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• • 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
  • A61K47/58—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 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

• the present invention relates to soluble synthetic polymer-bound anthracyclines, to their preparation and to pharmaceutical compositions containing them.
• Doxorubicin, 4'-epidoxorubicin and 4-demethoxydaunorubicin are examples of anthracyclines which are known from the prior art and which are currently used in the clinical treatment of neoplastic malignancies; see for example, F.Arcamone: "Doxorubicin” Medicinal Chemistry monograph, vol 17, Academic Press 1981.
• doxorubicin Many polymeric derivatives of doxorubicin, endowed with antitumour activity, have been prepared. Amongst these, a particularly promising candidate for clinical development is soluble polymer-bound doxorubicin, which consists of hydrophilic moieties and peptide chains to which doxorubicin and 2-hydroxypropylamine are linked.
• This polymer-bound-doxorubicin derivative is prepared by condensing doxorubicin hydrochloride with a methacrylic polymeric precursor containing peptidyl chains, activated as the p-nitrophenyl ester, in dimethylsulfoxide in the presence of triethylamine followed by aminolysis of the remaining ester groups with 1-amino-2-hydroxypropane.
• the polymer-bound-anthracycline systems of the present invention are based on methacrylic polymers bearing hydrophilic moieties, peptidyl pendant chains linked only to the amino group of anthracycline and residues of
• anthracycline may be easily released from the polymer to which it is bound.
• the polymer-bound anthracyclines of the invention have broader antitumour activity and lower general toxicity than the corresponding free anthracyclines.
• the present invention provides a
• polymer-bound anthracycline of formula A which consists essentially of three units represented by formulae 1, 2 and 3:
• Gly represents glycine
• y is from 1 to 29 mol %
• z is from 1 to 29 mol %
• R 1 is a C 1 -C 6 alkyl group substituted by one or more hydroxy groups
• Y is an amino acid residue or a peptide spacer
• [NH-D] is the residue of an aminoglycoside
• Z is hydroxy or a residue of formula -NHR 1 as defined above.
• aminoglycoside anthracycline of which [NH-D] is a residue is represented herein as [NH 2 -D] wherein D denotes the structure of an anthracycline aminoglycoside minus the amino group of the sugar moiety.
• the polymer-bound anthracycline preferably contains the units 1 in a range of from 90 to 98 mol %, the units of formula 2 from 1 to 10 mol % and the units of formula 3 from 1 to 10 mol %.
• Suitable alkyl groups which R 1 may represent are C 1 -C 4 alkyl groups substituted by one or more hydroxy groups; examples include hydroxyethyl, 2-hydroxypropyl and 3-hydroxypropyl groups.
• the peptide spacer Y should be susceptible to
• the spacer may be resistant to extracelluar hydrolysis.
• the peptide spacer may be from 1 to 10, for example 2 to 4, amino acid residues long.
• the peptide spacer is a tripeptide or a
• Each of the constituent amino acid residues of the peptide spacer Y which is chiral may be present as either the D or the L optical isomer, or as a D/L mixture.
• the conventional three letter system of denoting amino acids is employed herein, wherein the symbols denote the L configuration of the chiral amino acid unless otherwise stated.
• the peptide spacer Y may be present as a racemic mixture or as an optically pure isomer.
• Y is selected from Gly-Phe-Gly, Gly-Leu-Gly, Phe-Leu-Gly, Gly-Phe-Leu-Gly, or Leu-Leu-Gly with the glycyl residue in each case being bound to the
• aminoglycoside anthracycline residue [NH-D] is suitably the residue of an anthracycline aminoglycoside [NH 2 -D] of the following formula Q:
• R 1 and R 11 are hydrogen and the other is a hydroxy group or iodine;
• R III is hydrogen or OCH 3 and
• R IV is hydrogen or a hydroxy group.
• aminoglycoside [NH 2 -D] are: doxorubicin, 4'-epidoxorubicin, 4-demethoxydaunorubicin, idarubicin and 4 , -iodo, 4 , -desoxy doxorubicin.
• the invention also provides a process for the preparation of a polymer-bound anthracycline A which consists essentially of the units 1, 2 and 3 as defined above.
• the process comprises:
• step (ii) when it is desired to prepare a polymer-bound anthracycline wherein Z is the unit of formula 3 is NHR 1 , reacting the product of step (i) wherein R 2 is a leaving group with a compound of formula NH 2 R 1 in which R 1 is as defined above.
