WO2021053185A1 - Formulations de melflufène et leur utilisation dans le traitement ou la prophylaxie d'un ostéosarcome - Google Patents

Formulations de melflufène et leur utilisation dans le traitement ou la prophylaxie d'un ostéosarcome Download PDF

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WO2021053185A1
WO2021053185A1 PCT/EP2020/076176 EP2020076176W WO2021053185A1 WO 2021053185 A1 WO2021053185 A1 WO 2021053185A1 EP 2020076176 W EP2020076176 W EP 2020076176W WO 2021053185 A1 WO2021053185 A1 WO 2021053185A1
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osteosarcoma
melflufen
doxorubicin
salt
administered
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Konstantin BYRGAZOV
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Oncopeptides Ab
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Priority claimed from GBGB2010259.6A external-priority patent/GB202010259D0/en
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Publication of WO2021053185A1 publication Critical patent/WO2021053185A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention relates to the treatment and/or prophylaxis of osteosarcoma.
  • the present invention also relates to dosage regimens and kits that find utility in the treatment and/or prophylaxis of osteosarcoma.
  • Osteosarcoma is a mesenchymal tumor histologically characterized by the presence of mesenchymal cells and production of the bone stroma (Abarrategi A, et al, Stem Cells Int 2016;2016:3631764). It is the most common bone tumor, predominantly occurring in children, adolescents and young adults (Bielack SS, et al, Advances in the management of osteosarcoma. FlOOORes 2016;5:2767).
  • the overall clinical outcome in osteosarcoma patients is determined by the success of chemotherapy (Smeland S, et al, Eur J Cancer 2019;109:36-50; Marina NM, et al, Lancet Oncol 2016;17(10):1396-408), and high-risk osteosarcomas display poor response to the treatment.
  • the last significant improvement in the management of osteosarcoma patients happened in the 1980s when combined therapy including surgery and multi-agent chemotherapy consisting of methotrexate, adriamycin/doxorubicin and cis-platin (MAP) had been introduced (Jaffe N, et al, Sarcoma 2013;2013:203531).
  • osteosarcoma has a high invasive and metastatic potential: osteosarcoma patients suffer from pulmonary metastasis, fatal in the majority of the patients (Bielack S, et al, Minerva Pediatr 2013;65(2):125-48; Kager L, et al, J Clin Oncol 2003;21(10):2011-8).
  • Melflufen (also known as melphalan flufenamide and L-Melphalanyl-4-fluoro-L- phenylalanine ethyl ester), is an anti-tumor agent useful in treatment of multiple myeloma. Melflufen is described in WO 01/96367 and WO 2014/065751. The structure of the hydrochloride salt of melflufen is shown in Scheme 1 below: ci
  • Melflufen is a potent and highly lipophilic alkylating agent and it achieves targeted delivery of alkylating metabolites to tumor cells. Due to its high lipophilicity, melflufen rapidly enters tumor cells where it is immediately cleaved by peptidases leading to entrapment and enrichment of alkylating payload (Stiller CA, et al. Cancer Epidemiol. 2018;56:146-153). Overexpression of peptidases is often seen in tumor cells and this might be responsible for high melflufen sensitivity (Stiller CA, et al. Cancer Epidemiol. 2018;56:146-153). Summary of the Invention
  • the present invention provides melflufen, and salt(s) thereof, for use in the treatment and/or prophylaxis of osteosarcoma.
  • the present invention further provides a method for the treatment or prophylaxis of osteosarcoma, comprising the step of administering melflufen, or a salt thereof to a patient with osteosarcoma.
  • the present invention further provides a pharmaceutical formulation comprising melflufen, or a salt thereof, for use in the treatment and/or prophylaxis of osteosarcoma.
  • the invention also provides the use of melflufen, or a salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of osteosarcoma.
  • the invention also provides a kit comprising melflufen and a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin or carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan; preferably doxorubicin.
  • the kits of the present invention find use in the treatment and/or prophylaxis of osteosarcoma.
  • melflufen and salts thereof, are surprisingly effective for the treatment and prophylaxis of osteosarcoma.
  • the present inventors have found that melflufen demonstrates superior anti-neoplastic activity in comparison to other chemothereapeutic agents, and in particular in comparison to other alkylating agents including cyclophosphamide, ifosfamide, busulfan, melphalan, and bendamustine.
  • the inventors have found that in vivo administration of melflufen reduced tumor growth and metastasis of highly aggressive osteosarcoma cells in a xenograft model.
  • meflfuen for treating osteosarcoma is especially surprising because, to the inventors' knowledge, there are no alkylating agents approved for use in the treatment of osteosarcoma.
  • Melflufen is the only alkylating agent to show high efficacy in preclinical osteosarcoma models.
  • melflufen synergizes with doxorubicin, a long-term first-line agent for the treatment of osteosarcoma.
  • the present invention further provides melflufen, or a salt thereof, and doxorubicin for use in the treatment and/or prophylaxis of osteosarcoma.
  • the present invention further provides a method for the treatment or prophylaxis of osteosarcoma, comprising the step of administering melflufen, or a salt thereof, and doxorubicin to a patient with osteosarcoma.
  • the present invention further provides a pharmaceutical formulation comprising melflufen, or a salt thereof, and doxorubicin for use in the treatment and/or prophylaxis of osteosarcoma.
  • the invention also provides the use of melflufen, or a salt thereof, and doxorubicin for the manufacture of a medicament for the treatment and/or prophylaxis of osteosarcoma.
  • the invention also provides a kit comprising melflufen and doxorubicin, and optionally further comprising methotrexate and cisplatin.
  • FIG. 1 Drug sensitivity profile of osteosarcoma cell lines:
  • A Drug sensitivity profile of osteosarcoma cell lines in a panel of drugs used or proposed for osteosarcoma treatment (the numbers are plC50 (-logio(ICso)), thus higher numbers represent higher drug sensitivity).
  • B, C Representative pictures of growth inhibition of 143B (B) and STA-OS-3 (C) by melflufen (MFL), melphalan (MPH) and doxorubicin (DXR).
  • D, E, F Growth inhibition of primary osteosarcoma cells isolated from cryo- preserved patient samples, including doxorubicin-resistant sample (D) with demonstrated sensitivity to melflufen.
  • G Expanded representation of drug response curves of a patient-derived multidrug-resistant cell line STA-OS-3.
  • FIG. 1 Melflufen's activity in relation to aminopeptidase activity and expression: Effect of aminopeptidase inhibition by bestatin on melflufen's cytotoxicity in STA-OS- 3 (A) and 143B (B) cells.
  • C Correlation between melflufen plC50 and expression of ANPEP/CD13 (molecules per cell) (the correlation analysis was done using Spearmann method).
  • D Representative flow cytometry analysis of ANPEP/CD13 on osteosarcoma cells.
  • FIG. 3 Analysis of ANPEP mRNA expression in murine and human osteosarcoma samples:
  • A,B MG63, MG63.3 and 143B osteosarcoma express higher ANPEP mRNA when isolated from pulmonary metastasis in xenograft when compared to the primary culture, GSE74320, no statistical test (not enough data points).
  • C ANPEP mRNA is up-regulated in the primary tumor samples of patients with pulmonary metastasis presented at diagnosis (MET) compared to the patients where no pulmonary metastasis was detected at 5 years upon diagnosis, GSE32981, ** p ⁇ 0.001.
  • ANPEP mRNA is up-regulated in murine osteosarcoma tumors with metastatic (MET) potential when compared to the tumors which did not produce pulmonary metastasis (No MET), GSE43281, ** p ⁇ 0.001 Mann-Whitney U test.
  • E The tumors capable of producing pulmonary metastasis in p53 murine model display higher ANPEP mRNA expression in the metastasis (Pulm Met) when compared to the primary bone tumor (Prim T), GSE43281, ** p ⁇ 0.001 Wilcoxon test.
