Patent Application
20150352246
Polymeric surgical sealants are used to provide leak-free closures around sutures and surgical anastomoses. Such sealants must have adequate tissue adherence and sufficient mechanical strength to withstand fluid pressure from the suture needle holes, and must be sufficiently flexible to maintain integrity and continue sealing during the post-surgery recovery process. Polymerization should be sufficiently rapid to allow quick wound closure during surgery. After recovery is complete and the suture wound has healed, the sealant should degrade and be reabsorbed. Sealants are also used as adhesion barriers, films, fabrics, gels and other materials that are applied between layers of tissues at the end of a surgery. An adhesion barrier acts as a physical barrier to separate tissue surfaces so that they do not adhere to one another while the tissue surfaces heal. The adhesion barrier should dissolve and be absorbed by the body after the healing process is complete. Other sealants are used as tissue adhesives for sutureless closure of wounds (Mizrahi, et al., “Tissue Adhesives as Active Implants,” in Active Implants and Scaffolds for Tissue Regeneration, M. Zilbermann, Ed., Springer, 2011, pp 39-56).
A number of sealants are currently marketed, including DuraSeal® (Confluent Surgical, Waltham, Mass.; Covidien), a 4-arm 20-kDa polyethylene glycol crosslinked with trilysine, used to prevent leakage of cerebrospinal fluid from dural sutures during spinal surgery; it is hydrolyzed and absorbed over a 4-8 week period. A newer formulation using a lower molecular weight polyethylene glycol, DuraSeal® Exact, has been reported to provide a tighter hydrogel matrix with less swelling than the original formulation. It is degraded by hydrolysis and reabsorbed over a 9-12 week period. In both cases, the hydrogel is thought to adhere to tissue by mechanical means. CoSeal® (Angiotech Pharmaceuticals, Vancouver, BC; Baxter), a mixture of a 4-arm PEG tetra-hydroxysuccinimide ester and a 4-arm PEG tetra thiol, each of approximate MW 10 kDa, used for arterial and vascular reconstruction. The resulting gel comprises thioester linkages that are hydrolytically labile, resulting in eventual gel degradation and resorption. Tissue adherence is provided by reaction of some of the reactive hydroxysuccinimide esters, and possibly some of the thioester groups, with protein amine groups in the tissue. CoSeal® is reported to remain effective at the application site for 7 days, and is fully degraded after 30 days. Hemaseel® (Haemacure Corporation, Montreal, Calif.), a fibrin-based sealant used between skin grafts and wound sites. The use of the fibrin sealant between the skin graft and the wound bed interface provides adhesive qualities allowing fixation of the graft without the use of staples or sutures and seals the tissue bed layer, thereby inhibiting seroma or hematoma formation without compromising the healing process, resulting in a higher percentage of graft take with a more acceptable cosmetic outcome than using mechanical fixation. Omnex® (Ethicon, Somerville, N.J.), a mixture of 2-octyl cyanoacrylate and butyl lactoyl cyanoacrylate, used in vascular reconstructions. Omnex® degrades by hydrolysis over approximately 36 months. While cyanoacrylates have also been used as tissue adhesives, for example DermaBond® (Omnex®), their use is limited by toxicity, such as tissue necrosis at the site of application. Progel® (Neomend, Irvine, Calif.), human serum albumin crosslinked with a bifunctional hydroxysuccinimidyl-polyethylene glycol (U.S. Pat. No. 6,899,889 B1), used for intraoperative sealing of pleural air leaks. A formulation using a recombinant albumin, Progel® Platinum Surgical Sealant, has been developed. Progel® AB is a hydrogel adhesion barrier sealant that can be sprayed onto general visceral organs during surgery to help prevent post-operative adhesions. Approximately 60% of Progel® is degraded after 1 day, and complete degradation is observed after 2 weeks. BioGlue® (CryoLife, Kennesaw, Ga.) is a mixture of albumin (supplied as a 45% solution) and glutaraldehyde (supplied as a 10% solution) used in cardiovascular surgery including arteriovenous access, aortobifemoral bypass, femoral popliteal bypass, endarterectomy, abdominal aortic aneurysm and aortotomies. Toxicity has been reported due to released glutaraldehyde (Fuerst & Banerjee, “Release of Glutaraldehyde From an Albumin-Glutaraldehyde Tissue Adhesive Causes Significant In Vitro and In Vivo Toxicity,” Ann. Thoracic Surg (2005) 79:1522-1528), and the stiffness of the polymerized material may cause mechanical issues with flexible tissues. FocalSeal-L® (Genzyme, Cambridge, Mass.) is a mixture of a polyethylene glycol capped with short segments of acrylate-capped poly(L-lactide) and poly(trimethylene carbonate) with a photoinitiator, eosin Y, and has been used to limit air leak after pulmonary resection. The solution polymerizes upon exposure to blue-green light to form a thin film hydrogel. The sealant does not bond covalently with tissue, and expands upon contact with bodily fluids over approximately 24 hours. Hydrolysis of the lactide and carbonate linkages allows for gel degradation and resorption. FocalSeal® has been used as a tissue adhesive. Adherus® Dural Sealant and Spinal Sealant (HyperBranch Medical Technology, Durham, N.C.), a mixture of poly(ethylene imine) crosslinked with a bifunctional PEG-hydroxy-succinimidyl ester, used in cranial and spinal surgery to prevent cerebrospinal fluid leakage and dural adhesions. OcuSeal® Liquid Ocular Bandage (HyperBranch Medical Technology, Durham, N.C.), a synthetic hydrogel that is applied directly to the ocular surface as a liquid, using a brush applicator.
U.S. Pat. No. 7,151,135 (issued 19 Dec. 2006) and U.S. Pat. No. 7,176,256 (issued 13 Feb. 2007) disclose crosslinked synthetic polymers for use as bioadhesives among other uses. U.S. Pat. No. 8,303,973 (issued 6 Nov. 2012) discloses sealants comprising PEG and chitosan. U.S. Pat. No. 6,602,952 (issued 5 Aug. 2003) discloses hydrogel sealants. U.S. Pat. No. 6,495,127 (issued 17 Dec, 2002) discloses sealants formed from multifunctional polymers together with a tensile strength enhancer. U.S. Pat. No. 6,458,147 (issued 1 Oct. 2002) discloses sealants formed from a protein such as albumin together with a polymer. U.S. Pat. No. 6,312,724 (issued 6 Nov. 2001) discloses sealants formed by reaction of a thiol-polymer and a thiol-reactive polymer.
Hydrogels offer several benefits for use as surgical sealants, such as high water content, tissue compatibility, good mechanical strength, and flexibility. A hydrogel is a 3-dimensional network of natural or synthetic hydrophilic polymer chains 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 may preserve the folding of a protein needed for its bioactivity. The high water content of the hydrogels render the material biocompatible and minimize inflammatory reactions of tissues in contact with the gel, and provide a flexibility comparable to that of living tissue. Hydrogels are thus of interest in biomedical engineering, as absorbent materials for wound dressings and disposable diapers, as carriers for extended drug release, and as flexible sealants for surgical procedures. Hydrogels have been prepared by physical or chemical crosslinking of hydrophilic natural or synthetic polymers. Examples of hydrogels formed from crosslinking of natural polymers include those formed from hyaluronans, chitosans, collagen, dextran, pectin, polylysine, gelatin or agarose. (See: W. E. Hennink and C. F. van Nostrum, Adv. Drug Del. Rev. (2002) 54:13-36; A. S. Hoffman, Adv. Drug Del. Rev. (2002) 43:3-12). These hydrogels consist of high-molecular weight polysaccharide or polypeptide chains. Some examples of their use include the encapsulation of recombinant human interleukin-2 in chemically crosslinked dextran-based hydrogels (J A. Cadee, et al., J. Control. Release (2002) 78:1-13) and insulin in an ionically crosslinked chitosan/hyaluronan complex (S. Surini, et al., J. Control. Release (2003) 90:291-301).
Examples of hydrogels formed by chemical or physical crosslinking of synthetic polymers include poly(lactic-co-glycolic)acid (PLGA) polymers, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronie), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A- PEO-PL(G)A copolymers, poly(ethylene imine), and others. (See for example A. S Hoffman, Adv. Drug Del. Rev (2002) 43:3-12). Examples of protein-polymer encapsulation using such hydrogels include the encapsulation of insulin in physically crosslinked PEG-g-PLGA and PLGA-g-PEG copolymers (B. Jeong, et al., Biomacromolecules (2002) 3:865-868) and bovine serum albumin in chemically crosslinked acrylate-PGA-PEO-PGA-acrylate macromonomers (A. S. Sawhney, et al., Macromolecules (1993) 26:581-587). Hydrogels formed by crosslinking 4-arm PEGs have been disclosed (K. Nishi, et al., “Kinetic Study for AB-Type Coupling Reaction of Tetra-Arm Polymers,” Macromolecules (2012) 45:1031-1036; T. Sakai, et al., “Highly Elastic and Deformable Hydrogel Formed from Tetra-arm Polymers,” Macromolecular Rapid Comm. (2010) 31:1954-1959; T. Sakai, et al., “Design and Fabrication of a High-Strength Hydrogel with Ideally Homogeneous Network Structure from Tetrahedron-like Macromonomers,” Macromolecules (2008) 41:5379-5384; X. Li, et al., “Precise Control and Prediction of Hydrogel Degradation Behavior,” Macromolecules (2011) 44:3567-3571; T. Matsunaga, et al., “Structure Characterization of Tetra-PEG Gel by Small-Angle Neutron Scattering,” Macromolecules (2009) 42:1344-1351; T. Matsunaga, et al., “SANS and SLS Studies on Tetra-Arm PEG Gels in As-Prepared and Swollen States,” Macromolecules (2009) 42:6245-6252.
PCT application PCT/US2012/54278 and Ashley, et al., “Hydrogel Drug Delivery System with Predictable and Tunable Drug Release and Degradation Rates,” Proc. Natl. Acad. Sci. USA (2013) 110:2318-2323) describe degradable hydrogels having precisely controlled rates of degradation and optionally having controlled drug release. The gels described in the PNAS paper do not contain moieties that permit them to couple to proteins on membranes or cells for which sealants are desired. Neither do the exemplified hydrogels in the above cited PCT application. However, the generic class included in the PCT application would include instances where, by virtue of the functional groups employed to carry out the crosslinking, it is possible that some of the resulting hydrogels, due to having unreacted functional groups of particular types, would have the possibility to couple to proteins under appropriate conditions. For example, although the exemplified hydrogels form crosslinks by reaction between an azide and a cyclooctatriene moiety, neither of which will couple to protein, alternative possible functional groups may have this capacity. The present invention assures the presence of suitable functional groups on the surface of the hydrogel to effect linking to proteins.
This invention provides degradable sealants having precisely controlled rates of degradation and optionally having controlled drug release and methods for their preparation and use. These sealants are expected to provide leak-free closures around sutures, surgical anastomoses, surgical implants, and wounds, as well as serve as adhesion barriers, tissue adhesives, and bandages. Controlled degradation rates are achieved through the use of beta-elimination linkers, which may be incorporated into the sealant crosslinks, drug attachment connectors, tissue attachment connectors, or combinations thereof.
In one aspect the invention provides sealants having controlled rates of degradation by virtue of crosslinkers that undergo β-elimination and optionally having controlled drug release. The sealants of the invention are crosslinked polymers wherein the crosslinks comprise groups that degrade by a pH-dependent elimination process thus allowing the sealant to be resorbed and wherein the sealants provide functional groups that promote tissue adherence. The protein-reactive functional groups that provide tissue adherence are linked to the polymer optionally via degradable linkers, and in some embodiments these linkers are biodegradable by elimination reactions. In some embodiments, the sealants of the invention further comprise drugs, wherein the drugs are covalently linked to the polymer optionally through linkers that degrade by a pH-dependent elimination process thereby releasing the drug.