• the leaving group which R 2 may represent is suitably a phenyloxy group which is substituted on the phenyl ring by one or more electron-withdrawing groups.
• suitable electron-withdrawing groups include nitro (-NO 2 ) and halogen.
• R 2 is preferably the leaving group wherein L is an electron withdrawing
• R 2 is a p-nitrophenoxy group or a 2,4-dichlorophenoxy group.
• n, R 1 and R 2 are as defined above.
• Polymeric intermediates consisting of units 1 and 4 in which R 2 represents hydroxy may be prepared by radical homogeneous polymerization.
• the peptidyl-anthracycline derivatives of formula 5 are a further aspect of the present invention. Methods for their preparation are known. For example, since it is important to react a suitable N-protected peptide with anthracycline, the N-protecting peptidyl group must be selected from those that are removed in conditions capable conferring stability on the anthracycline. An example is the triphenylmethyl group.
• the peptidyl anthracycline derivatives of formula 5 may be prepared by a process which comprises
• R 3 is an acid sensitive protecting amino group
• P is a leaving group
• Y is an amino acid residue or a peptide spacer as defined above, with an anthracycline aminoglycoside [NH 2 -D] as defined above to produce an intermediate of general formula 10:
• P may be a leaving group as defined and exemplified, above for R 2 .
• P may be a pentafluorophenyloxy or N-hydroxy-succinimido group. Examples of the acid
• sensitive protecting group R 3 include trityl and
• Peptidyl derivatives of formula 8 and 9 are prepared following standard synthetic procedures that are known from peptide literature. Protection of the amino function with an acid sensitive group such as triphenylmethyl is
• compound 9 may be reacted with an anthracycline hydrochloride salt in an anhydrous polar solvent such as dimethylformamide in the presence of equivalent amounts of an organic base such as triethylamine, for example at room temperature for 15 hours, to give an intermediate of formula 10 that is purified by chromatography and then deblocked to derivative of formula 5, for example in aqueous 75% acetic acid at room temperature.
• an anthracycline hydrochloride salt in an anhydrous polar solvent such as dimethylformamide in the presence of equivalent amounts of an organic base such as triethylamine, for example at room temperature for 15 hours, to give an intermediate of formula 10 that is purified by chromatography and then deblocked to derivative of formula 5, for example in aqueous 75% acetic acid at room temperature.
• anthracyclines bearing a hydroxy group at position C-14 such as doxorubicin and 4'-epidoxorubicin in the form of hydrochloride salt, with activated peptidyl derivatives of formula 9 , in the presence of the organic based needed to deblock the 3'-amino group of the anthracyclines, in a polar solvent, affords a mixture of derivative 10 and anthracyclines substituted both at the amino group of the sugar moiety and at the C-14 position.
• the bis (3'-N,14-O-peptidyl)derivatives are removed from the mixture by chromatography.
• Compounds of formula 10 may be also prepared by condensing an N-protected peptide of formula 8 with an anthracycline in the form of the hydrochloride salt, in a dry polar solvent such as dimethylsulfoxide in the presence of an equivalent amount of condensing agent such as 1-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. This procedure does not afford bis-peptidyl derivatives of anthracyclines bearing a C-14 hydroxy groups.
• Polymer-bound drugs of formula A in which residue Z of unit 3 represents a group of formula NHR 1 , as previously defined, are preferably prepared by reacting intermediate B in which R 2 is a leaving group as defined above, with an anthracycline derivative of formula 5 in an anhydrous polar organic solvent such as dimethylformamide or
• the reaction can typically be effected for from 8 to 24 hours.
• the reaction is typically carried out at a temperature from 15oC to 30oC, preferably at room temperature for 15 hours, then the remaining leaving groups are displaced by reacting the conjugate with a compound of formula NH 2 R 1 , as above defined, for a time of 0.5 to 3 hours at room temperature.
• Polymer-bound drugs of formula A in which residue Z of unit 2 represents a hydroxy group are preferably prepared by reacting intermediate B in which R 2 is hydroxy with an anthracycline derivative of formula 5 in an
• reaction can typically be effected for from 8 to 24 hours.
• the reaction is typically carried out at a temperature from 15 to 30oC, preferably at room temperature for 15 hours.
• anthracycline 5 at room temperature for 15 hours.
