Patent Application
20120276039
1. Field of the Invention
The present disclosure relates generally to healing of wounds and wound-treatment therapies. More particularly, but not by way of limitation, the present disclosure relates to modified materials, for example, silyl modified polyurethane foam for the reversible binding of factors in a wound bed.
2. Background Information
Clinical studies and practice have shown that providing a reduced pressure in proximity to a tissue site augments and accelerates the growth of new tissue at the tissue site. The applications of this phenomenon are numerous, but application of reduced pressure has been particularly successful in treating wounds. This treatment (frequently referred to in the medical community as “negative pressure wound therapy,” “reduced pressure therapy,” or “vacuum therapy”) provides a number of benefits, including faster healing and increased formulation of granulation tissue. One of the major clinical benefits of negative pressure wound therapy is its ability to effectively eliminate wound exudate, thereby reducing edema and allowing tissue decompression. Negative pressure wound therapy may not always be able to differentiate between harmful and beneficial factors removed from the wound. Coating used to address this such as collagen, PVA, PEG, and fibrinogen often suffer from not being covalent, uniform, or target specific. Typically they cannot bind proteins at a specific site and present them to cells in a manner that allows the active site to retain its function. Improvements that would allow the binding of molecules in a covalent manner, specify the types of compounds that could be bound, the chemical reactions with which to bind them, and/or the orientation with which the protein is presented to the cells, e.g., for use in a dressing, wound insert, pad, etc., would therefore be highly desirable.
The present disclosure provides novel materials, including aptamer (e.g., polypeptide) modified polymers, which may be used for the binding of factors, such as from a wound bed. In some embodiments, the factors are endogenous. In others they are exogenous. In some embodiments, the bind is reversible, in others it is irreversible.
In some embodiments a polypeptide is provided comprising an amino acid sequence identical to SEQ ID NO: 2 (P16; GHQGQH1GQMS), a sequence at least 90% identical to SEQ ID NO: 2 or a sequence comprising 1, 2, or 3 amino acid substitutions or deletions relative to SEQ ID NO: 2. In some aspects, the amino acid sequence further comprises an amino or carboxyl terminal Cys residue (e.g., the amino or carboxyl terminus of a polypeptide of the embodiments comprises a PEG spacer-cysteine residue). For example, the amino acid sequence can comprise the sequence AEEAc-Cys-NH2 at the C-terminus. In still further aspects, a wound dressing is provided comprising a polymer foam substrate conjugated to a polypeptide of the embodiments, such as a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 (GHQGQHIGQMS) or a sequence at least 90% identical to SEQ ID NO: 2.
In certain embodiments a wound dressing is provided comprising a polymer foam substrate conjugated to a polypeptide, wherein the polypeptide comprises an amino acid sequence identical to SEQ ID NO: 1 (P22; NAIQEARRLLNLSRD), a sequence at least 90% identical to SEQ ID NO:1 or a sequence comprising 1, 2, or 3 amino acid substitutions or deletions relative to SEQ ID NO: 1. In some aspects, the amino acid sequence further comprises an amino or carboxyl terminal Cys residue (e.g., the amino or carboxyl terminus of a polypeptide of the embodiments comprises a PEG spacer-cysteine residue). For example, the amino acid sequence can comprise the sequence AEEAc-Cys-NH2 at the C-terminus.
In some embodiments, a polypeptide of the embodiments is covalently attached to a polymer foam (e.g., to form a wound dressing). In some aspects, a polypeptide is attached to the polymer foam through a thioether linker. For example, the linker can be an EMCS-derived linker or a sulfo-EMCS-derived linker. In some aspects, the thioether linker comprises a Cys residue in the polypeptide, such as a Cys residue in a AEEAc-Cys-NH2 sequence of the polypeptide. In still further aspects, a linker between a polypeptide and a polymer foam can be a silyl derived linker (e.g., a substituted silyl derived linker derived from aminoundecyltriethoxysilane or aminopropyldiisopropylethoxysilane). In still further aspects, a polypeptide of the embodiments is attached to a polymer foam via a non-covalent binding, such as by a biotin-avidin binding.
In certain aspects, a wound dressing of the embodiments further comprises a growth factor, chemokine or cytokine bound to the wound dressing (e.g., bound to the wound dressing via an interaction with a polypeptide of the embodiments). In some aspects, the growth factor is granulocyte macrophage colony stimulating factor (GM-CSF). In certain aspects, the growth factor is vascular endothelial growth factor (VEGF).
In some embodiments, a polypeptide of the embodiments is attached to a polymer foam to form a wound dressing. For example, the polymer foam substrate can be a reticulated open-celled foam. Examples of polymer foams include, without limitation, foam substrates comprising polyvinyl alcohol, polyurethane, polypropylene, polystyrene, polyols, poloxamer, aminoglycosides, amino sugars, or a combination of one or more of these.
In further embodiments a method is provided for binding a growth factor, chemokine or cytokine comprising contacting a fluid (such as a body fluid) comprising the growth factor, chemokine or cytokine with a wound dressing according to embodiments, thereby binding the growth factor, chemokine or cytokine. In some aspects, a wound dressing of the embodiments is adapted for using in applying negative pressure to a wound site. In further aspects, a method comprises removing some or all of the fluid in contact with the wound dressing. In still further aspects, a method of the embodiments further comprises contacting the wound dressing with at least a second fluid wherein some or all of the growth factor, chemokine or cytokine bound to the wound dressing is eluted into the second fluid. In certain aspects, a method of the embodiments is defined as an in vitro method.
In yet a further embodiment, a composition is provided for the treatment of a wound, comprising a wound dressing according to the embodiments. In some aspects, a method is provided for treating a wound comprising contacting a wound site with a wound dressing of the embodiments. In certain aspects, a method of the embodiments further comprises applying a negative pressure to the wound site.
Any embodiment of any of the present systems and/or methods can consist of or consist essentially of—rather than comprise/include/contain/have—any of the described steps, elements, and/or features. Thus, in any of the claims, the term “consisting of” or “consisting essentially of” can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Note that simply because a particular compound is ascribed to one particular generic formula doesn't mean that it cannot also belong to another generic formula.
The following drawings illustrate by way of example and not limitation. For the sake of brevity and clarity, every feature of a given structure is not always labeled in every figure in which that structure appears. Identical reference numbers do not necessarily indicate an identical structure. Rather, the same reference number may be used to indicate a similar feature or a feature with similar functionality, as may non-identical reference numbers.
FIG. 18—VEGF in solution was passed over beads linked to an anti-VEGF antibody. After passing the solution over the beads, beads were spun down and washed. The washed beads were then used for endothelial cell migration assays. Results from this experiment showed that after 3 hours, 2 fold more cells had migrated towards the beads bound to the antibody (and VEGF) than beads with no antibody.
In some aspects, the present disclosure provides aptamer modified materials, which may be used, in some embodiments, as wound inserts for catching and concentrating specific factors in the wound environment, for example, factors triggering a biological response such as angiogenesis, or to deliver biomolecules or bioactive compounds to a wound bed. In some embodiments, the factors are endogenous. In others they are exogenous. In some embodiments the bind is reversible, in others it is irreversible.
In one aspect, the invention provides aptamer-functionalized wound dressings, comprising: (a) a substrate; and (b) an aptamer covalently attached to the polymer foam. In some embodiments, the substrate is a polymer foam. In some embodiments, the aptamer comprises a polypeptide, a small-molecule binder, a dendrimer, a nanoparticle, and/or an oligonucleotide. In some embodiments, said aptamer is a polypeptide. In some embodiments, the polypeptide is synthetic. In some embodiments, the polypeptide is natural.
In some embodiments, the polypeptide is covalently attached to the polymer foam. In some embodiments, C-terminus of the polypeptide comprises a PEG spacer-cysteine residue. In some embodiments, the aptamer is attached to the polymer foam through a linker. In some embodiments, the linker is an EMCS-derived linker or a sulfo-EMCS-derived linker. In some embodiments, the linker is a substituted silyl derived linker. In some embodiments, the substituted silyl derived linker is derived from compound 630 (aminoundecyltriethoxysilane). In some embodiments, the substituted silyl derived linker is derived from compound 602 (aminopropyldiisopropylethoxysilane). In some embodiments, the linker comprises a first and second linker, wherein the first linker is an EMCS-derived linker or a sulfo-EMCS-derived linker and the second linker is a substituted silyl derived linker.
In some embodiments, the linker is bound to the polymer foam through an oxygen atom. In some embodiments, the linker is bound to the polymer foam through a nitrogen atom. In some embodiments, the aptamer is configured to bind to endogenous or exogenous factors in a wound environment. In some embodiments, the binding is irreversible. In some embodiments, the binding is reversible. In some embodiments, the aptamer is configured to bind to vascular endothelial growth factor. In some embodiments, the aptamer is configured to bind to platelet derived growth factor. In some embodiments, the aptamer is configured to bind to fibroblast growth factor. In some embodiments, the aptamer is configured to bind to keratinocyte growth factor. In some embodiments, the aptamer is configured to bind to granulocyte macrophage colony stimulating factor.
In some embodiments, the substrate is a reticulated open-celled foam. In some embodiments, the substrate comprises polyvinyl alcohol, polyurethane, polypropylene, polystyrene, polyols, poloxamer, aminoglycosides, amino sugars, or a combination of one or more of these. In some embodiments, the polymer foam comprises a first monomer subunit. In some embodiments, the first monomer subunit is a diisocyanate. In some embodiments, the diisocyanate is toluene diisocyanate, methylene diphenyl diisocyanate, or ethyl lysine diisocyanate. In some embodiments, the polymer foam further comprises a second monomer subunit. In some embodiments, the second monomer is a triol. In some embodiments, the triol is a PEGylated glycerine. In some embodiments, the polymer foam further comprises a third monomer subunit. In some embodiments, the third monomer subunit is an aminoglycoside. In some embodiments, the aminoglycoside is chitosan. In some embodiments, the aminoglycoside is glucosamine. In some embodiments, the third monomer subunit is neomycin. In some embodiments, the third monomer subunit is dibekacin. In some embodiments, the third monomer subunit is kanamycin. In some embodiments, the third monomer subunit is tobramycin. In some embodiments, the third monomer subunit is streptomycin. In some embodiments, the third monomer subunit is gentamicin. In some embodiments, the polymer foam further comprises an oligomeric subunit. In some embodiments, the oligomeric subunit is a chitosan-based oligomer. In some embodiments, the oligomeric subunit is a glucosamine-based oligomer.