  • ANPEP mRNA is higher expressed in tumor samples of osteosarcoma patient with poor response to chemotherapy, GSE14827, **, p ⁇ 0.001 Mann-Whitney U test.
  • ANPEP mRNA is up-regulated in osteosarcoma tumors produced pulmonary metastasis (MET) within 5 years upon initial diagnosis when compared to the samples from patients where no metastasis was detected 5 years upon initial diagnosis (No MET), GSE14827 ** p ⁇ 0.001.
  • Figure 3H there is shown metastasis-free survival in the subsets of patients with high and low ANPEP expression within a cohort of HGOS patients from the GSE21257 study.
  • FIG. 4 Effect of melflufen on apoptosis and DNA damage in osteosarcoma cells:
  • A, B Melflufen is rapidly inducing apoptosis in a multidrug resistant STA-OS-3 (A) and 143B (B) osteosarcoma cell lines.
  • C, D Western blot analysis of osteosarcoma cells treated with vehicle control (DMSO), melflufen, melphalan and doxorubicin.
  • E Analysis of time to apoptosis saturation in melflufen- and doxorubicin-treated osteosarcoma cell lines.
  • Figure 5 Effect of doxorubicin on ANPEP mRNA expression in ANPEP/CD13-low U20S cell line: Analysis of previously published datasets (A) GSE46493 and (B) GSE84863.
  • Figure 6. Synergy testing of a combination of melflufen+doxorubicin in a two- dimensional drug sensitivity analysis of osteosarcoma cells.
  • Figure 7 Effect of doxorubicin on aminopeptidase expression and melflufen's efficacy:
  • A Synergy scores (BLISS and HSA) for a combination of doxorubicin and melflufen in the panel of osteosarcoma cell lines.
  • B The effect of doxorubicin on ANPEP/CD13 expression in osteosarcoma cells.
  • FIG. 8 In vivo evaluation of anti-osteosarcoma activity of melflufen:
  • A Schematic representation of the chick embryo system used in the study.
  • B Toxicity profile showing the number of dead chick embryos upon addition of the drugs (100 nM doxorubicin (DXR), 500 nM melflufen (MFL)).
  • C Average tumor weight isolated from the xenografts, ANOVA test, *** P ⁇ 0.001.
  • D Metastasis rate measured by the presence of tumor cells in the lower CAM, ANOVA test, * P ⁇ 0.05.
  • E Immunohistochemistry analysis of tumor sections stained for a DNA damage marker yH2AX and an apoptosis marker cleaved caspase-3.
  • Figure 9 Alkylator drug sensitivities of cell lines from NCI osteosarcoma panel. The data are adapted from the website sarcoma.cancer.gov and the values are presented as negative logio(ICso M).
  • melflufen is surprisingly effective for the treatment or prophylaxis of osteosarcoma.
  • melflufen was surprisingly potent in all cell lines. They further found that melflufen rapidly induces apoptosis in osteosarcoma cells, especially in CD13-high cells, thus eradicating the aggressive clones. They have found that melflufen can insert cytotoxic activity within the malignant cells where methotrexate and etoposide failed.
  • melflufen also did not show any significant toxicities at the concentration at which anti-neoplastic and anti-metastatic activity was demonstrated in osteosarcoma cells.
  • the efficacy of melflufen has been found to be especially high in osteosarcoma cells with expression of ANPEP/CD13.
  • Melflufen's cytotoxic activity positively correlated with ANPEP/CD13 surface expression and was reduced by bestatin, an aminopeptidase inhibitor, suggesting a contribution of aminopeptidase activity to melflufen's anti-osteosarcoma effect.
  • the inventors have also surprisingly found that melflufen synergizes with doxorubicin, a long-term first-line agent for osteosarcoma, in CD13-low cells due to the positive effect of doxorubicin treatment on CD13 expression.
  • the present inventors postulate that the surprisingly high potency of melflufen, as well as its synergistic effect with doxorubicin, will allow for the use of therapeutically effective doses that are safer and more effective than existing osteosarcoma treatments.
  • the use of melflufen for treating osteosarcoma is especially surprising because, to the inventors' knowledge, there are no alkylating agents approved for use in the treatment of osteosarcoma. Melflufen is the only alkylating agent that has been shown (by the current inventors) to have high efficacy in preclinical osteosarcoma models.
  • Aminopeptidase N (ANPEP/CD13) has been implicated in the pathogenesis and metastasis of several cancer types (Amin SA, et al, J Med Chem 2018;61(15):6468-90; Nohara S., et a I, Clin Res Hepatol Gastroenterol 2016;40(4):494-503; Zhang Q, et a I, J Cancer Res Ther 2015;ll(l):223-8).
  • Aminopeptidase activity of the enzyme was linked to activation of PI3K and MAPK pathways (Liang W, et al, Int J Oncol 2014;45(6):2475-85; Kido A, et al, Clin Exp Metastasis 2003;20(7):585-92), main contributors to osteosarcoma pathogenesis and metastasis (Loh AHP, et al, Cancer Lett 2019;442:262-70; Gupte A, et al, Clin Cancer Res 2015;21(14):3216-29).
  • High ANPEP/CD13 expression was linked to poor survival in osteosarcoma patients (Shuo Wang HX, et al, Int J Clin Exp Med 2016;9:22034-40).
  • the current inventors hypothesise that high ANPEP/CD13 expression in osteosarcoma may in part be responsible for the effectiveness of melflufen in the osteosarcoma models that have been investigated.
  • Melflufen also known as melphalan flufenamide and L-Melphalanyl-4-fluoro-L- phenylalanine ethyl ester
  • Melflufen, and salts thereof, especially the hydrochloride salt thereof are known from, for example, WO 01/96367 and WO 2014/065751.
  • the structure of the hydrochloride salt of melflufen is shown below:
  • melflufen when used, it includes salts of melflufen, as well as isotopic derivatives of melflufen, unless stated otherwise.
  • the mass of melflufen is the mass of the melflufen molecule excluding the mass of any counterion unless explicitly stated otherwise.
  • Salts of melflufen which are suitable for use in the present invention are those wherein a counterion is pharmaceutically acceptable.
  • Suitable salts include those formed with organic or inorganic acids.
  • suitable salts formed with acids according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids, or with organic sulfonic acids, such as (C 1 -C 4 ) alkyl or aryl sulfonic acids which are unsubstituted or substituted, for example by halogen.
  • Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxalic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine.
  • Preferred salts of melflufen include acid addition salts such as those formed from hydrochloric, hydrobromic, acetic, p-toluenesulfonic, tartaric, sulphuric, succinic, phosphoric, oxalic, nitric, methanesulfonic, malic, maleic and citric acid. More preferably, the salt of melflufen for use according to the present invention is the hydrochloride salt (i.e. the addition salt formed from hydrochloric acid).
  • solvates are described in Water-Insoluble Drug Formulation, 2 nd ed R. Lui CRC Press, page 553 and Byrn et al Pharm Res 12(7), 1995, 945-954.
  • the melflufen, or a salt thereof, for use in the present invention may be in the form of a solvate.
  • Solvates of melflufen that are suitable for use according to the present invention are those wherein the associated solvent is pharmaceutically acceptable. For example a hydrate is a pharmaceutically acceptable solvate.
  • melflufen, and salts thereof While it is possible for melflufen, and salts thereof, to be administered alone, it is preferable for it to be present in a formulation and particularly in a pharmaceutical formulations.
  • Pharmaceutical formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon, for example, the condition and disorder of the subject under treatment.
  • melflufen is administered as a pharmaceutical formulation suitable for oral or parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary) administration.
  • compositions of melflufen suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion.
  • the melflufen may also be presented as a bolus, electuary or paste.
  • Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit dosage or divided dosage containers, for example sealed ampoules and vials.
  • the formulation may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline, a physiologically acceptable solution or water-for-injection, immediately prior to use.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • the melflufen for use in the present invention comprises a lyophilized pharmaceutical preparation of a melflufen or a salt thereof.
  • lyophilized pharmaceutical preparation of a melflufen or a salt thereof is understood to mean that the melflufen or a salt thereof is free-dried ("lyophilization", “lyophilized” etc. may in the present context be used interchangeably with “freeze-drying", “freeze-dried” etc.).
  • a lyophilized pharmaceutical preparation of melflufen or a salt thereof as described herein may be a white, fluffy powder in contrast to a non-lyophilized melflufen or a pharmaceutically acceptable salt thereof, which is typically in the form of a dense, slightly yellowish powder.
  • a lyophilized pharmaceutical preparation of melflufen, or a salt thereof, for use in the present invention may comprise sucrose.
  • sucrose provides lyophilized preparation that is stable as such, and water-soluble, without the presence of an organic solvent, at a sufficient rate compared to the degradation rate, and is thereby useful in therapy and does not have toxicity brought about by the organic solvent.
  • a dissolved melflufen, or a salt thereof, solution such as a pharmaceutical composition comprising melflufen, or a salt thereof, which has a usefully high concentration of melflufen and which is substantially free from organic solvents.
  • Preparation of a lyophilized pharmaceutical preparation, a lyophilized pharmaceutical composition, and a kit for making such compositions, of melflufen or a salt thereof, is described in detail in WO 2012/146625 and WO 2014/065741, the contents of which are incorporated herein by reference.
  • a pharmaceutical formulation of melflufen, or a salt thereof, for use in the present invention may comprise a lyophilized pharmaceutical preparation comprising melflufen, or a salt thereof.
  • the formulation comprises sucrose. More preferably, the formulation comprises sucrose with a weight ratio (w/w) between melflufen and sucrose of about 1:25 to 1:75, for example 1:50.
  • the formulation is a pharmaceutical solution
  • it may be prepared from a lyophilized pharmaceutical preparation comprising melflufen, or a salt thereof, and further comprise a physiologically acceptable solvent(s), such as a glucose solution and/or a saline solution.
  • formulations for use in this invention may include other agents conventional in the art having regard to the type of formulation in question.
  • Melflufen, or a salt(s) thereof, and pharmaceutical formulations comprising melflufen find use in the treatment and/or prophylaxis of osteosarcoma, optionally in combination with doxorubicin.
  • the amount of melflufen which is required to achieve a therapeutic effect will vary with particular route of administration and the characteristics of the subject under treatment, for example the species, age, weight, sex, medical conditions, the particular disease and its severity, and other relevant medical and physical factors.
  • An ordinarily skilled physician can readily determine and administer the effective amount of melflufen required for treatment or prophylaxis of osteosarcoma.
  • Melflufen may be administered daily, every second or third day, weekly, every second, third or fourth week or even as a high single dose depending on the subject and severity of the osteosarcoma to be treated.
  • the melflufen, or salt thereof may be administered in an amount of about 1 to 150 mg (excluding the mass of any counterion).
  • the dosage of melflufen or salt thereof, per administration is 1 to 50 mg (excluding the mass of any counterion), for example 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg.
  • the dosage of melflufen or salt thereof, per administration is 1 to 40 mg (excluding the mass of any counterion), for example 1, 5, 10, 15, 20, 25, 30, 35, or 40mg.
  • the dosage of melflufen or salt thereof, per administration is 10 to 40 mg (excluding the mass of any counterion), for example 10, 15, 20, 25, 30, 35, or 40mg .
  • the dosage of melflufen or salt thereof, per administration may be 10 to 20 mg, 20 to 30 mg, or 30 to 40 mg.
  • Melflufen, or a salt thereof may be administered as a parenteral or oral dosage.
  • melflufen or a salt thereof is administered as a parenteral dosage.
  • pharmaceutical formulations useful according to the invention are those suitable for parenteral administration.
  • Parenteral administration includes intravenous (into a vein, for example a central or a peripheral vein) (bolus or infusion), intra-arterial (into an artery, for example a central or a peripheral artery), intraosseous infusion (into the bone marrow), intra muscular (into muscle), intradermal (into the dermis), and subcutaneous (under the skin) administration.
  • the dosage of the present invention is administered intravenously or intra-arterially, and more preferably by intravenous infusion (for example central intravenous infusion or peripheral intravenous infusion).
  • pharmaceutical formulations especially useful for the present invention are those suitable for intravenous administration, and more especially intravenous infusion.
  • the dosage of melflufen (excluding the mass of any counterion) is administered as a parenteral dosage at an infusion rate of around 0.3 to 1.8 mg/min, for example 0.5 to 1.8 mg/min, for example 0.8 to 1.8 mg/min, for example 1.0 to 1.8 mg/min, for example 1.1 to 1.8 mg/min, for example 1.1 to 1.7 mg/min, for example 1.1 to 1.6 mg/min, for example 1.2 to 1.6 mg/min, or for example 1.2 to 1.5 mg/min.
  • the dosage of melflufen (excluding the mass of any counterion) is administered as a parenteral dosage at an infusion rate of around 1.2 to 1.4 mg/min (for example 1.2, 1.3 or 1.4 mg/min).
  • a dosage of melflufen of around 1 to 50 mg (excluding the mass of the counterion) is administered as a parenteral dosage over around 5-35 minutes.
  • a dosage of melflufen of 40 mg (excluding the mass of the counterion) and as a parenteral dosage over around 30 minutes or for example, a dosage of melflufen of 20 mg (excluding the mass of the counterion) and as a parenteral dosage over around 15 minutes, or for
  • IB example a dosage of melflufen of 10 mg (excluding the mass of the counterion) and as a parenteral dosage over around 7.5 minutes.
  • the dosage of melflufen for use in the present invention when a mass of melflufen or a salt thereof is referred to, that is the mass when no counterion is included in the calculation of the dosage mass of the melflufen.
  • the molecular weight of counterion-free melflufen is 498.42 g/mol.
  • the actual dosage mass administered to the patient must take into account the mass of the counterion. This is routine for the person skilled in the art.
  • the equivalent dosage rate to 1.1 to 1.8 mg/min for melflufen hydrochloride (including the mass of the counterion) will be 1.2 to 1.9 mg/min.
  • the equivalent dosage of melflufen hydrochloride will be approximately 1.1 to 53.8 mg.
  • the equivalent dosage of melflufen hydrochloride will be approximately 10.7 to 48.3 mg.
  • the dosage of melflufen When melflufen, or a salt thereof, is administered as a parenteral dosage, the dosage of melflufen must be in the form of a liquid, for example a solution or suspension comprising the melflufen.
  • the melflufen, or salt thereof, of the present invention is taken as part of a treatment cycle.
  • the melflufen may be administered on day 1 of the cycle, wherein the cycle lasts X days, with no further melflufen administered for the next X-l days.
  • X may be, for example, from 1 to 42, from 5 to 42, from 7 to 42, from 10 to 42, or from 14 to 42.
  • X may be from 14 to 35 days, more preferably from 21 to 35 days, and more preferably 21 to 30 days; for example 21 days, 28 days, 29 days, 30 days or 35 days.
  • X may be, for example, 21 to 30 days, 21 to 28 days, 28 to 30 days, or 28 to 29 days.
  • a dose of melflufen administered in a treatment cycle may be a dose as described elsewhere in the present application, for example a dose of 1 to 50 mg melflufen.
  • melflufen, or a salt thereof is administered on day 1 of a 21 day cycle followed by 20 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 28 day cycle followed by 27 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 29 day cycle followed by 28 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 30 day cycle followed by 29 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 35 day cycle followed by 34 days of rest with no further melflufen being administered during that time.
