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WO2007103364A2 - Administration ciblée de médicament - Google Patents

Administration ciblée de médicament Download PDF

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Publication number
WO2007103364A2
WO2007103364A2 PCT/US2007/005679 US2007005679W WO2007103364A2 WO 2007103364 A2 WO2007103364 A2 WO 2007103364A2 US 2007005679 W US2007005679 W US 2007005679W WO 2007103364 A2 WO2007103364 A2 WO 2007103364A2
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Prior art keywords
drug
linker
conjugate
conjugates
oligopeptide
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PCT/US2007/005679
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Inventor
Ying Chau
Robert S. Langer
Ying Luo
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers

Definitions

  • US 2004-01 16348 to Chau et al. describes a targeted drug delivery system that makes use of a polymer-linker-drug conjugate (generally referred to herein as a "conjugate").
  • the linker includes a segment that is recognized and cleaved by a digestive enzyme that is overexpressed in a target tissue, preferably in the extracellular space of the target tissue.
  • the target tissue may be a diseased tissue (e.g., a tumor).
  • the recognition segment within the linker is thought to be cleaved by the digestive enzyme.
  • the active drug is thereby released from the conjugate and subsequently internalized by the cells of the target tissue.
  • the present invention provides compositions and methods that enable multiple drugs to be administered to patients in a safe and effective manner. Indeed, the treatment efficacy of many traditional combination therapies (e.g., cancer treatments that use two or more drugs) is often limited because the dose-limiting toxicities (DLTs) of the individual drugs are lower when the two drugs are administered in combination than when they are administered individually. In such cases, the dose of each drug needs to be reduced in the combination therapy, thereby reducing the individual drug contributions to overall treatment efficacy. In addition, this hampers the opportunities for identifying novel synergisms.
  • DLTs dose-limiting toxicities
  • the present invention solves this problem by using a conjugate as one or more of the combination therapeutics. Because conjugates deliver their drugs in a targeted manner, they have higher dose-limiting toxicities than the drugs themselves. By using a conjugate as one or more of the combination therapeutics one can therefore increase the dose of one or more of the drugs in the combination.
  • the present invention provides compositions and methods for improving the targeted delivery of drugs using conjugates.
  • conjugates are administered in combination with a secondary treatment that increases the concentration of the digestive enzyme within the target tissue.
  • a conjugate may be administered in combination with radiation that is known to increase the concentration of the digestive enzyme of interest within the target tissue.
  • the targeting of a conjugate sensitive to the digestive enzyme should improve as a result of the increased presence of enzyme within the target tissue.
  • administered in combination with encompass concurrent and sequential administration.
  • the schedule of administration will be selected to provide optimal therapeutic effect to the patient. For example, when two or more therapeutics are administered in combination, the schedule of administration may be selected to avoid antagonistic effects and/or to achieve synergistic treatment outcomes.
  • a conjugate is administered in combination with a secondary treatment that increases the concentration of the digestive enzyme within the target tissue, it will be appreciated that the conjugate is preferably administered so that it reaches the target tissue when the enzyme concentration is sufficiently increased to generate an improvement in delivery.
  • a “digestive enzyme” is an enzyme that cleaves polymers.
  • the cleaved polymers are oligopeptides or oligosaccharides.
  • Digestive enzymes of the present invention exhibit some form of specificity.
  • a digestive enzyme that cleaves oligopeptides will typically exhibit strong selectivity for oligopeptides that include one or a small subset of amino acid sequences called recognition sequences.
  • the specificity and recognition sequence of a particular digestive enzyme may be determined by comparing the rate at which it cleaves different polymers within a given family (e.g., oligopeptides or oligosaccharides having different sequences).
  • Dose-limiting toxicity As used herein, the “dose-limiting toxicity” or “DLT” of a drug is the dose at which side effects appear during treatment that are severe enough to prevent further increase in dosage of a drug.
  • the DLT of a given drug can be obtained from the Phase I clinical trial results.
  • the DLT of a drug may depend on the treatment regimen. Thus, a drug will often have a lower DLT when used in combination with another drug than when used alone.
  • an "oligopeptide” comprises a string of at least three amino acid residues linked together by peptide bonds.
  • the terms “oligopeptide”, “peptide”, “polypeptide” and “protein” may be used interchangeably.
  • Inventive oligopeptides preferably contain only natural amino acid residues, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • one or more of the amino acid residues in an inventive oligopeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • the modifications of the oligopeptide lead to a more stable oligopeptide (e.g., greater stability to digestion by enzymes in the gastrointestinal tract). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. If the modifications are made to an oligopeptide drug molecule, they should not substantially interfere with the desired biological activity of the oligopeptide.
  • an “oligonucleotide” comprises a string of at least three nucleotides linked together by phosphodiester bonds.
  • the terms “oligonucleotide”, “polynucleotide” and “nucleic acid” may be used interchangeably.
  • an oligonucleotide comprises at least three nucleosides.
  • Oligonucleotides may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine), chemically modified bases, biologically modified
  • oligosaccharide refers to a polymer of sugars.
  • the terms “oligosaccharide”, “polysaccharide” and “carbohydrate” may be used interchangeably.
  • an oligosaccharide comprises at least three sugars.
  • the polymer may include natural sugars (e.g., glucose, fructose, galactose, mannose, arabinose, ribose and xylose) and/or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose and hexose).
  • a digestive enzyme is “overexpressed” in a tissue sample if it is present in excess relative to the abundance of that enzyme in at least one other tissue sample (e.g., when comparing a diseased tissue with a healthy tissue from the same individual).
  • tissue sample e.g., when comparing a diseased tissue with a healthy tissue from the same individual.
  • overexpression at the RNA level and overexpression at the protein level.
  • detection of either mRNA or a corresponding polypeptide is generally sufficient to determine whether a particular polypeptide is overexpressed.
  • it may be more convenient and/or practical to detect mRNA while in other situations it may be more convenient and/or practical to detect polypeptides.
  • Toxic As used herein, a drug combination is "toxic” if it produces side effects during treatment that are severe enough to prevent further treatment at that dosage.
  • Figure 1 is a schematic that illustrates the overall structure of an embodiment of a conjugate that may be used according to the invention.
  • Figure 2 is a schematic that illustrates how substrate phage display methodology can be used to select suitable oligopeptide sequences for use in linkers of conjugates.
  • Figure 3 is a schematic that illustrates the chemical structure of the free drug doxorubicin.
  • Figure 4 is a schematic that illustrates the chemical structure of a CM-dextran- oligopeptide-doxorubicin conjugate.
  • Figures 5A-B are graphs that compare the digestion of four different CM-dextran- oligopeptide-doxorubicin conjugates by pure matrix-metalloproteinase II (MMP-2) at 37 C.
  • the conjugates each include a linker with an oligopeptide in the series IPVGLIG (diamond), IPVGLI (cross), IPVGL (square) and IPVG (triangle).
  • the graphs show the concentration of released peptidyl-doxorubicin as a function of digestion time.
  • FIGS. 6A-F are graphs that compare the cytotoxicity of free doxorubicin (empty squares) and the cleaved peptidyl-doxorubicin (i.e., LIG-doxorubicin, filled squares) on six different human tumor cell lines, namely HT- 1080, BT-20, U-87, PC-3, KK-47 and MGH- Ul.
  • the graphs show the % survival of tumor cells as a function of the concentration of the doxorubicin-equivalent concentration in the cell culture.
  • Figure 7 is a schematic that illustrates the chemical structure of a dextran- poly(ethyleneglycol)-oligopeptide-doxorubicin conjugate.
  • Figure 8 is a schematic that illustrates the chemical structure of a methoxy- poly(ethyleneglycol)-oligopeptide-doxorubicin conjugate
  • Figure 9 is a schematic that illustrates the chemical structure of the free drug methotrexate.
