JP2009510182A - Cycitol linker polymer conjugate - Google Patents

Abstract

  In order to improve the in vivo properties of biologically active molecules, it has been described to conjugate biologically active molecules with polymers, high molecular weight non-immunogenic and lipophilic compounds. The present invention relates to a conjugate comprising a polymer, a biologically active molecule, and cyclitol linking the polymer to the biologically active molecule. The invention also relates to an activated polymer composition comprising a polymer, an active functional group, and cyclitol linking the polymer to the active functional group.

Description

RELATED APPLICATION This application is to US Provisional Application No. 60 / 703,133, filed July 28, 2005, Attorney Docket No. EYE-034P, which is incorporated herein by reference in its entirety. Claim priority.

TECHNICAL FIELD This invention relates to conjugates comprising a polymer, a biologically active molecule, and a carbocyclic group linking the polymer to the biologically active molecule.

  In order to improve the in vivo properties of biologically active molecules, it has been described to conjugate biologically active molecules with polymers, high molecular weight non-immunogenic and lipophilic compounds. Biologically active molecules modified with polymers, high molecular weight non-immunogenic and lipophilic compounds have reduced immunogenicity / antigenicity and improved compared to those in an unmodified manner Shows pharmacokinetic properties such as stability. For example, US Pat. No. 6,011,020 discloses conjugating a high molecular weight non-immunogenic compound to an aptamer to increase the in vivo circulating half-life of the aptamer.

  The activated polymer is reacted with a biologically active molecule having a nucleophilic functional group that functions as a binding site. Polyalkylene glycols such as polyethylene glycol (PEG) belong to the most widely used polymers. U.S. Pat. No. 4,179,337 describes an initial model for attaching PEG to peptides or polypeptides. US Pat. No. 5,122,614 discloses that PEG molecules activated with an N-succinimide carbonate functional group can be attached under aqueous basic conditions by a urethane linkage to an amine group of a polypeptide. To do. US Pat. No. 5,932,462 describes activated multi-armed PEG molecules and multi-armed PEG conjugates. Multi-armed PEG conjugates contain two linear PEG units attached to a multifunctional central moiety such as lysine as a linker between the PEG chain and the biologically active molecule.

  There is a continuing need for new activated polymer derivatives useful for conjugation to biologically active molecules and new conjugates comprising biologically active molecules and polymers.

  The present invention relates to a conjugate comprising a polymer, a biologically active molecule, and cyclitol linking the polymer to the biologically active molecule.

  The invention also relates to an activated polymer composition comprising a polymer, an active functional group, and cyclitol linking the polymer to the active functional group.

  The invention also relates to a method of forming a conjugate comprising a polymer, a biologically active molecule, and a cyclitol that links the polymer to the biologically active molecule.

  In certain embodiments, the present invention provides compounds of the formula:

（Ａ_１は、生物学的に活性な部分又は活性な官能基であり；
Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ｃ、Ｒ_１ｄ及びＲ_１ｅが、−ＯＨ、−ＮＨ_２及び−Ｘ_２−Ｌ_２−Ａ_２からなる群から各々独立に選択され；
Ａ_２は、水素、ポリマー、生物学的に活性な部分及び活性な官能基からなる群から選択され；
Ｌ_１及びＬ_２は、結合及びスペーサー部分からなる群から各々独立に選択され；
Ｘ_１及びＸ_２は、−Ｏ−、−Ｓ−及び−ＮＲ_２−からなる群から各々独立に選択され；並びに
Ｒ_２は、水素及び低級アルキル基からなる群から選択される。）
を有する化合物に関する。

(A ₁ is a biologically active moiety or an active functional group;
R _1a , R _1b , R _1c , R _1d and R _1e are each independently selected from the group consisting of —OH, —NH ₂ and —X ₂ -L ₂ -A ₂ ;
A ₂ is selected from the group consisting of hydrogen, a polymer, a biologically active moiety and an active functional group;
L ₁ and L ₂ are each independently selected from the group consisting of a bond and a spacer moiety;
X ₁ and X ₂ are each independently selected from the group consisting of —O—, —S— and —NR ₂ —; and R ₂ is selected from the group consisting of hydrogen and lower alkyl groups. )
It relates to a compound having

  The present invention has several advantages. Cycitol is a versatile multifunctional moiety that can attach up to 5 polymers to a broad spectrum of biologically active molecules through a single linker unit. Cycitol is also a robust part that gives conjugates that have a stable bond to hydrolysis.

  The present invention relates to a conjugate comprising a polymer, a biologically active molecule, and cyclitol linking the polymer to the biologically active molecule.

  The invention also relates to an activated polymer composition comprising a polymer, an active functional group, and cyclitol linking the polymer to the active functional group.

  In certain embodiments, the composition has the formula:

（Ａ_１は、生物学的に活性な部分又は活性な官能基であり；
Ｒ_１ａ、Ｒ_１ｂ、Ｒ_１ｃ、Ｒ_１ｄ及びＲ_１ｅは、−ＯＨ、−ＮＨ_２及び−Ｘ_２−Ｌ_２−Ａ_２からなる群から各々独立に選択され；
Ａ_２は、水素、ポリマー、生物学的に活性な部分及び活性な官能基からなる群から選択され；
Ｌ_１及びＬ_２は、結合及びスペーサー部分からなる群から各々独立に選択され；
Ｘ_１及びＸ_２は、−Ｏ−、−Ｓ−及び−ＮＲ_２−からなる群から各々独立に選択され；並びに
Ｒ_２は、水素及び低級アルキル基からなる群から選択される。）
を有する。

(A ₁ is a biologically active moiety or an active functional group;
R _1a , R _1b , R _1c , R _1d and R _1e are each independently selected from the group consisting of —OH, —NH ₂ and —X ₂ -L ₂ -A ₂ ;
A ₂ is selected from the group consisting of hydrogen, a polymer, a biologically active moiety and an active functional group;
L ₁ and L ₂ are each independently selected from the group consisting of a bond and a spacer moiety;
X ₁ and X ₂ are each independently selected from the group consisting of —O—, —S— and —NR ₂ —; and R ₂ is selected from the group consisting of hydrogen and lower alkyl groups. )
Have

In one embodiment, A ₁ is a biologically active moiety. In another embodiment, A ₁ is a nucleic acid. In another embodiment, A ₁ is an aptamer. In another embodiment, A ₁ is an anti-VEGF aptamer.

In one embodiment, A ₁ is an active functional group. In another embodiment, A ₁ is an electrophilic functional group. In another embodiment, A ₁ is carboxylic acid, carboxylic acid halide, carboxylic acid chloride, halogen, N-succinimide carbonate, succinimidyl ester, 1-benzotriazolyl carbonate ester, N-hydroxymale. Active functional groups including midyl esters, vinyl sulfones, azalactones, cyclic amidothione carbonyls, imidazole carbonyls, isocyanates or isothiocyanates.

In one embodiment, at least one of R _1a , R _1b , R _1c , R _1d and R _1e is independently -X ₂ -L ₂ -A ₂ . In another embodiment, at least two of R _1a , R _1b , R _1c , R _1d, and R _1e are independently -X ₂ -L ₂ -A ₂ . In another embodiment, at least three of R _1a , R _1b , R _1c , R _1d, and R _1e are independently -X ₂ -L ₂ -A ₂ . In another embodiment, at least four of R _1a , R _1b , R _1c , R _1d, and R _1e are independently -X ₂ -L ₂ -A ₂ . In another embodiment, each of R _1a , R _1b , R _1c , R _1d and R _1e is independently -X ₂ -L ₂ -A ₂ .

In one embodiment, A ₂ is a polymer. In another embodiment, at least one A ₂ is a polymer. In another embodiment, A ₂ is a biologically active moiety. In another embodiment, A ₂ is an active functional group.

In one embodiment, L ₁ and L ₂ are each independently of the formula:
_{- (CH 2) n - (} X 3) m - (C (X 4)) p - (X 5) q - (CH 2) r -
(N is 0 to 10;
m is 0 or 1;
p is 0 or 1;
q is 0 or 1; and r is 0-10. )
It is a spacer part represented by.

