DI-FUNCTIONALIZED HYALURONIC ACID DERIVATIVES AND HYDROGELS THEREOF
20180291123 ยท 2018-10-11
Inventors
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
A61K49/0054
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61L27/3895
HUMAN NECESSITIES
C08B37/0072
CHEMISTRY; METALLURGY
C12N5/0622
CHEMISTRY; METALLURGY
A61L27/58
HUMAN NECESSITIES
A61L2430/38
HUMAN NECESSITIES
A61L27/3675
HUMAN NECESSITIES
A61K35/30
HUMAN NECESSITIES
A61L27/383
HUMAN NECESSITIES
A61L27/3878
HUMAN NECESSITIES
A61K35/545
HUMAN NECESSITIES
A61L27/50
HUMAN NECESSITIES
International classification
A61K51/06
HUMAN NECESSITIES
A61K35/545
HUMAN NECESSITIES
A61L27/36
HUMAN NECESSITIES
A61L27/58
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61K35/30
HUMAN NECESSITIES
A61L27/50
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
Abstract
Provided herein are di-functionalized hyaluronic acids, such as molecules including (or that have been functionalized at) a thiol and azide side chain. Also, provided herein are hydrogels of these di-functionalized hyaluronic acids and methods of using these compounds to promote cell (e.g., neuronal cell) growth and development. In some aspects, the present disclosure also provides methods of treating injuries, including brain injuries such as stroke through the use of hydrogels of the compounds described herein and stem cells.
Claims
1. A compound of the formula: ##STR00016## wherein: R.sub.1 is Y.sub.1-L.sub.1, wherein: Y.sub.1 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.1 is a first linking group; R.sub.2 is Y.sub.2-L.sub.2, wherein: Y.sub.2 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.2 is a second linking group; R.sub.3 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); R.sub.4 is hydroxy, alkoxy.sub.(C6), or substituted alkoxy.sub.(C6); R.sub.5 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); X.sub.1, X.sub.2, and X.sub.3 are each independently hydrogen, alkyl.sub.(C6), substituted alkyl.sub.(C6), or a hydroxy protecting group; X.sub.4 is hydrogen, acyl.sub.(C6), substituted acyl.sub.(C6), or a monovalent amino protecting group; m is 1-20; and n is 1-5000; provided that the first linking group and the second linking group are different; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein the compound is further defined as: ##STR00017## wherein: R.sub.1 is Y.sub.1-L.sub.1, wherein: Y.sub.1 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.1 is a first linking group; R.sub.2 is Y.sub.2-L.sub.2, wherein: Y.sub.2 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.2 is a second linking group; R.sub.3 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); R.sub.4 is hydroxy, alkoxy.sub.(C6), or substituted alkoxy.sub.(C6); R.sub.5 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); m is 1-20; and n is 1-5000; provided that the first linking group and the second linking group are different; or a pharmaceutically acceptable salt thereof.
3. The compound of either claim 1 or claim 2, wherein the compound is further defined as: ##STR00018## wherein: R.sub.1 is Y.sub.1-L.sub.1, wherein: Y.sub.1 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.1 is a first linking group; R.sub.2 is Y.sub.2-L.sub.2, wherein: Y.sub.2 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.2 is a second linking group; R.sub.3 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); m is 1-20; and n is 1-5000; provided that the first linking group and the second linking group are different; or a pharmaceutically acceptable salt thereof.
4. The compound according to any one of claims 1-3, wherein Y.sub.1 is alkanediyl.sub.(C12) or substituted alkanediyl.sub.(C12).
5. The compound of claim 4, wherein Y.sub.1 is CH.sub.2CH.sub.2.
6. The compound according to any one of claims 1-5, wherein L.sub.1 is SH.
7. The compound according to any one of claims 1-5, wherein L.sub.1 is an acrylate or a methylacrylate group.
8. The compound of claim 7, wherein L.sub.1 is: ##STR00019##
9. The compound according to any one of claims 1-8, wherein Y.sub.2 is alkanediyl.sub.(C12) or substituted alkanediyl.sub.(C12).
10. The compound of claim 4, wherein Y.sub.2 is CH.sub.2CH.sub.2.
11. The compound according to any one of claims 1-11, wherein L.sub.2 is N.sub.3.
12. The compound according to any one of claims 1-11, wherein R.sub.3 is hydrogen.
13. The compound according to any one of claims 1-12, wherein m is 2, 3, or 4.
14. The compound according to any one of claims 1-13, wherein n is an integer between 1 and 1000.
15. The compound of claim 14, wherein n is an integer between 1 and 500.
16. The compound according to any one of claims 1-13, wherein the compound is further defined as: ##STR00020## or a pharmaceutically acceptable salt thereof.
17. A hydrogel comprising a compound in accordance with claim 1.
18. The hydrogel of claim 17, comprising two hyaluronic units comprising: a first hyaluronic unit of the formula: ##STR00021## wherein: R.sub.1 is Y.sub.1-L.sub.1, wherein: Y.sub.1 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.1 is a first linking group; R.sub.2 is Y.sub.2-L.sub.2, wherein: Y.sub.2 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.2 is a second linking group; R.sub.3 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); R.sub.4 is hydroxy, alkoxy.sub.(C6), or substituted alkoxy.sub.(C6); R.sub.5 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); X.sub.1, X.sub.2, and X.sub.3 are each independently hydrogen, alkyl.sub.(C6), substituted alkyl.sub.(C6), or a hydroxy protecting group; X.sub.4 is hydrogen, acyl.sub.(C6), substituted acyl.sub.(C6), or a monovalent amino protecting group; m is 1-20; and n is 1-5000; and a second hyaluronic unit of the formula: ##STR00022## wherein: R.sub.1 is Y.sub.3-L.sub.3, wherein: Y.sub.3 is a covalent bond, alkanediyl.sub.(C12), alkenediyl.sub.(C12), alkynediyl.sub.(C12), arenediyl.sub.(C12), or a substituted version of any of these groups; and L.sub.3 is a third linking group; R.sub.2 and R.sub.3 are each independently hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); R.sub.4 is hydroxy, alkoxy.sub.(C6), or substituted alkoxy.sub.(C6); R.sub.5 is hydrogen, alkyl.sub.(C6), or substituted alkyl.sub.(C6); X.sub.1, X.sub.2, and X.sub.3 are each independently hydrogen, alkyl.sub.(C6), substituted alkyl.sub.(C6), or a hydroxy protecting group; X.sub.4 is hydrogen, acyl.sub.(C6), substituted acyl.sub.(C6), or a monovalent amino protecting group; m is 1-20; and n is 1-5000; provided that the first, second, and third linking groups are all different; and wherein the hydrogel comprises a ratio of the first hyaluronic acid to the second hyaluronic acid from about 10:1 to about 1:10.
19. The hydrogel of claim 18, wherein the first linking group comprises a thiol unit.
20. The hydrogel of either claim 18 or claim 19, wherein the second linking group comprises an azide group.
21. The hydrogel according to any one of claims 18-20, wherein the third linking group is a methacrylate.
22. The hydrogel according to any one of claims 18-21, wherein the ratio is from about 1:1 to about 1:7.5.
23. The hydrogel of claim 22, wherein the ratio is from about 1:2 to about 1:5.
24. The hydrogel according to any one of claims 18-23, wherein the first linking group, the second linking group, or the third linking group is linked to a polypeptide.
25. The hydrogel of claim 24, wherein the polypeptide is derived from laminin, neuroligin-1, fibronectin, collagen, n-cadherin, cartilage oligomeric protein, neural cell adhesion molecule 1, victronectin, brain-derived neurotrophic factor, neurexin, insulin-like growth factor, link binding protein, bone morphgenic proteins, decoriin, or aggrecan binding peptides.
26. The hydrogel of claim 25, wherein the polypeptide comprises the sequence IKVAV (SEQ ID NO:1.
27. The hydrogel according to any one of claims 18-23, wherein the first linking group, the second linking group, or the third linking group is linked to an imaging agent.
