WO2008048079A1 - Formulation biopharmaceutique à action de longue durée - Google Patents

Formulation biopharmaceutique à action de longue durée Download PDF

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WO2008048079A1
WO2008048079A1 PCT/KR2007/005179 KR2007005179W WO2008048079A1 WO 2008048079 A1 WO2008048079 A1 WO 2008048079A1 KR 2007005179 W KR2007005179 W KR 2007005179W WO 2008048079 A1 WO2008048079 A1 WO 2008048079A1
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aptamer
chemical formula
hyaluronic acid
reacting
group
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PCT/KR2007/005179
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English (en)
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Sei-Kwang Hahn
Hyun-Gu Kang
Sung-Ho Ryu
Jung-Kyu Park
Eun-Ju Oh
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Postech Academy-Industry Foundation
Posco
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Publication of WO2008048079A1 publication Critical patent/WO2008048079A1/fr
Priority to US12/422,950 priority Critical patent/US8247493B2/en

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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
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds

Definitions

  • the invention relates to a long-acting formulation of biopharmaceutical, more specifically an aptamer therapeutics.
  • a branch-type PEG or a hyaluronic acid (HA) derivative of which degradation in vivo is regulated is linked by the bioconjugation with biopharmaceutical.
  • the new formulation of aptamer therapeutics improves the function of aptamer therapeutics and patient compliance, thereby improving quality of medical service.
  • Aptamers are nucleotide molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Aptamers, like peptides generated by phage display or monoclonal antibodies
  • mAbs are capable of specifically binding to selected targets and modulating the target's activity, e.g., through binding aptamers may block their target's ability to function.
  • SELEX systemic evolution of ligands by exponential enrichment
  • aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors.
  • a typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind to other proteins from the same gene family).
  • aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarities, hydrophobic contacts, steric exclusion) that drive affinity and specificity in antibody- antigen complexes.
  • the first FDA-approved aptamer therapeutics of Macugen (pegaptanib) by Pfizer and Eyetech Co. in 2004 was a PEGylated vascular endothelial growth factor inhibitor for the treatment of age-related macular degeneration. It is reported that the risk of sightless person administered by Macugen ® decreases to a half after the administration.
  • the branched PEGylated aptamer therapeutics of MACUGEN ® is represented by following chemical structure.
  • n is approximately 450.
  • the good aptamer therapeutics When the good aptamer therapeutics are administered parenterally in vivo, they are removed by the degradation with nuclease and by the rapid renal clearance in a few minutes. To overcome the short half-life, a chemical conjugation of the aptamer to poly-L-Lysine, polyamide, long chained alcohol, cholesterol and other steroids, phospholipid and peptide, etc. has been reported. In addition, PEG conjugation of the aptamer increases 10 times of half-life in vivo, and thus the aptamer can be used for therapeutically-active therapeutics. PEG includes a moiety of HO-(-CH 2 CH 2 O)n-H, and has been widely used for a long-acting formulation of biopharmaceutical .
  • PEGylation reagent where polyethylene glycol is linked to two amine residues of lysine via a peptide bond is commercially sold by Nektar. Urethane bond instead of the peptide bond in such product largely increases the stability of aptamer therapeutics (US 2003/0114647).
  • Commercial PEGylated interferon of PEGASYS (trademark) produced by ROCHE is a PEGylated product with the branch-typed PEGlyation reagent sold by Nektar to largely increase bioavailability.
  • the present invention provides a new long-acting formulation of biopharmaceuticals such as aptamer specifically binding to a target molecule, protein and peptide.
  • An object of the present invention is to provide a PEGylation reagent for producing a branch-type PEGylated compounds by introducing bis-amine group to glycine or ethanolamine with cyanoethylation, and linking PEG to each amine group.
  • the present invention relates a compound represented by chemical formula 1, and more preferable PEGylated aptamer conjugate, and a method therefore.
