Protease Triggered Release Of Molecules From Hydrogels

Abstract

The invention concerns compositions comprising: (i) biocompatible hydrogel, comprising a plurality of cross-linkers connecting backbone components of the hydrogel; wherein the hydrogel is cross-linked utilizing a cross-linker comprising a peptide sequence that is capable of being degraded by a matrix metalloproteinase; said inhibitor being effective as a treatment of a condition related to the presence of the matrix metalloproteinase; wherein the hydrogel encapsulates and retains the inhibitor within the intact hydrogel through non-covalent interactions; wherein the hydrogel comprises one or more of hyaluronic acid, sulfated hyaluronic acid, sulfonated hyaluronic acid, dextran, dextran sulfate, sulfonated dextran, chondroitin sulfate, heparin and heparan sulfate; (ii) pentosan polysulfate (PPS) and (iii) optionally, a therapeutic agent comprising an inhibitor of matrix metalloproteinase.

Claims

1. A composition comprising: (i) biocompatible hydrogel, comprising a plurality of cross-linkers connecting backbone components of said hydrogel; wherein said hydrogel is cross-linked utilizing a cross-linker comprising a peptide sequence that is capable of being degraded by a matrix metalloproteinase; said inhibitor being effective as a treatment of a condition related to the presence of said matrix metalloproteinase; wherein said hydrogel encapsulates and retains the inhibitor within the intact hydrogel through non-covalent interactions; wherein said hydrogel comprises one or more of hyaluronic acid, sulfated hyaluronic acid, sulfonated hyaluronic acid, dextran, dextran sulfate, sulfonated dextran, chondroitin sulfate, heparin and heparan sulfate; (ii) pentosan polysulfate (PPS) and (iii) optionally, a therapeutic agent comprising an inhibitor of matrix metalloproteinase.

2. The composition of claim 1, comprising a therapeutic agent comprising an inhibitor of matrix metalloproteinase.

3. The composition of claim 1, wherein said peptide sequence is incorporated into the cross-linker via reaction of thiol groups of cysteines with acrylates, methacrylates or maleimide groups.

4. The composition of claim 1, where said inhibitor of matrix metalloproteinase is TIMP-3.

5. The composition of claim 1, where said inhibitor of matrix metalloproteinase is a hydroxymate based compound such as ilomastat

6. The composition of claim 1, where said inhibitor of matrix metalloproteinase is a tetracycline based compound such as doxycycline or a modified doxycycline

7. The composition of claim 1, wherein said matrix metalloproteinase is MMP-13 or MMP-2

8. The composition of claim 1, wherein said matrix metalloproteinase is MMP-1, MMP-8, or MMP-9

9. The composition of claim 1, wherein said cross-linker comprises said peptide sequence and at least one of hyaluronic acid or polysaccharides.

10. The composition of claim 1, wherein said hydrogels comprise at least one of hyaluronic acid or other polysaccharide.

11. The composition of claim 1, wherein the composition is such that encapsulated inhibitors are released from the hydrogel and into the extracellular matrix of tissue in the presence of pathological levels of matrix metalloproteinase.

12. The composition of claim 1, wherein said peptide comprises a sequence GPQGIAGQ (SEQ ID NO: 4), GPQGIWGQ (SEQ ID NO: 3), GCRDGPQGIWGQDRCG (SEQ ID NO: 5), GGPQGIWGQGCG (SEQ ID NO: 6), or GCGQGWIGQPGGG (SEQ ID NO: 7).

13. A process for treating myocardial infarction, osteoarthritis, meniscal repair, ligament repair or treating aortic aneurisms compromising administering to a patient in need of such treatment a composition of claim 1.

14. The process of claim 13, wherein said patient is a mammal.

15. The process of claim 13, wherein said patient is a human.

16. The process of claim 13, wherein said peptide sequence is incorporated into the cross-linker via reaction of thiol groups of cysteins with acrylates or methacrylates.

17. The process of claim 13, wherein said hydrogels comprise at least one of hyaluronic acid and polysaccharides.

18. A process for treating myocardial infarction, osteoarthritis, meniscal repair, ligament repair or treating aortic aneurisms compromising administering to a patient in need of such treatment a composition of claim 2.

19. The process of claim 18, wherein said patient is a mammal.

20. The process of claim 18, wherein said patient is a human.

21. The process of claim 18, wherein said peptide sequence is incorporated into the cross-linker via reaction of thiol groups of cysteins with acrylates or methacrylates.

