When less than 20%, preferably less than 10%, more preferably less than 2% of the spacer is degraded or cleaved in the circulating blood (especially by enzymes), or in human blood at 37 ° C. the spacer exceeds 2 hours When maintained, the spacer is said to be “stable in the circulatory tissue”. Advantageously, because of the separation of group A from structure B and its favorable hydrophilic character, the spacer allows or facilitates structure B to be cleaved near or on the target cell; This allows or facilitates the release of I or the release of I having a B group. The spacer may have a length corresponding to 1 to 100 amino acids.

  As another embodiment of the present invention, the size of the spacer can be changed according to the molecular weight of the group A. In this embodiment, the larger the spacer, the greater the molecular weight of A.

  The spacer of the present invention is composed of or comprises at least one group selected from: amino acid sequence; peptidomimetic agent; pseudopeptide; peptoid; substituted alkyl, aryl or arylalkyl chain; Polyalkyl glycols; polysaccharides; polyols; polycarboxylates; and poly (hydro) esters. The spacer may contain a combination of at least two of these groups.

  In an advantageous form of the invention, the spacer is a conformational D natural amino acid, a non-genetically encoded amino acid or a synthetic amino acid that cannot be cleaved by an enzyme present in the circulating tissue, such as a β amino acid or a γ amino acid. It consists of 1 to 100, preferably 1 to 20, very preferably 2 to 10 homologous or heterologous amino acids selected from the group comprising or contains such amino acids. “Conformation D natural amino acid” means an amino acid normally encoded in the genetic code, and is not an amino acid synthesized to conformation D despite being conformation L in nature. . In general, amino acids that are not genetically encoded can be produced by synthesis or derived from natural products.

  Preferred among the naturally occurring amino acids in conformation D are hydrophilic amino acids selected from the following: D-glutamine, D-asparagine, D-aspartic acid, D-glutamic acid, D-lysine, D-arginine, D Histidine, particularly preferably D-serine and D-threonine.

  In a preferred method of the invention, the spacer is composed of or comprises a homologous amino acid sequence selected from: (D-serine) x or (D-threonine) x, where x is An integer of 1-20, preferably an integer of 2-10, very preferably an integer of 2-6.

In particular, the spacer is:
(D-serine)-(D-serine)-(D-serine)-(D-serine), which is the same as D-seryl-D-seryl-D-seryl-D-seryl. ,
(D-threonine)-(D-threonine)-(D-threonine)-(D-threonine), which is the same as D-threonyl-D-threonyl-D-threonyl-D-threonyl- is there.

  In the present invention, amino acids are represented by either a three-letter code or a one-letter code known to those skilled in the art.

  Group A is a group that prolongs the half-life of BI in the circulating tissue in vivo. This task is achieved especially when A reduces the renal excretion of the preferred substance I or compound BI, where excretion means the ultrafiltration of the compound in the kidney according to size. . Thus, the larger the compound, the slower the excretion, and the compound having a molecular weight of at least 50,000 daltons is not excreted by the kidneys. This task can also be achieved by reducing the degradation of the compounds of the invention due to hepatic metabolism. In other words, to increase the half-life, the mean residence time of the compound in blood can be increased or the blood or plasma clearance can be decreased.

  “Circulating tissue” means bodily fluids, particularly blood, preferably mammalian circulating tissue.

  The group A is preferably hydrophilic or amphiphilic.

Less than 20%, preferably less than 2% of the compounds of the formula A which are stable in the circulatory tissue (i.e. the formula (A) _p- (EB) _n- (I) _m ) When enzymatically degraded or cleaved, the compound is maintained in human blood at 37 ° C. for more than 2 hours), which is non-toxic to normal cells, non-immunogenic, non- It is preferably aggregating or masking (ie preventing the beneficial substance (I) from acting on the cell surface until the beneficial substance (I) is released from the prodrug).

Advantageously, the group A may have one or more of the following properties: preventing non-specific cleavage and / or degradation of the linking group E-B; until the advantageous substance is released from the prodrug Inhibits the biological action of advantageous substances; improves the stability of the compound in the circulating tissue; formula (A) _p- (EB) _n- (I in water, blood and / or serum ) Solubility (or increase solubility) of _m compounds; Targeting properties (or enhance targeting) of compounds of formula (A) _p- (EB) _n- (I) _m to target cells.

“Targeting property of a compound represented by the formula (A) _p- (EB) _n- (I) _m to a target cell” means that the group A is represented by the formula (A) _p- (EB) _n- (I) Means allowing accumulation of _m compounds near or on the target cell. Such a group A is said to be “biologically specific”, ie, such a group A can develop specific biological interactions, so Recognized as In particular, by grafting a peptide such as an antibody, antigen or amino acid group (eg arginine-glycine-aspartic acid (RGD)) to the surface of the group A, the compounds of the invention can be attached to the surface of a particular cell line. Can be increased selectively. The group A may also include biological specific copolymers obtained by functionalizing existing polymer chains with suitable chemical groups for identification purposes or obtained by copolymerizing functional monomers.

  In particular, the group A is selected from: a polypeptide (eg polyglutamate, polyaspartate), immunoglobulin, albumin, polysaccharide, polymer or copolymer.

  Polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxy-ethyl-aspanamide-phenol, poly (ethylene oxide) -polylysine substituted with palmitoyl residues, poly (lactic acid), poly (epsilon) -Caprolactone), poly (hydroxybutyric acid), polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinkable or amphiphilic arrayed hydrogel copolymers.

  The group A is preferably selected from the following: polyalkylene glycols, polyalkylene oxides, polyalkyleneimines and vinyl chloride copolymers. For example, if the group A is a vinyl chloride copolymer, the presence of a sulfonate group or sulfonate and a carboxylate is essential so that the polymer does not exhibit aggregating activity.

  The group A is also preferably selected from: polyethylene oxide, polyethyleneimine, sodium styrene sulfonate (NaSS), sodium maleate and butyl maleate (MMBE), hydroxypropyl methacrylate or N- (2-hydroxypropyl) ) Methacrylamide (HPMA), Methyl methacrylate (MMA), Poly- [N- (2-hydroxyethyl) -L-glutamine] (PHEG), and Poly- [N- (hydroxyethyl) -DL-aspartamide] (PHEA).

The group A is particularly preferably polyethylene glycol (PEG). The size of the PEG may be 200 to 50,000 Da, preferably 350 to 20,000 Da, very preferably 1,000 to 10,000 Da. The presence of such PEG improves pharmacokinetic properties (Duncan et al., 1994) and pharmacodynamic properties, and as a result can reduce renal excretion of the compounds of the invention. Yet another advantage known in the prior art is that PEG can accumulate well within the tumor. In fact, PEG having a molecular weight of 10 ⁴ Da or more is known to accumulate more significantly in tumors than normal tissues (Greenwald et al., 2003, Seymour et al., 1995).

  Group A may also be a drug with therapeutic activity or a drug with diagnostic activity.

  Advantageously, the group A exhibiting the properties of a diagnostically active agent is a paramagnetic element, ie an element having a single electron in its electron layer, in particular gadolinium, which enhances contrast in magnetic resonance imaging (MRI). May have elements such as manganese and iron. An example is iron-bonded polystyrene sulfonate.

A compound using one or more targeting substances capable of directing the traveling direction of the compound represented by the formula (A) _p- (EB) _n- (I) _m to the target cell is also an object of the present invention. is there. For example, targeting agents can be antibodies, antigens and liposomes.

The targeting substance is preferably bound to the compound of formula (A) _p- (EB) _n- (I) _m with one or more groups A.

  In the present invention, structure B can be selectively cleaved by an enzyme that is present alone or preferably present in the target cell environment.

  “Target cell” means in particular a cell that exhibits pathological involvement or a cell that has advantages in terms of treatment or diagnosis. Such target cells may be primary or secondary (metastatic) tumor cells, stromal cells of primary or secondary tumors, angiogenic endothelial cells of tumors or metastatic tumors, macrophages, monocytes, lymphocytes or tumors And preferably selected from the group consisting of polynuclear bodies capable of invading metastatic tumors.

  “Selectively cleavable” means in particular that the sequence to be cleaved is cleaved. In other words, the sequence to be cleaved is desirably recognized by an enzyme present in the target cell environment and is slightly degraded or not degraded at all in the circulating tissue and near the non-target cell. The expression “in the target cell environment” means that the enzyme is present alone or near or on the target cell. Even if the cleavage does not occur near or only on the target cell, the cleavage occurs rather (or in most cases or in most cases) near or on the target cell. Note that cutting is considered optional if there is a fact that it is. In other words, cleavage is said to be selective when the enzyme is at a high concentration near or on the target cell relative to other parts of the body.

  Furthermore, “enzyme” means in particular a hydrolase. The enzyme may be selected from the group consisting of peptidases, endopeptidases, lysosomal enzymes, lipases and glycosidases.

  In a preferred method of the invention, the enzyme is a peptidase specific for tumor cells, tumor stromal cells, angiogenic endothelial cells, macrophages or monocytes. By “specific enzyme” is meant an enzyme that is a membrane enzyme or is secreted only from the target cell or mostly to the extracellular medium containing the target cell. For example, if the enzyme is specific for tumor cells, the latter is from neprilysin (CD10), thimetrigopeptidase (TOP), prostate specific antigen (PSA), plasmin, legumain, collagenase, urokinase, cathepsin, and the substrate metallopeptidase. It may be selected from the group consisting of:

  The choice of structure B will of course depend on the enzyme present in the target cell environment. Thus, if the enzyme is a peptidase, structure B is considered to contain an amino acid sequence (or oligopeptide) that can be cleaved by that peptidase; if the enzyme is a glycosidase (eg, a saccharase), structure B is cleaved by that glycosidase. If the enzyme is a lipase, structure B is believed to contain a lipid chain that can be cleaved by the lipase, etc.

