Intradiscal treatment of cartilage endplates to improve solute transport and disc nutrition

Abstract

Compositions and methods for treating degenerative disc disease in a subject in need thereof are provided. Specifically, the methods include locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to a cartilage endplate to increase the permeability of the cartilage endplate and improve disc nutrient transport.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of Provisional Patent Application No. 63 / 433,641, filed December 19, 2022, which is incorporated herein by reference in its entirety.

[0002] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers P30 AR066262, R01 AR070198, and R01 AR063705 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Low back pain is a leading cause of disability and a major public health problem worldwide (Fernandez-Moure et al., 2018; Haralson III & Zuckerman, 2009; Matta et al., 2017; Vassilaki & Hurwitz DC, 2014). In certain subgroups of patients with low back pain, degenerative disc disease can cause pain (Chou et al., 2011). Current medical interventions for degenerative disc disease are surgical in nature and often fail, prompting the development of non-surgical alternatives. Non-surgical approaches to regenerate the disc and reduce pain include delivering genes (Woods BI 2011), growth factors (Bae & Masuda 2011, Derek G Ju 2022), cells (Kregar Velikonja N 2014, Derek G Ju 2022), or other small molecules (Gawri et al. 2013, Wang Z 2013) to promote cell proliferation, stimulate anabolic activity, and reduce catabolism and inflammation. Importantly, all of these approaches require abundant nutrient supplies to sustain higher cell numbers or metabolic rates (Fernandez-Moure et al. 2018, Horner & Urban 2001). However, human disc degeneration is typically accompanied by limited nutrient supply (Fields et al., 2018, Huang et al., 2014, Jill P. G. Urban 2004), which may limit the clinical utility and effectiveness of these therapies (Huang et al., 2014) (Yong-Can Huang 2014, Zhu Q 2016, Binch 2021, Derek G. Ju 2022). However, there are no existing therapies to improve disc nutrient supply. Therefore, developing therapeutic strategies to improve disc nutrition may help slow or reverse degeneration and enhance the regenerative potential of biologic therapies that increase nutrient demand within the disc. Summary of the Invention

[0004] Compositions and methods for treating degenerative disc disease in a subject in need thereof are provided. Specifically, the methods include locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to a cartilage endplate to increase the permeability of the cartilage endplate and improve disc nutrient transport.

[0005] In one aspect, a method is provided for treating degenerative intervertebral disc disease in a subject in need thereof, the method comprising locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to a cartilage endplate (CEP).

[0006] In certain embodiments, the treatment increases the permeability of CEP and intervertebral disc nutrient transport.

[0007] In certain embodiments, one or more of the matrix-modifying enzymes have proteolytic activity against collagen, proteoglycan, or a combination thereof. In some embodiments, the one or more matrix-modifying enzymes include collagenase, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. In some embodiments, the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof. In some embodiments, the MMP8 is full-length MMP8. In some embodiments, the treatment comprises administering more than one matrix-modifying enzyme.

[0008] In certain embodiments, one or more matrix-modifying enzymes are encapsulated in the hydrogel. In some embodiments, the one or more matrix-modifying enzymes encapsulated in the hydrogel include collagenase. In some embodiments, the collagenase is collagenase P, MMP8, or a combination thereof. In some embodiments, the MMP8 is full-length MMP8. In some embodiments, the hydrogel maintains delivery of the one or more matrix-modifying enzymes for at least 2 days. In some embodiments, the hydrogel includes alginate. In some embodiments, the concentration of alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%). In some embodiments, the alginate is at least partially oxidized. In some embodiments, about 4% to about 10% of the alginate is oxidized.

[0009] In certain embodiments, one or more matrix-modifying enzymes are encapsulated in or conjugated to the surface of the liposome. In some embodiments, the one or more matrix-modifying enzymes encapsulated in or conjugated to the surface of the liposome include collagenase. In some embodiments, the collagenase is collagenase P, MMP8, or a combination thereof. In some embodiments, the MMP8 is full-length MMP8. In some embodiments, the liposome comprises a phospholipid. Exemplary phospholipids include, but are not limited to, phosphatidylcholine and phosphatidylethanolamine. In some embodiments, the liposome further comprises cholesterol. In some embodiments, the liposome further comprises polyethylene glycol.

[0010] In certain embodiments, the one or more matrix-modifying enzymes are administered in a volume small enough so as not to substantially increase intradiscal pressure, hi some embodiments, the one or more matrix-modifying enzymes are administered in a volume of 50 μL or less.

[0011] In certain embodiments, one or more matrix-modifying enzymes are administered within or adjacent to the CEP.

[0012] In certain embodiments, the one or more matrix-modifying enzymes are administered with an epidural needle. For example, the epidural needle may have a curved end such that the matrix-modifying enzyme exits the epidural needle laterally at a 45-degree angle.

[0013] In certain embodiments, the method further includes using medical imaging to guide the local administration of one or more matrix-modifying enzymes to CEPs or to select which intervertebral discs may benefit from treatment with one or more matrix-modifying enzymes. For example, one or more matrix-modifying enzymes may be locally administered to target CEPs using fluoroscopic guidance, computed tomography (CT) guidance, combined CT-fluoroscopic guidance, or ultrasound guidance.

[0014] In certain embodiments, the one or more matrix-modifying enzymes are administered locally to the CEP without exposing the nucleus pulposus to the one or more matrix-modifying enzymes.

[0015] In certain embodiments, the method further comprises administering a growth factor.

[0016] In certain embodiments, the method further comprises administering an analgesic or anti-inflammatory agent.

[0017] In certain embodiments, multiple cycles of treatment are administered to the subject.

[0018] In another aspect, a composition comprising one or more matrix-modifying enzymes is provided for use in a method for treating degenerative disc disease, the method comprising locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to a cartilage endplate (CEP).

[0019] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0020] In certain embodiments, one or more of the matrix-modifying enzymes in the composition have proteolytic activity against collagen, proteoglycan, or a combination thereof. In some embodiments, the one or more matrix-modifying enzymes include collagenase, and the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. In some embodiments, the collagenase is collagenase P, MMP8, or a combination thereof. In some embodiments, the MMP8 is full-length MMP8.

[0021] In certain embodiments, one or more matrix-modifying enzymes in the composition are encapsulated in a hydrogel. In some embodiments, the hydrogel comprises alginate. In some embodiments, the concentration of alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%). In some embodiments, the alginate is at least partially oxidized. In some embodiments, about 4% to about 10% of the alginate is oxidized.

[0022] In certain embodiments, one or more matrix-modifying enzymes in the composition are encapsulated in a liposome or conjugated to the surface of the liposome. In some embodiments, the liposome comprises a phospholipid. Exemplary phospholipids include, but are not limited to, phosphatidylcholine and phosphatidylethanolamine. In some embodiments, the liposome further comprises cholesterol. In some embodiments, the liposome further comprises polyethylene glycol.

[0023] In certain embodiments, the composition further comprises a growth factor.

[0024] In certain embodiments, the composition further comprises an analgesic or anti-inflammatory agent.

[0025] In another aspect, a method for increasing intervertebral disc solute transport in a subject in need thereof is provided, the method comprising locally administering an effective amount of one or more matrix-modifying enzymes to the cartilage endplate (CEP).

[0026] In certain embodiments, prior to locally administering one or more matrix-modifying enzymes to the CEP, the method further includes detecting whether intervertebral disc solute transport is inhibited in the CEP to determine whether the subject will benefit from the treatment. [Brief explanation of the drawings]

