WO2012098222A1 - Conjugués de biphosphonate-polyaspartamide en tant que vecteurs polymères pour le ciblage d'os par un médicament - Google Patents

Conjugués de biphosphonate-polyaspartamide en tant que vecteurs polymères pour le ciblage d'os par un médicament Download PDF

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WO2012098222A1
WO2012098222A1 PCT/EP2012/050854 EP2012050854W WO2012098222A1 WO 2012098222 A1 WO2012098222 A1 WO 2012098222A1 EP 2012050854 W EP2012050854 W EP 2012050854W WO 2012098222 A1 WO2012098222 A1 WO 2012098222A1
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phea
polymer
formula
integer
abp
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Massimo Fresta
Gaetano Giammona
Gennara Cavallaro
Mariano Licciardi
Donatella Paolino
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Universita' Degli Studi Magna Graecia Di Catanzaro
Universita' Degli Studi Di Palermo
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • 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
    • A61K47/54—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 compound
    • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
    • 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
    • A61K47/59—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 otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L77/00—Compositions of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Compositions of derivatives of such polymers
    • C08L77/04—Polyamides derived from alpha-amino carboxylic acids

Definitions

  • the present invention concerns biphosphonate-polymer conjugates, where the polymer has a poly-hydroxy-aspartamide backbone structure; the invention further relates to the use thereof as prodrugs or pharmaceutical carriers for the targeting to the bones of active agents, chemically linked or physically entrapped into biphosphonate-polymer conjugate; the invention relates alsoto a method for preparing of the above mentioned biphosphonate-polymer conjugates.
  • Biphosphonate (BP) are essential drugs for the treatment of both benign or malignant bone cancers.
  • biphosphonates accumulate to bones and there act by specifically inhibiting osteoclasts growth, with mechanism still not well known.
  • BP are analogous, metabolically stable, of pyrophosphate (P-O-P), an endogenous regulator of bone mineralization, able to adsorb onto hydroxylapatite crystals, causing bone stabilization and inhibition of its dissolution.
  • P-O-P pyrophosphate
  • P-C-P carbon atom
  • BP are more resistant against acid hydrolysis respect pyrophosphates.
  • the highly hydrophilic biphosphonate group is the moiety responsible of the high affinity for the bone hydroxylapatite and thanks to its higher hydrolysis stability, can accumulate into cells.
  • BPs effects on bone cells, specially on osteoclasts, can be distinguished four different actions: 1 ) inhibition of osteoclasts recruitment; 2) inhibition of osteoclasts adhesion on bone matrix; 3) reduction of osteoclasts life time and 4) direct inhibition of osteoclasts activity [1 ].
  • BPs represent now the principal class of drugs used for the treatment of osteolysis and pain associated to bone metastases.
  • Recently, the ability of BPs of linking with high affinity to hydroxylapatite has been used; this affinity makes them able to spontaneously accumulate into bone tissue and thus to act as targeting agents for the bones.
  • BPs are currently clinically used as targeting agents only for radiolabeled drugs.
  • BP-radiolabeled-drug conjugates are highly clinically used for image diagnosis and pain treatment of bone pathologies [2].
  • Different BP-radiolabeled-drug complexes such as for example 99mTc-idroxyethylidene biphosphonate, 99mTc-methylene biphosphonate and 99mTc-idroxymethylene biphosphonate [2] are currently used in the bone scanning for identification and evaluation of bone metastases, bone tumors, bone metabolic diseases, Paget disease, bone infections, traumatisms, arthritis, exc.
  • Another BP-radiolabeled-drug complex, samarium (153Sm) lexidronam has been approved by FDA for the treatment of bone metastases pain [3].
  • biphosphonate alendronate was successfully conjugated to PEG and Hydroxypropylmethacrylamide (HPMA), one for each polymeric chain [4].
