Classifications

• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the invention deals with the structure and properties of water-soluble polymeric therapeutic agents (pro-drugs) based on derivatives of paclitaxel (PTX), docetaxel (DTX) and larotaxel (LTX), destined mainly for treatment of solid tumours in therapy of tumour diseases in human medicine.
• PTX paclitaxel
• DTX docetaxel
• LTX larotaxel
• Polymeric conjugates of cancerostatic agents with soluble polymers were prepared and studied where the drug with an anti-cancer effect was attached to the polymer with a non-cleavable covalent bond, hydrolytically instable ionic bond and/or covalent bond allowing controlled release of the drug and thus its activation on the basis of enzymatic or plain chemical hydrolysis of this bond.
• Polymeric carrier systems are generally constructed in such a way to be able to release the therapeutically active cancerostatic agent from the carrier either in the tumour, or more specifically, directly in a tumour cell.
• polymeric therapeutic agents An important group of polymeric therapeutic agents is represented by polymeric drugs prepared on the basis of copolymers of N-(2- hydroxypropyl)methacrylamide (HPMA), a number of which are actively directed to tumours by means of a targeting structure attached to the polymer (antibody, lectin, hormone).
• HPMA N-(2- hydroxypropyl)methacrylamide
• a targeting structure attached to the polymer antibody, lectin, hormone
• HPMA copolymers as passively targeted high-molecular earners is due to their non-cleavable carbon chain and only polymers not exceeding the molecular weight of 40 - 50,000 g/mol can be excreted from the organism. This means that if accumulation of the polymer in the organism after repeated administration of the drug is not to occur and if the molecular weight of the carrier is to be as high as possible to make the passive targeting as efficient as possible, the polymeric carrier must be constructed as degradable in the organism.
• Literature mentions a lot of information about the preparation and studying of properties of polymers carrying a cancerostatic agent, attached to the polymer by a bond susceptible to hydrolysis in an aqueous environment [Kratz 1999].
• HPMA copolymers carrying the cancerostatic agent doxorubicin, bound to the polymeric chain by a hydrolytically cleavable hydrazone bond take an important position [Etrych 2002, Ulbrich 2004a, Ulbrich 2004b, Ulbrich pat CZ 293787 B6].
• This bond is relatively stable in the bloodstream environment (in the course of transport in the organism) and hydrolytically instable in the slightly acidic environment of the living cell.
• Paclitaxel and docetaxel belong to the group of taxanes, the anti-cancer drugs routinely used for the treatment of ovarian tumours and breast, lung, prostate and other tumours [Vanhoefer at al., 1997]. Besides their secondary toxicity, common for cancerostatic agents, they have another disadvantage of very low solubility in aqueous solutions, resulting in the necessity of administration in various excipients, especially in Cremophor EL, which also leads to other side effects of the dosage form. In some administration schemes the administration of taxanes in Cremophor EL may even significantly reduce the efficiency of the therapy [Ng at al., 2006].
• paclitaxel was covalently bound to poly( ethylene glycol) (PEG) by a non-cleavable ester or 7-carbamate bonds (C2 -OH group), or by means of a hydrolytically instable amino-acid spacer (Ala, GIy) [Greenwald et al., 1995; Greenwald, 2001; Greenwald et al., 2003; Pendri et al., 1998]. It has been shown that the anti-tumour activity of the conjugate depends on the structure of the bond used between the polymer and the drug and that the molecular weight of the carrier is another important factor. The conjugate with PTX was tested in phase I of clinical trials [Satchi-Fainaro et al., 2006], but apparently with little success.
• conjugate of paclitaxel with poly(glutamic acid) appears to be much more successful in clinical trials [Winter 2005, Kratz et al., 2008].
• PTX is bound to a polyamino-acid earner by an ester bond via the -OH group in position 2.
• the drug is released due to degradation of the polymeric chain, formation of the GIu derivative of PTX and its subsequent hydrolysis.
• this conjugate is in phase III of clinical trials.
• Paclitaxel has also been bound to HPMA copolymers by an ester bond using a biodegradable oligopeptide GlyPheLeuGly spacer. It has been demonstrated that PTX, after incubation with lysosomal enzymes, is released from the carrier and this release is important for achieving the anti-cancer activity in vivo.
• PNU166945 is a conjugate of a HPMA copolymer with ester-bound PTX, which was clinically tested in phase I [Terwogt et al., 2000; Terwogt et al., 2001], however, after this phase further testing was stopped.