[0027] [Figure 1] Intradiscal delivery of collagenolytic enzyme treatment via a 17G Tuohy needle into the superior CEP of (Figure 1A) the bovine coccyx and (Figure 1B) the human lumbar intervertebral disc under fluoroscopic guidance. [Figure 2] Experimental setup (Figure 2A) and compression testing protocol (Figure 2B) of an intact whole intervertebral disc while immersed in fluorescein tracer solution. [Figure 3] (Figure 3A) Before compression testing, the entire intervertebral disc was isolated from the surrounding soft tissue, and the bony endplates were removed with a deburring tool. (Figure 3B) After compression testing, the discs were flash-frozen in liquid nitrogen, and two 5 mm-wide midsagittal slabs were removed for further biochemical and fluorescein measurements. The circles indicate the locations of the CEP punches taken for biochemical measurements. (Figure 3C) Midsagittal slab showing NP tissue sample locations (three 2 mm punches per inferior-superior position) for treated and untreated CEPs. (Figure 3D) Two 4 mm diameter punches were taken from the central region of each CEP in the midsagittal slab (Figure 3B). [Figure 4](Figure 4A) The mean fluorescein concentration (mean ± standard deviation, n = 3 discs / group) was significantly higher at position A in bovine NPs proximal to the superior CEP treated with collagenase (ap < 0.0001 vs. control buffer, bp < 0.0001 vs. sham, p > 0.10 for all other comparisons). (Figure 4B) The mean fluorescein concentration (mean ± standard deviation, n = 5 discs / group) was significantly higher at position A in human NPs proximal to the superior CEP treated with collagenase (ap < 0.0001). [Figure 5] (Figure 5A) In bovine intervertebral discs treated with collagenase, the sGAG content in treated (upper) CEPs was significantly lower than that in untreated (lower) CEPs (p=0.03, lower vs. upper paired t-test; mean ± standard deviation; n=3 discs / group). (Figure 5B) In human intervertebral discs treated with collagenase, the sGAG content in treated (upper) CEPs was significantly lower than that in untreated (lower) CEPs (p<0.01, lower vs. upper paired t-test; mean ± standard deviation; n=5 discs / group). [Figure 6] (Figure 6A) In bovine intervertebral discs treated with collagenase, the collagen content in the treated (upper) CEP was similar to that in the untreated (lower) CEP (p=0.21, paired t-test; mean ± standard deviation; n=3 discs / group). (Figure 6B) In human intervertebral discs treated with collagenase, the collagen content in the treated (upper) CEP was similar to that in the untreated (lower) CEP (p=0.07, paired t-test; mean ± standard deviation; n=5 discs / group). [Figure 7] Whole disc compressive creep strain (top) and elastic modulus (bottom) were similar in (Figure 7A) bovine and (Figure 7B) human discs with collagenase-treated CEPs versus control CEPs. p values ​​are from one-way ANOVA. [Figure 8]Scatter plots showing the relationship between sGAG content in human CEPs and fluorescein concentration in adjacent NP tissue. For collagenase-treated CEPs, each marker represents the average sGAG content in one of four CEP biopsies taken from the central region of the superior (treated) CEP and the average fluorescein concentration in site-matched NP tissue at location A. Error bars indicate the standard error for five intervertebral discs. For control CEPs, each marker represents the average sGAG content in one of four CEP biopsies taken from the central region of the superior and inferior untreated CEPs and the average fluorescein concentration in site-matched NP tissue. [Figure 9] Cumulative release (%) of 40 kDa dextran from alginate gels with 10%, 5%, and 0% oxidation over 24 h. Different alginate gels mixed with fluorescent dextran (total volume 50 μL) were placed in opaque Eppendorf tubes containing 2 mL of ddH2O, and aliquots were assayed for fluorescence at 0.2, 1, 2, 4, 20, and 24 h using a microplate reader (494 nm / 524 nm ex / em). [Figure 10] CEPs from bovine intervertebral discs were superficially treated with collagenase or control buffer (n = 3 discs / group). After treatment, the discs were compressed between porous platens while immersed in a fluorescein tracer solution (376 Da, 0.1 mg / mL). At the end of the compression test, the discs were flash-frozen in liquid nitrogen, coronally slabbed, and assayed for fluorescein concentration. (Figure 10A) Fluorescein concentrations were 3.3-fold higher in the NPs proximal to collagenase-treated CEPs. (Figure 10B) Collagenase-treated CEPs showed greater sGAG release during the 18-hour treatment period than buffer-treated CEPs (p = 0.01), consistent with the higher fluorescein levels at location A. (Figure 10C) Whole-disc creep behavior was similar in discs with collagenase- and buffer-treated CEPs. [Figure 11] Collagen content in CEP after treatment. n = 3-4 samples / group, mean ± standard error. [Figure 12] C = C downstream / C upstream concentration ratio from a pilot study (0.6–1.0 MPa at 0.5 Hz for 80 min) of CEP treated with full-length MMP-8 or buffer. [Figure 13] Effect of MMP-8 proteolysis on cytokine production by CEP cells after 72 hours (fold difference relative to "basal" control). Mean ± standard error for 5 patients. Release from proteolysis by full-length MMP-8 (0.2 U / mL [200 ng / mL], 2.0 U / mL [2000 ng / mL]). ^p<0.05 vs. basal. [Figure 14] (Figure 14A) Cell viability in NP and CEP tissue from bovine intervertebral discs with treated CEPs (mean ± standard deviation of one disc per group, in five projections per disc). "Day 0" indicates the mean ± standard deviation of five projections per disc for two discs. (Figure 14B) Fluorescein concentration (mean ± standard error of 12 samples, six per position for each disc × 2 discs / group) was significantly higher at position A in NPs close to MMP-8-treated CEPs (p<0.0001). Positions A–D of NP tissue correspond to Figure 12C. DETAILED DESCRIPTION OF THE INVENTION

[0028] Compositions and methods for treating degenerative disc disease in a subject in need thereof are provided. Specifically, the methods include locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to a cartilage endplate to increase the permeability of the cartilage endplate and improve disc nutrient transport.

[0029] Before the methods, devices, and compositions of the present disclosure are described, it is to be understood that this invention is not limited to the particular methods, devices, or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0030] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each of the ranges in which the smaller ranges include either, neither, or both limits is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publication is cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0032] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any described method can be carried out in the order of events described or in any other order which is logically possible.

[0033] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an intervertebral disc" includes a plurality of such discs, a reference to "a drug" includes a reference to one or more drugs and equivalents thereof (e.g., therapeutic agents, drugs, pharmaceuticals) known to those skilled in the art, and so forth.

[0034] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0035] definition The term "about" is meant to encompass a deviation of plus or minus 5%, particularly with respect to a given quantity.

[0036] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely cured. "Treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., halting its development, or (c) relieving the disease, i.e., causing regression of the disease. A therapeutic agent may be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease, where treatment stabilizes or reduces undesirable clinical symptoms in the patient. Desirably, such treatment occurs before complete loss of function in the affected tissue. The subject therapies may be administered during, and in some cases after, the symptomatic stage of the disease.

[0037] A "therapeutically effective amount" refers to the amount of an active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" is an amount that induces, alleviates, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological conditions associated with a disease, or improves resistance to a disease.

[0038] The term "pharmacologically effective amount" or "therapeutically effective amount" of a matrix-modifying enzyme refers to an amount of matrix-modifying enzyme sufficient to provide a desired response, such as improved nutrient transport across cartilage endplates. Additionally, a therapeutically effective amount of matrix-modifying enzyme may prevent, slow, or reduce intervertebral disc degeneration, alleviate pain caused by intervertebral disc degeneration, improve spinal mobility, improve spinal flexibility, and / or increase disc height and / or intervertebral separation. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the condition being treated, the particular drug(s) used, the mode of administration, etc. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0039] "Treatment" or "treating" of degenerative disc disease includes (1) preventing degenerative disc disease, i.e., causing disc degeneration to not occur or to occur to a lesser extent in a subject who may be prone to developing degenerative disc disease but who has not yet experienced or exhibited degenerative disc disease; (2) inhibiting disc degeneration, i.e., halting its development, slowing down, or reversing disc degeneration; and (3) alleviating the symptoms of degenerative disc disease, i.e., reducing the severity of degenerative disc disease, improving spinal flexibility, or alleviating back pain experienced by a subject.

[0040] "Substantially" or "essentially" means nearly completely or entirely, eg, 95% or more of some given amount.

[0041] "Substantially purified" generally refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that it constitutes the majority percentage of the sample in which it is present. Typically, in a sample, the substantially purified component comprises 50%, preferably 80%-85%, and more preferably 90%-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation by density.

[0042] "Isolated," when referring to a polypeptide or peptide, means that the indicated molecule is separate and distinct from the whole organism in which it is found in nature, or exists in the substantial absence of other biological macromolecules of the same type. With reference to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or part of sequences that are normally associated with it in nature, or a sequence that is naturally occurring but has heterologous sequences associated with it, or a molecule that is dissociated from the chromosome.

[0043] As used herein, the terms "increase," "increasing," "enhance," and "enhancing" (and grammatical variations thereof) describe a detectable increase compared to a reference value, unless the context dictates otherwise. An increase can include an increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to another measurable property or amount (e.g., a control value).

[0044] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition. These terms also include post-expression modifications of the polypeptide, such as phosphorylation, glycosylation, acetylation, hydroxylation, oxidation, and the like.

[0045] As used herein, a "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence, for example, as a segment of a full-length protein or a protein fragment. Reference sequences can include, for example, sequences identifiable in databases such as GenBank and UniProt, as well as other sequences identifiable to one of skill in the art.

[0046] Algorithms and programs for comparing primary biological sequence information between any two sequences are readily identifiable to those skilled in the art. Non-limiting examples of such mathematical algorithms include the Myers and Miller algorithm (Myers and Miller 1988), the Smith et al. local homology algorithm (Smith and Waterman 1981), the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch 1970), the Pearson and Lipman similarity search method (Pearson and Lipman 1988), the Karlin and Altschul algorithm (Karlin and Altschul 1990), and modifications of Karlin and Altschul et al. (Karlin and Altschul 1993). Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Such implementations include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, CA); the ALIGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA (Pearson and Lipman 1988)); and TFASTA (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA) in the Wisconsin Genetics Software Package, version 8. Alignments using these programs can be performed using default parameters and allow users to identify database sequences that are similar to a reference sequence (query sequence) above a certain confidence threshold.