In one aspect, the sealants of the invention comprise a biodegradable hydrogel coupled to a multiplicity of functional groups reactive with protein,
wherein said protein-reactive functional groups are coupled to the hydrogel through linkers of the formula
wherein
n is 0 or 1;
X2 is a group that will allow attachment to the hydrogel or to protein;
at least one or both R1 and R2 is independently CN; NO2;
wherein R1 and R2 may be joined to form a 3-8 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, (CH2CH2O)p wherein p=1-1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
wherein at least one of R1, R2, and R5 is substituted with X2 wherein one and only one of X2 is a group that binds to the hydrogel and is not capable of binding to protein unless already coupled thereto and the other is part of a protein-reactive group, P. Thus, as shown, P would be represented by
One example of formula (1) would then be
wherein P is OCOSu or OCO—NH—(CH2)x-R7; wherein R7 is maleimide, alpha-halocarbonyl, vinylsulfonyl, vinylsulfonamide, and the like; and wherein one R1, R2 or R5 can couple to hydrogel.
Alternatively, or in addition, the sealants comprise a biodegradable hydrogel coupled to a multiplicity of protein-reactive groups wherein the hydrogel comprises macromonomers which are coupled by crosslinkers of the formula
wherein
n is 0 or 1;
wherein one of R1, R2, and R5 is substituted with X3, wherein X3 is a functional group for binding the hydrogel and is not a protein-reactive group and R1 , R2 and R5 are otherwise as defined in formula (1) and/or
of the formula
wherein one of R1, R2 and R5 in at least two of the t moieties shown within the bracket comprises said functional group X3 and R1, R2 and R5 are otherwise as defined in formula (1) and wherein
n is 0 or 1;
m is 0-1,000;
s is 0-2;
t is 2, 4, 8, 16 or 32,
W is O(C═O)O, O(C═O)NH, O(C═O), S,
R6 is H, alkyl (1-6C), aryl or heteroaryl; and
Q is a core group having a valency=t.
In some embodiments, the sealants may also contain a releasable drug coupled through a biodegradable linker. In some embodiments, the linker is of formula (1b)
wherein n is 0 or 1 and one of R1, R2 and R5 is substituted with X4 wherein one X4 is a hydrogel binding group and the other is a drug binding group and neither X4 is a protein-reactive group unless already coupled to drug and wherein R1, R2 and R5 are otherwise as defined in formula (1).
Thus, in one example of formula (1b), this would result in
wherein D is a drug and one of R1, R2 and R5 can couple to hydrogel.
In some embodiments, the linked drug is of the formula
wherein n, R1, R2 and R5 are as defined above,
D is a residue of a drug or prodrug coupled through O, S or N;
Y is absent and Z is O or S; or
Y is NBCH2 and Z is O;
wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
wherein one of R1, R2 and R5 or B is coupled to the hydrogel.
Methods for preparation are also part of the invention and depend on the nature of the functional groups, the sequence of reaction of crosslinkers with the various components of the sealant, and the stoichiometry desired. Such methods are described in more detail below.
In another aspect the invention provides multi-layer hydrogels or sealants. In one embodiment, the multilayer hydrogels or sealants are especially useful for drug delivery to tissue that is normally in contact with a bodily fluid. In this embodiment, the multilayer hydrogel sealants for this purpose comprise a layer in contact with the tissue to which drug is to be delivered with sufficient porosity to deliver the drug to the tissue and is overlain with a layer with smaller pore size which prevents the contact of degradation enzymes from the fluid normally in contact with the tissue from contact with the drug in the drug delivery layer and, in some cases, can prevent the leakage of drug into the surrounding fluid.
In another aspect the invention provides methods for the use of the sealants of the invention and of the multilayer hydrogels or sealants.
The invention provides degradable sealants having controlled rates of degradation and optionally having controlled drug release and methods for their preparation and use. These sealants are expected to provide leak-free closures around sutures, surgical anastomoses, surgical implants, and wounds, as well as serve as adhesion barriers, tissue adhesives, and bandages. Degradable sealants have been previously disclosed, although degradation has been achieved by hydrolytic reactions having rates that are poorly controlled and difficult to predict. In the present invention, controlled degradation rates are achieved through the use of beta-elimination linkers, which may be incorporated into the sealant crosslinks, drug attachment connectors, tissue attachment connectors, or combinations thereof.
By “a moiety capable of being cleaved by elimination under physiological conditions” is meant a structure comprising a group —CH—(CH═CH)nC—X wherein n is 0 or 1 and X is a leaving group, wherein an elimination reaction to remove the elements of HX can occur at a rate such that the half-life of the reaction is between 1 and 10,000 hours under physiological conditions of pH and temperature. Preferably, the half-life of the reaction is between 1 and 5,000 hours, and more preferably between 1 and 2,500 hours, more preferably between 1 and 1,000 hours under physiological conditions of pH and temperature. By physiological conditions of pH and temperature is meant a pH of between 7 and 8 and a temperature between 30 and 40° C.
It should be noted that when ranges are given in the present application, such as 1-2,500 hours, the intermediate interval numbers should be considered as disclosed as if specifically and explicitly set forth. This avoids the necessity of long list of numbers and applicants clearly intend to include any arbitrary range between the outer boundaries. For example, the range 1-1,000 also includes 1-500 and 2-10.
A “hydrogel” is a three-dimensional, predominantly hydrophilic polymeric network comprising a large quantity of water, formed by chemical or physical crosslinking of natural or synthetic homopolymers, copolymers, or oligomers. The components of the hydrogel that are crosslinked together may be multi-armed polymers. In describing the makeup of the hydrogels of the invention, the components whether single polymers or multi-arm polymers will be referred to as “macromonomers” because they constitute the individual elements in the overall crosslinked structure which is the hydrogel or sealant. Hydrogels may be formed through crosslinking polyethylene glycols (considered to be synonymous with polyethylene oxides), polypropylene glycols, poly(N-vinylpyrrolidone), poly-methacrylates, polyphosphazenes, polylactides, polyacrylamides, polyglycolates, polyethylene imines, agarose, dextran, gelatin, collagen, polylysine, chitosans, alginates, hyaluronans, pectin, and carrageenan. A multi-armed polymer is formed of more than a single chain and typically has an even number of arms, each arm of which may bear one or more functional groups for further reaction. A single-armed polymer is a single chain which may have one or more functional groups at each end. A multi-armed polymer can support more than one or two functional groups at the terminus of each of the arms. Hydrogels may also be environment-sensitive, for example being liquids at low temperature but gelling at 37° C., for example hydrogels formed from poly(N-isopropylacrylamide).
The pore sizes characteristic of the polymer are also variable depending on the concentrations and nature of the reactants used to compose it. A “mesoporous” hydrogel is a hydrogel having pores between approximately 1 nm and approximately 100 nm in diameter. The pores in mesoporous hydrogels are sufficiently large to allow for free diffusion of biological molecules such as proteins. A “macroporous” hydrogel is a hydrogel having pores greater than approximately 100 nm in diameter. A “microporous” hydrogel is a hydrogel having pores less than approximately 1 nm in diameter.
In some embodiments, for example, of the multilayer sealants or hydrogels of the invention, the hydrogel in contact with tissue or intended to be in contact with tissue is a macroporous hydrogel and the upper layer in contact with fluid is the mesoporous hydrogel or a microporous hydrogel. Alternatively, for example, the layer in contact with a tissue may be a mesoporous hydrogel while the layer in contact with the fluid is the microporous hydrogel. Typically, the multilayer hydrogels or sealants will be formed in situ—i.e., the layer in contact with tissue is laid down first, followed by application of the overlaying hydrogel layer. Alternatively, the multilayer hydrogel may be pre-formed and the layer intended for tissue contact be provided with a protein-reactive set of functional groups so as to attach to the tissue itself.
A “biodegradable sealant” is a sealant that loses its structural integrity through the cleavage of component chemical bonds under physiological conditions of pH and temperature. Biodegradation may be enzymatically catalyzed or may be solely dependent upon environmental factors such as pH and temperature. Biodegradation results in formation of fragments of the polymeric network that are sufficiently small to be soluble and thus undergo clearance from the system through the usual physiological pathways. In many embodiments of the present invention, the degradation occurs through an elimination reaction effected by virtue of crosslinkers of formulas (1a) or (2) described above.
“Functional groups” refer to groups of atoms that are reactive towards other functional groups, most preferably under mild conditions compatible with the stability requirements of peptides, proteins, and other biomolecules. Suitable functional groups found in crosslinkers that couple the macromonomers and in the macromonomers themselves of the hydrogel include maleimides, thiols or protected thiols, alcohols, acrylates, acrylamides, amines or protected amines, amino ethers, carboxylic acids or protected carboxylic acids, azides, alkynes including cycloalkynes, 1,3-dienes including cyclopentadienes and furans, cyclooctenes, cyclopropenes, alpha-halocarbonyls, N-hydroxysuccinimide or N-hydroxysulfo-succinimide esters or carbonates, and 1,2,4,5-tetrazines.
Thus, functional groups capable of connecting to the macromonomers are functional groups that react to cognate functional groups of a reactive polymer to form a covalent bond to the macromonomer. These functional groups that are used to assemble the hydrogel are not protein-reactive. Examples of suitable functional groups are illustrated in the embodiments below.
A “protein-reactive” functional group is a group that is capable of reacting directly with a protein in situ. Typically, proteins contain SH groups, COOH groups, and NH2 groups that are available for reaction with the protein-reactive group. Since the sealant must be operable in situ, reactions that require additional crosslinking such as reaction which require carbodiimide, for example, are not considered “protein-reactive functional groups.” Examples of protein-reactive functional groups include succinimidyl carbonates, succinimidyl ester, maleimides, alpha-halo-carbonyl derivatives and the like. Thus, these protein reactive groups include a hydroxysuccinimide or sulfohydroxysuccinimide ester or carbonate; a substituted phenyl ester or carbonate; a maleimide, vinylsulfone, or vinylsulfonamide; or an alpha-halo ketone, alpha-halo carboxamide, or alpha-halo carboxylate, an aldehyde, or a perfluorohydrocarbyl group.
The “crosslinkers” or “linkers” are compounds comprising at least two functional groups that are capable of forming covalent bonds with one or more reactive macromonomers or other molecules. As used in the present application, “crosslinker” refers to the molecules that join the macromonomers to form the hydrogels and “linkers” refer to simple molecules that couple a protein-reactive group or a drug to the hydrogel. Typically, the reactive macromonomers are soluble, and crosslinking results in formation of an insoluble three-dimensional network or gel. The functional groups of the crosslinking reagent may be identical (homofunctional) or different (heterofunctional). The functional groups of the heterofunctional crosslinking reagent are chosen so as to allow for reaction of one functional group with a cognate group of the reactive macromonomers and reaction of the second functional group with a cognate group of the same or a different macromonomer. The functional groups of a multifunctional crosslinking reagent are chosen so that they are not reactive with themselves, i.e., are not cognates. In typical embodiments of the present invention, the crosslinkers used to construct the hydrogel will be linked via functional groups that are not “protein-reactive groups.” In this instance, there is no possibility that incompletely reacted crosslinkers would provide functional groups that could react with proteins and behave as sealants.
In one aspect the invention provides sealants having controlled rates of degradation and optionally having controlled drug release. The sealants of the invention are hydrogels of crosslinked macromonomers wherein the hydrogel also comprises a plurality of functional groups that promote tissue adherence, i.e., protein-reactive groups. These are linked to the hydrogel optionally through degradable linkers, and wherein the linkers may comprise groups that degrade by a pH-dependent elimination process thus allowing the sealant to be resorbed. In some embodiments, the sealants of the invention further comprise drugs, wherein the drugs are covalently linked to the hydrogel optionally through linkers that degrade by a pH-dependent elimination process thereby releasing the drug.