In another embodiment, L ₁ and L ₂ are each independently
_{- (CH 2) n - (} C (X 4)) p -,
_{- (C (X 4))} p - (CH 2) r -,
_{- (C (X 4))} p -, and - _{(CH 2) n} -
(N, p and r are as described above.)
A spacer moiety represented by a formula selected from the group consisting of:

In one embodiment, X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are each independently selected from the group consisting of —O— and —NH—. In another embodiment, X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are each —O—.

  In another embodiment, the conjugate is

（Ｄは、生物学的に活性な部分であり；
ＰＯＬＹは、ポリマーであり；
各Ｘ_３、Ｘ_４及びＸ_５は、−Ｏ−、−Ｓ−及び−ＮＲ_２−からなる群から各々独立に選択され；
Ｒ_２は、水素及び低級アルキル基からなる群から選択され；
ｍは、０又は１であり、及び
ｎは、０から１０までの整数である。）
からなる群から選択される式を有する。

(D is a biologically active moiety;
POLY is a polymer;
Each X ₃ , X ₄ and X ₅ is independently selected from the group consisting of —O—, —S— and —NR ₂ —;
R ₂ is selected from the group consisting of hydrogen and lower alkyl groups;
m is 0 or 1, and n is an integer from 0 to 10. )
Having a formula selected from the group consisting of

  In one embodiment, D is a nucleic acid. In another embodiment, D is an aptamer. In another embodiment, D is an anti-VEGF aptamer.

  In a specific embodiment, the conjugate is

（Ｚは、約４５０である。）
からなる群から選択される式を有する。

(Z is about 450.)
Having a formula selected from the group consisting of

  In another embodiment, the activated polymer composition comprises

（Ｇは、脱離基であり；
ＰＯＬＹは、ポリマーであり；
各Ｘ_３、Ｘ_４及びＸ_５は、−Ｏ−、−Ｓ−及び−ＮＲ_２−からなる群から各々独立に選択され；
Ｒ_２は、水素及び低級アルキル基からなる群から選択され；
ｍは、０又は１であり；並びに
ｎは、０から１０までの整数である。）
からなる群から選択される式を有する。

(G is a leaving group;
POLY is a polymer;
Each X ₃ , X ₄ and X ₅ is independently selected from the group consisting of —O—, —S— and —NR ₂ —;
R ₂ is selected from the group consisting of hydrogen and lower alkyl groups;
m is 0 or 1; and n is an integer from 0 to 10. )
Having a formula selected from the group consisting of

  In one embodiment, G is a leaving group selected from the group consisting of halide, chloride, N-succinimide, 1-benzotriazole, N-hydroxymaleimide, azalactone, cyclic amidothione and imidazole.

  In one embodiment, POLY is a high molecular weight polymer. In another embodiment, POLY is a polyether polyol, polyethylene glycol, polysaccharide, polyester, high molecular weight polyoxyalkylene, polyamide, polyurethane, polysiloxane, polyacrylate, polyol, polyvinyl pyrrolidone, polyvinyl alcohol, polyanhydride, It is a polymer selected from the group consisting of carboxymethylcellulose, other cellulose derivatives, chitosan, polyaldehyde and polyether. In another embodiment, POLY is a polyalkylene glycol. In another embodiment, POLY is polyethylene glycol (PEG).

  In another embodiment, POLY is a PEG having a molecular weight from 5 kDa to 100 kDa. In another embodiment, POLY is PEG having a molecular weight of about 20 kDa.

In one embodiment, each X ₃ , X ₄ and X ₅ is each independently selected from the group consisting of —O— and —NH—.

  In one embodiment, n is an integer from 0 to 6. In another embodiment, n is 5.

  In a specific embodiment, it is understood that there are two or more polymers, and each polymer can be the same or different.

  In specific embodiments, it is understood that there are two or more biologically active molecules, and each biologically active molecule can be the same or different.

  As used herein, “cyclitol” is a cycloalkane containing a hydroxyl group on one or more ring atoms. T.A. Hudricky et al. Summarizes known cyclitols and their derivatives (Cyclitols and Their Derivatives: A Handbook of Physical, Spectral and Synthetic Data, T & Hudricky and ul. The entirety is hereby incorporated by reference))).

  In one embodiment, cyclitol is a 6-membered cycloalkane. In one embodiment, cyclitol is a 5-membered cycloalkane. In one embodiment, cyclitol is tri-, tetra-, penta- and hexa-hydroxycyclohexane. Cyclitol derivatives optionally include substituents including but not limited to alkyl, amino, thio, carbonyl or halogen substituents.

In one embodiment, cyclitol is inositol. Inositol includes, but is not limited to, myo-inositol, D-chiroinositol, L-chiroinositol, mucoinositol, Scyllo inositol, alo-inositol, epiinositol, cis-inositol and neo-inositol. (See Inositol Phosphates and Derivatives Synthesis, Biochemistry, and Therapeutic Potential, Edited by A. B. Reitz, American Chemical Society, booked in 19 by U.S.).
As used herein, an “activated polymer” is a polymer that contains active functional groups. The activated polymer can be used to bind a biologically active molecule to form a conjugate.

  As used herein, an “active functional group” is a functional group that can rapidly react with electrophilic groups or nucleophilic groups on other molecules. For example, as understood in the art, the term “active ester” includes esters such as N-hydroxysuccinimidyl carboxylate esters that react rapidly with nucleophilic groups such as amines. Other examples of active esters include, but are not limited to, N-hydroxymaleimide carboxylate esters and 1-benzotriazolyl carbonate esters.

  The active functional group can include a leaving group. Examples of suitable leaving groups include, but are not limited to, halide, N-hydroxy-succinimide, N-succinimide, 1-benzotriazole, N-hydroxymaleimide, cyclic amidothione and imidazole.

  Examples of active functional groups that react with nucleophiles are carboxylic acid, carboxylic acid halide, carboxylic acid chloride, N-succinimide carbonate, succinimidyl ester, 1-benzotriazolyl carbonate ester, N- Including, but not limited to, hydroxymaleimidyl ester, vinyl sulfone, azalactone, cyclic amidothionecarbonylimidazole, isocyanate and isothiocyanate.

  Examples of active functional groups that react with electrophiles include primary amines, alcohols, hydrazine and hydrazide functional groups, acyl hydrazides, carbazates, semicarbazates and thiocarbazates, However, it is not limited to these.

  For example, to attach the polyalkylene oxide to a nucleophile, one of the hydroxyl end groups of the polyalkylene oxide is converted to an active functional group. This process is referred to as “activation” and the product is referred to as “activated polyalkylene oxide”.

  As used herein, the term “spacer” or “spacer moiety” refers to a moiety that covalently attaches cyclitol to a biologically active moiety or active functional group.

In one embodiment, the spacer has the formula:
_{- (CH 2) n - (} X 3) m - (C (X 4)) p - (X 5) q - (CH 2) r -
(N is 0 to 10;
m is 0 or 1;
p is 0 or 1;
q is 0 or 1; and r is 0-10. )
Represented by

In another embodiment, the spacer is
_{- (CH 2) n - (} C (X 4)) p -,
_{- (C (X 4))} p - (CH 2) r -,
_{- (C (X 4))} p -, and - _{(CH 2) n} -
(N, p and r are as described above.)
Represented by a formula selected from the group consisting of

A “lower alkyl group” is a C ₁ -C ₈ linear or branched hydrocarbon, or a C ₃ -C ₈ cyclic hydrocarbon. Examples of lower alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups.

  The term “hydrolytically stable” or “non-hydrolyzable” bond or linkage is used herein to refer to a bond or linkage that is substantially stable in water or does not substantially react with water. Used to represent. For example, a linkage that is stable to hydrolysis does not react under aqueous conditions for an extended period of time. A linkage that is stable to hydrolysis can exist indefinitely under aqueous conditions.

  The term “physiologically stable” bond or linkage is used herein to be substantially stable to in vivo cleavage or hydrolysis, but also stable in the presence of other in vitro factors. Used to refer to the resulting bond or linkage. A physiologically stable bond or linkage is stable to hydrolysis and stable to physiological processes in the patient's cells, organs, skin, membranes or other parts of the body. A link that is physiologically stable can exist indefinitely under physiological conditions.

  A bond or linkage that is “esterase resistant” or “stable to esterase” is stable in the presence of esterase.

