hydrogels with biodegradable crosslinking

专利摘要:
hydrogels with biodegradable crosslinking. hydrogels that degrade, under appropriate pH and temperature conditions, by virtue of the cross-linking compounds that cleave through an elimination reaction, are described. hydrogels can be used to release various agents, such as pharmaceuticals.
公开号:BR112014005390A2
申请号:R112014005390-1
申请日:2012-09-07
公开日:2021-01-26
发明作者:Gary W. Ashley；Daniel V. Santi；Jeffrey C. Henise
申请人:Prolynx Llc；
IPC主号:

专利说明:

[001] [001] This application claims the benefit of patent application having the serial number US 61 / 531,990 filed on September 7, 2011 which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION
[002] [002] A hydrogel is a three-dimensional network of chains of hydrophilic polymers, natural or synthetic, in which water (up to 99%) is the dispersion medium. Fragile macromolecules often require a well-hydrated environment for activity and structural integrity, and the high degree of hydration of a hydrogel can preserve the folding of a protein necessary for its bioactivity. The high water content of hydrogels makes the material biocompatible and minimizes the inflammatory reactions of tissues in contact with the gel, and provides flexibility comparable to that of a living tissue. Hydrogels are therefore of interest in biomedical engineering, as absorbent materials for dressings and disposable diapers, and as vehicles for prolonged drug release. Hydrogels have been prepared by chemical or physical cross-linking of natural or synthetic hydrophilic polymers.
[003] [003] Examples of hydrogels formed from crosslinking of natural polymers include those formed from hyaluronans, chitosans, collagen, dextran, pectin, polylysine, gelatin or agarose (see: Hennink, WE, et al., Adv. Drug Del. Rev. (2002) 54: 13-36; Hoffman, AS, Adv. Drug Del. Rev. (2002) 43: 3-12). These hydrogels consist of high molecular weight polysaccharides or polypeptide chains. Some examples for its use include the encapsulation of recombinant human interleukin-2 in chemically cross-linked dextran-based hydrogels (Cadee, JA, et al., J Control. Release (2002) 78: 1-13) and insulin in a complex ionically cross-linked chitosan / hyaluronan (Surini, S., et al., J. Control. Release (2003) 90: 291-301).
[004] [004] Examples of hydrogels formed by chemical or physical cross-linking of synthetic polymers include polymers of poly (lactic-co-glycolic acid) (PLGA), (meth) acrylate-oligolactide-PEO-oligolactide- (meth) acrylate, poly (ethylene) glycol) (PEO), poly (propylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronic®), poly (phosphazene), poly (methacrylates), poly (N-vinylpyrrolidone), PL (G) copolymers A-PEO-PL (G) A, poly (ethylene imine), and others (see, for example, Hoffman, AS, Adv. Drug Del. Rev. (2002) 43: 3-12). Examples of protein-polymer encapsulation using such hydrogels include insulin encapsulation in physically cross-linked PEG-g-PLGA and PLGA-g-PEG copolymers (Jeong, B., et al., Biomacromolecules (2002) 3: 865-868 ) and bovine serum albumin in chemically cross-linked acrylate-PGA-PEO-PGA-acrylate macromonomers (Sawhney AS, et al., Macromolecules (1993) 26: 581-587).
[005] [005] Depending on the pore size, degradation of a hydrogel is usually necessary for the release of the encapsulated compounds. The degradation increases the pore size, as the drug can diffuse out of the hydrogel into surrounding body fluids. Degradation is more desirable in order to remove the hydrogel from the body once the release of the drug is complete, just as surgical removal of the spent hydrogel vehicle is often painful. While many of the known hydrogels are theoretically biodegradable, in practice, degradation is uncontrolled and thus unpredictable. Thus, there is a need for new hydrogel materials that biodegrade at a predetermined rate.
[006] [006] In order to effect the degradation of the hydrogel, it is useful to have crosslinking agents that are cleavable under physiological conditions. In one approach, the enzymatic cleavage of the cross-linking agent as a substrate can affect this result. However, the dependence of enzyme degradation results on inter-patient variability, as well as the differences between in vivo and in vitro.
[007] [007] The present invention takes advantage of a cleavage mechanism described in a different context - namely for the release of drugs from macromolecular vehicles, which is described, for example in application US2006 / 0171920 and WO2009 / 158668, WO2011 / 140393, WO2011 / 140392 and WO2011 / 140376. The elimination reaction is based on a modulation group to control the acidity of the proton; ionization of this proton results in the release of the drug.
[008] [008] For the knowledge of the applicants, this mechanism was not used to establish a cleavable crosslinking agent for hydrogels that results in the degradation of the gel. Disclosure of the Invention
[009] [009] This invention provides hydrogels that break down into smaller components, soluble in a non-enzymatic process after exposure to physiological conditions and methods to prepare them. Hydrogels are prepared from crosslinking agents that are subjected to elimination reactions under physiological conditions, thus cleaving the crosslinking agent from the hydrogel's backbone. The invention also relates to the crosslinking agents themselves and intermediates in the formation of the hydrogels of the invention. Biodegradable hydrogels prepared according to the methods of the invention can be useful in several fields, including biomedical engineering, absorbent materials, and as vehicles for drug delivery.
[0010] [0010] Thus, in one aspect, the invention is directed to a hydrogel that is biodegradable under physiological conditions in which the hydrogel comprises one or more polymers cross-linked by a binder which decomposes by an elimination reaction. More specifically, hydrogels contain binders which, when disposed in the polymer residues of general formula (1): in which at least one of R1, R2 or R5, together with X, is coupled to said one or more polymers.
[0011] [0011] Alternatively, the binder is a residue of formula (2):
[0012] [0012] The definitions of R1, R2, R5, m, X, W, s, n, t and Q are defined here in detail, below. In the case of formula (2), the coupling can be through two R1 that exist in the same molecule of formula (2) or through an R1 and an R5, for example, in formula (2). This is the requirement that at least two of these substituents, as coupled to one or more polymers, simply mean that the crosslinking agent in the formula (2) itself, must have at least two attachment points. In some embodiments, the substituents R1, R2 and R5 are uniform in each of the "arms" t.
[0013] [0013] The hydrogel may also contain one or more drugs. The drug (s) can simply be contained in the pores of the hydrogel or can be coupled to a crosslinking agent, which in turn is coupled to the polymeric backbone of the hydrogel.
[0014] [0014] The invention also provides methods for preparing biodegradable hydrogels comprising simultaneously or sequentially contacting at least one reactive polymer and a cleavable cross-linking compound, wherein said cleavable cross-linking compound comprises a functional group which reacts with the reactive polymer and a portion that cleaves by elimination under physiological conditions,
[0015] [0015] Thus, drugs or another agent can simply be trapped in the hydrogel or can be included in the hydrogel by virtue of coupling through a ligand that releases the drug through an elimination reaction, thus, without the need for degradation of the gel in itself.
[0016] [0016] In another aspect, the invention provides cross-linking reagents that comprise a portion capable of being cleaved by elimination under physiological conditions, and further comprising reactive groups capable of forming covalent bonds with the reactive polymers.
[0017] [0017] In yet another aspect, the invention provides intermediates formed by the reaction of cross-linking reagents of the invention, with at least one reactive polymer. Brief Description of Drawings
[0018] [0018] Figure 1 illustrates an embodiment of the invention, in which hydrogels are formed by crosslinking a multi-arm polymer with a crosslinking agent of formula (1). A polymer of 4 arms, in which each arm is terminated with a cyclooctin (CO) and a crosslinking agent of formula (1) in which an R5 is (CH2) RN3 and X is O-CO-NH-CH2CH2 (OCH2CH2) p-N3 (Example 20) provides a 4x4 hydrogel comprising a beta-eliminative linker L at each crosslink. The rate of degradation of the hydrogel is controlled by the appropriate choice of the modulating group R1 in linker L. Also illustrated is the formation of (1) by reacting a succinimidyl carbonate with an amino-PEG-azide.
[0019] [0019] Figures 2A and 2B illustrate two embodiments of the invention, in which the hydrogels are formed by crosslinking of multi-arm polymers with the compounds of formula (2). Panel A shows crosslinking of a 4-arm polymer, where each arm is terminated with a cyclooctin (CO) with another 4-arm polymer of formula (2), where each arm is terminated with a beta-azide binder eliminative (L2-N3). The resulting 4x4 hydrogel comprises a beta-scavenger binder at each crosslink. The rate of degradation of the hydrogel is controlled by the appropriate choice of the L2 ligand. Panel B shows the crosslinking of a polymer of 8 arms, in which 4 arms are terminated with a cyclooctin (CO) and the remaining arms are connected to an erosion probe (EP) or a bound release drug (L1-D). Crosslinking with a 4-arm polymer, where each arm is terminated with a beta-eliminative azide ligand (L2-N3) provides a 4x8 hydrogel comprising an L2 beta-eliminative ligand in each cross-linking and comprising drug D covalently linked via another beta-eliminative L1 ligand. The rates of drug release from the hydrogel and the degradation of the hydrogel are controlled by adequate choices of the ligands L1 and L2, respectively.
[0020] [0020] Figure 3 shows the degradation of the PEG hydrogels of 4x4, at pH 7.4, 37 ° C, as measured by solubilized fluorescein-PEG fragments described in example 28; reverse gelation times using different modulators: R1 = (4-chlorophenyl) SO2, 30 hours, R1 = phenyl-SO2, 55 hours; R1 = O (CH2CH2) 2NSO2, 22 days; R1 = CN, 105 days. Solubilized fluorescein was used as an erosion probe, with defrost times being defined as the complete dissolution point.
[0021] [0021] Figure 4 shows the relationship between the degelification times measured in 4x4 hydrogels of example 28, and the release rate of 5- (aminoacetamido) fluorescein measured from soluble PEG conjugates using equivalent ligands.
[0022] [0022] Figure 5 shows the pH dependence for degelification of 4x4 PEG hydrogels of example 28, in which L2 has modulator R1 = (4-chlorophenyl) SO2.
[0023] [0023] Figure 6 shows the correlation between pH and defrosting time for the gels of example 28.
[0024] [0024] Figure 7 shows the release of the 5- (aminoacetamido) fluorescein substitute drug from the 4x8 PEG hydrogels of example 29.
[0025] [0025] Figure 8 shows the pH dependence of the release of the substituted drug 5- (aminoacetamido) fluorescein from 4x8 PEG hydrogels of example 29. The half-lives for the release were measured at pH 7.4 (23.0 H); pH 7.8 (14.0 h); pH 8.1 (6.9 h); pH 8.4 (3.2 h); pH 8.7 (1.9 h); and pH 9.0 (1.1 h).
[0026] [0026] Figure 9 shows the correlation between pH and half-lives for drug release from hydrogels 8x4 of example 29.
[0027] [0027] Figure 10 shows the release of the exenatide peptide (exendin-4) covalently linked through a releasable linker L1 having modulator R11 = CH3SO2 of a PEG hydrogel 8x4 cross-linked with degradable ligands L2 having modulator R1 = CN, to the pH 8.8, 37 ° C (example 33). Knowing the pH dependence of the ligand release and gel degradation, the scale corresponding to pH 7.4 is also given. Total solubilized exenatide (circles) is released with apparent t1 / 2 = 20.7h at pH 8.8 which corresponds to t1 / 2 = 21 days at pH 7.4. Degelification (squares = solubilized fluorescein erosion probe) was observed at 172h at a pH of 8.8, corresponding to pH 7.4 for 180 days.
[0028] [0028] Figure 11 illustrates an embodiment of the invention, in which the drug-releasing hydrogels are formed by the reaction of a first polymer comprising at least two orthogonal functional groups (B and C) are reacted with a drug linker of formula (3) wherein the drug linker comprises a functional group (B ') which reacts with only one of the orthogonal functional groups (B) present in the first polymer, linking the drug linker to the first polymer through the residue B*. The remaining orthogonal functional group (C) on the first resulting drug-loaded polymer is used to form a hydrogel by reaction with a compound of formula (1) or (2), where these compounds comprise a functional group (C ') that reacts with only the remaining orthogonal functional group present on the first drug loaded polymer to crosslink the hydrogel through residue C *. Modes for carrying out the invention
[0029] [0029] The hydrogels of the invention are crosslinked polymer (s) by binders that separate the polymer (s) by
[0030] [0030] In the elimination mechanism, the illustrated proton H is removed by a base; in an aqueous medium, the base is normally hydroxide ion such that the rate of elimination is determined by the pH of the medium. Under physiological conditions, the pH of the surrounding fluid and that permeates the hydrogel appears to be the predominant factor in controlling the elimination rate. Thus, when X and Y represent chains within a polymer matrix located in a physiological environment, pH-dependent elimination results in the rupture of the bond between X and Y and subsequent biodegradation of the polymeric matrix in a process that does not require the action of enzymes.