[0047] Algorithms and programs for comparing primary biological sequence information between any two sequences typically provide an output that includes the percent identity between the retrieved sequence and the reference sequence.

[0048] Those skilled in the art will understand that identity between sequences is typically measured by a process that includes aligning two polypeptide or polynucleotide sequences to form an aligned sequence, then finding the number of matched characters, i.e., similar or identical characters, between the two aligned sequences, and calculating the total number of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity result is expressed as a percentage of identity.

[0049] As used herein, "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of a polynucleotide sequence within the comparison window may contain additions or deletions (gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0050] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerated when administered to humans (or non-human animals, in the case of veterinary use) and typically do not produce allergic or similar adverse reactions, such as acute gastric upset or dizziness. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0051] A "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the present invention and that causes no significant adverse toxicological effects to the patient.

[0052] "Pharmaceutically acceptable salts" include, but are not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from any of the corresponding inorganic acid forms of the foregoing, such as hydrochloride, or salts prepared with organic acids, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, paratoluenesulfonate, palmate, salicylate, and stearate, as well as estolate, gluceptate, and lactobionate.Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0053] "Biocompatibility," as used herein, refers to the property of a material that allows for prolonged contact with cells or tissues without causing toxicity or significant damage.

[0054] The term "hydrogel" refers to a material formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that traps water molecules to form a gel. Generally, these polymers are at least partially soluble in aqueous solutions such as water, buffered salt solutions, or aqueous alcohol solutions with charged side groups, or their monovalent ionic salts. Biocompatible hydrogels refer to polymers that form gels that are not toxic to living cells.

[0055] "Subject" means any member of the subphylum Chordata, including, but not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species; livestock, such as cows, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs; domestic birds, such as chickens, turkeys, and other poultry birds, ducks, geese, and birds, including wild and game birds.

[0056] Increased disc solute transport Compositions and methods are provided for increasing solute transport in intervertebral discs. Insufficient nutrient transport through cartilage endplates contributes to disc degeneration and limits the effectiveness of therapies to promote disc regeneration. Nutrients entering the disc and metabolites leaving the disc must pass through the cartilage endplates. Solute passage can be slowed or blocked by changes in the composition of the CEP matrix caused, for example, by dehydration, mineralization, and / or fibrosis. Intradiscal delivery of matrix-modifying enzymes to the cartilage endplates can improve solute transport and disc nutrition, slowing or reversing disc degeneration and enhancing the regenerative potential of biologic therapies, particularly those that increase intradiscal nutrient demand.

[0057] In some embodiments, methods are provided for increasing intervertebral disc solute transport in a subject in need thereof, the methods comprising locally administering an effective amount of one or more matrix-modifying enzymes to a cartilage endplate. In certain embodiments, the one or more matrix-modifying enzymes have proteolytic activity against collagen, proteoglycans, and / or combinations thereof. In some embodiments, the one or more matrix-modifying enzymes include collagenase, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. Exemplary collagenases include, but are not limited to, microbial collagenases, such as bacterial collagenases from Vibrio (e.g., Vibrio alginolyticus, Vibrio pomeroyi, Vibrio mimicus, and Vibrio grimontia) and Clostridium (e.g., Clostridium histolyticum and Clostridium perfringens) bacteria, and matrix metalloproteinases (MMPs) with collagenase activity, such as, but not limited to, MMP1, MMP8, MMP13, and MMP18. In some embodiments, the collagenase is collagenase P, MMP8, or a combination thereof. In some embodiments, the collagenase is collagenase P from Clostridium histolyticum. In some embodiments, the MMP8 is full-length MMP8. Enzymatically reducing the amount of collagen in the impermeable cartilage endplates improves disc solute transport and nutrition. Collagenase can be derived from an organism, recombinantly produced, or chemically synthesized by methods well known in the art.Many collagenases are commercially available from, for example, Nordmark Pharma GmbH (Uetersen, Germany), VitaCyte LLC (Indianapolis, IN), Universal Biologicals (Cambridge, United Kingdom), Thermo Fisher Scientific (Waltham, MA), and Worthington Biochemical (Lakewood, New Jersey).

[0058] In some embodiments, one or more matrix-modifying enzymes are encapsulated in a hydrogel. Any suitable hydrogel polymer can be used to form the hydrogel. Exemplary hydrogel polymers include natural polymers, including polysaccharides, including hyaluronic acid, chitosan, heparin, alginate, cellulose, dextran, and agarose, and proteins, including fibrin, fibrinogen, collagen, elastin, gelatin, silk, laminin, fibronectin, albumin, thrombin, and keratin; modified natural polymers, including hydroxymethylcellulose, hydroxyethylcellulose, gelatin methacrylate, polyanionic N-carboxymethylchitosan, and polycationic N-trimethylchitosan; and synthetic polymers, including polyvinyl alcohol, N-vinylpyrrolidone, polyethylene glycol, poly(ethylene glycol), and the like. Examples of suitable polymers include, but are not limited to, cellulose acetate diacrylate, polyacrylamide, poly(N-isopropylacrylamide), sodium polyacrylate, acrylate polymers and copolymers such as hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, ethylene glycol di-methyl acrylate, methyl methacrylate, glycidyl methacrylate, and glycol methacrylate, poly(N-isopropylacrylamide-co-acrylic acid), polyesters, polyurethanes, nylons, synthetic polyamino acids, prolamines; and combinations thereof, and other such molecules, including recombinant versions of such polymers.

[0059] In exemplary embodiments, one or more matrix-modifying enzymes are encapsulated in an alginate hydrogel (see, e.g., Example 1 for a description of a biodegradable alginate hydrogel encapsulating collagenase P from Clostridium histolyticum). In some embodiments, the concentration of alginate in the hydrogel ranges from about 0.5 to about 10 weight percent (wt%), including any wt% within this range, e.g., 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 wt%. In some embodiments, the alginate is partially oxidized. For example, about 2% to about 10% of the alginate can be oxidized, including any percentage within this range, such as 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In one embodiment, the alginate in the hydrogel is about 4% to about 10% oxidized. In some embodiments, the alginate is at a concentration of about 1% by weight in the hydrogel. Such alginate hydrogels can maintain delivery of matrix-modifying enzymes for at least two days after administration to a subject (see, e.g., Example 1 and Figure 9).

[0060] Methods for preparing hydrogels are well known in the art. See, for example, Barbucci Hydrogels Biological Properties and Applications, Springer, 2009; Hydrogels in Cell-Based Therapies, edited by Connon and Hamley, Royal Society of Chemistry, 2014; and Tunable Hydrogels, edited by Lavrentieva, Pepelanov, and Seliktar, Springer Nature Switzerland AG, 2020, which are incorporated herein by reference. In certain instances, the hydrogel is crosslinked by chemical crosslinking. Exemplary chemical crosslinkers include, but are not limited to, N,N,N',N'-tetramethylethylenediamine (TEMED), ammonium persulfate, glutaraldehyde, formaldehyde, epoxy compounds, dialdehydes, N,N'-methylenebis(acrylamide) (MBA), ethylene glycol diacrylate (EGDA), ethylene glycol dimethyl acrylate (EGDMA), PEG diacrylate (PEGDA), glyoxal, epichlorohydrin, and sodium borate / boric acid. Crosslink density can vary depending on the type and concentration of the chemical crosslinker used. Alternatively, hydrogels can be photocrosslinked by exposure to light. UV-sensitive photoinitiators (i.e., polymerization is initiated upon exposure to UV light in the 200-400 nm range) or visible light-sensitive photoinitiators (i.e., polymerization is initiated upon exposure to visible light in the 400-800 nm range) can be used. Photoinitiators can be used to induce photopolymerization. The photopolymerization reaction may involve free radical-initiated chain polymerization or bioorthogonal click reaction. Exemplary photoinitiators include, but are not limited to, (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and eosin-Y. The crosslink density may vary depending on the intensity of the electromagnetic radiation applied to promote photopolymerization of the hydrogel, as well as the duration of irradiation.In some embodiments, the crosslinked hydrogel has a crosslink density of 1×10. -15 moles / cm 3 ~1×10 -3 moles / cm 3 In some embodiments, the hydrogel contains a divalent cation (e.g., Ca 2+ , Mg 2+ , Zn 2+ , or Ba 2+ ) or trivalent cations (e.g., Fe 3+ Or Al 3+ ) is ionically cross-linked.