28. The hydrogel of claim 27, wherein the imaging agent is a chelating group bound to a radioisotope.
29. The hydrogel of claim 27, wherein the imaging agent is fluorophore.
30. A composition comprising: (a) a hydrogel according to any one of claims 18-29; and (b) a cell.
31. The composition of claim 30, wherein the cell is a stem cell.
32. The composition of claim 31, wherein the stem cell is a human induced pluripotent stem cell.
33. The composition of claim 30, wherein the cell is a neural cell or a neural stem cell.
34. The composition according to any one of claims 30-33, wherein the cell is encapsulated in the hydrogel.
35. A method of treating a disease or disorder in a patient comprising implant a hydrogel or composition according to any one of claims 18-34 into a tissue of the patient.
36. The method of claim 35, wherein the disease or disorder is a neurological disease or disorder and wherein the hydrogel or composition is implanted in a neuronal tissue.
37. The method of claim 36, wherein the neurological disease or disorder is a brain injury.
38. The method of claim 36, wherein the neurological disease or disorder is a stroke.
39. The method of claim 36, wherein the neurological disease or disorder results in a neuronal lesion.
40. A method of promoting cell growth in a tissue lesion comprising implanting a hydrogel or composition according to any one of claims 18-34 into the tissue lesion under conditions sufficient to promote cell or tissue growth.
41. The method of claim 40, further defined as a method of promoting neuronal growth in a neuronal lesion comprising implanting the hydrogel or composition into the neuronal lesion under conditions sufficient to promote neuronal tissue formation.
42. The method of claim 40, wherein the method comprises implanting a composition.
43. A method of inducing differentiation in a stem cell comprising contacting the stem cell with a hydrogel according to any one of claims 18-29 under conditions sufficient to cause the stem cell to differentiate into a mature cell.
44. The method of claim 43, wherein the stem cell is a neuronal stem cell.
45. The method of claim 44, comprising encapsulating the neuronal stem cell with the hydrogel.
46. The method of claim 44, wherein the mature cell is a mature neuron, oligodendrocyte, or astrocyte.
47. The method according to any one of claims 35-46, wherein the method is performed in vitro.
48. The method according to any one of claims 35-46, wherein the method is performed in vivo.
49. The method according to any one of claims 35-48, wherein the method is performed in a patient.
50. The method of claim 49, wherein the patient is a mammal.
51. The method of claim 50, wherein the patient is a human.
52. The method according to any one of claims 35-39 and 43-51, wherein the method comprises implanting the hydrogel or the composition according to any one of claims 18-34 into a neuron or neuronal tissue.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0104] In some aspects, the present disclosure provides hyaluronic derivatives that contain two or more linking groups, such as a thiol and/or azide-based linking group. Also provided herein are hydrogels prepared using the di-functionalized hyaluronic acid and methods of using the hydrogels to induce cell growth and stem cell differentiation. For example, in some aspects hydrogels including the hyaluronic derivatives described herein may be used to promote cell growth and/or differentiation in a neurological tissue, such as brain tissue. In some embodiments, the di-functionalized hyaluronic shows reduced stiffness and allows for flexibility in the hyaluronic backbone while introducing two different functional groups with different reaction chemistries. Additionally, in some embodiments, the functionalization of the hyaluronic acid does not degrade the hyaluronic chain. These properties render the hydrogels detailed herein very useful, for example in growth of tissue matrices or repair of damaged tissues in vivo. For example, in some aspects, the hydrogels prepared using the di-functionalized hyaluronic acid are implanted in damaged neurons with stem cells in a manner sufficient to induce stem cell differentiation and the formation of neuronal tissue.
I. Brain Nerve Deficits and Stroke
[0105] Stroke is a leading cause of acquired disability in adults worldwide (Roger et al., 2012). Although survival after stroke has increased, little progress has been made in restoring neurological function (Group et al., 2009). Recently, stem cell therapy has emerged as a treatment with the potential to restore neurological function lost due to stroke.
[0106] Stroke causes changes in the ECM (
[0107] Native NSC, which migrate to the perimeter of the stroke lesion, have been found to differentiate to mature neurons, oligodendrocytes and astrocytes when isolated and cultured in vitro (Nakagomi et al., 2009). This indicates that the extracellular environment around the stoke lesion is inhibiting tissue repair. Modulating that environment to promote further migration and differentiation with an aECM will increase the restoration of neurological function beyond that achieve utilizing cells alone (Jin et al., 2010; Yasuda et al., 2010; Osanai et al., 2010).
II. Neuronal Tissue Formation and Extracellular Components
[0108] Synapse Formation and Angiogenesis in cerebral tissue involves multiple signaling pathways.
[0109] Laminin is a major constituent of the basal lamina, which surrounds the brain and blood vessels throughout the CNS (Timpl and Brown, 1996), and has been shown to work in conjunction with NL to promote the stabilization and maturation of newly formed neural microvasculature (Samarelli et al., 2014). Laminin, also, plays a role in synapse formation and structure (Egles et al., 2007) and has been associated with axon extension into the lesion in long term survivors of spinal cord injury (Buss et al., 2007). Other cellular effects of laminin in the CNS include increased cellular survival and proliferation, myelination, and modulation of neuronal electrical activity (Buttery, 1999; Meiners and Mercado, 2003; Liesi et al., 2001; Colognato, 2005; Garcia-Alonso and Fetter, 1996). When included in HA based aECM implanted in the brain of rats, laminin has increased neurite extension beyond that of aECM alone (Hou et al., 2005).
[0110] Neuroligin-1 (NL) is a synaptogenic adhesion protein (Ichtchenko et al., 1995; Irie et al., 1997; Sudhof, 2008), which early in development NL is expressed down the length of the neurites (Aiga et al., 2011) and promotes the survival of neurons (Schnell et al., 2014). The protein and its peptide fragments have been shown increase dendritic process formation and length and synapse formation in vivo and in vitro (Schnell et al., 2012; Gjrlund et al., 2012; Chen et al., 2012). The matrix metallopeptidase 9 cleaved fragment and the membrane tethered NL shortened bioactive fragments are biologically active both in vivo and in vitro (Gjrlund et al., 2012; Peixoto et al., 2012).
[0111] NL is widely expressed throughout the vascular system Bottos et al., 2009), and is thought to interact directly with the vascular niche participating in crosstalk between the neural and vascular system (Doetsch, 2003; Bottos et al., 2011). Although the extent of this crosstalk is not well understood, NL has been shown to promote angiogenesis through interactions with vascular endothelial growth factor A and fibroblast growth factor 2 (Bottos et al., 2009; Rissone et al., 2012) and promote vessel stabilization and maturation through interactions with 61 integrin and laminin Samarell et al., 2014).
[0112] Hyaluronic Acid (HA) is a high molecular weight glycosaminoglycan present in the ECM of the developing brain in higher amounts than the adult brain, but even at lower amounts is still major constituent in the adult ECM (Margolis et al., 1975; Dityatev et al., 2010). HA has multiple modification sites that can be modified by several methods to form a hydrogel and has been used extensively as a scaffolding base in tissue engineering for numerous tissues (Fujiwara et al., 2000; Prestwich et al., 1998; Gamini et al., 2002; Slaughter et al., 2009). HA hydrogels have been shown to be capable of supporting NSC growth and differentiation in vitro (Pan et al., 2009). When implanted in rat brains, HA hydrogels have increased cell infiltration, angiogenesis and integration with host tissue compared to control (Hou et al., 2005). HA in the perisynaptic ECM has been implicated in synapse plasticity through modulation of calcium channels (Kochlamazashvili et al., 2010).
III. Hyaluronic Acid Derivatives
[0113] The hyaluronic acid derivatives provided by the present disclosure are shown, for example, above in the summary of the invention section and in the claims below. They may be made using the methods outlined in the Examples section. These methods can 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 incorporated by reference herein.
[0114] Hyaluronic acid derivatives of the invention may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S or the R configuration.
[0115] Chemical formulas used to represent compounds of the invention will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.
[0116] Hyaluronic acid derivatives of the 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.
[0117] In addition, atoms making up the hyaluronic acid derivatives of the present disclosure are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include .sup.13C and .sup.14C.
[0118] It should be recognized that the particular anion or cation forming a part of any salt form of a compound provided herein 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.