  Although it is preferred that all bonds or linkages have a reasonable stability under aqueous or physiological conditions, in certain embodiments, the use of selectively hydrolyzable linkages or linkages is envisioned. A selectively hydrolyzable bond or linkage still has moderate stability in the circulation. A “selectively hydrolyzable” bond or linkage includes all releasable, cleavable or hydrolyzable linkages only under certain conditions or preferentially under certain conditions.

  Examples of selectively hydrolyzable bonds include, but are not limited to, disulfide and trisulfide bonds and acid labile bonds.

  “Biologically active molecule”, “biologically active moiety” or “biologically active agent” includes (but is not limited to) viruses, bacteria, fungi, plants, animals and humans. It can be any substance that can affect some physical or biochemical property of the organism. Biologically active molecules are intended for the purpose of diagnosing, alleviating, treating or preventing disease in humans or other animals, or to enhance the physical or mental health of humans or animals. It may contain all substances that do. Examples of biologically active molecules include, but are not limited to, nucleic acids, nucleosides, oligonucleotides, aptamers, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, cells, viruses, liposomes, microparticles, and micelles. It is not done. The classes of biologically active substances suitable for use in the present invention are antibiotics, fungicides, antiviral drugs, anti-inflammatory drugs, antitumor drugs, cardiovascular drugs, anxiolytic drugs, hormones, growth Including, but not limited to, factors, steroids and the like.

  By “treating” is meant medical management of a patient for the purpose of obtaining cure, remission, cessation or prevention of a disease, pathological condition or disease. This term includes aggressive treatments, i.e. treatments aimed at recovering from a disease, pathological condition or disease in particular and aims to eliminate the cause of treatment, i.e. the cause of the disease, pathological condition or disease. Including treatment. Furthermore, the term is intended to prevent palliative treatment, i.e. not cure the disease, pathological condition or disease, but rather to treat, preventive treatment, i.e. illness, pathological condition or disease intended to alleviate symptoms. And augmentation treatments, ie treatments used to supplement a disease, pathological condition or another specific therapy aimed at recovery from the disease. The term “treating” also includes symptomatic treatment, ie treatment for systemic symptoms, pathological conditions or diseases of the disease.

  In one embodiment, the methods of the invention provide a means for inhibiting or treating ocular neovascular diseases. In some embodiments, an ocular neovascular disorder susceptible to treatment or suppression by the methods of the present invention is ischemic retinopathy, iris neovascularization, intraocular neovascularization, age-related macular degeneration, corneal neovascularization, Retinal neovascularization, choroidal neovascularization, preterm infant retinopathy, retinal vascular occlusion, diabetic retinal ischemia, diabetic macular edema or proliferative diabetic retinopathy. In yet another embodiment, the method of the invention inhibits or treats psoriasis or rheumatoid arthritis in a patient in need thereof or a patient diagnosed or at risk for developing such a disease. Provide a means for

  As used herein, the terms “angiogenesis” and “angiogenesis” are used interchangeably. Angiogenesis and angiogenesis refers to the development of new blood vessels into cells, tissues or organs. The regulation of angiogenesis typically changes in certain disease states, and in many cases the pathological damage associated with the disease is altered, uncontrolled, or unregulated blood vessels. Associated with formation. Persistent and uncontrolled angiogenesis occurs in multiple disease states, including those characterized by abnormal proliferation by epithelial cells, and is associated with pathological damage seen in these conditions, such as vascular leakage and permeability. Be the basis.

  By “ocular neovascular disease” is meant a disease characterized by altered or uncontrolled angiogenesis in a patient's eye. Typical ocular neovascular diseases are optic disc neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreous neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, Diabetic macular edema, vascular retinopathy, retinal degeneration, uveitis, retinal inflammatory disease and proliferative vitreoretinopathy.

  A variety of anti-VEGF drugs that inhibit VEGF activity or production, including aptamers and VEGF antibodies, are available and can be used in the methods of the invention. Certain anti-VEGF drugs are described in US Pat. Nos. 6,168,778, 6,147,204, 6,051,698, 6,011,020, 5,958,651, 5,817,785, 5,811,533, 5,696,249, 5,683,867, 5,670,637 and 5,475,096 (by reference, VEGF nucleic acid ligands such as those described in their entirety herein. One specific anti-VEGF drug is pegaptanib sodium.

  The class of biologically active substances includes (but is not limited to) antibiotics, antivirals and antifungals, antiinfectives, analgesics, antiallergic drugs, mast cell stability. Agents, steroidal and non-steroidal anti-inflammatory drugs, decongestants, adrenergic drugs, beta-adrenergic blockers, alpha-adrenergic blockers, parasympathomimetics, cholinesterase inhibitors, carbonic anhydrase inhibitors, and prostaglandins Anti-glaucoma drugs, antioxidants, dietary supplements, angiogenesis inhibitors, antimetabolites, fibrinolytic agents, wound regulators, neuroprotective agents, angiogenesis-inhibiting steroids, including but not limited to Mydriatic drug, ciliary myalgia mydriatic drug, miotic drug, vasoconstrictor, vasodilator, anticoagulant, anticancer drug, immunomodulator, VEGF antagonist, immunosuppressant, and this These combinations and their prodrugs. Other biologically active substances include, but are not limited to, dyes, lipids, cells, viruses, liposomes, microparticles and micelles.

  Examples of biologically active substances are nucleic acids, nucleosides, oligonucleotides, antisense oligonucleotides, RNA, DNA, siRNA, RNAi, aptamers, antibodies, peptides, proteins, enzymes, fusion proteins, porphyrins, and small molecule drugs Including, but not limited to. Examples of antibodies include, but are not limited to, bevacizumab (Avastin®) and ranizumab (Lucentis ™), VEGF antibodies from Genentech, San Francisco, CA. Examples of aptamers include, but are not limited to, pegaptanib (Macugen®; (OSI) Eyetech, Inc., New York, NY). Examples of steroids include, but are not limited to, anecoltab acetate (Retane®; Alcon, Inc., Fort Worth, TX). Examples of fusion proteins include, but are not limited to, VEGF Trap ™ (Regeneron Pharmaceuticals, Inc. Tarrytown, NY). Examples of RNAi include, but are not limited to, Direct RNAi ™ (Anylam Pharmaceuticals, Cambridge, Mass.).

  The compositions of the invention can be administered by any suitable means that provides an effective concentration for the treatment of angiogenic diseases. For example, each composition can be mixed with a suitable carrier material and is generally present in an amount of 1 to 95% by weight of the total weight of the composition. The composition can be provided in dosage forms suitable for ophthalmic, oral, parenteral (eg, intravenous, intramuscular, subcutaneous), rectal, transdermal, nasal, or inhalation administration. . Thus, the composition is for example a tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel containing hydrogel, paste, ointment, cream, plaster, delivery device, suppository, enema, injection It can be in the form of a liquid, implant, spray or aerosol. A pharmaceutical composition containing a single antagonist or two or more antagonists can be formulated according to conventional pharmaceutical practice (see, eg, Remington: The Science and Practice of Pharmacy, (20th ed.) Ed. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, PA. And Encyclopedia of Pharmaceutical Technology, eds., J. Swarbrick and J. C. k.

  It is understood that pharmaceutically acceptable prodrugs of the biologically active substances disclosed herein are also included in the invention and can be used in the compositions and methods disclosed herein.

  It is understood that pharmaceutically acceptable salts of the biologically active substances disclosed herein are also included in the present invention and can be used in the compositions and methods disclosed herein.

  In certain embodiments of the invention, the anti-VEGF drug is given in a sustained release formulation. Examples of sustained release microparticles are described in US Provisional Patent Application No. 60 / 796,071, which is hereby incorporated by reference in its entirety.

  In one embodiment, the sustained release formulation comprises a biocompatible, biodegradable polymer selected from the group consisting of lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers.

  The formulations of the present invention can be used in the treatment of all diseases of the eye. The formulations of the present invention can also be used to induce anti-VEGF drugs into specific eye tissues such as the retina or choroid. The combination of anti-VEGF drug or biologically active substance is selected based on the disease, disorder or condition being treated. In addition to anti-VEGF drugs for certain conditions, other compounds, such as antibiotics to prevent microbial growth, may be included for secondary effects. The amount and dosage frequency of the formulation will depend on the disease, disorder or condition being treated and the biologically active substance used. One skilled in the art can make this determination.