[0061] In other embodiments, one or more matrix-modifying enzymes are encapsulated in liposomes or conjugated to the surface of liposomes. Liposome preparations for use in delivering matrix-modifying enzymes can contain cationic (positively charged), anionic (negatively charged), and neutral lipids. A variety of cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the Lipofectin trademark from GIBCO BRL, Grand Island, NY (see also Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, for example, Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198, PCT Publication No. 90 / 11092, for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Anionic and neutral liposomes are also readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include, among others, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE). These materials can also be mixed with DOTMA and DOTAP in appropriate ratios. Methods for making liposomes using these materials are well known in the art. The type of liposome selected is preferably of a size and composition suitable to limit the mobility of the matrix-modifying enzyme to the nucleus pulposus.

[0062] Any suitable bioconjugation method can be used to conjugate matrix-modifying enzymes to the surface of liposomes. Crosslinking agents that can be used include, but are not limited to, dimethylsuberimidate, N-hydroxysuccinimide, formaldehyde, and glutaraldehyde. Additionally, carboxyl-reactive chemical groups such as diazomethane, diazoacetyl, and carbodiimide can be used to crosslink carboxylic acids to primary amines. In particular, carbodiimide compounds such as 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N',N'-dicyclohexylcarbodiimide (DCC) can be used for conjugation with carboxylic acids. To improve the efficiency of the crosslinking reaction, N-hydroxysuccinimide (NHS) or a water-soluble analogue (e.g., sulfo-NHS) can be used in combination with the carbodiimide compound. Carbodiimide compounds (e.g., EDC or DCC) attach NHS to carboxyl groups to form NHS ester intermediates that readily react with primary amines at physiological pH. For descriptions of various cross-linking agents and techniques, see, for example, Wong and Jameson, "Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation" (CRC Press, 2nd edition, 2011), and Hermanson, "Bioconjugate Techniques" (Academic Press, 3rd edition, 2013), which are incorporated herein by reference in their entireties.

[0063] In certain embodiments, the matrix-modified enzyme is crosslinked to the surface of the liposome using click chemistry, which can be performed using suitable crosslinkers containing reactive azide or alkyne functional groups. For example, Kolb et al.,2004,Angew Chem Int Ed 40:3004-31, Evans,2007,Aust J Chem 60:384-95, Millward et al.(2013)Integr Biol(Camb)5(1):87-95, Lallana et al.(2012)Pharm Res 29(1):1-34, Gregoritza et al. (2015) Eur J Pharm Biopharm.97(Pt B):438-453, Musumeci et al. (2015) Curr Med Chem.22(17):2022-2050, McKay et al. (2014) Chem Biol 21(9):1075-1101, Ulrich et al. (2014) Chemistry 20(1):34-41, Pasini (2013) Molecules 18(8):9512-9530, and Wangler et al. (2010) Curr Med Chem. 17(11):1092-1116, which are incorporated by reference in their entireties.

[0064] Specifically, crosslinking can be performed using strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry, a Cu-free variant of click chemistry that is generally cell and biocompatible. SPAAC utilizes substituted cyclooctynes ​​with an internal alkyne in a strained ring system. The ring strain, along with electron-withdrawing substituents in the cyclooctyne, promotes a [3 + 2] dipolar cycloaddition with the azide functionality. SPAAC can be used for bioconjugation and crosslinking by attaching the azide and cyclooctyne moieties to molecules. For a description of SPAAC, see, for example, Baskin et al. (2007) Proc Natl Acad Sci USA 104(43):16793-16797, Agard et al. (2006) ACS Chem.Biol.1:644-648, Codelli et al. al. (2008) J.Am.Chem.Soc.130:11486-11493, Gordon et al. (2012) J.Am.Chem.Soc.134:9199-9208, Jiang et al. (2015) Soft Matter 11(30):6029-6036, Jang et al. (2012) Bioconjug Chem.23(11):2256-2261, Ornelas et al. (2010) J Am Chem Soc. 132(11):3923-3931, which are incorporated herein by reference in their entireties.

[0065] A physician may identify the location of target CEPs for matrix-modifying enzyme treatment, for example, by medical imaging. Medical imaging can also be used to select which intervertebral discs can benefit from matrix-modifying enzyme treatment. Exemplary medical imaging techniques include, but are not limited to, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), computed tomography (CT), ultrasound imaging (UI), optical imaging (OI), photoacoustic imaging (PI), fluoroscopy, and fluorescence imaging. In some embodiments, one or more matrix-modifying enzymes are administered locally to the target CEPs using fluoroscopic guidance, computed tomography (CT) guidance, combined CT-fluoroscopic guidance, or ultrasound guidance.

[0066] Pharmaceutical Composition One or more matrix-modifying enzymes (e.g., collagenase, full-length MMP8) can be formulated into pharmaceutical compositions, optionally containing one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Suitable excipients for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, buffers, and surfactants. Carbohydrates such as sugars, derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, myo-inositol, and the like.

[0067] The excipient may also include an inorganic salt or buffer, such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof. Those skilled in the art will readily recognize various buffers that can be used in the composition. Buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. For example, buffer components may be water-soluble materials such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Exemplary buffers include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0068] The compositions can also include antimicrobial agents to prevent or inhibit microbial growth. Non-limiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0069] Antioxidants can also be present in the composition. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of one or more matrix-modifying enzymes or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0070] Surfactants can be present as excipients. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80" and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids, and fatty esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc and other such suitable cations.

[0071] Acids or bases can be present in the composition as excipients.Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.Examples of suitable bases include, but are not limited to, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0072] In certain embodiments, one or more additional factors, such as nutrients, cytokines, growth factors, or immunosuppressants, may be added to the composition.

[0073] Exemplary growth factors include, but are not limited to, insulin-like growth factor (IGF), transforming growth factor beta (TGF-β), growth / differentiation factor 5 (GDF-5), bone morphogenetic protein-1, bone morphogenetic protein (BMP), epiregulin, fibroblast growth factor (FGF), epidermal growth factor (EGF), endothelial growth factor (ECGF), nerve growth factor (NGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), vascular endothelial growth factor ("VEGF"), and cholecystokinin octapeptide.

[0074] Exemplary immunosuppressants are well known and can be steroids (e.g., prednisone) or non-steroids (e.g., sirolimus (Rapamune, Wyeth-Ayerst Canada), tacrolimus (Prograf, Fujisawa Canada), and anti-IL2R daclizumab (Zenapax, Roche Canada). Other immunosuppressants include 15-deoxyspergualin, cyclosporine, methotrexate, rapamycin, Rapamune (sirolimus / rapamycin), FK506, or lisofylline (LSF).

[0075] The amount of matrix-modifying enzyme in a composition (e.g., when contained in a drug delivery system) will vary depending on many factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined empirically by repeatedly administering increasing amounts of the composition to determine which amount produces a clinically desired endpoint.

[0076] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and the specific needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing various amounts of the excipient (ranging from low to high), examining stability and other parameters, and then determining the range in which optimal performance is achieved without significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight of the excipient, preferably about 5% to about 98% by weight, more preferably about 15% to about 95% by weight, and most preferably at a concentration of less than 30% by weight. These aforementioned pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

[0077] The compositions include all kinds of formulations, especially formulations suitable for injection, such as powders or lyophilized products that can be reconstituted with a solvent before use, as well as ready-to-inject solutions or suspensions, dry insoluble compositions for combining with a vehicle before use, and emulsions and liquid concentrates for dilution before administration. Examples of diluents suitable for reconstituting solid compositions before injection include bacteriostatic water for injection, 5% dextrose in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. Regarding liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include compositions for local delivery to CEP.

[0078] The pharmaceutical formulations herein may also be housed in syringes, implantation devices, etc., depending on the intended mode of delivery and use. Preferably, the compositions comprising the matrix-modifying enzyme are in unit dosage form, meaning the amount of the conjugate or composition of the invention suitable for a single dose, in pre-measured or pre-packaged form.

[0079] The compositions herein may optionally contain one or more additional drugs for treating degenerative disc disease, such as analgesics or anti-inflammatory agents or other medications. For example, the combination formulation may contain acetaminophen, nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, and naproxen), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, and pethidine), steroids (e.g., hydrocortisone, prednisone, triamcinolone, methyl-prednisolone, and dexamethasone), gabapentin, memantine, pregabalin, fluticasone, fluoxetine ... The therapeutic agent may include at least one matrix-modifying enzyme and one or more other drugs for treating degenerative disc disease, such as analgesics and anti-inflammatory agents, including, but not limited to, valine, cannabinoids, tramadol, lamotrigine, carbamazepine, duloxetine, milnacipran, tricyclic antidepressants (e.g., amitriptyline, nortriptyline, and desipramine), and serotonin-norepinephrine reuptake inhibitors (e.g., duloxetine, venlafaxine, and milnacipran).