IV. DEFINITIONS
[0119] When used in the context of a chemical group: hydrogen means H; hydroxy means OH; oxo means O; carbonyl means C(O); carboxy means C(O)OH (also written as COOH or CO.sub.2H); halo means independently F, Cl, Br or I; amino means NH.sub.2; hydroxyamino means NHOH; nitro means NO.sub.2; imino means NH; cyano means CN; isocyanate means NCO; azido means N.sub.3; in a monovalent context phosphate means OP(O)(OH).sub.2 or a deprotonated form thereof; in a divalent context phosphate means OP(O)(OH)O or a deprotonated form thereof; mercapto means SH; and thio means S; sulfonyl means S(O).sub.2; hydroxysulfonyl means S(O).sub.2OH; sulfonamide means S(O).sub.2NH.sub.2; and sulfinyl means S(O).
[0120] In the context of chemical formulas, 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 formula
##STR00008##
includes
##STR00009##
And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol -, when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof. The symbol , when drawn perpendicularly across a bond (e.g.,
##STR00010##
for methyl) 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 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 geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.
[0121] When a group R is depicted as a floating group on a ring system, for example, in the formula:
##STR00011##
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:
##STR00012##
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.
[0122] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: Cn defines the exact number (n) of carbon atoms in the group/class. Cn 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/class in question, e.g., it is understood that the minimum number of carbon atoms in the group alkenyl.sub.(C8) or the class alkene.sub.(C8) is two. Compare with alkoxy.sub.(C10), which designates alkoxy groups having from 1 to 10 carbon atoms. Cn-n defines both the minimum (n) and maximum number (n) of carbon atoms in the group. Thus, alkyl.sub.(C2-10) designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms C5 olefin, C5-olefin, olefin.sub.(C5), and olefin.sub.C5 are all synonymous.
[0123] The term saturated when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term saturated is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
[0124] The term aliphatic when used without the substituted modifier signifies that the compound or chemical 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 carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).
[0125] The term aromatic when used to modify a compound or a chemical group atom means the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by an interaction of the bonds forming the ring.
[0126] 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 acyclic structure, and no atoms other than carbon and hydrogen. The groups CH.sub.3 (Me), CH.sub.2CH.sub.3 (Et), CH.sub.2CH.sub.2CH.sub.3 (n-Pr or propyl), CH(CH.sub.3).sub.2 (i-Pr, .sup.iPr or isopropyl), CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu), CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl), CH.sub.2CH(CH.sub.3).sub.2 (isobutyl), C(CH.sub.3).sub.3 (tert-butyl, t-butyl, t-Bu or .sup.tBu), and CH.sub.2C(CH.sub.3).sub.3 (neo-pentyl) are 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 acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups CH.sub.2 (methylene), CH.sub.2CH.sub.2, CH.sub.2C(CH.sub.3).sub.2CH.sub.2, and CH.sub.2CH.sub.2CH.sub.2 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 or alkyl. Non-limiting examples of alkylidene groups include: CH.sub.2, CH(CH.sub.2CH.sub.3), and C(CH.sub.3).sub.2. An alkane refers to the class of compounds having the formula HR, wherein R is alkyl as this 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, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2. The following groups are non-limiting examples of substituted alkyl groups: CH.sub.2OH, CH.sub.2Cl, CF.sub.3, CH.sub.2CN, CH.sub.2C(O)OH, CH.sub.2C(O)OCH.sub.3, CH.sub.2C(O)NH.sub.2, CH.sub.2C(O)CH.sub.3, CH.sub.2OCH.sub.3, CH.sub.2OC(O)CH.sub.3, CH.sub.2NH.sub.2, CH.sub.2N(CH.sub.3).sub.2, and CH.sub.2CH.sub.2Cl. The term haloalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. F, Cl, Br, or I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, CH.sub.2Cl is a non-limiting example of a haloalkyl. The term fluoroalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups CH.sub.2F, CF.sub.3, and CH.sub.2CF.sub.3 are non-limiting examples of fluoroalkyl groups.
[0127] 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, 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 include: CHCH.sub.2 (vinyl), CHCHCH.sub.3, CHCHCH.sub.2CH.sub.3, CH.sub.2CHCH.sub.2 (allyl), CH.sub.2CHCHCH.sub.3, and CHCHCHCH.sub.2. 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, a linear or branched 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 CHCH, CHC(CH.sub.3)CH.sub.2, CHCHCH.sub.2, and CH.sub.2CHCHCH.sub.2 are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms alkene and olefin are synonymous and refer to the class of compounds having the formula HR, wherein R is alkenyl as this term is defined above. Similarly the terms terminal alkene and -olefin are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule. When any of these terms are used with the substituted modifier one or more hydrogen atom has been independently replaced by OH, F, Cl, Br, I, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2. The groups CHCHF, CHCHCl and CHCHBr are non-limiting examples of substituted alkenyl groups.
[0128] The term alkynyl when used without the substituted modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups CCH, CCCH.sub.3, and CH.sub.2CCCH.sub.3 are non-limiting examples of alkynyl groups. The term alkynediyl 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, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon triple bond, no carbon-carbon double bonds, and no atoms other than carbon and hydrogen. The groups CC, CCCH.sub.2, and CH.sub.2CCCH.sub.2 are non-limiting examples of alkynediyl groups. It is noted that while the alkynediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. An alkyne refers to the class of compounds having the formula HR, wherein R is alkynyl. When any of these terms are used with the substituted modifier one or more hydrogen atom has been independently replaced by OH, F, Cl, Br, I, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2.
[0129] 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 or aralkyl groups (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, C.sub.6H.sub.4CH.sub.2CH.sub.3 (ethylphenyl), naphthyl, and a 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, aryl or aralkyl groups (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. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). Non-limiting examples of arenediyl groups include:
##STR00013##
An arene refers to the class of compounds having the formula HR, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the substituted modifier one or more hydrogen atom has been independently replaced by OH, F, Cl, Br, I, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2.
[0130] The term acyl when used without the substituted modifier refers to the group C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as those terms are defined above. The groups, CHO, C(O)CH.sub.3 (acetyl, Ac), C(O)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)CH(CH.sub.2).sub.2, C(O)C.sub.6H.sub.5, C(O)C.sub.6H.sub.4CH.sub.3, C(O)CH.sub.2C.sub.6H.sub.5, 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. The term aldehyde corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a CHO group. When any of these terms are used with the substituted modifier one or more hydrogen atom (including a hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) has been independently replaced by OH, F, Cl, Br, I, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2. The groups, C(O)CH.sub.2CF.sub.3, CO.sub.2H (carboxyl), CO.sub.2CH.sub.3 (methylcarboxyl), CO.sub.2CH.sub.2CH.sub.3, C(O)NH.sub.2 (carbamoyl), and CON(CH.sub.3).sub.2, are non-limiting examples of substituted acyl groups.
[0131] 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 include: OCH.sub.3 (methoxy), OCH.sub.2CH.sub.3 (ethoxy), OCH.sub.2CH.sub.2CH.sub.3, OCH(CH.sub.3).sub.2 (isopropoxy), OC(CH.sub.3).sub.3 (tert-butoxy), OCH(CH.sub.2).sub.2, O-cyclopentyl, and O-cyclohexyl. The terms cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkoxy, heteroaryloxy, heterocycloalkoxy, and acyloxy, when used without the substituted modifier, refers to groups, defined as OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term alkylthio and acylthio when used without the substituted modifier refers to the group SR, in which R is an alkyl and acyl, respectively. 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 ether corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group. 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, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2.
[0132] The use of the word a or an, when used in conjunction with the term comprising in the claims and/or the specification may mean one, but it is also consistent with the meaning of one or more, at least one, and one or more than one.
[0133] Throughout this application, the term about is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0134] The terms comprise, have and include are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as comprises, comprising, has, having, includes and including, are also open-ended. For example, any method that comprises, has or includes one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
[0135] The term effective, as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. Effective amount, Therapeutically effective amount or pharmaceutically effective amount when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.
[0136] A hydroxyl protecting group is well understood in the art. A hydroxyl protecting group is a group which prevents the reactivity of the hydroxyl group during a reaction which modifies some other portion of the molecule and can be easily removed to generate the desired hydroxyl. Hydroxyl protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference. Some non-limiting examples of hydroxyl protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; acyloxy groups such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, ,-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. When used herein, a protected hydroxy group is a group of the formula PG.sub.HO wherein PG.sub.H is a hydroxyl protecting group as described above.
[0137] As used herein, the term IC.sub.50 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.