• a polymeric drug in accordance with this invention is characterized in that a cancerostatic agent from the group of taxanes, paclitaxel (PTX), docetaxel (DTX), or larotaxel (LTX) (hereinafter drugs) is bound to a water-soluble polymeric carrier, prepared on the basis of a linear or grafted HPMA copolymer.
• the drug is bound to the polymeric chains via an ester group produced by acylation of the -OH group in position 2 by means of spacers, containing pH-sensitive hydrolytically cleavable hydrazone bonds.
• spacers may consist of acid residues of various oxo acids, by means of which the carbonyl group is introduced to the drug structure, linked with residues of individual amino acids, oligopeptides, or other structures, allowing termination of the side chains of the polymeric earner with the hydrazone group.
• the molecular weight of the polymeric chain is selected below the excretion limit of HPMA copolymers from the organism, preferably in the range of 10 - 50,000 g/mol.
• a grafted copolymer the molecular weight is selected in the range of 50 — 250,000 g/mol.
• the polymeric drug in accordance with the invention is destined for intravenous (injection or infusion) administration in a solution, but it may also be administered intratumourally or intraperitoneally and is intended for treatment of solid tumours.
• the polymer with the chemically bound cytostatic agent is constructed in such a way to be stable during circulation in the bloodstream and to prevent hydrolysis of the hydrazone bond between the given taxane and the polymer, or, possibly, to keep the hydrolysis rate in the course of transport through the organism as low as possible (at the pH value 7.4 in the bloodstream), so that no cytotoxic effect of the released drug or its derivative can be manifested.
• the entire system is constructed as a two-phase system.
• the polymeric conjugates with a targeted anti-cancer effect in accordance with the invention are characterized in that the cytostatic agent (taxol, docetaxel, larotaxel) is attached, by means of an ester bond and spacer, to the polymeric carrier formed by a linear [Etrych pat CZ 297827 B6, publ. 2008], or grafted [Etrych pat CZ 298945 (B6), publ.
• cytostatic agent taxol, docetaxel, larotaxel
• HPMA copolymer through a hydrolytically instable hydrazone group produced by a reaction of the carbonyl group of the molecule of the drug derivative with the hydrazide group of the polymeric carrier.
• the polymeric carriers are generally prepared by radical solution copolymerization of HPMA with comonomers corresponding to the desired composition.
• the binding of the corresponding taxane to the polymeric carrier results in considerable reduction of its cytotoxicity, considerable increase of the molecular weight of the drug, and thus extension of the circulation time in the bloodstream; i.e. extension of the total residence time of the drug in the organism and hence increase of its bioavailability.
• the polymeric drug in accordance with the invention is further characterized in that the bond of the drug to the polymeric carrier is relatively stable in the course of the transport in the bloodstream and in body liquids and is hydrolytically cleavable in the slightly acidic environment of the tumour and especially inside the target tumour cells in endosomes characterized by slightly acidic pH.
• the bond of the drug to the polymeric carrier is relatively stable in the course of the transport in the bloodstream and in body liquids and is hydrolytically cleavable in the slightly acidic environment of the tumour and especially inside the target tumour cells in endosomes characterized by slightly acidic pH.
• the drug is transported via the bloodstream in an inactive form bound to the polymer and it is released and activated predominantly after the penetration into the target tumour cells.
• the fact that the drug is only activated in the target cells eliminates the side effects of otherwise toxic cytostatic agents and directs their effect preferentially to the tumour cells.
• the efficiency of accumulation in the tumour tissue may be controlled by the changes in the structure of the skeleton of the polymeric carrier (non- degradable linear polymer, high-molecular biodegradable grafted polymer) that is responsible for the targeted (passive) transport to the tumour or tumour cells.
• the polymeric carrier non- degradable linear polymer, high-molecular biodegradable grafted polymer
• the sphere of application of the presented invention includes the use of polymeric drugs of the invention for the treatment of solid tumours in malignant diseases in the human medicine.
• the basic monomers are synthesized: HPMA, methacryloylated derivatives of amino acids and of oligopeptides, terminated with the hydrazide (CONHNH 2 ) group, or possibly terminated with the hydrazide group protected with the t-butyloxycarbonyl group (Boc).
• polymeric precursors are synthesized, i.e. HPMA copolymers carrying the functional groups (random copolymers), serving as the polymeric carriers for the drugs.