  • the treatment cycle is 28 days; or administered on day 1 of a 42 day cycle followed by 41 days of
  • the cycle may be repeated one or several times depending on the category (for example high grade or low grade), class (for example metastatic, at risk of being metaststic, relapsed, refractory, etc.) or stage of the osteosarcoma.
  • the cycle may be repeated from 1 to 15 times, for example from 2 to 12 times, for example 2 to 7 times, for example 2, 3, 4, 5, 6 or times.
  • the cycle may be repeated,
  • An ordinarily skilled physician or clinician can readily determine the number of cycles of melflufen, or a salt thereof, required to treat, prevent, counter or arrest the progress of the osteosarcoma.
  • the dosage regimens of the invention are particularly safe and effective for the treatment and prophylaxis of osteosarcoma in patients with osteosarcoma, or at risk of developing osteosarcoma, or at risk of developing metastatic osteosarcoma.
  • melflufen Whilst melflufen, or a salt thereof, may be used as the sole active ingredient in the present invention, it is also possible for it to be used in combination with one or more further therapeutic agent(s), and the use of such combinations provides one preferred embodiment of the invention.
  • further therapeutic agents may be agents useful in the treatment or prophylaxis of osteosarcoma, or other pharmaceutically active materials. Such agents are known in the art.
  • chemotherapeutic agent for example a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan; preferably doxorubicin
  • an immunotherapeutic agent for example a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan; preferably doxorubicin
  • an immunotherapeutic agent for example a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan.
  • the one or more further therapeutic agent(s) may be used simultaneously, sequentially or separately with/from the administration of the dosage of the melflufen, or salt thereof.
  • the individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
  • At least one of the one or more further therapeutic agent(s) is a chemotherapeutic agent.
  • a chemotherapeutic agent for example, methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan.
  • at least one of the one or more further therapeutic agent(s) is doxorubicin.
  • the further therapeutic agent(s) include doxorubicin, cisplatin and methotrexate.
  • the further therapeutic agent(s) include doxorubicin, cisplatin and methotrexate, and optionally etoposide and/or ifosfamide.
  • At least one of the one or more further therapeutic agent(s) is an immunotherapeutic agent.
  • an immunotherapeutic agent for example, monoclonal antibodies, checkpoint inhibitors, non-specific immunotherapies (for example interferons and interleukins), adoptive cell therapies (for example chimeric antigen receptor T-cell (CAR T-cell) therapies and tumor-infiltrating lymphocyte (TIL) therapies) and cancer vaccines.
  • checkpoint inhibitors include agents or antibodies that inhibits one or more of CTLA4, PD-1, PD-L1, LAG-3, B7-H3, B7-H4, TIM3, VISTA and KIR.
  • PD-1 inhibitors include cemiplimab, nivolumab and pembrolizumab.
  • Examples of PD-L1 inhibitors include atezolizumab, avelumab and durvalumab.
  • An example of a CTLA-4 inhibitor is ipilimumab.
  • the simultaneous, sequential or separate administration of a chemotherapeutic agent such as doxorubicin, with melflufen further enhances the effectiveness of melflufen in treating osteosarcoma.
  • a chemotherapeutic agent such as doxorubicin
  • melflufen further enhances the effectiveness of melflufen in treating osteosarcoma.
  • the osteosarcoma is osteosarcoma with low expression of peptidase, for example low surface expression of ANPEP/CD13.
  • the dose of doxorubicin is from 1 mg/m 2 to 100 mg/m 2 , preferably 5 mg/m 2 to 80 mg/m 2 , more preferably 10 mg/m 2 to 60 mg/m 2 (for example 10, 20,
  • the dose of doxorubicin is 25 mg/m 2 to 37.5 mg/m 2 .
  • the doxorubicin is administered as an intravenous dose.
  • a dose of doxorubicin is administered on the same day as the administration of the melflufen.
  • the doxorubicin may be administered on the same day simultaneously, sequentially or separately from administration of the melflufen.
  • doxorubicin is administered separately from and on the same day as the melflufen.
  • doxorubicin may be administered before the melflufen, separately from and on the same day as the melflufen (for example from 15 minutes to 12 hours before the melflufen, and in particular from 30 minutes to 8 hours before the melflufen (e.g. 30 minutes, 1 hour,
  • the melflufen, or a salt thereof, for use in the present invention may be taken as part of a treatment cycle (for example melflufen, or a salt thereof is administered on day 1 of a cycle lasting X days, with no further melflufen taken for the next X-l days), the doxorubicin may be administered simultaneously, sequentially or separately on the same day as the melflufen is administered (i.e. on day 1).
  • X may be, for example, 1 to 42, from 5 to 42, from 7 to 42, from 10 to 42, or from 14 to 42.
  • X may be from 14 to 35 days, more preferably from 21 to 35 days, and more preferably 21 to 30 days; for example 21 days, 28 days, 29 days, 30 days, or 35 days.
  • a dose of doxorubicin administered in a treatment cycle may be a dose as described elsewhere in the present application, for example a dose of 10 mg/m 2 to 60 mg/m 2 of doxorubicin.
  • melflufen is administered on day 1 of a 21 day cycle, and doxorubicin is administered simultaneously, sequentially or separately on day 1 of the cycle, followed by 20 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 28 day cycle, and doxorubicin is administered simultaneously, sequentially or separately on day 1 on the cycle, followed by 27 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 29 day cycle, and doxorubicin is administered simultaneously, sequentially or separately on day 1 on the cycle, followed by 28 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 30 day cycle, and doxorubicin is administered simultaneously, sequentially or separately on day 1 on the cycle, followed by 29 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 35 day cycle, and doxorubicin is administered simultaneously, sequentially or separately
  • a dose of doxorubicin is administered 1, 2, or 3 days before the day on which the melflufen is administered. For example, it is administered separately from and 1 day before the administration of the melflufen.
  • the doxorubicin may be administered 1, 2, or 3 days before the day as the administration of the melflufen (i.e. on day 0, -1 or -2).
  • X may be, for example, from 1 to 42, from 5 to 42, from 7 to 42, from 10 to 42, or 14 to 42.
  • X may be from 14 to 35 days, more preferably from 21 to 35 days, and more preferably 21 to 30 days; for example 21 days, 28 days, 29 days, 30 days or 35 days.
  • melflufen is administered on day 1 of a 21 day cycle, and doxorubicin is administered separately on day 0 of the cycle, followed by 20 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 28 day cycle, and doxorubicin is administered separately on day 0 on the cycle, followed by 27 days of rest with no further melflufen being administered during that time; or administered on day 0 of a 29 day cycle, and doxorubicin is administered separately on day 0 on the cycle, followed by 28 days of rest with no further melflufen being administered during that time; or administered on day 0 of a 30 day cycle, and doxorubicin is administered separately on day 1 on the cycle, followed by 29 days of rest with no further melflufen being administered during that time; or administered on day 0 of a 35 day cycle, and doxorubicin is administered separately on day 1 on the cycle, followed by 35 days of rest with no further melfluflu
  • melflufen is administered on day 1 of a 21 day cycle, and doxorubicin is administered separately on day -1 of the cycle, followed by 20 days of rest with no further melflufen being administered during that time; or administered on day 1 of a 28 day cycle, and doxorubicin is administered separately on day -1 of the cycle, followed by 27 days of rest with no further melflufen being administered during that time; or administered on day 0 of a 29 day cycle, and doxorubicin is administered separately on day -1 of the cycle, followed by 27 days of rest with no further melflufen being administered during that time; or administered on day-1 of a 30 day cycle, and doxorubicin is administered separately on day 1 on the cycle, followed by 29 days of rest with no further melflufen being administered during that time; or administered on day-1 of a 35 day cycle, and doxorubicin is administered separately on day 1 on the cycle, followed by 34 days of rest with no further melflufen
  • doxorubicin is administered on day -2, -1, 0 or 1 in a treatment cycle, followed by one or more further dose(s) of doxorubicin are administered on one or more of days 1, 2, 3, 4, 5, 6, 7 and/or 8 of a cycle lasting X days, for example a cycle lasting 21 days, 28 days, 29 days, 30 days, 35 days or 42 days.