[0138] An isomer of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
[0139] 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.
[0140] 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.
[0141] 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).
[0142] The term pharmaceutically acceptable carrier, as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
[0143] 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.
[0144] 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, [CH.sub.2CH.sub.2].sub.n, the repeat unit is CH.sub.2CH.sub.2. 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.
[0145] A stereoisomer or optical isomer is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. Enantiomers are stereoisomers of a given compound that are mirror images of each other, like left and right hands. Diastereomers are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2.sup.n, where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and/or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase substantially free from other stereoisomers means that the composition contains 15%, more preferably 10%, even more preferably 5%, or most preferably 1% of another stereoisomer(s).
[0146] Treatment or treating includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
[0147] The above definitions supersede any conflicting definition in any 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.
V. EXAMPLES
[0148] 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.
[0149] In some embodiments, provided di-functional HA can act as linker molecule with broad application to biomedicine and tissue engineering. Examples of such applications include, but are not limited to, as a multi-functional scaffold base material for neural tissue engineering applications, as a replacement or supplement to those tissues that are largely composed of hyaluronic acid, such as but not limited to cartilage and thus diHA has many uses in orthopedic medicine including, but not limited to a role as an injectable lubricant. In additional embodiments, diHA provides a broadly applicable tissue engineering matrix that could be injected and form the final matrix within the body, for example as dermal filler useful for reconstructive and cosmetic applications. In other embodiments, diHA can also be used as a therapeutic delivery agent in non-tissue engineering applications. In still other embodiments, diHA also provides a method to increase the persistence of additional signal or therapeutic molecules (drugs, bioactive signals, proteins) in injections.
[0150] In some embodiment the diHA describe herein, utilizes a single functional chemistry for tethering and gelation, the addition of a second chemistry increases the total concentration and control of tether and gelation. The thiol and azide chemistries do not interact with each other meaning functionalization of one chemistry dose on reduce the availability of the other chemistry to interact with additional bioactive signals or gelation chemistries. Functionalization based on classical reactions used in polysaccharide chemistry typically have unsatisfactory efficiency and harsh conditions leading to polymer chain degradation or was not cleaved during the synthesis (Mergy et al., 2012), the current diHA method does not have these disadvantages. Sufficient functionalization efficiency for hydrogel formation is achieved without HA backbone degradation. Functionalization of HA polymer backbone has also been associated with chain stiffing, which reduces the ability of the material to interact with cells reducing attachment (Camci-Unal et al., 2013). DiHA allows for the addition of two functional chemistries which maintaining enough flexibility in the HA backbone to allow for cellular attachment and differentiation. Thus a surprising and unexpected aspect was that two functional chemistries could be put on one backbone without breaking the polymer backbone and that the backbone remained flexible enough to allow cellular attachment and differentiation without further functionalization.
[0151] In some embodiments, provided is one-pot synthesis of diHA, a strategy that improves efficiency of the chemical reaction by subjecting to successive chemical reactions in just one reactor, thus avoiding a lengthy separation process and purification of the intermediate chemical compounds, saving time and resources while increasing chemical yield.
Example 1Synthesis of Difunctional HA
[0152] Synthesis of Di-Functional HA
[0153] Hyaluronic acid (0.04 g, M.sub.W=60,000 kDa) was dissolved in 8 mL ultrapure water (DDW) (0.5% w/v solution). The pH of the solution was raised to 10 with 1M NaOH. A two-folder molar excess of ethylene sulfide was added to the HA solution very slowly and the reaction mixture was stirred overnight in the chemical hood at room temperature. The solution was filtered through 3-cm bed of Celite 545. A five-fold molar excess of DTT was added to filtered solution, and raised a pH of 8.5 with 1M NaOH. The reaction was stirred overnight in chemical hood at room temperature. 78.4 mg of MES (2-(N-morpholino)-ethanesulfonic acid buffer) (50 mM, pH 4), 95.6 mg of EDC-HCl, 57.4 mg of NHS ((N-hydroxysuccinimide), and 154 L of 11-azido-3,6,9-trioxaundecan-1-amine were added on 8 mL of HA-sulfhydryl solution, and the reaction of mixture was stirred 24 hrs in the chemical hood at room temperature. The solution was dialyzed (cutoff=12-14 kDa) against 1M NaCl for 1 day, and then an additional DDW for 5 days. DW was changed every day. The solution was lyophilized to obtain HA-SH-AA (di-functional HA) powder for use in hydrogel formation.
[0154] The step-by-step process is shown below in Table 1.
TABLE-US-00001 TABLE 1 Step by Step Process for Formation of Di-Functionalized Hyaluronic Acid A. HASH (HA-sulfhydryl) 1. 0.04 g (60k sodium HA) + 8 ml DW (0.5% HA in DW) 2. pH 10 using 1M NaOH 3. Add 192ul ES/8 ml HA solution (5-fold molar excess ethylene sulfide) slowly - dropwise 1 drop/2-3 min 4. Reaction overnight at RT under chemical hood 5. Filter 3 cm bed of Celite 545 6. Add 0.1M stock solution 32ul/8 ml HA + ES (5-fold DTT) 7. pH 8.5 using 1M NaOH 6. Reaction overnight (24 hrs) at RT under chemical hood 7. pH 3.5 using 6N HCl Optionally, dialysis with a 10K membrane was then conducted for 5 days (everyday DW change), and then lyophilizer (power), however this was only in the case that reaction should be terminated with only the thiol modification. B. HAAA 8. 0.04 g HA/ES/DTT/8 ml DW + 0.0784 g MES (50 mM, pH 4) 9. Add 0.0956 g EDC-HCl/154ul AA/0.0574 g NHS 10. Reaction 24 hrs at RT 11. Dialysis (12 kDa) at NaCl for 1 day 12. Dialysis (12 kDa) at DW for 5 days (everyday water change) 13. Freeze 14. Dry using lyophilizer 15. Gelling test and structure analysis using by analytical techniques such as nuclear magnetic resonance, mass, and infrared spectroscopy. (HA = hyaluronic acid; DW = ultrapure water; ES = ethylene sulfide; DTT = dithiothreitol; MES = 2-morpholinoethanesulfonic acid; EDC-HCl = N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS = N-hydroxysuccinimide; AA = 11-azido-3,6,9-trioxaundecan-1-amine)
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Example 2Optimal Concentrations of Bioactive Peptides
[0155] To individually determine the optimal concentration of bioactive peptides, such as but not limited to, laminin and neuroligin-1 (NL) needed to increase hiPSC derived NSC survival, axon extension, and synapse formation continuous gradient samples are used for two and three dimensional in vitro culture. Laminin and NL have not been widely studied in an artificial extracellular matrix (aECM) for the brain. The therapeutic concentrations and combinations of peptide fragments are not known. Previously, the inventors have utilized a continuous gradient pump system (
[0156] Peptides from laminin and NL (Table 2) will be synthesized using solid phase Fmoc chemistry and will be coupled with methacryloyl chloride (Johnson et al., 2010). Peptide concentration gradients will be made in a manner similar to previously described using the gradient system in
TABLE-US-00002 TABLE2 Bioactivepeptidefragmentsfromlamininandneuroligin-1. Protein BioactiveSignalingPeptides Source SEQIDNO: Laminin IKVAV JBiolChem.1989;264(27):16174-82. 1 YIGSR Biochemistry.1987;26(22):6896-900. 2 RNIAEIIKDI ACSChemicalBiology,2007.2(5):347-55. 3 neuroligin-1 SEGNRWSNSTKGLFQRA FASEBJ201226(10):4174-86. 4 LRE Neuron.200756(6):979-91. 5
[0157] For cell experiments, hiPSC derived NSC will be encapsulated in the peptide gradient hydrogels (Smith Callahan et al., 2013a; Smith Callahan et al., 2013b). Cell laden hydrogels are maintained in NSC maintenance media (DMEM:F12 containing 1N2, 1B27, 20 ng/mL bFGF, 2 g/mL heparin, 2 mM Glutamax, 1% non-essential amino acid, and 55 mM 2-mercaptoethanol) in order to support hiPSC derived NSC survival, but permit an assessment of biological effect of the peptides due to the lack of cytokines driving further differentiation in the media. Over a 8 week time course, cell survival and differentiation will be assessed as described in Table 3 according to previously published protocols (see Smith Callahan et al., 2013c; Liu et al., 2002).