Thus, some alternative embodiments of the invention are those where a protein-reactive functional group is coupled to a biodegradable hydrogel through linkers that undergo elimination reactions to control release of the moiety containing the protein-reactive functional group when the sealant has been bound to protein, but wherein the remainder of the polymer is degradable by conventional methods.
In some embodiments, the protein-reactive functional group containing moiety is coupled to the hydrogel not so as to be releasable under physiological conditions, but the hydrogel itself is biodegradable by virtue of crosslinkers that undergo elimination reactions under physiological conditions. In other embodiments, both the moiety containing the protein-reactive functional group and the hydrogel itself are crosslinked through groups that undergo biodegradation through an elimination reaction. In those embodiments wherein a drug is coupled to the hydrogel, unless the hydrogel is completely biodegradable, the drug should be releasably linked to the hydrogel, optionally, though not necessarily, through a linker that undergoes elimination to release the drug.
An illustrative embodiment of the sealants of the invention is shown in
Some of the crosslinkers or macromonomers are bound to tissue adherence functional groups—i.e., protein-reactive groups designated “P” in the figure. These are coupled to the remainder of the gel typically by bifunctional linkers. If the biodegradable hydrogel does not contain a plurality of crosslinkers that degrade through elimination as described above, the linker coupled to the protein-reactive group should be capable of degradation through the elimination reaction described. In the illustration shown in
It is optional, but the matrix may also include a drug, symbolized by “D” which itself may be coupled to the gel through a linker which is optionally and preferably biodegradable, more preferably through cleavage by an elimination reaction. Preferably, the bifunctional linkers shown as T1 in the figure that couple D to the remainder of the polymer are biodegradable through an elimination reaction—i.e., are of formula (1b). If at least the linker coupling P or a majority of the linkers coupling the polymers of the hydrogel are linked by crosslinkers that degrade by the elimination reaction, the crosslinker shown as T1 binding D to the remainder of the hydrogel may be biodegradable by other mechanisms.
Of course, more than one type of drug can be included in the structure and more than one type of protein-reactive group may be included. It should be noted as well that some of the crosslinkers, labeled T2, may form part of the hydrogel itself by virtue of their nature—i.e., the 4 arms shown as T2 may be coupled to a polymeric center; i.e., T2 may be of formula (2).
In
The complicated-looking hydrogel structures of the invention in
(MTx)y (3)
where M is a multivalent polymer (i.e., two or more reactive functional groups),
T is a crosslinker,
x is an integer of 2-20 or 2-40, and
y is an integer that results in the hydrogel.
The hydrogel itself can then be coupled to protein-reactive groups, P, and optionally to drug, D.
As is evident from
In each embodiment M may be homopolymeric or copolymeric poly(ethylene glycol)s or poly(ethylene oxide)s (PEG or PEO), polypropylene glycols (PPG), poly(N-vinyl-pyrrolidone), polymethacrylates, polyphosphazenes, polylactides, polyacrylamides, polyglycolates, poly(ethyleneimine)s, agaroses, dextrans, gelatins, collagens, polylysines, chitosans, alginates, hyaluronans, pectins, or carrageenans that either comprise suitable reactive functionalities in their native state or have been derivatized so as to comprise suitable reactive functionalities X1. Native polymers that do not comprise an effective multiplicity of reactive groups can be transformed by reaction with reagents that introduce an effective multiplicity of reactive groups prior to formation of the hydrogel using methods well known in the art. The macromonomer may comprise multivalent branched structures. Examples include multivalent star-shaped polymers, for example those based on pentaerythritol, and comb-shaped polymers, for example those based on derivitization of hexaglycerin or tripentaerythritol (see core structures below). The number of monomer units comprising the macromonomer can be 10-1,000 or intermediate values such as 20, 50, 100, etc. This listing is intended to include all intermediate integers between 10 and 1,000, as most commercially available polymers are mixtures comprising a distribution of different monomer numbers. M is typically of molecular weight between 1,000 and 150,000; preferably between 1,000 and 70,000. In some embodiments, M is a protein, for example an albumin or fibrin, having a multiplicity of reactive amine groups from surface lysine residues. It may be necessary to provide an adaptor to supply a functional group cognate to a functional group that is not protein-reactive in this case. For instance, a heterofunctional linker wherein one group reacts with amines, sulfhydryl and carboxy and a second group such as an azide can be used.
M may also comprise multiple arms wherein each arm is terminated with at least two functional groups X1, wherein each X1 on an arm may be the same or different. In one embodiment, each arm is terminated with two functional groups X1. As examples, one X1 may be an azide and the other an aldehyde (if already bound to protein), or one X1 may be a cyclooctyne while the other is a cyclopropene, or one X1 may be a highly reactive cyclooctyne such as DBCO while the other is a relatively unreactive cyclooctyne such as MOFO. In the first example, the azide can be used to couple or crosslink using 1,3-dipolarcycloaddition reactions. In the second example, the cyclooctyne can be used to couple or crosslink with an azide using 1,3-dipolarcycloaddition reactions while the cyclopropene may be used to couple or crosslink to a tetrazine using a Diels-Alder reaction. In the third example, the two different cyclooctynes can be used to couple to two different azides based on the differential rates of reaction. Other such combinations will be apparent. In another embodiment, each arm of M is terminated with three functional groups X1, allowing for control over sealant crosslinking, protein attachment group linking, and drug linking. For example, each arm of M may be terminated with a cyclooctyne, a cyclopropene, and an aldehyde group.
In some embodiments, the functional groups X1 and X3 employed to form the matrix itself will not include those wherein one of the cognates is a group found in protein so that adherence to the tissue would be effected by any leftover reactive groups. Thus, the cognate pairs in the formation of the hydrogels will be those that are not interactive with any carboxyl, amino, or sulfhydryl groups. Examples of these groups include those wherein one of X1 and X2 has a terminal acetylene, 1,1,1-trifluoro-propyne, or cyclooctyne moiety and the other is a group capable of undergoing a 1,3-dipolar cycloaddition, such as N3 resulting in formation of a triazole linkage, or a nitrone resulting in isoxazoline formation (see, for example, Ning, et al., “Protein Modification by Strain-promoted Alkyne-Nitrone Cycloaddition,” Ang. Chem. Int. Ed.
(2010) 49:3065-3068). Examples of moieties comprising cyclooctynes include dibenzocyclooctynes (DBCO, DIBO, BARAC, DIBAC), fluorocyclooctynes (MOFO, DIFO, DIFO2, DIFO3), strained bicyclic cyclooctynes such as bicyclononynes (BCN), and others known in the art (see, for example, Marjoke F. Debets, et al., “Bioconjugation with Strained Alkenes and Alkynes,” Accounts of Chemical Research (2011) 44:805-815, incorporated herein by reference). The 1,1,1-trifluoropropyne may be generated in situ from the corresponding Diels-Alder adduct with furan. When one group is a terminal acetylene, the reaction is catalyzed by addition of a metal ion such as copper.
Another example is that wherein one of X1 and X3 comprises a 1,2,4,5-tetrazine, and the other is a trans-cyclooctene, norbornene, or cyclopropene (see for example, Karver, et al., “Bioorthogonal Reaction Pairs Enable Simultaneous, Selective, Multi-target Imaging,” Ang. Chem. Int. Ed. (2012) 51:920-922; Yang, et al., “Live-Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine Cycloadditions,” Ang. Chem. Int. Ed. (2012) 51:7476-7479; and Devaraj, “Advancing Tetrazine Bioorthogonal Reactions through the Development of New Synthetic Tools,” Synlett (2012) 23:2147-2152, each of which is incorporated herein by reference). These groups react via Diels-Alder reaction to provide a stable pyridazine linkage.
However, with respect to any cleavable linker that provides a protein-reactive group, X2 may be such a group. For example, if X2 comprises a hydroxysuccinimide ester or carbonate moiety it can bind to a thiol or an amine, resulting in formation of a thioester, thiocarbonate, amide, or carbamate linkage, respectively. If X2 comprises a maleimide, vinylsulfone, vinylsulfonamide, acrylate, or acrylamide, it can bind a thiol, resulting in formation of a thioether linkage.
If X2 comprises an aldehyde it can bind an amine, resulting in formation of an imine or it can bind an NH2CHCH2SH moiety of a cysteine, resulting in formation of an amide linkage via native chemical ligation or a pseudoproline linkage via pseudoproline peptide ligation (Hu, et al., “Hydrogels cross-linked by native chemical ligation,” Biomacromolecules (2009) 10:2194-2200; and Wathier, et al., “Hydrogels formed by multiple peptide ligation reactions to fasten corneal transplants,” Bioconjugate Chem. (2006) 17:873-876).
Bifunctional crosslinkers that undergo 0 elimination have been previously disclosed, for example in Santi, et al., “Predictable and Tunable Half-life Extension of Therapeutic Agents by Controlled Chemical Release from Macromolecular Conjugates,” Proc. Natl. Acad. Sci USA (2012) 109:6211-6216; PCT publications WO2009/158668 and WO2011/140393; and PCT application US/2012/54293, which are hereby incorporated by reference and are of the formula
wherein
n is 0 or 1;
X is a group that binds with the components of the hydrogel or other moiety;
at least one or both R1 and R2 is independently CN; NO2;
wherein R1 and R2 may be joined to form a 3-8 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, (CH2CH2O)p wherein p=1-1000, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted; and
wherein at least one of R1, R2, and R5 is substituted with X, wherein X is a functional group for binding to X1. In some embodiments, X does not bind directly to protein.
The crosslinking reagents for the hydrogel also include multivalent compounds of the formula
wherein one of R1, R2 and R5 in at least two of the t moieties shown within the bracket comprises the functional group X3 wherein in some embodiments X3 does not react with protein and R1, R2 and R5 are otherwise defined above and wherein
n is 0 or 1;
m is 0-1,000;
s is 0-2;
t is 2, 4, 8, 16 or 32,
W is O(C═O)O, O(C═O)NH, O(C═O), S,
wherein R6 is as defined above; and
Q is a core group having a valency=t.
The core group Q is a group of valency =t which connects the multiple arms of the crosslinking reagent. Typical examples of Q include an ethylene core CH2CH2 (t=2), pentaerythritol core C(CH2)4 (t=4); a hexaglycerin core (t=8); and a tripentaerythritol core (t=8).
Compounds of formula (2) may be prepared by the reaction of a multi-arm polyethylene glycol with a suitable reagent as disclosed in PCT application US2012/54278, which is incorporated herein by reference. A variety of multi-arm polyethylene glycols are commercially available, for example from NOF Corporation and JenKem Technologies.
The linkers of formulas (1), (1a), (1b) and (2) degrade through a non-hydrolytic elimination mechanism, with the rates of release being controlled primarily by the groups R1 and R2, and to a lesser extent R5. The properties of R1 and R2 may be modulated by the optional addition of electron-donating or electron-withdrawing substituents. By the term “electron-donating group” is meant a substituent resulting in a decrease in the acidity of the R1R2CH; electron-donating 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 on non-aromatic moieties.) 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-withdrawing group” is meant a substituent resulting in an increase in the acidity of the R1R2CH group; electron-withdrawing groups are typically associated with positive Hammett σ or Taft σ* constants and are well-known in the art of physical organic chemistry. Examples of suitable electron-withdrawing substituents include but are not limited to halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C(=O)—RX, wherein RX is H, lower alkyl, lower alkoxy, or amino, or S(O)mRY, wherein 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 upon the position of the substituent. For example, an alkoxy substituent on the ortho- or para-position of an aryl ring is electron-donating, and is characterized by a negative Hammett σ constant, while an alkoxy substituent on the meta-position of an aryl ring is electron-withdrawing and is characterized by a positive Hammett σ constant. A table of Hammett σ and Taft σ* constants values is given below.