• B is suitably employed at 14% w/v and 5 at 2.3% w/v.
• a compound of formula NH 2 R 1 as defined above, is then added,
• dimethylsulfoxide is treated with a peptidyl anthracycline 5 at room temperature for 15 hours.
• B is suitably employed at 14% w/v and 5 at 2.3% w/v.
• the conjugate is then precipitated with acetone, dissolved in absolute ethanol, typically at a concentration of 8% (w/w) and precipitated again with acetone to give a polymer-bound anthracycline of formula A as defined above.
• the anthracycline content of the conjugates A is determined by analysis of the aglycone released from bound anthracycline by means of acid hydrolysis; thus,
• adriamycinone is the aglycone moiety of doxorubicin and 4'-epirubicin and 4-demethoxydaunomycinone is that of 4-demethoxydaunorubicin.
• Polymer-bound-anthracyclines of the present invention exhibit good water solubility, biocompatibility, stability at physiological pH and release of the free active drug, D-NH 2 , after incubation with lysosomal enzymes.
• the polymer-bound anthracyclines of formula A have anti-tumour activity.
• a human or animal can therefore be treated by a method comprising administering thereto a therapeutically effective amount of a polymer-bound
• anthracycline of formula A The condition of the human or animal patient can thus be improved.
• the dosage range adopted will depend on the route of administration and on the age, weight and condition of the patient being treated.
• the polymer-bound anthracyclines of formula A are typically administered by the parenteral route, for example intramuscularly, intravenously or by bolus infusion.
• a suitable dose range is from 5 to 800 mg/m 2 of anthracycline equivalent, for instance from 20 to 500 mg/m 2 .
• a suitable regime entails administering a solution of 25 mg anthracycline equivalent /m2
• the polymer-bound anthracyclines of formula A may be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier or diluent.
• polymer-bound anthracyclines are formulated for parenteral administration, for example by dissolution in sterile water or water for injection.
• Examples 1-6 relate to synthetic procedures for the preparation of nonomers of formula 6 and 7 and polymeric intermediates of formula B.
• Methacryloylglycyl p-nitrophenyl ester (7a: 5.28 g, 20 mmol), prepared as described in Makromol.Chem. 178, 2159 (1977), was dissolved in anhydrous tetrahydrofurane (20 ml) and treated with 1-amino-2-hydroxypropane (3.2 ml, 40 mmol). After 20 minutes at room temperature, the solvent was removed under reduced pressure and the title compound 6b (3.3 g, yield 82.5%) was recovered after crystallization with acetone/ethyl ether. TLC on Kieselgel plate F 254 (Merck), eluting system methylene chloride/acetone (90:10 by volume) R f 0.47.
• the title compound 7b was prepared from methacryloylglycine (2.66 g, 20 mmol), prepared as described in Makromol. Chem. 178, 2159 (1977), and 2,4-dichlorophenol (3.26 g, 20 mmol) in anhydrous tetrahydrofurane (50 ml) and in presence of DCC (4.2 g, 21 mmol).
• Compound 2b (4.7 g, yield 82%) was crystallized from ethyl acetate and n-hexane.
• N-methacryloylamide-2-hydroxypropane (25.2 g, 0.18 mol), methacryloylglycine (2.86 g, 20 mmol) and ⁇ , ⁇ '-azoisobutirronitrile (5.9 g) were dissolved in anhydrous methanol (164 ml) The mixture was kept at 60°C under nitrogen for 20 hours, then the reaction mixture was added to acetone (2000 ml) under stirring. The precipitate was collected, washed with acetone and dried to constant weight to give the title polymer B2 (26 g). Content of carboxy groups (w): 10 mol%
• the title polymeric intermediate B4 was prepared from N-methacryloylamide-2-hydroxyethane (6c: 23.2 g, 0.18 mol), methacryloylglycine (2.86 g, 20 mmol) and ⁇ , ⁇ '-azoisobutirronitrile (5.9 g) in anhydrous methanol (164 ml) as described in Example 2. Content of carboxy groups (w): 10 %
• Examples 7-12 relate to methods for the preparation of peptidyl-anthracyclines of formula 5
• N-trityl-L-Phenylalanine (20.3 g, 50 mmol), prepared as described in J.Org.Chem. 47, 1324 (1982) was dissolved in anhydrous terahydrofurane (150 ml) and added with anhydrous N-hydroxybenzotriazole (8 gr).