In another aspect, the invention provides aptamer-modified polymers, wherein the polymer backbone comprises a first repeat unit of the formula:
wherein:
U1 is the unmodified portion of the first repeat unit;
L1 is a linker molecule or a bond; and
L2 is a linker molecule or a bond.
In some embodiments, L1 is a bond and the formula of the first repeat unit is further defined as:
In some embodiments, L2 is a bond and the formula of the first repeat unit is further defined as:
In some embodiments, L1 and L2 are bonds and the formula of the first repeat
In some embodiments. U1 is of the formula:
wherein the point of attachment of the side chain connects to L1.
In some embodiments, U1 is of the formula:
wherein the point of attachment of the side chain connects to L1.
In some embodiments. U1 is of the formula:
wherein the point of attachment of the side chain connects to L1.
In some embodiments, U1 is of the formula:
wherein:
n1, n2 and n3 are each independently from 0 to 20; and
the point of attachment of the side chain connects to L1.
In some embodiments, U1 is based on a triol-based monomer. In some embodiments, the triol-based monomer is a PEGylated glycerine molecule having a molecular weight between 500 and 5,000 g/mole.
In some embodiments, L1 is of the formula —O—Si(R1)(R2)—R3—X—, wherein:
In some embodiments, R1 and R2 are isopropyl. In some embodiments, R1 and R2 are hydroxy. In some embodiments, R3 is alkanediyl(C3-11). In some embodiments, R3 is —(CH2)3—. In some embodiments, R3 is —(CH2)11—. In some embodiments, X is —NH—.
In some embodiments, L1 or L2 is an amino thio linker. In some embodiments, L1 or L2 is an NHS-Maleimide crosslinker. In some embodiments, L1 or L2 is an NHS-Haloacetyl crosslinker. In some embodiments, L1 or L2 is an NHS-Pyridyldithiol crosslinker.
In some embodiments, the amino thio linker is derived from a reagent selected from the group consisting of SM(PEG)24, SM(PEG)12, SM(PEG)8, SM(PEG)6, SM(PEG)4, Sulfo-LC-SMPT, SM(PEG)2, Sulfo-KMUS, LC-SMCC, LC-SPDP, Sulfo-LC-SPDP, SMPH, Sulfo-SMPB, SMPB, SMPT, SLAB, Sulfo-SIAB, Sulfo-EMCS, EMCS, Sulfo-SMCC, SMCC, MBS, GMBS, Sulfo-GMBS, Sulfo-MBS, SPDP, SBAP, BMPS, AMAS, and SIA.
In some embodiments, the aptamer-modified polymer comprises a second repeat unit. In some embodiments, the second repeat unit is a chitosan-based oligomer.
In some embodiments. U1 is of the formula:
wherein the point of attachment of the side chain connects to L1.
In some embodiments, L1 is of the formula:
In some embodiments, L2 is of the formula:
In some embodiments, L2 is of the formula —C(O)(CH2)5-S-Cys-AEEAc-, wherein AEEAc is:
In some embodiments, the aptamer-modified polymer further comprises a second repeat unit.
In some embodiments, the second repeat unit has the formula:
In some embodiments, aptamer is an the oligopeptide having 8 to 12 residues. In some embodiments, the oligopeptide has the sequence GHQGQHIGQMS. In some embodiments, aptamer is P16. In some embodiments, aptamer is P22.
In some embodiments, the aptamer-modified polymer further comprises a third repeat unit of the formula:
wherein X is:
-alkanediyl(C≦12)-;
-arenediyl(≦12)-;
-arenediyl(≦6)-CH2-arenediyl(≦6)-; or
substituted versions of any these groups.
In some embodiments, X is further defined as:
In some embodiments, the third repeat unit is a polyurethane repeat unit. In some embodiments, the third repeat unit is an ether-based polyurethane repeat unit. In some embodiments, the third repeat unit is an ester-based polyurethane repeat unit. In some embodiments, the polymer is an open-celled foam.
In another aspect, the invention provides an aptamer-modified polymer or co-polymer comprising:
In some embodiments, the first repeat unit has two hydroxy groups.
In some embodiments, the first repeat unit has the formula:
In some embodiments, R1 and R2 are isopropyl. In some embodiments, wherein R1 and R2 are hydroxy. In some embodiments, R3 is alkanediyl(C3-11). In some embodiments, R3 is —(CH2)3—.
In some embodiments, the second repeat unit has the formula:
In some embodiments, R3 is —(CH2)11—. In some embodiments, the second repeat unit has the formula:
In some embodiments, X is —NH—. In some embodiments, the linker is further defined by the formula:
wherein the carbonyl carbon is attached to X.
In some embodiments, the aptamer is bound through the linker through the mercapto group of a cysteine residue. In some embodiments, the aptamer is granulocyte-macrophage colony stimulating factor (GM-CSF).
In some embodiments, the aptamer-modified polymer further comprises a third repeat unit of the formula:
wherein X is:
-alkanediyl(C≦12)-;
-arenediyl(≦12)-;
-arenediyl(≦6)-CH2-arenediyl(≦6)-; or
substituted versions of any these groups.
In some embodiments, X is further defined as:
In some embodiments, the third repeat unit is a polyurethane repeat unit. In some embodiments, the third repeat unit is an ether-based polyurethane repeat unit. In some embodiments, the third repeat unit is an ester-based polyurethane repeat unit. In some embodiments, the polymer is an open-celled foam.
In another aspect, the invention provides a wound insert comprising an aptamer-modified polymer or co-polymer as described above or below. In another aspect, the invention provides a wound dressing comprising the wound insert and a drape configured to be coupled to skin adjacent a wound of a patient. In some embodiments, the wound dressing further comprises a fluid delivery pad configured to be coupled to the drape and a fluid source such that the fluid source is actuatable to deliver a fluid to a wound through the wound dressing.
In another aspect, the invention provides a wound-treatment apparatus comprising the wound dressing and a fluid source configured to be coupled to the wound dressing such that the fluid source is actuatable to deliver a fluid to the wound dressing. In some embodiments, the apparatus further comprising a vacuum source configured to be coupled to the wound dressing such that the vacuum source is actuatable to apply negative pressure to the wound dressing. In some embodiments, the fluid comprises a functional solution that enhances target binding or facilitates target release into the wound bed. In some embodiments, the fluid comprises saline solutions, solutions with slightly acidic pH, slightly basic pH, solutions with various surfactants (e.g., polysorbate), EDTA and/or EGTA. In some embodiments, the fluid comprises hypochlorous acid and hypochlorite ion.
As used herein, the term “aptamer” refers not only to oligonucleic acid or peptide molecules that bind to a specific target molecule, but also to any ligand that binds to a target molecule or ion. For example, as used herein, “aptamer” would include EDTA (ethylene-diaminetetraacetic acid), which binds to Ca2+.
When used in the context of a chemical group, “hydrogen” means —H; “hydroxy” means —OH; “oxo” means ═O; “halo” means independently —F, —Cl, —Br or —I; “amino” means —NH2 (see below for definitions of groups containing the term amino, e.g., alkylamino); “hydroxyamino” means —NHOH; “nitro” means —NO2; imino means ═NH (see below for definitions of groups containing the term imino, e.g., alkylimino); “cyano” means —CN; “azido” means —N3; in a monovalent context “phosphate” means —OP(O)(OH)2 or a deprotonated form thereof; in a divalent context “phosphate” means —OP(O)(OH)2 or a deprotonated form thereof; “mercapto” means —SH; “thio” means ═S; “thioether” means —S—; “sulfonamido” means —NHS(O)2— (see below for definitions of groups containing the term sulfonamido, e.g., alkylsulfonamido); “sulfonyl” means —S(O)2— (see below for definitions of groups containing the term sulfonyl, e.g., alkylsulfonyl); and “sulfinyl” means —S(O)— (see below for definitions of groups containing the term sulfinyl, e.g., alkylsulfinyl).
The symbol “—” means a single bond, “═” means a double bond, and “≡” means triple bond. The symbol “- - - -” represents an optional bond, which if present is either single or double. The symbol “” represents a single bond or a double bond. Thus, for example, the structure
includes the structures
As will be understood by a person of skill in the art, no one such ring atom forms part of more than one double bond. The symbol “”, when drawn perpendicularly across a bond indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in rapidly and unambiguously identifying a point of attachment. The symbol “
” means a single bond where the group attached to the thick end of the wedge is “out of the page.” The symbol “
” means a single bond where the group attached to the thick end of the wedge is “into the page”. The symbol “
” means a single bond where the conformation (e.g., either R or S) or the geometry is undefined (e.g., either E or Z).
Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to the atom. When a group “R” is depicted as a “floating group” on a ring system, for example, in the formula:
then R may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a group “R” is depicted as a “floating group” on a fused ring system, as for example in the formula:
then R may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals —CH—), so long as a stable structure is formed. In the example depicted, R may reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter “y” immediately following the group “R” enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.
For the groups and classes below, the following parenthetical subscripts further define the group/class as follows: “(Cn)” defines the exact number (n) of carbon atoms in the group/class. “(C≦n)” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl(C≦8)” or the class “alkene(C≦8)” is two. For example, “alkoxy(C≦10)” designates those alkoxy groups having from 1 to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both the minimum (n) and maximum number (n′) of carbon atoms in the group. Similarly, “alkyl(C2-10)” designates those alkyl groups having from 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms)).
The term “saturated” as used herein means the compound or group so modified has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. The term does not preclude carbon-heteroatom multiple bonds, for example a carbon oxygen double bond or a carbon nitrogen double bond. Moreover, it does not preclude a carbon-carbon double bond that may occur as part of keto-enol tautomerism or imine/enamine tautomerism.