The subject of the present invention is a preferred structure B composed of or comprising an oligopeptide that can be selectively cleaved by an enzyme present in the tumor cell environment. Such oligopeptides preferably contain 2 to 10 amino acids, more preferably 3 to 7 amino acids. For example, the subject of the present invention is the following sequence (conformation L is preferred):
Ala-Phe-Lys (SEQ ID No. 1), Ala-Leu-Ala-Leu (SEQ ID No. 2) or βAla-Leu-Ala-Leu, Ala-Leu-Lys-Leu-Leu (SEQ ID No. 1) 3), Ala-Tyr-Gly-Gly-Phe-Leu (SEQ ID No. 4), His-Ser-Ser-Lys-Leu-Gln-Leu (SEQ ID No. 5), Gly-Pro-Leu-Gly -Ile-Ala-Gly-Gln (SEQ ID No. 6) and Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID No. 7).

  However, those skilled in the art will recognize other amino acid sequences that can be selectively cleaved by enzymes specific for tumor cells, such as WO 96/05863, 00/33888, 01/68145, 01/91798. Familiarity with the amino acid sequences described in the PCT patent specifications of No. 01/95943, 01/95945, 02/00263, 02/100353, 02/07770 or 99/28345 ing.

  Since the enzyme of the present invention can selectively cleave structure B, it is possible to release I or I having group B. The expression “release of I with group B” will be explained below with an example. When structure B is an amino acid sequence, the sequence is Ala-Leu-Ala-Leu, an advantageous substance is doxorubicin (where B-I is Ala-Leu-Ala-Leu-Dox) and the enzyme is CD10 This enzyme is thought to cleave the amino acid sequence between Ala-Leu-Ala and Leu and release the product Leu-doxorubicin. This product is defined as "I with group B".

“Extra-blood reactivation” or “reactivation in the extra-blood compartment” means a peptide bond of a prodrug having a structure represented by the formula (A) _p- (EB) _n- (I) _m It means that B is cleaved by a specific endopeptidase that is present in any (eg normal or tumor) organ or tissue other than blood, preferably present in the target cell. When structure B (eg a peptide) is cleaved, the active form of active substance I (eg a therapeutic agent) is released.

  The “active substance (I) advantageous for the target cell” means a substance whose site of action is present on the surface or inside of the target cell or whose effect affects the surface or inside of the target cell. For example, such advantageous substances include chemicals, polypeptides, proteins, nucleic acids (DNA, sense RNA or antisense RNA, single or double stranded, complementary DNA, interfering RNA, etc.), antibiotics, viruses Alternatively, it can be selected from the group consisting of markers to which vector substances (eg antibodies) are optionally bound.

  Said advantageous substance (I) is preferably a drug having therapeutic activity, more preferably a drug having anti-tumor, anti-angiogenic or anti-inflammatory therapeutic activity. Such agents may have a target (eg, a receptor) or have a site of action extracellularly or intracellularly. Such agents may also include penetrating peptide sequences such as those described in PCT Patent Specification No. WO01 / 64738. For example, I is selected from the group of drugs with anti-tumor therapeutic activity including: vinca alkaloids such as vincristine, vinblastine, vindesine, vinorelbine; taxanes or taxoids such as paclitaxel, docetaxel, 10-deacetyltaxol. 7-epi-taxol, baccatin III, lexylosyl taxol; alkylating agents such as ifosfamide, melphalan, chloroaminophen, procarbazine, chlorambucil, thiophosphoramide, busulfan, dacabazine (DTIC), mitomycin including mitomycin C Nitroso-urea and derivatives thereof (eg estramustine, BCNU, CCNU, fotemustine); platinum derivatives such as cisplatin (eg carboplatin, Anti-metabolites such as methotrexate, aminopterin, 5-fluorouracil, 6-mercaptopurine, raltitrexed, cytosine arabinoside (or cytarabine), adenosine arabinoside, gemcitabine, cladribine, pentostatin, fludarabine phosphate, hydroxy Urea; inhibitors of topoisomerase I or II, such as camptothecin derivatives (eg irinotecan and topotecan or 9-dimethylaminomethyl-hydroxy-camptothecin hydrochloride), epipodophyllotoxins (eg etoposide, teniposide), amsacrine; mitoxan L-canavanine; antibiotics such as anthracyclines such as adriamycin or doxorubicin, THP-adriamycin Daunorubicin, idarubicin, rubidazone, pirarubicin, zorubicin and aclarubicin, analogues of anthracyclines such as epiadriamycin (4 ′ epi-adriamycin or epirubicin), mitoxantrone, bleomycin, actinomycin including actinomycin D, streptozotocin, calicheamicin, duo L-asparaginase; hormone; pure inhibitor of aromatase; androgen, analog of LH-RH-antagonist; cytokines such as interferon alpha (IFN-alpha), interferon gamma (IFN-gamma) , Interleukin 1 (IL-1), IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, tumor necrosis factor-α (TNF-α), IGF -1 antagonist (insulin-like growth factor); proteasome inhibitor; farnesyl-transferase inhibitor (FTI); epothilone; maytansinoid; discodermolide; fostriecin; BH3 peptide; P53 peptide; caspase; Monoclonal antibodies, such as rituximab, tastuzumab; inhibitors of tyrosine kinases, such as STI571 (imatinib mesylate); andstatins; proteins, peptides and anti-inflammatory cytokines.

  “Marker” means an enzyme, antibody, fluorescent or phosphorescent chemical molecule, molecule that can be used in scintigraphy. For example, coumarin, 7-amido-trifluoromethylcoumarin, paranitroanilide, 8-naphthylamide and 4-methoxynaphthylamide, fluorescein, biotin, rhodamine, tetramethylrhodamine, GFP (green fluorescent protein), as a radioisotope Drugs used in scintigraphy, and derivatives of these compounds.

  The subject of the invention is furthermore the basic or acidic addition salts, hydrates, solvates, precursors, metabolites or stereoisomers of the compounds of the invention which are pharmaceutically available.

  “Pharmaceutically available salt” means a non-toxic salt of a compound of the invention, generally prepared by reacting the free base of the compound of the invention with a suitable organic or inorganic acid. To do. These salts retain the biological potency and properties of the free base. Representative examples of such salts include water-soluble and water-insoluble salts such as acetate, ansonate (4,4-diaminostilbene-2,2'-disulfonate), benzenesulfonate, benzonate, carbonate. , Bisulfate, bitartrate, borate, bromide, butyrate, calcium editate, cansylate, carbonate, chloride, citrate, clavalariat, dichlorohydrate, editate, editate, estrate, esylate, fumarate, glutaceptate, gluconate, glutamate, glycolylarsani Lato, hexafluorophosphate, hexyl resorcinate, hydrabamine, bromohydrate, chlorohydrate, hydroxynaphthoate, iodide, Sothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucato, napsilate, nitrate, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, Pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, emboate), pantothenate, phosphate / diphosphate, picrate, polyglucuronate (eg polygalacturonate and polyglucuronate), propionate, p-toluenesulfo Nart, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, sulamate, tannate, tartrate, teok Over DOO, tosylate, triethiodide, valerate and N- methylglucamine ammonium salt.

  The subject of the invention is also a composition comprising at least one compound according to the invention as an active ingredient. Furthermore, the use of such compositions in formulations and products related to biology, pharmaceuticals, cosmetics, agriculture, diagnosis or tracking is also a challenge.

  More particularly, the object of the present invention is a preparation containing at least one compound of the present invention, wherein said preparation is combined with a pharmaceutically available excipient, vector, diluent or pharmaceutical additive. It may be.

  A subject can be administered a pharmaceutically effective amount of a compound of the invention. “Pharmaceutically effective amount” means the amount that an engaged researcher or doctor expects to be able to produce a biological or medical response to a tissue, system, animal or human being.

  The composition may also contain at least one other pharmaceutically active ingredient together with the compounds of the present invention or adjuvants known to those skilled in the art (vitamin C, antioxidants) for the purpose of improving therapy and expanding the therapeutic range. Etc.) may be contained or combined.

  This composition is very useful because it has very little or no toxicity.

  The preparation of the present invention can be used for the prevention or treatment of, for example, viral infection, metastasis, cell apoptosis (degenerative disease, tissue ischemia, etc.), infection (viral infection, bacterial infection, mycosis, etc.), cancer and abnormal angiogenesis. Can be used in vivo for purposes.

  Administration of the compounds of the present invention can be carried out by any administration method possible for therapeutic agents. Such methods include systemic administration, such as oral, nasal, parenteral or topical administration, such as transdermal absorption or central administration, such as administration via the intracranial surgical route, or administration into the eye. included.

  Oral administration can be performed using tablets, capsules, soft capsules (including delayed or extended release formulations), pills, powders, granules, elixirs, dyes, suppositories, syrups and emulsions. . The presented dosage forms are also particularly suitable for passage through the intestinal barrier.

  Parenteral administration is usually performed by subcutaneous injection, intramuscular injection, intravenous injection or perfusion. Injectable compositions can be made into common dosage forms, ie, suspensions or solutions, or solid forms that can be readily dissolved in liquids.

  For parenteral administration, a sustained or extended release system may be introduced that can maintain a constant dose, for example, as described in US Pat. No. 3,710,795.

  For intranasal administration, suitable intranasal excipients can be used.

  For transdermal absorption, transdermal skin patches known to those skilled in the art can be used. Sustained administration is possible with a transdermal release system.

  Other preferred topical formulations include creams, medicated ointment formulations, lotions, aerosol sprays and gels.

  Depending on the designated method of administration, the compound may be solid, semi-solid or liquid.