  The term “oligomer” refers to a polymer having a molecular weight that is too small to be considered a polymer. Oligomers typically have hundreds of molecular weights, while polymers typically have molecular weights of thousands or more.

  The term “oligonucleotide” refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (main chain) linkages. The term also includes modified or substituted oligomers containing non-naturally occurring monomers or portions thereof that function similarly. The incorporation of substituted oligomers is based on factors such as enhanced cellular uptake or enhanced nuclease resistance and is selected as is well known in the art.

  As used herein, the term “aptamer” refers to any polynucleotide or salt thereof that has a selective binding affinity for a molecule that is not a polynucleotide through a non-covalent physical interaction. Means. Aptamers can be polynucleotides that bind to a ligand in a manner similar to antibody binding to an epitope of the antibody. The target molecule can be any interesting molecule. An example of a molecule that is not a polynucleotide is a protein. Aptamers can be used to bind to the ligand binding domain of a protein, thereby preventing the interaction between the natural ligand and the protein.

  “Anti-VEGF aptamer” or “VEGF aptamer” is intended to include polynucleotide aptamers that bind to VEGF and inhibit the activity of VEGF. Such anti-VDGF aptamers can be RNA aptamers, DNA aptamers or aptamers with mixed (ie, RNA and DNA) compositions. Such aptamers can be identified using known methods. For example, in vitro evolution or SELEX methods are described in US Pat. Nos. 5,475,096 and 5,270,163, which are incorporated herein by reference in their entirety. Can be used. Anti-VEGF aptamers are hereby incorporated by reference in their entirety, U.S. Patent Nos. 6,168,778, 6,051,698, 5,859,228 and 6,426,335. Contain the sequences described in each of the above. The sequence can be modified to include a 5'-5 'inverted cap and / or a 3'-3' inverted cap. (See Adamis, AP et al., Published application WO2005 / 014814, the entire contents of which are hereby incorporated by reference in their entirety).

  Suitable anti-VEGF aptamers of the invention are the nucleotide sequence GAAGAAUUGG (SEQ ID NO: 1), or the nucleotide sequence

を含む。

including.

  Examples of anti-VEGF aptamers include, but are not limited to:

(I) An anti-VEGF aptamer having the sequence CGGAAUCAGUGAAUGCUUAAUACAUUCCG (SEQ ID NO: 4 as set forth in US Pat. No. 6,051,698, which is incorporated herein by reference in its entirety). Each C, G, A and U each represents the natural nucleotides cytidine, guanidine, adenine and uridine, or modified nucleotides corresponding thereto, preferably
(Ii) sequence

を有する抗ＶＥＧＦアプタマー。

Anti-VEGF aptamer having

  An example of a PEGylated aptamer is the structure

を有するペガプタニブ（ＥＹＥ００１；ＭａｃＩ）である。

Pegaptanib (EYE001; MacI).

  Examples of 5'-5 'capped anti-VEGF aptamers have the structure

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Wherein “G _m ” represents 2′-methoxyguanylic acid, “A _m ” represents 2′-methoxyadenylic acid, and “C _f ” represents 2 ′ - represents fluorocytidine Jill acid, _{"U f"} represents 2'-fluoro uridyl acid, "Ar" represents a Riboadeniru acid, _{"T d"} represents a deoxyribonucleotide deoxythymidylic acid (Adamis, a. P. et al., Published application WO2005 / 014814 (incorporated herein by reference in its entirety).

  The polymers of the present invention include polyether polyols (such as polyethylene glycol), polysaccharides (such as carboxymethylcellulose, other cellulose derivatives, glycosaminoglycans, hyaluronan and alginate), polyesters (such as PLURONIC®) Including, but not limited to, high molecular weight polyoxyalkylene ethers, polyurethanes, polysiloxanes, polyacrylates, polyols, polyvinylpyrrolidone, polyvinyl alcohol, polyanhydrides, chitosan, polyaldehydes or polyethers.

  In one embodiment, the polymer of the present invention is water soluble. In another embodiment, the polymer is a non-peptidic polymer. In another embodiment, the polymer is a non-nucleic acid polymer. In another embodiment, the polymer is a high molecular weight steric group.

  In one embodiment, the polymer is a polyether polyol. In one embodiment, the polymer is a polyalkylene glycol. In one embodiment, the polymer is polyethylene glycol (PEG). The PEG can have a free hydroxyl group or can be alkylated. In one embodiment, the end of the PEG that is not attached to a linker has cyclitol, an active functional group or a biologically active compound has a methoxy group. PEG containing a terminal methoxy group may also be referred to as “mPEG”. In another embodiment, the polyalkylene glycol is poly (propylene glycol) (“PPG”) and copolymers thereof (eg, copolymers of ethylene glycol and propylene glycol), their terpolymers, mixtures thereof, etc. It is.

The terms “polyethylene glycol”, “poly (ethylene glycol)” or “PEG” refer to all polymers of the general formula H (OCH ₂ CH ₂ ) _n OH. In one embodiment, n is 4 or greater. In one embodiment, n is from about 4 to about 4000. In another embodiment, n is from about 20 to about 2000. In one embodiment, n is about 450. In one embodiment, the PEG has a molecular weight of about 800 Daltons (Da) to about 100,000 Da. In further embodiments, the polyethylene glycol is 20 kDa PEG, 40 kDa PEG, or 80 kDa PEG.

  The average relative molecular weight of polyethylene glycol is sometimes indicated by the number of suffixes. For example, PEG having a molecular weight of 4000 Daltons (Da) can be referred to as “polyethylene glycol 4000”. A product conjugated with PEG may be referred to as a PEGylated product.

  The use of the word PEG is inclusive and is not intended to be exclusive. It should be understood that other related polymers are suitable for use in the practice of the present invention. The term PEG includes polyethylene glycol in all of its forms and includes bifunctional PEGs, multi-branched PEGs, forked PEGs, branched PEGs and pendant PEGs (ie, one or more suspended on a polymer backbone). PEG having functional groups or related polymers).

  PEG has several advantages. PEG is typically clear, colorless, odorless, water soluble, heat stable, inert to many chemicals, does not hydrolyze or degrade, and is generally non- Toxic. PEG is considered biocompatible. For example, PEG can coexist with living tissue or organs without causing harm. More specifically, PEG is substantially non-immunogenic. For example, PEG does not tend to produce an immune response in the body. When attached to a molecule with some desirable function, such as a biologically active substance in the body, PEG tends to shield the substance, so that the organism can tolerate the presence of the substance, It is possible to reduce or eliminate all immune responses. PEG conjugates tend not to produce a substantial immune response or tend to produce clotting or other undesirable effects. Furthermore, the addition of soluble high molecular weight steric groups such as PEG can improve the antagonist properties of aptamers (US patent application Ser. No. 11 / 105,279, which is incorporated herein by reference in its entirety. See)).

  In one embodiment, the polymer is a polysaccharide. Examples of polysaccharides include, but are not limited to, dextran, cellulose, chitosan, polyglucosamine and derivatives thereof. The reducing end of the polysaccharide is available for conjugation to the amine group of the molecule by Schiff base chemistry in conjugation. Polysaccharides can be coupled by reductive amination to amines such as amine-substituted cyclitols or amine termini of biologically active compounds.

  In another embodiment, the polymer is dextran. The dextran can be a linear or branched dextran. In another embodiment, the dextran is carboxymethyl dextran (CMDex).

  In another embodiment, the polymer is a cellulose derivative. In another embodiment, the polymer is carboxymethylcellulose (CMC).

  In another embodiment, the polymer is a polyaldehyde. In a further embodiment, the polyaldehyde group can be obtained synthetically or by oxidation of oligosaccharides.

  In another embodiment, the polymer is alginate. In a specific embodiment, the alginate group is an anionic alginate group provided as a salt having a cationic counter ion such as sodium or calcium.

  In another embodiment, the polymer is a polyester. In a specific embodiment, the polyester group may be a polyester group of a co-block polymer.

  In another embodiment, the polymer is polylactic acid (PLA) or polylactic acid coglycolide (PLGA). Suitable PLGA groups and methods for conjugating PLGA groups are described in J. Am. H. Jeong et al. Bioconjugate Chemistry 2001, 12, 917-923; E. Oh et al. , Journal of Controlled Release 1999, 57, 269-280 and J. Org. E. Oh et al. No. 6,589,548, the entirety of each of which is incorporated herein by reference.