  • Chemical formula 1
  • mPEGa and mPEGb are independently monomethoxy polyethylene glycol having molecular weight of 100 to 100,000 Da; nl is an integer of 1 to 10; n2 is an integer of 0 to 10; and Z is -OH, -COOH, -O-
  • the polymer further contains protein, enzyme, nucleotide, lipid, liposome, solid surface, or particle including a functional group being capable of reacting with Z, and more preferably an oligonucleotide aptamer in length of 30 to 45 nucleotides.
  • Another object of the present invention is to provide a method of preparing a
  • PEGylation reagent comprising the steps of: performing an amine group of glycine with cyanoethylation to produce two cyano moieties; reducing the cyano moieties to amine groups; forming urethane bond by reacting each amine group with monomethoxy polyethylene glycol (mPEG) succinimidyl carbonate; and obtaining PEGylation reagent by reacting with N-hydroxysuccinimide (NHS).
  • mPEG monomethoxy polyethylene glycol
  • NHS N-hydroxysuccinimide
  • Further object of the present invention is to provide a method of preparing a PEGylation reagent comprising the steps of: performing an amine group of ethanolamine with cyanoethylation to produce two cyano moieties; reducing the cyano moieties to amine groups; forming urethane bond by reacting each amine group with monomethoxy polyethylene glycol (mPEG) succinimidyl carbonate; substituting terminal alcohol group with acid chloride group by reacting with triphosphazine; and obtaining the PEGylation reagent by reacting with N-hydroxysuccinimide (NHS).
  • mPEG monomethoxy polyethylene glycol
  • NHS N-hydroxysuccinimide
  • Still further object of the present invention is to provide by introducing two or more functional groups to phosphoramidite compound, synthesizing oligonucleotide aptamer by using the same, and linking PEG to the terminus of the synthesized oligonucleotide to obtain long-acting formulation of aptamers.
  • the fourth object of the present invention is to provide a new long-acting formulation by using a hyaluronic acid (HA) derivative, of which degradation in vivo is suppressed or regulated, linked by the bioconjugation with biopharmaceutical aptamer.
  • the present invention provides a hyaluronic acid-aptamer conjugate by linking an aptamer with HA derivative of which a terminal carboxyl group is reacted with adipic acid dihydrazide (ADH), tris(2-aminoethyl)amine (TREN), 2-aminoethyl methacrylate, or N-(3-Aminopropyl) methacrylamide hydrochloride (APMAm).
  • ADH adipic acid dihydrazide
  • TREN tris(2-aminoethyl)amine
  • APMAm N-(3-Aminopropyl) methacrylamide hydrochloride
  • the fifth object of the present invention is to provide a long-acting formulation by using HA-ADH derivative which is prepared by reacting ADH to a carboxyl group of hyaluronic acid in a mixed solvent of water and an organic solvent.
  • the degradation in vivo of HA-ADH is regulated or suppressed.
  • the hyaluronic acid is modified with 2- aminoethyl methacrylate (AEMA) or-(3-Aminopropyl) methacrylamide hydrochloride (APMAm) in an organic solvent to produce HA-AEMA or HA-APMAm.
  • AEMA 2- aminoethyl methacrylate
  • APMAm 3-(3-Aminopropyl) methacrylamide hydrochloride
  • HA-AEMA or HA-APMAm is linked to an aptamer containing thiol group with Michael addition to obtain a long-acting aptamer therapeutic.
  • FIG. 2 is RP-HPLC result of PEGylation reagent(A), unreacted aptamer(B), and aptamer PEGylated with phosphoramidite derivative represented by chemical formula 5 in accordance with Example 2.
  • FIG. 3 is a schematic drawing showing synthesis of PEGylated anti-VEGF T-
  • FIGs 4A to 4C are SPR result showing a binding force of anti-VEGF aptamer to VEGF coated on BIAcore chip: FIG. 4A for anti-VEGF 2'-0Me-RNA aptamer, FIG. 4B for PEGylated anti-VEGF 2'-0Me-RNA aptamer, and FIG. 4C for anti-VEGF DNA aptamer.