The term “aliphatic” when used without the “substituted” modifier signifies that the compound/group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single bonds (alkanes/alkyl), or unsaturated, with one or more double bonds (alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl). When the term “aliphatic” is used without the “substituted” modifier only carbon and hydrogen atoms are present. When the term is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3.
The term “alkyl” when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, and no atoms other than carbon and hydrogen. Thus, as used herein cycloalkyl is a subset of alkyl. The groups —CH3 (Me), —CH2CH3 (Et), —CH2CH2CH3 (n-Pr), —CH(CH3)2 (iso-Pr), —CH(CH2)2 (cyclopropyl), —CH2CH2CH2CH3 (n-Bu), —CH(CH3)CH2CH3 (sec-butyl), —CH2CH(CH3)2 (iso-butyl), —C(CH3)3 (tert-butyl), —CH2C(CH3)3 (neo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arc non-limiting examples of alkyl groups. The term “alkanediyl” when used without the “substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, —CH2— (methylene), —CH2CH2—, —CH2C(CH3)2CH2—, —CH2CH2CH2—, and
are non-limiting examples of alkanediyl groups. The term “alkylidene” when used without the “substituted” modifier refers to the divalent group ═CRR′ in which R and R′ are independently hydrogen, alkyl, or R and R′ are taken together to represent an alkanediyl having at least two carbon atoms. Non-limiting examples of alkylidene groups include: ═CH2, ═CH(CH2CH3), and ═C(CH3)2. When any of these teens is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. The following groups are non-limiting examples of substituted alkyl groups: —CH2OH, —CH2Cl, —CF3, —CH2CN, —CH2C(O)OH, —CH2C(O)OCH3, —CH2C(O)NH2, —CH2C(O)CH3, —CH2OCH3, —CH2OC(O)CH3, —CH2NH2, —CH2N(CH3)2, and —CH2CH2Cl. The term “fluoroalkyl” is a subset of substituted alkyl, in which one or more hydrogen has been substituted with a fluoro group and no other atoms aside from carbon, hydrogen and fluorine are present. The groups, —CH2F, —CF3, and —CH2CF3 are non-limiting examples of fluoroalkyl groups. An “alkane” refers to the compound H—R, wherein R is alkyl.
The term “alkenyl” when used without the “substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples of alkenyl groups include: —CH═CH2 (vinyl), —CH═CHCH3, —CH═CHCH2CH3, —CH2CH═CH2 (allyl), —CH2CH═CHCH3, and —CH═CH—C6H5. The term “alkenediyl” when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups, —CH═CH—, —CH—C(CH3)CH2—, —CH═CHCH2—, and
are non-limiting examples of alkenediyl groups. When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, are non-limiting examples of substituted alkenyl groups. An “alkene” refers to the compound H—R, wherein R is alkenyl.
The term “aryl” when used without the “substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, —C6H4—CH2CH3 (ethylphenyl), naphthyl, and the monovalent group derived from biphenyl. The term “arenediyl” when used without the “substituted” modifier refers to a divalent aromatic group, with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Non-limiting examples of arenediyl groups include:
When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. An “arene” refers to the compound H—R, wherein R is aryl.
The term “aralkyl” when used without the “substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples of aralkyls are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the “substituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.
The term “heteroaryl” when used without the “substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of an aromatic ring structure wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the aromatic ring or any additional aromatic ring present. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), methylpyridyl, oxazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, thienyl, and triazinyl. The term “heteroarenediyl” when used without the “substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, said atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not preclude the presence of one or more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Non-limiting examples of heteroarenediyl groups include:
When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —1, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3.
The term “acyl” when used without the “substituted” modifier refers to the group —C(O)R, in which R is a hydrogen, alkyl, aryl, aralkyl or heteroaryl, as those terms are defined above. The groups, —CHO, —C(O)CH3 (acetyl, Ac), —C(O)CH2CH3, —C(O)CH2CH2CH3, —C(O)CH(CH3)2, —C(O)CH(CH2)2, —C(O)C6H5, —C(O)C6H4CH3, —C(O)CH2C6H5, —C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group —C(O)R has been replaced with a sulfur atom, —C(S)R. When either of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. The groups, —C(O)CH2CF3, —CO2H (carboxyl), —CO2CH3 (methylcarboxyl), —CO2CH2CH3, —C(O)NH2 (carbamoyl), and —CON(CH3)2, are non-limiting examples of substituted acyl groups.
The term “alkoxy” when used without the “substituted” modifier refers to the group —OR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkoxy groups include: —OCH3, —OCH2CH3, —OCH2CH2CH3, —OCH(CH3)2, —OCH(CH2)2, —O-cyclopentyl, and —O-cyclohexyl. The terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as —OR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and acyl, respectively. Similarly, the term “alkylthio” when used without the “substituted” modifier refers to the group —SR, in which R is an alkyl, as that term is defined above. The term “alkoxydiyl” when used without the “substituted” modifier refers to the divalent group —O-alkanediyl-. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group.
The term “alkylamino” when used without the “substituted” modifier refers to the group —NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkylamino groups include: —NHCH3 and —NHCH2CH3. The term “dialkylamino” when used without the “substituted” modifier refers to the group —NRR′, in which R and R′ can be the same or different alkyl groups, or R and R′ can be taken together to represent an alkanediyl. Non-limiting examples of dialkylamino groups include: —N(CH3)2, —N(CH3)(CH2CH3), and N-pyrrolidinyl.
The terms “alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”, and “alkylsulfonylamino” when used without the “substituted” modifier, refers to groups, defined as —NHR, in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is —NHC6H5. The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group —NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is —NHC(O)CH3. The term “alkylimino” when used without the “substituted” modifier refers to the divalent group ═NR, in which R is an alkyl, as that term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. The groups —NHC(O)OCH3 and —NHC(O)NHCH3 are non-limiting examples of substituted amido groups.
The terms “alkylsulfonyl” and “alkylsulfinyl” when used without the “substituted” modifier refers to the groups —S(O)2R and —S(O)R, respectively, in which R is an alkyl, as that term is defined above. The terms “alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”, “aralkylsulfonyl”, and “heteroarylsulfonyl”, are defined in an analogous manner. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3.
The term “glycoside” refers to a compound in which a sugar group is bound to a non-carbohydrate moiety. Typically the sugar group (glycone) is bonded through its anomeric carbon to another group (aglycone) via a glycosidic bond that has an oxygen, nitrogen or sulfur atom as a linker.
A “simple sugar” are the basic structural units of carbohydrates, which cannot be readily hydrolyzed into simpler units. The elementary formula of a simple monosaccharide is CnH2nOn, where the integer n is at least 3 and rarely greater than 7. simple monosachharides may be named generically according on the number of carbon atoms n: trioses, tetroses, pentoses, hexoses, etc. Simple sugars may be open chain (acyclic), cyclic or mixtures thereof. In these cyclic forms, the ring usually has 5 or 6 atoms. These forms arc called furanoses and pyranoses, respectively—by analogy with furan and pyran. Simple sugars may be further classified into aldoses, those with a carbonyl group at the end of the chain in the acyclic form, and ketoses, those in which the carbonyl group is not at the end of the chain. Non-limiting examples of aldoses include: glycolaldehyde, glyceraldehydes, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose. Non-limiting examples of aldoses include: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose. The ‘
An “amino sugar” or “aminoglycoside” refers to a derivative of a sugar, deoxy sugar, sugar acid or sugar alcohol, where one or more hydroxy group(s) has been replace with one more amino group(s). A “simple amino sugar” refers to a derivative of a simple sugar, simply deoxy sugar, simply sugar acid or sugar alcohol, where one or more hydroxy group(s) has been replace with one more amino group(s). These terms also cover N- and O-acetyl derivatives thereof. Non-limiting examples include N-acetylglucosamine, galactosamine, glucosamine and sialic acid.
The term “deoxy sugar” refers to a sugar derivative where one of the hydroxy groups of a carbohydrate has been replaced with a hydrogen atom. A “simple deoxy sugar” is a deoxy sugar derived from a simple sugar, as defined herein. These terms also cover O-acetyl derivatives thereof. Non-limiting examples of simple deoxy sugars are deoxyribose (based upon ribose), fucose, and rhamnose.
The term “sugar acid” refers to a sugar derivative where an aldehyde functional group or one or more hydroxy functional groups has been oxidized to a carboxyl group. Aldonic acids are those in which the aldehyde functional group of an aldose has been oxidized. Ulosonic acids are those in which the first hydroxyl group of a 2-ketose has been oxidized creating an α-ketoacid. Uronic acids are those in which the terminal hydroxyl group of an aldose or ketose has been oxidized. Aldaric acids are those in which both ends of an aldose have been oxidized. Non-limiting aldonic acids include glyceric acid (3C), xylonic acid (5C), gluconic acid (6C), and ascorbic acid (6C, unsaturated lactone). Non-limiting examples of ulosonic acids include neuraminic acid (5-amino-3,5-dideoxy-
The term “sugar alcohol” refers to a sugar derivative whose carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Non-limiting examples of sugar alcohols include: glycol (2-carbon), glycerol (3-carbon), erythritol (4-carbon), threitol (4-carbon), arabitol (5-carbon), xylitol (5-carbon), ribitol (5-carbon), mannitol (6-carbon), sorbitol (6-carbon), dulcitol (6-carbon), iditol (6-carbon), isomalt (12-carbon), maltitol (12-carbon), lactitol (12-carbon) or polyglycitol. A “simple sugar alcohol” is a sugar alcohol derived from a simple sugar. These terms also cover O-acetyl derivatives thereof.
As used herein, the term “monosaccharide group” refers to a monovalent carbohydrate group, with a carbon atom as the point of attachment. The term covers the groups resulting from removal of a hydroxyl radical from a simple sugar (e.g., glucose), simple deoxy sugar (e.g., fucose), simple sugar acid (e.g., gluconic acid), simple sugar alcohol (e.g., xylitol) or simple amino sugar (e.g., glucosamine). Typically the monosaccharide group is bonded through its anomeric carbon to another group (aglycone) via oxygen atom linker. In some cases the linker may be a nitrogen or sulfur atom.