  • HPMA Hydroxypropylmethacrylamide
  • principal objective of the present invention is that to provide alternative molecules in which biphosphonate group is conjugated to biocompatible multifunctional polymers, and not to scanning agents; this with the aim to:
  • ABP aminobiphosphonate
  • NRD neridronate
  • PHEA -poly(N-2-hydroxyethyl)-DL-aspartamide
  • PNFC paranitrophenylcarbonate
  • EDC ethyldimethylaminopropylcarbodiimide
  • the present invention provide a solution to the above mentioned problems by means of a polymer with polyaspartamidic structure having formula (I)
  • G 2 is absent or is chosen from the group consisting of -(CH 2 ) q -CO-, -NH-(CH 2 ) r - CO-, amino acid and peptide having a number of amino acids between 2 and 4, where q and r independently from each other are an integer between 1 and 6.
  • G3 is -(CH 2 )s- where s is an integer between 0 and 6;
  • G 4 is H or -OH
  • G5 is -OH or a drug or pro-drug
  • x and y are the numbers of alpha and beta repeating units of the polymer, respectively, and are between 63 and 380.
  • the present invention provides polymeric carriers, to which BPs are covalently linked, able to act as drug vectors targeted to bones, using polymer with structure , -poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) or its polyaspartamidic derivatives.
  • BPs anti-plastic drugs used in the treatment of bone metastases
  • other drugs among which antineoplastic agents, anti-inflammatory steroids or non steroids, anti-angiogenic factors, growth factors, hormones, biphosphonates, vitamines or diagnostic agents), chemically linked or physically entrapped, selectively to bones.
  • Another advantage of the present invention is that to use, in the production of drug delivery systems for bones, polyaspartamide copolymers having: high biocompatibility, easy production method with high yields, reproducibility and low costs; versatility in terms of drug content and bond type between drug and polymer or BP and polymer.
  • compositions containing a polymer of formula (I) as above described are also subject-matter of the invention.
  • x and y are the numbers of alpha and beta repeating units of the polymer, respectively, and are between 63 and 380.
  • Figure 1 Binding essays with hydroxylapatite: the graph shows the weight amount (mg) of the copolymer PHEA not modified or modified according with the invention, linked to hydroxylapatite.
  • FIG. 1 Binding assays with rabbit bone: the graph shows the weight amount (mg) of the copolymer PHEA not modified or modified according with the invention, linked to bone.
  • Figures 3-6 Radioactivity values (reported as ⁇ of 1 ,2- 3 H-ethanolamine hydrochloride) produced by PHEA, PHEA-NRD and PHEA-ABP in samples of: blood, heard, lungs, liver, spleen, kidneys, sternum and femur, after 1 h ( Figure 3), 4h ( Figure 4), 8h ( Figure 5) and 24h ( Figure 6). All values were normalized respect weight average of examined organs and total biodistribution volume.
  • aminoacids mean natural aminoacids, not-natural or modified usually known in the state of the art (see for example Tables 3 and 4 of OJ EPO 2007 Sp.Ed. n.3 pag. 66-67).
  • G2 groups in formula (I) are linked to the polymer by the left side of how are they reported, thus for example, the group -(CH 2 ) q -CO- is linked to the polymer by the side of methylene group, while carboxylic group is linked to amine group of the molecule bearing BP groups.
  • G2 is an aminoacid it means that aminic group is linked to the polymer while carboxylic group is linked to the molecule bearing BP groups.
  • G1 is -CH2-CH2- that is the starting polymer , -poly(N-2-hydroxyethyl)-DL- aspartamide (PHEA).
  • preferred polymers of formula (I) are those where G2 is absent, that is where molecules bearing BP groups are directly conjugated to the polymer by uretanic linkage.
  • G2 When G2 is present it acts as a linker (or spacer) between polymer and molecules bearing BP groups.
  • G5 when G5 is a drug or prodrug, it is chosen among antineoplastic agents, antiinflammatory steroidic or not, antiangiogenetic factors, growth factors, hormones, BPs, vitamins or diagnostic agents.