  • FIG. 5 is comparison of in vitro whole blood clotting times (WBCT) of (A) PBS as a reference, (B) anti-thrombin DNA aptamer and (C) PEGylated anti-thrombin DNA aptamer.
  • WBCT in vitro whole blood clotting times
  • FIG. 6 is 1 H NMR spectrum of HA-ADH which is prepared by linking adipic acid dihydrazide (ADH) to carboxyl group of HA.
  • FIG. 7 is a graph showing a modification degree of ADH depending on ethanol content of reacting solvent of HA modification.
  • FIG. 8 is 1 H NMR spectrum of HA-AEMA.
  • FIG. 9 is a chromatography (GPC) analysis result of HA-AEMA-aptamer conjugate which is prepared by linking HA-AEMA to aptamer containing a thiol group: A is HA-AEMA-aptamer conjugate and B is unreacted aptamer.
  • GPC chromatography
  • FIG. 10 is 1 H NMR spectrum of HA-APMAm which is prepared by linking APMAm to carboxyl group of HA.
  • the present invention relates to a PEGylation reagent for aptamer, a method of preparing PEGylated aptamer using phosphoramidite derivatives, HA derivatives with regulated degradation in vivo such a HA- ADH, HA-TREN, HT-AEMA and HA-APMAm, a conjugate of biopharmaceutical and the HA derivatives.
  • a PEGylation reagent for aptamer a method of preparing PEGylated aptamer using phosphoramidite derivatives, HA derivatives with regulated degradation in vivo such a HA- ADH, HA-TREN, HT-AEMA and HA-APMAm, a conjugate of biopharmaceutical and the HA derivatives.
  • Two cyano moieties are produced by performing cyanoethylation at a bis-amine group of ethanolamine or glycine, reduced into amine groups, and reacted with polyethylene glycol succinimidyl carbonate to produce strong urethane bond.
  • the carboxyl group is activated with NHS, and conjugated with aptamer having amine group.
  • the present invention relates to a method of preparing a PEGylation reagent comprising the steps of: performing an amine group of ethanolamine with cyanoethylation to produce two cyano moieties; reducing the cyano moieties to amine groups; forming urethane bond by reacting each amine group with monomethoxy polyethylene glycol (mPEG) succinimidyl carbonate; substituting terminal alcohol group with acid chloride group by reacting with triphosphazine; and obtaining the PEGylation reagent by reacting with N-hydroxysuccinimide (NHS).
  • the representative branch-type PEGylation reagent is a compound of chemical formula 1:
  • mPEGa and mPEGb are independently monomethoxy polyethylene glycol having molecular weight of 100 to 100,000 Da; nl is an integer of 1 to 10; n2 is an integer of 0 to 10; and
  • Z is -OH, -COOH, -0-C(O)-O-Cl, or
  • the preferred compound represented by chemical formula 1 is compounds of chemical formula 2a, chemical formula 2b, chemical formula 3a, chemical formula 3b, and chemical formula 3c.
  • the branch-type PEGylation reagent is prepared by introducing bis-amine group to glycine or ethanolamine with cyanoethylation, and reacting with polyethylene glycol.
  • mPEGa and mPEGb is the same or different monomethoxy polyethylene glycol, and independently have a molecular weight of 100 to 100,000 Da, and more preferably 10,000 Da to 60,000 Da, for examples 10,000 Da, 20,000 Da, 40,000 Da, or 60,000Da.
  • Chemical formula 2a is the same or different monomethoxy polyethylene glycol, and independently have a molecular weight of 100 to 100,000 Da, and more preferably 10,000 Da to 60,000 Da, for examples 10,000 Da, 20,000 Da, 40,000 Da, or 60,000Da.
  • the polymer further comprises protein, enzyme, nucleotide, lipid, liposome, solid surface, or particle including a functional group being capable of reacting with Z, and more preferably aptamer.
  • the aptamer of the invention is any kind of aptamer but not limited to.
  • Exemplary aptamer of oligonucleotide includes modified or non-modified DNA, RNA, or DNA/RNA hybrid, but not limited thereto.