  In solid preparations such as tablets, pills, powders or intact granules or capsules loaded with granules, the active ingredient can be combined with pharmaceutical additives, eg: a) diluents such as lactose, dextrose Sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants such as silica, talc, stearic acid, its magnesium and calcium salts and / or polyethylene glycol; c) binders such as magnesium silicate and aluminum Cart, starch paste, gelatin, tragacanth gum, methylcellulose, carboxymethylcellulose containing sodium carbonate and / or polyvinylpyrrolidone if necessary, d) disintegrants such as starch, agar, algin Or a sodium salt or a foaming agent; and / or e) absorbents, colorants, flavors and sweeteners.

  In semi-solid formulations such as suppositories, for example, fatty emulsions or suspensions or additives based on polyalkylene glycols such as polypropylene glycol can be used as pharmaceutical additives.

  Liquid preparations, especially liquid preparations for injection or soft capsule loading, may contain compounds of the invention such as water, sodium chloride (Nacl) saline, physiological serum, hydrous dextrose, glycerol, ethanol, oils. It can be produced by dissolving or dispersing in a pharmaceutically pure solvent.

  The compounds of the invention can also be administered in the form of liposomes or lipoplex-type release systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, including cholesterol, stearylamine or phosphatidylchlorins. In one form, as described in US Pat. No. 5,262,564, a liquid component film can be hydrated with an aqueous pharmaceutical solution to form a lipid layer encapsulating the pharmaceutical.

  The compositions of the invention can be sterilized and / or non-toxic auxiliaries and auxiliary substances such as preservatives, stabilizers, wetting agents or emulsifiers; solubilizers; salts for regulating osmotic pressure and / or A buffer may be included. It may further contain other substances that provide a therapeutic benefit. The compositions are individually manufactured through common mixing, granulating and coating methods.

  The dosage of the compound of the present invention depends on the type, strain, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the method of administration; the state of the renal and liver function of the subject and the use It is selected according to various y factors, including the nature of the compound or salt. Typically, an experienced doctor or veterinarian can easily determine and prescribe the effective amount of a compound needed to prevent, prevent, or stop the progression of a medical condition to be treated it is conceivable that.

  The aforementioned arbitrary preparation contains 0.1 to 99%, preferably 1 to 70%, of the active ingredient.

  For example, the oral dosage of the compounds of the present invention used to obtain the desired result is considered to be in the range of 0.05 to 5000 mg / day, preferably 5 to 1000 mg / day, orally with the active ingredient. Of tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg It is preferably administered in the form. For parenteral administration, effective levels of the compounds of the invention are considered to be in the range of 1 kg body weight and 0.002 mg to 500 mg per day.

  The compounds of the present invention may be administered once a day, or the total daily dose may be divided into 2, 3 to 4 times daily.

  In particular, the present invention relates to diagnostic pharmaceuticals used in vitro, consisting of at least one of the compounds of the invention or comprising at least one of the compounds of the invention. The compounds of the invention further have a marker as an advantageous substance (I). Such diagnostic drugs can also be used in vivo.

  Therefore, the subject of the present invention is also a diagnostic kit containing the diagnostic drug. In particular, the diagnostic kit contains a predetermined amount of the composition of the present invention in one or more containers.

  Other advantages and features of the invention will become apparent from the following examples, which are referred to in the following drawings.

  FIG. 1 is a diagram showing an outline of two synthesis methods of a PEGylated prodrug of doxorubicin.

2, _{doxorubicin, βALAL-Dox, PEG 2000 -βALAL} -Dox, PEG 2000 -DSer-βALAL-Dox and _PEG 2000 for MCF-7/6 cells - shows the _(DSer) cytotoxicity test of 4 -βALAL-Dox FIG. Cell viability was evaluated by a cell viability test (WST-1, Roche Molecular Diagnostics). Graphs 2A, B, C, D and E show doxorubicin (A), βALAL-Dox (B), PEG ₂₀₀₀ -βALAL-Dox (C), PEG ₂₀₀₀ -DSer-βALAL-Dox (D) and PEG _{2000, respectively.} -The viability (% of control) of MCF7 / 6 cell line with respect to the logarithm of the concentration of (DSer) ₄ -βALAL-Dox (E) is shown.

FIG. 3 graphically illustrates the variation in mean body weight of xenograft mice with LS-174-T tumors. The results are expressed as a percentage of the weight measured at the time of treatment: (●) NaCl, (■) doxorubicin 6.69 μmol / kg, (▲) doxorubicin 8.6 μmol / kg, (◯) Su-βALAL-Dox 45 μmol / kg , (+) Su-βALAL- Dox50μmol / kg, (×) PEG 2000 - (DSer) 4 -βALAL-Dox 1 × 45; 3 × 25μmol / kg, (*) PEG 2000 - (DSer) 4 -βALAL-Dox 1 × 50; 3 × 35 μmol / kg.

FIG. 4 shows daily changes in the mean value of relative tumor volume (RTV) of athymic mice with LS-147-T human colon cancer tumors treated with drug versus control (NaCl). FIG. 5 is a graph that is expressed in percentages based on the day: (●) NaCl, (■) doxorubicin 6.69 μmol / kg, (▲) doxorubicin 8.6 μmol / kg, (◯) Su-βALAL-Dox 45 μmol / kg, (+) Su-βALAL-Dox50μmol / kg, (×) PEG 2000 - (DSer) 4 -βALAL-Dox 1 × 45; 3 × 25μmol / kg, (*) PEG 2000 - (DSer) 4 -βALAL-Dox 1 × 50; 3 × 35 μmol / kg.

Figure 5 is a control NaCl0.9% (w / v) solution, doxorubicin _{(Dox), PEG 2000 - (} DSer) 4 -βALAL-Dox, Su-βALAL-Dox each 6.69μmol / kg, 1 × It is a figure which compares the inhibition of the 1st growth phase of a LS-174T tumor at the time of administering at 50 + 3 * 35 micromol / kg and 50 micromol / kg, and a 2nd growth phase. All mice received 4 intravenous injections on days 0, 7, 14, and 21. The minimum T / C ratio (T / Cmin.) Of the average value of the relative tumor volume (RTV) is an indicator of the maximum effect. Differences in doubling time (TC) and SGD (specific growth delay) of the mean RTV values of the drug-treated group relative to the control group were calculated from linear regression of the growth phase, and were determined by EORTC. Determine the activity along the line. The linear ratio of the regression slope (T / C slope) of the variation in the RTV average value of the drug-treated group relative to the control group is expressed in percent and is determined using the growth rate comparison parameter.

6, in a serum _{medium, βALAL-Dox (A),} PEG 2000 -βALAL-Dox (B) and _{PEG 2000 - (DSer) 4 -βALAL} -Dox of stability testing during in vitro of (C) FIG. The results show the complex concentration measured by HPLC over time.

7, compound ALAL-Dox (A), βALAL -Dox (B), PEG 2000 -βALAL-Dox (C) and _{PEG 2000 - (DSer) 4 -βALAL} -Dox in human whole blood of the (D) It is a figure of a stability test. The results show the variation in complex and metabolite concentrations that can be formed as measured by HPLC over time.

FIG. 8 is a graphic representation of the change in mean body weight of athymic mice with HCT-116 human colon cancer tumors. The results are expressed as a percentage of body weight measured during drug treatment: (●) NaCl, (+ ) Su-βALAL-Dox30μmol / kg; (▲) PEG 2000 - (DSer) 4 -ALAL-Dox53μmol / kg; ( _{*) PEG 2000 - (DSer)} 4 -ALAL-Dox110μmol / kg.

FIG. 9 shows the inhibition of HCT-116 tumor growth from the change in the ratio of the average RTV values of the drug-treated group (T) and the control group (C), expressed as a percentage (T / C (%)). Is done. Treatment: (●) NaCl, (+ ) Su-βALAL-Dox30μmol / kg; (▲) PEG 2000 - (DSer) 4 -ALAL-Dox53μmol / kg; (*) PEG 2000 - (DSer) 4 -ALAL-Dox110μmol / kg.

FIG. 10 is a graph showing the variation in survival rate of xenograft mice with HCT-116 tumors (expressed in%). _{Treatment: NaCl (- ● -),} PEG 2000 - (DSer) 4 -ALAL-Dox200μmol / kg (...... ▲ ......), 300μmol / kg (- ▲ -), 400μmol / kg (- ▲ -); PEG ₂₀₀₀ -ALAL-Dox 200 μmol / kg (…… ■ ……), 300 μmol / kg (-■-), 400 μmol / kg (-■-).

FIG. 11 shows growth inhibition of HCT-116 tumor from the change in the ratio of the average RTV values of the drug-treated group (T) and the control group (C), and is expressed as a percentage (T / C (%)). . _{: NaCl (- ● -),} PEG 2000 - (DSer) 4 -ALAL-Dox200μmol / kg (...... ▲ ......), PEG 2000 -ALAL-Dox200μmol / kg (...... ■ ......).

FIG. 12 is a graph showing changes in survival rate of transplanted mice having B16-BL6 (%) melanoma tumors. Drug Treatment: Control _{PBS (●); (PEG 5000} -ALAL) n -TNFα (■); (PEG 5000 - (DSer) 4 -ALAL) n -TNFα (▲); (PEG 5000) n -TNFα (◆) .

FIG. 13 is a graph showing changes in average body weight of transplanted mice having B16-BL6 melanoma tumors. Results are expressed as a percentage of body weight measured during drug treatment. Control _{PBS (●); (PEG 5000} -ALAL) n -TNFα (■); (PEG 5000 - (DSer) 4 -ALAL) n -TNFα (▲); (PEG 5000) n -TNFα (◆).

FIG. 14 shows the growth inhibition of B16-BL6 tumor from the change in the ratio of the average RTV values of the drug-treated group (T) and the control group (C), expressed as a percentage (T / C (%)). . : Control _{PBS (●); (PEG 5000} -ALAL) n -TNFα (■); (PEG 5000 - (DSer) 4 -ALAL) n -TNFα (▲); (PEG 5000) n -TNFα (◆).