• a polymeric precursor, carrying functional hydrazide groups along the chain can be prepared either by radical copolymerization of the above-mentioned functional monomers with HPMA, or by polymer-analogous transformation of the basic copolymer carrying the functional groups.
• Grafted copolymers are prepared from multivalent and semitelechelic HPMA copolymers in accordance with the procedure described in [Etrych 2008 pat, publ.].
• the basic copolymer is a copolymer of HPMA and methacryloylated hydrazides of amino acids or of oligopeptides selected from the group of glycyl, glycylglycyl, ⁇ -alanyl, 6-aminohexanoyl (AH), 4-aminobenzoyl, or a mixed acyl derived from the oligopeptides GlyPheGly, GlyLeuGly, GlyLeuPheGly and GlyPheLeuGly), characterized in that it contains 70 - 98 mole % of HPMA and 2 - 30 mole % of units with hydrazide functional groups (see Scheme 2 with the spacer consisting of 6-aminohexanoyl).
• a drug derivative is a compound of the drug (PTX, DTX or LTX) produced by acylation of the hydroxyl group in position 2 of the drug with the corresponding oxo acid.
• the following oxo acids have been preferably used: levulic acid, 4-(2-oxopropyl) benzoic acid, 4-oxo-pent-2-enoic acid and 5-oxo-hex-2-enoic and 6-oxo-hept-2-enoic acids (see Scheme 1).
• a polymeric conjugate is a compound of a polymeric precursor with a drug derivative, wherein the drug derivative is bound to the polymeric precursor by a hydrazone bond prepared by reaction of the carbonyl group of the drug derivative with the hydrazide groups of the polymer, characterized in that it contains 70 - 98 mole % of HPMA, 1.5 - 29.5 mole % of units with hydrazide functional groups and 0.5 - 10 mole % of units with the hydrazone-bound drug derivative (see Scheme 3; with the spacer consisting of 6-aminohexanoyl and levulic acid).
• FIGURES Fig. 1 Diagram of the release rate of PTX and its derivatives from linear polymeric conjugates in a buffer of pH 5 (an intracellular environment model).
• Fig. 2 Diagram of the release rate of PTX and its derivatives from linear polymeric conjugates in a buffer with pH 7.4 (a bloodstream model).
• the drug doses were 2x20 mg of the DTX equivalent/kg
• PHPMA- AH-NH-N DTX-LEV linear polymeric conjugates with the DTX content of 8.2 % and 16.3 % in the dose of 2x30 mg of the DTX equivalent DTX i.v., on days 9 and 13.
• HPMA was prepared in accordance with the previously described procedure [Ulbrich et al., 2000].
• the product was chromatographically pure.
• the ester of levulic acid and paclitaxel (in the -OH 2 position) was prepared by reaction of levulic acid with paclitaxel by means of the carbodiimide method (dicyclohexylcarbodiimide, DCC) in A ⁇ iV'-dimethylformamide (DMF).
• Levulic acid (19.4 mg, 0.167 mmol) and DCC (37.5 mg, 0.182 mmol) were dissolved each in 0.15 mL of DMF at the laboratory temperature. Both solutions were cooled to -18 0 C and mixed.
• the product was purified of low-molecular admixtures by means of chromatography on a column (60cm x 4cm) filled with silica gel in ethyl acetate.
• the fraction containing the PTX-LEV product was collected and concentrated to 0.4 mL and the product was precipitated with 20 mL diethyl ether.
• the product was aspirated, washed with a small amount of diethyl ether and dried in vacuum until the constant weight.
• the yield was 98 mg of the product (84 %) with the melting point of 136 to 138 0 C.
• TLC ethyl acetate : hexane 1:1
• one spot at Rf 0.15.
• MALDI-TOF MS 970 (M+Na).
• Levulic acid 38 mg, 0.327 mmol
• DCC 100 mg, 0.487 mmol
• Both solutions were cooled to -18 0 C and mixed.
• a solution of docetaxel 200 mg, 0.247 mmol
• iV,N-dimethylaminopyridine 28 mg, 0.229 mmol
• the reaction went on at -18 0 C for 30 minutes and at 4 0 C for 16 h.