Alkyl R5 groups slow the elimination reaction slightly relative to aryl R5 groups, and so may also be used to tune the rates of elimination and degradation.
Tissue adherence of the sealant is enhanced by reaction of a protein-reactive functional group P with the tissue matrix. P is embodied in one X2 in formula (1). For example, reaction of P with complementary functional groups on tissue proteins may provide adherence of the sealant with the tissue. Most commonly, the available protein functional groups will be amines, such that P is a group reactive with amines, for example N-hydroxysuccinimide ester or carbonate. Other groups reactive with amines may be used, including 1,3-diketones, aldehydes, and ketones. P may also be a group reactive towards protein thiols, including maleimide, vinylsulfone, vinylsulfonamide, disulfide, haloacetyl, haloacetamide, acrylate, and acrylamide.
A variety of drugs D may optionally be attached to the sealant in order to enhance wound healing. In particular, antibiotics including antibacterials, antifungals, and antivirals; hormones including steroids such as triamcinolone, triamcinolone acetonide, dexamethasone, betamethasone, prednisone, prednisolone, rimexolone, and derivatives thereof; immunosuppressants including FK506 and rapamycin; cytostatic agents including 5-fluorouracil and tubulin inhibitors such as paclitaxel, docetaxel, vincristine, and epothilones; peptides and proteins including growth factors, coagulating agents, and antibodies; and nucleic acids including aptamers and siRNA may be used. A variety of growth factors have been found to play a role in wound healing and thus may be used in the invention, including platelet-derived growth factors (PDGF), bone morphogenetic factors such as BMP-2 and BMP-7, epidermal growth factors (EGF), fibroblast growth factors such as bFGF and FGF-2, transforming growth factors like TGF-β1, vascular endothelial growth factors (VEGF), hepatocyte growth factors (HGF), keratinocyte growth factors (KGF), and insulin-like growth factors like IGF-1. The preparation of linker-drugs X2-L2-D is detailed in PCT publications WO2009/158668 and WO/2011/140393, which are hereby incorporated by reference.
In some embodiments, an adapter unit A may be present to introduce multiple functionality at the end of each arm of a reactive polymer M-(T)q. Unit A comprises a functional group X5 that is reactive with functional groups terminating the arms of reactive polymer M together with at least two functional groups, which may be the same or different:
Typical examples of suitable adapters A include derivatives of lysine, aspartic acid, or glutamic acid. If these are used as crosslinkers, further conversion of amino, carboxyl or sulfhydryl groups to cognates of groups that are not protein-reactive is needed.
In other aspects the invention provides methods for the preparation of the sealants of the invention. The sealant forming reactions may be performed in a variety of suitable solvents, for example water, alcohols, acetonitrile, or tetrahydrofuran, but are preferably performed in aqueous medium optionally in the presence of small amounts of organic cosolvents. Formation of the sealants may be performed in a stepwise or a concerted fashion. Thus, in one embodiment of the invention, a first solution comprising the hydrogel is mixed with a second solution comprising the moiety comprising the protein-reactive group, preferably of formula (1). The compound of formula (1) containing drug D may also be included. Any order of reaction may be used.
The molar ratios of the components in the polymerization mix may be adjusted to control the physical properties of the sealant, the drug content, and the attachment of the sealant to tissue. For example, the physical properties of the sealant may be controlled through appropriate selection of hydrogel with the degree of its crosslinking, and the ratio of the moiety comprising the protein-reactive functional group.
The nature of the sealant as related to the nature of the hydrogel itself is controlled by the level of crosslinking. This is determined by the value of q in the crosslinker of formula (2). Generally speaking, as q increases the sealant becomes stiffer and more durable, with the upper bound of q being determined by the difference in the number of reactive groups on M and the sum of the number of drugs D and tissue adherent groups P concurrently attached to M. It is not necessary for all reactive groups on M to be bonded to T, D, and P groups, and given the polymeric nature of the sealants of the invention it is not necessary for every M unit in the sealant to be bonded to a P and/or D group, such that taken on average across the bulk polymeric sealant, the number n of D groups and m of P groups per M unit may be non-integer ratios. In some embodiments, the number of D groups is 0.
The polymer content of the final water-swollen sealants may be between 1 and 50% w/v. In some embodiments, the polymer content is between 1 and 25% w/v. In some embodiments, the polymer content is between 1 and 10% w/v.
Some illustrative methods are shown in
In
The components of the polymerization mixture may be supplied as dried solids or as suspensions or solutions, for example as aqueous solutions optionally in the presence of buffers, antioxidants, or pharmaceutically acceptable excipients. When supplied as dry solids, the components may also contain excipients in dry form and may be reconstituted with sterile water prior to use or may be dissolved directly into a solution comprising other components of the sealant mixture. In some embodiments, the reactivity of one or more component is modulated by control of the pH of the solution. Pharmaceutically acceptable dyes may be added to enhance visualization of the sealant.
The sealant is applied to the site requiring sealing and allowed to set. Application may be as a bulk liquid, for example by extrusion from a syringe onto the wound, or by aerosol, for example using a spray device. Two solutions may be premixed or mixed during application using a multi-channel device, for example a multi-barrel syringe wherein each barrel contains a component of the polymerization mixture and the reactive components are mixed upon extrusion into the syringe tip or needle. Devices for application of surgical sealants have been disclosed, for example in U.S. Pat. No. 8,343,183 (issued 1 Jan. 2013) and U.S. Pat. No. 8,262,608 (issued 11 Sep. 2012).
In an alternate embodiment of the invention, the sealant may be preformed and then used as a surgical implant. The sealant may be formed into specific shapes through molding or cutting, and then applied to the wound in the polymeric form.
In another aspect the invention provides multi-layer hydrogels and sealants. The multi-layer hydrogels or sealants of the invention are degradable hydrogels comprising at least two layers, wherein each layer is a hydrogel formed from polymers, some of which are multi-armed polymers coupled through biodegradable linkages and wherein the layers of the multilayer hydrogel or sealant are coupled covalently to each other.
In various embodiments of the invention, the successive layers of the multi-layer hydrogel have different degradation rates, or successive layers have different elastic moduli or other physical characteristics such as polymer molecular weight or wt % polymer, or successive layers comprise different releasable drugs D, or successive layers comprise one or more drugs D attached via different releasable linkers or one layer comprises a releasable drug D while the other does not. In one specific embodiment of the invention, one layer of the sealant comprises a tissue-reactive group P while the other comprises a peptide or protein drug D.
In some embodiments, the coupling is through functional groups themselves used to form the individual layers, and which are unreacted in the hydrogel formation of each layer. In some embodiments, at least one of the layers contains a plurality of protein-reactive functional groups so that the multilayer hydrogel behaves as a sealant. In some embodiments, one of the layers comprises a drug. The plurality of protein-reactive functional groups is coupled to at least a first layer through a biodegradable linkage and, in some embodiments, this linkage is degradable through an elimination reaction. The hydrogels themselves are typically biodegradable and the crosslinkers conferring capability for biodegradation may respond to enzymatic or other types of cleavage and in some embodiments are biodegradable by virtue of crosslinkers that are cleavable by an elimination reaction. Thus, the hydrogels and sealants described in the cited PCT application PCT/US2012/54278 and the sealants described in the present application may form the first and second layers. Typically, a first layer intended to be adjacent to tissue will comprise a sealant (i.e., contains said plurality of protein-reactive functional groups) and the second layer, intended to overlay the first layer and in contact with a biological fluid ordinarily in contact with the tissue would also comprise a hydrogel as above described. Such an arrangement is especially useful for drug delivery; the first layer would thus contain drug in a form that can be released into the tissue and the second layer serves a protective function with respect to the drug. Thus, the multilayer hydrogel or sealants of the invention in one embodiment comprise at least a first layer having a first pore size and a second layer overlaying the first layer and coupled thereto said second layer having a different pore size from the first layer.
Where the second layer is intended to shield the first layer which may be adjacent a tissue, the pore size of the second layer will be smaller than that of the first. For example, the first layer may have a pore size with an average diameter of >100 nm and the second layer has a pore size with an average diameter of 1-100 nm. Alternatively, the first layer may have a pore size with an average diameter of 1-100 nm and the second layer a pore size of <1 nm average diameter. In some embodiments, the first layer has a pore size of average diameter of more than 100 nm and the second layer has a pore size of an average diameter of 1-100 nm. In multilayer sealants or hydrogels with more than two layers, varying pore sizes may also be present in each layer.
The multilayer hydrogels and sealants of the invention may be synthesized ex vivo. If so, they may be implanted as such in a subject for sealing tissue or drug delivery or other medical purposes. Alternatively, the multilayer hydrogels or sealants may be formed in situ laying a first layer over the tissue and a second layer atop the first and so on.
Various coupling techniques may be employed; essentially the layers are coupled through cognate functional groups. The cognate functional groups are typically selected from those set forth above for a formation of hydrogels and binding of drugs or protein-reactive groups thereto. The functional groups may constitute those useful in formation of the hydrogels that have been left unreactive by hydrogel formation. For this purpose, in forming at least a bilayer, the cognate functional groups need not exclude protein-reactive groups.
As noted above, in one particularly useful embodiment, a first layer with larger pores than the second will comprise at least one drug coupled thereto by a biodegradable linkage and in some cases by a linker of formula (1b).
The bilayers of the invention are typically biodegradable and preferably biodegradable by virtue of a plurality of crosslinking molecules that are cleavable by an elimination reaction.
All references cited herein are hereby incorporated by reference in their entirety. The invention is further illustrated but not limited by the following examples.