Example 1 Materials and Methods 1.1) Cell lines MCF7 / 6 cells: MCF-7 strain (Michigan Cancer Foundation, Engel et al., 1978) variant obtained from pleural effusion of a patient with lung cancer in 1970 (Soule et al., 1973). The cells are obtained from Professor Mallile's laboratory in Ghent (Experimental Cancer Laboratory, Ghent University Hospital, Belgium).

  LNCap cells: Isolated from biopsy material of supraclavicular lymph nodes of patients with metastatic prostate cancer in 1977 by Horosezewic et al. This strain is obtained from ATCC (American Type Culture Collection, USA).

  LS-174T cell line: A mutant of the LS180 line obtained from a woman suffering from colon cancer. These cells form tumors at a very fast rate when inoculated into athymic mice. The cells are obtained from ECACC (European Collection of Cells Cultures, UK).

  HCT-116 strain: A cell line established from a primary culture of human colon cancer cells. These cells form tumors when injected subcutaneously into athymic mice. The cells are obtained from ATCC (American Type Culture Collection, USA).

1.2) Anticancer agent used for chemotherapy test Doxorubicin is provided by Meiji Seika Co., Ltd. (Tokyo, Japan); succinyl-βAla-Leu-Ala-Leu-doxorubicin (Su-βAla-Leu-Ala-Leu-Dox) ) (Fernandez, _{etc., 2001), PEG 2000 -Ala-} Leu-Ala-Leu-Dox and _{PEG 2000 - (DSer) 4 -Ala} -Leu-Ala-Leu-Dox were synthesized.

1.3) Animals NMRI or SWISS female mice, nude / nude (5 weeks after transport from JANVIER breeding facility) were used.
The handling of animals relates to their use and health of animals during experimental chemotherapy trials, including UKCCCR (United Kingdom Coordinating Committee on Cancer Research; Workman et al., 1998) and FELASA (Federation of European Laboratories Laboratories Laboratories Laboratories; Rehbinder et al., 2000; Rehbinder et al., 1996).

2) Synthesis of PEGylated derivative prodrugs of doxorubicin The synthesis of PEGylated derivative prodrugs of doxorubicin was carried out in two different ways “A” and “B” (FIG. 1).

PEGylated derivative prodrugs of doxorubicin synthesized by either of the two methods are:
_{-PEG 2000 - (DSer) 4 -βAla} -Leu-Ala-Leu- doxorubicin (1)
_{-PEG 2000 - (DSer) 4 -Ala} -Leu-Ala-Leu- doxorubicin (2)
_{-PEG 2000 - (DSer) -βAla-} Leu-Ala-Leu- doxorubicin (3)
-PEG ₂₀₀₀ -βAla-Leu-Ala-Leu-Doxorubicin (4)
_{-PEG 2000 -Ala-Leu-Ala-} Leu- doxorubicin (5).

  Compounds 1, 3 and 4 were synthesized by method “A” and compounds 2 and 5 were synthesized by method “B”.

  Note: Ala-Leu-Ala-Leu-doxorubicin and βAla-Leu-Ala-Leu-doxorubicin prodrugs were described in the patent specification WO 96/05863.

2.1) Synthesis method “A”
The principle of the method, NH 2 _- peptides - is a PEG of a solution of doxorubicin compound. 1.5 mol equivalent (Nektar) of mPEG ₂₀₀₀ -SPA (mPEG ₂₀₀₀ -succinimidyl propionate ester) and 5 μl of diisopropylethylamine (DIPEA) dissolved in chloroform / methanol mixture (4: 1) were mixed with chloroform / methanol mixture ( 4: 1) dissolved in NH2-peptide-doxorubicin product (product previously obtained after carrying out the three steps of the synthesis method, see FIG. 1).

  After about 48-72 hours, the final product of the reaction is analyzed using HPLC chromatography (see paragraph 2.2 below).

  Products (3) and (4) are extracted with dichloromethane at pH 4 (using lactic acid). The product accumulates in the organic phase, which is concentrated by vacuum evaporation and then lyophilized.

2.2) Synthesis Method “B”: Peptide Synthesis on Solid Material by Fmoc (Fluoroenyl-Lmethoxycarbonyl) Chemistry (SPPS)
The principle of the method is the synthesis of PEGylated peptide sequence on solid polymer material (Wang type resin) and subsequent conjugation of PEG ₂₀₀₀ -peptide-OH product and doxorubicin.

  The principle of SPPS is to sequentially bind various amino acids of a given peptide to a solid phase material (Wang type resin), which is performed in a manner known to those skilled in the art (Merifield, 1963 and 1965; Steward and Young, 1969).

Peptide synthesis on solid phase materials Synthetic peptides are obtained, for example, by synthesizing peptides on solid phase materials using a chemical means using the Fmoc group in a manual synthesis reactor (AnaSpec). All resins (AnaSpec or Novabiochem) used in the synthesis on solid phase material are Wang type resins, which are 0.4-0.7 mmol per gram of resin with the first substitution by the supplier Having a first protected amino acid previously conjugated. Fmoc amino acids are provided by AnaSpec or Novabiochem and have a protecting group in the side chain, eg: Trityl (Asn, Cys, Gln, and His), Acm (Cys), Boc (Lys and Trp), O-tert -Butyl (Asp and Glu), tert-butyl (Ser, Thr, Tyr) and Pbf (Arg). Various polyethylene glycols (PEG-SPA) having the desired molecular weight are obtained from Nektar in the form of pre-activated hydroxysuccinimidyl (OSu) ester. Deprotection of the Fmoc group is performed by treating piperidine in dimethylformamide (DMF). The binder used during the synthesis is HCTU (1H-benzotriazoium 1- [bis (dimethylamino) methylene] -5 chlorohexafluorophosphate (1-) 3-oxide) in DMF or HBTU (2 -(1H-benzotriazol-1-yl) -1, -1,3, -3-tetramethyluronium hexafluorophosphate) or HATU (O- (7-azabenzotriazol-1-yl) -N, -N, -N ', -N'-tetramethyluronium hexafluorophosphate). A typical amino acid binding cycle is performed as follows: 3 × 30 seconds, washed with DMF; 3 × 2 minutes, then 1 × 7 minutes, deprotected with piperidine diluted to 20% with DMF Washed with DMF for 5 × 20 seconds; 2 × 15 minutes, combined with amino acids (2 equivalents), binder (2 equivalents) and DIPEA (4 equivalents), then washed with DMF for 3 × 30 seconds. Substituents of the original resin at the time of commercialization are reduced by adding 0.5 to 0.3 molar equivalents of the first Fmoc-AA-OH to be bound, depending on the desired sequence (and ultimately desired The ratio of the substituents is 0.1-0.22 mmol / g) and the remaining amino group is acetylated (Viender et al., 1981). The binding of each amino acid is confirmed by a Kaiser test (Kaiser et al., 1970) known to those skilled in the art. After all amino acids are coupled according to the desired sequence, PEGylation occurs on Wang type solid phase material by coupling active ester PEG-SPA (Nektar) and DIPEA for 2 × 2 days.

  Chemical cleavage of the covalent bond linking the PEGylated peptide to a Wang type solid phase material is a mixture comprising trifluoroacetic acid (TFA): water: triisopropylsilane (TIS) at 95: 2.5: 2.5. Is added to the completely dry resin in a manual synthesis reactor for 3 hours. The PEGylated peptide is precipitated with cold ethanol, centrifuged, washed with ethanol and lyophilized.

Analysis by HPLC chromatography Using a VYDAC-type column (reverse phase) (C8.5 μm, 250 × 4.6 mm), a 0.1% solution of trifluoroacetic acid (TFA) in water was used as the first eluent (eluent A). ) And a 0.1% solution of TFA in acetonitrile (ACN) as the second eluent (eluent B), and the eluent B is allowed to flow with a gradient of 0% to 70% over 25 minutes. analyzed.

2.3) Synthesis _{Example: PEG 2000 - (DSer) 4} -Ala-Leu-Ala-Leu- synthetic doxorubicin 1.2 moles of doxorubicin (2) and _{PEG 2000 -Ala-Leu-Ala-} Leu- doxorubicin (5) eq, HCl and DIPEA3.2 molar equivalents dissolved in DMF, _PEG obtained by synthesizing a solid phase material on ₂₀₀₀ - peptide _{-OH (PEG 2000 - (DSer)} 4 -Ala-Leu-Ala- Leu-OH or PEG ₂₀₀₀ -Ala-Leu-Ala-Leu-OH). The mixture is protected from light, stirred at ambient temperature for 15 minutes, and 1.3 molar equivalents of HATU (binder, PE Biosystem) are added. The reaction is carried out for about 3 hours until HPLC chromatographic analysis (see section 2.3 above). At the end of the reaction, the solvent is evaporated in vacuo. The remaining product is taken up in water and extracted with dichloromethane. Product (5) is initially recovered in the aqueous phase, but product (2) rather accumulates in the organic phase. The organic phase containing the product (2) is concentrated by vacuum evaporation and the product is precipitated in ether at ambient temperature. The resulting precipitate is dissolved in water and lyophilized. The aqueous phase containing product 5 is recovered and frozen in a bath of acetone and dry ice and then lyophilized. Prior to lyophilization, the product was analyzed by HPLC (see section 2.2 above).

3) The product doxorubicin to be used for cell culture experiments in the preparation in vitro of the product solution to be used for cell culture, βAla-Leu-Ala-Leu- _{doxorubicin, PEG 2000 - (DSer) 4} -βAla-Leu-Ala- _{Leu-Dox (1), PEG} 2000 - (DSer) -βAla-Leu-Ala-Leu-Dox (3), PEG 2000 -βAla-Leu-Ala-Leu-Dox (4), PEG 2000 -Ala-Leu- Ala-Leu-Dox (5).