TABLE-US-00003 TABLE 3 Strategy for testing neural stem cell response to aECM. Each gradient sample will be divided into at least 5 discrete samples for analysis. Groups Gradient Control: Tissue Samples: laminin peptide, & Analysis Culture Plastic neuroligin-1 peptide Cell Survival DNA Content (Horechest dye) & Tunnel Staining Differentiation Immunofluorescence: quantification of TUJ1 espressing neurite lengths & Percent of cells expressing TUJ1, NeuN & synapsin. qPCR: Pax6, TUJ1, MAP2, NeuN, NF & synapsin.
[0158] All statistical analysis will be performed using one-way analysis of variance and a comparison-wise Tukey test at 95% confidence. Cell culture experiments will be conducted twice using quadruplicate samples.
[0159] It is expected that peptides at specific concentrations will augment NSC survival, axon extension and synapse formation. However it is possible that the selected peptides do not produce the desired effects in hiPSC derived NSC. In that case, additional media supplements will be added to facilitate differentiation, longer linkers will be used to tether peptides to limit steric hindrance or additional peptides from laminin and NL will be examined in the gradients.
Example 3Hyaluronic Acid Matrices
[0160] Hyaluronic acid matrices capable of presenting bioactive peptides from laminin and neuroligin-1 at independent concentrations will be fabricated and characterized. The presentation of multiple signaling elements in an aECM will provide synergistic signaling to overcome the inhibitory signals in the CNS and facilitate the reestablishment of synaptic connections in the lesion. Laminin signaling will facilitate the extension of cells into CNS lesions (Buss et al., 2007). When combined with NL and HA, the advanced aECM proposed here will expedite the restoration of neurological function through increased survival, axon extension and synapse formation.
[0161] High molecular weight HA (75 kDA) will be modified with a thiol and azide group as described in
[0162] For gelation via Michael addition, high molecular weight HA will methacrylated as described in
[0163] It is expected that HA will remain biologically active following both the chemical derivation and polymerization. If the extent of chemical derivation is low or the biological activity of the peptides is significantly tempered in the hydrogel, alternative strategies will be pursued including alternative purifications and inclusion of longer linkage segments to attach the peptide to the backbone. Additionally, the examination of the oligodendrocyte response and microvasculature invasion in the aECM, may direct further modification of the backbone to tri-functionalization, or development of hybrid matrices using additional polymers and peptides to further emulate aspects of the native brain ECM.
Example 4Evaluate hiPSC Derived NSC Survival, Axon Extension and Synapse Formation
[0164] The hiPSC derived NSC survival, axon extension and synapse formation in hyaluronic acid matrices established using the bioactive peptides from laminin and neuroligin-1 in vivo will be evaluated. To establish the safety and efficacy of therapies that use such advanced biomaterials either alone or in combination with cell therapy in an animal stroke model in order to develop the combination therapies necessary to generate clinical functional recovery, more studies must be done. The inventors will implant the optimized matrices developed using the methods described herein in rodent models to establish their value in improving hiPSC survival and maturation beyond that of the standard stem cell therapy procedures.
[0165] A moderate transient focal ischemia is induced in all SCID mice by right middle cerebral artery occlusion (MCAO) using the intraluminal thread method as previously described (Li et al., 2010). Seven days after the MCAO, a neurological deficit score (Table 4) will be determined and behavioral testing (Table 5) will be conducted as previously described (see for example, Hatcher et al., 2002; Hua et al., 2002; Cao et al., 2010; Xiong et al., 2011). Mice are assigned to transplant groups (Table 5) and 200,000 GFP+hiPSC derived NSC will be mixed in with 10 L of saline or HA solutions from Example 3 with and without peptides will be injected into the infarcted striatum and cortex. Behavioral testing will be conducted weekly for 4 weeks (Table 5). After the final behavior test, the mice in each group will subdivided into 3 groups. Subgroup 1 (containing 5 mice) will be stained with triphenyltetrazolium chloride to quantify the infract volume. Subgroup 2 (containing 5 mice) will be used for Evans blue extravasation to examine the integrity of the blood brain barrier via spectrometry. The 3rd subgroup (containing 6 mice) will be used for cresyl violet staining to identify the infarct and immunofluorescence (Table 5).
TABLE-US-00004 TABLE 4 Neurological Deficit Scoring System. Score Neurological Deficit 0 no deficit 1 flexion of contralateral torso and forlimb upon lisfting of the whole animal by tail 2 circling to the contralateral side, when helped by tail with feet on floor 3 spontaneous circling to the contralateral side 4 no spontaneous motor acitivity
TABLE-US-00005 TABLE 5 In vivo aECM Biological Testing. Groups NSC + HA + Analysis NSC NSC + HA Laminin + NL Survival Total number of GFP.sup.+ NSC Proliferation % of GFP.sup.+ and Ki67.sup.+ NSC Differentiation length of TUJ1/GFP neurites, % of TUJ1/GFP, % of NeuN/GFP & % of synapsin/GFP Behavioral Neurological Deficit Score, Testing Rotarod Test, Corner Turning Test & SmartCage System
[0166] A priori power analysis using G*Power 3.1.9 software with a statistical power of 0.8 ( error<0.05) indicates 45 animals will be necessary for the study based on the effect size of a previously published dataset (Jin et al., 2010). All statistical analysis of collected data will be performed using one-way analysis of variance and a comparison-wise Tukey test at 95% confidence.
[0167] The HA matrices with multiple signaling peptides are anticipated to provide superior microenvironments for hiPSC derived NSC survival, axon extension and synapse formation compared to hydrogels containing lesser or no peptides signals. If this fail, strategies such as those described in Examples 2 and 3 will be utilized to further optimize the aECM. Should the integration of human cells in animal models pose a problem, mouse NSC will be differentiated from mouse iPSCs. Typically, optimal conditions for human stem cells cultured on matrices that have worked for similar rodent stem cells with little alteration (Smith et al., 2010; Smith et al., 2009), so if a change in species be necessary to facilitate animal studies it should not pose a major challenge to progress.
Example 5Characterize Spatial Location of Di-Functional Hyaluronic Acid within Gradient Polyethylene Glycol Hydrogels by Radioactive Detection
[0168] The concentrations and spatial location of bioactive signal and polymer content are rarely quantified in tissue engineering and device coating applications and when done, fluorescent or colorimetric readouts are typically used, which provide little information about the spatial location of these signaling and structural elements in the 3D culture. (Zustiak et al. 2010, Smith Callahan, Ganios, et al. 2013). Utilizing radiotracers enables quantitative analysis of HA components and significantly improves both the determination of concentrations and spatial locations beyond currently available assays. HA functionalized with reactive moieties for Michael addition and click chemistry as developed by the inventors (
[0169] High molecular weight HA (75 kDA) is modified with a thiol and azide group as shown in
[0170] Modified HA is then be dissolved in 10% polyethylene glycol dimethacrylate (PEGDM). HA gradient is then made in a manner similar to previous described using the computer controlled gradient making system in
[0171] If the current does not allow for greater sensitivity in spatial and moiety characterization than currently utilized fluorometric and colorimetric assays, the radiotracers will be bound to the HA backbone prior to matrix formation, the radiotracer concentration will be increased, or alternative chemistries will be utilized for coupling of labels, near-infrared fluorescence detection agent will be added to radiotracer, and/or HA concentration within gradient will be altered further to determine the assays range of sensitivity.
Example 6Determine Effects on Radiotracer Binding on Hyaluronic Acid Matrix Degradation, Mechanics and Material Properties
[0172] Modification of HA backbone has been shown to alter the degradation rate, mechanical properties and material properties of HA matrices (Baier Leach et al. 2003). Previously described methods of hydrogel characterization (see
[0173] Modified HA with and without coupled radiotracers are mixed with methacrylated HA at concentrations that contain an equal ratio of available thiol to methacrylate and allowed to form a gel overnight via Michael addition. For degradation studies, samples will be incubated in PBS with 2.6 u/mL hyaluronidase at 37 C. for 1 week (Khetan et al. 2009). Supernatant will be collected daily and the amount of uronic acid, a degradation product of HA, in the supernatant will be quantified using the carbazole reaction technique (Bergman et al. 1970). A gamma counter will also be used to determine the amount of radioactivity in the supernatant to further quantify degradation. The Young's and shear modulus are determined with a TA.TX texture analyzer as previously described, while material properties (mesh size, swelling ratio, and water content) are determined as previously described in Baier Leach et al., 2003, and Smith Callahan, Policastro et al. 2013 (incorporated herein by reference).