[0031] [0031] By "a portion capable of being cleaved by elimination under physiological conditions" means a structure comprising an HC group (CH = CH) mCX in which m is 0 or 1 and X is an leaving group, in which a reaction Elimination, as described above to remove the HX elements, can occur at such a rate that the reaction half-life is between 1 and 10,000 hours under physiological pH and temperature conditions. Preferably, the reaction half-life is between 1 and 5000 hours, and more preferably between 1 and 1000 hours, under physiological conditions of pH and temperature. For physiological conditions of pH and temperature it means a pH value between 7 and 8 and a temperature between 30 and 40 ° C.
[0032] [0032] It should be noted that when the ranges are given in this application, such as 1 to 1000 hours, the numbers of intermediate intervals must be considered as described as if specifically and expressly defined. This avoids the need for a long list of numbers and applicants intend to clearly include any arbitrary gap between the outer limits. For example, the range from 1 to 1,000 also includes 1 to 500 and 2 to 10.
[0033] [0033] By hydrogel means a three-dimensional polymeric network, predominantly hydrophilic comprising a large amount of water, formed by chemical or physical cross-linking of natural or synthetic homopolymers, copolymers, or oligomers. Hydrogels can be formed by crosslinking polyethylene glycols (considered to be synonymous with polyethylene oxides), polypropylene glycols, poly (N-vinyl pyrrolidone), polymethacrylates, polyphosphazenes, polylactides, polyacrylamides, polyglycolates, polyethylene imines, agarose, agarose, agarose , gelatin, collagen, polylysine, chitosans, alginates, hyaluronans, pectin, carrageenan. The polymer can be a multi-arm polymer, as illustrated below.
[0034] [0034] Hydrogels can also be sensitive to the environment, for example, being liquid at low temperature but gelling at 37 ° C, for example, hydrogels formed from poly (N-isopropylacrylamide).
[0035] [0035] By mesoporous hydrogel means a hydrogel with pores between approximately 1 nm and approximately 100 nm in diameter. The pores are mesoporous hydrogels large enough to allow free diffusion of biological molecules, such as proteins. By macroporous hydrogel is meant a hydrogel with pores larger than approximately 100 nm in diameter. By microporous hydrogel is meant a hydrogel with pores smaller than about 1 nm in diameter.
[0036] [0036] By reactive polymer and reactive oligomer is meant a polymer or oligomer containing functional groups that are reactive with other functional groups, most preferably under mild conditions compatible with the requirements of peptides, proteins and other stability biomolecules. Suitable functional groups found in reactive polymers include maleimides, protected thiols or thiols, alcohols, acrylates, acrylamides, protected amines or amines, carboxylic acids or protected carboxylic acids, azides, alkines including cycloalkines, 1,3-dienes including cyclopentadienes and furans, alpha -halocarbonyls, and N -hydroxy-succinimidyl, N -hydroxysulfosuccinimidyl, or nitrophenyl esters or carbonates.
[0037] [0037] By functional group capable of binding to a reactive polymer is meant a functional group that reacts with a corresponding functional group of a reactive polymer to form a covalent bond to the polymer. Suitable functional groups capable of binding to a reactive polymer include maleimides, protected thiols or thiols, acrylates, acrylamides, protected amines or amines, carboxylic acids or protected carboxylic acids, azides, alkines including cycloalkines, 1,3-dienes including cyclopentadienes and furans , alpha-halocarbonyls, and N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or nitrophenyl esters or carbonates.
[0038] [0038] By biodegradable hydrogel means a hydrogel that loses its structural integrity through the cleavage of chemical bonds of the components under physiological conditions of pH and temperature. Biodegradation can be enzymatically catalyzed or it can only be dependent on environmental factors such as pH and temperature. Biodegradation results in the formation of fragments of the polymeric network that are small enough to be soluble and, therefore, subjected to the clearance of the system through normal physiological pathways.
[0039] [0039] By cross-linking reagent means a compound comprising at least two functional groups that are capable of forming covalent bonds with one or more reactive polymers or oligomers. Typically, reactive polymers or oligomers are soluble, and the cross-linking results in the formation of an insoluble three-dimensional mesh or gel. The two functional groups of the cross-linking reagent can be identical (homobifunctional) or different (hetero). The functional groups of the heterobifunctional cross-linking reagent are chosen so as to allow the reaction of a functional group with a cognate group of the reactive polymer or oligomer and the reaction of the second functional group with a cognate group of the same or a different reactive polymer or oligomer. The two functional groups of a bifunctional cross-linking reagent are chosen so that they are not reactive with themselves, that is, they are not cognates.
[0040] [0040] Examples of reactive cognate pairs of functional groups include: Azide + acetylene, cyclooctin, maleimide Thiol + maleimide, acrylate, acrylamide, vinyl sulfone, vinyl sulfonamide, halocarbonyl Amine + carboxylic acid, activated carboxylic acid Maleimide + 1,3-diene cyclopentadiene furan
[0041] [0041] Thus, as an example of a heterobifunctional cross-linking reagent it can be prepared having an azide and an amine group, but not an azide and a cyclooctin group.
[0042] [0042] "Substituted" means an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group with one or more substituent groups in place of one or more hydrogen atoms. Substituent groups can generally be selected from halogen including F, Cl, Br and I; lower alkyl including linear, branched, and cyclic; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy including linear, branched and cyclic; SH; lower alkylthio including linear, branched, and cyclic; amino, alkylamino, dialkylamino, silyl including alkylsilyl,
[0043] [0043] The properties of R1 and R2 can be modulated by the optional addition of electron removal substituents or electron donors. By the term "electron donor group" is meant a substituent resulting in a decrease in the acidity of R1R2CH; electron donor groups are typically associated with negative Hammett σ or Taft σ * constants and are well known in the art of physical organic chemistry (Hammett constants refer to aryl / heteroaryl substituents, Taft constants refer to substituents in non-aromatic portions). Examples of suitable electron donating substituents include, but are not limited to, lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl. Similarly, by "electron removal group" is meant a substituent resulting in an increase in the acidity of the R1R2CH group; electron removal groups are typically associated with positive Hammett constants σ or Taft σ * and are well known in the art of physical organic chemistry. Examples of suitable electron removal substituents include, but are not limited to, halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C (= O) -Rx, where Rx is H, lower alkyl, lower alkoxy, or amino, or - S (O) mRY, where m = 1-2 and Ry is lower alkyl, aryl, or heteroaryl. As is well known in the art, the electronic influence of a substituent group may depend on the position of the substituent. For example, an alkoxy substituent in the ortho- or para- position of an aryl ring is an electron donor, and is characterized by a negative Hammett constant σ, while an alkoxy substituent group in the meta position of the aryl ring is a receptor of electrons and is characterized by a positive Hammett constant σ. A table of constant values for Hammett σ and Taft σ * is given below.
[0044] [0044] "Alkyl", "alkenyl" and "alkynyl" include linear, branched or cyclic hydrocarbon groups of 1-8 carbon atoms or 1-6 carbon atoms or 1-4 carbon atoms where alkyl is a hydrocarbon saturated, alkenyl includes one or more carbon-carbon double bonds and alkynyl includes one or more carbon─carbon triple bonds. Unless otherwise stated, they contain 1-6 carbon atoms.
[0045] [0045] The term "aryl" includes aromatic hydrocarbon groups of 6-18 carbon atoms, preferably 6-10 carbon atoms, including groups such as phenyl, naphthyl, and anthracenyl. "Heteroaryl" includes aromatic rings comprising 3-15 carbon atoms that contain at least one N, O or S atom, preferably 3-7 carbon atoms containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.
[0046] [0046] "Halogen" includes fluorine, chlorine, bromine and iodine.
[0047] [0047] "Maleimide" is a group of the formula
[0048] [0048] The terms "protein" and "peptide" are used interchangeably, regardless of the length of the chain, and these terms further include pseudo peptides which comprise bonds except amide bonds, such as CH2NH2 bonds, as well as peptideomimetics.
[0049] [0049] The terms "nucleic acids" and "oligonucleotides" are also used interchangeably regardless of the length of the chain. The nucleic acids or their oligonucleotides can be single-stranded or double-sided, or they can be DNA, RNA, or their modified forms with altered bonds, such as phosphodiesters, phosphoramidates and the like. For both proteins and nucleic acids useful as drugs in the invention, these terms also include those with side chains not found in nature, in the case of proteins and bases that are not found in nature, in the case of nucleic acids.
[0050] [0050] Small molecules in the context of drugs is a well-understood term in the art, and is intended to include compounds other than proteins and nucleic acids that are either synthesized or isolated from nature and, in general, do not resemble proteins or nucleic acids. Typically, they have molecular weights <
[0051] The present invention provides cross-linking reagents that comprise a portion capable of being cleaved by elimination under physiological conditions, and further comprising reactive groups capable of forming covalent bonds with the reactive polymers. In one embodiment, the cross-linking reagents are of formula (1) m is 0 or 1; X comprises a functional group capable of binding to a reactive polymer that is susceptible to elimination from the binder under physiological conditions and a second reactive group Z2 that couples to a reactive polymer; wherein at least one of R1, R2, R5 and comprises a first functional group Z1 capable of bonding to a polymer; at least one or both R1 and R2 independently represents CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R9 is independently, H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted; wherein R1 and R2 can be joined to form a 3- to 8-membered ring; and wherein one and only one of R1 and R2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (OCH2CH2) p O-alkyl, where p = 1 to 1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted.
[0052] [0052] The crosslinking reagents of formula (1) comprise a group capable of being cleaved by elimination under physiological conditions. Thus, hydrogels formed using crosslinking reagents of formula (1) are biodegradable under physiological conditions. The elimination mechanism is dependent on the pH and temperature of the medium. Although the cross-linking reagents are stable in the sense of cleavage by eliminating low pH and temperature, at physiological values of pH (approximately 7.4) and temperature (approximately 37 ° C) the elimination occurs at a rate that is essentially controlled by the groups R1 and R2, and to a lesser extent by groups R5.
[0053] [0053] The elimination reaction rates are predictable based on the structures of the groups R1, R2, R5. Groups R1 and R2 that remove electrons accelerate the elimination reaction, while groups R1 and R2 electron donors that delay the elimination reaction, so that the obtained rates can be varied in order to provide ligands having elimination half-lives at from minutes to years. R5 alkyl groups delay the elimination reaction slightly compared to R5 aryl groups. By changing groups R1 and R2, it is thus possible to control the rate at which elimination occurs, and consequently, the hydrogel's biodegradation rate can be controlled across a wide range. Hydrogels formed using crosslinking reagents of formula (1) are thus expected to find use in applications where a temporary gel matrix is required, for example, as vehicles or drug delivery tanks or as temporary supports for regeneration of the fabric. X embodiments
[0054] [0054] X comprises a functional group capable of binding to a reactive polymer and is also susceptible to elimination under physiological conditions. Typically, the resulting HX acid will have a pKa of 10 or less, preferably a pKa of 8 or less. Examples of suitable groups X thus include carbonates, carbonyl halides, carbamates, thioethers, esters, and optionally substituted phenols. In an embodiment of the invention, X is an activated carbonate, such as succinimidyl carbonate, sulfosuccinimidyl carbonate, or nitrophenyl carbonate. In another embodiment of the invention, X is a carbonyl halide, such as O- (C = O) Cl or O (C = O) F. In another embodiment of the invention, X is a carbamate of the formula where T * is O, S or NR6 where R6 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl or optionally substituted heteroarylalkyl; z is 1 to 6; and Y is absent or is OR7 or SR7, where R7 is optionally substituted by alkylene, optionally substituted phenylene or (OCH2CH2) p, where p = 1 to 1000, and Z2 is a functional group capable of bonding with a reactive polymer. In a particular embodiment of the invention, Y is (OCH2CH2) p, where p = 1 to 1000; or Y is (OCH2CH2) p, where p = 1 to 100; or Y is (OCH2CH2) p, where p = 1 to 10.
[0055] [0055] In another embodiment, X is OR7 or SR7, where R7 is optionally substituted alkylene, optionally substituted phenylene or (OCH2CH2) p, where p = 1 to 1000, and Z2 is a functional group capable of bonding with a reactive polymer.
[0056] [0056] In certain embodiments, the invention provides cross-linking reagents of formula (1) in which R5 is the substituent between R1, R2 and R5, which further comprises a functional group capable of binding to a polymer. In more particular embodiments, the invention provides cross-linking reagents of formula (1) in which one of R5 further comprises a functional group capable of binding to a polymer and the other R5 is H.