• ester of 4-(2-oxopropyl)benzoic acid and paclitaxel (in the -OH 2 position) (OPB-PTX) was prepared by the same above-described method for the preparation of LEV-PTX, namely by reaction of 4-(2-oxopropyl) benzoic acid with paclitaxel using the conjugation reagent DCC in
• the ester of 4-oxo-pentenoic [3-acetylacrylic] acid and paclitaxel (in the -OH 2 position) was prepared by the same above-described method for the preparation of LEV-PTX, namely by reaction of 4-oxo-pentenoic acid with paclitaxel using DCC in DMF.
• the ester of 5-oxo-hexenoic acid and paclitaxel (in the -OH 2 position) was prepared by the same above-described method for the preparation of PTX-LEV, namely by reaction of 5- oxo-hexenoic acid with paclitaxel using the conjugation reagent DCC in DMF.
• the ester of 5-oxo-hexenoic acid and docetaxel (in the -OH 2 position) was prepared by the same above-described method for the preparation of DTX-LEV, namely by reaction of 5-oxo-hexenoic acid with docetaxel using the conjugation reagent DCC in DMF.
• Example 2 Synthesis of a polymeric precursor - a copolymer of HPMA with MA-AH-NHNH 2
• the PoIy(HPMA-Co-MA-AH-NHNH 2 ) copolymer was prepared by solution radical copolymerization of HPMA and MA-AH-NHNH 2 in methanol at 60 0 C in accordance with a previously described procedure [Etrych patent].
• Copolymers with derivatives of PTX, DTX and LTX linked to the PHPMA carrier by a hydrolytically cleavable hydrazone bond were prepared by reaction of polymeric precursors containing hydrazine groups with the corresponding drug derivative in methanol under catalysis with acetic acid.
• the polymeric fraction was isolated, concentrated in a vacuum evaporator and the product was precipitated with 50 niL of ethyl acetate, isolated by filtration on frit S4, washed with 150 mL of ethyl acetate and dried until the constant weight.
• the content of total PTX or its derivative in the polymeric conjugate was determined by the HPLC method (HPLC Shimadzu system) after complete hydrolysis of the polymeric conjugate in an HCl solution (pH 2) at 37 °C for 1 hour and extraction of the PTX derivative with chloroform.
• ⁇ M W > and the molecular weight distribution were determined by means of liquid chromatography (TSKGeI 4000 column (300x10 mm), 20% 0.3 M acetate buffer (CH 3 COONa/CH 3 COOH; pH 6.5; 0.5 g/L Of NaN 3 ) and 80% methanol, flow rate 0.5 mL/min, detection with a differential refractometer, a light dispersion detector (DAWN-DSP-F, Wyatt Technology, USA) and a UV detector (250 nm).
• TSKGeI 4000 column 300x10 mm
• 20% 0.3 M acetate buffer CH 3 COONa/CH 3 COOH; pH 6.5; 0.5 g/L Of NaN 3
• flow rate 0.5 mL/min
• Amounts of PTX, DTX or their derivatives released from the polymeric conjugates were measured after their incubation in a phosphate buffer with pH 5.0 (0.1M phosphate buffer containing 0.05M of NaCl), modelling the intracellular environment, and a phosphate buffer with pH 7.4, modelling the bloodstream environment.
• the amounts of the released drugs or their derivatives in the incubation solutions were determined by means of HPLC (Shimadzu).
• the spacer consisting of levulic acid or 4-(2-oxopropyl)benzoic acid allows very fast release of the drug derivative at pH 5; however, hydrolysis of the ester bond between the drug and the acid occurs only very slowly.
• the spacer consisting of 4-oxo-pentenoic acid stabilizes both the hydrazone and the ester bond, so there is no significant release of the drug or its derivative.
• partial hydrolysis of the hydrazone bond occurs in the case of polymeric conjugates with a spacer consisting of levulic acid or 4-(2-oxopropyl)benzoic acid, with a significantly lower rate as compared to the environment with pH 5.
• Example 5 Demonstration of in vitro biological activity of linear polymeric conjugates of docetaxel and palitaxel during incubation with cells of tumour lines; EL-4 T cell lymphoma and 4Tl mammary gland carcinoma.
• Example 6 Demonstration of in vivo biological activity of linear polymeric conjugates of docetaxel and palitaxel in mice inoculated with EL4 T cell lymphoma
• mice of the C57BL/6 strain females
• mice of the C57BL/6 strain were subcutaneously implanted with 1x10 5 EL-4 tumour cells on day 0.
• the drugs were administered intravenously (i.v.), in two doses administered on day 8 and day 12 after the transplantation of the tumour cells.