  • a biopharmaceuticals of the present invention, particularly aptamer contained in a therapeutic or pharmaceutical composition is referred to active or therapeutic material dissolved or dispersed in a pharmaceutically acceptable carrier or diluant in an effective amount.
  • the carriers or diluents include any solvent, dispersing medium, coating, antibiotic agent, antifungal agent, isotonic agent, and absorption retarding agent, and the like.
  • a supplemental active agent can be contained in the composition of the present invention.
  • the pharmaceutical composition can be prepared by a skilled person in this art in accordance with the general preparation method of pharmaceutical composition.
  • the composition can be formulated in forms of solution or suspension; injectable solution, solid formulation, or suspension; tablet, or solid formulation suitable for enteral administration; time release capsule; cream, lotion, salve, or inhalant.
  • the formulation can be administered in a pharmaceutically effective matter according to a general method.
  • the formulation can be administered in various routes, for examples injection or capsule.
  • the administration amount of active agent can be varied depending on the subject.
  • the amount of active agent can be determined by a doctor.
  • the oligonucleotide aptamer is synthesized and linked with biopolymer such as PEG at terminal functional group to obtain a long-acting formulation of aptamer therapeutics.
  • branched phosphoramidite is dendrimeric phosphoramidite having terminal hydroxyl group provided by Glen research.
  • the dendrimeric phosphoramidite is linked with PEGylation reagent to form ester bond which is easily degraded in vivo.
  • two functional groups in three functional groups of core molecule are attached by PEG, and one functional group is combined with biopharmaceuticals such as aptamer, peptide, and protein to produce a long- acting formulation.
  • branched oligonucleotide aptamer is synthesized by using two phosphoramidite derivatives. More specifically, aptamer conjugate linked with two PEG is prepared by coupling symmetric doubler phosphoramidite to 5 '-terminus of an oligonucleotide aptamer synthesized using solid phase phosphoramidite chemistry, introducing a terminal amine group of the linked doubler phosphoramidite, and combining the PEG with the terminal amine group.
  • reaction scheme 3 two functional groups are introduced to aptamer by using phosphoramidite having branch-type amine groups, and then two PEG molecules are reacted.
  • the preparation method is performed by coupling symmetric doubler phosphoramidite to 5'-terminus of an oligonucleotide aptamer synthesized using solid phase, reacting with 5'-amino modif ⁇ er-C ⁇ , and linking PEG to terminus of phosphoramidite to produce branched aptamer conjugate having two PEG molecules at its terminus.
  • x is an integer of 170 to 350.
  • branched 40KD PEG is introduced to the oligonucleotide aptamer by reacting with two 20KD mPEG succinimidyl propinonate.
  • symmetric doubler phosphoramidite(l 0-1920-02) provided by Glen research is introduced to the oligonucleotide aptamer, and then, reacted with 5'-amine-modifier-C6-TFA(10-1016- 02) to obtain the aptamer having two amine groups.
  • HA derivative is obtained by reacting carboxyl group of hyaluronic acid with adipic acid dihydrazide (ADH), tris(2-aminoethyl)amine (TREN), 2- aminoethyl methacrylate, or N-(3-Aminopropyl) methacrylamide hydrochloride (APMAm), and then conjugated with aptamer to produce a hyaluronic acid-aptamer conjugate.
  • ADH adipic acid dihydrazide
  • TREN tris(2-aminoethyl)amine
  • APMAm N-(3-Aminopropyl) methacrylamide hydrochloride
  • Hyaluronic acid of the present invention is not particularly limited, for examples hyaluronic acid with molecular weight of 20,000 Da to 4,000,000 Da.
  • x+y is about 50 to 10,000 in following chemical formulae where x and y are integer of equal to or more than 1.
  • Reaction scheme 4 shows the conjugating reaction of HA-ADH and aptamer activated by N-hydroxysuccinimide(NHS) at its terminus.
  • HA-ADH with regulated degradation in vivo is prepared by reacting ADH to a carboxyl group of hyaluronic acid in a mixed solvent of water and an organic solvent.
  • HA-aptamer conjugate is prepared by using HA-ADH.