  • Figure 10 is a graph that compares the cytotoxicity of methotrexate-PVG (empty squares), methotrexate-IPVG (empty triangles) and free methotrexate (empty circles) on the human tumor cell line HT-1080.
  • the graphs show the % survival of tumor cells as a function of the concentration of the methotrexate-equivalent concentration in the cell culture.
  • Figures HA-C are graphs that compare the cytotoxicity of free methotrexate (empty circles) and the cleaved peptidyl-methotrexate (i.e., PVG-methotrexate, filled circles) on three different human tumor cell lines, namely HT-1080, BT-20 and RT-112.
  • the graphs show the % survival of tumor cells as a function of the concentration of the methotrexate- equivalent concentration in the cell culture.
  • Figures 12A-C depict a synthetic scheme for preparing dextran-oligopeptide- methotrexate conjugates.
  • Figure 13 is a graph comparing the tumor progression after different groups of SCID mice bearing HT-1080 tumors were treated with free methotrexate, a dextran-methotrexate conjugate, a dextran-oligopeptide-methotrexate conjugate, modified dextran or phosphate buffered saline (PBS).
  • Figure 14 is a graph comparing the body weight change after different groups of
  • SCID mice bearing HT-1080 tumors were treated with free methotrexate, a dextran- methotrexate conjugate, a dextran-oligopeptide-methotrexate conjugate, modified dextran or PBS.
  • Figure 15 is a graph comparing the tumor progression after two different groups of SCID mice bearing HT- 1080 tumors were treated with two dextran-oligopeptide- methotrexate conjugates having different backbone negative charges.
  • Figure 16 is a table that lists the cleavage motifs for a range of secreted or membrane bound proteases that are overexpressed in certain tumor tissues.
  • the present invention relates to novel uses of polymer-linker-drug conjugates including those described in US 2004-0116348 to Chau et al.
  • the conjugates, including their polymer, linker and drug components, are described in greater detail below.
  • the present invention provides compositions and methods that enable multiple drugs to be administered to patients in a safe and effective manner.
  • the treatment efficacy of many traditional combination therapies e.g., cancer treatments that use two or more drugs
  • DLTs dose-limiting toxicities
  • the present invention solves this problem by using a conjugate as one or more of the combination therapeutics. Because conjugates deliver their drugs in a targeted manner, they have higher dose-limiting toxicities than the drugs themselves.
  • a conjugate as one or more of the combination therapeutics one can therefore increase the dose of one or more of the drugs in the combination.
  • two or more conjugates that carry different drugs are administered in combination.
  • a conjugate is administered with one or more non-conjugated drugs.
  • one can increase the dose of just one or several drugs in the combination e.g., one or both drugs in a combination of two drugs. It is also to be understood that one can increase the dose of a drug which is conjugated and/or the dose of a drug which is non-conjugated.
  • compositions of the present invention are in no way limited to specific drugs, specific drug combinations or specific diseases.
  • Exemplary drugs that may be used according to the present invention in conjugated or non-conjugated form are discussed in this section, the section below describing the drug component of conjugates and further in the Examples.
  • a conjugate will be used to replace one or more non- conjugate drugs in a known combination therapy.
  • certain metastatic breast cancers are currently treated with a combination of cyclophosphamide, methotrexate and fluorouracil (CMF) or a combination of cyclophosphamide, doxorubicin and fluorouracil (CAF).
  • methotrexate in the CMF combination could be replaced with a methotrexate conjugate (e.g., without limitation, the MMP-sensitive methotrexate conjugates described in the Examples).
  • cyclophosphamide or fluorouracil could also be replaced by a corresponding conjugate.
  • doxorubicin in the CAF combination could be replaced with a doxorubicin conjugate (e.g., without limitation, the MMP-sensitive doxorubicin conjugates described in the Examples).
  • two or all three drugs in these combination therapies could be replaced by corresponding conjugates.
  • Bladder, head and neck and endometrial cancers could similarly be treated by replacing one or more of the individual drugs in M-VAC (methotrexate, vinblastin, adriamycin, cisplatin) or CMV (cisplatin, methotrexate, vinblastin) with a corresponding conjugate.
  • M-VAC metalhotrexate, vinblastin, adriamycin, cisplatin
  • CMV cisplatin, methotrexate, vinblastin
  • Replacing methotrexate or another one of the drugs with a conjugate in these regimens could potentially increase the effectiveness of the combination while reducing toxicity.
  • the doses of individual drugs are generally reduced in a traditional combination therapy because their dose-limiting toxicities (DLTs) are lower when administered in combination than when administered individually.
  • DLTs dose-limiting toxicities
  • conjugates deliver their drugs in a targeted manner, they have higher dose-limiting toxicities than the drugs themselves.
  • a conjugate as one or more of the combination therapeutics one can therefore increase the dose of one or more of the drugs in the combination.
  • the dose of just one of the drugs in the combination is increased (e.g., one drug in a combination of two or more drugs).
  • the dose of several drugs in the combination is increased (e.g., two, three, four, etc. drugs in a combination of two or more drugs).
  • the present invention allows the overall dosage of the drugs in the combination to be such that the combination would be toxic but for the presence of the one or more conjugates.
  • a traditional combination of drug A and drug B that is non-toxic when the drugs are administered at concentrations [Al] and [Bl] but toxic when they are administered at concentrations [Al] and [B2] (where [B2] > [Bl]).
  • the present invention would allow drug A and drug B to be administered at concentrations [Al] and [B2] without toxicity.
  • the methods and compositions of the present invention do not require the dose of one or more of the component drugs to be increased as compared to a traditional therapy. Indeed, by including a conjugate in an inventive combination therapy without changing the dosage (or even reducing a dosage) one will still obtain a beneficial increase in treatment efficacy and decrease in toxicity.
  • the present invention would allow drug A and drug B to be administered at concentrations [Al] and [Bl] with improved efficacy and/or decreased toxicity.
  • the administration of drug A and drug B at concentrations [Al] and [Bl] may be slightly or even severely toxic. According to the present invention by replacing drug A and/or drug B with a corresponding conjugate the same combination could be administered at concentrations [Al] and [Bl] without toxicity. In another embodiment the administration of drug A and drug B at concentrations [Al] and [Bl] may produce low efficacy. According to the present invention by replacing drug A and/or drug B with a corresponding conjugate the same combination could be administered at concentrations [Al] and [Bl] with increased efficacy.
  • the present invention provides compositions and methods for improving the targeted delivery of drugs using conjugates.
  • conjugates are administered in combination with a secondary treatment that increases the concentration of the digestive enzyme within the target tissue.
  • the conjugate is administered in such a way that it reaches the target tissue at or around the time when the digestive enzyme concentration reaches a peak. It will be appreciated that this may require the conjugate to be administered before, with or after the secondary treatment depending on the kinetics involved. When the secondary treatment yields a rapid spike in enzyme concentration then the conjugate may need to be administered before the secondary treatment. Conversely, when the secondary treatment leads to a delayed increase in enzyme concentration then the conjugate may be advantageously administered some time after the secondary treatment.
  • the conjugate is administered in combination with radiation that is known to increase the concentration of the digestive enzyme of interest within the target tissue.
  • radiation that is known to increase the concentration of the digestive enzyme of interest within the target tissue.
  • MMP-2 matrix- metalloproteinase II
  • Several conjugates that are recognized and digested by MMP-2 are described in the Examples herein. When combined with ionizing radiation, the targeting of these conjugates should improve as a result of the increased presence of MMP-2 within the target tissue. It will be appreciated that this particular combination is simply illustrative of the broader invention.
  • traditional radiation therapies e.g., external or internal ionizing radiation used to kill cancer cells, ultraviolet light therapy used to treat psoriasis, etc.