  • G5, like molecules bearing BP groups, can be directly linked to the polymer (when G2 is absent) or can be linked by mean of a linker (when G2 is present).
  • G2 is preferably chosen among -CH 2 -CH 2 -CO-, -NH-(CH 2 ) 4 - CO-.
  • G4 is H or OH
  • G5 is -OH or a drug or prodrug chosen among antineoplastic agents, antiinflammatory steroidic or not, antiangiogenetic factors, growth factors, hormones, BPs, vitamins or diagnostic agents.
  • Polymer functionalized according with invention can be obtained starting from a polymer of formula (II):
  • x and y are the numbers of alpha and beta repeating units of the polymer, respectively, and are between 63 and 380;
  • G3 is -(CH 2 )s- where s is an integer between 0 and 6;
  • G 4 is H or -OH
  • G 3 is -(CH 2 ) S - where s is an integer between 0 and 6;
  • G 4 is H or -OH
  • G2 is chosen from -NH-(CH 2 ) r -CO-, or amino acid or peptide having a number of amino acids between 2 and 4 where r is an integer between 1 and 6; and wherein G 5 is -OH;
  • G3 is -(CH 2 )s- where s is an integer between 0 and 6;
  • G 4 is H or -OH
  • reaction (b) when G2 is absent, reaction (b) is followed by another activation reaction with a carbonylating agent and subsequent reaction with a drug or prodrug bearing -
  • reactions (b") or (c') are followed by reactions with a drug or prodrug bearing -NH 2 or -OH groups available to react with a carboxyl group in presence of an appropriate activating agent of carboxyl group, forming amidic or ester linkages.
  • Reactions (a) or (a') are preferably carried out in aprotic polar solvent, for example DMF.
  • Carbonylating agent is preferably a phenyl-bis-carbonate, such as for example PNFC.
  • Reactions (b), (b') are preferably carried out by adding an aqueous solution of a compound of formula (III) or an aminoacid, a peptide of 2-4 aminoacids or a molecule of formula H 2 N-(CH 2 )rCOOH where r is an integer between 1 and 6, to a polymer solution activated by a carbonylating agent.
  • Reactions (c') or (b") are carried out preferably in presence of appropriate carboxylic group activating agents (for example EDC or HOBT).
  • appropriate carboxylic group activating agents for example EDC or HOBT.
  • Molecules of formula (III) as above described are molecules bearing BP groups, known in the literature, and as example can be cited pamidronate, neridronate, alendronate.
  • activation degree of carboxyl groups on polymer backbone is depending on the molar ratio between starting polymer repeating units (R.U.) and moles of activating agent, and reaction time.
  • R.U. starting polymer repeating units
  • polymer functionalization degree can be varied by modulating concentration of solution containing the molecule of formula (III) or aminoacid or peptide.
  • molecules bearing BP groups such as aminobiphosphonate (ABP) and neridronate (NRD) have been conjugated to polyaspartamide polymeric carriers, such as for example , -poly(N-2- hydroxyethyl)-DL-aspartamide (PHEA), directly (obtaining PHEA-ABP and PHEA- NRD) or through a spacer (such as dicarboxylic acid or aminoacid), for example succinic acid or 6-aminocaproic acid, obtaining conjugates (PHEA-succinate-ABP and PHEA-caproylcarbammate-ABP).
  • polyaspartamide polymeric carriers such as for example , -poly(N-2- hydroxyethyl)-DL-aspartamide (PHEA), directly (obtaining PHEA-ABP and PHEA- NRD) or through a spacer (such as dicarboxylic acid or aminoacid), for example succinic acid or 6-aminocaproic acid, obtaining
  • ABP has been conjugated directly to PHEA through urethanic linkage
  • the type of linkage depends by the spacer used itself.