A 1.6 M solution of n-butyllithium (3.1 mL, 5.0 mMol) in hexane was added dropwise to a stirred solution of R1CH3 (5.0 mMol) in anhydrous tetrahydrofuran (THF) (15 mL) cooled to −78° C. After 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 back 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 aq. NH4Cl was added and the mixture was allowed to continue warming to ambient 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. Compounds prepared according to this method include:
1-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptanol (R1=4-(trifluoro-methyl)phenyl-SO2): from 4-(trifluoromethyl)phenyl methyl sulfone (1.73 g, 94%): 1H-NMR (400 MHz, CDCl3): δ8.08 (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 (R1=4-chlorophenyl-SO2): from 4-chlorophenyl methyl sulfone; colorless oil (1.49 g, 90% yield): 1H-NMR (400 MHz, d6-DMSO): δ7.90 (2H, d, J=8.8-Hz), 7.70 (2H, d, J=8.8-Hz), 4.83 (1H, d, J=6-Hz), 3.86 (1H, m), 3.39 (2H, m), 3.29 (2H, t, J=6.8-Hz), 1.2˜1.5 (8H, m);
1-(phenylsulfonyl)-7-azido-2-heptanol (R1=phenyl-SO2): from phenyl methyl sulfone; pale yellow oil (1.25 g, 85%): 1H-NMR (400 MHz, d6-DMSO): δ7.89 (2H, m), 7.72 (1H, m), 7.63 (2H, m), 4.84 (1H, d J=6-Hz), 3.86 (1H, m), 3.33 (2H, m), 3.28 (2H, t, J=6.8-Hz), 1.47 (2H, m), 1.2˜1.4 (6H, m);
1-(4-methylphenylsulfonyl)-7-azido-2-heptanol (R1=4-methylphenyl-SO2): from 4-(methylsulfonyl)toluene; colorless oil (1.39 g, 85% yield): 1H-NMR (400 MHz, d6-DMSO): δ7.76 (2H, d, J=6.4-Hz), 7.43 (2H, d, J=6.4-Hz), 4.82 (1H, d, J=6-Hz), 3.85 (1H, m), 3.31 (2H, m), 3.28 (2H, t, J=6.8-Hz), 2.41 (3H, s), 1.4˜1.5 (2H, m), 1.2˜1.4 (6H, m);
1-(4-methoxyphenylsulfonyl)-7-azido-2-heptanol (R1=4-methoxyphenyl-SO2): from 4-methoxyphenyl methyl sulfone (1.53 g, 94% yield): 1H-NMR (400 MHz, CDCl3): δ7.85 (2H, d, J=8.8-Hz), 7.04 (2H, d, J=8.8-Hz), 4.13 (1H, m), 3.90 (3H, s), 3.24 (2H, t, J=6.8-Hz), 3.20 (1H, dd, J=8.8, 14.4-Hz), 3.14 (1H, dd, J=2.4, 14.4-Hz), 2.47 (3H, s), 1.57 (2H, m), 1.5˜1.3 (6H, m);
1-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptanol ((R1=2,4,6-trimethylphenyl-SO2): from (2,4,6-trimethyl)phenyl methyl sulfone (1.30 g from 4.0 mMol reaction; 96%): 1H-NMR (400 MHz, CDCl3): δ6.99 (2H, s), 4.30 (1H, m), 3.49 (1H, d, J=2-Hz), 3.25 (2H, t, J=6.8-Hz), 3.18 (1H, d, J=1-Hz), 3.17 (1H, s), 2.66 (6H, s), 2.31 (3H, s), 1.59 (2H, m), 1.5˜1.3 (6H, m);
1-(morpholinosulfonyl)-7-azido-2-heptanol (R1=O(CH2CH2)2N-SO2): from 1-morpholino methylsulfonamide (1.36 g from 10 mMol reaction, 89%): 1H-NMR (400 MHz, d6-DMSO): δ4.99 (1H, d, J=6.4 Hz), 3.88 (1H, m), 3.62 (4H, t, J=4.8-Hz), 3.32 (2H, t, J=6.8-Hz), 3.20˜3.15 (6H, overlap), 1.53 (2H, m), 1.46˜1.25 (6H, m);
1-(methylsulfonyl)-7-azido-2-heptanol (R1=CH3-SO2): from dimethylsulfone; colorless oil (880 mg, 75%): 1H-NMR (400 MHz, d6-DMSO);
1-cyano-7-azido-2-heptanol (R1=CN): from acetonitrile;
1-(methylsulfonyl)-7-azido-2-heptanol (R1=CH3-SO2): from dimethylsulfone; colorless oil (880 mg, 75%): 1H-NMR (400 MHz, CDCl3): δ4.29 (1H, m), 3.28 (2H, t, J =7.2 Hz), 3.17 (1H, dd, J=9.6, 14.4 Hz), 3.07 (1H, dd, J=1.2, 14.4 Hz), 3.02 (3H, s), 2.90 (1H, d, J=3.6), 1.35˜1.7 (8H, m);
1-cyano-7-azido-2-heptanol (R1=CN): from acetonitrile; colorless oil (320 mg, 0.98 mMol, 98%). 1H-NMR (400 MHz, CDCl3): δ5.18 (1H, d, J=5 Hz), 3.69 (1H, m), 3.32 (2H, t, J=6 Hz), 2.60 (1H, dd, J=4.8, 16.4 Hz), 2.51 (1H, dd, J=6.4, 16.4 Hz), 1.55 (2H, m), 1.42 (2H, m), 1.30 (4H, m);
1-(N,N-diethylaminosulfonyl)-7-azido-2-heptanol; from N,N-diethyl methane-sulfonamide, colorless oil [0.471 g (1.6 mMol) from 4.0-mMol reaction, 40% yield]; 1H-NMR (400 MHz, CDCl3): δ1.20 (6H, t, J=7.2 Hz), 1.41 (6H, m), 1.59 (2H, m), 2.95; and (2H, m), 3.26 (6H, m), 3.39 (1H, d, J=2.2 Hz), 4.15 (1H, m).
Pyridine (160 μL) was added dropwise to a stirred solution of the azidoalcohol (Preparation A, 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.
Pyridine (300 μL) was added dropwise to a stirred solution of the above chloroformate 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 the crude succinimidyl carbonate. Purification by silica gel chromatography provided the purified product as an oil which typically spontaneously crystallized. Recrystallization could typically be effected using ethyl acetate/hexane.
A stirred solution of an azido-linker alcohol of Preparation A (1 mMol) in 1 mL of tetrahydrofuran (THF) was treated with a 1.0 M solution of trimethyl-phosphine in THF (1.2 mL) for 1 hour at ambient 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 was dried over MgSO4, filtered, and evaporated to provide the amino-alcohol.
A solution of the amino-alcohol (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 was 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 BOC-amino-alcohol.
BOC-amino alcohols were converted into the chloroformates and succinimidyl carbonates using the methods of Preparation B above.
A solution of the succinimidyl carbonate of Preparation B (0.27 mMol) in 2 mL of acetonitrile was treated with 11-azido-3,6,9-trioxaundecan-l-amine (65 mg, 0.30 mMol) for 10 min at ambient temperature. After evaporation of the solvent, the residue was dissolved in 1 mL of CH2C12 and chromatographed on a 4-g column of silica gel using a step gradient of hexane, 3:1 hexane/ethyl acetate, 1:1 hexane/ethyl acetate, and 1:2 hexane/ethyl acetate. The product-containing fractions were pooled and evaporated to provide the product.
In a typical example, a solution of 25 μMol of the azido-linker-HSC (Preparation B) in 1 mL of acetonitrile was added to 5 μMol (100 mg) of 20-kDa 4-arm PEG-tetraamine hydrochloride in 1 mL of water and 40 μL of 1.0 M NaHCO3. After 1 h at ambient temperature, TNBS assay indicated <2% of the amine groups remained, and the solution was dialyzed against 1 L of 50% methanol followed by 1 L of methanol (12 kDa cutoff membrane). After evaporation, the residue (109 mg) was dissolved in 2.12 mL of sterile-filtered 10 mM NaOAc, pH 5.0, and stored frozen at −20° C. The azide concentration was determined spectrophotometrically by reaction with DBCO acid.
In a typical example, a solution of 26 mg (50 μMol) of the BOC-amino-linker-HSC (Preparation C), 200 mg of 20-kDa 4-arm PEG-tetraamine hydrochloride (10 μMol; 40 μMol amine), and 17 μL (100μMol) of diisopropylethylamine in 2 mL of tetrahydrofuran was stirred for 1 h at ambient temperature. TNBS assay indicated <1% of the amine groups remained, and the product was precipitated by slow addition of 10 mM of methyl t-butyl ether. The BOC-protected product was collected by vacuum filtration, washed with MTBE, dried, and stored frozen at −20° C.
The BOC-protected product (100 mg, 5 μMol) was dissolved in 2 mL of CF3CO2H, kept for 1 h, and then evaporated to dryness. The residue was washed twice with 5 mL portions of ether, then dissolved in 2 mL of THF and precipitated with 10 mL of MTBE to provide the PEG-(linker-amine)4 as the trifluoroacetate salt. TNBS assay indicated 91% of the theoretical amine content by weight.
PEG-linker-X2 crosslinkers wherein X2 is other than azide or NH2 may be prepared by derivitization of the PEG-linker-amine crosslinkers of Preparation F using the appropriate reagents. Thus, crosslinkers wherein X2=SH may be prepared by reaction with a reagent such as the succinimidyl ester of 3-(2-pyridyldithio)propionate so as to prepare the intermediate 2-pyridyldisulfide, followed by reduction to the thiol using standard reagents like phosphines (TCEP, trimethylphosphine, triphenylphosphine, etc.). Reaction with a reagent such as the succinimidyl ester of 3-(maleimido)propionate will provide the crosslinkers wherein X2=maleimide.
PEG20 kDa-(DBCO)4: A 60 mM solution of freshly chromatographed DBCO-NHS (Click Chemistry Tools) in acetonitrile (0.5 mL, 30 μMol, 1.5 eq) was added to a solution of 20 kDa 4-arm PEG-amine hydrochloride (pentaerythritol core, JenKem Technologies; 100 mg, 5 μMol), and diisopropylethylamine (0.010 mL, 57 μMol) in acetonitrile (1 mL). After stirring 2 h at ambient 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 50 mg/mL stock (10 mM DBCO by spectrophotometric assay), which was stored frozen at −20° C.
PEG40 kDa-(DBCO)8: One mL of 40 mM solution (40 μMol) of DBCO-NHS in THF was added to a solution of 168 mg (4.2 μMol) of 40-kDa 8-arm PEG-amine hydrochloride (tripentaerythritol core, JenKem Technologies) and 12.9 μL diisopropylethylamine (74 μMol) in 0.6 mL of ACN, and the mixture was kept at ambient 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 water and stored frozen at −20° C. The DBCO concentration determined spectrophotometrically was 16 mM.
PEG40 kDa(BCN)8: A solution of 200 mg of 40 kDa 8-arm PEG-amine.HCl (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 ambient 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, 1:1 methanol/water, and methanol using a 12 kDa membrane. After evaporation, the residue was dissolved in 2 mL of THF and precipitated with 10 mL of methyl tbutyl ether. The product was collected and dried (190 mg).
One method for preparation of multi-arm crosslinkers of formula (2) is illustrated wherein R1=4-morpholino-SO2, R2=H, n=0, one R5=h and the other R5 =(CH2)5NH—CO—CH2O—NH2, W═O(C═O)NH, Q=C(CH2)4, m˜110, s=2, and t=4. A solution of 4-arm 20 kDa PEG-tetraamine hydrochloride (200 mg, 10 uMol, JenKem Technologies), 7-(tBOC-amino)-1-(4-morpholinosulfonyl)-2-heptyl succinimidyl carbonate (26 mg, 50 uMol), and N,N-diisopropylethylamine (17 uL, 100 uMol) in 2 mL of THF was kept for 1 h. The product was precipitated by addition of 10 mL of methyl t butyl ether (MTBE) and dried. Analysis by TNBS assay indicated 99% derivitization of PEG amine groups. A solution of PEG20 kDa-[NHCO2—CH(CH2SO2N(CH2CH2)2O))(CH2)5NHtBOC]4 (94 mg) in 1:1 CF3CO2H/CH2Cl2 (2 mL) was kept at room temperature for 1.5 h then concentrated to dryness under vacuum. The resulting residue was dissolved in 2 mL of MeOH and precipitated with Et2O (15 mL) to give the product (36 mg) as a white solid. A solution of PEG20 kDa-[NHCO2-CH(CH2SO2N(CH2CH2)2O))(CH2)5NH3+CF3CO2−]4 (34 mg, 0.0068 mMol (NH2), 1 equiv) was treated with a solution of 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylaminooxy)acetate (2.35 mg, 0.0082 mMol, 1.2 equiv) and DIPEA (0.0012 mL, 0.9 mg, 0.0070 mMol, 1 equiv). The resulting mixture was kept at room temperature for 2 h then assessed for free amines by TNBS assay: A sample of the reaction mixture (0.020 mL) was incubated in 100 mM pH 9.4 borate buffer (1 mL) containing 0.04% w/v picrylsulfonic acid. The absorbance of this solution was monitored at 420 nm until stable (˜1 h) and compared to a reaction containing PEG20 k-[SO2Morph-linker-NH2.TFA]4 at the same concentration. Less than 2% amines remained. The reaction mixture was then diluted with Et2O (15 mL) and the precipitated product was collected by filtration to give the product (29 mg) as a white solid. A solution of PEG20 kDa-[NHCO2-CH(CH2SO2N(CH2CH2)2O))(CH2)5NH—CO—CH2ONHtBOC]4 (39 mg) in 1:1 CF3CO2H/CH2Cl2 (2 mL) was kept at room temperature for 1.3 h then concentrated to dryness under vacuum. The resulting residue was triturated with Et2O (2×10 mL) to give the product (29 mg) as a white solid.