  • radiation therapies may be used to provide the necessary radiation (e.g., see “Principles and Practice of Radiation Oncology,” Edited By Perez et al., Lippincott Williams & Wilkins, 2003).
  • such radiation therapies will have a dual benefit to the patient, namely their primary purpose (e.g., killing cancer cells) and a novel secondary purpose (i.e., enhancing the release of drugs from conjugates).
  • any form of radiation that increases the expression of a digestive enzyme of interest within a target tissue may be used with a conjugate that is sensitive to that digestive enzyme.
  • the present invention makes use of polymer-linker-drug conjugates that are designed to preferentially deliver drugs to target tissues that overexpress a digestive enzyme.
  • the linker includes a segment that is recognized and cleaved by a digestive enzyme that is overexpressed in the extracellular space of the target tissue.
  • the recognition segment is preferably an oligopeptide or oligosaccharide segment.
  • the linker may also include a spacer that separates the recognition segment from the polymeric carrier and/or drug.
  • the polymeric carrier is preferably hydrophilic, biodegradable and biocompatible. In preferred embodiments the polymeric carrier is greater in size than the renal excretion limit. The physiochemical features of the polymeric carrier allow the conjugate to circulate longer in plasma by decreasing renal excretion and liver clearance.
  • the polymeric carrier may be loaded with any number of drug molecules. In particular it is to be understood that the conjugate may include a single drug molecule or a plurality of drug molecules each attached to the polymeric carrier via an inventive linker.
  • any drug molecule whether a small molecule drug or a biomolecular drug (e.g., a therapeutic protein or nucleic acid) may be delivered using a conjugate prepared according to the invention.
  • a conjugate prepared according to the invention may be delivered using a conjugate prepared according to the invention.
  • the following sections describe certain embodiments of the polymeric, linker and drug components of the conjugates.
  • the polymeric carrier allows conjugates and hence drugs to circulate longer in plasma by decreasing renal excretion and liver clearance.
  • Strategies for minimizing body clearance have focused on the liver and kidney because they are known to be the major elimination sites in the body.
  • Hashida and Takakura have reviewed the current status of macromolecular drug delivery systems (Hashida and Takakura, Journal of Controlled Release 31:163, 1994). They related the physiological features of the liver and kidney to the clearance data obtained with macromolecules.
  • the polymeric carrier may be designed to be greater than this renal excretion limit. It is to be understood however that the inventive conjugates may also include polymer carriers that are smaller than the renal excretion limit.
  • CM- dextran carboxymethyl-dextran
  • EPR enhanced permeability and retention
  • the PEG brush also inhibits proteins from interacting with the core through steric hindrance (VertutDoi et al., Biochimica Biophysica Acta - Biomembranes 1278:19, 1996; Gref et al., Colloids Surfaces B - Biointerfaces 18:301, 2000). These features prolong the circulation of polymeric carriers in the circulation.
  • the polymeric carriers used in a conjugate are biocompatible.
  • Biocompatible polymers are not significantly toxic to cells.
  • In order to prevent chromic accumulation of polymeric carriers that are larger than the renal excretion limit are preferably both biocompatible and biodegradable.
  • Biodegradable polymers are broken down by the cellular machinery and/or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect.
  • a biodegradable polymer and its biodegradation byproducts are biocompatible. It is to be understood that any known biodegradable polymer may be incorporated in a conjugate.
  • Preferred polymeric carriers are hydrophilic, e.g., they may include polar groups, such as hydroxyl or amine groups; anionic groups, such as carboxylate, sulfonate, sulphate, phosphate, or nitrate groups; or cationic groups, such as protonated amine, quaternary ammonium, or phosphonium groups.
  • Suitable hydrolytically degradable polymers known in the art include for example, certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes and polyphosphoesters.
  • biodegradable polymers include, for example, certain carbohydrates, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, poly(amino acids), polyacetals, polyethers, biodegradable polycyanoacrylates and biodegradable polyurethanes.
  • specific biodegradable polymers that may be used include but are not limited to alginate, carboxymethyl-alginate, cellulose, polylysine, poly(lactic acid), poly(glycolic acid), poly(caprolactone), poly(lactide- co-glycolide), poly(lactide-co-caprolactone) and poly(glycolide-co-caprolactone).
  • conjugates may comprise block co-polymers, graft co-polymers, or adducts of these and other polymers.
  • any drug whether a small molecule drug or a biomolecular drug may be delivered using a conjugate.
  • the term "small molecule drug” refers to a molecule, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that has a relatively low molecular weight and that is not a protein, a nucleic acid, or a carbohydrate.
  • small molecule drugs are monomelic and have a molecular weight of less than about 1500 g/mol.
  • the drug is one that has already been deemed safe and effective for use by the appropriate governmental agency or body.
  • drugs for human use listed by the FDA under 21 C.F.R. ⁇ 330.5, 331 through 361 and 440 through 460; drugs for veterinary use listed by the FDA under 21 C.F.R. ⁇ 500 through 589, incorporated herein by reference, are all considered acceptable for use in conjugates.
  • Classes of small molecule drugs that can be used in the practice of the present invention include, but are not limited to, anti-AIDS drugs, anti-cancer drugs, antibiotics, immunosuppressants, anti-viral drugs, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-inflammatory drugs, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson drugs, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti -protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or anti
  • biomolecular drug refers to a molecule, whether naturally- occurring or artificially created (e.g., via synthetic or recombinant methods) that has a relatively high molecular weight and that is a protein, a nucleic acid or a carbohydrate. Typically, though not necessarily, biomolecular drugs have a molecular weight of more than about 1500 g/mol.
  • Classes of biomolecular drugs that can be used in the practice of the present invention include, but are not limited to, therapeutic proteins (e.g., enzymes, neurotransmitters, hormones, cytokines, cell response modifiers such as growth factors and chemotactic factors, antibodies, haptens, toxins, interferons, etc.) and therapeutic nucleic acids (e.g., aptamers, ribozymes, anti-sense agents, gene vectors, etc.).
  • therapeutic proteins e.g., enzymes, neurotransmitters, hormones, cytokines, cell response modifiers such as growth factors and chemotactic factors, antibodies, haptens, toxins, interferons, etc.
  • therapeutic nucleic acids e.g., aptamers, ribozymes, anti-sense agents, gene vectors, etc.
  • the conjugates include a linker with at least a first and a second end.
  • the linker includes at least one segment that is cleaved when exposed to a digestive enzyme that is expressed in the target tissue.
  • the digestive enzyme is overexpressed in the target tissue.
  • the cleavable segment includes an oligopeptide or an oligosaccharide sequence.
  • the first end of the linker is associated with the polymeric carrier and the second end is associated with the drug molecule.
  • two entities are "associated with" one another as described herein, they are linked by a covalent or ligand/receptor type interaction.
  • the association is covalent.
  • a whole host of synthetic methods exist for covalently linking oligopeptide or oligosaccharide segments with the polymeric carriers and drugs of the present invention.
  • the Examples presented below describe exemplary synthetic methods in greater detail.
  • any ligand/receptor pair with a sufficient stability and specificity to operate in the context of the inventive system may be employed to associate two entities.
  • the ligand/receptor interaction should be sufficiently stable to prevent premature release of the drug molecule from an inventive conjugate (i.e., prior to enzymatic cleavage of the linker).
  • a drug may be covalently linked with biotin and the linker with avidin.
  • ligand/receptor pairs include antibody/antigen, protein/co-factor and enzyme/substrate pairs.
  • biotin/avidin these include for example, biotirt/streptavidin, FK506/FK506-binding protein (FKBP), rapamycin/FKBP, cyclophilin/cyclosporin and glutathione/glutathione transferase pairs.