  • the organic solvent amount of mixed solvent is 1 to 90mol%, and more preferably 25mol% to 85 mol%.
  • the exemplary organic solvent is ethanol.
  • Reaction scheme 5 shows the conjugating reaction of HA-TREN and aptamer activated by N-hydroxysuccinimide(NHS) at its terminus.
  • HA-TREN with regulated degradation in vivo is prepared by reacting TREN to a carboxyl group of hyaluronic acid in a mixed solvent of water and an organic solvent.
  • HA-aptamer conjugate is prepared by using HA-TREN.
  • the organic solvent amount of mixed solvent is 1 to 90mol%, and more preferably 25mol% to 85 mol%.
  • the exemplary organic solvent is ethanol.
  • HA-AEMA is produced by chemically modifying hyaluronic acid with 2-aminoethyl methacrylate (AEMA), and is linked to an aptamer having thiol group at its terminus in the presence of Tri(2-carboxyethyl)phosphine hydrochloride (TCEP) to produce a long-acting formulation of aptamer therapeutics.
  • AEMA 2-aminoethyl methacrylate
  • TCEP Tri(2-carboxyethyl)phosphine hydrochloride
  • HA-APMAm is produced by chemically modifying hyaluronic acid with APMAm, and is linked to an aptamer having thiol group at its terminus in the presence of Tri(2-carboxyethyl)phosphine hydrochloride (TCEP) to produce a long-acting formulation of aptamer therapeutics.
  • TCEP Tri(2-carboxyethyl)phosphine hydrochloride
  • ⁇ PEGylated aptamer or hyaluronic acid derivative-aptamer conjugate is filled into HA hydrogel or PLGA microsphere to produce slow-released formulation, thereby largely extending a half-life of aptamer.
  • HA derivatives with regulated degradation in vivo can be developed as a delivery system of biopharmaceuticals such as aptamer and protein.
  • EXAMPLE 1 Preparation of PEGylation reagent and PEGylated aptamer using the reagent
  • Branched PEGylated alcohol 120 mg was dissolved in DMF 5ml reacted with triphosgene (5 mmol) and N-hydroxysuccinimide (3 mmol) at 50 degree for 3 hours The residue was re-crystallized form 2-propanol to obtain compound of chemical formula 3a.
  • PEGylation reagents obtained from Examples 1.1 and 1.2 were used for performing PEGylation of the compounds of chemical formula 2a and chemical formula 3a.
  • Anti-thrombin DNA aptamers were synthesized using solid phase phosphoramidite chemistry with an automated oligonucleotide synthesizer.
  • the aptamer sequence was 5'- d(GGTTGGTGTGGTTGG)-3' (SEQ ID NO:1).
  • 5'-amino modifier-C ⁇ was diluted as 0.1M solution in acetonitrile for terminal modification.
  • the oligonucleotide was deprotected with ammonium hydroxide/methylamine (1:1) at room temperature for 12 hours and purified by ion exchange HPLC. Aptamer was dissolved to 2 mM in 100 mM sodium carbonate buffer, pH 8.5, and was reacted for 1 hour with a 2.5 molar excess of compound 1-1 (MW 40 kDa) in equal volumes of acetonitrile. The resulting products were then purified by reverse phase HPLC on Vydac Cl 8 columns with acetonitrile, 50 mM TEAA as an eluant.
  • DNA and modified phosphoramidite were purchased from Glenresearch, and mPEG-SPA was purchased from NOF Co., (Tokyo, Japan). The polymers were dried under vacuum prior to use. Organic synthesis reagents and solvents were purchased from Aldrich chemical Co. (Milwaukee, WI) and used without further purification.
  • Anti-thrombin DNA aptamers were synthesized using solid phase phosphoramidite chemistry with an automated oligonucleotide synthesizer.
  • the aptamer sequence was 5'- d(GGTTGGTGTGGTTGG)-3' (SEQ ID NO:1).
  • the symmetric doubler phosphoramidite (Glenresearch. Cat. No. 10-1920) at a concentration of 0.1 M in acetonitrile was coupled to the terminal aptamer sequence.