  In another embodiment, the polymer is a biologically active molecule. In another embodiment, the polymer is a nucleic acid, nucleoside, oligonucleotide, aptamer, peptide or protein.

  In another embodiment, the polymer is a glycosaminoglycan, hyaluronan, hyaluronic acid (HA), alginate, high molecular weight polyoxyalkylene ether, PLURONIC® (a block copolymer based on ethylene oxide and propylene oxide). Polymer), polyamide, polyurethane, polysiloxane, polyacrylate, polyvinylpyrrolidone, polyvinyl alcohol, polyanhydride, polyether, polycaprolactone or polypeptide.

  In another embodiment, the polymer is a dendron. The dendron can be composed of any combination of monomers and surface modifications. Examples of useful monomers include, but are not limited to, polyamidoamine (PAMAM). Examples of useful surface modifications include, but are not limited to, cationic ammonium, N-acyl and N-carboxymethyl groups. The dendron may be multivalent anionic, polyvalent cationic, hydrophobic or hydrophilic. In certain specific embodiments, the dendron has from about 1 to about 256 surface modifying groups. In another specific embodiment, the dendron has about 4, 8, 16, 32, 64 or 128 surface modifying groups.

  The term “dendron” refers to a molecule that represents half of a dendrimer structure. Dendrons are typically built on half of the dendrimer core or by cleavage of the dendrimer core after dendrimer construction. A dendron can be composed of any combination of monomers and surface modifications. Examples of useful monomers include, but are not limited to, polyamidoamine (PAMAM). Examples of useful surface modifications include, but are not limited to, cationic ammonium modifications, N-acyl modifications, and N-carboxymethyl modifications. Alternative surface modifications allow a wide variety of properties. For example, the dendron can be multivalent anionic, polyvalent cationic, hydrophobic or hydrophilic. Dendrons can be reasonably tailored so that the exact number of monomers and surface modifying groups can be determined by the generation of dendrons. (G1, G2, G3, G4, G5 and G6 have 4, 8, 16, 32, 64 and 128 groups, respectively). Examples of dendron and dendrimer conjugation techniques are described in US Pat. No. 5,714,166 and US Patent Application Publication Nos. 2005/0009988 and 2002/0123609, which are incorporated herein by reference in their entirety. Embedded in the book.) Construction of dendron-biologically active molecule conjugates with 1: 1 stoichiometry can be achieved by reduction of disulfides in dendrimers containing a cystamine core. This reduction results in the formation of a single orthogonal sulfhydryl functionality that can be attached to all biologically active molecules that have been modified to contain a single thiol reactive group. . This can be accomplished by reacting a biologically active molecule containing an amine with a bifunctional linker containing an amine reactive group at one end and a thiol reactive group at the other end. Examples of dendritic polymers and dendritic polymer conjugates containing disulfides are found in US Pat. No. 6,020,457, which is incorporated herein by reference in its entirety. It is done. Examples of star-shaped polymer compounds having polyols that form micelles in solution can be found in US Patent Application Publication No. 2004/198641, which is incorporated herein by reference in its entirety. In one embodiment of the invention, the cyclitol moiety itself serves as the core for the dendron.

  In another embodiment, the soluble high molecular weight steric group is bovine serum albumin (BSA). Since there is a free thiol on the BSA, the aptamer containing the amine is conjugated to the BSA by using a bifunctional linker containing a thiol reactive group at one end and an amine reactive group at the other end. It becomes possible.

The polymer main chain may be linear or branched. Branched polymer backbones are generally known in the art. Typically, the branched polymer has a central branched core portion and a plurality of linear polymer chains linked to the central branched core. PEG is universally used in branched forms that can be prepared by adding ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch can also be derived from several amino acids such as lysine. Branched poly (ethylene glycol) may be represented in the general form as W (-PEG-OH) _x , where W is derived from a central portion such as glycerol, glycerol oligomers or pentaerythritol, where x is Represents the number of branches.

  One skilled in the art will recognize that the foregoing list of polymers is by no means exclusive, is exemplary only, and all polymeric materials having the aforementioned qualities are envisioned.

  The polymers of the present invention may have a molecular weight from about 800 Da to about 3,000,000 Da. In one embodiment, the polymer has a molecular weight of about 20 kilodaltons (kDa) to about 1000 kDa. In another embodiment, the polymer has a molecular weight from about 5 kDa to about 100 kDa. In certain specific embodiments, the polymer has a molecular weight of about 20 kDa. In another specific embodiment, the polymer has a molecular weight of about 40 kDa. In another specific embodiment, the polymer has a molecular weight of about 80 kDa.

  The polymers of the present invention can be conjugated to nucleic acids. The polymer can be conjugated to the nucleic acid through the 5 'end of the nucleic acid, through the 3' end of the nucleic acid, or through all positions along the nucleic acid sequence between the 5 'and 3' ends. Examples of internal nucleic acid sequence positions suitable for conjugation to the polymer (ie, positions that are neither the 5 'end nor the 3' end) are exocyclic amino groups on the base of the nucleic acid sequence, position 5 of pyrimidine nucleotides, It contains position 8 of the purine nucleotide, the hydroxyl group of phosphate, or the hydroxyl group of ribose group.

  The terminal activating group or cyclitol can also include a spacer moiety. The spacer moiety can be located near the polyalkylene oxide or near the terminal activating group. The spacer moiety can be a heteroalkyl group, an alkoxy group or an alkyl group containing up to 18 carbon atoms and including 18 carbon atoms or even further polymer chains. The spacer moiety can be added using standard synthetic techniques.

  Specific compounds of the present invention can be obtained as stereoisomers (eg, diastereomers and enantiomers) and the present invention includes all of the disclosed compounds (including pure isomers and racemic mixtures thereof). It is understood that the molecular species forms and mixtures thereof.

  It is understood that the specific compounds of the invention can be obtained as various pharmaceutically acceptable salts. The term “pharmaceutically acceptable salts” refers to non-toxic acid addition salts and alkaline earth metal salts of the compounds of the present invention. The salts can be prepared in situ during the final isolation and purification of such compounds, or individually by reacting the free base or acid functionality with, for example, a suitable organic acid or base. Typical acid addition salts are hydrochloride, hydrobromide, sulfate, disulfate, acetate, valerate, oleate, palmitate, stearate, laurate, borate , Benzoate, lactate, phosphate, tosylate, mesylate, citrate, maleate, fumarate, succinate, tartrate, glucoheptonate, lactobionate, lauryl sulfate Etc., but is not limited to these. Representative alkali metal and alkaline earth metal salts include, but are not limited to, sodium, calcium, potassium and magnesium salts.

  The invention also relates to a method of forming a conjugate comprising a polymer, a biologically active molecule, and a cyclitol that links the polymer to the biologically active molecule.

  Inositol can be prepared such that the position with the PEG chain can be selectively functionalized.

  Conditol can be used as a precursor to inositol fragments. The synthesis of chondritol is described in Balci, Pure App. Chem. 1997, 69, 97-104, Subbyaz et al. , J .; Chem. Soc. Chem. Commun. 1988, 1330-1331 (incorporated herein by reference in its entirety) and references therein.

  An example of the preparation of PEG-VEGF aptamer conjugate 1 linked by inositol is shown in FIG. 1 and Example 3. Two linkages to the activated ester derivative of 20 kDa mPEG were performed in an organic solvent (DMF / DCM 2: 1) to give the desired 40 kDa conjugate. Alternative conditions containing an aqueous buffer are optionally used. The synthesis of 2 in 1 step, 1 container, is active in situ with compound 3 by excess amounts of N, N, N ′, N′-tetramethyl-O— (N-succinimidyl) uronium (TSU) tetrafluoroborate. This was achieved by adding an aptamer after conversion.

  The preparation of inositol linker 2 and inositol activated polymer 3 is shown in FIG. 2 and Examples 1-2. Alkylation of myo-inositol-1,3,5-orthoformate gives compound 5. The presence of a symmetric element in 5 simplifies confirmation of the regiochemical reaction by NMR and ultimately eliminates the formation of diastereomeric products when conjugated to optically active APIs. The Removal of the silyl protecting group from 5 gave intermediate 6. Alternative electrophiles can be used. Next, upon successive deprotection of 7, the desired linker 2 is prepared. This was achieved by first refluxing in 0.1 M methanolic HCl for 15 minutes, then refluxing in 0.4 M methanolic hydrazine for 60 minutes.