  • the further doses of doxorubicin may independently be 1 mg/m 2 to 100 mg/m 2 , preferably 5 mg/m 2 to 80 mg/m 2 , more preferably 10 mg/m 2 to 60 mg/m 2 (for example 10, 20, 25, 30, 25, 37.5, 40, 50, or 60 mg/m 2 ), for example, the dose of doxorubicin is 25 mg/m 2 to 37.5 mg/m 2 .
  • the further one or more dose(s) of doxorubicin are administered as an oral dose or an intravenous dose.
  • the doxorubicin is administered simultaneously, sequentially or separately from the melflufen on day 1, followed by a second separate dose of doxorubicin on day 2 of a cycle lasting X days.
  • melflufen is administered on day 1 of a X day cycle, followed by X-l days of rest with no further melflufen being administered during that time, and doxorubicin is administered simultaneously, sequentially or separately on day 1 on the cycle, followed by a second separate dose on day 2 of the X day cycle.
  • X may be 7 to 42 days, for example 7 days, 14 days, 21 days, 28 days, 29 days, 30 days, 31 days, 35 days or 42 days.
  • the doxorubicin is administered separately from the melflufen on day 0, followed by a second separate dose of doxorubicin on day 1 of a cycle lasting X days.
  • melflufen is administered on day 1 of a X day cycle, followed by X-l days of rest with no further melflufen being administered during that time, and doxorubicin is administered separately on day 0 of the cycle, followed by a second separate dose on day 1 of the X day cycle.
  • X may be 7 to 42 days, for example 7 days, 14 days, 21 days, 28 days, 29 days, 30 days, 31 days, 35 days or 42 days.
  • melflufen is administered in combination with cisplatin. In another embodiment of the invention, melflufen is administered in combination with methotrexate.
  • melflufen is administered in combination with: cisplatin, doxorubicin, and methotrexate (MAP); or or MAP plus ifosfamide; or or MAP plus etoposide; or or MAP plus ifosfamide and etoposide (MAPIE).
  • MAP methotrexate
  • MAPIE MAP plus ifosfamide and etoposide
  • MAP may be administered in a 6 week (42 day) treatment cycle as follows: administration of cisplatin 120 mg/m 2 , doxorubicin 37.5 mg/m 2 per day on days 1 and 2 followed 3 weeks later (day 21) by high-dose methotrexate 12 g/m 2 over 4 h.
  • ifosfamide may be administered as a high- dose ifosfamide (14 g/m 2 ) at 2.8 g/m 2 per day with equidose mesna uroprotection on days 1 to 5.
  • ifosfamide may be administered at a dose of 100 mg/m 2 per day over 1 h on days 1 to 5.
  • MAPIE may be administered in a treatment cycle as follows: administration of cisplatin 120 mg/m 2 , doxorubicin 37.5 mg/m 2 per day on days 1 and 2 (on weeks 1 and 6) followed 3 weeks later by high-dose methotrexate 12 g/m 2 over 4 h, high-dose ifosfamide (14 g/m 2 ) at 2.8 g/m 2 per day with equidose mesna uroprotection, followed by etoposide 100 mg/m 2 per day over 1 h on days 1 to 5.
  • melflufen may be administered on one or more of days 1, 2, 3, 4 and/or 5.
  • melflufen may be administered on day 1, or on day 2, or on days 1 and 2, or on days 1 to 5.
  • melflufen may be administered on day 21.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the cisplatin. For example, it is administered separately from and on one or more of the same days as the cisplatin (e.g. day 1, day 2, or day 1 and 2).
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the doxorubicin. For example, it is administered separately from and on one or more of the same days as the doxorubicin (e.g. day 1, day 2, or day 1 and 2).
  • the melflufen may be administered on the same day simultaneously, sequentially or separately from administration of the methotrexate.
  • the melflufen may alternatively be administered on a different day to any of the MAP components, for example on day 3, 4 or 5; or it may be administered the day before the doxorubicin/cisplatin is administered.
  • melflufen may be administered on one or more of days 1, 2, 3, 4 and/or 5.
  • melflufen may be administered on day 1, or on days 1 and 2, or on days 1 to 5.
  • melflufen may be administered on day 21.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the cisplatin. For example, it is administered separately from and on the same days as the cisplatin.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the doxorubicin. For example, it is administered separately from and on the same days as the doxorubicin.
  • the melflufen may be administered on the same day simultaneously, sequentially or separately from administration of the methotrexate. For example, it is administered separately from and on the same day as the methotrexate.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the ifosfamide. For example, it is administered separately from and on one or more of the same days as the ifosfamide (e.g. day 1, day 2, day 3, day 4, day 5, or days 1 to 5).
  • the melflufen may alternatively be administered on a different day to any of the MAP components and ifosfamide, for example on day 6, 7 or 8; or it may be administered the day before the doxorubicin/cisplatin is administered.
  • melflufen may be administered on one or more of days 1, 2, 3, 4 and/or 5.
  • melflufen may be administered on day 1, or on days 1 and 2, or on days 1 to 5.
  • melflufen may be administered on day 21.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the cisplatin. For example, it is administered separately from and on the same days as the cisplatin.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the doxorubicin. For example, it is administered separately from and on the same days as the doxorubicin.
  • the melflufen may be administered on the same day simultaneously, sequentially or separately from administration of the methotrexate. For example, it is administered separately from and on the same day as the methotrexate.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the etoposide. For example, it is administered separately from and on one or more of the same days as the etoposide (e.g. day 1, day 2, day 3, day 4, day 5, or days 1 to 5).
  • the melflufen may alternatively be administered on a different day to any of the MAP components and etoposide, for example on day 6, 7 or 8; or it may be administered the day before the doxorubicin/cisplatin is administered.
  • melflufen may be administered on one or more of days 1, 2, 3, 4 and/or 5.
  • melflufen may be administered on day 1, or on days 1 and 2, or on days 1 to 5.
  • melflufen may be administered on day 21.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the cisplatin. For example, it is administered separately from and on the same days as the cisplatin.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the doxorubicin. For example, it is administered separately from and on the same days as the doxorubicin.
  • the melflufen may be administered on the same day simultaneously, sequentially or separately from administration of the methotrexate. For example, it is administered separately from and on the same day as the methotrexate.
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the ifosfamide. For example, it is administered separately from and on one or more of the same days as the ifosfamide (e.g. day 1, day 2, day 3, day 4, day 5, or days 1 to 5).
  • the melflufen may be administered on one or more of the same days simultaneously, sequentially or separately from administration of the etoposide. For example, it is administered separately from and on one or more of the same days as the etoposide (e.g. day 1, day 2, day 3, day 4, day 5, or days 1 to 5).
  • the melflufen may alternatively be administered on a different day to any of the MAPIE components, for example on day 6, 7 or 8; or it may be administered the day before the doxorubicin/cisplatin is administered.
  • a dose of melflufen administered with MAP, MAP plus ifosfamide, MAP plus etoposide, or MAPIE may be a dose as described elsewhere in the present application, for example a dose of 1 to 40 mg melflufen.
  • a cycle of an embodiment of the present invention may be repeated one or several times depending on the category (for example high grade or low grade), class (for example metastatic, at risk of being metaststic, relapsed, refractory, etc.) or stage of the osteosarcoma.