  • the coupling time was about 15 minute for the doubler phosphoramidite addition.
  • Anti-thrombin DNA Aptamer was used as a model for various aptamer therapeutics.
  • a branch-type phosphoramidite and the following amine modified phosphoramidite synthon were successfully introduced to the final sequence of anti-thrombin aptamer.
  • the conventional linear PEGylation reagents were conjugated to the terminal amine group of oligonucleotide resulting in the branch-type PEGylated anti-thrombin DNA aptamer.
  • the product was thought to be comparable to that by using the branch-type PEGylation reagent of NEKTAR Therapeutics.
  • FIG. 2 shows the RP-HPLC of reaction products in an hour.
  • FIG. 2 shows the RP-HPLC of reaction products in an hour.
  • EXAMPLE 3 Change in binding affinity of PEGylation 3.1. Surface plasmon resonance (SPR) of anti-VEGF aptamer samples to VEGF coated on the BIAcore chip
  • Anti-VEGF 2'-OMe-RNA aptamer was synthesized using a solid phase phosphoramidite chemistry with an automated oligonucleotide synthesizer.
  • the sequence of the aptamer was 5'-AmUmGmCmAmGmUmUmUmGmAmGmAmAmGmUmCm GmCmGmCm AmU-3 '(SEQ ID NO: 2).
  • DNA aptamer was also synthesized with the same sequence. (5'-AmTmGmCmAmGmTmTmTmTmGmAmGmTmCm GmCmGmCmAmT-3')(SEQ ID NO:3).
  • FIG. 3 is SPR result showing a binding force of anti-VEGF aptamer to VEGF coated on BIAcore chip.
  • the biological activity of PEGylated anti-VEGF 2'-0Me-RNA aptamer was assessed by the measurement of binding affinity using surface plasmon resonance (SPR) analysis.
  • SPR has been widely used for the detection of bio-affinity adsorption between the biomolecules such as DNA, RNA, and protein.
  • the 2OK PEGylated anti-VEGF 2' -OMe- RNA aptamer, purified by RP-HPLC fractionation method, was dissolved in phosphate buffered saline (PBS, pH 7.4) at a concentration of 500 nM.
  • SPR analysis was performed using a BIAcore 2000 instrument.
  • a series of diluted solutions of anti-VEGF 2'-OMe-RNA aptamer, PEGylated anti-VEGF 2'-0Me-RNA aptamer, and anti-VEGF DNA aptamer were passed over the immobilized VEGF on the chip.
  • the adsorption of aptamers onto the VEGF resulted in the formation of aptamer- VEGF complex, which was detected by SPR. before each injection, the surface of the chip was regenerated with 0.03% sodium dodecyl sulfate (SDS) and 50 mm sodium hydroxide (NaOH) containing 0.5 m sodium chloride (NaCl).
  • SDS sodium dodecyl sulfate
  • NaOH sodium hydroxide
  • Figs. 4a to 4c show the binding affinity of anti-VEGF aptamer samples to VEGF coated on the biacore chip.
  • concentration of each VEGF aptamer sample varied from 30 to 500 nM. While the Kd value of non-modified anti-VEGF 2'-OMe-RNA aptamer was 1.87 x 10 '9 M, the Kd value of PEGylated anti-VEGF 2'-OMe-RNA aptamer was 8.7 x 10 "8 M. Despite of the slightly increased Kd value after PEGylation, we could confirm the considerable binding affinity of PEGylated anti-VEGF 2'-0Me-RNA aptamer to VEGF (Figs. 4A and 4B).
  • the bioactivity of PEGylated anti-thrombin DNA aptamer was assessed by measuring the whole blood clotting time (WBCT) in vitro. Previously, the half-life of anti- thrombin DNA aptamer was reported to be 108 seconds and the WBCT to be extended by 26 to 43 seconds in human plasma. PBS without aptamer was used as a control and the WBCT was about 340 seconds. The WBCT of non modified anti-thrombin DNA aptamer was 382 seconds, 42 seconds longer than that of PBS treated sample. PEGylated DNA aptamer was the longest 456 seconds reflecting its anti-thrombin bioactivity. FIG. 6 shows the WBCT of three different samples. .