[0057] [0057] Thus, the invention provides cross-linking reagents of formula (1a) where m is 0 to 1; r is 2 to 8; and R1, R2, R5, m, X and Z are as defined above. In a more particular embodiment, the invention provides cross-linking reagents of formula (1a) where R5 is H. In an even more specific embodiment, the invention provides cross-linking reagents of formula (1a) in which R1 is CN or R8SO2 , where R8 is optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or OR9 or NR92 where each R9 is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; R2 and R5 are H, and m = 0.
[0058] [0058] In another embodiment, the invention provides cross-linking reagents of formula (1a) where X is of the formula where T * is O, S or NR6 where R6 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl or optionally substituted heteroarylalkyl; z is 1 to 6; and Y is absent or is OR7 or SR7, where R7 is optionally substituted by alkylene, optionally substituted phenylene or (OCH2CH2) p, where p = 1 to 1000, and Z2 is a functional group capable of bonding with a reactive polymer. In a particular embodiment of the invention, Y is (OCH2CH2) p, where p = 1 to 1000; or Y is (OCH2CH2) p, where p = 1 to 100; or Y is (OCH2CH2) p, where p = 1 to 10.
[0059] [0059] In another embodiment of the invention, X is OR7 or SR7, where R7 is optionally substituted by alkylene, phenylene or (OCH2CH2) p optionally substituted, where p = 1 to 1000, and Z2 is a functional group capable of bond with a reactive polymer.
[0060] [0060] In one embodiment, the invention provides cross-linking reagents of formula (1b) where m is 0 to 1 and R1, R2, R5, m, X and Z2 are as defined above. In a more particular embodiment, the invention provides cross-linking reagents of formula (1b) where R5 is H. In an even more specific embodiment, the invention provides cross-linking reagents of formula (1b) in which R1 is CN or R8SO2 , where R8 is optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or OR9 or NR92 where each R9 is independently H or an alkyl group, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; R2 and R5 are H, and m = 0.
[0061] [0061] The methods for the preparation of compounds of formula (1) in which X is OH, Cl or O-succinimidyl has previously been disclosed in patent publications WO2009 / 158668, WO2011 / 140393 and WO2011 / 140392. Compounds of formula (1) in which X is a carbamate of formula can be prepared from compounds of formula (1) in which X is Cl or O-succinimidyl by reaction with amines of the formula. R6-NH- (CH2) ZY-Z2 using methods illustrated in the working examples below.
[0062] [0062] In another embodiment of the invention, the multivalent cross-linking reagents of formula (2) are provided in which at least one of R1, R2 and R5 comprises a functional group Z1 capable of binding to a polymer, and are of otherwise defined as in formula (1); where m is 0 or 1; n is 1 to 1000; s is 0 to 2; t is 2, 4, 8, 16 or 32, W is O (C = O) O, O- (C = O) NH, O (C = O), S,, or; and Q is a nucleus group having a valence = t, where t = 2, 4, 8, 16, or 32.
[0063] [0063] The nucleus Q is a group of valence = t, which connects the multiple arms of the cross-linking reagent. Typical examples of Q include C (CH2) 4 (t = 4), in which the multiple arm reagent is prepared based on a pentaerythritol core; (t = 8), in which the multi-arm reagent is prepared based on a hexaglycerin core; and (t = 8), in which the multi-arm reagent is prepared based on a tripentaerythritol core.
[0064] [0064] The compounds of formula (2) can be prepared by reacting a polyethylene glycol of multiple arms, with a reagent of formula (1). A variety of multi-arm polyethylene glycols are commercially available, for example, from NOF Corporation and JenKem Technologies.
[0065] [0065] In a particular embodiment of the present invention, t is 4. In another embodiment of the invention, t is 8. Preparation of hydrogels
[0066] [0066] In another aspect, the invention provides methods for the preparation of biodegradable hydrogels comprising or simultaneously or sequentially contacting at least one reactive polymer and a cleavable crosslinking compound wherein said cleavable crosslinking compound comprises a functional group which reacts with the reactive polymer and a portion that cleaves by elimination under physiological conditions.
[0067] [0067] In an embodiment of the invention, biodegradable hydrogels are formed by reacting a single reactive polymer and a cleavable crosslinking compound wherein said cleavable crosslinking compound comprises a functional group that reacts with the reactive polymer and a portion also including a functional group that reacts with a reactive polymer that cleaves by elimination under physiological conditions. In this embodiment, the reactive polymer will be multivalent, in order to allow the formation of nodes in the three-dimensional matrix of the hydrogel. As an illustration of this process, a multi-arm PEG in which each arm is terminated with a reactive functional group Z3, as defined below, is allowed to react with a crosslinking reagent of formula (1) or (2) to form a hydrogel. Multi-arm PEGs are commercially available in a variety of sizes and with a variety of reactive functional groups, for example, from NOF Corporation and JenKem Technologies. As another illustration of this method, a linear polymer comprising multiple copies of a reactive functional group Z3 is allowed to react with a crosslinking reagent of formula (1) or (2) to form a hydrogel. Illustrations of such linear polymers that contain several Z3 groups are hyaluronic acid, carboxymethyl cellulose, polyvinyl alcohol, poly (2-hydroxyethyl methacrylate), dextran, collagen, chitosan, alginate and agarose.
[0068] [0068] In another embodiment, the invention provides methods for the formation of biodegradable hydrogels by reacting a first reactive polymer, a second reactive polymer, and a cleavable cross-linking compound comprising a first functional group that reacts with the first reactive polymer, a second functional group that reacts with the second polymer, and a portion that cleaves by elimination under physiological conditions. The first and second functional groups can be the same or different. For the formation of a three-dimensional gel network of the reactive components (first reactive polymer, second reactive polymer if any) will be multi-arms and, thus, serve to form nodes in the gel matrix. In preferred embodiments of the invention, this anode-reactive component comprises at least 3 arms and more preferably at least 4 arms.
[0069] [0069] In each embodiment, the reactive polymers can be homopolymeric or copolymeric polyethylene glycols, polypropylene glycols, poly (N-vinylpyrrolidone), polymethacrylates, polyphosphazenes, polylactides, polyacrylamides, polyglycolates, polyethylene imines, agarose, gelatines, gelatines, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins, gelatins. , polylysines, chitosans, alginates, hyaluronans, pectins, carrageenans that comprise or reactive functionalities suitable in their native state or have been derived in order to understand suitable reactive functionalities.
[0070] [0070] In some embodiments, the polymers include multivalent branched structures of the formula [Z3- (CH2) s- (CH2CH2O) n] tQ, where Z3 is a reactive functional group selected from the options indicated above for Z1 and Z2, s is 0 to 2, Q is a group of the multivalent nucleus having valence t, where t is 2, 4, 8, 16 or
[0071] [0071] The gel forming reactions can be carried out in a variety of suitable solvents, for example, water, alcohols, acetonitrile, or tetrahydrofuran, and are preferably carried out in an aqueous medium.
[0072] [0072] The formation of hydrogels can be carried out step by step or in a concerted manner. Thus, in an embodiment of the invention, a first reactive polymer is allowed to react with a cross-linking reagent of formula (1) or (2) in order to form an intermediate non-cross-linked combination, which is optionally isolated. This uncrosslinked combination is then allowed to react with the second reactive polymer to form the final crosslinked gel. In another embodiment of the invention, the first reactive polymer, second reactive polymer, and crosslinking reagent of formula (1) or (2) are combined and allowed to react and form the hydrogel, in a single operation.
[0073] [0073] In one embodiment, the invention provides methods for the formation of hydrogels by crosslinking a polymer with a crosslinking reagent of formula (1). Depending on the functionality present, the polymer may be in its native state or it may first be derived by methods known in the art to introduce a functionality that is reactive by crosslinking with the functionality in the compound of formula (1). In this embodiment, the two functional groups capable of reacting with a polymer of the compound of formula (1) are typically the same. An example of this embodiment is illustrated in figure 1. As shown, a cleavable crosslinking agent of formula (1) with two functional groups azide cross-links a polymer with 4 arms with functional cyclooctin groups. Alternative gels with other embodiments as noted above for Z1, Z2 and Z3 are prepared to provide similar or identical results.
[0074] [0074] In another embodiment, the invention provides methods for the formation of hydrogels by crosslinking two differently substituted polymers one of which comprises a crosslinking agent susceptible to elimination. Two examples of this embodiment are illustrated in figure 2. Panel A shows crosslinking of a first 4-arm polymer, where each arm is terminated with a cyclooctin (CO) with a second 4-arm polymer, where each arm is terminated with a beta-eliminative azide binding compound of formula (1) (L2-N3) which is thus a compound of 4 arms of formula (2). The resulting 4x4 hydrogel comprises a beta-eliminative binder at each crosslink. The gel therefore contains alternating nodes derived from the 4-arm polymer and formula (2).
[0075] [0075] As illustrated in Panel B, this method can also use polymers with a greater number of arms. As shown, some of the 8-arm polymer arms can be derived for a drug by coupling to a compound of formula (3) below. In addition, or instead, one or more of the arms can be coupled to a marker compound, such as a fluorescent dye, in order to assess the rate of disintegration of the gel as a function of environmental conditions and / or as a function of nature of R1, R2 and / or R5. This "erosion probe" allows design of gels with desired disintegration rates.
[0076] [0076] In one aspect of the design, a drug can simply be included in the gel pores through gel formation in the presence of the drug and the rate of drug release is controlled by the appropriate choice of substituents in the cross-linking compounds that result in the formation of gel.
[0077] [0077] Gels can also be prepared that contain drug both included in the pores and coupled to the polymer through a bond as shown in formula (3) below. Release rates from the connection and pores can be compared.
[0078] [0078] In the third alternative, the drug can be supplied simply, in the form of formula (3) so that the release rate from the gel is determined exclusively by the drug elimination reaction from the gel.
[0079] [0079] In another aspect, the invention provides hydrogels that are formed according to the above methods. These hydrogels can comprise a variety of hydrophilic polymers, included as described above, native or modified forms of polyethylene glycols, polypropylene glycols, poly (N-vinylpyrrolidone), polymethacrylates, polylactides, polyphosphazenes, polyacrylamides, polyglycolates, polyethylene imines, gelatines, dextrates , collagens, polylysines, chitosans, alginates, hyaluronans, pectins, carrageenans, or the illustrated multibrace polymers, and are characterized by their crosslinking which includes at least a portion capable of being cleaved by elimination under physiological conditions. These hydrogels are therefore biodegradable through a pH-dependent process.
[0080] [0080] Through appropriate choice of reagents and stoichiometry, the pore size of the resulting hydrogels can be determined. The hydrogels of the invention can be microporous, mesoporous, or macroporous, and can have a range of biodegradation rates that are determined by the nature of the cross-linking reagents used in their preparation.
[0081] [0081] The hydrogels of the invention may also contain residual reactive groups that were not consumed during the gelation process, either through the chosen stoichiometry, through incomplete crosslinking, or through the incorporation of functional groups that do not participate in the gelation process due orthogonal reactivity. These residual reactive groups can be used to further modify the resulting hydrogel, for example, by covalent bonding of drugs or prodrugs. In an embodiment of the invention, residual reactive groups are used to fix prodrugs that comprise a drug attached to a linker that subsequently releases the drug from the hydrogel matrix. In a more particular embodiment of the invention, the drug is released from the hydrogel matrix via an elimination mechanism. The use of elimination binders for drug conjugation is described, for example, in PCT publications WO2009 / 158668 and WO2011 / 140393, which are incorporated herein by reference.
[0082] [0082] An embodiment of degradable drug-releasing hydrogels of the invention is illustrated in figure 2B and exemplified in working examples 29 and 33 below. The reaction of a subset of the functional groups on a first polymer with a releasable drug-linker, wherein the linker comprises a first modulator group that controls the rate of drug release, provides a drug-loaded intermediate polymer; the residual functional groups are reacted with a cross-linking reagent of formula (1) or (2) which comprises a second modulator group that controls the rate of degradation of the hydrogel to provide a degradable drug-loaded hydrogel. By properly selecting the modulating groups present in the drug binder and the cross-linking reagent, the rates of drug release and hydrogel degradation can be controlled. In a method of the invention, the first polymer is treated with the drug-linker in a first step; the drug loaded intermediate polymer is optionally isolated; and the hydrogel is formed by reacting with the cross-linking reagent in a separate step. In a second method of the invention, the first polymer, drug-linker and cross-linking reagent are combined in one step. If all the reactive functionalities in the polymers are not consumed by any binding to the binding drug or the crosslinking, the excess of functionalities can be optionally capped by reaction with suitable reagents. For example, excess cyclooctins can be capped by reacting with azides-PEG, such as azido-heptaethylene glycol.