A “disaccharide group” is a monovalent carbohydrate group consisting of two monosaccharide groups, wherein the second monosaccharide group replaces a hydrogen on a hydroxy group of the first monosaccharide group. Non-limiting examples of disaccharide groups include those derived from sucrose, lactulose, lactose, maltose trehalose and cellobiose.
A “trisaccharide group” is a monovalent carbohydrate group consisting of three monosaccharide groups, wherein the second monosaccharide group replaces a hydrogen on a hydroxy group of the first monosaccharide group and the third monosaccharide group replaces a hydrogen on a hydroxy group of either the first or the second monosaccharide groups.
An oligosaccharide is a monovalent carbohydrate group consisting of three to ten, preferably three to six monosaccharide groups, wherein the second monosaccharide replaces a hydrogen on a hydroxy group of the first monosaccharide, the third monosaccharide replaces a hydrogen on a hydroxy group of either the first or the second monosaccharide groups, and subsequent monosaccharide groups replace hydrogens on any previously joined monosaccharide groups, thus forming either a linear or branched structure.
The term “silyl” when used without the “substituted” modifier refers to the group —SiR3, where each R is independently hydrogen or unsubstituted alkyl, as that group is defined above. The term “substituted silyl” refers to the group, —SiR3, wherein at least one of the R groups and as many as all of the R groups, is independently a substituted alkyl or —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —N(CH3)2, —C(O)NH2 or —OC(O)CH3. Any remaining R groups of the substituted silyl group are independently hydrogen or unsubstituted alkyl. The term “silylated” or “silanated” indicates that a given compound has been derivatized to contain a silyl and/or substituted silyl group. The abbreviation “-Sil” refers to silyl and/or substituted silyl groups.
The term “coupled” is defined as connected, although not necessarily directly, and not necessarily mechanically; two items that are “coupled” may be integral with each other. The terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise. The terms “substantially,” “approximately,” and “about” are defined as largely but not necessarily wholly what is specified, as understood by a person of ordinary skill in the art.
The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”) and “contain” (and any form of contain, such as “contains” and “containing”) are open-ended linking verbs. As a result, a method that “comprises,” “has,” “includes” or “contains” one or more steps possesses those one or more steps, but is not limited to possessing only those one or more steps. Likewise, a wound dressing that “comprises,” “has,” “includes” or “contains” one or more elements possesses those one or more elements, but is not limited to possessing only those elements. For example, in a wound dressing that comprises a wound insert and a drape, the wound dressing includes the specified elements but is not limited to having only those elements. For example, such a wound dressing could also include a connection pad configured to be coupled to a wound-treatment apparatus.
The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.
As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.
“PBS” is phosphate buffered saline; “phr” is parts per hundred resin; “OPA” is ortho-pthaldialdehyde; “RT” is room temperature.
As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
“Pharmaceutically acceptable salts” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
“Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
As used herein, the term “polymer” includes “copolymers.”
A “repeat unit” is the simplest structural entity of certain materials, for example, frameworks and/or polymers, whether organic, inorganic or metal-organic. In the case of a polymer chain, repeat units are linked together successively along the chain, like the beads of a necklace. For example, in polyethylene, —[CH2CH2]n—, the repeat unit is —CH2CH2—. The subscript “n” denotes the degree of polymerization, that is, the number of repeat units linked together. When the value for “n” is left undefined or where “n” is absent, it simply designates repetition of the formula within the brackets as well as the polymeric nature of the material. The concept of a repeat unit applies equally to where the connectivity between the repeat units extends three dimensionally, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc.
The term “reticulated open cell foam” or ROCF refers to a foam material with a porous structure consisting of an interconnected network of solid struts. The open cells are formed by the reticulation process, which is in turn defined as in the form of a network or having a network of parts. Because all struts are connected, the open cell porosity is also connected creating a continuously porous material. The ROCF can be defined specifically by three independent properties; pore size, relative density, and base material. In some embodiments, ROCF is made from polyurethane.
The above definitions supersede any conflicting definition in any of the reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.
Further, a device or structure that is configured in a certain way is configured in at least that way, but it can also be configured in other ways than those specifically described.
In one aspect the present invention provides a covalent system for catching and concentrating specific factors in the wound environment, including factors that trigger a significant biological response, such as proliferation, differentiation, or angiogenesis. This technology may be used, for example, to deliver biomolecules or bioactive compounds to the wound bed. Aptamer modified materials provided herein may be used in some embodiments as a system for catching and concentrating specific factors in the wound environment, for example, factors triggering a biological response such as angiogenesis, or to deliver endogenous or exogenous biomolecules or bioactive compounds to the wound bed.
In one aspect, the aptamer modified materials are provided comprising at least three components: (1) a polymer, (2) at least one linker covalently attached to the polymer, e.g., directly, through a side chain of the polymer, or through another linker molecule connected to the polymer, and (3) at least one aptamer molecule, which may be covalently attached to the polymer backbone, but will be more typically covalently attached to a linker.
1) Suitable Polymers
Polymers for use with the present invention include hydrophobic or hydrophilic polyurethanes, chitosan, crosslinked and/or uncrosslinked polyolefins, polyols, ethylene vinyl acetate (EVA), elastomers such as acrylonitrile butadiene (NBR), polychloroprene (PCP or CR), ethylene propylene rubber (EPR & EPDM), poloxamers, silicones, and/or fluoro carbon polymers. For example, in some embodiments, a chitosan-based polyurethane polymer may be used.
Polyurethanes are reaction polymers. A urethane linkage is produced by reacting an isocyanate group, —N═C═O with a hydroxy group, and polyurethanes are produced by the polyaddition reaction of a polyisocyanate with a diol or a polyol, typically in the presence of a catalyst and other additives. A polyisocyanate is a molecule with two or more isocyanate functional groups, R—(N═C═O)n, wherein n≧2 and a polyol is a molecule with two or more hydroxyl functional groups, R′—(OH)n, wherein ≧2. The reaction product is a polymer containing the urethane linkage, —RNHCOOR′—. Polyurethanes may be produced by reacting a liquid isocyanate with a liquid blend of polyols, catalyst, and other additives. The blend of polyols and other additives may also be called a resin or a resin blend. In some embodiments, resin blend additives may include chain extenders, cross linkers, surfactants, flame retardants, blowing agents, pigments, and/or fillers. The synthesis of breathable or air-permeable, open cell, flexible urethane polymers is taught for example by U.S. Pat. No. 5,686,501, which is incorporated by reference herein in its entirety.
Molecules that contain two isocyanate groups are called diisocyanates. Isocyanates may be classed as aromatic, such as diphenylmethane diisocyanate (MDI), diphenylethane diisocyanate (EDI), or toluene diisocyanate (TDI); or aliphatic, such as hexamethylene diisocyanate (HDI) or isophorone diisocyanate (IPDI). An example of a polymeric isocyanate is polymeric diphenylmethane diisocyanate, which is a blend of molecules with two-, three-, and four- or more isocyanate groups. Isocyanates can be further modified by partially reacting them with a polyol to form a prepolymer. A “quasi-prepolymer” is formed when the stoichiometric ratio of isocyanate to hydroxyl groups is greater than 2:1. A “true prepolymer” is formed when the stoichiometric ratio is equal to 2:1. Important characteristics of isocyanates include their molecular backbone, % NCO content, functionality, and viscosity.
Molecules that contain two hydroxyl groups are called diols. Examples include, ethylene glycol (EG), 1,4-butanediol (BDO), diethylene glycol (DEG). Molecules that contain three hydroxyl groups are called triols. Examples include glycerol. Polyols may themselves be polymers. For example, they may be formed by base-catalyzed addition of propylene oxide (PO), ethylene oxide (E0) onto a hydroxy or amino-containing initiator, or by polyesterification of a di-acid, such as adipic acid, with glycols, such as ethylene glycol or dipropylene glycol (DPG). Polyols extended with PO or EO are typically called polyether polyols. Polyols formed by polyesterification are typically called polyester polyols. The choice of initiator, extender, and molecular weight of the polyol will typically affect its physical state, and the physical properties of the resulting polyurethane. Important characteristics of polyols are their molecular backbone, initiator, molecular weight, % primary hydroxyl groups, functionality, and viscosity.
One attribute of polyurethanes is its ability to be turned into foam. Small amounts of water may also be added to accomplish this in some embodiments. Water will react with isocyanate to create carbon dioxide gas, which fills and expands cells created during the mixing process. Blowing agents may also be used, including certain halocarbons such as HFC-245fa (1,1,1,3,3-pentafluoropropane) and HFC-134a (1,1,1,2-tetrafluoroethane), and hydrocarbons such as n-pentane. In some embodiments, surfactants may be used to modify the characteristics of the polymer during the foaming process.
Though the properties of the polyurethane are typically determined mainly by the choice of polyol, the diisocyanate exerts some influence. For example, the cure rate will generally be influenced by the reactivity of a given functional group and the number of functional isocyanate groups.
Softer, elastic, and more flexible polyurethanes typically result when linear difunctional polyethylene glycol segments, commonly called polyether polyols, are used to create the urethane links. More rigid products typically result if polyfunctional polyols are used, as these create a three-dimensional cross-linked structure which, again, can be in the form of a low-density foam. Control of viscoelastic properties, for example, by modifying the catalysts and polyols used can lead to memory foam, which is much softer at skin temperature than at room temperature.
In some embodiments of the present invention, the polyurethane foam is formed by the polymerization of isocyanates and polyols, typically toluene diisocyanate and a multi arm polyether polyol. These components may be indexed, such that the isocyanate and hydroxyl group are in a one to one ratio.
Chitosan is a linear polysaccharide derived from the exoskeletons of crustaceans, which can be represented in some embodiments by the following formula:
In some embodiments, chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). The presence of the hydroxy groups and amino groups allows the utilization of a wide variety of conjugation chemistries. For example, chitosan may be polymerized and/or co-polymerized with diisocyanate or triisocyanate molecules, including diisocyanate or trisiocyanate prepolymers, into a polyurethane polymer or co-polymer.