  • the linkage polymer-spacer is an ester bond and the linkage spacer- drug is an amidic bond
  • the linkage polymer- spacer is an urethanic bond while the linkage spacer-drug is an amidic bond.
  • conjugate -poly(N-2-hydroxyethyl)-co-[N-2-(aminobiphosphonate- N-carbamate)ethylene]-DL-aspartamide (PHEA-ABP) was synthesized starting from the hydrosoluble polymer , -poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) having average molecular weight (Mw) between 10 and 70 kDa (preferably 50 kDa). Hydroxyl groups present in the PHEA side chain were activated by reacting with p-nitrophenil-bis-carbonate (PNFC), in anhydrous DMF solution at 40 °C.
  • PNFC p-nitrophenil-bis-carbonate
  • Molar ratio between PHEA repeating units (RU) and moles of activating agent, and reaction time determine activation degree of hydroxyl groups of polymer. For example, by using a RU/PNFC moles ratio of 0.5 and an activation time of 4h it was obtained an activation degree of hydroxyl groups of PHEA equal to 40%. After activation reaction, activated PHEA solution was added dropwise to an ABP bidistilled water solution with appropriate ABP concentration and mixture maintained at 25 °C for 24h. Reaction product was purified by exhaustive dialysis and lyophilized. PHEA-ABP was obtained with a yield of 95% respect starting PHEA.
  • Conjugation degree of ABP to PHEA was determined by phosphates essay (Ames assay) and can be between 1 and 20 mole% respect PHEA RU. For example by using a 0.23 imM ABP concentration solution, a 4.4 mol% ABP derivatization degree in PHEA-ABP conjugate was obtained.
  • Average molecular weight (Mw) of PHEA-ABP conjugate was determined by aqueous (phosphate buffer pH 9) SEC, and can be between 25000 and 50000 Da (preferably 30000 Da), calculated by comparison with a calibration curve obtained by using PEG molecular weight stardards ranging from 1 000 to 145000 Da.
  • PHEA-CS -poly(N-2-hydroxyethyl)-co-[N-2-(2'-carboxysuccinate)ethylene]-DL- aspartamide
  • Average molecular weight (Mw) of PHEA-CS conjugate was determined by aqueous (CaCI 2 0.05M solution) SEC, and can be between 20000 and 55000 Da (preferably 45000 Da), calculated by comparison with a calibration curve obtained by using PEG molecular weight stardards ranging from 1000 to 145000 Da.
  • ABP conjugation reaction to PHEA-CS was carried out in bidistilled water in presence of carboxyl groups activating agents hydroxybenzotriazole (HOBT) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC HCI) at pH 6.8, 25 °C for 18h.
  • carboxyl groups activating agents hydroxybenzotriazole (HOBT) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC HCI) at pH 6.8, 25 °C for 18h.
  • ABP conjugation degree in PHEA-CS-ABP was determined by phosphate assay (Ames assay). Average molecular weight (Mw) of PHEA-CS- ABP conjugate was determined by aqueous (phosphate buffer pH 9) SEC, and can be between 45000 and 65000 Da (preferably 55000 Da), calculated by comparison with a calibration curve obtained by using PEG molecular weight stardards ranging from 1000 to 145000 Da.
  • Copolymer -poly(N-2-hydroxyethyl)-co-[N-2-(N'-6-carboxycaproyl- carbamate)ethylene]-DL-aspartamide (PHEA-CAPROYLCARBAMATE) was synthesized starting from the hydrosoluble polymer , -poly(N-2-hydroxyethyl)-DL- aspartamide (PHEA) having average molecular weight (Mw) between 10 and 70 kDa (preferably 50 kDa). Hydroxyl groups present in the PHEA side chain were activated by reacting with p-nitrophenil-bis-carbonate (PNFC), in anhydrous DMF solution at 40 °C.