  • FKBP FK506/FK506-binding protein
  • rapamycin/FKBP rapamycin/FKBP
  • cyclophilin/cyclosporin glutathione/glutathione transferase pairs.
  • Other suitable ligand/receptor pairs would be recognized by those skilled in the art.
  • the chemical composition of the cleavable segment (e.g., the length and sequence of amino acids or sugars) will depend for the most part on the motif that is recognized and cleaved by the digestive enzyme in the target tissue.
  • the cleavage motifs are known for a number of proteases.
  • the table provided in Figure 16 lists the cleavage motifs for a range of secreted or membrane bound proteases that are overexpressed in certain tumor tissues. These are all potential target enzymes that could be used to trigger release of drug molecules from conjugates.
  • the Examples describe the optimization of a cleavage segment that is recognized by matrix-metalloproteinase II (MMP-2), a proteinase that is also overexpressed in a variety of rumors.
  • MMP-2 matrix-metalloproteinase II
  • a variety of methods are also known in the art that can be used to determine the cleavage motif of a target enzyme when it is not yet known. These include substrate phage display libraries (Matthews and Wells, Science 260:1113, 1993); positional-scanning peptide libraries (Rano et al., Chem. Biol. 4:149, 1996); and mixture-based peptide libraries (Turk et al., Nature Biotechnology 19:661, 2001). Positional-scanning synthetic peptide libraries are based on the detection of cleavage by the release of a C-terminal fluorogenic group. The technique is rapid and enables analysis of all possible peptide sequences.
  • the cleavage site motif C- terminal to the cleavage site is first determined by partial digestion and N-terminal sequencing of a completely random peptide mixture. Information from this first round of screening is then used to design a second library in which strong selected amino acids are fixed, allowing data on sites N-terminal to the cleavage site to be obtained. Reiteration of this process allows an optimal recognition sequence to be determined.
  • the method has recently been used to determine the cleavage motifs of a variety of matrix-metalloproteinases (Turk et al., Nature Biotechnology 19:661, 2001).
  • the phage display method has been used to determine peptide substrates for a number of proteases, for example, plasmin (Hervio et al., Chemistry & Biology 7:443, 2000); tissue- type plasminogen activator (Ding et al., Proc. Natl. Acad. Sci USA 92: 7627, 1995; Ke et al., J. Biol. Chem. 272: 16603, 1997); prostate-specific antigen (Coombs et al., Chemistry & Biology 5:475, 1998); and membrane type-1 matrix metalloproteinase (Ohkubo et al.,
  • Each phagemid codes for a phage coat protein and a tether with a protease substrate sequence linking the two.
  • the substrate sequence is randomized by site-directed mutagenesis.
  • Each fusion protein is displayed on the surface of a phage particle.
  • the phage particles are incubated with the protease of interest.
  • the entire digest is then captured using a support with affinity for the tether. Phage with digestion resistant sequences bind the support via the tether.
  • the tether can be an epitope for monoclonal antibody, a histidine tag or a protein-binding peptide.
  • Phage with labile peptide sequences lack the tether and are not bound by the support. Phage with resistant sequence are subsequently released under elution conditions that disrupt binding between the tether and affinity support. Phage with desired sequences, either labile or resistant to the protease of interest, are propagated in bacteria and the cycle is repeated for enrichment.
  • the incubation conditions can be changed to vary the stringency of the screening criteria. For example, highly labile sequences can be identified by selecting for phage that are released at lower protease concentrations or after shorter incubation times.
  • sequences which are labile to target enzyme but resistant to serum proteins can be preferentially selected from a library of peptides. For example, in a first screen, phage that are released upon incubation with the target enzyme are enriched. These phage are then incubated with serum proteins. Phage which remain on the affinity support are then enriched. The DNA sequence of the phagemids can be determined and translated into an amino-acid sequence.
  • phage display screening technique One potential disadvantage of the phage display screening technique is that the peptides may adopt a different conformation when displayed on the phage surface. The peptides may therefore lose their specificity when used as a linker in the polymer-drug conjugate. Hence it may be necessary to study the kinetics of cleavage of the screened peptides in conjugation with a polymeric carrier of choice, in order to confirm their specificity towards the target enzyme. For example, in order to evaluate whether a particular linker is suitable for use in an inventive conjugate, a set of polymer-linker-drug conjugates or polymer-linker-dye conjugates may be synthesized for kinetic analysis.
  • mPEG-linker-pNA conjugates where mPEG is methoxy poly(ethyleneglycol) and pNA is the dye p-nitroanilide.
  • pNA is the dye p-nitroanilide.
  • the stability of the test conjugates in circulation may be assessed by incubating these with serum (e.g., as described in Trouet, Masquelier et al., Proc. Natl. Acad. ScL USA 79:626-629, 1982).
  • the digestion mixtures are then analyzed by size exclusion HPLC which separates large polymers from small dye or drug molecules. Dye or drug molecules that have been leaked are detected using a spectrophotometer or fiuorometer.
  • the linker sequences are also preferably cleaved at much higher rates by the target enzyme than by non-specific enzymes in normal tissues. Fulfilling this condition is preferred in order to ensure site-specific release of the active drug molecules.
  • the test conjugates are first incubated with the target enzyme(s) and the initial rates of release are measured. ⁇ similar experiment is then performed with the non-specific enzymes.
  • the kinetics can be modeled using the Michaelis-Menten equation, that is,
  • linker can also include spacer segments that increase the distance between the bulky polymeric carrier and the drug molecule.
  • a spacer segment can be placed between the cleavage recognition sequence and the polymeric carrier and/or between the cleavage recognition sequence and the drug component.
  • the spacer segment(s) may have any chemical composition. In certain embodiments they can be constructed from a polymer, e.g., without limitation a synthetic polymer such as poly(ethylene glycol) or a natural oligosaccharide or oligopeptide (i.e., whereby additional amino acid or sugar residues are added on either side of the recognition segment).
  • the spacer segments may also result from the specific covalent or non-covalent means used to associate the polymeric carrier, linker and drug components.
  • a spacer segment is not susceptible to cleavage by the digestive enzyme of interest.
  • a spacer segment may be modified to increase its stability to non-specific enzymatic digestion. For example, for an oligopeptide spacer these modifications may include cyclization of the peptide, incorporation of D-amino acids, etc. It is also worth noting that unless the drug molecule is associated with the linker at the cleavage site, the cleavage product will necessarily include certain additional residues as compared to the free drug molecule. Under certain circumstances these additional residues may interfere with the biological activity of the drug.
  • an attachment site on the drug molecule for the linker that minimizes any such reduction in biological activity. These sites are commonly referred to as “bulk tolerant" sites.
  • the linkers of the present invention need not necessarily include a digestible oligopeptide segment.
  • the linkers may alternatively include digestible oligosaccharide sequences.
  • the chemical composition of the recognition segment e.g., the length and sequence of sugar residues
  • the cleavage motifs of glycosidases have not been characterized in as much detail as those of proteases, a number of general synthetic routes have been developed in recent years for preparing specific oligosaccharides.
  • Chemical coupling methods have become increasingly sophisticated to fine-tune reactivity of reagents by fortuitous choices of anomeric activating group and protecting groups.
  • Heparanase is an endo- ⁇ -D-glucuronidase that catalyzes the hydrolytic cleavage of the ⁇ -l,4-glycosidic bond between a D-glucuronate and a D- glucosamine in heparan sulfate (Pikas et al., J. Biol. Chem.
  • heparanase has been implicated in many important physiological and pathological processes including tumor cell metastasis, angiogenesis and leukocyte migration (Vlodavsky et al., Israel Medical Association Journal, 2:37, 2000; Zcharia et al., Journal of Mammary Gland Biology and Neoplasia, 6:311, 2001 ).