  The product is dissolved in very little water and filter sterilized through a die with a hole size of 0.22 μm. The concentration of the solution is determined by measuring the absorbance (measured at 495 nm based on the molar extinction coefficient of doxorubicin, ε = 10837 M-1 cm-1).

4) Cell culture The medium used for the following experiments is RPMI 1640 medium containing glutamax-1 containing 10% fetal bovine serum (SBF: Gibco-BRL).

5) Cytotoxicity test The cytotoxicity test of doxorubicin and derivatives was performed using MCF-7 / 6 cells and LNCaP cells.

  Cells are detached with trypsin, counted and seeded into 96-well plates containing 200 μl of serum medium.

Cells are incubated at 37 ° C. for 24 hours and test product is added:
Doxorubicin,
βAla-Leu-Ala-Leu-Dox,
PEG ₂₀₀₀ -Ala-Leu-Ala-Leu-Dox (5),
PEG ₂₀₀₀ -βAla-Leu-Ala-Leu-Dox (4)
PEG ₂₀₀₀ -DSer-βAla-Leu-Ala-Leu-Dox (3)
_{PEG 2000 - (DSer) 4 -βAla} -Leu-Ala-Leu-Dox (1)
This is diluted to various concentrations with serum medium. After 48 hours of incubation in the presence of various compounds, the cells are finally post-incubated for 24 hours at 37 ° C. in the presence of serum medium alone. Following this incubation, cell viability testing is performed (WST-1, Roche Molecular Biochemical).

6) Stability and hydrolysis test of PEGylated derivative prodrug of doxorubicin 6.1) Effect of PEGylation when doxorubicin prodrug βALAL-Dox is reactivated by peptidase secreted from tumor cells (MCF-7 / 6, LS-174T, LNCaP) mixed and subcultured, washed twice with a saline phosphate buffer solution and containing no serum and 0.02% bovine serum albumin Is added (100 μl / cm ² ). After incubation for 24 hours, the conditioned medium is removed, centrifuged at 300 g for 10 minutes, buffered with 1M Tris-HCl buffer (pH 7.5) (buffer 1 volume + medium 19 volumes), and concentrated 20 times by ultrafiltration. (Filtration limit is 10 kDa) (Tris: trishydroxymethyl-aminomethane).

  Comparison of reactivation of a PEGylated peptide prodrug of doxorubicin derivative (βALAL-Dox) by peptidase secreted by tumor cells with reactivation of the first prodrug, βALAL-Dox, where βALAL-Dox Is hydrolyzed to Leu-Dox and doxorubicin during incubation in conditioned medium with tumor cells. Compounds are diluted to 20 μmol with conditioned medium and incubated for 6-18 hours at 37 ° C. in a constant temperature bath. Quantify compound hydrolysis by HPLC analysis (see section 7.1 below)

The following PEGylated prodrug is used.
PEG ₃₅₀ -βALAL-Dox
PEG _750- βALAL-Dox
PEG ₂₀₀₀ -βALAL-Dox
PEG ₅₀₀₀ -βALAL-Dox.

6.2) Stability in media _{PEG 2000 - (DSer) 4 -βAla} -Leu-Ala-Leu-Dox (1), PEG 2000 -βAla-Leu-Ala-Leu-Dox (4) and betaAla-Leu- Ala-Leu-Dox (control) is diluted to 20 μmol with 500 μl of medium (serum medium) containing 10% fetal bovine serum. Samples are distributed into 24-well plates and incubated at 37 ° C. in a saturated atmosphere containing 5% CO ₂ . For each product, extraction (extraction method with acetonitrile, see section 7.3 below) is performed in triplicate at 0 hours, 1, 2, 6 and 24 hours after incubation. The sample is then analyzed by HPLC chromatography (see item 7.3 below).

6.3) Stability in human whole blood _{PEG 2000 - (DSer) 4 -βAla} -Leu-Ala-Leu- doxorubicin _{(1), PEG 2000 -βAla-} Leu-Ala-Leu-Dox (4), βAla- Leu-Ala-Leu-Dox and Ala-Leu-Ala-Leu-Dox are diluted to 20 μmol with 500 μl of human whole blood (after collection, stored at 4 ° C. in a 5 ml sterile citrated tube). Incubate the tube containing the sample at 37 ° C. Extraction of each product (extraction method with acetonitrile, see item 7.3 below) is performed in triplicate at 0 hours, 1, 2, 4 and 6 hours after incubation.

7) Analytical method 7.1) Basic extraction Tube containing 25 μl sample to be extracted, 500 μl water and 100 μl internal standard (prolyl-daunorubicin 3.45 μmol), 2.5 ml organic chloroform / methanol mixture (4: 1) Add to. After the addition of basic buffer (0.5 M borate buffer 600 μl, pH 9.8), the tube is rapidly stirred for 5 seconds and centrifuged at 1800 g for 10 minutes.

  The organic phase is recovered and dried under a stream of air. Add 500 μl of a mixture containing 30% acetonitrile and 10% ammonium formate buffer (pH 4) to dissolve the sample. The sample is then filtered and analyzed by HPLC chromatography (see item 7.3 below).

7.2) Extraction with acetonitrile 10 μl of internal standard (prolyl-daunorubicin) and 150 μl of 50 μmol of acetonitrile are added to a tube containing 50 μl of the sample to be extracted. The tube is agitated and centrifuged at 13000 g for 10 minutes. The supernatant is diluted 4-fold with formate buffer (pH 4), centrifuged, and analyzed by HPLC chromatography (see item 7.3 below).

7.3) Chromatography Analysis All product samples by _{HPLC (PEG 2000 -βAla-Leu-} Ala-Leu-Dox (4), PEG 2000 - (DSer) 4 -βAla-Leu-Ala-Leu-Dox (1 ), PEG ₂₀₀₀ -Ala-Leu-Ala-Leu-Dox (5), βAla-Leu-Ala-Leu-Dox and Ala-Leu-Ala-Leu-Dox), VYDAC (C8.5 μm, 250 × 4. HPLC chromatography analysis is performed using a HP1100 system (Agilent) using a column (reverse phase) such as 6 mm). Doxorubicin and its derivatives are detected with fluorescence (Lambda _exc. = 235 nm; Lambda _em. = 560 nm) and are identified by comparing the respective relative residence times to the internal standard. The concentration is calculated on the basis of the integrated surface of peaks found in HPCL chromatography.

  The resulting stability curve (concentration variation over time) allows for product stability assessment, and the hydrolysis test should use a table summarizing the amount of Leu-Dox and Dox obtained after 6 hours of incubation. Thus, the degree of hydrolysis of the PEGylated derivative by the enzyme secreted from the tumor cell can be evaluated.

8) Tumor reactivation study of PEGylated derivative prodrug of doxorubicin (in vivo)
Tumor reactivation of a PEGylated derivative prodrug of doxorubicin (Su-βALAL-Dox) was measured in athymic mice xenografted with LS-174T human colon cancer. The following PEGylated derivatives were used: PEG ₂₀₀₀ -βALAL-Dox and PEG ₂₀₀₀ -βALAL Dox.

  Mice were injected with various compounds in an intravenous bolus (intravenous) in an amount of 69 μmol / kg. The animals died 2 to 72 hours after the injection, and the active molecules (Leu-Dox + Dox) accumulated in the tumor were quantified by HPLC chromatography (see the above item 7.3).

9) Chemotherapy test of doxorubicin PEGylated derivative prodrug using ectopic xenograft model subcutaneously An experiment using athymic mice previously transplanted subcutaneously with tumor LS-174T or HCT116 (human colon cancer) of human origin Chemotherapy was performed.

  Mice were selected to distribute tumor volumes equally and distributed to different groups. Different therapeutic agents were randomly assigned to the formed group.

  The drug is administered intravenously into the tail vein. During the injection, the animals are given a constant volume of 10 μl / g of product (Workman et al., 1998). After injection, clinical symptoms are followed in a timely manner (every hour on the first day, every day until the end of the study), and body weight and tumor volume (mm3) are measured simultaneously. Tumor growth is monitored twice a week by measuring the two orthogonal diameters (“diagonals”) (longest and shortest “diagonal”) of the tumor using a scissors scale.

  In order to measure the variation in tumor volume in different groups, the average value of relative tumor volume (RTV = relative tumor volume) is calculated as a variation relative to the average tumor volume on day 0 and expressed as a percentage. The average RTV ratio (T / C (%)) of the drug-treated group (T) and the control group (C) expressed as a percentage is calculated, and the effectiveness of various therapeutic drugs is evaluated.

  The minimum value of the T / C ratio is an index for estimating the maximum effect of the product used.

  Tumor growth inhibition time is evaluated by calculating the delay (TC) of doubling growth (doubling time = DT = Doubling Time) of the average RTV value between the drug-treated group and the control group (in FIG. 5, control). The group doubling time is displayed as 200%). Specific growth delay (SGD = Specific Growth Delay) is also calculated from the time required to double the average RTV in the various groups involved in the study. SGD is therefore equal to (DT treatment group-DT control group) / (DT control group).

  The activity of the various products tested was also evaluated based on the recommended criteria of EORTC (European Organization for Research and Treatment of Cancer) (Boven et al., 1992; Langdon et al., 1994) and the results are shown in Table I. It was described in. However, since the criteria proposed in the literature (Boven et al., 1992) apply only to tumors that exhibit one linear growth phase, the method of estimating compound efficacy is an adaptation.

  The LS-174-T cells used are characterized by two growth phases, the final growth phase (phase 2) being much faster than the first growth phase (phase 1). For this reason, growth delay (TC) and specific growth delay (SGD) were calculated by linear regression of different phases characterized by tumor growth.