22. The process of claim 18, wherein said hydrogels comprise at least one of hyaluronic acid and polysaccharides.

23. A process for delivery of an inhibitor of matrix metalloproteinase comprising: administering a hydrogel of claim 1 to a patient; allowing said hydrogel to contact matrix metalloproteinase; said contact resulting in the release of at least a portion of said inhibitor of matrix metalloproteinase.

24. The process of claim 23, wherein the delivery is accomplished through a syringe or catheter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 illustrates synthesis of HA-aldehyde (Synthesis 1) for use in forming a hydrogel.

[0023] FIG. 2 presents analytical analysis of the HA-aldehyde shown in FIG. 1.

[0024] FIG. 3 illustrates synthesis of HA-peptide hydrazide for use in forming the hydrogel. The product has a hyaluronic acid backbone modified with either an aldehyde or a peptide with a hydrazide end. FIG. 3 discloses SEQ ID NO: 8.

[0025] FIG. 4 illustrates synthesis of HA-hydrazide (Synthesis 2). The example depicted in the figure has 33% modification. FIG. 4 discloses SEQ ID NO: 6.

[0026] FIG. 5 illustrates formation of a hydrogel by mixing precursors containing a hyaluronic acid backbone modified with either an aldehyde or a peptide with a hydrazide end. By using a peptide that is MMP sensitive, the gel will form an enzyme sensitive hydrogel.

[0027] FIG. 6 illustrates gel formation through mixing of Synthesis 1 and Synthesis 2: hydrazone formation via hydrazide-aldehyde reaction. Gelation/properties can be controlled by HA modification and ratio of HA-aldehyde to HA-hydrazide.

[0028] FIG. 7 illustrates a MMP-specific gel with a peptide crosslinker that responds to MMP-1, MMP-2. FIG. 7 discloses SEQ ID NO: 7.

[0029] FIG. 8 illustrates mass loss with the gel of FIG. 7 as a function of time upon exposure to MMP (at day 1 or day 3).

[0030] FIG. 9 illustrates the crosslinking of an acrylated hyaluronic acid with a peptide (containing thiols on each end). FIG. 9 discloses SEQ ID NOS 9 and 5, respectively, in order of appearance.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0031] The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms a, an, and the include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term plurality, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about, it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0032] It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, reference to values stated in ranges include each and every value within that range.

[0033] Hydrogels containing degradable cross-linkers can be utilized in the delivery of therapeutic and/or diagnostic agents to a specified site within a patient. In the present invention, the cross-linkers comprise a peptide sequence that is degradable by particular enzymes. When one utilizes a hydrogel containing a therapeutic agent to a condition that is associated with the presence of a particular enzyme, one can selectively deliver the agent to a specified target within the patient because the enzyme will cause degradation of the cross-links which will result in release of the agent.

[0034] Any peptide sequence containing linking group that is capable of being degraded by the desired enzyme can be utilized. In some embodiments, the peptide is at least two units in length. While the peptide has no maximum length so long as it is degradable by the desired enzyme, in certain embodiments, the peptide is up to 20, 30, 50, 100, 250 500 or 1000 units in length. Some peptides are at least 5 or 10 units in length. Each of these upper and lower limits are intended to be combinable to reflect some preferred peptide lengths. Examples of suitable peptides include those containing the QGIWGQ (Seq. ID No. 1) or QGIAGQ (Seq. ID No. 2) sequence from collagen including GPQGIWGQ (Seq. ID No. 3), GPQGIAGQ (Seq. ID No. 4), GCRDGPQGIWGQDRCG (Seq. ID No. 5), GGPQGIWGQGCG (Seq. ID No. 6), GCGQGWIGQPGGG (Seq. ID No. 7) and collagen or gelatin itself.

[0035] Polysulfated polysaccharides can be utilized as binding groups for matrix metalloproteinase inhibitors encapsulated in the hydrogel, but also as active agents to inhibit matrix metalloproteinases in the target tissue. For example, pentosan polysulfate (PPS) has been shown to inhibit MMP activity by stimulating production of TIMPs, preventing endocytosis of TIMPs, and enhancing the activity of TIMPs [M Takizawa et al., Calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 (aggrecanase-1) in osteoarthritic chondrocytes. FEBS Letters 582 (2008) 2945-2949; M Takizawa et al., Production of Tissue Inhibitor of Metalloproteinases 3 is Selectively Enhanced by Calcium Pentosan Polysulfate in Human Rheumatoid Synovial Fibroblasts. Arthritis & Rheumatism, Vol. 43, No. 4, April 2000, pp 812-820 and L Troeberg et al., Calcium pentosan polysulfate is a multifaceted exosite inhibitor of aggrecanases. FASEB J. 2008 October; 22(10): 3515-35241 As such, PPS can be used with or without an additional inhibitor of matrix metalloproteinase.