  • the inventive conjugates may be modified to include targeting agents that will direct an inventive conjugate to a particular cell type, collection of cells, or tissue.
  • the targeting agents are associated with the polymeric carrier.
  • suitable targeting agents are known in the art (Cotten et al., Methods Enzym. 217:618, 1993; Torchilin, Eur. J. Pharm. Sci. 11:881, 2000; Garnett, Adv. Drug Deliv. Rev. 53:171, 2001).
  • any of a number of different materials which bind to antigens on the surfaces of target cells may be employed. Antibodies to target cell surface antigens will generally exhibit the necessary specificity for the target.
  • suitable immunoreactive fragments may also be employed, such as the Fab, Fab', or F(ab') 2 fragments.
  • Fab fragment antigen binding fragment
  • Fab' fragment antigen binding fragment
  • F(ab') 2 fragments fragment antigen binding fragments
  • ligands for any receptors on the surface of the target cells may suitably be employed as targeting agent. These include any small molecule or biomolecule, natural or synthetic, which binds specifically to a cell surface receptor, protein or glycoprotein found at the surface of the desired target cell.
  • compositions may be combined with pharmaceutically acceptable carriers to form one or more pharmaceutical compositions. If several different conjugates (e.g., with different conjugated drugs) are to be administered simultaneously then they may be combined into a single pharmaceutical composition. Alternatively, they may be prepared as separate compositions that are then mixed or simply administered one after the other. If several different conjugates (e.g., with different conjugated drugs) are to be administered at different times then they are preferably prepared as separate compositions. If non-conjugated drugs are going to be included in an inventive combination therapy they can be added to one or more of these conjugate pharmaceutical compositions or prepared as separate compositions. As would be appreciated by one of skill in this art, the carriers in the pharmaceutical compositions may be chosen based on the route of administration as described below, the location of the target issue, the drug or drugs being delivered, the time course of delivery of the drug or drugs, etc.
  • the term "pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Remington 's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, PA, 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEENTM 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-
  • compositions of this invention can be administered to a patient by any means known in the art including oral and parenteral routes.
  • patient refers to humans as well as non-humans, including, for example, mammals, birds, reptiles, amphibians and fish.
  • the non-humans are mammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig).
  • parenteral routes are preferred since they avoid contact with the digestive enzymes that are found in the alimentary canal.
  • inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).
  • injection e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection
  • rectally rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).
  • sterile injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • conjugates and/or drugs are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEENTM 80.
  • the injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the inventive conjugate with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the inventive conjugate.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the inventive conjugate.
  • Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches.
  • the conjugates or drugs are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulations, ear drops and eye drops are also contemplated as being within the scope of this invention.
  • the ointments, pastes, creams and gels may contain, in addition to the conjugates or drugs of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the inventive conjugates in a proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the inventive conjugates in a polymer matrix or gel.
  • Powders and sprays can contain, in addition to the conjugates or drugs, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these drugs. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
  • the conjugates or drugs are preferably, but not necessarily, encapsulated.
  • suitable encapsulation systems are known in the art ("Microcapsules and Nanoparticles in Medicine and Pharmacy,” Edited by Doubrow, M.., CRC Press, Boca Raton, 1992; Mathiowitz and Langer J. Control.
  • conjugates or drugs are encapsulated within biodegradable polymeric microspheres or liposomes.
  • Examples of natural and synthetic polymers useful in the preparation of biodegradable microspheres include carbohydrates such as alginate, cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes, polypropylfumarates, polyethers, polyacetals, polycyanoacrylates, biodegradable polyurethanes, polycarbonates, poly anhydrides, polyhydroxyacids, poly(ortho esters) and other biodegradable polyesters.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.
  • phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.
  • compositions for oral administration can be liquid or solid.
  • Liquid dosage forms suitable for oral administration of inventive compositions include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsif ⁇ ers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as
  • the oral compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvant refers to any compound which is a nonspecific modulator of the immune response.
  • the adjuvant stimulates the immune response. Any adjuvant may be used in accordance with the present invention.
  • a large number of adjuvant compounds is known in the art (Allison, Dev. Biol. Stand. 92:3, 1998; Unkeless et al., Annu. Rev. Immunol. 6:251, 1998; and Phillips et al., Vaccine 10:151, 1992).
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the encapsulated or unencapsulated conjugate is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
  • the exact dosage of the conjugates or drugs is chosen by the individual physician in view of the patient to be treated. In general, dosage and administration are adjusted to provide an effective amount of the conjugates or drugs to the patient being treated.
  • the "effective amount" of a conjugate or drug refers to the amount necessary to elicit the desired biological response.
  • the effective amount of conjugate or drug may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc.
  • the effective amount of conjugate containing an anti-cancer drug might be the amount that results in a reduction in tumor size by a desired amount over a desired period of time.
  • Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; other drugs in use; reaction sensitivities; and tolerance/response to therapy.
  • Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.
  • the conjugates or drugs of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of conjugate or drug appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity of conjugates or drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the following Examples describe the synthesis and characterization of conjugates that release anti-cancer drugs in the presence of matrix-metalloproteinase II (MMP -2), a widely studied proteinase that is overexpressed in a number of cancers (Woessner and Nagase, Matrix Metalloproteinases and TIMPS, Oxford University Press, 2000).
  • MMP-2 matrix-metalloproteinase II
  • the conjugates include linkers with different fractions of an oligopeptide motif that is recognized and cleaved by MMP-2.
  • the conjugates are characterized by comparing the rate of drug release in the presence of MMP-2 and fetal bovine serum (an in vitro control for a normal circulation environment).
  • the cytotoxicity of the conjugates is also compared by exposing various human tumor cell lines to the cleavage products. In vivo experiments with a mice model are also described.
  • MMP-2 has been found to be elevated in a number of human epithelial cancers, including breast (Davies et al., British Journal of Cancer 67:1126, 1993); prostate (Hamdy et al., British Journal of Cancer 69:177, 1994); colon (Levy et al., Cancer Research 51 :439, 1991); ovary (Naylor et al., International Journal of Cancer 58:50, 1994); bladder (Davies et al., British Journal of Cancer 67:1126, 1993); and gastric carcinoma (d'Errico et al., Mod. Pathol. 4:239, 1991). Elevated expression has been found in both malignant epithelial cells and the surrounding stromal fibroblasts. Moreover, MMP-2 has been implicated with the angiogenic and metastatic potential of cancer.
  • Doxorubicin belongs to the class of anthracyclines which kill cells by intercalating within DNA molecules.
  • the structure of doxorubicin is illustrated in Figure 3.
  • Free doxorubicin has a clinical dose of 60-80 mg/m . After intravenous injection, the drug molecules distribute ubiquitously throughout the body before being quickly eliminated by renal excretion (Cassidy et al., Cancer Surveys 17:315, 1993). Major toxicity is observed within the haemolymphopoietic system, gastro-intestinal tract, skin, testes and heart.
  • Doxorubicin is used widely in the treatment of cancers, including breast, ovarian, bladder, lung cancers, non-Hodgkin's lymphoma, Hodgkin's disease and sarcoma.
  • Carboxymethyl-dextran-oligopeptide-doxorubicin conjugates were synthesized using traditional techniques of peptide coupling and dextran modification. Oligopeptides of four to seven amino acids were used as linkers between carboxymethyl- dextran (CM-dextran) and doxorubicin. They are listed below in Table 1. The potential cleavage site is between Pl and PF (using the nomenclature of Schechter and Berger, Biochem. Biophys. Res. Comm.