[0083] Thus, in an embodiment of the invention, a method for the formation of degradable drug-releasing hydrogels is provided consisting of the steps of: (a) reacting a first multivalent polymer comprising reactive functionalities with a substoichiometric amount of a drug binder that has the formula (3)
[0084] [0084] The preparation of binding drugs of formula (3) is detailed in PCT publications WO2009 / 158668 and WO / 2011/140393, which are incorporated herein by reference.
[0085] [0085] The linked drug D can be a small molecule or a polypeptide, including peptides and proteins. Working example 32 below describes the preparation of a degradable drug-release hydrogel, where D is the exenatide peptide, which has the sequence: H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Leu-Ser- Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Fen-Ile-Glu-Trp-Leu -Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly- Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 1).
[0086] [0086] In an embodiment of the invention, the exenatide peptide is coupled to the linker via an amino group to provide where R1, R2, R5 and m are as defined for formula (3) above. In certain embodiments, m = 0, R2 is H, R5 is H, and the other R5 is (CH2) nY where n = 1 to 6 or CH2 (OCH2CH2) pY where p = 1 to 1000 and Y is a group comprising an N3, SH, StBu, maleimide, 1,3-diene, cyclopentadiene, furan, alkaline, cyclooctin, acrylate, acrylamide, vinyl sulfone, or vinyl sulfonamide group. In certain embodiments of the invention, R1 is CN or SO2R3, where R3 is optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, OR9, or N (R9) 2, where each R9 is independently optionally substituted alkyl , optionally substituted aryl, optionally substituted heteroaryl, and where N (R9) 2 can form a heterocyclic ring. The linker can be attached to any free amino group on the peptide, i.e., the N-terminal amine or any side chain amine, such as epsilon-amino groups on lysine.
[0087] [0087] In a specific embodiment of the invention, the linker drug of formula (3) comprises a reactive group of azide in an R5. A substoichiometric amount of the binding drug is thus reacted with a multi-arm polymer comprising reactive cyclooctin groups at the end of each arm. Examples of reactive cyclooctin groups include those effective in copper-free 1,3-dipolar cycloaddition reactions with azides, including, for example, dibenzocyclooctins, dibenzoazacyclooctins (DBCO), difluorocyclooctins (DIFO) and tense bicyclic cyclooctins such as bicyclononines (BCN) .
[0088] [0088] In an embodiment of the invention, the first polymer comprises at least 8 arms, each arm terminated with a reactive functional group. As shown in figure 2B, 3 arms of the first polymer are used for crosslinking to compounds of formula (1) or (2). In a preferred embodiment of the invention, at least 4 arms of the first polymer are used for the cross-linking of compounds of formula (1) or (2). Thus, the substoichiometric amount of the binding drug used can vary between 0.01 to 5 molar equivalents with respect to the first polymer, which leads to a charge of 0.01 to 5 molecules of drug D per first 8-arm polymer. In an embodiment of the invention, the stoichiometric amount of binding drug used can vary from 0.1 to 5 molar equivalents with respect to the first polymer. In another embodiment of the invention, the stoichiometric amount of binding drug used can vary from 1 to molar equivalents with respect to the first polymer.
[0089] [0089] Thus, in certain embodiments of the invention, a degradable exenatide-releasing hydrogel is prepared by reacting a first multivalent polymer comprising a cyclooctin group at the end of each arm with a substoichiometric amount of a binding drug of formula ( 4)
[0090] [0090] The first polymer loaded with exenatide is then reacted with a cleavable compound of formula (1) or (2) to form the degradable hydrogel for exenatide release. In certain embodiments of the invention, the first exenatide releasing polymer is an 8-arm polyethylene glycol, and the cleavable compound used for the formation of hydrogel is a compound of formula (2). In certain embodiments of the invention, the cleavable compound used for the formation of hydrogel is a compound of formula (2) where m is 0, n is 10 to 150, s is 0, t is 4, and Q is C ( CH2) 4.
[0091] [0091] As described above, the rates of drug release and hydrogel degradation are mainly controlled by the choice of groups R1 and R2 in the binding drugs and cross-linking agents, respectively. The chosen drug release rate is typically determined by the desired pharmacokinetics of the drug, for example, the maximum and / or minimum concentrations of free drug over the duration of administration, as already described in
[0092] [0092] In another embodiment of the invention, degradable drug-releasing hydrogels are prepared by a method in which a first multi-arm polymer in which each arm is terminated by a group comprising at least two orthogonal functional groups is reacted with a binding drug of formula (3) wherein the binding drug comprises a functional group which reacts with only one of the orthogonal functional groups present in the first polymer. The remaining orthogonal functional group in the first resulting drug-loaded polymer is used to form a hydrogel by reaction with a compound of formula (1) or (2), wherein these compounds comprise a functional group that reacts with only one of the remaining functional groups orthogonal elements present in the first drug loaded polymer. This method is advantageous in that it must provide biodegradable drug-releasing hydrogels of a more regular structure than those formed by the stoichiometric control of the components. This method is illustrated in working example 37 below. The first multi-arm polymer, where each arm is terminated by a group comprising at least two orthogonal functional groups, can be prepared from multi-arm polymers, where each arm ends with a single functional group by condensation with an adapter. suitable multifunctional. This situation is illustrated in figure 11.
[0093] [0093] The hydrogels of the invention can be prepared in vitro, then implanted, as needed. Gels can be expressed in specific forms, or they can be prepared as microparticles or microspherical suspensions for injection. Alternatively, hydrogels can be formed by in situ gelation, in which case pharmaceutically acceptable formulations of the hydrogel components are prepared; mixing of the components is followed by injection or application before gelation. The injection can be subcutaneous, intramuscular, intraocular, intratumor, or intravenous. The hydrogels of the invention can be applied topically, for example, in an in situ gelation of the mixed components after application to the skin or to surgical wounds. The hydrogels of the invention can also be applied as coatings on medical devices or surgical dressings.
[0094] [0094] All references cited herein are hereby incorporated by reference in their entirety. The invention is further illustrated but not limited by the following examples. Example 1 Preparation of 6-azidohexanal
[0095] [0095] (1) 6-Azido-1-hexanol: a mixture of 6-chloro-1-hexanol (25 g, 183 mmol) and sodium azide (32.5 g, 500 mmol) in 200 ml of water was heated to reflux for h, then cooled to room temperature and extracted 3x with ethyl acetate. The combined extracts were washed with brine, dried over MgSO4, filtered, and concentrated to produce the product as a pale yellow oil (28.3 g).
[0096] [0096] (2) 6-Azidohexanal: solid trichloroisocyanuric acid (4.3 g) was added in small portions to a vigorously stirred mixture of 6-azido-1-hexanol (7.15 g), TEMPO (50 mg), and sodium bicarbonate (5.0 g) in dichloromethane (100 ml) and water (10 ml). The mixture was stirred for an additional 30 minutes after the addition, then filtered through a pad of Celite. The organic phase was separated and washed, successively, with saturated aqueous NaHCO3 solution and brine, then dried over MgSO4, filtered and concentrated to provide the product (5.8 g), which was used without further purification. Example 2 Preparation of ω-azido-PEG-aldehydes
[0097] [0097] Solid trichloroisocyanuric acid (60 mg) was added to a vigorously stirred mixture of O- (2-azidoethyl) heptaethylene glycol (n = 7, 250 mg), 1 mg TIME, 100 mg NaHCO3, 2 ml CH2Cl2 , and 0.2 ml of water. The mixture turned orange and, after approximately 30 minutes, a white suspension was formed. TLC analysis (1: 1 acetone / hexane) indicated the formation of a product that was stained with phosphomolybdic acid. The mixture was diluted with 10 ml of CH2Cl2, dried by stirring with MgSO4, filtered and evaporated to obtain the crude product. This was dissolved in CH2Cl2 and loaded onto a column of 4 g of silica gel balanced with hexane, which was eluted sequentially with 25 ml each of hexane, 75:25 hexane / acetone, 50:50 hexane / acetone and 25: 75 hexane / acetone. Fractions containing the product were combined and evaporated to provide the purified product. Example 3 Preparation of azidoalcohols
[0098] [0098] A 1.6M solution of n-butyllithium (3.1 ml, 5.0 mmol) in hexane was added dropwise to a stirred solution of R-SO2CH3 (5.0 mmol) in tetrahydrofuran anhydrous (THF) (15 ml) cooled to -78 ° C. After the addition, the cooling bath was removed and the mixture was allowed to warm slowly to 0 ° C over approximately 30 min. The mixture was then cooled again to -78 ° C, and 6-azidohexanal (5.5 mmol) was added. After stirring for 15 minutes, the cooling bath was removed and the mixture was allowed to warm. At the point where the mixture became clear, 5 ml of saturated aqueous NH4Cl solution was added and the mixture was allowed to continue to warm to room temperature. The mixture was diluted with ethyl acetate and washed successively with water and brine, and then dried over MgSO4, filtered and evaporated to provide the crude product as an oil. Chromatography on silica gel using a gradient of ethyl acetate in hexane provided the purified products.
[0099] [0099] The compounds prepared according to this method are: 1- (4- (trifluoromethyl) phenylsulfonyl) -7-azido-2-heptanol: from 4- (trifluoromethyl) phenyl methyl sulfone 1 (1.73 g, 94%): H- NMR (400 MHz, CDCl3): δ 8.08 (2H, d, J = 8.4 Hz), 7.87 (2H, d, J = 8.4 Hz), 4.21 (1H, m), 3.25 (2H, t, J = 6.8 Hz), 3.28 (1H, dd, J = 8.8, 14.4 Hz), 3.20 (1H, dd, J = 2.0, 14.4 Hz), 3.12 (1H, d, J = 2.8 Hz), 1.58 (2H, m), 1.5 ~ 1.3 (6H, m); 1- (4-chlorophenylsulfonyl) -7-azido-2-heptanol; from 4-chloro-phenyl-methyl-sulfone; colorless oil (1.49 g, 90% 1 yield): H-NMR (400 MHz, d6-DMSO): δ 7.90 (2H, d, J = 8.8 Hz), 7.70 (2H, d, J = 8.8 Hz), 4.83 (1H, d, J = 6 Hz),
[00100] [00100] A 1.6 M solution of n-butyllithium (3.1 ml, 5.0 mmol) in hexane is added dropwise to a stirred solution of R-SO2CH3 (5.0 mmol) in tetrahydrofuran. anhydrous hydrofuran (THF) (15 ml) cooled to -78 ° C. After the addition, the cooling bath is removed and the mixture is allowed to warm slowly to 0 ° C over approximately 30 min. The mixture is then cooled again to -78 ° C, and ω-azido-heptaethylene glycol aldehyde (n = 7, 1.2 g) is added. After stirring for 15 minutes, the cooling bath is removed and the mixture is allowed to warm up. At the point where the mixture becomes clear, 5 ml of saturated aqueous NH4Cl solution is added and the mixture is allowed to continue to warm to room temperature. The mixture is diluted with ethyl acetate and washed successively with water and brine, then dried over MgSO4, filtered and evaporated to provide the crude product. Chromatography on silica gel provides the purified products. Example 5 Preparation of chloroformate azido-ligand
[00101] [00101] Pyridine was added (160 µl) dropwise to a stirred solution of the azidoalcohol of example 3 (1.0 mmol) and triphosgene (500 mg) in 15 ml of anhydrous THF. The resulting suspension was stirred for 10 minutes, then filtered and concentrated to provide the crude chloroformate, as an oil.
[00102] [00102] The compounds prepared according to this method are: 1- (4- (trifluoromethyl) phenylsulfonyl) -7-azido-2-heptyl chloroformate; 1- (4-chlorophenylsulfonyl) -7-azido-2-heptyl chloroformate; 1- (phenylsulfonyl) -7-azido-2-heptyl chloroformate; 1- (4-methylphenylsulfonyl) -7-azido-2-heptyl chloroformate; 1- (4-methoxyphenylsulfonyl) -7-azido-2-heptyl chloroformate; 1- (2,4,6-trimethylphenylsulfonyl) -7-azido-2-heptyl chloroformate; 1- (4-morpholinosulfonyl) -7-azido-2-heptyl chloroformate; (methanesulfonyl) -7-azido-2-heptyl chloroformate.