[0080] Administration of matrix-modifying enzymes At least one therapeutically effective cycle of treatment with a composition containing one or more matrix-modifying enzymes (e.g., collagenase, full-length MMP8) is administered to a subject for the treatment of degenerative disc disease. A "therapeutically effective dose or amount" of a matrix-modifying enzyme refers to an amount that, when administered, results in a positive therapeutic response, such as improved transport of nutrients through cartilage endplates to the intervertebral disc. Additionally, a therapeutically effective amount of a matrix-modifying enzyme may prevent, delay, or reduce the progression of, disc degeneration, alleviate pain caused by disc degeneration, improve spinal mobility, improve spinal flexibility, and / or increase disc height and / or intervertebral separation. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the condition being treated, the particular type of matrix-modifying enzyme used, the mode of administration, etc. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0081] Therapy may be administered to one or both CEPs of one or more intervertebral discs in a vertebrate subject. The treated disc may be any disc in the spinal column. In some embodiments, the subject is a human. A human subject has 25 intervertebral discs, including seven cervical discs, twelve thoracic discs, five lumbar discs, and one sacral disc, any of which may be treated. In other embodiments, the subject is a non-human vertebrate species. The vertebrate may be treated, for example, for veterinary or research purposes. Non-human vertebrate subjects include, but are not limited to, non-human primates such as chimpanzees and other apes and monkey species; livestock such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; and birds, including chickens, turkeys, and other poultry birds, domestic birds such as ducks and geese, and wild and game birds.

[0082] In some embodiments, the subject has been diagnosed with degenerative disc disease. For example, the subject may be diagnosed using radiography, computed tomography, magnetic resonance imaging, or discography. In some embodiments, the subject has spinal pain, e.g., lower back pain or neck pain.

[0083] In other embodiments, pharmaceutical compositions containing one or more matrix-modifying enzymes are administered prophylactically, for example, to prevent intervertebral disc degeneration. Such prophylactic use is particularly valuable for subjects at high risk for developing intervertebral disc degeneration due to insufficient nutrient transport through cartilage endplates, a genetic predisposition to developing intervertebral disc degeneration (e.g., due to mutations in the COL1A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, ACAN, TIMP1, COX2, or THSD2 genes), age, spinal deformity, spinal injury, physically demanding occupations (e.g., requiring heavy weight carrying), obesity, or smoking. In some cases, subjects are at risk for developing intervertebral disc degeneration due to their age. For example, in humans, subjects over 45 years of age may be at risk for developing intervertebral disc degeneration. In some embodiments, the subject is over 45 years of age, over 50 years of age, over 55 years of age, or over 60 years of age.

[0084] In certain embodiments, multiple therapeutically effective doses of a composition containing one or more matrix-modifying enzymes and / or one or more other therapeutic agents, such as one or more drugs for treating degenerative disc disease or other pharmaceuticals, are administered. For example, a combination formulation may be administered containing at least one matrix-modifying enzyme for treating degenerative disc disease and one or more drugs, such as acetaminophen, nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, and naproxen), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, and pethidine), steroids (e.g., hydrocortisone, prednisone, triamcinolone, Other medications may include analgesics and anti-inflammatory agents, including, but not limited to, methylprednisolone, and dexamethasone), gabapentin, memantine, pregabalin, cannabinoids, tramadol, lamotrigine, carbamazepine, duloxetine, milnacipran, tricyclic antidepressants (e.g., amitriptyline, nortriptyline, and desipramine), and serotonin-norepinephrine reuptake inhibitors (e.g., duloxetine, venlafaxine, and milnacipran), or other medications are administered. Compositions comprising one or more matrix-modifying enzymes are typically, but not necessarily, administered locally within or adjacent to the CEPs in need of treatment.

[0085] The pharmaceutical formulations may be in the form of a liquid solution or suspension immediately prior to administration, but may also take other forms such as syrups, creams, ointments, tablets, capsules, powders, gels, liposomes, matrices, etc. Pharmaceutical compositions containing one or more matrix-modifying enzymes and / or other agents may be administered according to any medically acceptable method known in the art, using the same or different routes of administration.

[0086] Compositions comprising one or more matrix-modifying enzymes are suitable for localized delivery to target CEPs. A physician may identify the location of injected CEPs, for example, by medical imaging. Exemplary medical imaging techniques include, but are not limited to, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), computed tomography (CT), ultrasound imaging (UI), optical imaging (OI), photoacoustic imaging (PI), fluoroscopy, and fluorescence imaging. In some embodiments, one or more matrix-modifying enzymes are administered locally to target CEPs using fluoroscopic guidance, computed tomography (CT) guidance, combined CT-fluoroscopic guidance, or ultrasound guidance. In some embodiments, a contrast agent is included in the composition comprising the matrix-modifying enzyme, allowing for confirmation of localization of the composition to CEPs by medical imaging after administration. In some embodiments, the contrast agent is a microbubble (e.g., for use in ultrasound) or a radiopaque contrast agent (e.g., for use in radiography). The imaging agent may be contained in the same composition as the matrix-modifying enzyme or in a different composition, and may be used before or after administration of the matrix-modifying enzyme, hi certain embodiments, the composition is injected into or adjacent to the CEP.

[0087] In another embodiment, pharmaceutical compositions containing one or more matrix-modifying enzymes and / or other drugs and / or other agents for treating degenerative disc disease are administered in sustained-release formulations (e.g., sustained-release hydrogel carriers) or sustained-release devices. Such devices are well known in the art and include, for example, miniature implantable pumps that can provide continuous, steady-state drug delivery over time at various doses to achieve a sustained-release effect in non-sustained-release pharmaceutical compositions.

[0088] Those skilled in the art will understand which conditions a matrix-modifying enzyme or derivative thereof can effectively treat. The actual dose administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the health care professional, and the conjugate being administered. A therapeutically effective amount can be determined by one of skill in the art and will be adjusted according to the specific requirements of each particular case.

[0089] In certain embodiments, compositions comprising multiple therapeutically effective doses of one or more matrix-modifying enzymes will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered one day per week, two days per week, three days per week, four days per week, or five days per week, etc. By "intermittent" administration, it is intended that a therapeutically effective dose can be administered, for example, every other day, every two days, every three days, weekly, every other week, etc. For example, in some embodiments, a composition comprising a matrix-modifying enzyme will be administered once per week, twice per week, or three times per week for an extended period of time, such as 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks. By "twice per week" or "twice per week," it is intended that two therapeutically effective doses of an agent are administered to a subject within a seven-day period, starting on day 1 of the first week of administration, with a minimum of 72 hours between doses and a maximum of 96 hours between doses. By "three times per week" or "three times per week," it is intended that three therapeutically effective doses are administered to a subject within a seven-day period, allowing a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of administration is referred to as "intermittent" therapy. According to the methods of the present invention, a subject can receive intermittent therapy (i.e., once-weekly, twice-weekly, or three-times-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agent can be administered by any acceptable route of administration, as described herein below. The dosage will depend on the potency of the one or more matrix-modifying enzymes and / or other agents administered, the magnitude of the desired effect, and the route of administration.

[0090] The matrix-modifying enzyme (again, preferably provided as part of a pharmaceutical formulation) can be administered alone or in combination with one or more other therapeutic agents, such as other agents for treating degenerative disc disease or other pharmaceutical agents used to treat a particular condition or disease, according to a variety of administration schedules depending on the clinician's judgment, the patient's needs, etc. Specific administration schedules are known by those skilled in the art or can be determined empirically using routine methods. Exemplary administration schedules include, but are not limited to, five times daily, four times daily, three times daily, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions require administration no more than once daily.

[0091] One or more matrix-modifying enzymes can be administered before, simultaneously with, or after the other agent. When provided simultaneously with the other agent, the one or more matrix-modifying enzymes can be provided in the same composition or in different compositions. Thus, one or more matrix-modifying enzymes and one or more other agents can be presented to an individual through combination therapy. "Combination therapy" refers to administration to a subject such that the therapeutic effect of the combination of substances is achieved in the subject receiving the therapy. For example, combination therapy can be achieved by administering a pharmaceutical composition containing one or more matrix-modifying enzymes in a dose according to a specific dosing regimen and a pharmaceutical composition containing at least one other agent, such as another drug for treating degenerative disc disease, which together constitute a therapeutically effective dose. Similarly, one or more matrix-modifying enzymes and one or more other therapeutic agents can be administered in at least one therapeutic dose. The separate pharmaceutical compositions can be administered simultaneously or at different times (i.e., on the same or different days, sequentially in any order), as long as the therapeutic effect of the combination of substances is achieved in the subject receiving the therapy.

[0092] Toxicity can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50(the dose lethal to 50% of the population) or LD 100 The dose ratio between toxic and therapeutic effects can be determined by determining the therapeutic index (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to further optimize and / or define therapeutic and / or sub-therapeutic dose ranges (e.g., for use in humans). The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0093] Diagnostic methods Diagnostic methods for determining whether a subject requires treatment to increase disc solute transport are also provided. Diagnostic methods generally involve detecting abnormal thickness, abundance, or composition of the cartilage layer in vertebral endplates adjacent to a degenerated disc as a marker of transport inhibition. In one embodiment, the cartilage condition (e.g., thickness, abundance, and / or composition) of the endplates adjacent to a degenerated disc is assessed in a patient. If one or both endplates appear to have abnormal cartilage condition, this indicates that inhibition of permeability of the endplate(s) is involved in a degenerative disc condition and that the patient could benefit from a permeability-enhancing treatment, as described herein.