  • Carboxymethyl-dextran was prepared by exposing dextran with an average MW of 40,000 Da to chloracetic acid. Dextran was selected in part because of its biocompatibility (Sgouras and Duncan, J. Mater. Sci.: Mat. Med. 1 :61, 1990) and biodegradability (Vercauteren, Schacht et al., J. Bioactive and Compatible Polymers 7:346 1992). In addition, from a synthetic standpoint, the hydroxyl groups on the dextran backbone provide convenient sites for covalent association with the oligopeptide recognition segment.
  • the oligopeptides were synthesized by conventional solid-phase techniques (Bodansky and Bodansky, The Practice of Peptide Synthesis, Springer 1994) and N-terminal protected with an FMOC group.
  • the N-terminal protected FMOC-peptides (2 eq.) were conjugated with doxorubicin (1 eq.) in the presence of N,N"-diisopropylethylamine (5 eq.) in DMF using PyBop (1.9 eq.) and HOBT (2 eq.) as coupling reagents. The reactions were held at room temperature overnight.
  • the aminoribosyl group on doxorubicin allows the drug molecule to be covalently associated with the oligopeptide linker via an aminolysis reaction.
  • Reversed-phase chromatography employing an acetonitrile gradient in 0.2% trifluoroacetic on a Vydac C 18 column was used to isolate the FMOC- oligopeptide-doxorubicin. Purity as analyzed by HPLC was greater than 95%. Deprotection of FMOC was completed in 5 minutes using 10% piperidine in DMF followed by quenching on ice with a mixture of trifluoroacetic acid/pyridine/DMF (3:7:20). After deprotection, oligopeptide-doxorubicin was covalently linked to pre-activated dextran carrier in the presence of N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ).
  • EEDQ N-ethoxycarbonyl-2-ethoxy-l,2-di
  • the optimal MMP-2 cleavage motif determined by Turk et al. was Ile-Pro-Val-Ser-Leu-Arg-Ser (P4 through P3 ⁇ shown on the bottom of Table 1).
  • P4 through P3 ⁇ shown on the bottom of Table 1.
  • the presence of reactive side chains on serine and arginine i.e., at positions Pl, P2' and P3 ⁇ shown in bold in Table 1
  • the fact that doxorubicin is labile to harsh deprotection procedures, posed problems for synthesis.
  • doxorubicin is linked to the oligopeptide adjacent to the cleavage site (i.e., at the Pl' position), the released molecule is free doxorubicin — a fully active drug.
  • the cytotoxicity may probably be attenuated if doxorubicin is linked further away from the cleavage site (e.g., at the P2' or P3' positions).
  • past studies on matrix-metalloproteinase specificity have implied that substrate sites from P3 to P3' are all required for cleavage to occur.
  • K m k_i/k
  • k ⁇ t is the turnover rate constant
  • k cat /K m is termed the specificity constant and provides a measure of the rate of enzymatic cleavage.
  • Salt concentration (NaCl, M) kct/K,,, (M 1 . s 1 )
  • CM-dextran-oligopeptide- doxorubicin conjugates of Example 1 were incubated at 37 C with fetal bovine serum for up to 24 hours. All four conjugates were relatively stable in the serum, exhibiting less than 5% release of doxorubicin.
  • CM-dextran-IPVGLIG-doxorubicin i.e., the conjugate most sensitive to cleavage by MMP -2 in this series
  • the cleavage product was cytotoxic to a number of human tumor cell lines, namely HT- 1080 (a human fibrosarcoma line, Figure 6A), BT-20 (a human breast carcinoma line, Figure 6B), U-87 (a human glioblastoma cell line, Figure 6C), PC-3 (a human prostate tumor cell line, Figure 6D), KK-47 (a human bladder tumor cell line, Figure 6E) and MGH-Ul (a human bladder cell line, Figure 6F).
  • Open circles in Figures 6A-F represent free doxorubicin and closed circles represent LIG- doxorubicin.
  • MTT assay e.g., see Alley et al., Cancer Res. 48:589, 1988.
  • the tetrazolium salt MTT is metabolized by NAD-dependent dehydrogenase to form a colored reaction product and the amount of dye formed directly correlates with the number of viable cells.
  • the formula for calculating % survival is given by:
  • T; Net MTT reading of test growth in the presence of drugs
  • T 2 Net MTT reading at time zero with respect to the drug test
  • C Net MTT reading of control growth
  • LIG-doxorubicin was less potent than the free drug doxorubicin.
  • Doxorubicin exerts its cytotoxicity by intercalating DNA and the ribosyl amino group is important in stabilizing DNA binding through hydrogen bonding. Modification of this amino group may therefore explain the reduction in activity.
  • the polymeric backbone is dextran grafted with poly(ethyleneglycol) (PEG).
  • Doxorubicin is linked to PEG via a cleavable oligopeptide sequence.
  • PEG is biocompatible (Sgouras and Duncan, J. Mater. Sci.: Mat. Med. 1 :61, 1990).
  • dextran it is not biodegradable and hence the use of this polymer is limited to a size below the renal excretion limit (Yamaoka et al., J. Pharm. Sci. 83:601, 1994).
  • Pegylation is known to prolong circulation time in the blood stream, reduce immunogenicity and increase solubility. These advantages are highly dependent on the hydrophilicity imparted by the polymer. With these benefits, PEG-L-asparaginase has been approved for clinical use in the United States to treat leukemia (Keating et al., Leukemia Lymphoma 10:153, 1993).
  • the oligopeptide linkers are synthesized by conventional solid-phase techniques (Bodansky and Bodansky, The Practice of Peptide Synthesis, Springer 1994) and the N- terminus is protected by FMOC.
  • the ribosyl amino group of doxorubicin is condensed with the carboxyl terminus of the oligopeptide in the presence of PyBop and HOBT in DMF.
  • the FMOC protection group on the oligopeptide is then removed using 10% piperidine to expose the free N-terminus, which is then attached to a heterogeneously substituted bivalent PEG- FMOC-NH-PEG-NHS- using EDC as the conjugating reagent in aqueous medium.
  • the ratio of PEG to PEG-oligopeptide-doxorubicin on the dextran core may affect the relative rates at which the final conjugate is engulfed by cells or cleaved by digestive enzymes in the extracellular space.
  • the surface hydrophobicity of the conjugate may have a bearing on the endocytosis rate of the conjugate.
  • Hydrophobic particles are generally engulfed more quickly than hydrophilic particles. For this reason, micro- and nanoparticles in drug delivery applications are frequently coated with hydrophilic PEG to elongate their circulation by evading the engulfment by the retinoendothelial system (RES).
  • RES retinoendothelial system
  • the conjugates With dextran and PEG as the polymer backbone, the conjugates will have a hydrophilic nature. However, loading of hydrophobic drugs could alter the surface characteristics of the conjugates and increase the clearance by the RES (Kataoka, p. 49 in "Targetable polymeric drugs. Controlled drug delivery: challenges and strategies" Ed. by Park, American Chemistry Society, 1997).
  • the hydrophobicity of conjugates can be compared by measuring the contact angle with water on a surface coated with the conjugates.
  • the endocytotic rate of conjugates can also be measured by placing radioactively labeled conjugates in a Kupffer cell culture. Kupffer cells are chosen since they are the major phagocytes in the RES. At specified time points, cell samples are removed, washed extensively and homogenized. The amount of conjugate engulfed is then proportional to the radioactivity detected by a scintillation counter.
  • DPVGLIG-doxorubicin complex was prepared following the procedures described in Example 1 and then covalently attached to a linear flexible polymer, namely methoxy- poly(ethyleneglycol)-NHS ester (mPEG-NHS, nominal MW of ⁇ 20,000 Da from
  • the digestion kinetics of mPEG-IP VGLIG-doxorubicin conjugate by MMP-2 was measured and found to be equivalent to that of IP VGLIG-doxorubicin complex, thus much faster than that of CM- dextran-IPVGLIG-doxorubicin conjugate (see Example 2).