[00103] [00103] Other chloroformates can be prepared according to this general method. Example 6 Preparation of azido-chloroformate binders
[00104] [00104] Pyridine (160 µl) is added dropwise to a stirred solution of the azidoalcohol of example 4 (1.0 mmol) and triphosgene (500 mg) in 15 ml of anhydrous THF. The resulting suspension is stirred for 10 minutes, then filtered and concentrated to provide the crude chloroformate. Example 7 Preparation of succinimidyl carbonate binder azido
[00105] [00105] Pyridine (300 µl) was added dropwise to a stirred solution of chloroformate from example 5 (1.0 mmol) and N-hydroxysuccinimide (350 mg) in 15 ml of anhydrous THF. The resulting suspension was stirred for 10 minutes, then filtered and concentrated to provide crude succinimidyl carbonate. Purification by silica gel chromatography gave the purified product as an oil which crystallized spontaneously. Recrystallization can be carried out using ethyl acetate / hexane.
[00106] [00106] The compounds prepared according to this method are: O- [1- (4- (trifluoromethyl) phenylsulfonyl) -7-azido-2-heptyl] -O'-succinimidyl carbonate: crystals from 1 40: 60 ethyl acetate / hexane (280 mg, 55%): H-NMR (400 MHz, d6-DMSO): δ 8.12 (2H, m), 8.04 (2H, m), 5.18 ( 1H, m), 4.15 (1H, dd, J = 9.2, 15.2), 3.96 (1H, dd, J = 2.4.15.2), 3.29 (2H, t , J = 6.8), 2.80 (4H, s), 1.68 (2H, m), 1.47 (2H, m), 1.27 (4H, m); O [1- (4-chlorophenylsulfonyl) -7-azido-2-heptyl] - O'-succinimidyl carbonate: crystals from 40:60 1 ethyl acetate / hexane (392 mg, 83%): H-NMR (400 MHz, d6-DMSO): δ 7.85 (2H, m), 7.72 (2H, m), 5.14 (1H, m), 4.04 (1H, dd, J = 9.6 , 15.6), 3.87 (1H, dd, J = 2.4, 15.6), 3.29 (2H, t, J = 6.8), 2.81 (4H, s), 1 , 68 (2H, m), 1.47 (2H, m), 1.27 (4H, m); O- [1- (phenylsulfonyl) -7-azido-2-heptyl] -O'-succinimidyl carbonate: crystals from 40:60 1 ethyl acetate / hexanes (391 mg, 89%): H-NMR (400 MHz, d6-DMSO): δ 7.91 (2H, m), 7.76 (1H, m), 7.66 (2H, m), 5.12 (1H, m), 3.96 ( 1H, dd, J = 8.8,15.2), 3.83 (1H, dd, J = 2.8, 15.2), 3.29 (2H, t, J = 6.8), 2 , 81 (4H, s), 1.69 (2H, m), 1.47 (2H, m), 1.27 (4H, m); O [1- (4-methylphenylsulfonyl) -7-azido-2-heptyl] - O'-succinimidyl carbonate: crystals at rest after 1 chromatography (402 mg, 89%): H-NMR (400 MHz, d6- DMSO): δ 7.77 (2H, d, J = 8.0); 7.45 (2H, d, J = 8.0); 5.11 (1H, m),
[00107] [00107] Other succinimidyl carbonates can be prepared according to this general method.
[00108] [00108] Pyridine (300 µl) is added dropwise to a stirred solution of chloroformate from example 6 (1.0 mmol) and N-hydroxysuccinimide (350 mg) in 15 ml of anhydrous THF. The resulting suspension is stirred for 10 minutes, then filtered and concentrated to provide the crude succinimidyl carbonate. Purification by chromatography on silica gel provides the purified product. Example 9 Preparation of azido-sulfosuccinimidyl carbonate binder
[00109] [00109] A stirred suspension of sodium N-hydroxy sulfonate (1 mmol) in N, N-dimethylformamide (10 ml) is treated with pyridine (3 mmol) and chloroformate from the example
[00110] [00110] A stirred solution of an alcohol azido-binder of example 3 (R = phenyl, 1 mmol) in 1 ml of tetrahydrofuran (THF) was treated with a 1.0 M solution of trimethylphosphine in THF ( 1.2 ml) for 1 hour at room temperature. Water (0.1 ml) was added, and the mixture was allowed to stir for an additional 1 hour, then the mixture was evaporated to dryness using a rotary evaporator. The residue was dissolved in ethyl acetate, washed with water and brine, then dried over MgSO4, filtered, and evaporated to provide the product.
[00111] [00111] Other amino alcohol binders can be prepared according to this general method. Example 11 t Preparation of Boc-amino-ligand of alcohols
[00112] [00112] A solution of the alcohol amino binder of example 10 (R = phenyl, 1.0 mmol) in 2 ml of THF was treated with di-tert-butyl dicarbonate (1.5 mmol) for 1 hour, and then evaporated to dryness. The residue was dissolved in ethyl acetate, washed with water and brine, then dried over MgSO4, filtered, and evaporated to provide the product. Chromatography on silica gel using a gradient of ethyl acetate in hexane, provided the purified product. t
[00113] [00113] Other Boc-amino-alcohol binders can be produced according to the same general method. Example 12 Preparation of 4- (N, N-diethylcarboxamido) aniline
[00114] [00114] (1) N, N-diethyl-4-nitrobenzamide: Diethylamine (5.6 ml) was added to an ice-cooled solution of 4-nitrobenzoyl chloride (5.0 g) in 100 ml DCM. After 1 h, the mixture was washed successively with water, saturated aqueous NaHCO3 solution, and brine, then dried over MgSO4, filtered and evaporated to provide a colorless liquid which crystallized on standing. Recrystallization from ethyl acetate / hexane gave the product as light yellow crystals (4.6 g).
[00115] [00115] (2) 4- (N, N-diethylcarboxamido) aniline: A mixture of N, N-diethyl 4-nitrobenzamide (4.44 g) and 10% palladium on carbon (0.2 g) in 100 ml of methanol was treated with ammonium formate (4.0 g) for 2 h at room temperature. The mixture was filtered through Celite and concentrated. The residue was redissolved in DCM,
[00116] [00116] Were also prepared according to the same procedure was 4- (morpholinocarbonyl) aniline, replacing diethylamine with morpholine. Example 13 Preparation of azidocarbamates
[00117] [00117] The crude chloroformate prepared from 2.5 mmol of azidoalcohol according to the procedure of example 5 was dissolved in 20 ml of THF, and aniline (2.5 mmol) and triethylamine (0.7 ml, 5.0 mmol) were added. After 1 h, the mixture was diluted with ethyl acetate, washed successively with 1 N HCl, water, saturated NaHCO3 solution, and brine, then dried over MgSO4, filtered and evaporated. The residue was subjected to chromatography on silica gel using ethyl acetate / hexane to provide the carbamate product.
[00118] [00118] The compounds prepared according to this method are: O- [1- (phenylsulfonyl) -7-azido-2-heptyl] -N- [4- (diethylcarboxamido) phenyl carbamate;
[00119] [00119] A mixture of the azidocarbamate of example 13 (1.0 mmol), paraformaldehyde (45 mg), chlorotrimethylsilane (1 ml), and THF (1 ml) in a sealed 20 ml flask, was heated in a bath at 55 ° C for 17 h. After cooling to room temperature, the flask was opened and the mixture was concentrated on a rotary evaporator to a thick oil, which was taken up in ethyl acetate and concentrated again. The residue was dissolved in 2: 1 ethyl acetate / hexane, filtered, and concentrated to provide N-chloromethyl carbamate, which was used without further purification.
[00120] [00120] The compounds prepared according to this method are:
[00121] [00121] The N-chloromethyl carbamate of example 14 (0.4 mmol) was dissolved in 5 ml of dry methanol. After 1 h, the mixture is evaporated to dryness, and the residue was subjected to chromatography on silica gel (ethyl acetate / hexanes) to provide the product.
[00122] [00122] The compounds prepared according to this method are: O- [1- (phenylsulfonyl) -7-azido-2-heptyl] -N- [4- (diethylcarboxamido) phenyl] -N-methoxymethyl carbamate; O- [1- (morpholino) -7-azido-2-heptyl] -N- [4- (diethylcarboxamido) phenyl] -N-methoxymethyl carbamate; and O- [1- (methanesulfonyl) -7-azido-2-heptyl] -N- [4- (diethylcarboxamido) phenyl] -N-methoxymethyl carbamate. Example 16 7- (Terc-butoxycarbonylamino) -2- (R1-sulfonyl) -1-heptanol
[00123] [00123] P-Toluenesulfonyl chloride (1 mmol) is added to a solution of 6-azido-1-hexanol (example 1.1 mmol) in pyridine (2 ml) cooled on ice.
[00124] [00124] A solution of a heterobifunctional amino-thiol PEG in THF is treated with an excess of di-tert-butyl dicarbonate until the reaction is complete, and the di-BOC product is isolated by chromatography. Thiocarbonate is cleaved by treatment with an equivalent of NaOMe in methanol, and 2-bromoethanol is added to form the hydroxyethyl thioether, which is oxidized with peracetic acid to form the product. Example 18
[00125] [00125] These compounds can be prepared by a method analogous to that described for methoxy-PEG-hydroxyethyl sulfone (Morpurgo, et al., Bioconjugate Chemistry (1996) 7: 363-368, incorporated herein by reference). For example, a solution of 11-azido-3,6,9-trioxaundecan-1-ol (x = 3) (3 mmol) in toluene is dried by azeotropic distillation. After dissolution in CH2Cl2, methanesulfonyl chloride is added followed by triethylamine to form the mesylate. A solution of the mesylate in water is treated with 2-mercaptoethanol and 2N NaOH to form the hydroxyethyl sulfide. The sulfide is subsequently oxidized to the sulfone, for example, using hydrogen peroxide in the presence of a tungsten acid catalyst or, alternatively, using peracetic acid. Hydroxyethyl sulfone is then activated as the succinimidyl carbonate according to the methods described in the examples above. Example 19 Example 20 Preparation of crosslinkers of formula (1)
[00126] [00126] A solution of 7-azido-1- (phenylsulfonyl) -2-heptyl succinimidyl carbonate (119 mg, 0.27 mmol) in 2 ml of acetonitrile was treated with 11-azido-3,6,9-
[00127] [00127] A 40-kDa solution of 4-arm polyethylene glycol with aminopropyl end groups that has a core of pentaerythritol (NOF America, PTE400PA) (500 mg, 12.5 mmol), triethylamine (20 ml), and ester of succinimidyl of 6-aza-5, 9-dioxo-9- (1,2-didehydrodibenzo [b, f] azocin-5 (6H) -yl) nonanoic acid ("DBCO-NHS",
[00128] [00128] A solution of 4.5 mg of PEG- [DBCO] 4 of 4 arms (example 21) in 100 ml of 10 mM acetate buffer, pH 5, was treated with 5.0 µl of a solution of 40 mg / ml of diazide crosslinking agent of example 20. The solution quickly set out to provide an elastic hydrogel.
[00129] [00129] Likewise, a solution of 4.5 mg PEG- [DBCO] 4 of 4 arms (example 21) in 100 µl of 10 mM acetate buffer, pH 5, was treated with 2.5 µl of a 40 mg / ml solution of diazide crosslinking agent from example 20. The solution gelled to produce a viscous hydrogel. Example 23 Preparation of cross-linking reagents of PEG- (binder-azide) x multivalent of formula (2)
[00130] [00130] The preparation of multivalent PEG- (binder-azide) x cross-linking reagents is exemplified by the preparation of a compound of formula (2) in which m = 0, n = approximately 100, s = 0, t = 4, W = O (C = O) NH, Q = C (CH2) 4, R1 = PhSO2, R2 = H, one R5 = H and the other R5 = (CH2) 5N3. Other compounds of formula (2) were prepared using the same method by substituting the appropriate succinimidyl azide-binder-carbonate of example 7. Also, if necessary, succinimidyl azide-binder-carbonates analogous to other examples.
[00131] [00131] Thus, a solution of 25 µmol of succinimidyl azido-binder-carbonate (example 7) in 1 ml of ACN was added to a mixture of 5 µmol (100 mg) of 20-kDa of amine hydrochloride-PEG of 4 arms (pentaerythritol core, JenKem Technologies) in 1 ml of water and 40 µl of 1.0 M NaHCO3 (40 µmol). After 1 hour at room temperature, the solution was dialyzed (12-14 k MWCO) against 1 l of 50% methanol followed by 1 l of methanol. After evaporation, the residue (109 mg) was dissolved in 2.12 ml of sterile, filtered, mM NaOAc solution, pH 5.0, and stored frozen at -20 ° C. The concentration of azide determined by reaction with DBCO-acid was 9.5 mM. Example 24 Preparation of PEG- (cyclooctins) x multivalent
[00132] [00132] PEG20kDa- (DBCO) 4: A 60 mM solution of freshly chromatographed DBCO-NHS (Click Chemistry Tools) in acetonitrile (0.5 ml, 30 mmol, 1.5 eq) was added to a 20 kDa solution 4-arm amine-PEG hydrochloride (pentaerythritol core, JenKem Technologies, 100 mg, 5 µmol), and diisopropylethylamine (0.010 ml, 57 µmol) in acetonitrile (1 ml). After stirring for 2 hours at room temperature, the mixture was evaporated to dryness under reduced pressure. The residue was dissolved in 50% aqueous methanol (4 ml) and dialyzed against 50% aqueous methanol, followed by methanol. After evaporation, the residue (100 mg) was dissolved in water to give a stock of 50 mg / ml (10 mM DBCO per spectrophotometric assay) which was stored frozen at -20 ° C.