  An example of the preparation of PEG-VEGF aptamer conjugate 10 linked by inositol is shown in FIG. 3 and Examples 4 and 5.

  A receptor binding assay to measure the ability of the VEGF aptamer conjugates of the invention to inhibit VEGF binding to VEGF-R1 (Flt-1) is described in Example 6. The results of the assay are shown in Example 7. The ability of inositol-linked VEGF aptamer conjugate 1 to inhibit VEGF binding to VEGF-R1 (Flt-1) is pegaptanib (MacI) and a 5'-5 'capped anti-VEGF aptamer It was compared with the ability of EYE002 (MacII). The PEG-VEGF aptamer conjugate linked by inositol showed an activity indistinguishable from that of pegaptanib.

EXAMPLES The following examples serve to illustrate specific useful embodiments and aspects of the invention and should not be construed as limiting the scope of the invention. Other materials and methods can be used to obtain similar results.

Synthesis of Myoinositol Linker (2) Compound 2 was prepared from commercially available myoinositol-1,3,5-orthoformate using the procedure described in Carbohydrate Research 2000, 325, 313-320.

  To a stirred solution of 4 (2.5 g, 8.2 mmol) in 75 mL of anhydrous N-methylpyrrolidone at 0 ° C. was added sodium hydride (1.6 g, 41 mmol of 60% dispersion in mineral oil). . The ice bath was immediately removed and the solution was stirred for 30 minutes at which time N- (3-bromopropyl) -phthalimide (11.0 g, 41 mmol) and tetrabutylammonium iodide (3.0 g, 8.2 mmol) were added. Added. The reaction was stirred for 18 hours at room temperature under an argon atmosphere and then quenched by the dropwise addition of 10 mL of ice-cold water. The resulting solution was extracted with chloroform (3 × 15 mL) and the organic layers were pooled and concentrated under vacuum. The crude material was eluted from the flash silica gel by using 7: 3 hexane / ethyl acetate in a Biotage Flash + chromatography system. Compound 5, 4.0 g was isolated (yield 72%).

  A solution of tetrabutylammonium fluoride (TBAF) (500 μL, 1 M) in tetrahydrofuran (THF) was added to a solution of 5 (200 mg, 295 μmol) in 2 mL of 1,4-dioxane. The solution was stirred at room temperature for 16 hours and then concentrated in vacuo. The oil obtained from above was purified by eluting from flash silica gel using 2: 3 hexane / ethyl acetate to give 52 mg (31% yield) of compound 6, oil.

Sodium hydride (65 mg, 1.61 mmol of 60% dispersion in mineral oil) was added to a stirred solution of 6 (91.0 mg, 161 μmol) in 4 mL of anhydrous DMF at 0 ° C. The ice bath was removed immediately and the solution was stirred for 30 minutes, at which point 8-bromooctanoic acid (144 mg, 645 μmol) was added. The reaction was stirred for 24 hours at room temperature under an argon atmosphere and then quenched by adding 5 mL of water dropwise at 0 ° C. The pH of the resulting solution was adjusted to 4 with 1M HCl and the solution was extracted with chloroform (4 × 4 mL). The organic layers were pooled and concentrated in vacuo. Use 1: 1 hexane / ethyl acetate (500 mL) followed by 1: 2 hexane / ethyl acetate (500 mL) followed by 100% ethyl acetate (750 mL) to remove crude material from flash silica gel. Eluted. The isolated yield of pure material was 35 mg (31% yield).
The crude product 7 was dissolved in 5 mL of methanolic 0.1 M HCl and refluxed for 15 minutes. The resulting solution was concentrated in vacuo and coevaporated 3 times with 5 mL of methanol. The obtained residue was dissolved again in 40 mL of methanol, and 503 μL of hydrazine was added. The solution was refluxed for 1 hour. The solution was then concentrated to half its original volume by collecting the distillate. The resulting solution was cooled and concentrated in vacuo. The resulting material was subjected to chromatography using a C-18 HPLC column (Waters Deltapak, 15 μm, 300, 18.5 min).

Synthesis of active PEG compound (3) To a solution of compound 2 (750 μg, 1.7 μmol) in 2 mL anhydrous dimethylformamide (DMF), mPEG-SPA (75 mg, 3.7 μmol, Nektar in 1 mL anhydrous dichloromethane (DCM). Therapeutics LN PT-01E-16) was added followed by 100 μL of pyridine. After stirring the solution for 28 hours at room temperature under an argon atmosphere, 8 mL of diethyl ether (Et ₂ O) was added to precipitate all PEG species. The PEG precipitate was deposited by centrifugation (1000 × g for 5 minutes) and the vessel was tilted to collect the solution. The resulting pellet was redissolved in ₄ mL DCM, reprecipitated with 36 mL Et ₂ O, centrifuged (1000 × g for 5 min) to pellet the PEG species. The PEG species were further dried under vacuum and then purified by SEC (Shodex KW-804, 100% PBS of uniform concentration as eluent). The 40 kDa fraction was lyophilized to a white powder and analyzed by MALDI-MS.

Synthesis of PEG-VEGF aptamer conjugate (1) linked by inositol Compound 3 (2.1 mg, 52.9 mmol) was dried by co-evaporation with toluene and then dissolved in 420 μL of DMSO. To this solution was added 16 μL of TSU solution (1 mg / mL in DMSO, 1.1 molar equivalent) with stirring under an argon atmosphere, followed by triethylamine (TEA) solution (8.9 μL / mL in DMSO, 2 0.0 eq) 1 μL was added and the reaction was stirred at room temperature for 1 h. Aptamer (1 mg) was added to the solution as a lyophilized powder and the reaction was stirred for an additional hour. To this reaction mixture, 1 mg of TSU dissolved in 100 μL of DMSO was added in 10 μL aliquots every 10 minutes until all the material was added. 5 μL of TEA was added at the completion of TSU addition.

"RPHPLC 1mLmin Compound 1 was purified using analytical HPLC (C-18, Hamilton PRP-1) using the “PDA” method (solvent A: 10 mM ammonium acetate, solvent B: acetonitrile). Two 100 μL injections were performed on the same column. The material eluted at 25.6 minutes in each trial was pooled, concentrated and then lyophilized to a white powder. This powder was redissolved in 50 μL of PBS and the aptamer concentration was measured by Beer's law calculation (260 nm) and confirmed against a standard curve of pegaptanib injection in HPLC.

Synthesis of activated myo-inositol orthoformate linker (8) To a stirred solution of myo-inositol-1,3,5-orthoformate (1.00 g, 5.26 mmol) in 20 mL of N-methylpyrrolidinone at 0 ° C. Sodium hydride (1.05 g, 26.3 mmol) was added. After the solution was stirred at 0 ° C. for 30 minutes, tetrabutylammonium iodide (10.0 mg) and allyl bromide (3.56 mL, 42.1 mmol) were added to the solution. The solution was kept in an ice bath at 0 ° C. and then warmed slowly as the ice melted. The solution was kept stirring for 14 hours at ambient temperature and then quenched by the dropwise addition of deionized water (20 mL). The reaction mixture was extracted with chloroform (3 × 20 mL), the organic layers were combined, and the product was concentrated at about 0.1 mm Hg and 30 ° C. Chromatography on silica gel (Biotage 40S flash column) using a binary mixture of hexane and ethyl acetate (85:15) and eluted. The desired product was observed on silica TLC at Rf 0.3, eluting using hexane and ethyl acetate (85:15). 1.12 g of the obtained diallyl compound was recovered (3.62 mmol, yield 69%). 1H-NMR and 13C-NMR. Calculated value 310.14, found value 311.3 (H + adduct) and 333.2 (Na + adduct) for ESI-MS (negative ion mode) C16H22O6.