  • the three-dimensional structure of the catalytic domain of MMP-2 was examined (available online in the Brookhaven protein bank).
  • the substrate binding site consists of a groove that is enclosed in the enzyme interior and has space for about six amino acids. Although the two ends of the groove are exposed, directly tethering the recognition segment to a large polymer backbone is likely to give rise to significant steric hindrance.
  • CM-dextran-IPVGLIG-doxorubicin and mPEG-IPVGLIG-doxorubicin suggests that incorporation of a flexible spacer (e.g., polyethylene oxide) between the digestible oligopeptide and the polymer backbone (e.g., dextran or CM-dextran) may improve release of drugs from such conjugates.
  • a flexible spacer e.g., polyethylene oxide
  • the polymer backbone e.g., dextran or CM-dextran
  • these results suggest that one could construct a conjugate using a PEG star polymer with oligopeptide-drugs linked to the outer ends for better presentation of the digestible oligopeptide.
  • the activity of a drug that is to be incorporated into a conjugate of the present invention is minimally affected by the covalent attachment of several amino acids (i.e., the peptidyl remnants of linker cleavage).
  • methotrexate an anti-metabolite that is used to treat cancer and arthritis
  • Bulk tolerance at the ⁇ -carboxyl of its glutamic acid
  • Methotrexate is effective against a number of human tumors, for example, lymphoblastic leukemia in children, chriocarcinoma and related trophoblastic tumors in women, osteosacroma and carcinomas of breast, head, neck, ovary and bladder.
  • the major side effect is toxicity in gastrointestinal tract and bone marrow (Chamber et al., p. 1389 in "Goodman and Gilman 's: The Pharmacological Basis of Therapeutics", Ed. by Hardman et al., McGraw-Hill, 2001).
  • the structure of methotrexate is illustrated in Figure 9.
  • the cytotoxicity of methotrexate and various peptidyl-methotrexates were compared in order to assess bulk tolerance.
  • the peptidyl-methotrexates were prepared by linking short peptides of three to four amino acids to methotrexate. The peptides were chosen to represent appropriate cleavage fragments from the MMP-2 sensitive substrate peptide IPVGLIG that was characterized in Examples 1-2. Methotrexate was covalently coupled to the N-terminal end of the peptides via the ⁇ -carboxyl of its glutamic acid (Nagy et al., Proc. Nat. Acad. Sci. USA 90:6373, 1993).
  • cytotoxicity results with HT-1080 tumor cells are presented in Figure 10 as a plot of % cell survival against equivalent drug concentration. Cytotoxicity was assayed as described above in Example 4. The cytotoxicity of methotrexate was still reduced when attached to the short peptides, but to a lesser degree that with doxorubicin (see Figures 6 A-F).
  • methotrexate-PVG was found to be cytotoxic when tested on three human tumor cell lines, namely HT-1080 (a human fibrosarcoma line, Figure 1 IA), BT-20 (a human breast carcinoma line, Figure 1 IB) and RT-112 (human urinary bladder transitional carcinoma line, Figure 11C), albeit with a certain degree of neutralization as compared to free methotrexate.
  • HT-1080 a human fibrosarcoma line, Figure 1 IA
  • BT-20 a human breast carcinoma line
  • RT-112 human urinary bladder transitional carcinoma line
  • Dextran-oligopeptide-methotrexate conjugates were synthesized following the route of Figures 12A-C. The scale of synthesis and the average step yield are also shown.
  • the synthesis route may be modified to prepare conjugates with different oligopeptide recognition segments and different backbone charges. By tuning these parameters, the new conjugate may be optimized in terms of sensitivity against a digestive enzyme of interest and stability in circulation.
  • Dextran T-70 (nominal MW of 70,000 Da) was obtained from Pharmacia, l-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and ethanolamine were obtained from Alfa Aesar.
  • EDC l-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride
  • ethanolamine obtained from Alfa Aesar.
  • O-Bis-(aminoethyl)ethylene glycol trityl resin, PyBop and HOBT were obtained from Nova Biochem.
  • Glutamic acid- ⁇ -OtBu was purchased from Bachem.
  • Trifluoroacetic acid (TFA), dimethylformamide (DMF), dimethylsulfoxide (DMSO) and acetonitrile (ACN) were obtained from EM Science. Chloroacetic acid, N,N'- diisopropyl(ethylamine) (DIPEA), BOP, 4-amino-4-deoxy-N 10 -methylpteroic acid (APA) and other chemicals were from Sigma. Purified active human MMP -2 was purchased from Calbiochem. Precast protein gels with 10% gelatin were obtained from Biorad. All cell culture media and reagents were ordered from Gibco. Human tumor cell lines HT- 1080, U-87 and BT-20 were obtained from American Type Cell Culture. Human bladder tumor cell line RT-112 was a gift from the laboratory of Professor Marsha Moses of the Children's Hospital (Boston, MA).
  • a Tosohaas PW3000 7.8 mm ID x 30 cm L column followed by a PW2000 7.5 mm ID x 30 cm L column were connected to an Agilent 1 100 series HPLC solvent delivery system equipped with a UV detector. Isocratic elution was performed with 20% acetonitrile and 3.6 X phosphate buffered saline in water at 0.8 ml/min. Detection for methotrexate- containing moieties was made at A307. The default sample injection volume was set at 10 ⁇ L. A calibration curve was constructed correlating the peak areas in chromatograms and concentrations of methotrexate standard.
  • Dextran, dextran-methotrexate conjugates and dextran-oligopeptide-methotrexate conjugates were dissolved in DI water at 10 mg/ml. Measurement was performed with a Zeta-analyzer from Brookhaven Instruments Co ⁇ .
  • Conjugates of interest were dissolved in assay buffer (10 mM CaCb, 0.2 M NaCl in 50 mM Tris pH 7.5) at a range of concentrations (5, 10, 20, 50 and 100 ⁇ M). For each substrate concentration, active MMP-2 at a final concentration of 9.7 nM was added and the mixture was incubated at 37 C. At specific time points, the reaction mixture was quenched with EDTA solution to a final concentration of 20 mM. The mixture was assayed by two- column size exclusion HPLC and the cleavage product peak was quantified to determine the reaction rate. The initial rate was based on the time taken to cleave the first 20% of the conjugate. Triplicate measurements were made at each time point for each substrate concentration. Linear regression was performed on a double reciprocal plot of 1/Vj versus 1/S 0 to determine the specificity constant k cat /K m according to the Michaelis-Menten equation.
  • MTX- ⁇ -(OtBu) was prepared from 4-amino-4-deoxy-N l0 -methylpteroic acid (APA) and glutamic acid- ⁇ -OtBu using BOP as the conjugating reagent, following the procedure of Nagy (Nagy et al., Proc. Natl. Acad. Sci. USA 90:6373, 1993). Crude MTX- ⁇ -(OtBu) was precipitated by centrifugation in a 1:1 mixture of cold ether/ethyl acetate. The material was further purified by HPLC on a Vydac C18 preparative scale column using a gradient from 0- 100% acetonitrile with 0.2% TFA. The organic solvent in product fractions was removed with RotaVap and the purified material was subsequently lyophilized. The product purity was confirmed to be > 95% and the amount was quantified by analytical scale reversed-phase HPLC. The average retention factor was 6.14.
  • OtBu (2.5 eq.) was dissolved in DMF with DIPEA (2.5 eq.). PyBop (2.5 eq.), HOBT (2.5 eq.) and another 2.5 eq. of DIPEA were added to activate the ⁇ -carboxyl of MTX- ⁇ -OtBu for about 15 minutes. More DMF was added such that the final concentration of MTX- ⁇ -OtBu was about 0.1 M. The mixture was added to the resins loaded with oligopeptide and stirred at room temperature overnight. The reaction was confirmed for completeness the next day using the ninhydrin test. Excessive reactants were washed away from resins with DMF, DCM and methanol. Resins were dried thoroughly.