[00133] [00133] PEG40kDa- (DBCO) 8: One ml of a solution of 40 mM (40 µmol) of DBCO-NHS in THF was added to a solution of 168 mg (4.2 µmol) of 40-kDa of 8 arms of amine hydrochloride-PEG (tripentaerythritol core, JenKem Technologies) and 12.9 µl diisopropylethylamine (74 mmol) in 0.6 ml ACN, and the mixture was kept at room temperature overnight. The reaction mixture was dialyzed against 2 l of 50% methanol followed by 1 l of methanol. After evaporation, the residue (149 mg) was dissolved in 1.49 ml of water and stored frozen at - 20 ° C. The DBCO concentration determined spectrophotometrically was 16 mM.
[00134] [00134] PEG40kDa- (BCN) 8: A 200 mg solution of 40 kDa of HCl • amine-PEG of 8 arms (JenKem Technologies, 40 µmol NH2), 20 mg of BCN p-nitrophenyl carbonate (SynAffix, 63 µmol ), and 20 µl of N, N-diisopropylethylamine (115 µmol) in 2 ml of DMF was stirred 16 h at room temperature. After quenching with 0.5 ml of 100 mM taurine in 0.1 M KPi, pH 7.5, for 1 h, the mixture was dialyzed sequentially against water, methanol / water 1: 1, and methanol using a membrane 12 kDa. After evaporation, the residue was dissolved in 2 ml of THF and precipitated with 10 ml of methylbutyl ether. The product was collected and dried (190 mg). Example 25 Preparation of BODIPY-azide erosion probe
[00135] [00135] A 100 mM solution of 11-azido-3,6,9-trioxaundecan-1-amine in acetonitrile (13 µl, 13 mmol) was added to a 12.8 mM solution of BODIPY TMR-X SE ( Invitrogen) in DMSO (100 µl, 1.28 mmol). After 30 min at room temperature, the mixture was diluted to 2 ml with 0.1 M KPi, pH 7.4, and loaded onto a 500 mg C18 BondElut ™ extraction column (Varian). The column was washed successively with 5 ml portions of water and 20% ACN / water, then eluted with 50% ACN / water and concentrated to dryness. The residue was dissolved in 1.0 ml of ACN and the concentration (1.0 mM) was determined using ε544 nm = 60000 M-1 cm-1. Example 26 Preparation of fluorescein-azide erosion probe
[00136] [00136] A 10 mg / ml solution of 5- (aminoacetamido) fluorescein (Invitrogen) in DMF (100 µl) was mixed with a 25 mM solution of 6-azido-hexyloxy succinimidyl carbonate (100 ml) for 1 h to provide 12.5 mM of fluorescein-azide erosion probe solution. Example 27 Preparation of hydrogels using multivalent cross-linking reagents of formula (2)
[00137] [00137] For the preparation of 4x4 hydrogels, a 50 mg / mL solution of PEG20kDa (DBCO) 4 (example 24, 250 µl, 2.5 µmol of terminal groups of DBCO) in water was mixed with 25 µl of a 10 mM solution of fluorescein-azide erosion probe in DMF (example 26, 0.25 µmol of azide) and maintained for 30 min at room temperature. Fifty µl aliquots (0.42 µmol DBCO) were mixed with 28 µl mM NaOAc, pH 5.0, followed by 42 µl 50 mg / ml PEG20kDa (ligand-azide) 4 (example 23; 0.42 mmol of azide).
[00138] [00138] Preparation of 4x8 hydrogels followed by the same method, using solutions of PEG40kDa (DBCO) 8 or PEG40kDa (BCN) 8 (example 24) instead of PEG20kDa (DBCO) 4 and adjusting the proportions of cyclooctin monomers of 8 arms and of 4-arm azide binder, in order to provide gels having the PEG in% in the desired total weight and degree of crosslinking. Example 28 Measurement of reverse gelation times
[00139] [00139] The gel disks (example 27) were suspended in buffer at 37 ° C, and OD493 in the solution was measured periodically to monitor fluorescein solubilization. The reverse gelation times (tRGEL) were those times when gels were completely dissolved. The pH dependence of the degelification time was determined using 4x4 gels (5% total PEG by weight), prepared from crosslinked PEG20kDa (DBCO) 4 using a compound of formula (2) where m = 0, n = approximately 100, s = 0, t = 4, W = O (C = O) NH, Q = C (CH2) 4, R1 = (4-chlorophenyl) SO2, R2 = H, one R5 = H and the other R5 = (CH2) 5N3. The gel discs were suspended in buffers from pH 7.8 to 9.0. Degelification curves are shown in figure 5, with times measured at pH 7.8 = 20.9 h, pH 8.1 = 10.9 h, pH 8.4 = 5.6 h, pH 8.7 = 2, 8 h, and pH 9.0 = 1.5 h. As shown in figure 6,
[00140] [00140] The effect of the ligand modulator R1 on the defrosting time was determined by preparing PEG20kDa (DBCO) 4 hydrogel discs cross-linked with compounds of formula (2) where m = 0, n = approximately 100, s = 0, t = 4, W = O (C = O) NH, Q = C (CH2) 4, R2 = H, one R5 = H and the other R5 = (CH2) 5N3, and where R1 or was (4-chlorophenyl) ) SO2, phenyl-SO2, morpholino-SO2, or CN. A control gel was prepared having no modulator (R1R2CH is absent). Degelification curves of the discs suspended in KPi, pH 7.4, 37 ° C, are shown in figure 3. As shown in figure 4, there is a linear correlation between the ligand cleavage half-life as determined by the release of 5- (aminoacetamido) fluorescein (see Santi, et al., Proc Nat Acad Sci USA (2012) 109: 6211-6216), incorporated herein by reference, and the corresponding hydrogel de-gel time. Example 29 Controlled drug release from hydrogels
[00141] [00141] The hydrogels were prepared from PEG40kDa- (DBCO) 8, in which a fraction of the cyclooctins were first reacted with a small amount of azide erosion probe and with an azide binder drug of formula (3) in which the linker comprised a modulation group R1, then cross-linked using a compound of formula (2) where m = 0, n = approximately 100, s = 0, t = 4, W = O (C = O) NH, Q = C (CH2) 4, R2 = H, one R5 = H and the other R5 = (CH2) 5N3, and where R1 was either (4-chlorophenyl) SO2, phenyl-SO2, morpholino-SO2, or CN. The azide-binding drug modulating groups of structural formula (3) and the compound of formula (2)
[00142] [00142] In one example, the gels were prepared using 5- (acetamido) fluorescein (AAF) as a drug substitute. The groups modulating R1 in formula (3) were varied as indicated below. Thus, a solution (99.6 µl) containing 50 µl of 100 mg / ml PEG40kDa- (DBCO) 8 (1.0 µmol of terminal groups DBCO) in water was mixed with 6.2 µl of 12.5 mM of AAF azide binder (0.078 µmol) in 1: 1 DMF: acetonitrile (where the binder comprised one of several modulators), 15 µl of 1.0 mM BODIPY-azide (0.015 µmol) in acetonitrile, as an erosion probe , 20 µl of 20 mM O- (2-azidoethyl) heptaethylene glycol (0.40 µmol) in water to cover excess cyclooctins, and 8.4 µl of water. After 10 min at room temperature, the solution containing 0.5 µmol of independent DBCO groups was mixed with 50 µl of a 50 mg / ml solution of the compound of formula (2) where R1 = CH3-SO2 (0.5 µmol azide groups) in 10 mM NaOAc, pH 5.0.
[00143] [00143] Duplicate fused gels were suspended in 0.1 M HEPES, pH 7.4, at 37 ° C, and OD493 for fluorescein and OD546 for BODIPY in the solution was measured periodically. The fluorescein release times in which R1 in formula 3 is of various groups were measured as shown in figure 7. The reverse gelation time, as determined by complete solubilization of the BODIPY erosion probe, was 630 ± 39 (SD) h ( n = 8). The fluorescein solubilization followed the first order speed law [F] t / Ftot = exp (-kobsdt) and gave apparent kobsd ± SE for the total fluorescein released from 0.021 ± 0.00014 h-1 for R1 = 4-ClPhH -SO2-, 0.011 ± 0.00031 h-1 for R1 = Ph-SO2-, 0.0053 ± 0.00022 h-1 for R1 = 4-MeO-Ph- SO2-, and 0.0033 ± 0.00010 h-1 for R1 = MeSO2-. Rate data were converted to batches for fluorescein released directly from the gel using equation S6 (example 30).
[00144] [00144] The pH dependence of the drug release was determined by observing the release of AAF from the gels prepared above using R1 = (4-chlorophenyl) SO2 between pH 7.4 and 9.0. As shown in figures 8 and 9, the drug release rate increases with increasing pH. Example 30 Modeling of drug release and gel erosion
[00145] [00145] Drug release and gel degradation occurs as follows, with the final products which are the drugs and free gel monomers: (Gel) - (Drug) n → Drug + EP-fragments of gel-drugs → Drug + EP-monomers
[00146] [00146] The drug substitute or drug released in solution can emanate directly from L1 cleavage from the gel, or from solubilized fragments that arise from erosion gel through L2 cleavages. To distinguish the drug released from the intact gel versus fragments of solubilized gel, it is necessary to determine the distribution of drug support nodes between the intact gel and solution at time t. In the present study, a modification of a reported approach was used to monitor and model gel degradation (2). The appearance of an EP erosion probe permanently connected to the gel nodes allows the calculation of the fraction of nodes in solution such as EP (t) / E∞; the concentration of drug initially present on these solubilized nodes, Ds (t), is therefore given by the equation S1. Ds (t) = D∞ * EP (t) / EP or (D∞ / EP∞) * EP (t) [S1]
[00147] [00147] The drug released from the intact gel at time t, Dg (t), is the difference between the total drug released, D (t), and the drug either contained in or released from solubilized gel fragments Ds ( t), as in equation S2. Dg (t) = D (t) - Ds (t) = D (t) - (D∞ / EP∞) * EP (t) [S2]
[00148] [00148] The calculation of the first order drug release rate from the intact gel nodes is not easy to measure D (t) as a function of the amount of erosion gel change, but it can be calculated based on the fraction of the drug that remains in the gel intact. Based on the EP (t) erosion probe released, the remaining gel fraction is 1-EP (t) / EP∞. The amount of drug originally transported by this amount of gel is given by D∞ * (1 - EP (t) / EP∞). As the drug that remains on the intact gel is D∞ -D (t), the fraction of drug that remains on the intact gel, Df, gel (t) is given as equations S3-S4. Df, gel (t) = [D∞ - D (t)] / [D∞ * (1- EP (t) / EP∞)] [S3] = [1 - D (t) / D∞] / [ 1 - EP (t) / EP∞] [S4]
[00149] [00149] To obtain a first-order drug release from the gel, Df, gel (t) will show an exponential decay having a KL1 velocity constant that describes the rate of drug release from the intact gel, equation S5 . Merging equation S4 and S5 provides S6 which can be used to experimentally estimate the rate of drug release directly from the intact gel.