  • PNFC p-nitrophenil-bis-carbonate
  • Molar ratio between PHEA RU and moles of activating agent, and reaction time determine activation degree of hydroxyl groups of polymer. For example, by using a RU/PNFC moles ratio of 0.5 and an activation time of 1 h it was obtained an activation degree of hydroxyl groups of PHEA equal to 15%.
  • activated PHEA solution was added dropwise to an ABP bidistilled water solution with appropriate 6-aminocaproic acid concentration and mixture maintained at 25 °C for 24h. Reaction product was precipitated in acetone and purified by exhaustive dialysis and lyophilized.
  • PHEA- CAPROYLCARBAMATE was obtained with a yield of 75% respect starting PHEA and characterized by FT-IR and 1 H-NMR analysis.
  • Conjugation degree of 6-aminocaproic acid in PHEA-CAPROYLCARBAMATE was determined by 1 H-NMR and can be between 5 and 30 mol% respect PHEA RU. For example by using a 0.56 imM 6-aminocaproic acid concentration solution, a 5 mol% caprilate derivatization degree in PHEA-CAPROYLCARBAMATE conjugate was obtained.
  • Average molecular weight (Mw) of PHEA-CAPROYLCARBAMATE conjugate was determined by aqueous (phosphate buffer pH 9) SEC, and can be between 20000 and 60000 Da (preferably 50000 Da), calculated by comparison with a calibration curve obtained by using PEG molecular weight stardards ranging from 1 000 to 145000 Da.
  • ABP conjugation reaction to PHEA-CAPROYLCARBAMATE was carried out in bidistilled water in presence of carboxyl groups activating agent N- (3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC HCI) at pH 6.8, 25°C for 1 8h.
  • EDC HCI carboxyl groups activating agent N- (3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
  • PHEA-CAPROYLCARBAMATE- ABP was obtained with a yield of 90% respect starting PHEA- CAPROYLCARBAMATE and characterized by FT-IR and 1 H-NMR analysis.
  • ABP conjugation degree in PHEA-CAPROYLCARBAMATE-ABP was determined by phosphate assay (Ames assay).
  • Average molecular weight (Mw) of PHEA- CAPROYLCARBAMATE-ABP conjugate was determined by aqueous (phosphate buffer pH 9) SEC, and can be between 20000 and 65000 Da (preferably 54000 Da), calculated by comparison with a calibration curve obtained by using PEG molecular weight stardards ranging from 1000 to 145000 Da.
  • Scheme 4 Structure of , -poly(N-2-hydroxyethyl)-co-[N-2-(N'-6-carboxycaproyl- carbamate)ethylene]-co-[N-2-(N'-6-biphosphonatecarboxyamidecaproyl- carbamate)ethylen]-DL-aspartamide (PHEA-CAPROYLCARBAMATE-ABP) conjugate.
  • Copolymer -poly(N-2-hydroxyethyl)-co-[N-2-(6-amino-caproyl-biphosphonate- N-carbamate)ethylene]-DL-aspartamide (PHEA-NRD) was synthesized starting from the hydrosoluble polymer PHEA having Mw between 10 and 70 kDa (preferably 50 kDa). Hydroxyl groups present in the PHEA side chain were activated by reacting with PNFC, in anhydrous DMF solution at 40 °C. Molar ratio between PHEA RU and moles of activating agent, and reaction time determine activation degree of hydroxyl groups of polymer.
  • an activation degree of hydroxyl groups of PHEA equal to 1 0%.
  • activated PHEA solution was added dropwise to a NRD bidistilled water solution with appropriate NRD concentration and mixture maintained at 25 °C for 24h. Reaction product was purified by exhaustive dialysis and lyophilized. PHEA- NRD was obtained with a yield of 98% respect starting PHEA.
  • Conjugation degree of NRD in PHEA-NRD conjugate was determined by 1 H-NMR and can be between 1 and 20 mol% respect PHEA RU. For example by using a 0.056 imM NRD concentration solution, a 5 mol% NRD derivatization degree in PHEA-NRD conjugate was obtained.