  • jeffamine-oligopeptide-MTX(OtBu) 0.5 mmole of jeffamine-oligopeptide-MTX(OtBu) [structure iii] was dissolved in 1.9 ml DI water and then added to 4 g of CM-dextran [structure iv] dissolved in 6.7 ml of DI water.
  • 4.7 g of EDC was added to the mixture with stirring. Reaction was held overnight at room temperature. Another 4.7 g of EDC was added the next day and the reaction continued overnight to form CM-dextran-oligopeptide-MTX(OtBu).
  • modified dextran-oligopeptide-MTX(OtBu) was resuspended in 40 ml strong trifluroacetic acid by vigorous stirring and held at room temperature for 3 hours.
  • the deprotection step was quenched on ice by diluting in 0.1 M phosphate buffer and neutralizing the pH with 5 N sodium hydroxide.
  • Extensive diafiltration was performed using Millipore Pellicon XL diafiltration cassettes of regenerated cellulose with 10,000 MWCO to remove excessive reactants and byproducts of reaction. After purification, the product in the retentate was concentrated and lyophilized for storage. The product purity was confirmed to be > 99% and the loading density was quantified by size exclusion HPLC. The loading density was estimated to be at 1 +/- 0.2% (mole % per unit glucose unit).
  • jeffamine- MTX(OtBu) was used instead of jeffamine-oligopeptide-MTX(OtBu).
  • methotrexate was selected to form the drug component of these inventive conjugates because it exhibits bulk tolerance at the ⁇ -carboxyl group.
  • the less tolerant ⁇ -carboxyl group was protected throughout the synthesis process by preparing MTX- ⁇ -(OtBu) [structure i] from APA.
  • the ⁇ -protected methotrexate was conjugated to the recognition oligopeptide in a solid phase reaction.
  • Trityl resin was chosen as the solid phase support so that the oligopeptide-drug product [structure iii] could be released from the resin using dilute acid that did not affect the tert-butyl protecting group on methotrexate.
  • the tert- butyl group was removed in the last reaction step using strong TFA cleavage (step 3A, Figure 12).
  • Dextran-PVGLIG-methotrexate and dextran-methotrexate conjugates from Example 8 were incubated with 14.7 nM active human MMP-2 in MMP-2 assay buffer at 37 C for 6 hours and assayed for percentage cleavage using size exclusion chromatography.
  • Dextran- PVGLIG-methotrexate conjugate released more than 90% of methotrexate whereas dextran- methotrexate did not show any significant release. The comparison confirms that the labile oligopeptide segment promotes cleavage of the conjugate. Without the recognition segment, the conjugate was stable even in the presence of a high concentration of MMP-2.
  • the small molecular weight cleavage product was collected from the digest of dextran-PVGLIG- methotrexate by size exclusion chromatography and analyzed by mass spectroscopy.
  • the molecular weight of 707.5 was consistent with the notion that methotrexate-Pro-Val-Gly (MTX-PVG) was the released product after cleavage.
  • the conjugates remain stable in blood circulation and that the drug stays attached to the polymer backbone before reaching the target tissue.
  • MMP-2 an elevated enzyme level in the blood stream
  • protease activity is inhibited by serum proteins such as ⁇ -2-macroglobulin (Woessner and Nagase, Matrix Metalloproteinases and TlMPs, Oxford University Press, 2000).
  • dextran-PVGLIG-methotrexate conjugates of Example 8 were therefore measured in both fetal bovine serum and fetal bovine serum spiked with active human MMP-2 (at 10 nM final concentration). After incubating the conjugates at 37 C in these conditions for 24 hours, no significant cleavage was detected as assayed by size exclusion chromatography.
  • Example 11 - 7 « vitro cytotoxicity ofpeptidyl methotrexate
  • Example 12 In vivo evaluation of anti-tumor efficacy ofdextran-oligopeptide-methotrexate conjugates
  • HT-1080 is a human fibrosacroma cell line that is known to express high level of MMP-2. After 7-10 days, tumors of size 150-300 mm 3 were established on each mouse.
  • Free methotrexate, dextran- oligopeptide-methotrexate (with MMP-2 labile oligopeptide segment Pro-Val-Gly-Leu-Ile- GIy) or dextran-methotrexate were injected intraperitoneally on day 1, 8 and 15 after a tumor was first established. Weight and tumor size were monitored three times a week. Tumor size was calculated as width 2 x length x 0.52. To ensure that the modified dextran carrier of the conjugates did not induce toxicity, a first control group of mice were injected with dextran that was charge neutralized with ethanolamine. As a second control, another group of mice were injected with phosphate buffered saline. Each group included three mice.
  • methotrexate Linking methotrexate to dextran increases the half-life of the small molecule drug due to decreased renal elimination, rendering the benefit of passive targeting. As described above, the leakiness of tumor blood vessels as compared to other normal tissue further increases the targeting ratio.
  • methotrexate must be released from the carrier to exert its effect on cell growth by inhibiting DNA synthesis. The release can happen in two ways, namely (1) by non-specific endocytosis, whereby conjugates are internalized by cells and methotrexate can be released by either acid hydrolysis or lysosomal enzyme digestion; and (2) by MMP-2 cleavage of the oligopeptide segment in the extracellular space of the tumor tissue. The latter route is only possible for conjugates with an MMP-2 labile oligopeptide segment between methotrexate and the dextran carrier.
  • mice The body weight of the mice was also monitored as an indicator of toxic side effects (see Figure 14).
  • methotrexate and dextran-oligopeptide-methotrexate caused similar percentage weight drops (13% and 14% respectively compared to initial weight).
  • the significantly better efficacy of dextran-oligopeptide-methotrexate here demonstrates the advantage of targeting.
  • the enhanced anti-tumor effect of dextran-oligopeptide-methotrexate is presumably a result of altered biodistribution of methotrexate, with more drug delivered to the targeted rumor tissue.
  • peptidyl-methotrexate is released from dextran-oligopeptide-methotrexate conjugates. Because of the lower cytotoxicity of the peptidyl-methotrexate versus the free drug, a lower toxic response is seen in normal tissues.

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Abstract

L'invention concerne des compositions et des méthodes pour améliorer la l'administration ciblée de médicaments au moyen de conjugués polymère-lieur-médicament. Les conjugués comprennent un lieur qui est reconnu et clivé par une enzyme digestive qui est surexprimée dans un tissu cible. Des compositions et des méthodes sont décrites qui permettent à plusieurs médicaments d'être administrés aux patients de façon sûre et efficace. Les conjugués polymère-lieur-médicament sont utilisés comme l'une des thérapeutiques de combinaison. Des méthodes sont aussi décrites pour rehausser l'administration du médicament en administrant les conjugués en combinaison avec un traitement secondaire qui augmente la concentration de l'enzyme digestive dans le tissu cible.
PCT/US2007/005679 2006-03-03 2007-03-05 Administration ciblée de médicament Ceased WO2007103364A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7943569B2 (en) 2005-05-16 2011-05-17 The Board Of Trustees Of The University Of Illinois Composition and method for providing localized delivery of a therapeutic agent
DE102014015625A1 (de) 2014-10-16 2016-04-21 Gonzalo Urrutia Desmaison Lösungsvermittelnde Zusammenssetzungen

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7943569B2 (en) 2005-05-16 2011-05-17 The Board Of Trustees Of The University Of Illinois Composition and method for providing localized delivery of a therapeutic agent
DE102014015625A1 (de) 2014-10-16 2016-04-21 Gonzalo Urrutia Desmaison Lösungsvermittelnde Zusammenssetzungen

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