[00150] [00150] The amount of drug released by the gel over time depends on the rate of release, KL1, along with the rate of erosion of the gel. If the solubilization of the erosion probe can be approximated by a first order process between the times of t = 0 and t1 with the ksol rate, the amount of drug released from the gel during that time can be approximated as equation S7. Dg (t1) = D∞ * (kL1 / (ksol)) * [1- e- (ksol) t1] [S7]
[00151] [00151] If the drug that remains on the intact gel is negligible at time t1, then the total drug fraction released directly from the gel is given by the equation S8 Dg (t1) / D∞ = kL1 / ksol = t1 /2,sol/t1/2.L1 [S8]. Example 31 Effect of crosslink density on defrost time
[00152] [00152] As indicated in Table 1, a mixture of 100 mg / m PEG40kDa- (BCN) 8 (20 mM BCN end groups) in water was combined with appropriate amounts of mM fluorescein-azide and the compound of formula (2) where m = 0, n = approximately 100, s = 0, t = 4, W = O (C = O) NH, Q = C (CH2) 4, R2 = H, an R5 = H and the other R5 = (CH2) 5N3, and R1 = (4-chlorophenyl) SO2 (10 mM azide) in water and 50 mM O-azidoethyl-heptaethylene glycol in water to prepare 4% of PEG hydrogels with 4, 5, 6, 7, or 7.8 crosslinks per 8 arm PEG monomer. Molded gels were placed in 1 ml of 0.1 M borate, pH 9.2, and maintained at 37 ° C. Dissolution of the gels was monitored for the appearance of OD493 in the supernatant. Table 1 Preparation and defrosting times for gels with different crosslinking densities Reticulations / PEG 4 5 6 7 7.8 8 arms PEG- (BCN) 8 40 µl 36.9 µl 34.3 µl 32.0 µl 30.4 µl Fluorescein- 1.5 µl 1.5 µl 1.4 µl 1.5 µl 1.5 µl azide Capped azide 7.7 µl 5.2 µl 3.1 µl 1.3 µl 0 µl PEG- (L2-N3) 4 40 µl 46.2 µl 51.4 µl 56.0 µl 59.2 µl Water 60.8 µl 60.2 µl 59.7 µl 59.2 µl 58.9 µl Time 0.62 h 0.77 h 0.83 h 0.88 h 0.97 h de-gelation (pH 9.2)
[00153] [00153] Gels dissolved at pH 9.2 with defrost times as shown in table 1, with defrost time at pH 7.4 calculated as (defrost time at pH 9.2) * 10 (9.2 -7.4) as determined in example 28. As expected, defrosting time increased with the increase in the number of crosslinks for each 8 arm monomer. Example 32 Preparation of a degradable hydrogel that releases exenatide
[00154] [00154] Exenatide linked to the α-terminal to an azide ligand having R1 = MeSO2- as a modulator was synthesized by solid phase peptide synthesis in AnaSpec (Fremont, CA) as previously described (Santi, et al. Proc. Nat Acad. Sci. EUA (2012) 109: 6211-6216), resulting in a compound of formula (3) where R1 = MeSO2,
[00155] [00155] A gel disc (example 32) was placed in 1.0 ml of 0.1 M borate buffer, pH 8.8, and maintained at 37 ° C. The solubilization of exenatide (either as free peptide or solubilized gel exenatide fragments) and gel erosion were monitored at 280 nm and 544 nm, respectively, by periodic sampling of the supernatant. These results are shown in figure 10. The release was calculated as adjusted solubilization for gel erosion. The solubilization of exenatide is a first order process with t1 / 2 = 20.7 h at pH 8.8, which, assuming the reaction is first order in the hydroxide ion, corresponds to a half-life of 520 hours (21 days ), at pH 7.4; a t1 / 2 of 23.6 h at pH 8.8, corresponds to 593 h (24.7 d) at pH 7.4 was calculated for the drug directly released from the gel
[00156] [00156] Stock solutions of ~ 90 OD280 / ml myoglobin (17.7 kDa), carbonic anhydrase (29.0 kDa), and BSA (66.4 kDa) were prepared in 0.1 M KPi, pH 7.4. PEG hydrogels (4%) were prepared by adding 100 mg / ml of PEG20kDa- (NHCO2 (CH2) 6N3) 4 (50 µl) to a mixture of 100 mg / ml of 20 kDa of PEG- (DBCO) 4 (50 µl), protein stock (50 µl), and 10x-PBS (100 µl). Molded gels were suspended in 2 ml of 0.1 M KPi, pH 7.4, at 37 ° C, and OD280 in the solution was periodically measured. The t1 / 2 values for solution release were ~ 20 min to 24 min for myoglobin for carbonic anhydrase and 150 min for BSA. Example 35 Preparation of derived hyaluronic acids
[00157] [00157] Sodium hyaluronate of molecular weight = 1.6 x 106 (Lifecore Biomedical, 10.4 mg, 0.0275 mmol of carboxylate) was treated with a 4- (4,6-dimethoxy-1) chloride solution , 3,5-triazin-2-yl) -4-methylmorpholine (DMTMM; 30.4 mg, 0.110 mmol, 4 equiv) in 1.05 ml of 0.1 M MES buffer, pH 5.5. The resulting mixture was stirred vigorously for 15 min to dissolve. A solution of DBCO-PEG4-NH2 (Click Chemistry Tools; 0.113 ml of 24.3 mM in 2: 1 ACN: MeOH, 0.00275 mmol, 0.1 equiv) in 0.3 ml of MES buffer was added. The resulting mixture was left to stand for 24 h, then analyzed for consumption of the free amine by the TNBS test at 3.5 and 24 h, as follows: 0.05 ml of the reaction mixture was diluted to 1 ml in 0.075 M borate buffer (pH 9.34) containing 0.004% w / v 2,4,6-trinitrobenzenesulfonic acid and 25% methanol. The absorbance of the reaction at 420 nm was followed until stable (~ 1 h). Reactions containing amounts of DMTMM, hyaluronic acid, or DBCO-PEG4-NH2 were used as controls. Upon completion, the reaction mixture was diluted with 8 ml of water and dialyzed (12,000-14,000 MWCO) five times against water and then once against methanol. The dialyzed product was concentrated to dryness under reduced pressure and dried in a high vacuum with P2O5 to give hyaluronic acid-DBCO (11 mg, ~ 0.029 mmol of disaccharide) as a clear dry glassy solid. This material was dissolved in 3 ml of water to give a very viscous, slightly greasy solution containing 0.276 mM DBCO (based on ε309 = 13.448 M-1 cm-1. This corresponds to a 2.9% degree of substitution (5 , 3% based on the amine consumed in the TNBS test.) Hyaluronic acids of different molecular weights can be derived with cyclooctin reagents, such as DIFO or BCN, according to this method.
[00158] [00158] Amine-derived hyaluronic acids were prepared according to the following method. To a solution of sodium hyaluronate of molecular weight = 76,000 (Lifecore Biomedical, 154 mg, 0.385 mmol of disaccharide / carboxylate) in water (4 ml) was added 1,3-diaminopropane (0.973 ml, 856 mg, 11.6 mmol , 30 equiv). The pH of the resulting solution was adjusted to 7.0 with 6 N HCl (final volume of ~ 7 ml), then solid N-hydroxysuccinimide was added (177 mg, 1.54 mmol, 4 equiv), followed by 1- (3-
[00159] [00159] To a solution of molecular weight = 1.6 x 106 7% DS propylamino HA (0.5 ml of 0.64 mM NH2, 320 nmol of NH2) in water was added 0.1 ml of 100 mM PBS , followed by a solution of DBCO-PEG4-NHS ester (Click Chemistry Tools; 0.0308 ml of 25 mM as determined by ε309 = 13449 M-1cm -1, 770 nmol, 2.4 equiv) in methanol. The resulting mixture was allowed to settle for 4 hours. TNBS test indicated loss of 81% of the available amines on the derived hyaluronic acid. The parallel reaction using 1.2 equivalents of DBCO-PEG4-NHS ester resulted in the consumption of 64% of the available amines. For purification, the two reactions were combined and dialyzed (12-14K MWCO) against PBS, then 5% w / v NaCl, then twice against water, then once against methanol. The dialysis mixture was concentrated to dryness to give 2.6 mg of a white glassy solid. This material was dissolved in 1 ml of water to give a 6.5 mM disaccharide solution and 0.31 mM DBCO based on ε309 = 13448 M-1cm-1, which corresponds to a 4.8% DBCO replacement and an acylation yield of 71%. Example 36 Preparation of Hyaluronic Acid Hydrogels
[00160] [00160] Hyaluronic acid hydrogels are prepared by cross-linking hyaluronic acid-cyclooctin (example 35) with diazide cross-linking agents of formula (1) where m = 0, X = O-CO-NH- (CH2CH2O) 3CH2CH2N3 , R1 = PhSO2, R2 = H, one R5 = H and the other R5 = (CH2) 5N3. Gel formation is usually carried out in water or buffered water using a 2: 1 molar ratio of cyclooctin to diazide crosslinker, optionally in the presence of a protein solution or small molecule to be encapsulated.
[00161] [00161] For the study of protein diffusion from the hyaluronic acid hydrogel matrix, a stable hydrogel was prepared by mixing a solution (0.065 ml) of DBCO-HA (example 35), 6.6% of DS DBCO , 3.9 mM DBCO) in water with a diazido-PEG solution, either PM = 2000 or 5000 (0.005 ml 25 mM, 0.5 equiv / DBCO). This 0.07 ml hydrogel master mix was immediately mixed with a protein solution or small molecule substrate (0.01 ml) for encapsulation at the bottom of a standard 2.5 ml plastic cuvette. The half-lives for the diffusion of the gels are shown in table 2 below: Table 2
[00162] [00162] Alternatively, drugs can be releasably linked to hyaluronic acid, prior to gel formation by reacting a subset of the available cyclooctins with azide linker drug, as described in example 29 and example 32 above. In this case, the amount of diazide crosslinking agent used for gel formation is calculated based on the available cyclooctins remaining after drug fixation. Fixation of 5- (aminoacetamido) fluorescein through a linker with RD = (4-chlorophenyl SO2 from a hyaluronic acid hydrogel that released AAF with t1 / 2 = 49 h at pH 7.4, 37 ° C. Example 37 Method for preparing hydrogels with controlled stoichiometries
[00163] [00163] As shown in figure 11, commercially available St-Butylthio-cysteine (H Cys (tBuS)) is acylated with a cyclooctin succinimidyl ester (e.g. DBCO-HSE or BCN-HSE) to give CO-Cys (tBUS) OH t (A '= COOH, B = cyclooctin; C = BUS). A 4-arm amino PEG (A = NH2) is acylated (for example, using a carbodiimide) with that CO-Cys (tBUS) OH to give the functionalized CO / tBUS-PEG. An azide binder drug (R11) is coupled to the cyclooctin residues, then the t BUS group is removed, for example, using a thiol such as dithiothreitol or with a phosphine such as TCEP, and the thiol derived PEG is purified from small thiols (for example, using dialysis or gel filtration chromatography) and reacted with a cyclooctin-maleimide, cyclooctin-haloacetamide, or cyclooctin-vinylsulfonamide to introduce exactly 4 gelling sites per molecule.
This intermediate is then cross-linked to form a hydrogel using a compound of formula (1) or (2) in which the reactive functional groups are azide.
Alternatively, the thiol-derived PEG (prior to the reaction with cyclooctin-maleimide) can also be polymerized with a compound of formula (1) or (2) in which the reactive functional group is a Michael acceptor or an alkylating agent, such as such as maleimide, vinyl sulfone, vinyl sulfonamide, acrylate, acrylamide, haloacetate or haloacetamide.
Orthogonally protected adapters except S-t-Butylthio-cysteine can also be used, for example suitably protected lysines, aspartates or glutamates or synthetic adapters not based on amino acids.

权利要求:
Claims (39)
[1]
1. Method of preparing a biodegradable hydrogel, characterized by the fact that said method comprises the reaction of at least one first reactive polymer with a cleavable cross-linking compound, wherein said cleavable cross-linking compound comprises a first functional group which reacts with the reactive polymer and a portion that is cleaved by elimination under physiological conditions, wherein said portion comprises a second functional group which reacts with a reactive polymer.
[2]
Method according to claim 1, characterized in that the cleavable crosslinking agent is composed of formula (1) in which m is 0 or 1; X comprises a functional group capable of binding to a reactive polymer which is susceptible to elimination under physiological conditions and said second functional group, Z2; wherein at least one of R1, R2 and R5 comprises said first functional group Z1 capable of bonding to a polymer; wherein one and only one of R1 and R2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted;
at least one or both R1 and R2 is, independently, CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted; wherein R1 and R2 can be joined to form a 3- to 8-membered ring; and each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (OCH2CH2) p O-alkyl, where p = 1 to 1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted.
[3]
3. Method according to claim 2, characterized by the fact that:
X is succinimidyl carbonate, sulfosuccinimidyl carbonate, nitrophenyl carbonate, chloroformate, fluoroformate, or where T * is O, S or NR6 where R6 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally arylalkyl substituted or optionally substituted heteroarylalkyl; z is 1 to 6; and Y is absent or is OR7 or SR7, where R7 is optionally substituted alkylene optionally substituted phenylene or (OCH2CH2) p, where p = 1 to 1000, and Z2 is a functional group capable of bonding with a reactive polymer.
[4]
4. Method according to claim 3, characterized in that each of Z1 and Z2 independently comprises N3, NH2, NH-CO2tBu, SH, StBu, maleimide, CO2H, CO2tBu, 1,3-diene, cyclopentadiene, furan, alkyne, cyclooctin, acrylate or acrylamide; and in which, when one Z comprises N3 the other does not comprise alkaline or cyclooctin; when one Z comprises SH the other does not comprise maleimide, acrylate or acrylamide; when one Z comprises NH2, the other does not comprise CO2H; when one Z comprises 1,3-diene or cyclopentadiene the other does not comprise furan.