In some embodiments a polyol other than chitosan may be used in addition to the chitosan. Addition of chitosan to a polyol resin creates urethane bonds between the isocyanate and the hydroxyl groups on the chitosan ring. The resulting foam contains chitosan segments to be available on the struts of the foam and gives the foam some of the properties of chitosan. In addition the addition of chitosan produces an over-indexed foam, with surplus hydroxyl groups and amine groups on the chitosan segments that are available for additional surface chemistry. For example,
Formulations of 5, 10, 20 and 30 PHR (grams of chitosan Per Hundred grams of polyurethane Resin) have been produced and tested. They have been characterized by the amount of surface amine available for bonding by conjugation of the foam with o-phthaldialdehyde. The relative fluorescence units RFU were found to be proportional to the primary amine available on the surface of the foam.
2) Linker-Modified Polymer Backbone
In some embodiments, the invention provides substituted silyl-modified polymers. Examples include those comprising repeat units based on polyurethane, which may be hydrophobic or hydrophilic, chitosan, crosslinked and/or uncrosslinked polyolefin, polyols, ethylene vinyl acetate (EVA), elastomers such as acrylonitrile butadiene (NBR), polychloroprene (PCP or CR), ethylene propylene rubber (EPR & EPDM), silicones, and/or fluoro carbon polymers. For example, in some embodiments, a chitosan-based polyurethane polymers or copolymers may be used. In some embodiments, the substituted silyl groups are attached to the polymer or co-polymer through an oxygen atom. In other embodiments, the substituted silyl groups are attached directly to a carbon atom of the polymer or co-polymer.
In some embodiments, the present invention provides polymeric materials modified with substituted silyl groups, for example, (H2N(CH2)3)(i-Pr)2Si— and (H2N(CH2)11)(EtO)2Si—. In some embodiments, the materials of the present invention comprise a polymeric foam. Examples of foam materials can include polyurethane-based foam, which may be hydrophobic or hydrophilic, including, for example a chitosan-based polyurethane foam material. Other foams that may be suitable include chitosan, crosslinked and/or uncrosslinked polyolefin's, polyols, ethylene vinyl acetate (EVA), and elastomers such as acrylonitrile butadiene (NBR), polychloroprene (PCP or CR), ethylene propylene rubber (EPR & EPDM), silicones, and fluoro carbon polymers. For example, in some embodiments, a chitosan-based polyurethane foam may be used. In some embodiments, the substituted silyl groups are attached to the foam through an oxygen atom. In other embodiments, the substituted silyl groups are attached directly to a carbon atom of the foam.
The silylated polyurethane foams disclosed herein may be made by silylating the hydroxy groups on a polymer comprising such groups. As used herein, silylation is the introduction of a substituted silyl group (R3Si—) to a molecule. It involves the replacement of a hydrogen on the compound, e.g., the hydrogen of a hydroxy group, with an substituted silyl group, for example, the compound 3-aminopropyldiisopropylethoxysilane will react with hydroxy groups under the appropriate conditions such groups to form a new covalent Si—O bond thereby linking the H2N(CH2)3Si(iPr)2— group to an oxygen which is in turn attached to the point of attachment on the molecule where the hydroxy group had been previously attached. Without being bound be mechanism, the oxygen atom of the product, may be the same oxygen atom of the hydroxy group reactant. See “How do I apply my Silane?” Gelest Catalog. 2006, pages 19-20, which are incorporated by reference herein in their entirety.
Example 1 below describes in detail how amino-substituted silyl groups may be deposited on 30 PHR chitosan foam. Example 2, describes how maleimide-thiol conjugation can be performed with Sulfo-EMCS, which has the formula:
In some embodiments, the N-hydroxysuccinimide ester (NHS ester) on one end of the Sulfo-EMCS molecule can react with free amino groups of the linker or polymer. The maleimide group on the other end of the molecule may be used to react, for example, with —SH groups on a peptide aptamer to form stable thioether bonds. In this manner the Sulfo-EMCS may be used to link the peptide to a polymer or copolymer, including a foam substrate.
Furthermore, the peptides used in maleimide-thiol conjugation typically have specially modified C-termini. For example, the C-termini may be “capped” with Cys residues bearing reactive —SH groups to facilitate the conjugation. In a Sulfo-EMCS conjugations, Sulfo-EMCS is typically used in excess, for example, in 50-100× molar excess relative to an organosilane crosslinkers to facilitate the reaction. Such reactions may be carried out, for example, at pH 7.0-7.5 and at room temperature. Such methods may be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is also incorporated by reference herein in its entirety. For example, other suitable amino-thio linkers may be used. These include, for example: SM(PEG)24, SM(PEG)12, SM(PEG)8, SM(PEG)6, SM(PEG)4, Sulfo-LC-SMPT, SM(PEG)2, Sulfo-KMUS, LC-SMCC, LC-SPDP, Sulfo-LC-SPDP, SMPH, Sulfo-SMPB, SMPB, SMPT, STAB, Sulfo-SIAB, EMCS, Sulfo-SMCC, SMCC, MBS, GMBS, Sulfo-GMBS, Sulfo-MBS, SPDP, SBAP, BMPS, AMAS, and SIA.
In some embodiments of the present invention, the silylated polymer will be further modified with additional linker molecules, for example, oligopeptide oligomers. In some embodiments, the additional linkers are covalently attached to the backbone of the silylated polymer or copolymer. In other embodiments, the linker is covalently attached to a side chain or side group of the polymer or copolymer. In some embodiments, the additional linker is attached via a function group (e.g., an amino group) of a substituted silyl group of the silylated polymer or copolymer, forming a material-linker1-linker2 modified material, wherein the linker1 is a substituted silyl group (a.k.a. heterobifunctional silane crosslinker) such as those discussed herein, and linker2 may be another a second substituted silyl group or another type of linker molecule, such an oligopeptide. In some embodiments, linker2 may formed from the reaction of a terminal amino or a mercapto group on linker1 with EMCS(N—[ε-maleimidocaproyloxy]succinimide ester). See Example 2 below.
Further functionalization of the interfacial layer created by, for example, silylation can be achieved by linking the other end of the silyl group or linker2 group to aptamers, including “capture peptides” as discussed further below.
3) Aptamer-Linker-Modified Polymer Backbone
In some embodiments, aptamers, including peptide-based aptamers, may be conjugated to a polymer or copolymer. Peptide aptamers also referred to as “capture peptides” herein, can be used to bind protein targets in the wound environment without inhibiting their signaling capabilities or other functions. Such capture peptides may also be used to capture and increase concentrations of biologically active proteins across a wound bed of a patient, for example, to strengthen or activate a targeted biological response. These and other uses are described in greater detail below.
In some embodiments, a linker is further connected to one or more different types of aptamers, forming a polymer backbone-linker-aptamer conjugate. In some embodiments, the linker is covalently attached to the backbone of the polymer. In other embodiments, the linker is covalently attached to a side group of the polymer. In some embodiments, the aptamer is directly linked to the backbone of the polymer, forming a polymer backbone-aptamer conjugate.
Poloxamer-EMCS-peptide constructs were also successfully used to bind aptamers to a foam. The hydrophobic portion of the poloxamer has strong hydrophobic interactions with the foam. The hydrophilic “arms” has amino groups, which were then linked to oligopeptides using and EMCS linker.
In some embodiments, the aptamer is a computationally selected or phage display derived “capture peptide.” Such “capture peptides” may be used to target specific proteins or protein families for binding. One example of such a peptide is P-16, defined by the amino acid sequence GHQGQHIGQMS-AEEAc-Cys-NH2, where the AEEAc-Cys-NH2 is a specially modified C-terminus for conjugation to maleimide groups provided by the EMCS linker. P-16 has been shown to bind to VEGF and PDGF, and potentially other members of the PDGF super-family.
Example 2 below reports the conjugation of an aptamer to 30 PHR chitosan foam via a Sulfo-EMCS ([N-ε-maleimidocaproyloxy]sulfosuccinimide ester) crosslinkers.
For example, once bound, the terminal amino group of the 3-aminopropyl-diisopropylethoxysilane remains accessible for capture peptide or aptamer binding. In some embodiments biologically active peptides may be bound to this terminal amine group in a manner as to their retain their biological activity. These attached peptides, when the material is used, for example, as part of a dressing, a wound insert or pad, may be used to capture and increase concentrations of biologically active proteins across a wound bed strengthening or activating the targeted biological response. For example, dressings may be prepared to concentrate proteins such as vascular endothelial growth factor in the wound bed, to trigger angiogenesis, and/or to trigger tubule formation. In this manner such materials may be used to better control various biological pathways in a wound environment
When a peptide or a factor is attached to the other end of the silane cross linker, it will have a dissociation constant (Kd), or binding affinity, that is specific to a given target molecule at a given set of conditions. In some embodiments, it may allow for reversible binding of one or more target(s). In some embodiments, the aforementioned target may be released back into a wound when used as part of the methods and devices contemplated herein. In some embodiments, ingredients may be added to the instillation solution to not only help dissociate the bound factor back into the wound environment but to also interact synergistically with the retained exudate element(s) for a modulating effect so that a favorable wound healing response is elicited. Types of instillation solutions are discussed in greater detail below.
Examples of instilled ingredients which may be used in some embodiments to dissociate bound molecules from the peptide linkers include: saline solutions, solutions with slightly acidic pH, slightly basic pH, solutions with various surfactants (i.e. polysorbate), EDTA or EGTA. In some embodiments, the fluid instilled to initiate the dissociation of the bound factors from the linker will depend upon the binding strength of the factor-linker complex, which is in turn determined by the dissociation constant. The dissociation constant may be modified by using knowledge of amino acid chemistry of the factor of interest to design the linker/peptide/aptamer.