[0036] As used herein, the term pentosan polysulfate or PPS is a semisynthetic drug that can be manufactured from Beechwood hemicellulose by sulfate esterification of the xylopyranose groups. Typically, PPS has a molecular weight in the range of from about 1,500 to about 6,000 daltons (about 4,000 to about 6,000 daltons) in some embodiments. PPS is also known as xylan hydrogen sulfate and xylan polysulfate. Other polysulfated polysaccharides are expected to have similar effects as MMP inhibitors including but not limited to naturally occurring molecules such as low molecular weight heparin, high molecular weight heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, fucoidan-1, fucoidan-2, including salts thereof, as well as synthetically sulfated polysaccharides including but not limited to dextran sulfate, chitosan polysulfate, sulfated beta-cyclodextrin, sulfated gamma-cyclodextrin, or any polysulfate polysaccharide produced through sulfate esterification, including salts thereof.

[0037] One application concerns treatment of myocardial infarction (MI). Left ventricular (LV) remodeling after myocardial infarction, for example, is a common structural event and contributes to increased morbidity and mortality. Matrix metalloprotease (MMPs) have been demonstrated to contribute to LV remodeling after MI by contributing to the breakdown of interstinal matrix proteins like collagen and elastin. We have demonstrated that upregulation of MMPs and down regulation of their naturally occurring inhibitors, tissue inhibitors of matrix metalloprotease (TIMPs), occur in a type, region and temporal specific manner within the myocardium after MI. The use of a broad spectrum, systemically delivered MMP inhibitor is associated with significant adverse reactions. The ability to inhibit specific MMPs in specific regions of the heart at specific times after MI will lead to improved outcomes after MI for a large number of patients.

[0038] Regional delivery of MMP inhibiting therapy that would be active only where MMP's are active can also be utilized in treatments of other conditions. Arthritis is an example of another disease where this approach could be useful. Any disease state where localized release of therapeutics where certain MMPs exist may be treated by the hydrogel systems of the invention.

[0039] One concept that is disclosed herein is the use of synthetic hydrogels that incorporate peptide sequences that degrade in the presence of certain enzyme/proteases. The degradation or breaking of these crosslinks in the hydrogel alters the crosslinking density, which in turn alters the material properties (i.e. mechanics), which alters the diffusion of molecules through the hydrogel and hence delivery into the affected tissue. One area where this approach would be important is in disease processes where there are region specific changes in the levels of MMPs. An important example of such a pathologic phenomenon is post-MI LV remodeling. There may be target molecules (e.g., MMP inhibitors) that can alter MMP activity and treat or prevent this disease. With a material such as this, the release of these molecules will be locally dependent on the level of protease activity at those sites. For example MMP-13 is known to be up-regulated in the pen-infarct region in the first 8 weeks after MI and TIMP-3 (an inhibitor of MMP-13) down regulated during this time period; a hydrogel that is degraded only in the presence of MMP-13 and released TIMP-3 locally as it was degraded would likely have a beneficial effect on LV remodeling.

[0040] Hydrogels are well known in the art and are generally formed by the reaction of a macromer having a biocompatible backbone with a cross-linking agent. Any suitable hydrogel may be utilized. Types of materials that could be used for this purpose include crosslinked synthetic hydrogels that are based on molecules like hyaluronic acid or polyethylene glycols. Suitable hydrogels also include those constructed using polyesters, polyurethanes, polysaccharides, proteins, and combinations thereof. Polyesters, poly(ethylene oxide) (PEO), proteins such as collagen or gelatin and the like are also suitable polymeric materials can also be used as a polymeric component of the hydrogel. General synthetic methods for making hydrogels can be found, for example in Burdick, et al, Soft Matter, 2009, 5, 1601-1606.

[0041] Some hydrogels comprises one or more of hyaluronic acid, sulfated hyaluronic acid, sulfonated hyaluronic acid, dextran, dextran sulfate, sulfonated dextran, chondroitin sulfate, heparin and heparan sulfate.

[0042] A partial listing of polysaccharides that are useful in the claimed invention includes hyaluronic acid, amylase, amylopectin, glycogen, cellulose, heparin, agarose, alginate, and the like. In some embodiments, hyaluronic acid or any combination thereof is particularly suitable for use in the instant invention. In some embodimentssuch as those embodiments that include hyaluronic acidthe biocompatible backbone unit is capable of enzymatic degradation.