[5]
Method according to claim 1, characterized in that the cleavable linker compound has the formula in which m is 0 or 1; n is 1 to 1000; s is 0 to 2; t is 2, 4, 8, 16 or 32; W is O (C = O) O, O (C = O) NH, O (C = O) S,, or
; Q is a nucleus group having a valence = t; wherein at least one of R1, R2, and R5 comprises a functional group Z1 capable of bonding to a polymer, and at least one or both R1 and R2 is independently CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or
OR9 or NR92 in which each R is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted; wherein R1 and R2 can be joined to form a 3- to 8-membered ring; and wherein one and only one of R1 and R2 can be H or can be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (OCH2CH2) p O-alkyl where p = 1 to 1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted.
[6]
6. Method according to claim 5, characterized in that Z1 comprises N3, NH2, NH-CO2tBu, SH, StBu, maleimide, CO2H, CO2tBu, 1,3-diene, cyclopentadiene, furan, alkaline, cyclooctin, acrylate or acrylamide.
[7]
Method according to claim 2, characterized in that Z1 comprises N3, NH2, NH-CO2tBu, SH, StBu, maleimide, CO2H, CO2tBu, 1,3-diene, cyclopentadiene, furan, alkaline, cyclooctin, acrylate or acrylamide.
[8]
8. Method according to claim 3, characterized in that each Z1 and Z2 independently comprises N3, NH2, NH-CO2tBu, SH, StBu, maleimide, CO2H, CO2tBu, 1,3-diene, cyclopentadiene, furan, alkyl, cyclooctin, acrylate or acrylamide.
[9]
9. Method according to claim 1, characterized in that the first and any second polymer is selected from the group consisting of homopolymeric or copolymeric polyethylene glycols, polypropylene glycols, poly (N-vinylpyrrolidone), polymethacrylates, polyphosphazenes , polylactides, polyacrylamides, polyglycolates, polyethylene imines, agaroses, dextrans, gelatines, collagens, polylysines, chitosans, alginates, hyaluronans, pectins, carrageenans or that comprise either reactive functionalities in their native state or have been derived in order to understand suitable reactive functionality , or is of formula [Z3- (CH2) s (CH2CH2O) n] tQ, wherein Z3 is a reactive functional group, and n is 10 to 1000 es, Q and t are as defined in claim 5.
[10]
Method according to claim 1, characterized in that a first and a second reactive polymer are reacted with said cleavable cross-linking compound, sequentially or simultaneously,
[11]
11. Method according to claim 10, characterized by the fact that the first and second functional groups are the same.
[12]
12. Hydrogel, characterized by the fact that it is produced by the method defined in claims 1 to 11.
[13]
13. Hydrogel according to claim 13, characterized by the fact that it further comprises a drug.
[14]
Hydrogel according to claim 13, characterized by the fact that the drug is contained in a residue of formula (3) in which at least one of R1, R2, R5 comprises a first functional group for binding to a polymer and wherein R1, R2, R5 and m are as defined in claim 2; D is the drug residue; and Y is NH or NBCH2 where B is H, alkyl, arylalkyl, heteroaryl, or heteroarylalkyl, each optionally substituted.
[15]
15. Hydrogel that is biodegradable under physiological conditions, characterized by the fact that the hydrogel comprises one or more polymers cross-linked by a binder that decomposes by an elimination reaction.
[16]
16. Hydrogel according to claim 13, characterized in that the binder, when disposed in the polymer, is a residue of formula (1) in which at least one of R1, R2, R5 is coupled to said one or more polymers when it is X and where R1, R2, R5, m and X are as defined in claim 2.
[17]
17. Hydrogel according to claim 15,
characterized by the fact that said binder is a residue of formula (2) in which at least two of said R1, R2, R5 are coupled to said one or more polymers and in which R1, R2, R5, m are as defined in claim 2 and n is 1 to 1000; s is 0 to 2; t is 2, 4, 8, 16 or 32; W is O (C = O) O, O- (C = O) NH, O (C = O) S,, or.
[18]
Hydrogel according to any one of claims 15 to 17, characterized in that it further comprises a drug.
[19]
19. Hydrogel according to claim 18, characterized in that said hydrogel includes a residue of formula (3) in which at least one of R1, R2, R5 comprises a first functional group for bonding to a polymer and in that R1, R2, R5 are as defined in claim 2; D is the drug residue; and Y is NH or NBCH2 where B is H, alkyl, arylalkyl, heteroaryl, or heteroarylalkyl, each optionally substituted.
[20]
20. Hydrogel according to claim 16 or 17, characterized by the fact that the polymer is of the formula [Z3- (CH2) s (CH2CH2O) n] tQ, where Z3 is a reactive functional group, n is 10 to 1000 es, Q et are as defined in claim 5.
[21]
21. Crosslinking compound of formula (4) characterized by the fact that at least one of R1, R2, R5 further comprises a first functional group Z1 capable of binding to a polymer; where m is 0 or 1; R6 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl or optionally substituted heteroarylalkyl; v is 1 to 6; Y is absent or is OR7 or SR7, where R7 is optionally substituted alkylene, optionally substituted phenylene, or (OCH2CH2) p, where p = 1 to 1000; Z2 is a functional group capable of bonding with a reactive polymer; at least one or both R1 and R2 is independently CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl;
COR3 or SOR3 or SO2R3 where R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted; wherein R1 and R2 can be joined to form a 3- to 8-membered ring; and wherein one and only one of R1 and R2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (OCH2CH2) p O-alkyl where p = 1 to 1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted.
[22]
22. A compound according to claim 21, characterized by the fact that each Z1 and Z2 comprises N3, NH2, NH-CO2tBu, SH, StBu, maleimide, CO2H, CO2tBu, 1,3-diene, cyclopentadiene, furan, alkaline , cyclooctin, acrylate, vinyl sulfone, vinyl sulfonamide, or acrylamide and wherein, when one Z comprises N3 the other Z does not comprise alkaline or cyclooctin; when one Z comprises SH the other Z does not comprise maleimide, acrylate or acrylamide; when one Z comprises NH2 the other Z does not comprise CO2H; when one Z comprises a 1,3-diene or cyclopentadiene the other Z does not comprise furan.
[23]
23. A crosslinking compound of formula (2) characterized by the fact that, in at least two cases, R1, R2, and / or R5 further comprises a functional group Z capable of bonding to a polymer; m is 0 or 1; n is 1 to 1000; s is 0 to 2; t is 2, 4, 89, 16 or 32; W is O (C = O) O, O- (C = O) NH, O (C = O) S,, or Q is a nucleus group having a valence = t; at least one or both R1 and R2 is independently CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where
R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted; wherein R1 and R2 can be joined to form a 3- to 8-membered ring; and wherein one and only one of R1 and R2 may be H or may be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (OCH2CH2) p O-alkyl where p = 1 to 1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted.
[24]
24. A compound according to claim 23, characterized in that Z1 comprises an N3, NH2, NH-CO2tBu, SH, StBu, maleimide, CO2H, CO2tBu, 1,3-diene, cyclopentadiene, furan, alkane, cyclooctin, acrylate, acrylamide, vinyl sulfone or vinyl sulfonamide.
[25]
25. A compound according to claim 23, characterized by the fact that Q is pentaerythritol, tripentaerythritol, or hexaglycerin.
[26]
26. Method of preparing a degradable drug release hydrogel, characterized by the fact that it comprises the steps of: (a) reaction of a first multivalent polymer comprising reactive functionalities with a substoichiometric amount of a binding drug of formula (3) in that one of R1, R2, and R5 is replaced with a group that is reactive with the reactive functionality present in the first polymer, where m is 0 or 1; D is the residue of a drug; Y is O, NH or NBCH2, where B is H, alkyl, arylalkyl, heteroaryl, or heteroarylalkyl, each optionally substituted; at least one or both R1 and R2 is, independently, CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where R3 is H or optionally substituted alkyl;
aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted; wherein R1 and R2 can be joined to form a 3- to 8-membered ring; and wherein one and only one of R1 and R2 can be H or can be alkyl, arylalkyl or heteroarylalkyl, each optionally substituted; and each R5 is independently H or is alkyl, alkenylalkyl, alkynylalkyl, (OCH2CH2) p O-alkyl, where p = 1 to 1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; so as to form a first drug loaded polymer; (b) optionally isolating the first drug loaded polymer; and (c) crosslinking the remaining reactive functionalities in the first drug loaded polymer with a cleavable crosslinking compound of formula (1) or formula (2), to form a hydrogel.
[27]
27. Degradable drug release hydrogel, characterized by the fact that it is prepared by the method defined in claim 26.
[28]
28. Degradable drug release hydrogel according to claim 27, characterized by the fact that the reactive functionality of the first polymer is a cyclooctin, and the reactive functionality in the compound of formulas (1), (2) and (3) is azide.
[29]
29. Degradable drug release hydrogel according to claim 27, characterized by the fact that D is exenatide.
[30]
30. Degradable drug release hydrogel according to claim 27, characterized in that the first polymer is an 8-arm polyethylene glycol, comprising a cyclooctin group at the end of each arm; and the binding drug of formula (3) has the formula where R1 = CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where
R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R9 is independently H or optionally substituted alkyl, or both groups R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; or SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted.
[31]
31. Hydrogel according to claim 30, characterized by the fact that the cleavable cross-linking compound is of formula (2).
[32]
32. Hydrogel according to claim 31, characterized by the fact that in said formula (2), m is 0, n is 90 to 110, s is 0, t is 4, W is O (C = O) NH, Q is C (CH2) 4, R1 is CN, R2 and one of R5 is H, and the other R5 is (CH2) 5N3.
[33]
33. Hydrogel according to claim 32, characterized by the fact that R1 in formula (3) is SO2R3 in which R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or
OR9 or NR92 in which each R9 is independently H or optionally substituted alkyl, or both R9 groups taken together with the nitrogen to which they are attached form a heterocyclic ring.
[34]
34. Hydrogel according to claim 33, characterized by the fact that R1 in formula (3) is MeSO2.
[35]
35. Degradable drug release hydrogel, characterized by the fact that a multi-arm polymer of the formula [Z3- (CH2) s (CH2CH2O) n] tQ, where Z3 is a reactive functional group, and n is 10 to 1000 es , Q and t are as defined in claim 5 is cross-linked by a cleavable cross-linking compound of formula (1) or formula (2), to which hydrogel is coupled a binding drug of formula (3) wherein R1 = CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; COR3 or SOR3 or SO2R3 where R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 where each R9 is independently H or optionally substituted alkyl, or both groups
R9 taken together with the nitrogen to which they are attached form a heterocyclic ring; or SR4 where R4 is optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; or heteroaryl or heteroarylalkyl, each optionally substituted.
[36]
36. Hydrogel according to claim 35, characterized in that the cleavable cross-linking compound is of formula (2).
[37]
37. Hydrogel according to claim 36, characterized by the fact that in said formula (2), m is 0, n is 90 to 110, s is 0, t is 4, W is O (C = O) NH, Q is C (CH2) 4, R1 is CN, R2 and one of R5 is H, and the other R5 is (CH2) 5N3.
[38]
38. Hydrogel according to any one of claims 35 to 37, characterized in that R1 in formula (3) is SO2R3 in which R3 is H or optionally substituted alkyl; aryl or arylalkyl, each optionally substituted; heteroaryl or heteroarylalkyl, each optionally substituted; or OR9 or NR92 in which each R9 is independently H or optionally substituted alkyl, or two both R9 groups, taken together with the nitrogen to which they are attached form a heterocyclic ring.
[39]
39. Hydrogel according to claim 38, characterized by the fact that R1 in formula (3) is MeSO2.

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CN113304307A|2021-05-21|2021-08-27|浙江大学|Polyphosphazene-based hydrogel wound dressing with antibacterial and wet surface adhesion properties and preparation method thereof|

法律状态:
2021-05-25| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI N? 10196/2001, QUE MODIFICOU A LEI N? 9279/96, A CONCESS?O DA PATENTE EST? CONDICIONADA ? ANU?NCIA PR?VIA DA ANVISA. CONSIDERANDO A APROVA??O DOS TERMOS DO PARECER N? 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL N? 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVID?NCIAS CAB?VEIS. |

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2021-07-06| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161531990P| true| 2011-09-07|2011-09-07|

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US61/531,990|2011-09-07|

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PCT/US2012/054278|WO2013036847A1|2011-09-07|2012-09-07|Hydrogels with biodegradable crosslinking|

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