[0094] Additionally, such assessment of endplate cartilage status can be used to monitor the effectiveness of permeability-enhancing treatments. In some embodiments, the status of cartilage at the endplate is assessed in a subject undergoing treatment. A reduction in abnormal cartilage indicators in a treated subject compared to an appropriate control indicates that the permeability-enhancing treatment is effective. An appropriate control, e.g., cartilage thickness, abundance, and / or composition in an untreated subject, can be used, or the same subject can be observed at an earlier time point (e.g., before or early in treatment). This method is useful for monitoring the effectiveness of treatment in individual patients and, if ineffective, altering the course of treatment. Furthermore, this method is useful, for example, for assessing the effectiveness of a putative treatment in a test subject or animal.

[0095] Assessment of cartilage status can be performed by any imaging modality capable of visualizing and quantifying abnormal cartilage indicators within the endplate, such as cartilage thickness or abundance, or excess collagen. For example, in one embodiment, MR imaging of the endplate is utilized to assess cartilage status. Endplate cartilage cannot be visualized using conventional MRI due to its short T2 relaxation time, and therefore, cartilage signals are not captured by conventional MR sequences with long echo times. To overcome this challenge, ultrashort echo time (UTE) sequences for MR imaging of the cartilage components of the endplate can be used. For example, in a related study, Fields et al., 2014, “Cartilaginous endplates: quantitative MR imaging with very short echo times—orientation dependence and correlation with biochemical composition,” Radiology 274(2):482-9, observed that relaxation times derived from UTE images correlated with cartilage water content and collagen content.

[0096] kit Kits comprising any of the compositions described herein are also provided. In certain embodiments, the kits comprise a pharmaceutical composition comprising one or more matrix-modifying enzymes. In certain embodiments, the one or more matrix-modifying enzymes have proteolytic activity against collagen, proteoglycans, or a combination thereof. In some embodiments, the one or more matrix-modifying enzymes comprise collagenase, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. In some embodiments, the collagenase is collagenase P, MMP8, or a combination thereof. In some embodiments, the collagenase is collagenase P from Clostridium histolyticum. In some embodiments, the MMP8 is full-length MMP8. In certain embodiments, the one or more matrix-modifying enzymes are encapsulated in a hydrogel. In some embodiments, the hydrogel comprises alginate. In certain embodiments, the one or more matrix-modifying enzymes are encapsulated in a liposome or conjugated to the surface of a liposome. In some embodiments, the liposome comprises a phospholipid. Exemplary phospholipids include, but are not limited to, phosphatidylcholine and phosphatidylethanolamine. In some embodiments, the liposome further comprises cholesterol. In some embodiments, the liposome further comprises polyethylene glycol.

[0097] The kit may include one or more containers of the compositions described herein. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, including glass or plastic. The containers may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). The kit may further include a container containing a pharmaceutically acceptable buffer solution, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. The kit may also include other materials useful to the end user, including other pharmaceutically acceptable formulated solutions, such as buffers, diluents, filters, needles, and syringes, or other delivery devices.

[0098] The kit may also provide a delivery device pre-filled with a pharmaceutical composition comprising one or more matrix-modifying enzymes. Formulations suitable for local administration to the CEP are of particular interest, and in such embodiments, the kit may include a syringe (e.g., with an epidural needle) or other device for achieving such administration.

[0099] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, such as in kit packaging, in a package insert, or the like, such as one or more sheets of paper on which the information is printed. Yet another form in which these instructions may be present is as a computer-readable medium having the information recorded thereon, such as a diskette, compact disc (CD), DVD, Blu-ray, flash drive, or the like. Yet another form in which these instructions may be present is a website address that may be used via the Internet to access the information at the removed site.

[0100] Examples of Non-Limiting Aspects of the Disclosure Aspects, including embodiments of the present subject matter described above, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above, certain non-limiting aspects of the present disclosure, numbered 1 through 80, are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. 1. A method for treating degenerative intervertebral disc disease in a subject in need thereof, comprising locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to the cartilage endplate (CEP). 2. The method of embodiment 1, wherein the treatment increases CEP and disc nutrient transport permeability. 3. The method of aspect 1 or 2, wherein the one or more matrix-modifying enzymes have proteolytic activity against collagen, proteoglycan, or a combination thereof. 4. The method of embodiment 3, wherein the one or more matrix-modifying enzymes comprises collagenase. 5. The method of aspect 4, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. 6. The method of aspect 5, wherein the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof. 7. The method of any one of aspects 4 to 6, wherein the MMP8 is full-length MMP8. 8. The method of any one of aspects 1-7, wherein the one or more matrix-modifying enzymes are encapsulated in a hydrogel. 9. The method of embodiment 8, wherein the hydrogel comprises alginate. 10. The method of embodiment 9, wherein the concentration of alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%). 11. The method of embodiment 10, wherein the concentration of alginate in the hydrogel is about 1% by weight. 12. The method of any one of aspects 9-11, wherein the alginate is at least partially oxidized. 13. The method of claim 12, wherein about 4% to about 10% of the alginate is oxidized. 14. The method of any one of aspects 8-13, wherein the hydrogel maintains delivery of the matrix-modifying enzyme for at least 2 days. 15. The method of any one of aspects 1 to 7, wherein the one or more matrix-modifying enzymes are encapsulated in or conjugated to the surface of the liposome. 16. The method of embodiment 15, wherein the liposome comprises a phospholipid. 17. The method of claim 16, wherein the phospholipid is phosphatidylcholine or phosphatidylethanolamine. 18. The method of any one of aspects 15-17, wherein the liposome further comprises cholesterol. 19. The method of any one of aspects 15-18, wherein the liposome further comprises polyethylene glycol. 20. The method of any one of aspects 1-19, wherein the one or more matrix-modifying enzymes are administered in a volume small enough so that intradiscal pressure does not increase substantially. 21. The method of embodiment 20, wherein the one or more matrix-modifying enzymes are administered in a volume of 50 μL or less. 22. The method of any one of aspects 1-21, wherein the one or more matrix-modifying enzymes are administered within or adjacent to the CEP. 23. The method of any one of aspects 1-22, wherein the one or more matrix-modifying enzymes are administered with an epidural needle. 24. The method of embodiment 23, wherein the epidural needle has a curved end such that the matrix-modifying enzyme exits the epidural needle laterally at an angle of about 45 degrees. 25. The method of any one of aspects 1-24, further comprising using medical imaging to guide local administration of one or more matrix-modifying enzymes to CEPs or to select which intervertebral discs may benefit from treatment with one or more matrix-modifying enzymes. 26. The method of embodiment 25, wherein locally administering one or more matrix-modifying enzymes to the CEP is performed under fluoroscopic guidance, computed tomography (CT) guidance, combined CT fluoroscopic guidance, or ultrasound guidance. 27. The method of any one of aspects 1-26, wherein the one or more matrix-modifying enzymes are administered locally to the CEP without exposing the nucleus pulposus to the one or more matrix-modifying enzymes. 28. The method of any one of aspects 1-27, further comprising administering a growth factor. 29. The method of any one of aspects 1-28, further comprising administering an analgesic or anti-inflammatory agent. 30. The method of any one of aspects 1-29, wherein multiple cycles of treatment are administered to the subject. 31. A composition comprising one or more matrix-modifying enzymes for use in a method for treating degenerative disc disease. 32. The composition of aspect 31, further comprising a pharmaceutically acceptable excipient. 33. The composition of aspect 31 or 32, wherein the one or more matrix-modifying enzymes comprises collagenase. 34. The composition of aspect 33, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. 35. The composition of aspect 34, wherein the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof. 36. The composition of any one of aspects 33 to 35, wherein the MMP8 is full-length MMP8. 37. The composition of any one of aspects 31-36, wherein the one or more matrix-modifying enzymes are encapsulated in a hydrogel. 38. The composition of aspect 37, wherein the hydrogel comprises alginate. 39. The composition of embodiment 38, wherein the concentration of alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%). 40. The composition of embodiment 39, wherein the concentration of alginate in the hydrogel is about 1% by weight. 41. The composition of any one of aspects 38-40, wherein the alginate is at least partially oxidized. 42. The composition of aspect 41, wherein about 4% to about 10% of the alginate is oxidized. 43. The composition of any one of aspects 37-42, wherein the hydrogel maintains delivery of the matrix-modifying enzyme for at least 2 days. 44. The composition of any one of aspects 31-36, wherein the one or more matrix-modifying enzymes are encapsulated in or conjugated to the surface of the liposome. 45. The composition of aspect 44, wherein the liposome comprises a phospholipid. 46. ​​The composition of aspect 45, wherein the phospholipid is phosphatidylcholine or phosphatidylethanolamine. 47. The composition of any one of aspects 44-46, wherein the liposome further comprises cholesterol. 48. The composition of any one of aspects 44-47, wherein the liposome further comprises polyethylene glycol. 49. The composition of any one of aspects 31-48, further comprising a growth factor. 50. The composition of any one of aspects 31-49, further comprising an analgesic or anti-inflammatory agent. 51. A method for increasing intervertebral disc solute transport in a subject in need thereof, comprising locally administering to the cartilage endplate (CEP) an effective amount of one or more matrix-modifying enzymes. 52. The method of aspect 51, wherein administering increases the permeability of CEP and intervertebral disc nutrient transport. 53. The method of aspect 51 or 52, wherein the one or more matrix-modifying enzymes have proteolytic activity against collagen, proteoglycans, or a combination thereof. 54. The method of embodiment 53, wherein the one or more matrix-modifying enzymes comprises collagenase. 55. The method of aspect 54, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof. 56. The method of aspect 55, wherein the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof. 57. The method of any one of aspects 54 to 56, wherein the MMP8 is full-length MMP8. 58. The method of any one of aspects 51-57, wherein the one or more matrix-modifying enzymes are encapsulated in a hydrogel. 59. The method of embodiment 58, wherein the hydrogel comprises alginate. 60. The method of embodiment 59, wherein the concentration of alginate in the hydrogel ranges from 0.5% to 10 weight percent (wt%). 61. The method of embodiment 60, wherein the concentration of alginate in the hydrogel is about 1% by weight. 62. The method of any one of aspects 59-61, wherein the alginate is at least partially oxidized. 63. The method of embodiment 62, wherein about 4% to about 10% of the alginate is oxidized. 64. The method of any one of aspects 58-63, wherein the hydrogel maintains delivery of the matrix-modifying enzyme for at least 2 days. 65. The method of any one of aspects 51 to 57, wherein the one or more matrix-modifying enzymes are encapsulated in or conjugated to the surface of the liposome. 66. The method of embodiment 65, wherein the liposome comprises a phospholipid. 67. The method of embodiment 66, wherein the phospholipid is phosphatidylcholine or phosphatidylethanolamine. 68. The method of any one of aspects 65-67, wherein the liposome further comprises cholesterol. 69. The method of any one of aspects 65-68, wherein the liposome further comprises polyethylene glycol. 70. The method of any one of aspects 51-69, wherein the one or more matrix modifying enzymes are administered in a volume small enough so that intradiscal pressure does not increase substantially. 71. The method of embodiment 70, wherein the one or more matrix-modifying enzymes are administered in a volume of 50 μL or less. 72. The method of any one of aspects 51-71, wherein the one or more matrix-modifying enzymes are administered within or adjacent to the CEP. 73. The method of any one of aspects 51-72, wherein the one or more matrix-modifying enzymes are administered with an epidural needle. 74. The method of embodiment 73, wherein the epidural needle has a curved end such that the one or more matrix-modifying enzymes exit the epidural needle laterally at an angle of about 45 degrees. 75. The method of any one of aspects 51-74, further comprising using medical imaging to guide local administration of one or more matrix-modifying enzymes to CEPs or to select which intervertebral discs may benefit from treatment with one or more matrix-modifying enzymes. 76. The method of embodiment 75, wherein locally administering one or more matrix-modifying enzymes to the CEP is performed under fluoroscopic guidance, computed tomography (CT) guidance, combined CT fluoroscopic guidance, or ultrasound guidance. 77. The method of any one of aspects 51-76, wherein the one or more matrix-modifying enzymes are administered locally to the CEP without exposing the nucleus pulposus to the one or more matrix-modifying enzymes. 78. The method of any one of aspects 51-77, further comprising administering a growth factor. 79. The method of any one of aspects 51-78, further comprising administering an analgesic or anti-inflammatory agent. 80. The method of any one of aspects 51-79, further comprising detecting whether intervertebral disc solute transport is inhibited in the CEP prior to locally administering one or more matrix-modifying enzymes to the CEP to determine whether the subject will benefit from treatment.