Catecholborane (2 mL) was added to a stirred solution of diallyl compound (130 mg, 419 μmol) in 15 mL of 1,4-dioxane. The solution was stirred at reflux for 1 hour and then quenched by adding dropwise an alkaline hydrogen peroxide solution (5 mL of 1M NaOH and 5 mL of 30% H 2 O 2) at 0 ° C. The reaction was warmed to room temperature and stirred for 14 hours, then extracted with chloroform (3 × 25 mL), the organic layers combined, and the product concentrated in vacuo. The crude oil was subjected to chromatography on silica gel (Biotage 25 + flash column) using a binary mixture of hexane and ethyl acetate (1: 2) and eluted. The desired product was observed on silica TLC at Rf 0.22, eluting using hexane and ethyl acetate (1: 2). 38.8 mg of the obtained borate compound was recovered. ESI-MS calculated value 371.20 about (negative ion _mode) C _{16 H} 25 _{O 9,} Found 371.23 (H + adduct).

To a stirred solution of borate compound (2 mg) in 2 mL of deionized water and 2 mL of 1M NaOH, silver oxide (10 mg) was added. Then acetonitrile (4 mL) was added and the solution was stirred at room temperature for 2 hours. The solution was then filtered through filter paper to remove any silver adduct that did not precipitate as a mirror on the glass flask wall. The filtrate was concentrated and analyzed by MS. ESI-MS calculated value 406.11 about (negative ion _mode) C _{16 H} 22 _{O 12,} Found 430.01 (Na + adduct).

Synthesis of PEG-VEGF aptamer conjugate (10) linked by inositol To a stirred solution of 8 (25 μmol) in MES buffer (2.5 mL, 0.14 M, pH 5.5) in acetonitrile (2.5 mL) Of 20 kDa mPEG-NH2 (1.0 g, 50 μmol) and solid EDC (95.8 mg, 500 μmol) were added. The solution was stirred at room temperature for 14 hours before diethyl ether (90 mL) was added. The resulting precipitate was isolated and dried on a Buchner funnel. MALDI-MS showed that this material was a mixture of 20 kDa and 40 kDa species (THAP-NH4 Citrate matrix). This mixture of PEG species was dissolved in anhydrous DMSO (10 mL) and DCC (100 mg) was added. The solution was stirred for 14 hours at room temperature under an argon atmosphere. The PEG species were precipitated again by adding diethyl ether (90 mL) and isolated on a Buchner funnel. The PEG adduct was dissolved in acetonitrile (8 mL) and then added to a solution of anti-VEGF aptamer (250 mg) containing the amino terminus in borate buffer (6 mL, 100 mM, pH 8.5) followed by 14 hours. Shake. The resulting solution was concentrated and diluted to 9 mL in deionized water. Analysis of this material by reverse phase HPLC showed co-elution of product and pegaptanib standard. Purification of compound 9 using multiple elutions from C-18 HPLC yielded 8.5 mg of a 50 kDa product that co-eluted with pegaptanib.

  Compound 9 is dissolved in methanolic 0.1 M HCl and refluxed. The resulting solution is concentrated in vacuo and coevaporated with 5 mL of methanol. The resulting material 10 is subjected to chromatography on a C-18 HPLC column (Waters Deltapak, 15 μm, 300).

Receptor Binding Assay Receptor Plate Coating For each set of binding experiments, one row of 96 well Isoplate Plate (12 wells) is used. Each of the 12 wells is first coated with 2 pmoles (300 nanograms (ng)) of anti-human IgG1 Fc fragment specific antibody in 100 microliters (μl) of PBS at 4 ° C. The next day, additional protein that binds to each well is blocked by washing three times with 300 μl of Super Block blocking buffer at room temperature for 5 minutes each time. Next, each well is washed twice with 300 μL of binding buffer (PBS with 1 mM calcium chloride, 1 mM magnesium chloride, 0.01% HSA, pH 7.4) at room temperature. For KDR / Fc, 0.25 pmol (85 ng) of chimeric receptor in 100 μL of binding buffer is added to the first 11 wells, whereas the 12th well contains human ICAM-1 / Fc chimeric protein 0.5 pmol (118 ng) is given. For Flt-1 / Fc, 0.125 pmol (30.8 ng) of the chimeric receptor in 100 μL of each binding buffer is added to the first 11 wells, whereas the background control (# 12) contains human ICAM -1 / Fc chimeric protein 0.5 pmol (118 ng) is given. For neuropilin-1 / Fc, 0.2 pmol (48 ng) of the chimeric receptor in 100 μL of binding buffer is added to all 12 wells. The chimeric receptor and human ICAM-1 / Fc protein are captured on the wells by binding to an anti-human IgG ₁ Fc fragment specific antibody immobilized in each well for 2 to 3 hours at room temperature. To remove free chimeric receptor and human ICAM-1 / Fc protein, each well is washed at room temperature using 300 μL of binding buffer.

Preparation of ¹²⁵ I-VEGF ₁₆₅ -pegaptanib binding mixture Ten 5-fold dilutions of pegaptanib (tubes 1-10) ranging from 1 μM (or 2 μM) to 0.512 picomolar (pM) (or 1.024 pM) Each set of materials was combined with binding buffer (1 mM calcium chloride, 1 mM magnesium chloride, 0.01% HSA, pH 7.4) in a non-burning 1.5 mL microcentrifuge tube, each with a final volume of 100 μL each. Mix with approximately 0.01 μCi of ¹²⁵ I-VEGF _{165 in} PBS). For tubes 11 and 12, only 0.01 μCi of ¹²⁵ I-VEGF ₁₆₅ is added without any pegaptanib to serve as positive and background controls, respectively. All 12 tubes are incubated at 37 ° C. (for KDR and Flt-1) or at room temperature (for neuropilin-1) for 15-20 minutes to allow pegaptanib to bind to VEGF and equilibrate. To reach. Next, 100 μL of the binding mixture from each tube is applied to the corresponding wells on the receptor-coated Isoplate. The plates are incubated at 37 ° C. (for KDR and Flt-1) or at room temperature (for neuropilin-1) for 2-3 hours to equilibrate binding. The plate is washed 4 times with binding buffer 300 μL / well with 0.05% Tween 20 (for KDR and Neuropilin-1) or without 0.05% Tween 20 (for Flt) at room temperature. To do. The plate is air dried for about 10 minutes and about 200 μL of scintillation fluid is added to each well. Radioactivity in each well is measured by scintillation counting. For the experimental negative control, polyethylene glycol molecular weight 40,000 (40 kDa PEG) is used at the same molar concentration to replace pegaptanib in the binding assay after all the steps described above for pegaptanib.

Measurement of effective concentration for 50% inhibition of VEGF receptor binding (IC ₅₀ ) The number of counts retained in the wells (# 1 to # 11) minus the background (# 12 of wells) Calculate the ¹²⁵ I-VEGF ₁₆₅ : receptor binding ratio in the wells as the maximum binding (positive control, well # 11) minus the background (well # 12) minus the background. The binding ratios obtained at different pegaptanib concentrations were analyzed by using non-linear regression with the GraphPad PRISM program (single site competition) and the resulting curves were used to inhibit pegaptanib in inhibition of receptor binding to VEGF ₁₆₅ The half-maximal inhibition (IC ₅₀ ) of is determined. Data from an experimental negative control using PEG is analyzed by the same method.

Comparative inhibition of VEGF-R1 (Flt-1) The ability of cyclitol-linked VEGF aptamer conjugates to inhibit VEGF binding to VEGF-R1 (Flt-1) was compared to lysine-linked VEGF aptamer conjugates and non-stereoscopic. Compared to the enhanced ability of the VEGF aptamer conjugate.

Using the assay described in Example 6, the Flt-1 receptor binding activity of compound (1) was measured. For each sample,% inhibition, IC ₅₀ , IC ₅₀ range and R ² were measured. Inositol-linked VEGF aptamer conjugate (1) assay was performed in duplicate and labeled as “inositol conjugate (1) -1” and “inositol conjugate (1) -2”. The results are shown in Table 1 and FIG. “Inositol conjugate (1) -1” and “Inositol conjugate (1) -2” have IC ₅₀ of 0.5890 nM and 0.8259 nM, respectively. MacI has an IC ₅₀ of 0.4569 nM. MacII has an IC ₅₀ of 0.375 nM. The results show that inositol-linked VEGF aptamer conjugate (1) is as effective as pegaptanib (EYE-001, MacI), a lysine-linked VEGF aptamer conjugate, whereas inositol-linked and lysine Both ligated VEGF aptamer conjugates are shown to be much more effective at inhibiting VEGF binding compared to non-enhanced VEGF aptamers such as EYE-002 (MacII).