  • the cycle may be repeated from 1 to 15 times, for example from 2 to 12 times, 1 to 9 times, and 2 to 7 times (for example 2, 3, 4, 5, or 6 times).
  • An ordinarily skilled physician or clinician can readily determine the number of cycles of melflufen, or a salt thereof, required to treat, prevent, counter or arrest the progress of the osteosarcoma.
  • the present invention provides a kit comprising melflufen, or a salt(s) thereof, and one or more further therapeutic agents that are useful in the treatment or prophylaxis of osteosarcoma.
  • further therapeutic agents for use in the present invention include chemotherapeutic agents and immunotherapeutic agents.
  • At least one of the one or more further therapeutic agent(s) included in the kit of the invention is a chemotherapeutic agent or an immunotherapeutic agents.
  • the chemotherapeutic agent is selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan. More preferably the chemotherapeutic agent is doxorubicin.
  • the kit may comprise melflufen, or a salt(s) thereof, doxorubicin, cisplatin and methotrexate, and optionally further comprise ifosfamide and/or etoposide.
  • immunotherapeutic agents include monoclonal antibodies, checkpoint inhibitors, non-specific immunotherapies (for example interferons and interleukins), adoptive cell therapies (for example chimeric antigen receptor T-cell (CAR T-cell) therapies and tumor-infiltrating lymphocyte (TIL) therapies) and cancer vaccines.
  • checkpoint inhibitors include agents or antibodies that inhibits one or more of CTLA4, PD-1, PD-L1, LAG-3, B7-H3, B7-H4, TIM3, VISTA and KIR.
  • PD-1 inhibitors include cemiplimab, nivolumab and pembrolizumab.
  • PD- LI inhibitors include atezolizumab, avelumab and durvalumab.
  • An example of a CTLA-4 inhibitor is ipilimumab.
  • the kit of the present invention finds use in the treatment and/or prophylaxis of osteosarcoma.
  • the melflufen, or salt thereof is present in a kit according to the present invention in a form and quantity suitable for use according to the present invention.
  • Suitable pharmaceutical formulations are described herein. The skilled person can readily determine a quantity of the melflufen, or a salt thereof, and the suitable the one or more further therapeutic agent(s) for the use according the present invention.
  • Melflufen, or a salt(s) thereof, according to the invention is useful for the treatment of osteosarcoma.
  • Osteosarcoma that a patient is suffering from may be classed as follows:
  • metastatic osteosarcoma • metastatic osteosarcoma or osteosarcoma at risk of becoming metastatic osteosarcoma; and/or
  • osteosarcoma with resistance to one or more of a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan; and/or
  • a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan; and/or
  • osteosarcoma with high surface expression of ANPEP/CD13 or osteosarcoma with low surface expression of ANPEP/CD13 can be defined as osteosarcoma wherein the cancer cells are slow-growing and look quite similar to normal cells.
  • High grade osteosarcoma can be defined as osteosarcoma wherein the cancer cells are fast growing and look very abnormal.
  • High grade osteosarcoma is more aggressive than low grade osteosarcoma, and more likely to metastasize than low grade osteosarcoma.
  • Intermediate grade osteosarcoma can be defined as osteosarcoma falling between high grade and low grade osteosarcoma.
  • Melflufen or a salt thereof, finds use in the treatment of high grade osteosarcoma, intermediate grade, and low grade osteosarcoma. It is especially useful in the treatment of high grade osteosarcoma.
  • Relapsed osteosarcoma can be defined as osteosarcoma that recurs following the last dosage of treatment.
  • Refractory osteosarcoma can be defined as osteosarcoma that is not responsive to currently available treatments.
  • Refractory-relapsed osteosarcoma can be defined as osteosarcoma that initially responds to treatment, but does not respond to treatment after relapse.
  • Osteosarcoma with resistance to one or more of a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan can be defined as osteosarcoma that does not respond to treatment with one or more of those chemotherapeutic agents.
  • Melflufen finds use in the treatment of refractory, relapsed and/or refractory-relapsed osteosarcoma.
  • melflufen finds use in the treatment or prophylaxis of refractory, relapsed and/or refractory-relapsed osteosarcoma in patients that have previously been treated with a currently available treatment, such as single or repeat treatment cycles of methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and/or topotecan.
  • melflufen finds use in the treatment or prophylaxis of osteosarcoma in patients with refractory, relapsed and/or refractory-relapsed osteosarcoma.
  • the prophylaxis may thus be a treatment of a patient who has been previously treated for osteosarcoma and made a recovery.
  • Melflufen finds use in the treatment or prophylaxis of osteosarcoma in patients that have metastatic osteosarcoma or are known or suspected to be at risk of developing metastatic osteosarcoma.
  • Metastatic osteosarcoma is osteosarcoma that has spread from the initially affected bone to one or more other sites in the body. The most common sites which osteosarcoma may metastasize, are the lungs, other bones, the brain, or other organs.
  • melflufen may be an effective adjunct treatment for osteosarcoma patients, and in particular osteosarcoma patients with high grade osteosarcoma and/or osteosarcoma that is or is at risk of becoming metastatic.
  • the invention provides melflufen, or a salt thereof, for use in the treatment or prophylaxis of osteosarcoma, which is high grade osteosarcoma, metastatic osteosarcoma or osteosarcoma at risk of becoming metastatic; refractory or relapsed osteosarcoma; and/or osteosarcoma with resistance to one or more of a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan.
  • a chemotherapeutic agent selected from methotrexate, doxorubicin, cisplatin, carboplatin, epirubicin, ifosfamide, cyclophosphamide, etoposide, gemcitabine and topotecan.
  • Osteosarcoma with high expression of peptidase may be defined as osteosarcoma in which the cancer cells have a higher level of peptidases or higher peptidase RNA expression compared to normal bone tissue, or compared to a sample of primary (i.e. non-metastatic) or pre-treatment osteosarcoma tissue.
  • Osteosarcoma with low expression of peptidase may be defined as osteosarcoma in which the cancer cells have a similar or a lower level or expression of peptidases compared to a normal bone tissue or compared to a sample of primary (i.e. non-metastatic) or pre treatment osteosarcoma tissue.
  • Osteosarcoma with high surface expression of ANPEP/CD13 may be defined as osteosarcoma in which the cancer cells have a "positive" expression value of the CD13, for example wherein the cancer cells have a staining proportion of cancer cells above 10% as determined by immune-histochemical staining, and/or wherein the cancer cells have increased intensity as accessed by flow cytometry for detection of CD13 compared to normal bone tissue or compared to a sample of primary (i.e. non metastatic) or pre-treatment osteosarcoma tissue, and/or wherein the cancer cells have elevated CD13 RNA expression compared to normal bone tissue or compared to a sample of primary (i.e. non-metastatic) or pre-treatment osteosarcoma tissue.
  • Osteosarcoma with low surface expression of ANPEP/CD13 may be defined as osteosarcoma in which the cancer cells have a "negative" expression value of the CD13, for example wherein the cancer cells have a staining proportion of cancer cells below 10% as determined by immune-histochemical staining, and/or wherein the cancer cells have a similar or decreased intensity as accessed by flow cytometry for detection of CD13 compared to normal bone tissue or compared to a sample of primary (i.e. non-metastatic) or pre-treatment osteosarcoma tissue, and/or wherein the cancer cells have a similar or low CD13 RNA expression compared to normal bone tissue or compared to a sample of primary (i.e. non-metastatic) or pre treatment osteosarcoma tissue.
  • the cancer cells have a similar or low CD13 RNA expression compared to normal bone tissue or compared to a sample of primary (i.e. non-metastatic) or pre treatment osteosarcoma tissue.
  • CD13 expression detected by immune-histochemical staining may be, for example, examined via imaging with a BX 50-32 scanner (Olympus, Union City, CA, USA).