  Table 1 below evaluates the efficacy of chemotherapeutic agents according to the criteria recommended by EORTC (Boven et al. 1992; Langdon et al., 1994). Activity level: − = no activity; (+) = very low activity; + = low activity; ++ = moderate activity; +++ = significant activity; +++++ = very significant activity.

10) Chemotherapy test of PEGylated derivative of TNF-α using intradermal ectopic transplantation model 10.1) Cell line B16-BL6 cells: mouse melanoma cells, in vivo bladder invasion ability and lung It is selected because of its high natural metastatic potential. The cells are obtained from "NCI-Frederic Cancer Research and Development Center", Maryland, USA.

10.2) Anticancer agent used in chemotherapy trials RhTNF-α (“recombinant human tumor necrosis factor α” is provided by Henogen (Gosley, Belgium).

Was synthesized _{- ((DSer) 4 -Ala-} Leu-Ala-Leu PEG 5000) n-TNFα conjugate _{(PEG 5000) n-TNFα (} PEG 5000 -Ala-Leu-Ala-Leu) n-TNFα and. “N” is 1 to 18 (18 is the total number of lysines when the TNFα molecule is a trimer, and TNF monomers are known to have 6 lysine residues). Synthesis of (PEG-peptide) n-TNFα derivatives is performed as follows: peptides are synthesized in solid phase and activated polyethylene glycol (PEG) in the presence of dimethylformamide (DMF) and diisopropylethylamine (DIPEA). ) To form an N-hydroxysuccinimide type. After purification, PEG-peptide-OH dissolved in DMF is present in the presence of a binder known to those skilled in the art (eg: TSTU, 2-succinimide-1,1,3,3-tetramethyluronium tetrafluoroborate) Activate below. Then, rhTNFα dissolved in 20 mmol phosphate buffer (pH 7.4) containing 1% mannitol is added and the reaction mixture is stirred overnight at ambient temperature. The coupled product is recovered and excess PEG-peptide-OH is removed by ultrafiltration. The concentration of the final product is determined from the protein mass.

10.3) Animals Male C57BL / 6J mice (5 weeks after transport from Charles River Laboratories, France, breeding ground) are used.

  The handling of animals relates to their use and health of animals during experimental chemotherapy trials, including the UKCCCR (United Kingdom Coordinating Committee on Cancer Research; Workingman et al., 1998) and FELASA (Federation of Europe Laboratories Laboratories Laboratories Laboratories; Rehbinder et al., 2000; Rehbinder et al., 1996).

  Experimental chemotherapeutic studies are performed using normal C57BL / 6J type male mice previously implanted intradermally with B16-BL6 tumors (mouse melanoma) of mouse origin.

  Mice are selected to distribute tumor volumes equally and distributed to different groups. Different therapeutic agents are randomly assigned to the formed group.

  The therapeutic agent is administered intravenously on days 0-3. During the injection, the animals are given a constant volume of 10 μl / g of product (Workman et al., 1998). After injection, clinical symptoms are followed in a timely manner (every hour on the first day, every day until the end of the study), and body weight and tumor volume (mm3) are measured simultaneously. Tumor growth is monitored twice a week by measuring the two orthogonal diameters (“diagonals”) (longest and shortest “diagonal”) of the tumor using a scissors scale.

  The anti-tumor effect is quantified from the value of the T / C ratio (the minimum value corresponds to the maximum effect). The measurement method is detailed in item 9 above.

Example 2: Effect of PEGylation when doxorubicin prodrug βALAL-Dox is reactivated by peptidase secreted from tumor cells Compound βALAL-Dox, PEG ₃₅₀ -βALAL-Dox, PEG ₇₅₀ -βALAL-Dox , PEG ₂₀₀₀ -βALAL-Dox and PEG ₅₀₀₀ -βALAL-Dox (20 μmol) were incubated in conditioned medium of MCF-7 / 6 or LS-174T tumor cells for 6 hours. Prodrug reactivation (release of doxorubicin and Leu-Dox) was measured by HPLC chromatography.

  Table 2 below shows the hydrolysis of the prodrug βALAL-Dox and its PEGylated derivatives. The results are expressed as the concentration of doxorubicin (Dox) and leucyl-doxorubicin (L-Dox or Leu-Dox) and are expressed as the mean value ± standard deviation (n = 9) obtained in three independent experiments.

  The results listed in the table above indicate reactivation of the prodrug and it can be seen that the release of the active molecule (Leu-Dox + doxorubicin) is specifically inhibited as the molecular weight of PEG increases. PEGylation of doxorubicin prodrug (βALAL-Dox) prevents its reactivation by tumor peptidase. This inhibition is related to the size of the PEG. The inventor therefore hypothesized that such interference is probably the result of a steric hindrance phenomenon that limits the accessibility of the cleavage site.

Example 3: In vivo studies on tumor reactivation of PEGylated derivatives of doxorubicin prodrugs LS-174T human colon cancer tumors are heterogeneous for in vivo tumor reactivation of PEGylated derivatives of doxorubicin prodrug (βALAL-Dox) Evaluation was performed using a transplanted athymic mouse model. Table 3 below shows the mean concentrations of Dox and Leu-Dox that accumulate in the tumor at 2 and 72 hours after injection of the test compound (average obtained from 18 mice).

From the above table, it can be seen that the two drugs PEG ₂₀₀₀ -βALAL-Dox and PEG ₂₀₀₀₀ -βALAL-Dox do not reactivate Dox and Leu-Dox compared to the control Su-βALAL-Dox. That is, PEGylation of doxorubicin prodrug strongly suppresses reactivation related to tumors.

Example 4: Effect of spacer insertion on reactivation by peptidase secreted from tumor cells of PEGylated prodrug of doxorubicin βALAL-Dox prodrug (20 μmol) conditioned medium of LS-174T or LNCap tumor cells And incubated for 6-18 hours. Prodrug reactivation (release of doxorubicin and Leu-Dox) was measured by HPLC chromatography.

Two types of spacers were tested:
Insertion of _four hydrophilic D-serine ((DSer) ₄ ) residues-6-aminohexanoic acid residues (or aminocaproic acid, which is a longer hydrophobic amino acid than serine), denoted Ahx.

  Table 4 below shows the hydrolysis of Su-βALAL-Dox (control) prodrug and PEGylated βALAL-Dox derivatives. The results are expressed as a percentage of the released doxorubicin and Leu-Dox compared to the control (n = 3).

  The results in the table again show that the hydrolysis of PEGylated derivatives is clearly reduced compared to Su-βALAL-Dox.

After incubating the compound in conditioned medium of LS-174T cells, inserting four D-serine residues between PEG and βALAL-Dox can result in no spacer or three Ahx residues (hydrophobic Compared to PEGylated derivatives of PEG ₂₀₀₀ with a spacer group), the release of doxorubicin and Leu-Dox is increased 2-fold.

  Insertion of an aminohexanoic acid residue (hydrophobic amino acid) does not promote reactivation of the PEGylated derivative of the prodrug, even compared to a derivative without a spacer.

Example 5: Doxorubicin professional PEG derivative drug cytotoxicity tests MCF-7/6 tumor cells _{doxorubicin, βALAL-Dox, PEG 2000 -βALAL} -Dox, PEG 2000 -DSer-βALAL-Dox or _PEG 2000 - (DSer ) _4- βALAL-Dox was incubated for 48 hours in serum medium containing a concentration gradient. It is then post-incubated for 24 hours in the presence of serum medium. The product cytotoxicity is examined by a cell viability test (WST-1, Roche Molecular Diagnostics).

  The results of three independent tests are shown in FIG.

The IC ₅₀ values (50% inhibitory concentration) of doxorubicin and βALAL-Dox compounds are 0.045 μM and 158.48 μM, respectively, demonstrating the nature of the βALAL-Dox prodrug. Since PEG ₂₀₀₀ -βALAL-Dox does not show cytotoxicity up to a concentration of 500 μmol, a comparison of the mean IC ₅₀ values exhibited by βALAL-Dox and PEG ₂₀₀₀ -βALAL-Dox compounds inhibits prodrug reactivation. You can see that A prodrug containing DSer between PEG and βALAL-Dox (PEG ₂₀₀₀ -DSer-βALAL-Dox) does not show cytotoxicity. On the contrary, prodrug having a molecular spacer _{(DSer) 4} between the PEG and _{βALAL-Dox (PEG 2000 - (} DSer) 4 -βALAL-Dox) shows the _{cytotoxicity,} PEG 2000 - _{(DSer) 4} -ΒALAL-Dox has just the same activity (IC50 = 251.19 μmol) as the non-PEGylated prodrug, βALAL-Dox.

  This result indicates that insertion of 4 D-serine residues between PEG and βALAL-Dox allows reactivation of the PEGylated prodrug. In contrast, PEGylated prodrugs without spacers cannot be reactivated. The cytotoxicity of the latter prodrug is mainly due to reactivation outside the blood, i.e. releases more doxorubicin than other test compounds.

Example 6: PEG derivatives of doxorubicin prodrug βALAL-Dox, efficacy studies doxorubicin in LS-174T human colon carcinoma xenograft model, succinyl -βAla-Leu-Ala-Leu- Dox and _PEG 2000 - _{(DSer) 4} -The anti-tumor activity of Ala-Leu-Ala-Leu-Dox was tested in an athymic mouse (nude / nude NMRI) model in which LS-174T type human tumors were ectopically xenografted. The product was injected intravenously (10 μl / g, 6 mice per group).

PEG ₂₀₀₀ - a _(DSer) 4 -ALAL-Dox, initially, and iv bolus injection in an amount of 50 [mu] mol / kg and 45μmol / kg. One animal died at two doses.

The dose was reduced from day 7. PEG ₂₀₀₀ - _(DSer) a 4 -ALAL-Dox were injected 35 [mu] mol / kg and 25 [mu] mol / kg (1 weekly, 3 injections). Another compound was administered as an intravenous bolus once a week on days 0, 7, 14 and 21 (doxorubicin 6.69 and 8.6 μmol / kg, Su-βALAL-Dox45 and 50 μmol / kg).