[0174] Methods of material characterization utilized in this example are well established. If problems arise, alternative methods, which are equally established, will be used. If uronic acid assay output does not yield a clear picture of degradation, the mass loss of the matrices can also be tracked (Baier Leach et al. 2003). For mechanical testing, dynamic mechanical analysis or rheological methods will be utilized (Khetan et al. 2009, Holloway et al. 2014).
Example 7Evaluating Ability of Radiotracers to Spatially Locate Di-Functional Hyaluronic Acid in Three Dimensional Cultures of Human Induced Pluripotent Stem Cell Derived Neural Stem Cells and the Effects of the Radiotracers on Cellular Survival, Neurite Extension, and the Gene Expression of Mature Markers of Neural Differentiation
[0175] To improve cell-modified HA interaction, additional bioactive signaling proteins are coupled to HA matrixes (Shu et al. 2004). Bioactive peptide fragments have similar effects on cellular behavior as proteins, but are easier to tether with regio- and chemo-selectivity and spatially disburse in 3D materials (Meiners et al. 2003). The fabrication of bioactive peptides containing matrices for human cell culture has been previously described (Smith Callahan, Ma, et al. 2013, Smith Callahan, Policastro, et al. 2013). A bioactive peptide fragment from laminin, IKVAV (SEQ ID NO:1), was selected for this study with the radiotracers due to the ability to promote cellular survival and proliferation, neurite guidance, myelination, and the modulation of neuronal electrical activity (
[0176] Laminin peptide, IKVAV (SEQ ID NO:1), is synthesized using standard Fmoc chemistry and coupled to methacryloyl chrloride (Elbert et al. 2001). A percentage of free thiols on the HA backbone will be consumed to couple the IKVAV (SEQ ID NO: 1) peptide to the backbone. The remainder of the free thiols are used to form the matrices described in Example 6.
[0177] NSC derived from the HDF51 hiPSC line will be encapsulated in HA matrices containing IKVAV (SEQ ID NO:1) with and without the radiotracers. NSC derived from the HDF51 hiPSC line on matrigel coated tissue culture plastic will be used as a control. Each sample will be cultured individually in wells of a 48 well plate for up to 2 weeks in DMEM:F12 media containing 1% Glutamax, 1% non-essential amino acids, 0.5% N2 supplement, 1% B27 supplement, 20 ng/mL brain-derived neurotrophic factor, 20 ng/mL glial cell-derived neurotrophic factor, 200 ng/mL ascorbic acid, and 1 M cAMP. After 3, 9, and 14 days, cellular survival, axon extension, and gene expression will be examined as described Example 2 (see Table 3 above) according to previously published protocols (Liu et al. 2002, Smith Callahan, Ma, et al. 2013).
[0178] All statistical analysis will be performed using one-way analysis of variance and a comparison-wise Tukey test at 95% confidence. Cell culture experiments will be conducted twice using quadruplicate samples.
[0179] It is expected that IKVAV (SEQ ID NO:1) will remain bioactive and radiotracers will not have significant effect on cellular behavior in long term culture. Should the incorporated IKVAV (SEQ ID NO:1) concentration not adequately support cellular attachment and survival longer linkers will be used to tether peptides to limit steric hindrance, the concentration of IKVAV (SEQ ID NO:1) is increased or additional bioactive peptides from laminin (YIGSR (SEQ ID NO:2)) is added to the matrix. If radiotracers inhibit cell material-interface longer, flexible linkers will be used, the radiotracer concentration will be reduced, or an alternative detection agent will be utilized.
Example 8Preliminary Results
[0180] Molecular structure of di-functional (diHA) and methacrylate HA (mHA) are shown in
[0181] The preliminary characterization of thiol group functional HA backbone is shown in
[0182] Characterization of azide group on HA is shown in
[0183] Characterization of diHA/mHA hydrogels of various concentrations is shown in
[0184] Step-growth polymerization in hydrogels can lead to higher mechanical properties then observed in chain growth polymerized hydrogels (Lin, et al., 2011). Increased step-growth polymerization in the 1:5 hydrogel compared to the 1:2 hydrogel could be contribution to the increase in observed Young's Modulus. As the 1:2 hydrogel has a higher thiol content, it is more likely to form oxidation induced disulfide bonds than the 1:5 hydrogel. The formation of these disulfide bonds during polymerization would reduce the concentration of total reactive moieties and skew covalent polymerization toward chain growth, both of which would lead to reduced mechanical properties. Oxidation induced crosslinking due to disulfide bond formation has been linked to a reduced swelling ratio in hydrogels (Shu, et al., 2002), which is consistent with the observed data (Table 6).
TABLE-US-00006 TABLE 6 Material properties of di-functional hyaluronic acid (dif HA)/ methacrylate hyaluronic acid (mHA) hydrogel of various mixing ratios. dif HA:mHA Properties 1:2 1:5 Young's modulus [kPa] 2.1 0.4 3.7 0.07 Swelling ratio 20.5 2.5 26.2 3.7 Water content [%] 94.9 0.6 95.9 0.5
[0185] Characterization of diHA hydrogels synthesized using different molecular weight HA for the process is shown in
[0186] A number of factors may contribute to the altered degradation rates observed between tested hydrogel formulations (
[0187] A time course of mouse embryonic stem cell growth over time under growth conditions in proliferation and differentiation media (
[0188] Several factors potentially contribute to the observed difference in cellular response between formulations. The increased early degradation in the 1:2 hydrogel compared to the 1:5 hydrogel (
[0189] Although the Young's Moduli for both formulations (Table 2) falls within the range reported for brain tissue (Moore and Sheetz, 2011), neural stems cells can be sensitive to alterations in mechanical properties of their substrate (Leipzig and Shoichet, 2009), causing changes in cellular attachment and neurite extension (Balgude, et al., 2001; Jiang, et al., 2008). Conflicting reports exist in the literature regarding the sensitivity of mES during neural differentiation to changes in Young's Modulus (McKinnon, et al., 2013: Ali, et al., 2015; Norman and Aranda-Espinoza, 2010). Neurite extension and mRNA gene expression of neural differentiation markers by embryonic neurons and mES differentiating toward the neuronal linage have been reported to be unaffected by changes in the Young's modulus of the substrate in two-dimensional (2D) culture (Ali, et al., 2015; Norman and Aranda-Espinoza, 2010), while another study found reduced neurite extension of with increased Young's Modulus in three-dimensional (3D) culture (McKinnon, et al., 2013). However, the 3D study used increases in the polymer mass fraction to increase the Young's Modulus, which would change additional material properties in the hydrogel that could have hindered neurite extension and complicates the assessment that the modulus change alone was the cause of the observed biological results (Smith Callahan, et al., 2013). The current study used a consistent polymer mass fraction between formulations that reduces the number of material properties that could change between formulations compared to the previous 3D study. The contribution of the Young's modulus change to the observed biological results remains unclear due to alterations in additional material properties that would have greater effect on the biological results in the present 3D study than the previous 2D studies (Smith Callahan, 2016).
[0190] Based on in vitro results, the 1:2 difHA:mHA formulation was selected for preliminary testing in a contusion model of rodent spinal cord injury. Compared to saline injection, the 1:2 hydrogel was found to significantly reduce ED1.sup.+ macrophages in the injured spinal cords 1 week after implantation (
[0191] The in vivo gelation relied solely on Michael addition for covalent bond formation. It is possible that not all the thiols were consumed during Michael addition (8; Shu, et al., 2004). Conflicting reports in the literature regarding whether HASH or other materials containing free thiols cause an inflammatory response (Zheng, et al., 2004, Khaing, et al., 2011; Lam, et al., 2014; Young, et al., 2013). Previous studies of HA matrices that did not contain a thiol have found ED1 expression to either not differ substantially from control (Mothe, et al., 2013) or to be reduced to control (Gupta, et al., 2006). The current study found ED1 expression in matrix injected spinal cords to be significantly reduce at 1 week compared to control, but similar to control after 4 weeks of implantation, indicating that the 1:2 dif HA:mHA matrix did not evoke a significant inflammatory response at the time points studied.