[0043] In other embodiments, the biocompatible backbone is capable of hydrolytic degradation. Those embodiments are considered useful where a user may desire a degradable macromer whose degradation is dependent primarily on exposure to aqueous medium without the additional complication of a macromer that is also susceptible to enzymatic degradation. In some embodiments, the macromer is capable of both enzymatic and hydrolytic degradation.

[0044] The macromers may include a range of polymerizing moietes, such as acrylates, methacrylates, and the like. In some embodiments, the polymerizing moiety includes a carbon-carbon double or triple bond. The moiety is suitably polymerized by photopolymerization, by free radical-initiation, or by other methods of polymerization known to those of skill in the art.

[0045] The peptide moieties can be incorporated into the cross-linkers by reaction of active hydrogen atoms. In some embodiments, the active hydrogen atoms can be part of hydroxy, thiol, or amine groups (including hydrazine). In some embodiments, the peptide can be incorporated as crosslinks through the addition reaction of thiols in cysteines in the peptides with acrylate or methacrylates, vinyl sulfones, or maleimides on these molecules.

[0046] Any inhibitor of MMPs can be utilized with the present invention. In some embodiments, hydroxymate based inhibitors (ilomastat, batimastat, or marimastat for example) or non-hydroxymate based inhibitor (doxycycline or modified doxycyclines for example).

[0047] In some embodiments, the therapeutic molecule may be directly encapsulated during the gelation process by mixing the molecule with the pre-cursor solutions.

[0048] The compositions of the instant invention may be administered by methods well known to those skilled in the art. Such methods include local or systemic administration. In some embodiments, administration is topical. Such methods include ophthalmic administration and delivery to mucous membranes (including vaginal and rectal delivery), pulmonary (including inhalation of powders or aerosols; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (including intrathecal or intraventricular, administration); or into the joint (including knee, hip or shoulder); or into the spine.

[0049] Pharmaceutical compositions and formulations for topical administration include but are not limited to ointments, lotions, creams, transdermal patches, gels, drops, suppositories, sprays, liquids and powders. Utilization of conventional pharmaceutical carriers, oily bases, aqueous, powder, thickeners and the like may be used in the formulations.

[0050] The pharmaceutical compositions may also be administered in tablets, capsules, gel capsules, and the like.

[0051] Penetration enhancers may also be used in the instant pharmaceutical compositions. Such enhancers include surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Such enhancers are generally described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference.

[0052] In addition to treatment of diseases or other conditions, compositions disclosed herein may also be useful prophylactically.

[0053] In some preferred embodiments, the hydrogels can be delivered locally either via implantation or as an injection procedure, potentially through syringes or catheters.

[0054] Due to the variety of therapeutic agents that can be utilized with the cross-linked hydrogel systems, a wide variety of diseases and disorders can be treated with the technology described herein. Post MI remodeling is one application of the proposed therapeutic approach. In addition, the disclosed concept could be applied in any ailment in which MMPs contribute to the pathophysiology of the disease. Treatment methods comprise administration of the instant compositions by any appropriate method to a patient in need of such treatment. In some embodiments, the patent is a mammal. In certain preferred embodiments, the patient is a human.

[0055] The invention is illustrated by the following examples which are intended to be illustrative and not limiting in scope.

EXAMPLES

[0056] Unless noted otherwise, all percentages are by weight.

Example 1: Synthesis of HA-Aldehyde

[0057] Hyaluronic acid (HA) is contacted with NaIO.sub.4 to produce the aldehyde derivative (Synthesis 1) depicted in FIG. 1. FIG. 2 presents analytical data for the HA-aldehyde.

Example 2: Synthesis of HA-Peptide Hydrazide

[0058] Hyaluronic acid (HA) is contacted with EDC/HoBT at ph 6.8 for 12-14 hours to produce the intermediate depicted in FIG. 3. The intermediate is then contacted with trifluoroacetic acid (TFA, 49%), trisopropyl silane (TIS, 1%), and water (50%) for 4 hours to produce the hydrazide depicted in FIG. 3. EDC is ethyl-(N,N-dimethylamino) propylcarbodiimide hydrochloride (EDC). HoBT is 1-hydroxybenzotriazole. One peptide that could be used is GCRDGPQGIWGQDRCG (Seq. ID No. 5), which cleaves in the presence of MMP-2, but somewhat nonspecifically. In the example, the cross-linker utilized was of the formula (SEQ ID NOS 8 and 8 disclosed, respectively, in order of appearance):

##STR00001##

the peptide being of the sequence GDGPQGIWGQDG.