• the first dose was administered at the time when the tumours were well developed, palpable, with the size of approx. 300 mm 3 .
• the tumour size, body weight of the mice, overall health condition and survival rate were observed in the experiment.
• the effect of the conjugate was always compared to the effect of the free drug (DTX, PTX) and its derivatives (DTX-LEV, PTX-LEV).
• the average time of survival of untreated control mice with EL-4 lymphoma was 31.3 days (SD (SD 3.66, mean survival time 30.5 days).
• DTX, PTX and the DTX-LEV, PTX-LEV derivatives were dissolved for the i.v. application in a mixture of Cremophor EL (Sigma, USA) and ethanol (1 :1); after dissolution of the drug the volume was complemented with 4 volume parts of PBS (phosphate-buffered physiological solution).
• A. Anti-cancer activity of the PHPMA-AH-NH-N DTX-LEV conjugate (Fig. 3) Free DTX induced complete regression of EL-4 tumours in 4 mice out of 7 tested mice. The LEV- DTX derivative had lower effect and induced complete regression of the EL-4 tumour in 1 out of 8 tested mice.
• mice that experienced complete regression of the tumour remained without any symptoms of tumour growth or toxicity until day 94, when they were transplanted EL-4 cells again in the same (i.e. lethal) dose and the mice were left without treatment. This second transplantation was carried out to prove immunologically mediated resistance against the tumour.
• the PHPMA-AH-NH-N DTX-LEV conjugate had a significant anti-cancer effect in C57BL/6 mice with the EL-4 lymphoma, its administration was not accompanied by toxic side effects and enabled establishing of resistance against the tumour in 71% of the cured individuals.
• B. Anti-cancer activity of the PHPMA-AH-NH-N PTX-LEV conjugate (Fig. 4)
• the PTX-LEV derivative (2x30 mg of PTX eq./kg) or the conjugate containing the PTX derivative (2x60 mg of PTX eq./kg) did not have any therapeutic effect in the treatment of syngenic EL-4 lymphoma in mice.
• Example 7 Demonstration of in vivo biological activity of a linear polymeric conjugate of paclitaxel in mice inoculated with the 4Tl mammary gland carcinoma (Fig. 5)
• a model of murine syngenic 4Tl mammary gland carcinoma was used.
• Mice of the BALB/c strain females
• the drugs were administered intravenously (i.v.) in two doses administered on day 8 and day 12 after the transplantation of the tumour cells.
• the first dose was administered at the time when the tumours were well developed, palpable, with the size of approx. 300 mm 3 .
• the tumour size, body weight of the mice, the overall health condition and survival rate were observed in the experiment.
• the effect of the conjugate was always compared to the effect of the free drug (PTX) and its derivative (PTX-LEV).
• the PHPMA-AH-NH-N PTX-LEV conjugate completely cured 3 out of 8 tested mice.
• PTX-LEV derivative After administration of the PTX-LEV derivative, regression of the 4Tl tumour occurred in 1 out of 8 mice.
• Administration of the free drug (PTX) was accompanied by significant side effects: the i.v. administration of the second dose induced a severe reaction (spasms, later poor overall condition - bristled hair, slackness, which took at least 24 hours) in the first of the mice.
• the second dose was reduced by 10% and administered intraperitoneally instead of i.v. Only one mouse was cured that received the dose by i.v. injection, in the other mice no therapeutic effect of PTX was manifested. In no test group any weight loss was recorded as a drug toxicity indicator.
• mice were transplanted again 129 days after the first transplantation of tumour cells.
• IxIO 5 4Tl cells were injected s.c. and the mice were left without treatment.
• the tumours did not grow, which means that these mice were resistant to the given tumour.
• Example 8 Demonstration of in vivo biological activity of linear polymeric conjugates of docetaxel with a variable content of the drug in mice inoculated with the EL4 T cell lymphoma (Fig. 6)
• the tumour model of the EL-4 lymphoma was used.
• the conjugate with the lower content of the drug (8.2 % of DTX) cured 1 out of 8 tested mice and in the other ones it extended the survival time in a statistically significant way (untreated controls: average survival time 27.25 days, SD 1.64, median 28 days; treated mice: average 41.75 days, SD 6.54, median 45 days; p ⁇ .01).
• the conjugate with the higher drug content (16.3 % DTX) also cured 1 out of 8 tested mice but the survival of the other mice in the group was not significantly extended.

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