• the mixture was cooled at 0oC and treated with 1,3-dicyclohexylcarbodiimide (11.7 gr, 50 mmol) and, after 10 minutes, added dropwise with a solution of L-Leucylglycine ethylester p-toluensulphonate salt (20 g, 50 mmol) in in a mixture of anhydrous tetrahydrofurane (100 ml) and N-methylmorpholine (7 ml). The reaction mixture was kept at 0oC for one hour and overnight at room temperature, then was filtered and the solvent was removed under reduced pressure.
• 1,3-dicyclohexylcarbodiimide (8.24 g, 40 mmol) and kept overnight at 4°C. After that, the reaction mixture was filtered and the solvent removed under reduced pressure.
• the title compound 5c (3 g, yield 75%) was prepared from 4-demethoxydaunorubicin hydrochloride (2.9 g, 5 mmol) and N-trityl-Phenylalanylleucylglycyl p-nitrophenylester
• the title compound 5d (3.25 g, yield 86%) was prepared from 4 , -epidoxorubicin hydrochloride (2.9 g, 5 mmol) and
• Polymeric precursor B1 (10 g, 2.7 ⁇ 10 eq of p-nitrophenyl ester), prepared as described in Makromal.Chem. 178,
• Examples 13-18 illustrate the procedures for the preparation of polymer-bound-anthracyclines of formula A.
• Polymeric precursor B2 (7,15 g, 5 mmol of -COOH), prepared as described in Example 4, and 3'-N-(Glycyl-leucyl- L-phenylalanyl)doxorubicin (5a: 2,36g, 2,5 mmol) were dissolved in anhydrous dimethylformamide (100 ml), then added with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquino1ine (0.7 g, 2,5 mmol). The mixture was kept under stirring at room temperature for 24 hours, then poured in ethyl ether (800 ml). The precipitate was dissolved with ethanol (100 ml), precipitated with acetone (800 ml) and dried to constant weight to give the title compound A1 (7 g).
• the title compound was prepared as described in Example 13, from polymeric intermediate B2, 4'-epi-3'-N-(Glycylleucyl-L-phenylalanyl)doxorubicin (5e) and N-ethoxycarbonyl-2-iethoxy-1,2-dihydroquinoline, in anhydrous dimethylformamide.
• the title doxorubicin polymeric prodrug M was prepared by condensing polymeric intermediate B4 and 3'-N-(Glycyl-leucyl-L-phenylalanyl)doxorubicin (5e) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, in anhydrous dimethylformamide as described in Example 13.
• Treatment was administered i.v. in a volume of 10 ml/Kg body weight at days 5,9 and 15.
• Anthracyclines were dissolved in sterile water and the concentration was checked spectrophotometrically.
• Lyophylized polymers were dissolved in water to give a starting solution of 25 mg anthracycline equivalent/ml according to the reported concentration, further dilutions were performed in water.
• M5076 murine reticulosarcoma was obtained by serial i.m. passage and transplanted (5 ⁇ 10 5 cells/mouse) s.c. in C75 B1/6 mice to evaluate the activity on primary tumor.
• TFD tumor growth delay
• Toxicity was evaluated on the basis of body weight reduction and gross finding main of spleen and liver reduction.
• Neurotoxicity is determined as number of mice with lack of motor function.
• TDG Tumor Growth Delay
• the antitumor activity was tested with the same treatment schedule for 4'-epidoxorubicin.HCl and compound A4.
• TDG Tumor Growth Delay
• Toxicity was evaluated in healthy C57B1F mice, treatment i.v., with single doses of 4'-epidoxorubicin.HCl (13.2 - 16.15 - 19 -20.6 - 25.2 and 33.2 mg/kg) and compound A4 (50 - 63 - 79 - 100 -120 - 140 mg/kg)
• the therapeutic index was calculated using the following formula:
• Dose effective 50 is the dose causing 50% of tumor growth reduction. Animal safety was observed for 90 days.
• the LD 10 and LD 50 values in C57 Bl/F mice are as follows:
• the low toxicity of compound A4 allows to administer higher doses of product and to reach equivalent or better results than with 4'-epidoxorubicin.HCl and to obtain a better therapeutic index.
• the terapeutic index for 4'-epidoxorubicin.HCl and compound A4 are respectively 3 and 40.

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