Example 3: HA-Hydrazide Synthesis

[0059] HA-hydrazide synthesis (Synthesis 2) having 33% modification was performed as depicted in FIG. 4. The peptide utilized was of the formula (Seq ID No. 6):

##STR00002##

which is represented by the shorthand

##STR00003##

[0060] Analytical analysis of the product is also presented in FIG. 4.

Example 4: Hydrogel Formation

[0061] Hydrogel formed by mixing the aldehyde of Example 1 with the hydrazide of Example 2 to form the hydrogel depicted in FIG. 5.

Example 5: Gel Formation Through Mixing of Synthesis 1 and Synthesis 2

[0062] Gel was formed through a mixing of the products of synthesis 1 and synthesis 2 as presented in FIG. 6. Gelation/properties can be controlled by HA modification and ratio of HA-aldehyde to HA-hydrazide. Differences in time are illustrated by the plots presented in FIG. 6.

Example 6: MMP-Specific Gel Synthesis

[0063] FIG. 7 shows a MMP-specific gel with a peptide crosslinker that responds to MMP-1, MMP-2. In this example, the peptide has the sequence GCGQGWIGQPGGG (Seq. ID No. 7). Response to MMP of this gel is illustrated in FIG. 8.

Example 7: Crosslinking of an Acrylated Hyaluronic Acid

[0064] Schematic of the crosslinking of an acrylated hyaluronic acid with a peptide (containing thiols on each end) if depicted in FIG. 9.

Example 8: Triggered Release of Ilomastat

[0065] Ilomastat was purchased from Sigma Aldrich. Ilomastat is also known as galardin or GM6001 and is of the following formula.

##STR00004##

[0066] Ilomastat was ground into a fine powder of uniform microparticles. These microparticles were suspended in the dissolved HA-aldehyde and HA-peptide-hydrazide solutions at 10 g per 100 L of polymer solution. At this concentration ilomastat remains a solid particle within the polymer solution. HA-aldehyde and HA-peptide-hydrazide polymers were mixed 1:1 aldehdye:hydrazide to induce crosslinking into a solid gel. The gels were incubated in phosphate buffered saline at 37 C. After 14 days less than 10% of the ilomastat was released from the gels due to hydrophobic interactions of the ilomastat in the microparticles. The gels were then exposed to collagenase 200 U/mL and the gels degraded. Once the gels were degraded, ilomastat was then solubilized in the larger volume of the buffer as evidenced by HPLC.

Example 9: Administration of Compositions

[0067] Compositions described herein are administered to a patient for treatment of myocardial infarction, osteoarthritis, meniscal repair, ligament repair, or aortic aneurisms

Example 10: Administration of PPS Compositions

[0068] An MMP degradable peptide is contacted with EDC/HoBT in the presence of excess adipic acid dihydrazide at pH 5 to produce a positively charged peptide-hydrazide. PPS is mixed with peptide-hydrazide to form an electrostatic complex. The peptide/PPS is then contacted with HA-aldehyde to produce a crosslinked hydrogel with PPS at a final concentration of 1 wt %. A composition comprising a hydrogel described herein and PPS is administered to a patient for treatment of myocardial infarction, osteoarthritis, meniscal repair, ligament repair, or aortic aneurisms.

Example 11: Administration of PPS and Inhibitor of Matrix Metalloproteinase Compositions

[0069] Lyophilized recombinant TIMP-3 is resuspended with peptide/PPS solution. The peptide/PPS/TIMP-3 is then contacted with HA-aldehyde to produce a crosslinked hydrogel containing PPS and TIMP-3 at a final concentration of 1 wt % and 0.1% respectively. A composition comprising a hydrogel described herein, PPS and TIMP-3 is administered to a patient for treatment of myocardial infarction, osteoarthritis, meniscal repair, ligament repair, or aortic aneurisms

Example 12: Administration of PPS and Inhibitor of Matrix Metalloproteinase Compositions

[0070] A doxycycline hyclate solution at 5 wt % is contacted with peptide/PPS solution. The peptide/PPS/doxycycline is then contacted with HA-aldehyde to produce a crosslinked hydrogel containing PPS and doxycycline each at a final concentration of 1 wt %. A composition comprising a hydrogel described herein, PPS and doxycycline is administered to a patient for treatment of myocardial infarction, osteoarthritis, meniscal repair, ligament repair, or aortic aneurisms