One method for the preparation of a multi-arm M wherein each arm is terminated with an adapter unit comprising two differentially-reactive groups is illustrated by preparation of a 4-arm 20-kDa PEG having azide and aldehyde groups.
(S)-6-azido-2-(4-formylbenzamido)hexanoic acid. A solution of Boc-Lys(N3)—OH (Anaspec, 109 mg, 0.4 mMol) in 1:1 DCM:TFA (2 mL) was kept at room temperature for 1.5 h then concentrated to dryness under vacuum to give H-Lys(N3)—OH (95 mg, 83%) as a white solid (TFA salt). A solution of H-Lys(N3)—OH (19 mg, 0.066 mMol, 1 equiv) and DIPEA (0.035 mL, 26.0 mg, 0.20 mMol, 3 equiv) in DMF with 20% water (1.2 mL) was treated with a solution of 2,5-dioxopyrrolidin-1-yl 4-formylbenzoate (49.3 mg, 0.20 mMol, 3 equiv) in DMF (2 mL). The reaction was allowed to stir for 20 h prior to dilution with EtOAc (15 mL). The resulting mixture was extracted with 0.25 M NaHCO3 (3×6 mL). The combine NaHCO3 extracts were acidified to pH 2.5 with using 6 N HCl and extracted with EtOAc (4×6 mL). The combine EtOAc extracts were washed with water (3×5 mL), then brine (2 mL), and dried over MgSO4 to give a white solid (30.4 mg). This material was further purified by C18 HPLC 20-85% ACN with 0.1% TFA linear gradient elution (5 mL/min) as the mobile phase. The combine product containing fractions were concentrated under vacuum to 50% of their original volume then extracted with EtOAc (5×10 mL). The EtOAc extracts were washed with water (5×10 mL) then brine (5 mL) and concentrated to dryness to give (S)-6-azido-2-(4-formylbenzamido)hexanoic acid (10.2 mg, 52%) as a clear oil. C18 HPLC 20-100% ACN with 0.1% TFA linear gradient elution RV=5.8 mL.
(S)-2,5-dioxopyrrolidin-1 -yl 6-azido-2-(4-formylbenzamido)hexanoate. A solution of N,N′-dicyclohexylcarbodiimide (6.81 mg, 0.033 mMol, 1 equiv) in THF (0.7 mL) was added to a solution of (S)-6-azido-2-(4-formylbenzamido)hexanoic acid (10.0 mg, 0.034 mMol, 1 equiv) and N-hydroxysuccinimide (3.8 mg, 0.033 mMol, 1 equiv) in THF (0.7 mL) at 4° C. The resulting mixture was kept at 4° C. for 17 h then filtered. The filtrate was concentrated to dryness to give 18 mg of residue. This crude material (S)-2,5-dioxopyrrolidin-1-yl 6-azido-2-(4-formylbenzamido)hexanoate (13 mg, max yield) was used to acylate PEG20 k-(NH2)4 without further purification. C18 HPLC 20-100% ACN with 0.1% TFA linear gradient elution RV=6.8 mL.
PEG20 k-[Lys(N3)-CHO]4. A solution of PEG20 k(NH2.HCl)4 (JenKem, 27 mg, 0.0054 mMol (NH2), 1 equiv) and DIPEA (0.0028 mL, 3 mg, 0.0162 mMol, 3 equiv) in acetonitrile (0.5 mL) was treated with a solution of (S)-2,5-dioxopyrrolidin-1-yl 6-azido-2-(4-formylbenzamido)hexanoate (˜6.5 mg, 0.0162 mMol, 3 equiv) in DMF (0.5 mL). The resulting mixture was kept for 2 h then assessed for free amines by TNBS assay: A sample of the reaction mixture (0.018 mL) was incubated in 100 mM pH 9.4 borate buffer (1 mL) containing 0.04% w/v picrylsulfonic acid. The absorbance of this solution was monitored at 420 nm until stable (˜1 h) and compared to a reaction containing PEG20 k-(NH2.HCl)4 at the same concentration. Less than 2% amines remained. The reaction mixture was filtered to remove a small amount of insoluble material then it was then diluted with Et2O (15 mL) and the precipitated product was collected by filtration to give PEG20 k-[Lys(N3)-CHO]4 (26 mg) as a white solid.
One method for formation of X-linker-Drugs useful in attaching to the sealants of the invention is illustrated using D-NH2=5-(aminoacetamido)fluorescein as a model drug. A solution of 5-(aminoacetamido)fluorescein (Invitrogen, 0.1 mL, 21.7 mM, 0.0022 mMol, 1 equiv) in DMF was mixed with a solution of the linker of formula (1b) wherein R1=phenylsulfonyl, R2=H, n=0, one R5=H, the other R5=(CH2)5NHtBOC, and X4=O-succinimidyl (0.087 mL, 25 mM, 0.0022 mMol, 1 equiv). The resulting mixture was kept at room temperature for 1.5 h then it was acidified with 0.001 N HCl (5 mL), a yellow precipitate forms, and extracted with EtOAc (2×5 mL). The combine EtOAc extracts were washed with water (2×3 mL), then brine (2 mL), then dried over MgSO4, and concentrated to dryness under vacuum to give ˜2 mg of a yellow residue. This material was treated with 1:1 DCM:TFA (1 mL) for 1 h at room temperature. The resulting mixture was then concentrated to dryness under vacuum to give the amine intermediate as a yellow residue. This material was dissolved in acetonitrile (1 mL) to give a solution containing 1.4 mM (0.0014 mMol, 64% yield) fluorescein based on ε495=80000 M−1 cm−1. C18 HPLC 20-100% ACN with 0.1% TFA linear gradient elution RV=5.3 mL. Replacement of the 5-(aminoacetamido)fluorescein with an amine-containing drug allows for analogous preparation of linker-drug units. A solution of the linker-drug unit in acetonitrile (1 mL, 1.4 mM, 0.0014 mMol, 1 equiv) was treated with a solution of DBCO-PEG4-NHS ester (Click Chemistry Tools) in acetonitrile (0.0372 mL, 37.6 mM, 0.0014 mMol, 1 equiv) and a solution of N,N-diisopropylethylamine (DIPEA) in acetonitrile (5.74 mM, 0.50 mL, 0.0028 mMol, 2 equiv). The resulting mixture was kept at room temperature for 2.5 h then acidified with 0.001 N HCl (20 mL) and extracted with EtOAc (3×5 mL). The combine EtOAc extracts were washed with water (3×5 mL), then brine (2 mL), and concentrated to dryness. The resulting residue was dissolved in 0.3 mL DMF to give a solution containing 2.8 mM fluorescein (0.00084 mMol, 60%) based on ε495=80000 M−1 cm−1. C18 HPLC 20-100% ACN with 0.1% TFA linear gradient elution RV =7.5 mL. This method can be used to prepare analogous X-linker-drugs through appropriate choice of the X-NHS reagent.
To a solution of 7-(diethylamino)coumarin-3-carboxylic acid N-succinimidyl ester (Fluka 36801, 20.9 mg, 0.056 mMol, 1 equiv) in DMSO (1.4 mL) was added a solution of N-Boc-1,4-diaminobutane (Sigma 15404, 11.7 mg, 0.062 mMol, 1.1 equiv) in DMSO (0.72 mL), followed by Et3N (24 μL, 17.4 mg, 0.17 mMol, 3 equiv). The resulting mixture was allowed to stir at room temperature for 1.5 h, at which time the reaction was complete by TLC (Silica, 2:1 EtOAc:Hexanes, Rf(coumarin-NHS)=0.29, Rf(BOC-product)=0.43). The reaction mixture was then diluted with EtOAc (20 mL), washed with water (5×4 mL), then brine (2 mL), then dried over solid MgSO4, filtered through silica, and concentrated to dryness under reduced pressure. The resulting residue was dissolved in DCM (1 mL), treated with TFA (1 mL) for 1 h at room temperature, at which time the reaction was complete by TLC (Silica, EtOAc, Rf(BOC)=0.63, Rf(amine)=0). The reaction mixture was concentrated to dryness under reduced pressure. The resulting residue was triturated with Et2O (3×5 mL portions) then dried under vacuum to give DEAC-NH2 (26.3 mg, 0.059 mMol, 105%) as a yellow solid (TFA salt). C4 HPLC 0-100% ACN 0.1% TFA linear gradient elution (RVamine=5.8 mL). A solution of DEAC-NH2(TFA) (3.88 mg, 0.0087 mMol, 1 equiv) in DMF (0.5 mL) was treated with DIPEA (0.0033 mL, 2.5 mg, 0.0019 mMol, 2 equiv) and a solution of DBCO-NHS ester (Click Chem. Tools A102, 43.6 mM, 0.200 mL, 0.0087 mMol, 1 equiv). The resulting mixture was allowed to stir overnight (a small amount of DBCO-NHS remains as determined by C18 HPLC) then treated with 10 mM taurine in pH 7.5 HEPES. The resulting mixture was allowed to stir for 4 h then it was diluted with 0.5 N NaHCO3 (10 mL) and extracted with EtOAc (3 x 6 mL). The combine EtOAc extracts DEAC-DBCO (4.7 mg, 84%). This material was dissolved in DMF (0.7 mL) to give a 7.9 mM solution based on ε430=44800 M−1cm−1.
N-{7-Azido-1-[N-methyl-N-(2-methoxyethyl)aminosulfonyl]-2-heptyloxycarbonyl}-Glu(OtBu)-OH. N,N-Diisopropylethylamine (164 μL, 942 μMol) and a solution of 7-azido-1-[N-methyl-N-(2-methoxyethyl)aminosulfonyl]-2-heptyl succinimidyl carbonate (385 mg, 857 μMol) in 4 mL of acetonitrile were added to a suspension of H-Glu(OtBu)-OH (191 mg, 942 μMol) in 4 mL of acetonitrile. Suspended H-Glu(OtBu)-OH did not dissolve upon addition of reagents; thus, 8 mL of DMF was added. The solid did not immediately dissolve. Next, 2 mL of water was added, and the suspended solid dissolved within 2 min. After 30 min at ambient temperature the reaction was judged to be complete by TLC analysis, and the mixture was partitioned between 150 mL of 1:1 EtOAc:KHSO4 (½ sat aq). The layers were separated, and the organic phase was successively washed with KHSO4 (2.5% aq), water, and brine (1×100 mL each). The organic layer was then dried over MgSO4, filtered, and concentrated by rotary evaporation. The resulting crude colorless oil was purified by silica gel column chromatography (4 g) eluting with dichloromethane (40 mL) followed by a gradient of acetone in dichloromethane: 15% (40 mL), 30% (40 mL), and 65% (40 mL). Mixed fractions were rechromatographed eluting with dichloromethane (30 mL) followed by a gradient of acetone in dichloromethane: 3% (30 mL), 6% (30 mL), 9% (30 mL), 12% (30 mL), and 15% (30 mL). Clean product containing fractions from both columns were combined and concentrated to provide 316 mg (69%) of the title compound as a thick colorless oil.