[0101] It will be apparent to those skilled in the art that various changes and modifications can be made to the present invention without departing from the spirit or scope of the present invention.

[0102] experiment The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0103] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0104] The present invention has been described in terms of particular embodiments discovered or proposed by the inventors to include preferred modes for carrying out the invention. Those skilled in the art will understand, in light of this disclosure, that numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be within the scope of the appended claims.

[0105] Example 1 Intradiscal treatment of cartilage endplates to improve solute transport and disc nutrition Proper disc nutrition involves the exchange of nutrients and metabolites between nucleus pulposus (NP) cells and vertebral capillaries (Maroudas et al., 1975; Nachemson et al., 1970; Ohshima et al., 1989; Urban et al., 1977). Several factors can impair normal patterns of solute exchange. For example, nutrients entering and metabolites leaving the disc must pass through the cartilage endplate (CEP), and solute passage can be slowed or blocked by changes in the composition of the CEP matrix, including dehydration (Antoniou et al., 1996), mineralization (Wong et al., 2019; Benneker 2005; Grant 2016), and fibrosis (Antoniou et al., 1996; Wong et al., 2019). For example, researchers found that CEPs with higher amounts of collagen, sulfated glycosaminoglycans (sGAGs), and minerals hindered nutrient diffusion, thereby impairing the survival and function of NP cells within the diffusion chamber (Wong et al., 2019). Furthermore, these same deficiencies in CEP composition were also associated with significantly worsened disc degeneration in patients with low back pain (Bonnheim et al., 2022).

[0106] Motivated by these findings, we explored a matrix modification strategy to improve disc nutrition, which entailed enzymatically reducing the amount of collagen and aggrecan in impermeable and fibrous CEPs (Dolor et al., 2019). Treatment of human cadaveric CEPs with recombinant human MMP-8 enzyme, a matrix metalloproteinase with high specificity for type II collagen and aggrecan, dose-dependently reduced sGAG content. Importantly, reduction of CEP matrix content by MMP-8 treatment improved NP cell viability in diffusion chambers, indicating improved CEP permeability to nutrients (Dolor et al., 2019). While diffusion chambers represent a useful model system for studying the effects of CEP permeability enhancement, the chambers do not mimic the complex microenvironment or loading of intact intervertebral discs. Therefore, the overall impact of CEP permeability enhancement on solute transport in intact discs remains unknown. The objectives of this study were 1) to develop a model for controlled intradiscal delivery of matrix-modifying enzymes to CEPs and 2) to measure solute transport into whole human and bovine intervertebral discs after intradiscal treatment of CEPs with matrix-modifying enzymes.

[0107] method Bovine tissue: Nine intact bovine coccygeal motion segments were harvested from three bovine tails (three segments per bovine tail) obtained within two hours of slaughter from a local slaughterhouse. After harvesting the motion segments, a 17G Crawford needle with a plasma-mediated coblation wand (ArthroCare 2000) was inserted through the posterior annulus toward one CEP of each intervertebral disc. As the needle advanced into the distal annulus, the wand was applied at a low power setting (2 W) for 10 seconds, and the coblated tissue was debrided with PBS. This coblation step was necessary to overcome the high back pressure within the otherwise healthy bovine intervertebral disc. After coblation, the wand was removed with the needle in place, and the motor segments were randomly assigned to three groups (n = 3 motor segments / group, Table 2): collagenase—0.15 U in 50 μL of slow-release alginate carrier, control buffer—HBSS in 50 μL of alginate carrier, or sham—needle insertion only.

[0108] Human tissue: Five human cadaveric lumbar vertebrae (age range: 38-66 years, mean age: 56 ± 10 years) were obtained from donors with no history of musculoskeletal disorders (UCSF Willed Body Program). From these vertebrae, 10 intact motion segments (two motion segments per vertebra) were harvested and randomly assigned to two groups (n = 5 motion segments / group, Table 1): collagenase-0.15 U in 50 μL of alginate carrier or control buffer-HBSS in 50 μL of alginate carrier.

[0109] Preparation of alginate: For alginate purification, sodium alginate (10 g, Protanal LF 20 / 40) was dissolved in 1000 ml of ultrapure deionized water (diH2O), dialyzed against diH2O (MWCO 3500, Spectrum Laboratories Inc.) for 3 days, treated with activated charcoal (0.5 mg / 100 ml, 50–200 mesh, Fisher) for 30 min, filtered (0.22 μm filter), and lyophilized.