  • CD13 expression detected by flow cytometry may use commercially available ANPEP/
  • Melflufen finds use in the treatment or prophylaxis osteosarcoma with high expression of peptidase, for example osteosarcoma with high surface expression of ANPEP/CD13, and osteosarcoma with low expression of peptidase, for example osteosarcoma with low surface expression of ANPEP/CD13.
  • melflufen, or a salt(s) thereof finds use in the treatment or prophylaxis osteosarcoma with high expression of peptidase, for example osteosarcoma with high surface expression of ANPEP/CD13. According to the inventors' analysis, higher expression levels of the aminopeptidase ANPEP mRNA were attributable to osteosarcoma cells with higher metastatic potential.
  • melflufen, or a salt(s) thereof finds use in the treatment or prophylaxis osteosarcoma with raised expression level of aminopeptidase ANPEP mRNA.
  • the invention provides the treatment or prophylaxis of osteosarcoma in a patient in whom a raised expression level of aminopeptidase ANPEP mRNA has been detected.
  • the invention thus finds use in the treatment or prophylaxis of osteosarcoma with high expression of peptidase (for example aminopeptidase ANPEP) or high surface expression of ANPEP/CD13.
  • peptidase for example aminopeptidase ANPEP
  • ANPEP aminopeptidase ANPEP
  • Melflufen, or a salt(s) thereof, and doxorubicin finds use in the treatment or prophylaxis osteosarcoma with high expression of peptidase, for example osteosarcoma with high surface expression of ANPEP/CD13, and osteosarcoma with low expression of peptidase, for example osteosarcoma with low surface expression of ANPEP/CD13.
  • melflufen, or a salt(s) thereof, and doxorubicin finds use in the treatment or prophylaxis osteosarcoma with low expression of peptidase, for example osteosarcoma with low surface expression of ANPEP/CD13.
  • Melflufen or a salt(s) thereof, also finds use useful for the treatment of osteosarcoma in patient groups considered to be a "high risk" group that may not tolerate or respond positively to existing osteosarcoma treatments.
  • High risk patients include, for example, patients that have high grade osteosarcoma and/or metastatic osteosarcoma.
  • the present inventors postulate that the surprisingly high potency of melflufen will enable the effective treatment of osteosarcoma patient groups that have not responded to existing osteosarcoma treatments or that are considered unsuitable for existing osteosarcoma treatments, for example patients having refractory and/or relapsed osteosarcoma, and osteosarcoma that are categorised, for example, as "high-risk" osteosarcoma patients.
  • Osteosarcoma cell lines U20S, SaOS-2, CAL-72, MG-63, HOS, 143B/HOS and MNNG/HOS were obtained from ATCC (USA).
  • Primary osteosarcoma cell lines STA- OS-1, -2, -3, and -5 were previously established in St. Anna Children's Cancer Research Institute (Stock C, et al, Genes Chromosomes Cancer 2000;28(3):329-36). Cells were cultured in DMEM medium supplemented with 10 % heat inactivated fetal calf serum, 1 % Pen-Strep, 1 mM sodium pyruvate (all ThermoFisher Scientific). Regular mycoplasma testing was performed using MycoAlert assay (Lonza).
  • Melflufen was obtained from Oncopeptides AB.
  • Other drugs including melphalan, cyclophosphamide, ifosfamide, bendamustine, busulfan, etoposide, doxorubicin, methotrexate, bestatin, niraparib, olaparib, talazoparib were obtained from SelleckChem (Houston, USA). All the drugs were dissolved in DMSO as 10 mM or lOOmM stock solutions.
  • Serial drug dilutions (0.01-100 mM range) were prepared in phenol red-free cell culture medium. Cells were seeded in 96-well plates at 5 x 10 5 cells/mL density and incubated with the drugs for 72 h at 37°C and 5% CO2. Cell viability was assessed using the Vybrant s MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific) according to a protocol provided by the manufacturer. To estimate the half maximal inhibitory concentration (IC50) smoothed dose-response curves were fitted using GraphPad Prism (GraphPad Software, San Diego, USA). The IC50 values were calculated by determining the mean of three independent experiments.
  • Ex vivo chemosensitivity in primary osteosarcoma cells was assayed using the fluorometric microculture cytotoxicity assay (FMCA) (Blom K, et a I, J Lab Autom 2016;21(l):178-87). Briefly, tumour cells from the patient samples were prepared by enzyme treatment of minced samples, followed by density gradient separation to enrich for tumour cells (>70%). The cells from cryopreserved samples were seeded in 384-well microtiter plates (in 45 pL culture medium). Drugs were added immediately after cell seeding using acoustic droplet ejection with an Echo ® 550 (Labcyte Inc., Sunnyvale, CA, USA).
  • FMCA fluorometric microculture cytotoxicity assay
  • Flow cytometry analysis was performed on LSR Fortessa (BD, USA). Cells were grown in the medium indicated above, harvested by using Stem Cell Pro Accutase (Invitrogen, USA). PE-conjugated monoclonal antibodies WM15 directed against CD13 (Invitrogen, USA) were used for detection of the surface CD13. Isogenic control was included. Quantification was performed using BD QuantibriteTM PE-conjugated beads (BD Biosciences, USA) according to the manufacturer's protocol.
  • Apoptosis was assayed using DAPI (ThermoFisher Scientific, USA) and Annexin-APC (ThermoFisher Scientific, USA). The results were obtained on LSR Fortessa (BD Biosciences, USA) device and processed using BDiva software. Apoptosis kinetics was assessed using Real Time-Glo Annexin V Apoptosis and Necrosis assay (Promega,
  • Osteosarcoma cells were lysed using high-salt buffer (20 mM Tris*HCI, 400 mM NaCI, 0.5% NP40, 0.3% Triton X100) with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein content was assessed using the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA, USA).
  • GSE3362, GSE14827, GSE21257, GSE32981, GSE43281, GSE74230 were downloaded from GEO Omnibus and processed using a standard package where appropriate. The statistical significance between different samples was evaluated using Mann-Whitney test for unpaired samples and Wilcoxon test for the paired samples. Spearman correlation analysis was employed for the correlation analysis.
  • Fertilized White Leghorn eggs were incubated at 37.5°C with 50% relative humidity for 9 days (Inovotion INC, France).
  • the chorioallantoic membrane CAM
  • Osteosarcoma cells 143B/HOS were detached with trypsin, washed with complete medium and suspended in graft medium.
  • An inoculum of 500K cells was added onto the CAM of each egg. Eggs were then randomized into 8 groups.
  • tumors became detectable, and were treated with either vehicle (1% DMSO in lx PBS), doxorubicin at 110 nM per egg, or melflufen at 440 nM per egg. Concentration of melflufen chosen was based on achievable safe plasma concentration. For all conditions, the injection volume of 100 mI/egg, was dropped onto the tumor. On day E18 the upper portion of the CAM containing tumor was removed, washed in PBS and then directly transferred in PFA (fixation for 48h). The tumor was then washed, carefully cut away from normal CAM tissue and weighed. Analysis of metastasis was done in parallel.
  • a 1cm 2 portion of the lower CAM was collected to evaluate the number of metastasis cells.
  • Genomic DNA is extracted from the CAM and analyzed by qPCR with specific primers for human Alu sequences. Calculation of Cq for each sample, mean Cq and relative amount of metastasis for each group was performed with the Bio- Rad ® CFX Maestro ® software. To estimate toxicity, eggs were checked at least every two days, for the viability and visible macroscopic abnormalities. The number of dead embryos counted on day E18, combined with reported abnormalities was used to evaluate total toxicity.