N-{7-Azido-1-[N-methyl-N-(2-methoxyethyl)aminosulfonyl]-2-heptyloxycarbonyl}-Glu(OtBu)-OSu. N,N′-Disuccinimidyl carbonate (196 mg, 764 μMol) and 4-(dimethylamino)pyridine (0.20 M in acetonitrile, 0.30 mL, 60 μMol) were successively added to a solution of N-{17-azido-1-[N-methyl-N-(2-methoxyethyl)-aminosulfonyl]-2-heptyloxycarbonyl}-Glu(OtBu)-OH (316 mg, 589 μMol) in 5.6 mL of acetonitrile. The reaction mixture was stirred at ambient temperature while monitoring progress by TLC. After 15 min, the reaction was judged to be complete, and the mixture was partitioned between 200 mL of 1:1 EtOAc:NaHCO3 (½ sat aq). The layers were separated, and the organic phase was successively washed with water, KHSO4 (2.5% aq), water, and brine (1×100 mL each). The organic layer was then dried over MgSO4, filtered, and concentrated by rotary evaporation and high vacuum to provide 353 mg (94% crude) of the crude title compound as a colorless oil. The product was used without further purification.
{N-[7-Azido-1 -(N-methyl-N-(2 -methoxyethyl)aminosulfonyl)-2-heptyloxycarbonyl]-Glu(OtBu)}4-PEG20 kDa. N,N-Diisopropylethylamine (164 μL, 942 μMol) and N-{17-azido-1-[N-methyl-N-(2-methoxyethyl)aminosulfonyl]-2-heptyloxycarbonyl}-Glu(OtBu)-OSu (137 μM in MeCN, 2.85 mL, 390 μMol) were successively added to a solution of PEG20 kDa-(NH2.HCl)4 in 12 mL of acetonitrile. The reaction mixture was stirred at ambient temperature while monitoring progress by TLC. After 20 min, the starting succinimidyl ester was not observed by TLC. More N-{7-azido-1-[N-methyl-N-(2-methoxyethyl)aminosulfonyl]-2-heptyloxycarbonyl}-Glu(OtBu)-OSu (137 μM in MeCN, 0.57 mL, 78 nMol) was added, and the reaction mixture was stirred for 1.7 h more (2 h total). Acetic anhydride (28 μL, 0.30 mMol) was added to cap any unreacted amines, and stirring was continued for 20 min more. The reaction mixture was concentrated to 6 mL then added to 80 mL of tent-butyl methyl ether. The resulting suspension was stirred for 30 min then vacuum filtered. Solids were washed with tert-butyl methyl ether (3 x 20 mL) then dried under vacuum to provide 1.48 g (89%) of the title compound as a white powder.
{N-[7-Azido-1-(N-methyl-N-(2-methoxyethyl)aminosulfonyl)-2 -heptyloxycarbonyl]-Glu}4-PEG20 kDa. Trifluoroacetic acid (7 mL) was added to a solution of {N-[7-azido-1-(N-methyl-N-(2-methoxyethyl)aminosulfonyl)-2-heptyloxycarbonyl]-Glu(OtBu)}4-PEG20 kDa (1.48 g, 67.0 μMol PEG) in 7 mL of dichloromethane. The reaction mixture was stirred at ambient temperature while monitoring progress by C18 HPLC. After 2 h, the reaction mixture was concentrated to dryness. The crude residue was redissolved in 8 mL of tetrahydrofuran then added dropwise to 80 mL of diethyl ether. The resulting suspension was stirred for 30 min then vacuum filtered. Solids were successively washed with diethyl ether (3×30 mL) and 30 mL of tert-butyl methyl ether then dried under vacuum to provide 1.34 g (91%) of the title compound as a white powder.
{N-[7-Azido-1-(N-methyl-N-(2-methoxyethyl)aminosulfonyl)-2-heptyloxycarbonyl]-Glu(OSu)}4-PEG20 kDa. N,N′-Disuccinimidyl carbonate (9.3 mg, 36 μMol) and 4-(dimethylamino)pyridine (0.2 M in MeCN, 14 μL, 2.8 μMol) were successively added to a solution of {IN-[7-azido-1-(N-methyl-N-(2-methoxyethyl)aminosulfonyl)-2-heptyloxycarbonyl]-Glu}4-PEG20 kDa (140 mg, 6.4 μMol PEG, 26 μMol CO2H) in 1.4 mL of acetonitrile. The reaction mixture was stirred at ambient temperature while monitoring progress by C18 HPLC. After 45 min, the reaction mixture was added dropwise to 18 mL of tert-butyl methyl ether. The resulting suspension was stirred for 30 min, and the supernatant was decanted. The solid was resuspended in 10 mL of tert-butyl methyl ether then vacuum filtered. The solids were washed successively with 2-propanol (2×8 mL) and tert-butyl methyl ether (2×8 mL) then dried under vacuum to provide 122 mg (86%) of the title compound as a white powder.
A mixture of 8-arm 20-kDa PEG-octaamine.HCl (4.10 g, 205 μMol, 1640 μMol amine; tripentaerythritol core, JenKem), succinic anhydride (400 mg, 4000 μMol), and N,N-diisopropylethylamine (700 uL, 4000 μMol) in 40 mL of anhydrous acetonitrile was stirred for 30 min, at which time there were no remaining amine groups by TNBS assay. The polymer was precipitated by slow addition to 200 mL of stirred 2-propanol. The solid was collected by vacuum filtration and dried under vacuum (4.13 g). This material was dissolved in 20 mL of anhydrous acetonitrile and treated with disuccinimidyl carbonate (1.4 g, 5.5 mMol) and 4-(dimethylamino)pyridine (50 mg, 0.41 mMol) for 16 h. The solvent was evaporated under vacuum, and the residue was dissolved in 20 mL of THF and precipitated by slow addition to 150 mL of stirred MTBE. The solid was collected by vacuum filtration, washed once with 2-propanol, and dried under vacuum. The dried material was similarly precipitated once from 2-propanol, dried, then a second time from MTBE and dried to provide the product octa(succinimidyl ester) as a white solid, 4.0 g (90%). To analyze the product, a sample (2.7 mg) was reacted with 200 uL of 10 mM 4-nitrobenzylamine hydrochloride and 20 mM N,N-diisopropyl-ethylamine in 800 uL of acetonitrile for 30 min, and the mixture was analyzed by reversed-phase HPLC with integration of peaks detected at 275 nm, which indicated 7.9±0.2 HSE groups/PEG. The product was further analyzed using a published method (Gao, et al., Chemistry Central J. (2012) 6:142) that indicated 106 ±14% of the expected HSE content. Use of these materials rather than the ester-linked PEG-succinimidyl succinate (PEG-OCO—CH2CH2—COOSu)8 or PEG-succinimidyl glutarate (PEG-OCO—CH2CH2CH2—COOSu)8 provide sealants that are resistant to hydrolytic degradation due to the lack of ester linkages in the final sealants.
A general method for preparation of PEG-(linker-succinimidyl carbonate)8 macromonomers is illustrated by the specific preparation of the compound wherein R1 is SO2N(Me)(CH2CH2OMe). 7-(BOC-amino)-1-(N-methyl-N-(2-methoxyethyl)-aminosulfonyl)-2-heptanol (192 mg, 500 μMol; Preparation C) was dissolved in 2 mL of 1:1 CH2Cl2/CF3CO2H+1% triethylsilane. After 30 min, the mixture was evaporated to dryness and the residue was washed 3x with ethyl ether. The residue was dissolved in 5 mL of THF and treated with 8-arm 20-kDa PEG-octa(succinimidyl succinate) (1.00 g, 50 μMol PEG, 400 μMol amine, Preparation N) and N,N-diisopropylethylamine (100 uL, 575 uMol). Reaction progress was monitored by assay of aliquots in the 4-nitrobenzylamine assay described above. After complete consumption of HSE groups (1 h), the mixture was added slowly to 100 mL of stirred 2-propanol and the precipitated product was collected, washed with MTBE, and vacuum dried to give 1.01 g (84% yield) of PEG-(linker-alcohol)8.
The PEG-(linker-alcohol)8 (1.0 g, 42 μMol PEG) was dissolved in anhydrous acetonitrile (5 mL) and treated with N,N′-disuccinimidyl carbonate (256 mg, 1000 μMol) followed by a solution of 4-(dimethylamino)pyridine (100 mg, 820 μMol) in 1 mL of acetonitrile. The resulting clear solution was stirred for 6 h, and then ether was added to precipitate the product. The precipitate was collected and dried, and the product was purified by repeated precipitations.
Two solutions are prepared. In one method, the first solution comprises the polymer MX1x dissolved in water or buffer, optionally with a pharmaceutically acceptable excipient. The second solution comprises X2-(1′)-P, X2-1b′-D if present, where 1′ and 1b′ represent reacted forms of formulas 1 and 1b, and the crosslinker T in the appropriate molar ratios dissolved in water or buffer, optionally with a pharmaceutically acceptable excipient. The molar ratios are calculated to provide the desired loading of P and D groups and degree of gel crosslinking. If necessary, excess X1 groups may be capped by inclusion of an appropriate capping reagent such that the total concentrations of X1 and X2 groups are equal in the final polymerization mixture. Alternately, excess X1 and X2 groups may be used in the final polymerization mixture if subsequent sealant layers are to be applied.
In a second method, the first solution comprises the macromonomer, M, comprising functional groups X1 together with X2-(1′)-P and X2-(1b′)-D if present, dissolved in water or buffer, optionally with a pharmaceutically acceptable excipient. The second solution comprises the crosslinker T dissolved in water or buffer, optionally with a pharmaceutically acceptable excipient.
To prepare the sealant in either method, the appropriate amounts of the first and second solutions are mixed and applied to the site of the wound, suture, or anastomosis. The polymerization mixture is applied to the wound site and allowed to set. Application may be as a bulk liquid, for example by extrusion from a syringe onto the wound, or by aerosol, for example using a spray device. The two solutions may be premixed or mixed during application using a multi-channel device, for example a multi-barrel syringe wherein each barrel contains a component of the polymerization mixture and the reactive components are mixed upon extrusion into the syringe tip or needle.
A solution comprising an 8-arm PEG-(cyclooctyne)8 (Preparation G) dissolved in water is mixed with a freshly prepared solution comprising an azide-linker-succinimidyl carbonate (Preparation D) and a 4-arm PEG-(linker-azide)4 crosslinker (Preparation H) in 10 mM acetate buffer, pH 5 so as to form a polymerization mixture. The proportions of the three components are adjusted so as to provide the required tissue adhesion (controlled by the concentration of azide-linker-succinimidyl carbonate) and the mechanical properties of the sealant (controlled by the concentration of crosslinker and the total concentration of PEG). The polymerization mixture is applied to the site requiring sealing and allowed to set. Application may be as a bulk liquid, for example by extrusion from a syringe onto the wound or through application using a brush, or by aerosol, for example using a spray device. The two solutions may be premixed or mixed during application using a multi-channel device, for example a multi-barrel syringe wherein each barrel contains a component of the polymerization mixture and the reactive components are mixed upon extrusion into the syringe tip or needle.
A solution comprising a 4-arm PEG-(N-hydroxysuccinimidyl ester)4 is mixed with a 4-arm PEG-(linker-SH)4 (Preparation J) and the resulting polymerization mixture is applied to the site requiring sealing to provide a degradable sealant wherein the sealant matrix is formed through thioester bonds. The proportion of the two components is adjusted such that the N-hydroxysuccinimidyl ester groups are present in molar excess over thiol groups, thus providing tissue adherence.
A solution comprising an albumin is mixed with a freshly-prepared solution of PEG-(N-hydroxysuccinimidyl ester)x wherein x=2-8 in buffer at a pH value between 6 and 8, and the resulting polymerization mixture is applied to the site requiring sealing.