  • Mw of PHEA-NRD conjugate was determined by aqueous (phosphate buffer pH 9) SEC, and can be between 30000 and 60000 Da (preferably 45000 Da), calculated by comparison with a calibration curve obtained by using PEG molecular weight stardards ranging from 1 000 to 145000 Da.
  • Hydroxylapatite binding assay is a indirect method to determine the ability of biphosphonate bearing molecules to interact with inorganic component of bones that is calcium phosphate or hydroxylapatite.
  • Fifty mg of conjugates PHEA-ABP, PHEA-CS-ABP, P H E A-CA P RO YLCA R B AM AT E -A B P and PHEA-NRD having an amount of linked biphosphonate between 3 and 7 weight % (and starting PHEA polymer used as control) were dissolved in 2 ml of phosphate buffer at pH 7.4 and than 50 mg of pure hydroxylapatite were added to these solution. Obtained suspensions were gently stirred at 37°C for 4h.
  • Binding assay on samples of lyophilised bones is a ex vivo method to determine the ability of biphosphonate bearing molecules to interact with bone tissue explanted from healthy experiment animals. This experiment was carried out on each of derivatives object of this invention.
  • 50 mg of conjugates PHEA-ABP, PHEA-CS-ABP, P H E A-CA P RO YLCA R BA MATE -A B P and PHEA- NRD having an amount of linked biphosphonate between 3 and 7 weight % (and starting PHEA polymer used as control) were dissolved in 2 ml of phosphate buffer at pH 7.4 and than each solution was taken into contact with 100 mg of rabbit bone (femur) lacking of organic tissues, lyophilised and crushed.
  • copolymers object of this invention and PHEA, as control, were administered intravenously in isotonic solution through the tail vein.
  • In vivo experiments have been conducted on male rats in good health conditions, weighing 300 g, wistar type. The animals were left free to feed up until the time of the experiment, maintained and sacrificed according with the national and European regulations.
  • PHEA-NRD reaches maximum concentration into bones (summing either amount accumulated into sternum and femur) after 8h (figure 5); PHEA-ABP reaches maximum concentration into bones (summing either amount accumulated into sternum and femur) only after 24h (figure 6); Obtained data demonstrate that the presence of molecules bearing biphosphonate groups conjugated to PHEA (in particular biphosphonate neridronate, used to obtain the polymeric derivative PHEA-NRD) confers to the correspondent copolymers ability to target and localize for long times into bone tissue, thus constituting an efficient tool to specifically target drugs to bones.
  • biphosphonate neridronate used to obtain the polymeric derivative PHEA-NRD

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Abstract

Cette invention concerne des conjugués de biphosphonate-polymère dont le polymère possède un squelette de poly-hydroxy-aspartamide, pouvant s'accumuler dans les tissus osseux et être utilisés en tant que tels comme vecteurs pour le ciblage d'os par des agents actifs sont liés chimiquement ou emprisonnés dans un conjugué de biphosphonate-polymère, ou en tant que promédicaments macromoléculaires pouvant s'accumuler dans des tissus osseux et y libérer les biphosphonates.
PCT/EP2012/050854 2011-01-21 2012-01-20 Conjugués de biphosphonate-polyaspartamide en tant que vecteurs polymères pour le ciblage d'os par un médicament WO2012098222A1 (fr)

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ITFI2011A000012 2011-01-21
ITFI2011A000012A IT1403567B1 (it) 2011-01-21 2011-01-21 Vettori polimerici della poliaspartammide coniugati a bisfosfonati per il direzionamento di farmaci alle ossa.

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2020163637A1 (fr) * 2019-02-06 2020-08-13 Oregon Health & Science University Composés liés à des bisphosphonates

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Publication number Priority date Publication date Assignee Title
WO2020163637A1 (fr) * 2019-02-06 2020-08-13 Oregon Health & Science University Composés liés à des bisphosphonates

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