Incorporation by Reference The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. All patents, patent applications and published references cited herein are hereby incorporated by reference in their entirety.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein. Such equivalents are intended to be encompassed by the following claims.

FIG. 1 is a schematic representation for the synthesis of aptamer conjugates containing two PEG moieties and an inositol linker. FIG. 2 is a schematic representation for the synthesis of a trifunctional inositol binder. FIG. 3 is a schematic representation for the synthesis of aptamer conjugates containing two PEG moieties and an inositol linker. FIG. 4 shows VEGFR-1 (Flt) using a pegylated VEGF antagonist aptamer with an inositol linker (Compound 1), pegaptanib (MacI), and a 5′-5 ′ capped anti-VEGF aptamer (MacII). -1) A graphical representation of the results for the inhibition assay. FIG. 5 is a schematic representation of the chemical structure of a PEGylated VEGF antagonist aptamer (compound 1) having an inositol linker. FIG. 6 is a schematic representation of the chemical structure of a PEGylated VEGF antagonist aptamer (compound 10) having an inositol linker.

Claims (41)

  A conjugate comprising a polymer, a biologically active molecule, and a cyclitol group linking the polymer to the biologically active molecule.   The conjugate according to claim 1, wherein the cyclitol group is a 6-membered cycloalkane or a 5-membered cycloalkane.   The conjugate according to claim 1, wherein the cyclitol group is selected from the group consisting of tri, tetra, penta and hexahydroxycyclohexane.   The conjugate according to claim 1, wherein the cyclitol group comprises a substituent selected from the group consisting of alkyl, amino, thio, carbonyl and halogen substituents.   The conjugate according to claim 1, wherein the cyclitol group is inositol.   6. The inositol is selected from the group consisting of myo-inositol, D-kiloinositol, L-kiloinositol, mucoinositol, Scyllo inositol, alo-inositol, epiinositol, cis-inositol and neo-inositol. Conjugate.   The biologically active molecule is selected from the group consisting of nucleic acids, nucleosides, oligonucleotides, aptamers, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, cells, viruses, liposomes, microparticles and micelles. Item 2. The conjugate according to Item 1.   2. A conjugate according to claim 1 wherein the biologically active molecule is an oligonucleotide.   The conjugate of claim 1, wherein the biologically active molecule is an aptamer.   The conjugate according to claim 1, wherein the biologically active molecule is an anti-VEGF aptamer. Biologically active molecules are arranged

The conjugate according to claim 1, which is an anti-VEGF aptamer having   2. A conjugate according to claim 1 wherein the polymer is water soluble.   The conjugate of claim 1, wherein the polymer is a non-peptidic polymer.   The conjugate of claim 1, wherein the polymer is a non-nucleic acid polymer.   The conjugate according to claim 1, wherein the polymer is a high molecular weight steric group.   The polymer is a polyether polyol, polyethylene glycol, polysaccharide, carboxymethyl cellulose, cellulose derivative, glycosaminoglycan, hyaluronan, alginate, polyester, high molecular weight polyoxyalkylene ether, polyamide, polyurethane, polysiloxane, polyacrylate, polyol, The conjugate of claim 1, selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, polyanhydride, chitosan, polyaldehyde and polyether.   The conjugate of claim 1, wherein the polymer has a molecular weight of about 800 Da to about 100,000 Da.   The conjugate of claim 1, wherein the polymer has a molecular weight selected from the group consisting of about 20 kDa, 40 kDa and 80 kDa. formula:

(Where
A ₁ is a biologically active moiety or an active functional group;
R _1a , R _1b , R _1c , R _1d and R _1e are each independently selected from the group consisting of —OH, —NH ₂ and —X ₂ -L ₂ -A ₂ ;
A ₂ is selected from the group consisting of hydrogen, a polymer, a biologically active moiety and an active functional group;
L ₁ and L ₂ are each independently selected from the group consisting of a bond and a spacer moiety;
X ₁ and X ₂ are each independently selected from the group consisting of —O—, —S— and —NR ₂ —;
R ₂ is each independently selected from the group consisting of hydrogen and lower alkyl groups. )
And pharmaceutically acceptable salts thereof. A ₁ is a biologically active moiety, A compound according to claim 19. A ₁ is the active functional group, a compound of claim 19.   20. A compound according to claim 19, wherein the active functional group is an electrophilic functional group.   The active functional group is carboxylic acid, carboxylic acid halide, carboxylic acid chloride, halogen, N-succinimide carbonate, succinimidyl ester, 1-benzotriazolyl carbonate ester, N-hydroxymaleimidyl ester, vinyl 20. A compound according to claim 19 which is an electrophilic functional group selected from the group consisting of sulfone, azalactone, cyclic amidothione carbonyl, imidazole carbonyl, isocyanate and isothiocyanate. R _{1a, R 1b,} at least one of R _{1c, R 1d} and _{R 1e,} but -X _{2 -L} 2 -A 2, A compound according to claim 19. _{R 1a, R 1b, R 1c} , at least two _{R 1d} and _{R 1e,} but each is _-X 2 _-L 2 -A ₂ independently The compound of claim 19. _{R 1a, R 1b, R 1c} , at least three _{R 1d} and _{R 1e,} but each is _{-X 2 -L} 2 -A 2 independently The compound of claim 19. _{R 1a, R 1b, R 1c} , at least four _{R 1d} and _{R 1e,} but each is _{-X 2 -L} 2 -A 2 independently The compound of claim 19. _{R 1a, R 1b, R 1c} , is _{-X 2 -L} 2 -A 2 each _{R 1d} and _{R 1e} are independently a compound of claim 19. L ₁ and L ₂ are each independently of the formula:
_{- (CH 2) n - (} X 3) m - (C (X 4)) p - (X 5) q - (CH 2) r -
(N is from 0 to 10,
m is 0 or 1,
p is 0 or 1;
q is 0 or 1, and r is 0 to 10. )
20. A compound according to claim 19 which is a spacer moiety represented by: L ₁ and L ₂ are each independently
_{- (CH 2) n - (} C (X 4)) p -;
_{- (C (X 4))} p - (CH 2) r -;
_{- (C (X 4))} p -; and - _{(CH 2)} n -;
(N is 0 to 10;
p is 0 or 1; and r is 0-10. )
20. A compound according to claim 19 which is a spacer moiety represented by a formula selected from the group consisting of: Is _{X 1, X 2, X 3} , X 4 and _{X 5,} independently, are selected from the group consisting of -O- and -NH-, compound of claim 19. _{X 1, X 2, X 3} , X 4 and _{X 5} are, are each -O-, A compound according to claim 19.

(D is a biologically active moiety;
POLY is a polymer;
X ₃ , X ₄ and X ₅ are each independently selected from the group consisting of —O—, —S— and —NR ₂ —;
R ₂ is selected from the group consisting of hydrogen and lower alkyl groups;
m is 0 or 1; and n is an integer from 0 to 10. )
A compound having a formula selected from the group consisting of and pharmaceutically acceptable salts thereof.   34. The compound of claim 33, wherein D is a nucleic acid.   34. The compound of claim 33, wherein D is an aptamer.   34. The compound of claim 33, wherein D is an anti-VEGF aptamer. D is an array

34. The compound of claim 33, which is an anti-VEGF aptamer having

(G is a leaving group;
POLY is a polymer;
X ₃ , X ₄ and X ₅ are each independently selected from the group consisting of —O—, —S— and —NR ₂ —;
R ₂ is selected from the group consisting of hydrogen and lower alkyl groups;
m is 0 or 1; and n is an integer from 0 to 10. )
A compound having a formula selected from the group consisting of and pharmaceutically acceptable salts thereof.   40. The compound of claim 38, wherein G is a leaving group selected from the group consisting of halide, chloride, N-succinimide, 1-benzotriazole, N-hydroxymaleimide, azalactone, cyclic amidothione and imidazole. Construction:

(In the formula, Z is about 450.)
And pharmaceutically acceptable salts thereof. Construction:

(Z is about 450.)
And pharmaceutically acceptable salts thereof.

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