Trilysine is reacted with excess BOC-amino-linker-succinimidyl carbonate (Preparation E) to produce the (BOC-amino-linker-carbamoyl)4-trilysine intermediate, which is dissolved in trifluoroacetic acid to remove the BOC protection and provide (amino-linker-carbamoyl)4-trilysine as the trifluoroacetate salt. A solution of the (amino-linker-carbamoyl)4-trilysine salt in aqueous buffer is mixed with a freshly-prepared aqueous solution of a 4-arm PEG-(succinimidyl ester)4 such that the final pH is between 7 and 8, and the resulting polymerization mixture is applied to the site requiring sealing.
In one method, a solution of poly(ethylene imine) is mixed with an azido-linker-succinimidyl carbonate to produce a polymer comprising azido-linker-carbamates. This polymer solution is mixed with a solution of a bifunctional PEG-cyclooctyne and applied to the site requiring sealing to produce a degradable sealant.
In a second method, a solution of poly(ethylene imine) is mixed with a solution comprising azido-linker-succinimidyl carbonate and a bifunctional PEG-cyclooctyne and applied to the site requiring sealing to produce a degradable sealant.
To illustrate one embodiment of the invention, a hydrogel containing 5% w/v total PEG was made by mixing an aqueous solution of PEG20 k-[CHO-Lys(N3)]4 (100 mg/mL, 20 mM CHO and N3, 0.025 mL, 0.0005 mMol, 1 equiv) with 0.5 M pH 4.5 acetate buffer (0.0106 mL, 0.5 M), a solution of AAF-(SO2Ph-linker)-PEG4-DBCO in DMF (2.8 mM, 0.018 mL, 0.000050 mMol, 0.1 equiv), and a solution of DEAC-DBCO in DMF (7.9 mM, 0.0114 mL, 0.000090 mMol, 0.18 equiv). The resulting mixture was kept at room temperature for 20 min then an aqueous solution of PEG20 k-[SO2Morph-linker-O—NH2.TFA]4 (100 mg/mL, 20 mM 0-NH2, 0.025 mL, 0.0005 mMol, 1 equiv) and an aqueous solution of aniline (pH 4.5, 1 M, 0.010 mL, 0.010 mMol, 20 equiv) were added. To form the sealant into a defined size and shape for further study, the resulting mixture was immediately placed into a 64 uL (9×1 mM) circular rubber perfusion chamber (Grace Bio-Labs) mounted on a silanized glass microscope slide, and allowed to cure overnight. In order to add tissue-attachment groups P to the above gel together with releasable drug D, a mixture of D-linker-PEG4-DBCO and P-DBCO or P-linker-DBCO would be used.
A two-layer hydrogel was prepared as follows. A first 5% w/v hydrogel (A) comprising excess cyclooctyne groups was prepared by mixing PEG40 kDa-(BCN)8 (50 uL of 100 mg/mL in H2O; 1000 nMol BCN), 0.1 M MES, pH 6.0 (60 uL), and 4-azidobutyryl-Lys(DNP)-OH (15 uL of 10 mM in MeOH; 150 nMol N3), then adding PEG20kDa-(NH—CO—O—CH(CH2SO2N(CH2CH2)2O)(CH2)5N3)4 (25 uL of 100 mg/mL in H2O; 500 nMol N3), vortexing, centrifuging to remove air bubbles, and pipetting into gel molds. A second 5% w/v hydrogel (B) comprising excess azide groups was prepared by mixing PEG40 kDa-(BCN)8 (37.5 uL of 50 mg/mL in H2O; 750 nMol BCN), 0.1 M MES, pH 6.0 (60 uL), and 4-azidobutyryl-AAF (2.5 uL of 30 mM in MeOH; 75 nMol N3), then adding PEG20 kDa-(NH—CO—O—CH(CH2CN)(CH2)5N3)4 (50 uL of 100 mg/mL in H2O; 1000 nMol N3). Once the gels had set (30 minutes), they were removed from the molds and suspended in water. Junctions between gels were prepared by stacking one gel on the other followed by application of slight pressure to remove interfacial air and liquid. The junctions were allowed to sit for 1 hour, and then examined for adhesion by physical separation. Junctions prepared from equivalent disks, i.e., A-A and B-B, were found to readily separate into the intact component disks. The A-B junction prepared from complementary disks, however, was found the form a cohesive inseparable bond.
After curing for 18 h at room temperature the hydrogel disc from Example 7 was placed into 10 mL of 10 mM pH 5.5 Acetate (2×30 min) to remove aniline catalyst then it was placed into 2.5 mL of pH 9.4 borate buffer at 37° C. in a divided cuvette. The OD of the buffer was monitored over time to measure drug release (fluorescein (AAF) 495 nm, solid circles) or gel degradation (coumarin (DEAC) 430 nm, solid squares). At pH 9.4, drug release was observed with t1.2=1 h, while gel degradation was minimal up to the degelation point at ˜4 h. As both rates have been shown to be first-order in hydroxide concentration, the rates observed at pH 9.4 can be translated into a half-life of 100 h for drug release and a degelation time of 400 h (16 days) at pH 7.4 as shown in
In one formulation, macromonomer solutions were prepared by dissolving PEG-(NHCO—CH2CH2—COOSu)8 in 0.01 M phosphate, pH 5, and PEG-(NHCO2—CH(CH2R1)—(CH2)5NH2)4.(CF3CO2H)4 (Preparation I) in 0.1 M phosphate, pH 8. Sealants were prepared by mixing the two macromonomer solutions at appropriate volume ratios to provide the desired total PEG concentration, crosslinking density, and concentration of residual succinimidyl esters for tissue adhesion. For example, using macromonomers prepared using 20-kDa PEGs, mixing of equal volumes of the two 50 mg/mL macromonomer solutions provides a sealant having total PEG concentration of 5%, an average crosslinking density of 4 crosslinks and an average of 4 residual NHS esters per 8-armed node (5 mM residual NHS esters in the sealant). Mixing of equal volumes of these two macromonomer solutions (the 8-armed macromonomer at 40 mg/mL and the 4-armed macromonomer at 60 mg/mL) provides a sealant having total PEG concentration of 5%, an average crosslinking density of 6 crosslinks and an average of 2 residual NHS esters per 8-armed node (2 mM residual NHS esters in the sealant). Mixing of equal volumes of these two macromonomer solutions (each at 100 mg/mL) provides a sealant having total PEG concentration of 10%, an average crosslinking density of 4 crosslinks and an average of 4 residual NHS esters per 8-armed node (10 mM residual NHS esters in the sealant).
In a second formulation, macromonomer solutions were prepared by dissolving PEG-(NHCO—CH2CH2—CONH-linker-O(CO)OSu)8 (Preparation O) in 0.01 M phosphate, pH 4, and PEG-(NH2)4 in 0.1 M phosphate, pH 8.5. Sealants were prepared as described above.
Gel time was determined by placing a small magnetic stir bar and the first gel component solution in a 1.5-mL vial, then adding the second component and measuring the time required for the gel to set and stop rotation of the stir bar. For gels formed by triazole formation using a PEG-DBCO and a PEG-azide, the first component was the PEG-DBCO. The concentrations of the gel component solutions were determined by assay in the case of PEG-azide (by UV measurement of the DBCO consumed in the presence of excess DBCO) or by UV absorbance at 308 nm (using e=13,500 M−1 cm−1) in the case of PEG-DBCO. Room temperature was measured as 23° C. Monomer solutions were as follows:
For sealants formed using the reaction of a succinimidyl ester or carbonate with an amine to form an amide or carbamate linkage, respectively, the gelation time is a function of the pH of the gel mixture, with gel formation occurring more quickly as the pH increases. For 5% PEG sealants prepared from PEG-(NHCO—CH2CH2—CONH-linker-O(CO)OSu)8 and PEG5kDa-(NH2)4, gel times were measured as 9 sec at pH 9.4 and 72 sec at pH 8.4.
A device for measuring burst pressure was fabricated from 6061-T6 aluminum. This device allows the application of pressure from air, gas, or liquid buffer to a collagen sausage casing (Weston, 19 mm snack sticks) sealed against a Buna-N O-ring (OD=1 1/16″ (1.066″) 1/16″ width). Pressure is provided by a hand operated 50 to 100 mL syringe and sensed by a NSCDANN100PGUNV gauge pressure sensor (Honeywell). Pressure vs. time data was recorded by a custom computer application written in DAQ Factory (AzeoTech) using a Lab Jack USB DAQ.
Circles approximately 32 mm in diameter were cut from collagen sausage casing (Weston, 19 mm snack sticks). The center of these circles was pierced with the tip of a pasture pipette to give a hole approximately 1.5 mm in diameter. The resulting casings were soaked in PBS. Gels (9 mm diameter×1 mm thick) were then formed over the hole defect using rubber perfusion chamber molds. Test sealants were allowed to cure for 30 minutes then assessed for burst pressure either immediately or after swelling in PBS for 24 h. Sealant swelling ratios were measured by weighing sealant discs immediately after preparation and then following equilibration in PBS for 24 h. Hydrogel sealant properties were compared to previously reported sealants. Comparator A: 10-kDa PEG-(succinimidyl glutarate)4+10-kDa PEG-(thiol)4 (1:1 HSE:thiol) at 20% total PEG. Comparator B: 10-kDa PEG-(succinimidyl glutarate)4+trilysine (1:1 HSE:amine) at 9.4% PEG. PR a-b-c-d series sealants comprised PEG-(linker-HSE)8+PEG-(amine)4 wherein a=average mw of the 8-arm PEG, b=average molecular weight of the 4-arm PEG, c =HSE:amine ratio, and d =total % PEG (w/v) in the pre-equilibrium gel mixture. PE x-y-z series sealants comprised PEG-(DBCO-Lys(HSE))4+PEG-(linker-N3)4, wherein x=average mw of the bifunctional PEG, y=average mw of PEG-(linker-N3)4, and z=total % PEG (w/v) in the pre-equilibrium gel mixture are shown in the table below.
Hydrogels comprising degradable linkers that varied only in R1 were prepared by mixing solutions of PEG20kDa-(DBCO)4 and PEG20 kDa-(NH—CO2—CH(CH2R1)(CH2)5N3)4. A small fraction of a fluorescent erosion probe was added to allow measurement of gel solubilization. Thus, a 50-mg/mL solution of PEG20kDa-(DBCO)4 (250 uL, 2.50 uMol DBCO end-groups) in water was mixed with 25 μL of a 10-mM solution of the azide-linker-aminoacetylfluorescein (AAF) (0.25 μMol azide) erosion probe in DMF and kept 30 min at ambient temperature. Aliquots (50 uL, 0.42 uMol DBCO) were mixed with 28 uL of 10 mM NaOAc, pH 5.0, followed by 45 uL of 50 mg/mL PEG20kDa-(NH—CO2—CH(CH2R1)(CH2)5N3)4 (0.42 uMol azide). Gels were formed in 1×9 mm circular diffusion molds. For degradation assays, the gels were suspended in 2 mL of 0.1 M KPi, pH 7.4, at 37° C., and the OD493 of the supernatant was periodically measured to monitor fluorescein solubilization. The degelation time Tdg was defined as the point at which maximum OD493 was observed, indicating complete solubilization of the hydrogel. Results are given in Table 2 and compared with previously reported half-lives for release of acetamidofluorescein (Santi, et al., Proc. Natl. Acad. Sci. USA (2012) 109:6211-6216. It was observed that Tdg correlates with the reported half-lives for release of acetamidofluorescein, thus allowing for prediction of the rates of sealant degradation.
This application claims benefit of U.S. application Ser. No. 61/755,405 filed 22 Jan. 2013 and U.S. application Ser. No. 61/774,498 filed 7 Mar. 2013 which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/12571 | 1/22/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61755405 | Jan 2013 | US | |
| 61774498 | Mar 2013 | US |