One example of a modified dressing or wound insert is one capable of binding Ca2+, wound derived or otherwise, and retaining it at the wound site. During the early phases of wound healing, Ca2+ ions are typically released from the cells locally into the extracellular space. The resulting high Ca2+ concentration is believed to be a positive effector of many cellular processes involved in wound healing such as adhesion, migration and differentiation. When a high Ca2+ concentration is required or is beneficial to the wound bed, instillation or flushing with a solution containing a chelator (e.g., EDTA) may be used to disrupt the binding of Ca2+ to the dressing and into the wound bed.
Another factor for use with the current invention is transferrin, a blood plasma protein for iron binding. Chronic wound fluid has been shown to have significantly lower transferrin levels indicating that oxidative stress occurs in chronic wounds. It is known that free iron can play a role in the formation of free radicals. Without being bound by theory, high levels of free iron may contribute to exacerbation of tissue damage and delayed wound healing. Binding and concentrating transferrin onto the dressing can be used to sequester free iron in the wound bed with subsequent release with an appropriate instillation solution and subsequent removal with the exudate following the use of negative pressure. This specific time-dependent modulation of transferring and iron levels can provide a significant benefit to the patient. In an alternate view, the affinity of transferrin for iron is very high but is reversible in that it decreases progressively with decreasing pH below neutrality. If a need for localized iron concentration is necessary, instillation of a low pH solution can be used to unbind or release iron from transferrin.
Hyaluronan, or hyaluronic acid, is another possible target contemplated for use with the current invention. Without being bound by theory, immobilizing and concentrating hyaluronan to the wound bed before instillation with an appropriate solution for release may be used to contribute to keratinocyte proliferation and migration and reduce collagen deposition, which in turn is known to lead to reduced scarring. Hyaluronan is also known for its free-radical scavenging function that could be beneficial as it is bound to the foam on the wound site.
Lactoferrin, known for its antimicrobial and anti-inflammatory properties, is another possible target for some of the embodiments of the present invention. Secreted by endothelial cells, lactoferrin has been shown to have a synergistic effect with FGF2 in that there is a marked increase in their ability together to effect fibroblast migration and proliferation. Specifically designed aptamers can be used to bind both LF and FGF2 and release them with an appropriate instillation solution in an opportune therapeutic timeframe.
TGFB-3 can be another target another possible target for some of the embodiments of the present invention. This protein promotes reorganization of matrix molecules, resulting in an improved dermal architecture and reduced scarring. TGFB-3 is secreted in latent form that requires activation before it is functional. Activation of latent TGFB-3 occurs via binding to thrombospondin-1 (TSP-1). Therefore, TSP-1, may be used in some embodiments, as an ingredients in the instillation fluid to modulate TGFB-3 activity.
Possible other targets include calmodulin, S-100, thyroxine and cholate receptors, amongst many others.
The modified polymers described above and materials made therefrom may be used for a variety of purposes, including to (a) capture and concentrate biological targets in the wound environment, (b) specify the chemical nature of the binding, and/or (c) dictate the orientation with which the target factors are presented to the cells. As discussed in greater detail in the Examples section below, aptamer-modified polyurethanes, including peptide modified GranuFoam™ (GF), a type of ROCF, may be used to capture specific protein targets in vitro. For example, peptides P16, an anti VEGF peptide, and P22, anti-GM-CSF peptide, were covalently bound to ROCF, and it was demonstrated that these aptamers capture target proteins. Examples 2 and 3 provides results quantifying the amount of GM-CSF captured by ROCF coated with P22, and Example 4 presents results quantifying the amount of VEGF captured by ROCF coated with P16.
In some embodiments the aptamer-modified polymers may be used to bind protein targets in the wound environment without inhibiting their signaling capabilities or other functions. Such aptamer-modified polymers may be used as part of a dressing or wound insert, for example, to capture and increase concentrations of biologically active proteins across a wound bed of a patient, for example, to strengthen or activate a targeted biological response.
In some embodiments, such a dressing may be used to concentrate proteins of interest such as vascular endothelial growth factor in the wound bed to trigger angiogenesis and tubule formation. Additionally, peptides may be designed to antagonize or sequester proteins that adversely affect the healing process, such as matrix metalloproteinases. Dressings made of such aptamer-modified polymers may thus be used, in some embodiments, to modulate various biological pathways or to manage the presence of unwanted bioactive molecules or enzymes in the wound environment.
In addition to dressings, aptamer-modified polymers, including those using a heterobifunctional silyl-modified linker and a polyurethane-based polymer or copolymer, may also be used in a wide array of other materials, matrixes and biomedical devices, including catheters. In such embodiments, they may be used to conjugate a variety of aptamers, or other compounds, including antimicrobials. The application of these materials to a negative pressure-based therapy is discussed in greater detail below.
Materials of this invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
In another aspect, foam-based polymers may be physically and/or chemically treated, coated or manipulated before or after they are covalently linked to an aptamer. Some embodiments of making modified wound inserts comprise: compressing (and/or felting) at least a portion of a foam. Some embodiments comprise: treating (e.g., by applying heat, or activating a coating that has been applied to) the compressed foam such that the foam remains substantially compressed in the absence of an external compressive force. For example, in some embodiments, treating comprises heating the foam (e.g., foam) to an elevated temperature sufficient to reduce resiliency of the foam. For example, the foam can be heated to a temperature at which resiliency of the foam is reduced and/or relaxed, but that is below the melting temperature of the foam (e.g., such that the foam is not degraded by the elevated temperature). In this way, the foam can be compression set using heat and pressure (compressive force) to relax compressive strains developed in the foam. Generally, high temperatures are used to achieve the compression set. To achieve the desired “set” such that resiliency of the foam is reduced and/or the foam remains substantially compressed in the absence of a compressive force, temperatures can range from 158 degrees Fahrenheit to 482 degrees Fahrenheit (e.g., equal to, less than, greater than, or between any of: 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500 degrees Fahrenheit, depending upon the particular foam used). The foam may also be put through a cooling cycle to help retain the set introduced. For example, the foam may be cooled to a temperature below room or ambient temperature (e.g., to or in a temperature equal to, less than, greater than, or between any of: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 degrees Fahrenheit). In some embodiments of the present methods of forming a modified wound insert, the foam is disposed between two heated plates or platens (e.g., in a plate or platen press and/or where the plates are heated to a temperature sufficient to reduce the resiliency of the foam); and the press is actuated to move the plates toward one another (e.g., perpendicular to thickness 320 of thick portions 304) such that the foam is compressed to the desired overall thickness or degree of compression). Such a press can be electrically, mechanically, and/or hydraulically operated.
Some embodiments of the present methods of making modified wound inserts also comprise: cooling the foam (e.g., after heating the foam) such that the compressed portion of the foam remains substantially compressed at room temperature (e.g., at a temperature of 72 degrees Fahrenheit) in the absence of a compressive force. In other embodiments, cooling the foam includes cooling a coating that has been applied to the foam such that the compressed portion remains substantially compressed in the absence of a compressive force at a temperature or temperature range equal to, less than, greater than, or between any of: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and/or 150 degrees Fahrenheit.
Thick and thin regions in the foam can be formed by any suitable methods, such as, for example, laser cutting or the like. Such methods are taught, for example, by U.S. Patent Application Publication 2011/0178451, which is incorporated herein by reference.
In such embodiments, the coating can be dispersed through the foam, such as, for example, by spraying the foam with the coating, dipping the foam in the coating, and/or any other suitable way of dispersing the coating in the foam. In some embodiments, for example, the foam can be coated with a material that has a transition temperature (e.g., melting point, glass transition, etc.) that occurs at a relatively low temperature (e.g., lower than the foam alone), or that develops stiffness as it dries. In some embodiments, the coating can be configured to enable the foam to be compressed (and/or compression set) at lower temperatures (e.g., without heating), such that the coating becomes stiff or otherwise resistant to expansion as it cools or dries to hold the foam in its compressed configuration. For example, a fluid adhesive may be applied to thick portions before compressing the foam and permitted to dry before removing the compressive force, such that the dried adhesive will resist expansion from the compressed thickness. In other embodiments, the coating can be configured to compression set the foam such that the compression is reversible (e.g., at least partially and/or completely reversible) such that the foam can expand (e.g., after placing in or on a wound) as it warms or absorbs water. In some embodiments, the coating comprises a cross-linkable polymer and/or activating comprises exposing the coating to light and/or elevated temperature (e.g., above ambient temperature, such as, for example, a temperature sufficient to cause at least part of the cross-linkable polymer to cross-link) to cause at least some portion of the cross-linkable polymer to become modified.
Examples of suitable coatings include cross-linkable polymers that contain n-methylol acrylamide (NMA). NMA is a monomer that may be co-polymerized with many other monomers (such as acrylics & vinyls). On heating, (e.g., to about 140° C.), NMA reacts with itself and other hydroxyl-containing groups (e.g., carboxyl). Similarly, urea formaldehyde, melamine formaldehyde, and/or phenol formaldehyde can be caused to react with themselves and other hydroxyl-containing polymers to form crosslinks. Other crosslinking agents may include, for example, modified ethylene ureas, which react with hydroxyl-containing polymers at elevated temperatures to crosslink them. Other crosslinking agents can include peroxides which will crosslink most polymers at elevated temperatures. Polymers containing hydroxyl and carboxyl groups may also be combined, and, when heated, may form polyester crosslinks. Additionally, epoxy prepolymers can be used that have low reactivity at room temperatures, and when heated, react quickly to form an epoxy polymer with crosslinks. Similarly, polymeric isocyanates may be used that will only react significantly fast at elevated temperatures and in presence of hydroxyl groups, amines, or moisture to form polyurethanes or polyureas.