[0192] Typically, inclusion of a HA matrix leads to reduction of lesion volume (Mothe, et al., 2013; Gupta, et al., 2006; Austin, et al., 2012). Increases in axons post injury with HA matrices compared to control have, also, been observed (Austin, et al., 2012). However, neither was observed in the present study. However, axons were found to interact with the eosin-tagged HA matrix at the 1 week time point (
[0193] In order for the matrix to be beneficial crosslinking must occur (Liang, et al., 2013). It is possible that Michael's addition did not sufficiently occur and oxidation induced crosslinking established the initial gel structure in vivo (Tingaut, et al., 2011). These oxidative induced disulfide bonds would be susceptible to changes in pH (Shu, et al., 2002) and water absorption (Choh, et al., 2011). As the lesion environment is known to change over time (von Leden, et al., 2016; Volpato, et al., 2013), the lesion environment could become unsupportive to the maintenance of disulfide bonds, which would speed the clearance of the matrix from the area and limit its effects on tissue regeneration (
Example 9Methods and Materials
[0194] Materials and Analytical
[0195] HA was purchased from Lifecore (Chaska, Minn.). .sup.1H and .sup.13C NMR spectra (Bruker, Billerica, Mass.) were recorded at 600, or 150 MHz in D.sub.2O and were referenced to the residual proton. The degree of functionalization was probed by NMR analysis upon normalization to the integrals of the HA backbone methyl peak. Fourier Transform Infrared Spectroscopy (FT-IR) spectra were recorded on Nicolet iS10 with Attenuated Total Reflectance system (Thermo Fisher Scientific, Waltham, Mass.) using powder samples, and scanned 32 times from 600 cm.sup.1 to 4000 cm.sup.1, with a 2 cm.sup.1 resolution. Radiolabeled samples were analysed by radio-thin-layer chromatography with an AR-2000 scanner (Eckert & Ziegler Radiopharma, Hopkinton, Mass.). Chemicals were purchased from Thermo Fisher Scientific (Waltham, Mass.) or VWR International (Radnor, Pa.) unless specified otherwise.
[0196] Synthesis of Methacrylate HA (mHA)
[0197] HA was modified with methacrylate groups in a manner similar to previously described (
[0198] Colorimetric Quantification of Thiol Content
[0199] The thiol concentration within the HASH and dif HA products was determined in a manner similar to previously described using Ellman's Method (Serban, et al., 2008). Briefly, HA, HASH, or dif HA was dissolved in 5,5-Dithiobis-2-nitrobenzoic acid (DTNB, 2 mg/mL in PBS) and the resultant solution was stirred overnight at room temperature. The DTNB-HA, -HASH and -dif HA solution were dialyzed (MWCO=8-10 kDa) against DDW for 3 days with DDW changed daily, frozen overnight at 80 C., and then lyophilized. DTNB-HA, -HASH and -dif HA were dissolved in PBS (2 mg/mL), and 1% (w/v) DTT was added. The absorbance at 412 nm was read using a microplate reader (Tecan Infinite M1000, Maennedorf, Switzerland). The value of DTNB-HA derivatives was normalized to unmodified HA as a control.
[0200] Quantification of Azide Content by Radio-Thin Layer Chromatography
[0201] A modified dibenzocyclooctyne (DBCO) analog of the widely used bifunctional chelating agent, 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid (DOTA), was synthesized to enable Cu-free click cycloaddition and subsequent radiometric isotopic dilution to quantify the number of azide moieties on the dif HA backbone (Scheme 2). Briefly, DBCO-amine was reacted with DOTA-NHS (Macrocyclics, Dallas, Tex.) to prepare DOTA-DBCO. The product was purified by high performance liquid chromatography (Hitachi, Japan) and characterized by mass spectrometry. Next, a 5-fold molar excess of DOTA-DBCO was reacted with the azide-functionalized HA via click chemistry in a water/DMSO (1:1) mixture at 37 C. for 6 hours, and then continuously stirred for 2 days at room temperature. The reaction was purified by size-exclusion chromatography with a Zeba spin column (MWCO=7K) to remove unreacted DOTA-DBCO. DOTA-dif HA was then mixed with a carrier-added solution of .sup.64CUCl.sub.2 (Washington University School of Medicine, St. Louis, Mo.), as previously described (Hall, et al., 2012; Ghosh, et al., 2013; Ghosh, et al., 2015). Briefly, DOTA-dif HA was added to a solution containing a 10-fold molar excess of non-radioactive CuCl.sub.2 spiked with .sup.64Cu. After heating at 50 C. for 1 h, the reaction mixture was analysed by radio-thin-layer chromatography with Whatman chromatography paper (Sigma-Aldrich, St. Louis, Mo.) and a mobile phase of 0.1 M ammonium acetate/0.05 M EDTA.
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[0202] In Vitro Hydrogel Fabrication
[0203] Solutions of various ratios of dif HA and mHA were prepared. The total amount of HA in each solution was a constant 1.0% (w/v) in DDW. Hydrogels were formed via photopolymerization by adding 0.1% (w/v) Irgacure 2959 (Ciba Specialty Chemicals, Basel, Switzerland) to the solutions with exposure to 2.3 mJcm.sup.2 UVA lights for 5 mins.
[0204] Hydrogel Characterization
[0205] Hydrogel disks formed without cells were evaluated for swelling ratio, water content, and mechanical properties as previously described (Yang, et al., 2015). Briefly, the original mass of the hydrogel disks was measured immediately after fabrication and wet mass of the disks was measured after overnight incubation in neural differentiation media at 37 C., 5% CO.sub.2. The dry mass of the disks was measured after completely drying in a lyophilizer. The swelling ratio was calculated by normalization of the wet mass to the dry mass, while the water content was the ratio of the water mass to the wet mass (Cruise, et al., 1998; Canal and Peppas, 1989).
[0206] Young's modulus of the hydrogel disks were quantified using a TA.XTplus Texture Analyzer (Stable Micro Systems, Surrey, UK) with a spherical probe, which compressed the samples at a rate of 0.01 mms.sup.1 until 10% strain was reached. Young's modulus (E) was calculated using the following equations (Lin, et al., 2009; Edwin, et al., 2012):
a=R.sup.1/2.sup.1/2
=F/(a2)
E=3(12)/20
Force (F), depth of indentation () and strain () data were recorded to calculate the contact radius (a), indentation stress (), and Young's modulus (E). R is the radius of the spherical probe. is the Poisson's ratio (Khabarov and Selyanin, 2014) (0.5).
[0207] Hydrogel Degradation
[0208] HA disks (1 mm thickness, 9 mm diameter) were prepared in a manner similar to that described above. Samples (n3 per experiment group per time point) were degraded in 100 U hyaluronidase (Sigma Aldrich) per mL of PBS at 37 C. on an orbital shaker at 200 rpm. The amount of uronic acid released during degradation was measured using a previous established carbazole reaction technique from the supernatant until complete dissolution of the hydrogel (Burdick, et al., 2005). Briefly, 100 l of the degradation solution was added to a 0.025M sodium tetraborate.10H.sub.2O in sulfuric acid solution and heated to 100 C. for 10 min. After adding 100 l of 0.125% carbazole in absolute ethanol and heating to 100 C. for 15 min, the solution absorbance at 530 nm was measured. The amount of uronic acid was determined using solutions of known concentrations of the 75 kDa HA as a standard. The total amount of uronic acid by standard curve released at each time point is reported.