  • FIG. 5 is comparison of in vitro whole blood clotting times (WBCT) of (A) PBS as a reference, (B) anti-thrombin DNA aptamer and (C) PEGylated anti-thrombin DNA aptamer.
  • WBCT whole blood clotting times
  • HA with a molecular weight of 200,000 was obtained from Denkikagaku Kogyo Co. (Tokyo, Japan).
  • Adipic acid dihydrazide (ADH) and l-ethyl-3-[3-(dimethylamino)- propyl] carbodiimide (EDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • HA-ADH was prepared according to the method of Y. Luo, K. Kirker and GD.
  • Prestwich J. Control. ReI. Vol. 69 (2000), p.169. Briefly, 100 mg of HA was dissolved in 20 mL and 50 mL of water to give HA solutions of 5 mg/mL and 2 mg/mL, respectively. Forty times molar excess of solid ADH (1.736 g) was added to each solution and mixed for 10 min for complete dissolution. Then, ethanol was added and mixed for 30 min. The content of ethanol was varied by 0, 25, and 50%. The pH of the reaction mixture was adjusted to 4.8 by the addition of 1 N HCl. After that, four times molar excess of EDC (0.191 g) was added in solid form. The pH of the reaction mixture was maintained at 4.8 by the addition of 1 N HCl.
  • reaction was stopped by raising the pH of reaction mixture to 7.0 with 1 N NaOH.
  • the reaction mixture was poured into the pre-washed dialysis membrane tube (MWCO of 7 kDa) and dialyzed against large excess amount of 100 mM NaCl aqueous solution, followed by dialysis against 25 % ethanol and pure water. The solution was finally lyophilized for three days.
  • the purity of HA-ADH was determined by GPC analysis, and the degree of ADH modification was measured by 1 H NMR analysis (DPX300, Bruker, Germany).
  • FIG. 6 shows 1 H NMR spectrum of HA-ADH which is prepared by linking adipic acid dihydrazide (ADH) to carboxyl group of HA.
  • FIG. 7 is a graph showing a modification degree of ADH depending on ethanol content of reacting solvent of HA modification.
  • Fig. 6 and 7 show the peak assignments of HA-ADH in 1 H NMR spectra and the degree of ADH modification determined as described elsewhere.
  • the degree of ADH modification increased up to 85 mol% with increasing ethanol content in reaction solvent.
  • the addition of ethanol appeared to contribute for high degree of ADH modification. Therefore, as the amount of ethanol in mixed reaction solvent is higher, the long-acting activity of the aptamer increases.
  • Anti-thrombin DNA aptamer(5'-d(GGTTGGTGTGGTTGG)-3' (SEQ ID NO: I)) havaing a carboxyl group at 5'-terminus was activated with EDC/NHS, and reacted with HA-ADH to obtain HA-ADH-aptamer conjugate conjugate.
  • HA-TREN was prepared by the substantially same method of Example 4.1 except for use of TREN instead of ADH.
  • anti-thrombin DNA aptamer (5'- d(GGTTGGTGTGGTTGG)-3' (SEQ ID NO:1)) was conjugated with HA-TREN instead of HA-ADH according to the substantially same method of Example 4.2.
  • TSA-OH tetra-n- butylammonium hydroxide
  • HA- TBA was dissolved in DMSO. Then, (benzotriazol-1-yloxy) tris(dimethylamino) phosphonium hexafluorophosphate (BOP), 2-aminoethyl methacrylate hydrochloride (AEMA), and N, N-diisopropylethylamine (DIPEA) were added to the solution and mixed overnight. Finally, the reaction product was dialyzed against water and lyophilized for three days.