  • Immunohistochemical staining was performed as follows: 4-microns-thick sections were cut from formalin-fixed paraffin-embedded tissue blocks and were dried in a 37°C oven overnight. The sections were baked and dewaxed; antigen retrieved and rehydrated using the Deparaffinization protocol on the Leica Bond autostainer. This involved the incubation of the sections through a number of pre-programmed cycles at temperatures of up to 96°C using ER1 epitope retrieval buffer (pH6). The sections were then automatically rinsed with Bond wash buffer. Bond polymer refine peroxidase block was applied for 5 minutes then automatically rinsed with Bond wash buffer.
  • Dako serum free protein block was applied for 10 minutes without washing the slides, primary antibodies (anti-gH2AX Rabbit monoclonal Abeam (ab81299) at 0.025 (pg/mL) and anti-CC3 Rabbit monoclonal Cell Signalling (#9579) at 0.275 (pg/mL)) were applied for 30 minutes at ambient temperature, after which sections were rinsed with bond wash buffer. Polymer refine (anti-rabbit HRP) was subsequently applied to the slides for 15 minutes. All sections were then rinsed with bond wash buffer. Polymer refine DAB was then applied for 10 minutes.
  • Aminopeptidase-enhanced cytotoxic agent melflufen demonstrates higher potency against osteosarcoma cells than other alkylating agents
  • the present inventors studied the anti-neoplastic activity of novel aminopeptidase- enhanced alkylating agent melflufen in a panel of osteosarcoma cell lines including low passaged patient derived neoplastic cells (Stock C, et a I, Genes Chromosomes Cancer 2000;28(3):329-36).
  • the present inventors also compared cytotoxic activity of melflufen with a panel of commonly used alkylating agents such as melphalan, cyclophosphamide, ifosfamide, busulfan and bendamustine.
  • the inventors included compounds which are used within the standard of care protocol (doxorubicin, methotrexate, cytarabine), and employed in the recent EURAMOS-1 studies (etoposide and cyclophosphamide) (Marina NM, et a I, Lancet Oncol 2016;17(10):1396-408) or proposed for further clinical evaluation (PARP inhibitors, CDK4/6 inhibitors, MEK inhibitor, NF-KB inhibitor) (Holme H, et a I, Sci Rep 2018;8(1):10614; Engert F, et al. Oncotarget 2017;8(30):48794-806).
  • melflufen was also able to induce cytotoxicity in a primary cell line STA- OS-3 resistant to both methotrexate and etoposide (Figure 1C, Figure 1G).
  • STA-OS-3 cell line was established from a patient that poorly responded to the standard chemotherapy and died soon after the diagnosis (Stock C, et a I, Genes Chromosomes Cancer 2000;28(3):329-36).
  • Three other etoposide-resistant cell lines, SaOS-2, U20S and STA-OS-5 also displayed sensitivity to melflufen (Figure 1A).
  • melflufen also demonstrated anti-neoplastic activity in primary cultures of patient derived osteosarcoma cells, including specimen resistant to doxorubicin ( Figure ID).
  • Figure ID specimen resistant to doxorubicin
  • aminopeptidase activity influences melflufen cytotoxicity in osteosarcoma cells
  • the present inventors employed bestatin, a known aminopeptidase inhibitor (Kido A, et a I, Clin Exp Metastasis 1999;17(10):857-63; Hitzerd SM, et al, Amino Acids 2014;46(4):793-808; Wickstrom M, et a I, Mol Cancer Ther 2007;6(9):2409-17).
  • ANPEP/CD13 One of the most studied aminopeptidases in osteosarcoma is ANPEP/CD13. High expression of this gene has been linked to an aggressive osteosarcoma with high invasive potential (Liang W, et a I, Int J Oncol 2014;45(6):2475-85; Kido A, et a I, Clin Exp Metastasis 2003;20(7):585-92; Kido A, et a I, Clin Exp Metastasis 1999; 17(10) :857-63; Shuo Wang HX, et al, Int J Clin Exp Med 2016;9:22034-40).
  • the present inventors have evaluated expression of ANPEP/CD13 in the osteosarcoma cell lines used in this study using quantitative flow cytometry analysis (Figure 2D) and have found a positive correlation between sensitivity to melflufen and ANPEP/CD13 expression (Figure 2C).
  • ANPEP/CD13 expression is elevated in aggressive osteosarcoma
  • ANPEP/CD13 has been previously linked to poor survival in osteosarcoma (Shuo Wang HX, et al, Int J Clin Exp Med 2016;9:22034-40). Therefore, the present inventors have analyzed ANPEP mRNA expression in several previously published gene expression datasets of various osteosarcoma models including xenograft (Morrow JJ, et al, Nat Med 2018;24(2):176-85) and orthotopic (Zhao S, et al, Oncogene 2015;34(39):5069-79) mouse models as well as patient samples (Odagiri H, et al, Sci Signal 2014;7(309):ra7).
  • RNA-sequencing data shows that ANPEP mRNA expression is up-regulated in highly invasive osteosarcoma cell lines 143B and MG63.3, metastatic derivatives of well-known HOS and MG63 cell lines (Ren L, et al, Oncotarget 2015;6(30):29469-81; Khanna C, et al, Clin Exp Metastasis 2000;18(3):261-71) (Figure 3A-C).
  • GSE21257 Buddingh EP et al., Clin Cancer Res.
  • melflufen The kinetics of apoptosis and necrosis induction by melflufen was also studied.
  • melflufen was able to induce rapid apoptotic response in osteosarcoma cells with a maximum at 5 to 10 hours after treatment start, followed by the accumulation of late apoptotic/necrotic cells.
  • the indicated HGOS cell lines were cultivated in the presence of 1 mM melflufen and analysed at 0, 1, 2, 4, 6, 10,
  • Doxorubicin treatment up-regulates ANPEP/CD13 expression and synergizes with melflufen in killing osteosarcoma cells
  • ANPEP mRNA is up-regulated upon doxorubicin treatment of osteosarcoma cells U20S ( Figure 6) (Menendez D, et la, Nucleic Acids Res 2013;41(15):7286-301; Zanotto-Filho A, et al, Mol Cancer Ther 2016;15(12):3000-14). Moreover, ANPEP mRNA is up-regulated in doxorubicin-resistant subline of highly aggressive osteosarcoma cell line 143B (Rajkumar T, et al Anticancer Drugs 2008;19(3):257-65).
  • the present inventors first tested if addition of doxorubicin could up-regulate expression of aminopeptidase N in our panel of osteosarcoma cell lines. FACS analysis has demonstrated that indeed doxorubicin treatment promotes expression of ANPEP/CD13 ( Figure 7A). Second, the present inventors tested if doxorubicin pre treatment potentiates the cytotoxic effect of melflufen. Synergy scoring (lanevski A, et al Bioinformatics 2017;33(15):2413-5) in two-dimensional survival assays and apoptosis analysis indicate that doxorubicin enhances the anti-neoplastic effect of melflufen (Figure 7B). Melflufen shows high anti-neoplastic and anti-metastatic activity in osteosarcoma in vivo

Abstract

L'invention concerne du melflufène, ou un sel de celui-ci, destiné à être utilisé dans le traitement ou la prophylaxie d'un ostéosarcome, en particulier un ostéosarcome de haut grade, un ostéosarcome métastatique ou un ostéosarcome à risque de devenir métastatique; un ostéosarcome réfractaire ou récidivant; et/ou un ostéosarcome ayant une résistance à un ou plusieurs agents chimiothérapeutiques choisis parmi le méthotrexate, la doxorubicine, le cisplatine, le carboplatine, l'épirubicine, l'ifosfamide, le cyclophosphamide, l'étoposide, la gemcitabine et le topotécan. En particulier, l'invention est utile dans le traitement ou la prophylaxie d'un ostéosarcome ayant une expression élevée de peptidase ou une expression de surface élevée de ANPEP/CD13. Le traitement peut être avec un agent thérapeutique supplémentaire, par exemple la doxorubicine.
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