In some embodiments, a combination of high-density regions and low-density regions cooperate to provide various characteristics for the present modified wound inserts. For example, the high-density regions have a smaller aggregate cell size and increased cell density, such that the high-density regions have improved wicking function and more-effectively transmit fluid (e.g., draw fluids away from the wound surface and/or communicate fluid from a fluid source to the wound surface more effectively than the low-density regions. The high-density regions are generally also mechanically stronger than the low-density regions, such that the high-density regions can provide structural support for the low-density regions and/or the modified wound insert as a whole (e.g., such that the modified wound insert is resistant to tearing in directions that are not parallel to the low-density regions). Additionally, the low-density regions have a larger effective cell or pore size such that the low-density regions are less-susceptible to clogging. Especially when a negative pressure is applied to draw fluid and/or exudate away from the wound and through the modified wound insert, the larger pore size of the low-density regions may permit fluids to be drawn through the low-density regions at a higher velocity than the fluid is drawn through the high-density regions, such that particulate and granular matter are drawn to and/or through the low-density to discourage and/or decrease the likelihood of clogging in the high-density regions. In some embodiments, the foam can also be coated with a hydrophilic material to improve wicking properties of the modified wound insert.
The low-density regions may also be configured to permit the wound dressing to bend and/or otherwise conform to a wound. For example, the low-density regions can be relatively easier to bend (and/or less resilient when the modified wound insert is bent or folded along a low-density region) such as to double over a modified wound insert, and/or to conform a modified wound insert to additional hardware such as plates, pins, or the like.
Typical single-density foam modified wound inserts are isotropic such that under negative pressure, a typical single-density foam modified wound insert will contract proportionally in all directions. In some embodiments, the present modified wound inserts may also be configured to be anisotropic, such that the present modified wound inserts can be configured to mechanically assist with wound closure. For example, low-density regions are less-dense (and will compress more under negative pressure) than high-density regions. As such, if negative pressure is applied to modified wound insert, low density regions will contract more than high-density regions, such that high-density regions will be drawn together and modified wound insert will contract laterally more than longitudinally. In other embodiments, the present modified wound inserts can be configured to have alternating and sequentially larger closed ring-shaped high-density regions and low-density regions, such that under negative pressure, the modified wound insert will contract laterally inward to its own center.
In some embodiments, thick portions, thin portions, high-density regions, and/or low-density regions can be coated and/or printed (either before or after compression) to enhance the hydrophilic or hydrophobic properties of individual regions of the foam or of the foam as a whole. Such coated regions may also contain and/or be coated with other additives, such as antibiotics, or blockage-reducing agents.
In some embodiments of the present invention, wound dressings comprise a wound dressing configured to be positioned on a wound (e.g., 26) of a patient (e.g., 30) and/or on or in contact with the wound surface (e.g., 42).
Some embodiments of the present wound-treatment methods comprise: positioning a modified wound insert (e.g., any of the present modified wound inserts such as 34a, 34b) on a wound (e.g., 26) of a patient (e.g., 30), where the modified wound insert comprises a foam (e.g., 300) having high-density regions (e.g., 404) and low-density regions (e.g., 408) having a density that is less than the density of the high-density regions. In some embodiments, the foam is sterile (e.g., substantially free of microbes and/or bacteria). Some embodiments further comprise: coupling a drape (e.g., 38) to skin (e.g., 46) adjacent the wound such that the drape covers the modified wound insert and the wound, and forms a space between the drape and the wound. Some embodiments comprise: applying negative pressure to the wound through the wound dressing (e.g., through the modified wound insert). In some embodiments, applying negative pressure to the wound comprises activating a vacuum source (e.g., apparatus 14 of
Some embodiments of the present wound-treatment systems comprise either embodiment of system 10 (or any subset of components of either embodiment of system 10), and one or more of the present modified wound inserts and/or wound dressings.
The various illustrative embodiments of devices, systems, and methods described herein are not intended to be limited to the particular forms disclosed. Rather, they include all modifications and alternatives falling within the scope of the claims.
The claims are not intended to include, and should not be interpreted to include, means-plus- or step-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase(s) “means for” or “step for,” respectively.
It will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments. It will further be understood that reference to ‘an’ item refers to one or more of those items, unless otherwise specified.
The steps of the methods described herein may be carried out in any suitable order, or simultaneously where appropriate.
Where appropriate, aspects of any of the examples described above may be combined with aspects of any of the other examples described to form further examples having comparable or different properties and addressing the same or different problems.
It will be understood that the above description of preferred embodiments is given only way of example only and that various modifications may be made by those skilled in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments. Although various embodiments have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention.
The aptamer-modified polymers described above and materials made therefrom may be used for a variety of purposes, including to (a) capture and concentrate biological targets in the wound environment, with the option to release back into the wound bed, (b) specify the chemical nature of the binding, and/or (c) dictate the orientation with which the target factors are presented to the cells.
A dressing or wound insert made for an aptamer-modified polymer may be used together with negative pressure wound therapy. In some embodiments, compatible foam coated with aptamers or small peptide linkers (ligands) which select for beneficial molecules in the wound fluid as it passes through the foam would thereby prevent their removal into the negative pressure wound therapy canister. Appropriate molecules for selection from wound fluid include metabolites, growth factors, chemokines and cytokines which would not impede fluid flow through the negative pressure wound therapy dressing. In some embodiments, appropriate linkers would bind the wound fluid molecules in such an orientation that the “active site” of the wound fluid molecule is still available for eliciting a biological response. For example, one such molecule may be VEGF. VEGF has specific sites on the molecule that bind to cellular receptors. The binding of the VEGF molecule to the cellular receptor may be used to initiate a biological response, typically angiogenesis.
In some embodiments, the methods taught herein may be used to bind to molecules chemotactic to macrophages such as MCP1 (
Experiments were conducted whether aptamers bound to a peptide (in this case an antibody) could still maintain biological activity. VEGF in solution was passed over beads linked to an anti-VEGF antibody. After passing the solution over the beads, beads were spun down and washed. The washed beads were then used for endothelial cell migration assays. Results from this experiment showed that after 3 hours, 2 fold more cells had migrated towards the beads bound to the antibody (and VEGF) than beads with no antibody. See
Referring now to the drawings, and more particularly to
Apparatus 14 can comprise, for example, a vacuum source configured to be actuatable (and/or actuated) to apply negative pressure (e.g., via conduit 22) to wound dressing 18, a fluid source configured to be actuatable (and/or actuated) to deliver (e.g., via conduit 22) a fluid (e.g., an installation fluid such as a medicinal fluid, antibacterial fluid, irrigation fluid, and or the like) to wound dressing 18. System 10 can be implemented and/or actuated and/or coupled to patient 30 in any of various configurations and/or methods similar to those described in the prior art. For example, various wound therapy systems and components are commercially available through and/or from KCl USA, Inc. of San Antonio, Tex., U.S.A., and/or its subsidiary and related companies (collectively, “KCI”).
Conduit 22 can comprise a single lumen conduit (e.g., switched between a vacuum source and/or a fluid source and apparatus 14), or can comprise multiple single-lumen conduits or a multi-lumen conduit such that, for example, fluid can be delivered and/or negative pressure can be applied to wound dressing 18 individually and/or simultaneously. Additionally, conduit 22 can comprise, for example, a first lumen for the application of negative pressure and/or fluid delivery, and at least one additional lumen for coupling to pressure sensor(s) to sense pressure or negative pressure between drape 38 and surface 42. In some embodiments, conduit 22 can comprise multiple lumens (e.g., as in a single conduit with a central lumen for application of negative pressure and/or fluid delivery, and one or more peripheral lumens disposed adjacent or around the central lumen such that the peripheral lumens can be coupled to a pressure sensor to sense a pressure or negative pressure between drape 38 and surface 42 (e.g. in space 50). The lumens may be arranged with a central lumen and other lumens disposed radially around the central lumen, or in other suitable arrangements. The lumens may also be provided in separate conduits. In the embodiment shown, system 10 further comprises a wound dressing connection pad 54 configured to be coupled (and is shown coupled) to conduit 22. One example of a suitable connection pad 54 is the “V.A.C. T.R.A.C.® Pad,” commercially available from KCl. One example of a suitable drape 38 includes the “V.A.C.® Drape” commercially available from KCl.
Referring now to
Embodiments of the present wound treatment methods may be better understood with reference to
In the embodiment shown, apparatus 14 further comprises a pressure release valve 236 coupled to conduit 232. Further, in the embodiment shown, canister 204 and vacuum source 200 are coupled to wound dressing 18 by way of conduit 240; and/or canister 204 can comprise a filter 244 at or near an outlet of canister 204 to prevent liquid or solid particles from entering conduit 208. Filter 244 can comprise, for example, a bacterial filter that is hydrophobic and/or lipophilic such that aqueous and/or oily liquids will bead on the surface of the filter. Apparatus 14 is typically configured such that, during operation, vacuum source 200 will provide sufficient airflow through a filter 244 that the pressure drop across filter 244 is not substantial (e.g., such that the pressure drop will not substantially interfere with the application of negative pressure from wound dressing 18 from vacuum source 200).
In the embodiment shown, apparatus 14 further comprises a fluid source 248 coupled to wound dressing 18 by way of a conduit 252 that is coupled to conduit 240 such as, for example, by way of a tee- or other suitable fitting 256. In some embodiments, tee fitting 256 can comprise a switch valve and/or the like such that communication can be selectively permitted between wound dressing 18 and vacuum source 200, or between wound dressing 18 and fluid source 248. In some embodiments apparatus 14 comprises only one of vacuum source 200 and fluid source 248. In embodiments of apparatus 14 that comprise only fluid source 248, canister 204 and/or pressure sensor 212 can also be omitted. In various embodiments, such as the one shown, conduit 232 and/or conduit 240 and/or conduit 252 can be combined and/or comprised in a single multi-lumen conduit, such as is described above with reference to
In various embodiments, such as the one shown in
Apparatus 14 can also be configured to apply negative (or subatmospheric) pressure (e.g., continuously, intermittently, and/or periodically) to the wound site, and/or such that pressure relief valve 236 enables pressure at the wound site to be brought to atmospheric pressure rapidly. Thus, if apparatus 14 is programmed, for example, to relieve pressure at ten-minute intervals, at these intervals pressure relief valve 236 can open for a specified period, allow the pressure to equalize at the wound site, and then close to restore the negative pressure. It will be appreciated that when constant negative pressure is being applied to the wound site, valve 236 remains closed to prevent leakage to or from the atmosphere. In this state, it is possible to maintain negative pressure at the wound site without running and/or operating pump 200 continuously, but only from time to time or periodically, to maintain a desired level of negative pressure (i.e. a desired pressure below atmospheric pressure), which is sensed by transducer 216. This saves power and enables the appliance to operate for long periods on its battery power supply.