FIG. 3 shows the percent change in average body weight from the start of each day's administration. Doxorubicin and _PEG 2000 _- (DSer) 4 significant weight reduction up to 2 weeks in the treated group the -ALAL-Dox is not observed. Slight weight loss is observed in the group treated with Su-βALAL-Dox. In the latter case, the maximum weight loss is 10% and is observed on day 32 in the two treatment groups.

  FIG. 4 shows the variation of the mean value of the relative tumor volume (RTV) of the drug-treated mouse group (T) compared to the control (C) (NaCl). Despite administering less than the maximum tolerated dose (MTD), all test products have anti-tumor activity.

During the drug treatment, two tumor growth phases were observed (Figure 5). In the first growth _{phase, PEG 2000 - (DSer) 4} -ALAL-Dox (1 × 50 + 3 × 35μmol / kg) activity is equivalent to doxorubicin (6.69μmol / kg), succinyl -βALAL-Dox (50μmol / kg) lower. On the contrary, in the second growth _{phase, PEG 2000 - (DSer) 4} -ALAL-Dox is more active than doxorubicin, it is equivalent to the Su-βALAL-Dox. Overall, Su-βALAL-Dox product (T / Cmin = 26.6%; specific growth delay (SGD = 3.02)) and _{PEG 2000 - (DSer) 4 -ALAL} -Dox (T / Cmin = 38%; SGD = 1.58) was found to be more active than doxorubicin.

Example 7; in serum medium PEG derivatives of doxorubicin prodrug stability _{βALAL-Dox, PEG 2000 -βALAL-} Dox and _PEG 2000 _- stability (DSer) 4 -ALAL-Dox compounds, fetal calf serum Measurement was carried out after incubation for 24 hours at 37 ° C. in a medium containing 10%. The concentrations of initial product and metabolites that can be formed were quantified by HPLC. The three test complexes are stable in serum medium. No degradation products were detected (Figure 6).

Example 8: Stability in human whole blood of the PEG derivatives of doxorubicin prodrug _{βALAL-Dox, PEG 2000 -ALAL-} Dox, PEG 2000 -βALAL-Dox and _{PEG 2000 - (DSer) 4 -ALAL} -Dox compound Was diluted to 20 μmol with human citrate blood and incubated at 37 ° C. At various time points, the concentration of complexes and metabolites that could be formed were measured by HPLC. FIG. 7 shows the variation of product concentration versus incubation time.

ALAL-Dox compound was used as a control. The latter is not stable in the blood. After 1 hour, 80% of the starting material is decomposed into Leu-Dox and Dox. The other three test complexes are stable in human whole blood for 6 hours (Figure 7). 6 hours, βALAL-Dox (Dox8% and L-Dox10%) and _{PEG 2000 - (DSer) 4 -ALAL} -Dox (Dox5% and Leu-Dox5%) was only been only slightly hydrolyzed. This result shows that substitution of the terminal amine of the oligopeptide with β-alanine prevents hydrolysis of the sequence by peptidases in the blood. The stability of the PEGylated derivatives tested is believed to be due to the presence of non-natural amino acids (β-alanine or d-serine) and PEG used as a stabilizing group.

Example 9 Efficacy study of PEGylated derivative βALAL-Dox of doxorubicin prodrug in HCT116 human colon cancer xenograft model PEG ₂₀₀₀ using a xenograft model in which HCT-116 human colon cancer was transplanted intradermally into Swiss nude / nude mice The anti-tumor effect of-(DSer) ₄ -ALAL-Dox was compared to succinyl-βALAL-Dox. Animals succinyl -βALAL-Dox30μmol / kg or _{PEG 2000 - (DSer) 4 -ALAL} -Dox53 and 110Myumol / kg to 5 times (5 consecutive days) was intravenously bolus (6 mice / group). No death was confirmed in the drug-treated group. The results in FIG. 8 represent the change in animal weight. _PEG 2000 regardless dose _- (DSer) 4 Toxicity to -ALAL-Dox was observed, approximately 20% decrease in (at the end of the study the body weight was decreased in mice treated with succinyl -βALAL-Dox, 49 days From this, it can be seen that the maximum tolerated dose (MTD) is reached in this group. The anti-tumor effect is shown by a T / C curve showing two phases (FIG. 9). In the first phase (up to 21 _{days), PEG 2000 - (DSer)} 4 -ALAL-Dox (110μmol / kg) group showed activity, this is also true succinyl -βALAL-Dox (30μmol / kg) . In the second phase, the activity of the PEGylated derivative (110 μmol / kg) decreases compared to the activity of succinyl-βALAL-Dox. At 53 μmol / kg, tumor regression was induced in the week treated with the PEGylated derivative, rather than suppressing tumor growth. In summary, these _{results, PEG 2000 - (DSer) 4} -ALAL-Dox showed lower toxicity than succinyl -βALAL-Dox, which means that it is activated by the first phase of the test. In this test, the PEGylated derivative has not reached MTD.

Example 10 In HCT-116 human colon cancer xenograft _{model, PEG 2000 - (DSer) 4} -ALAL-Dox vs. _PEG 2000 -ALAL-Dox chemotherapy trials HCT-116 heterologous human colon carcinoma were implanted subcutaneously into Swiss nude / nude mice with transplantation _model, PEG 2000 _- tumor effect of anti _{PEG 2000 -ALAL-Dox - - (} DSer) anti 4 -ALAL-Dox compared to tumor effect. The animals were injected bolus (5 mice / group) with the compound 5 times (5 consecutive days) in amounts of 200, 300 and 400 μmol / kg. In contrast to PEG ₂₀₀₀ -ALAL-Dox, PEG 2000- (DSer) ₄ -ALAL-Dox is toxic at 300 and 400 μmol / kg, leading to weight loss and animal death (FIG. 10). Such toxicity _PEG 2000 _- suggests (DSer) 4 that towards -ALAL-Dox is significantly reactivated in the extracellular compartment than _PEG 2000 -ALAL-Dox. In toxic dose _{(200μmol / kg), PEG 2000} - - Non-equimolar 1 (DSer) 4 -ALAL-Dox is strong anti than _PEG 2000 -ALAL-Dox - shows tumor effect (Figure 11). This result again supports the hypothesis that a molecular spacer-bearing compound with four serines in conformation D leads to stronger tumor reactivation. This data clearly demonstrates that a positive effect is obtained by inserting four D-serine between sequences that can be cleaved upon reactivation of PEG and prodrug.

Example 11: Efficacy study of PEGylated derivatives of TNF-α Using a general male mouse model of type C57BL / 6J in which a B16BL6 mouse tumor (mouse melanoma) was transplanted ectopically (intradermally) _{(PEG 5000 - (DSer) 4} -ALAL) of n-TNFa anti - tumor activity _was compared with _{(PEG 5000) n -TNFα} and _{(PEG 5000 -Ala-Leu-Ala} -Leu) n -TNFα.

  The complex was administered intravenously twice a week for 1 week (0 and 3 days) at an equivalent of 2000 μg TNF-α per kg. There were no immediate signs of toxicity at this dose.

FIG. 12 shows the variation in animal survival. The drug treatment _{group, (PEG 5000 - (DSer)} 4 -ALAL) n -TNFα is 40% of mice on day 6 days and the deaths of 60% on day 10. The mortality rate of another group including the control group did not exceed 20% (1 out of 5 mice), which is thought to be due to the invasiveness and metastasis of B16-BL6 type tumors.

FIG. 13 is a diagram showing a change in average body weight with respect to time since the start of treatment of a medicine. On the day of the initial injection _(PEG 5000 _-ALAL) n -TNFα and _{(PEG 5000 - (DSer) 4} -ALAL) marked weight loss in the group treated with n-TNFa is observed (respectively 15 and 28%) . After the second _{injection, -} it is progressing further weight loss in the group treated with (PEG 5000 (DSer) 4 -ALAL ) n -TNFα. In the latter case, the maximum weight loss is 32% on day 6. In contrast, in the group treated with (PEG _5000- ALAL) _n -TNFα, the body weight recovered after the second injection (day 3). In the group treated with (PEG ₅₀₀₀ ) _n -TNFα, no weight loss was observed.

FIG. 14 shows the anti-tumor effect obtained based on the variation of T / C (ratio of average RTV of drug-treated group (T) and control group (C)) (expressed as a percentage). The results clearly show that the activation of the TNFα prodrug required to release the active form of the protein varies depending on the structure of the complex. _{(PEG 5000) n} -TNFα _{inert, (PEG 5000 -ALAL) n -TNFα} (T / Cmin = 51.7% (j3); SGD = 1.66) is _{(PEG 5000 - (DSer) 4} -ALAL ) The activity is lower than that of _n- TNFα (T / Cmin = 16.7% (j6); SGD = 3.72). This result shows that the activation and toxicity of the complex are correlated. If there is no peptide between PEG and TNFα, cytotoxicity is low or zero (determined by weight loss). Inserting an ALAL bond between these two sites makes the product more toxic, reflecting its high extra-blood reactivation. Extending this peptide (ALAL) by inserting a hydrophilic spacer (DSer) ₄ significantly increases the toxicity of the complex, but clearly this is a result of improved ability to reactivate outside the blood compartment.