[0209] D3 Mouse Embryonic Stem Cells Culture in Three-Dimensional Hydrogel
[0210] mES (D3, passage 68) were encapsulated in the HA solutions at a density of 110.sup.6 cells/mL during photopolymerization. The cell-laden hydrogels were cultivated with 500 L of pluripotent proliferation media (Dulbecco's modified Eagle's media supplemented with 10% FBS, 0.1 mM -mercaptoethanol, 0.224 g/mL L-glutamine, 1.33 g/mL HEPES, and 1000 units/mL human recombinant Leukemia inhibitory factor) for 1 day, and then cultivated with 500 L of neural differentiation media (80% F-12, 20% neurobasal medium, 1% penicillin/streptomycin, 0.8N2, 0.2B27, 10 mM sodium pyruvate and 2 M retinoic acid) to induce neural differentiation for the duration of the experiments. Pluripotent proliferation media for cell growth testing was changed every day, and neural differentiation media for differentiation experiments was changed every other day. Cell-laden hydrogel disks were harvested at days 3 and 6. mES from the original population before neural differentiation cultured on 0.1% gelatin-coated tissue culture plastic in pluripotent proliferation media was used as a control group
[0211] Capacity for Proliferation and Neural Differentiation of mES in 3D HA Hydrogels In Vitro
[0212] To assess the cell proliferation in HA hydrogel disks, a MTS luminescence assay was used on day 0, 3, and 6 according to the manufacturer's protocol (Promega, Madison, Wis., USA). The doubling time of mES in hydrogels was calculated as previously described (Lim, et al., 2015). At designated time points, mES containing HA hydrogel disks were fixed with 4% paraformaldehyde for 30 mins, and the cell membranes then were permeabilized using PBS with 1 Tween20 for 1 hr. Nonspecific antibody binding was minimized with incubation in 5% donkey serum in phosphate buffered saline (PBS) for 1 hr. The hydrogel disks were then incubated in purified rabbit anti-mouse neuron-specific class III-tubulin antibody (Covance, Princeton, N.J., TUJ1, 1:500) in PBS at 4 C. overnight. Fluorescently conjugated donkey anti-rabbit IgG secondary antibody (Life Technologies, Carlsbad, Calif., 1:500) and nuclei stain draq5 (1:1000) were applied for 1 hr. All incubation steps were carried out on an orbital shaker agitated at 60 rpm with several PBS washes between each step. Images were taken using a confocal microscope (Leica, Surrey, Canada), and percentage of TUJ1 positive cells were quantified using a LAS AF Lite program (Leica, Surrey, Canada). A minimum of 400 cells per sample and 3 different samples for each group were used for quantification of TUJ1 positive cells.
[0213] RNA was extracted from the homogenized lysate of the cell-laden HA hydrogel using Trizol reagent (Life Technologies, Carlsbad, Calif.) according to the manufacturer's protocol. Following RNA extraction, 200 ng of isolated RNA from each sample was reverse transcribed into cDNA using amfiRivert Platinum One cDNA Synthesis Master Mix (GenDEPOT, Houston, Tex.) according to the manufacturer's protocol. For real-time polymerase chain reaction (PCR), 10 L of SYBR Green Master Mix (Applied Biosystems, Foster City, Calif.), 2 pM of each forward and reverse primer, and a cDNA template corresponding to 10 ng of total RNA was placed in 96-well plate. Every reaction was run in duplicate to assure technical reproducibility. The primer sequences of the candidate genes and GAPDH (internal control) are shown in Table 7. SYBR Green PCR conditions were 95 C. for 10 min, followed by 40 cycles of 95 C. for 10 sec, 58 C. for 50 sec, and 72 C. for 20 sec. The 2.sup.Ct method was used for analysis of the relative changes in gene expression.
TABLE-US-00007 TABLE7 Primersequencesforreal-timePCR Gene Sequences SEQIDNO GAPDH 5-TGTGTCCGTCGTGGATCTGA-3 6 5-CCTGCTTCACCACCTTCTTGA-3 7 PAX6 5-AAATCCGAGACAGATTACTTCGC-3 8 5-AAGTCCCGGGATACCAACCA-3 9 TUJ1 5-TGAGGCCTCCTCTCACAAGT-3 10 5-GTCGGGCCTGAATAGGTGTC-3 11 MAP2 5-CTCTCTACCTCCGCTTCCCT-3 12 5-GCAGAGCTAGAGCTTCTCCG-3 13
[0214] Implantation of HA Hydrogel in Rat Spinal Cord Injury
[0215] All animal care and surgical interventions were undertaken in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, Guide for the Care and Use of Laboratory Animals and with the approval of the Animal Welfare Committee at the University of Texas Health Science Center at Houston. Eighteen female Fisher 344 rats received 150 kdyne injuries (Cao, et al., 2005; Kar, et al., 2016; Royce Hynes, et al., 2007). Rats were anesthetized via intraperitoneal injection of Nembutal (50 mg/kg) and a dorsal laminectomy was performed at the ninth thoracic vertebra level (T9) to expose the spinal cord. The exposed vertebral column was stabilized by clamping the rostral T8 and caudal T10 vertebral bodies with forceps. Before placing the animal under the IH impactor and securing it for contusion injury, the forceps were carefully adjusted to level the spinal cord in the horizontal plane. After the injury, actual injured forces were recorded and there was less than 0.05% variation between these values. After surgery, animals were given 10 cc of sterile saline subcutaneously and 0.1 cc of gentamycin intramuscularly before being placed on an 87Z heating pad until full recovery from anesthesia. Postoperative care included the manual expression of bladders twice a day for 10 days after which the bladder function returned and injections of Gentamycin (0.1 cc) once a day for up to 1 week. Two weeks post injury the rats were anesthetized and the spinal cords re-exposed at T9. A single 10 L volume of either sterile saline or 1:2 dif HA:mHA was injected into the injured spinal cord at a rate of 0.5 l/min through a glass micropipette with outer diameter 50-70 um and the tip sharp-beveled to 30-50. One and four weeks post-implantation of the hydrogel, spinal cords were collected and fixed using 10% neutral-buffered formalin for 24 h and transferred to 30% sucrose solution for an additional 24 h. The spinal cord was placed in Tissue-Tek OCT, frozen in 80 C., cross-sectioned (20 um), and then washed, blocked in 5% BSA for 1 h at room temperature. Antibodies against EDI (Millipore, Billerica, Mass., USA), 1:200), GFAP (Immunostar, Hudson, Wis., USA, 1:100), and pan-axonal neurofilament (Biolegend, San Diego, Calif., USA, 1:200) were applied overnight at 4 C. The next day, sections were washed 3 times with PBS, and Alexa Fluor secondary antibodies were applied at a dilution of 1:2000 for 4 h at room temperature. Next, slides were washed 3 times with PBS and slides were mounted using a mounting solution containing 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher) to assess nuclear staining. In order to quantify the staining of markers at the injury site, a series of images for whole spinal cord were captured using 4 objective using an inverted fluorescence microscope (Nikon TE2000-E, Tokyo, Japan), and then merged by Adobe Photoshop. The lesion site was determined by staining for GFAP, and lesion border measured from a series of longitudinal slices taken every 200 m across the spinal cord. The lesion volume was then determined using the Cavalieri method, where the sum of the cross-sectional areas is multiplied by the distance between tissue sections to determine the volume (Cao, et al., 2005; Michel and Cruz-Orive, 1988; Fan, et al., 2012). A previously reported custom Matlab script determined the injury area and positively stained areas above a user defined threshold for ED1, GFAP, and pan-axonal neurofilament from slices taken across the spinal cords every 200 m (Wilems, et al., 2015; Wilems and Sakiyama-Elbert, 2015; McCreedy, et al., 2014). The exposure time and gain were not changed during imaging for individual stains.
[0216] To confirm HA hydrogel persistence in vivo, eosin-Y was conjugated to both difHA and mHA through the following procedure. 1% dif HA and 1% mHA solution in 0.1M sodium bicarbonate buffer (pH 9) were prepared, separately. To each solution, 250 L of eosin Y (12 mg/mL) in anhydrous dimethyl sulfoxide was added, and reacted overnight at 4 C. with continuously stirring. The reaction was stopped with 263 L of 1M ammonium chloride solutions and then incubated for 2 hrs at 4 C. The solutions were dialyzed in ultrapure water for 2 days using 500 kDa molecular weight cutoff membrane, and then lyophilized. Eosin-Y labelled 1:2 dif HA:mHA hydrogel precursor solution was injected into spinal cord injuries and then sectioned and stained with additional antibodies after animal sacrifice as described above.
[0217] Statistical Analysis Statistical data is presented as meanstandard error of the mean. One- or Two-way ANOVA followed by Tukey's or Bonferroni's multiple comparisons test, respectively, were performed using GraphPad Prism version 5.0 for Mac (GraphPad Software, La Jolla, Calif.). Significance was defined as p<0.05 in all tests.
[0218] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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