In some embodiments, factors may be removed, or their concentration modulated, using electrical pulses, light, ultrasound and temperature.
In some embodiments dressing made from the aptamer modified polymers disclosed herein may be used together with wound instillation solutions, for example in the application of a negative pressure treatment to a patient's wound. In some embodiments, the instillation solution comprises ingredients to help release or modulate the release of the factors bound to the foam.
Examples of instilled ingredients which may be used in some embodiments to dissociate bound molecules include: saline solutions, solutions with slightly acidic pH, solutions with slightly basic pH, solutions with various surfactants (i.e. polysorbate), solutions with slight ionic charge, EDTA or EGTA. In some embodiments, the fluid instilled to initiate the dissociation of the bound factors from the linker will depend upon the binding strength of the factor-linker complex, which is in turn determined by the dissociation constant. The dissociation constant may be modified by using knowledge of amino acid chemistry of the factor of interest to design the linker/peptide.
In some embodiments, the instillation solution comprises hypochlorous acid (HOCl) and hypochlorite ion. Both are examples of effective antimicrobial agents for biocidal action. For example, HOCl is typically capable of killing a broad spectrum of microbes (e.g., fungus, bacteria, viruses, fungus, yeast, and the like); often in a relatively short period of time (e.g., is capable of killing greater than 99% of microbes within a period of less than 10 seconds). Such antimicrobial agents can be generated or formed by a combination of the present reactive agents and fluid (e.g., water and/or aqueous solution, such as, for example, saline solution) and may be more effective and/or more versatile than antibiotics and other commonly used antimicrobial agents used in wound treatment in the past. For example, antibiotics may be bacteria-specific such that testing may be required to determine a suitable antibiotic to use for a specific wound or infection; and/or such that antibiotics may have only limited effectiveness for individual wounds and/or infections (e.g., where testing is not performed and/or where a wound is infected with a plurality of different bacteria). Such testing may take as long as several days to determine an appropriate antibiotic, delaying treatment or selection of an effective antibiotic. Additionally, bacteria may develop resistance to antibiotics, such that antibiotics may have reduced effectiveness after an amount of time. Further, antibiotics are typically administered intravenously (systemically) such that antibiotics may kill beneficial bacteria (e.g., in a patient's digestive system) and/or may cause organ damage (e.g., to a patient's liver).
In contrast, the reactive agents (and/or antimicrobial products of the reactive agents) of the present embodiments can be configured to have a broad-spectrum killing power that will kill a variety of microbes (e.g., fungus, bacteria, viruses, fungus, yeast, etc.). Additionally, the present reactive agents (and/or antimicrobial products of the reactive agents) can be delivered locally (preventing systemic damage or other side effects to organs and the like).
However, due to the reactivity of HOCl or OCl− with oxidizable organic substances, its utility in wound care applications has previously been limited. For example, prior art methods of generating hypochlorous acid have required electrolysis of saltwater or the like (e.g., with expensive equipment at a patient's bedside). By way of another example, commercially available chemicals (e.g., bleach) have a hypochlorous acid concentration of 5% or greater, which is too high to permit medical uses (e.g., will cause cytoxicity). Additionally, at suitable medical concentrations (e.g., 2-20 mM hypochlorous acid solutions), approximately 99% or more of the solution is water, such that shipping is more expensive and/or more difficult than necessary. Further, storage of hypochlorous acid solutions is difficult, as reactions with containers typically degrade or reduce the concentration of the hypochlorous acid solution. However, the present wound inserts can be deposited with reactive agents (have reactive agents deposited in the foam of the wound inserts) such that upon application of a fluid such as saline or water, OCl (and/or ClO−) is released (e.g., to form hypochlorous acid) and delivered to a wound for biocidal action.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
In this procedure, a heterobifunctional silane crosslinker, 3-amino-propyldiisopropylethoxysilane, was deposited on 30 per hundred resin (30 phr) chitosan foam, which was produced using 0.3 g of chitosan (Sigma Product Number 448877) per 1.0 g of an aromatic diisocyanate and tri-branched polyol mix. A plurality of the —OH groups of the copolymer were then silylated with 3-aminopropyldiisopropylethoxysilane (Gelest™ compound SIA0602.0). This was accomplished using the protocol further below.
The resulting silylated 30 phr chitosan foam was compared with silylated reticulated open cell foam using an o-phtaldialdehyde (OPA) assay that compares relative fluorescence units (RFU). OPA fluoresces when it binds to primary —NH2 groups. Therefore, RFUs for 30 phr chitosan foam should be greater than, for example, OPA alone indicating the presence primary of —NH2. The results from this test are shown in Table 1 below and in
The equipment used in carrying out these experiments include:
The test procedure used included the following steps:
A 30 per hundred resin (30 phr) chitosan prototype was formulated using 0.3 g of chitosan (Sigma P/N 448877) to 1.0 g polyurethane. The following test apparatuses, materials and procedures were used in carrying out these experiments.
The materials used in carrying out these experiments include:
The materials used in carrying out these experiments include:
The test procedure used included the steps listed below: All buffers and reagents were prepared in advance, except the Sulfo-EMCS, which was reconstituted immediately before use. See Sulfo-EMCS Product Instructions (Thermo Scientific-Pierce), which is incorporated by reference herein in its entirety.
In this study ROCF was substituted for the chitosan substrate described in the examples above. DyLight Fluor Texas Red (Thermo Scientific P/N 46412) was used instead of OPA. It is an NH2-reactive dye as well; however, it fluoresces at a different wavelength.
Gelest Compound 630 was used instead of Gelest Compound SIA0602.0. Compound 630 has a shorter alkane chain and no propyl groups but carries out the same basic function.
Fluorometric Analysis of -Sil bound to ROCF. In order to determine the amount of silyl or substituted silyl groups bound to the ROCF, Texas Red fluorescent dye, an NH2-reactive fluorophore, was conjugated to the 630 linker for visualization and quantification. 630's heterobifunctionality enables its Si atom at one end to bind to —OH groups on the foam while the other end presents a —NH2 group that facilitates NH2-reactive chemistries. Once the 630 was deposited onto the ROCF, incubation with Texas Red identified the presence of the crosslinkers on the ROCF. Fluorescent analysis showed successful deposition of silyl or substituted silyl groups compound 630 to ROCF (
With the -Sil bond, a “capture peptide” was next conjugated to the amino-functionalized ROCF. To do so, Sulfo-EMCS chemistry developed in Example 2 was used to conjugate a commercially available GM-CSF antagonist capture peptide, P22, to ROCF. The P22-linker-foam construct was incubated with GM-CSF, washed stringently and eluted. The eluted GM-CSF was quantified via ELISA (
Elution results for the P22/GM-CSF experiment: P22 was obtained from the Bachem Americas Inc. 2010 peptide catalog. P22 is a GM-CSF antagonist that binds GM-CSF in an inhibitory manner. Briefly, 330 nmol P22 was conjugated to ROCF via Sulfo-EMCS chemistry. The P22-linker-foam construct was incubated with 1 μg GM-CSF overnight, washed five times, and subjected to stringent elutions. The wash preceding the elutions showed that non-specifically bound protein was reduced to negligible levels (<100 pg/mL). Upon elution, the sample with the most capture peptide, S3, outperformed ROCF alone by 49%. The total amount of GM-CSF bound to ROCF/P22, 608 pg/mL, was greater than the amount of GM-CSF bound non-specifically to the negative control, Cl, 376 pg/mL. Samples S1-S3 all consisted of peptide-linker-foam with Sulfo-EMCS concentration increasing from S1 to S3. There were no positive controls available for this experiment, as no technology has yet been proven to covalently conjugate peptides to polyurethane. However, positive controls were later developed for subsequent experiments based on experimental results. Data are given as mean±standard error.
P16, an anti-VEGF peptide, was designed de novo. The design was based on the dimeric X-ray crystallography structure of VEGF dimmer. The amino residues in the true biological design can be modified to enhance or reduce binding affinities.
P16 was designed de novo. P16 was based on the X-ray crystallography structure of the VEGF dimer, registered as accession number 1vpf in the Protein Data Bank. The P16 peptide design mimics the natural phenomenon of dimerization that takes place between two VEGF homodimers.
P16 consists of an 11 aa sequence that mimics the dimeric interface of Chain C in Protein Data Bank structure 1vpf. The P16 sequence consists mostly of hydrophobic residues with a few charged residues scattered throughout. The sole substitution is at aa1, where we substituted a glycine for a proline to minimize steric interference from the proline.
Further experiments demonstrate that P16 captures the protein target VEGF. ELISA results from P16/VEGF experiments indicated that P16 captured its protein target, VEGF (
The results summarized in both Examples 3 & 4 show that -Sil groups were successfully conjugated to ROCF. Fluorometric quantification of the positive test group indicated 21,702.5 RFUs at an Ex/Em of 584/630, whereas the negative control, ROCF without -Sil, yielded a total of 2,586.5 RFUs due to background levels of ROCF/dye interaction. P16 and P22 were successfully conjugated to the linkers deposited on the ROCF substrate. Finally, it was possible to quantify the effectiveness of P16 and P22 capturing their target proteins. In the case of P16, the elution data indicate that the positive sample bound 82% more VEGF than the greatest negative control, silanated ROCF. The positive sample bound 584 pg/mL, whereas the negative sample bound 321 pg/mL. For P22, the sample with the most capture peptide outperformed ROCF alone by 49%. The total amount of GM-CSF bound to ROCF/P22, 608 pg/mL, was greater than the amount of GM-CSF bound non-specifically to the negative control, 376 pg/mL.
The present application claims priority to U.S. Provisional Application Ser. No. 61/518,148 filed Apr. 29, 2011, the entire content of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61518148 | Apr 2011 | US |