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BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline of two synthesis methods of the PEGylated prodrug of doxorubicin of this invention. It is a figure which shows the cytotoxicity test with respect to MCF-7 / 6 cell of the prodrug of this invention. It is a figure which shows the fluctuation | variation of the average body weight of the xenograft mouse | mouth which has LS-174-T tumor at the time of the prodrug process of this invention. It is a figure which shows the fluctuation | variation of the average value of the relative tumor volume (RTV) of the athymic mouse which has the LS-147-T human colon cancer tumor at the time of the prodrug process of this invention. It is a figure which compares the inhibition of the 1st growth phase of the LS-174T tumor at the time of the prodrug treatment of this invention, and a 2nd growth phase. It is a figure which shows the stability in serum culture medium of the prodrug of this invention. It is a figure which shows the stability in human whole blood of the prodrug of this invention. It is a figure which shows the fluctuation | variation of the average body weight of the athymic mouse | mouth with a HCT-116 human colon cancer tumor at the time of the prodrug process of this invention. It is a figure which shows the growth inhibition of HCT-116 tumor from the fluctuation | variation of the ratio of the RTV average value of the prodrug processing group (T) of this invention, and a control group (C). It is a figure which shows the fluctuation | variation of the survival rate of the xenograft mouse | mouth with a HCT-116 tumor at the time of the prodrug process of this invention. It is a figure which shows the growth inhibition of HCT-116 tumor from the fluctuation | variation of the ratio of the RTV average value of the prodrug processing group (T) of this invention, and a control group (C). It is a figure which shows the fluctuation | variation of the survival rate of the transplanted mouse | mouth with a B16-BL6 melanoma tumor at the time of the prodrug process of this invention. It is a figure which shows the fluctuation | variation of the average body weight of the transplanted mouse | mouth with a B16-BL6 melanoma tumor at the time of the prodrug process of this invention. It is a figure which shows the growth inhibition of B16-BL6 tumor from the fluctuation | variation of the ratio of the RTV average value of the prodrug processing group (T) of this invention, and a control group (C).

Claims (33)

[A compound represented by the formula (A) _p- (EB) _n- (I) _m ,
Where
I is an active substance that favors the target cell,
A is a group that extends the half-life of BI during blood circulation;
EB is a group for linking A and I;
B is a structure that can be selectively cleaved by an enzyme present alone or preferably by an enzyme present near or on the target cell;
E is a hydrophilic group that is stable in the circulating tissue, which allows or facilitates cleavage of B near or on the target cell by pulling A away from B; As a result, release of I or release of I with the group B is possible or facilitated.
n is an integer from 1 to the total number of reactive groups I to which the linking group EB can bind, or up to the total number of reactive groups A to which the linking group EB can bind,
m is an integer from 1 to the total number of reactive groups A to which the linking group EB can bind;
p is an integer from 1 to the total number of reactive groups I to which the linking group EB can be attached;
n = m when p = 1, and n = p when m = 1]. A compound represented by the formula (A) _p- (EB) _n- (I) _m .   2. A compound according to claim 1, characterized in that I is directly bound to one or more structures B by a covalent bond.   2. A compound according to claim 1, characterized in that I is bound to one or more structures B via a linking arm.   4. A compound according to any one of claims 1 to 3, characterized in that the size of E is equal to 1 to 100 amino acids.   E is composed of or comprises 1 to 100, preferably 1 to 20, very preferably 2 to 10 identical or different amino acids, said amino acids being in conformation D natural 5. A compound according to any one of claims 1 to 4, characterized in that it is selected from the group comprising amino acids, said amino acids not being genetically encoded.   The amino acid is selected from D-glutamine, D-asparagine, D-serine, D-histidine, D-threonine, D-aspartic acid, D-glutamic acid, D-lysine and D-arginine. Item 6. The compound according to Item 5.   E is composed of or includes an amino acid sequence selected from (D-serine) x, (D-threonine) x, and x is an integer of 1 to 20, preferably 2 to 10 7. A compound according to any one of claims 1 to 6, characterized in that it is an integer, very preferably an integer from 2 to 6.   6. A compound according to claim 4 or 5, characterized in that E is a peptidomimetic drug, a pseudopeptide or a peptide.   E is composed of or contains at least one group selected from substituted alkyl chains, polyalkyl glycols, polysaccharides, polyols, polycarboxylates and poly (hydro) esters The compound according to any one of claims 1 to 4.   The compound according to any one of claims 1 to 9, wherein the size of E increases as the molecular weight of A increases.   E consists of one or more amino acids according to any of claims 5 to 8 and at least one group according to claim 9 or 10, or comprises such amino acids and groups A compound according to any one of claims 1 to 4, characterized in that A further shows the solubilization properties of the compound of formula (A) _p- (EB) _n- (I) _m in water, blood or serum. 12. The compound according to any one of 11 to 11. A further shows the targeting properties of the compound of formula (A) _p- (EB) _n- (I) _m to the target cell, characterized in that The compound according to any one of the above.   14. A compound according to any one of claims 1 to 13, characterized in that A is further a drug with therapeutic activity or a drug with diagnostic activity.   15. A compound according to any one of claims 1 to 14, characterized in that A is hydrophilic or amphiphilic.   16. A compound according to any one of claims 1 to 15, characterized in that A is selected from polypeptides, immunoglobulins, albumin, polysaccharides, polymers and copolymers.   17. A compound according to claim 16, characterized in that A is selected from polyalkylene glycols, polyalkylene oxides, polyalkyleneimines, and vinyl chloride copolymers.   A is polyethylene glycol, polyethylene oxide, polyethyleneimine, sodium styrenesulfonate (NaSS), sodium maleate and butyl maleate (MMBE), hydroxypropyl methacrylate or N- (2-hydroxypropyl) methacrylamide (HPMA), Selected from methyl methacrylate (MMA), poly- [N- (2-hydroxyethyl) -L-glutamine] (PHEG), and poly- [N- (hydroxyethyl) -DL-aspartamide] (PHEA) 18. A compound according to claim 17, characterized in that   18. A compound according to claim 17, characterized in that A is a polyethylene glycol with a size of 200 to 50,000 Da, preferably 350 to 20,000 Da, very preferably 1,000 to 10,000 Da.   In a cellular environment wherein B is selected from the group consisting of tumor cells, tumor stromal cells, tumor or metastatic tumor angiogenic endothelial cells, macrophages, monocytes, lymphocytes or multinucleated bodies capable of invading tumor and metastatic tumor 20. A compound according to any one of claims 1 to 19, characterized in that it can be cleaved by an existing enzyme.   21. A compound according to any one of claims 1 to 20, characterized in that B is selected from oligopeptides, oligosaccharides and lipid chains.   Structure B is selected from the following peptide sequences: L-alanine-L-leucine-L-alanine-L-leucine, L-alanine-L-tyrosine-L-glycine-L-glycine-L-phenylalanine-L-leucine The compound according to claim 21, wherein   23. A compound according to any one of claims 1 to 22, characterized in that B can be cleaved by an enzyme selected from peptidases, endopeptidases, lysosomal enzymes, lipases, and glycosidases.   24. B can be cleaved by peptidases that are selectively present in the environment of tumor cells, tumor stromal cells, angiogenic endothelial cells, macrophages, monocytes, lymphocytes or polynuclear bodies. Compound described in 1.   The tumor cell specific peptidase is selected from the group consisting of neprilysin (CD10), thyme oligopeptidase (TOP), prostate specific antigen (PSA), plasmin, legumain, collagenase, urokinase, cathepsin, and substrate metallopeptidase. 25. A compound according to claim 24, characterized in that   26. Any one of claims 1 to 25, characterized in that I is selected from the group consisting of chemicals, polypeptides, proteins, nucleic acids, antibiotics, viruses or markers optionally linked to vector material. The compound according to item 1.   27. A compound according to claim 26, characterized in that I is an agent having anti-tumor therapeutic activity, anti-angiogenic activity or anti-inflammatory activity.   I is anthracycline, doxorubicin, daunorubicin, folic acid derivative, vinca alkaloid, calicheamicin, mitoxantrone, cytosine arabinoside, adenosine arabinoside, fludarabine phosphate, melphalan, bleomycin, mitomycin, L-canavanine, taxoid, camptothecin 9-dimethylaminomethyl-hydroxy-camptothecin hydrochloride, proteasome inhibitor, farnesyl transferase inhibitor (FTI), epothilone, maytansinoid, discodermolide, fostriecin, platinum derivative, duocarmycin, combretastatin, Epipodophyllotoxin, BH3 peptide, P53 peptide, caspase, granzyme B; ribozyme, tumor necrosis factor-α (TNF-α ), Interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin 1 (IL-1), IL-2, IL-6, IL-12, IL-15 and IGF-1 antagonists 28. A compound according to claim 27, characterized in that it is selected from the group consisting of:   I is a group consisting of coumarin, 7-amido-trifluoromethylcoumarin, paranitroanilide, 8-naphthylamide and 4-methoxynaphthylamide, fluorescein, biotin, rhodamine and their derivatives and drugs used in scintigraphy 27. Compound according to claim 26, characterized in that it is a more selected marker. And further comprising one or more targeting substances capable of directing the compound of formula (A) _p- (EB) _n- (I) _m to the target cell. Item 30. The compound according to any one of Items 1 to 29. 31. The targeting substance according to claim 30, characterized in that the targeting substance is bound on one or more groups A with a compound of formula (A) _p- (EB) _n- (I) _m . Compound.   32. A diagnostic or follow-up medicinal product comprising at least one compound according to any one of claims 1 to 31 as an active ingredient. Compound:
Polyethylene glycol-D-seryl-D-seryl-D-seryl-D-seryl-L-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin polyethylene glycol-D-seryl-D-seryl-D-seryl -D-Ceryl-L-alanyl-L-leucyl-L-alanyl-L-leucyl-TNFα
Polyethylene glycol-D-seryl-D-seryl-D-seryl-D-seryl-L-alanyl-L-tyrosyl-L-glycyl-L-glycyl-L-phenylalanyl-L-leucyl-doxorubicin polyethylene glycol-D -Ceryl-D-seryl-D-seryl-D-seryl-L-alanyl-L-leucyl-L-alanyl-L-leucyl-TNFα
A compound selected from the group comprising:

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