  • BOP benzotriazol-1-yloxy tris(dimethylamino) phosphonium hexafluorophosphate
  • AEMA 2-aminoethyl methacrylate hydrochloride
  • DIPEA N, N-diisopropylethylamine
  • aptamer conjugate anti-thrombin DNA aptamer (5'-d(GGTTGGTGTGGTTGG)-3' (SEQ ID NO: I)) was dissolved in phosphate buffer (20OmM, pH8.74), and then a 10-fold molar excess of Traut's reagent (Pierce, Rockford, IL, USA) to aptamer was dissolved in the aptamer solution. After reaction for 2 hrs, the solution was eluted through PD-IO desalting column to remove unreacted Traut's reagent. 100-fold molar excess of TCEP (Sigma-Aldrich, St.
  • HA-AEMA aminoethyl methacrylate
  • phosphate buffer 20OmM, pH8.74
  • the molar ratio of MA to aptamer was 10.
  • the HA-AEMA solution was added to the aptamer solution, mixed immediately, and incubated at 37 °C overnight.
  • the reaction mixture was poured into the pre-washed dialysis membrane tube (MWCO of 1OkDa) and dialyzed against large excess amount of 10OmM NaCl aqueous solution, followed by dialysis against 25% ethanol and pure water.
  • the solution was finally lyophilized for three days.
  • the purity of HA-aptamer conjugate was determined by GPC analysis and the extent of HA-aptamer conjugation was measured by 1 H NMR analysis.
  • FIG. 9 is a chromatography (GPC) analysis result of HA-AEMA-aptamer conjugate which is prepared by linking HA-AEMA to aptamer containing a thiol group: A is HA-AEMA-aptamer conjugate and B is unreacted aptamer.
  • GPC chromatography
  • HA-APMAm was prepared by the substantially same method of Example 6.1 except for use of APMAm instead of AEMA.
  • FIG. 10 shows 1 H NMR spectrum of HA-APMAm which is prepared by linking APMAm to carboxyl group of HA. As a result of NMR analysis, substitution rate of APMAm was 55.7 mol%.
  • This preparation method of HA-APMAm aptamer conjugate was performed as the substantially same as that of HA-AEMA described in Example 6.2 except for use of N-(3- Aminopropyl) methacrylamide hydrochloride (APMAm) in stead of AEMA HA-AEMA.
  • APMAm N-(3- Aminopropyl) methacrylamide hydrochloride

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Abstract

L'invention concerne une formulation biopharmaceutique à action de longue durée, en particulier une thérapeutique à base d'aptamère. Pour obtenir la formulation à action de longue durée précitée, on procède à la bioconjugaison d'un aptamère PEGylé ramifié ou d'un dérivé de l'acide hyaluronique (HA) dont la dégradation in vivo est régulée et d'un agent biopharmaceutique.
PCT/KR2007/005179 2006-10-20 2007-10-22 Formulation biopharmaceutique à action de longue durée WO2008048079A1 (fr)

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WO2017094897A1 (fr) 2015-12-04 2017-06-08 全薬工業株式会社 Aptamère anti-il-17 présentant une meilleure rétention dans le sang
EP3385383A4 (fr) * 2015-12-04 2019-07-24 Zenyaku Kogyo Co., Ltd Aptamère anti-il-17 présentant une meilleure rétention dans le sang
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US11013756B2 (en) 2015-12-04 2021-05-25 Zenyaku Kogyo Co., Ltd. Anti-IL-17 aptamer having improved retention in blood
US10308943B2 (en) 2016-02-08 2019-06-04 Vitrisa Therapeutics, Inc. Compositions with improved intravitreal half-life and uses thereof
EP3414330A4 (fr) * 2016-02-08 2019-07-03 Vitrisa Therapeutics, Inc. Compositions à demi-vie intravitréenne améliorée et leurs utilisations
WO2018148333A1 (fr) * 2017-02-08 2018-08-16 Vitrisa Therapeutics, Inc. Compositions à demi-vie intravitréenne améliorée et utilisations associées
CN111848922A (zh) * 2020-07-23 2020-10-30 深圳飞扬兴业科技有限公司 一种水性异氰酸酯固化剂、制备方法及其应用

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