[0110] Oxidized alginate (OA) was prepared by reacting sodium alginate (Protanal LF20 / 40, 196,000 g / mol, FMC biopolymer) with sodium periodate (Sigma) using a previously described method (Jeon et al., 2017). Briefly, sodium alginate (10 g) was dissolved in ultrapure deionized water (diH2O, 900 ml) overnight. Sodium periodate (543 mg and 1085 mg) was dissolved in 100 ml of diH2O and added to the separate alginate solutions with stirring at room temperature in the dark for 24 h to achieve different degrees of theoretical alginate oxidation (5% and 10%, respectively). OA was purified by dialysis against diH2O (MWCO 3500, Spectrum Laboratories Inc.) for 3 days, treated with activated charcoal (0.5 mg / 100 ml, 50-200 mesh, Fisher) for 30 min, filtered (0.22 μm filter), and lyophilized. Actual oxidation (4.2 ± 0.5 and 8.5 ± 0.3%) was determined using the Amplite™ colorimetric aldehyde quantitation kit (AAT Bioquest Inc.) according to the manufacturer's instructions.

[0111] Intradiscal CEP Treatment: Enzyme treatment or control buffer was delivered to the CEP under fluoroscopic guidance. Briefly, a 17G Tuohy needle (Medline, item: PAIN8001) was inserted through the posterior annulus and advanced to the central region of the superior CEP of each intervertebral disc (Figure 1). After needle placement, 50 μL of either collagenase suspended in a sustained-release alginate carrier or control buffer was injected into the CEP. Then, fibrin sealant (TISSEEL, Baxter International Inc., CA, USA) was applied to the annulus while the needle was retracted. All motion segments were then incubated in a protease inhibitor cocktail (Sigma-Aldrich, catalog number P2714) at 37°C for 18 hours.

[0112] Transport Experiments: Using custom settings, the effect of CEP permeability enhancement on solute transport into the intervertebral disc was evaluated (Figure 2). To eliminate any potentially confounding effects of differences in bone endplate structure within and between species, the inferior and superior bone endplates were carefully removed from the motion segment using a deburring tool (Figure 3A). To ensure complete removal of all bone tissue and to calculate the cross-sectional area of ​​the isolated intervertebral disc, several X-ray images (Faxitron Cabinet X-ray System Model 43855A) were taken during the bone removal process (ImageJ, open-source software, Schneider, CA, 2012).

[0113] The isolated disc was then sandwiched between a porous stainless steel platen (0.8 mm hole diameter, 1.8 mm thickness, and 17.9% hole coverage) and an open-cell porous aluminum foam block (Duocel® Aluminum Foam Disc, 7-9% relative density, ERG Aerospace Corp., Oakland, CA) with porosity and stiffness similar to that of lumbar cancellous bone and immersed in 200 ml of fluorescein tracer solution (376 Da, 0.1 mg / mL in PBS) (Figure 2). A servo-hydraulic loading frame (MTS Bionix 858, MTS Systems Corp., Eden Prairie, MN) was used to apply a load that resulted in physiological disc pressure. Specifically, a 6-hour static compressive load representing the lower end of the range of intradiscal pressures measured during various static postures (0.2 MPa) was applied (Nachemson 1966; Wilke, et al. 1999), followed by 1.6 hours of cyclic compressive loading, resulting in a pressure range (0.4-0.8 MPa) and frequency (0.2 Hz) similar to that measured within the intervertebral disc during walking (Wilke, et al., 1999).

[0114] NP and CEP tissue sampling: Immediately after the transport experiment, the discs were flash-frozen in liquid nitrogen to prevent any further fluorescein dispersion. Next, two 5 mm-thick coronal slabs were cut using a low-speed diamond saw (IsoMet, Buehler, IL) (Figure 3B). From each slab, three NP samples were collected using a 2 mm biopsy punch at four superior-inferior positions (Figure 3C). The superior and inferior CEP layers were then carefully removed from the disc, and any remaining NP tissue was trimmed (Figure 3D). Two CEP samples were collected from each intact CEP using a 4 mm biopsy punch (Figure 3D). All NP and CEP samples were massed on a microbalance, dried by lyophilization, and thoroughly digested in papain for subsequent assays.

[0115] Fluorescein concentration: NP tissue digestion was assayed for fluorescence using a SpectraMax iD5 microplate reader (Molecular Devices, San Jose, CA) and fluorescence readings (ex. 485 nm, em. 525 nm) referenced to a standard curve of known fluorescein concentrations. Fluorescein concentrations within NP samples were calculated based on measured water content from lyophilization and compared between site-matched locations within the disc with collagenase-treated and buffer-treated CEPs.

[0116] CEP biochemical composition: CEP tissue digests were assayed for sGAG content using the dimethylmethylene blue assay (DMMB) (Farndale RW 1986, Sampson 2019). Digest absorbance was measured at 595 nm in a microplate reader and referenced to a standard curve of known chondroitin sulfate concentrations.

[0117] Aliquots of the CEP digest were also hydrolyzed in 6N HCl for 18 hours at 110°C. The neutralized acid hydrolysates were then assayed for total collagen content using the chloramine-T colorimetric assay (Fields A. 2014), referencing absorbance measurements against a standard curve of known hydroxyproline concentrations.

[0118] The total sGAG and collagen content in the CEP tissue was used to assess the effect of enzyme treatment on the CEP matrix composition.

[0119] Biomechanical properties of the whole disc: Axial force, crosshead displacement, and time data were collected during the transport experiments and processed with a custom script (MATLAB, Mathworks, Natwick, MA) to calculate the disc elastic modulus and total creep strain. The disc elastic modulus was considered to be the slope of the stress-strain curve at the maximum stress value in the linear portion of the static compression phase. The total creep strain was considered to be the ratio of the total displacement at the end of the static compression phase to the initial disc height.

[0120] statistics: A mixed-effects model, accounting for multiple measurements per subject via a random intercept, was used to estimate least-squares mean fluorescein concentration at each NP location, and least-squares mean sGAG and collagen content at the CEP. Differences between groups were tested using Tukey's HSD post-hoc test. One-way analysis of variance was used to compare whole disc compressive creep strain and elastic modulus between groups. Statistical analysis was performed in JMP Pro 15. p<0.05 was considered statistically significant.

[0121] result Effect of enzyme treatment on solute transport Intradiscal enzymatic treatment of CEP significantly improved solute transport into bovine coccygeal intervertebral discs (Figure 4A). Specifically, in NPs proximal to the supraspinal treated CEP, fluorescein tracer concentrations were 14-fold higher in discs with collagenase-treated CEP than in discs with control buffer-treated CEP (19.72 ± 5.17 µg / mL vs. 1.41 ± 1.11 µg / mL, p < 0.0001) and 7-fold higher than in discs with untreated CEP (2.81 ± 1.79 µg / mL, sham). In discs with buffer-treated CEPs and sham CEPs, fluorescein concentrations were generally highest near the CEPs, at locations A (buffer: 1.41 ± 1.11, sham: 2.81 ± 1.79 μg / mL) and D (buffer: 1.48 ± 1.66, sham: 1.35 ± 0.52 μg / mL), and lowest in the center of the disc, at locations B (buffer: 0.33 ± 0.14, sham: 0.81 ± 0.34 μg / mL) and C (buffer: 0.43 ± 0.27, sham: 0.71 ± 0.25 μg / mL).

[0122] Similarly, intradiscal enzymatic treatment of CEP improved solute transport into human lumbar intervertebral discs (Figure 4B). At location A, in the NPs proximal to the superiorly treated CEP, fluorescein concentrations were 10.8-fold higher in discs with collagenase-treated CEP than in discs with control buffer-treated CEP (55.27 ± 31.11 μg / mL vs. 5.14 ± 3.54 μg / mL, p < 0.0001). In discs with buffer-treated CEP, fluorescein concentrations varied by location; concentrations were generally highest adjacent to the CEP at locations A (5.14 ± 3.54 μg / mL) and D (7.14 ± 2.26 μg / mL) and lowest in the center of the disc at locations B (0.58 ± 0.18 μg / mL) and C (0.62 ± 0.19 μg / mL).

[0123] Effect of enzyme treatment on CEP matrix composition Intradiscal enzyme treatment significantly reduced the amount of solute-blocking matrix components in both bovine and human CEPs. Specifically, collagenase treatment reduced sGAG content, with treated bovine CEPs exhibiting an average of 40% lower sGAG content than their untreated counterparts (p=0.03, Figure 5A). Untreated CEPs and CEPs treated with control buffer had similar sGAG content. Similarly, the same enzyme dose delivered intradiscally to human CEPs significantly reduced sGAG content, with the upper CEPs exhibiting an average of 33.5% lower sGAG content than their lower CEP counterparts (p=0.01, Figure 5B).

[0124] Collagenase treatment tended to be associated with lower collagen content in treated CEPs, but the difference was not statistically significant. Specifically, mean collagen content was lower in the upper treated CEPs (156.79 ± 84.55 μg / mg dry weight) than in the lower CEPs of their pairs (233.39 ± 85.73 μg / mg dry weight, p = 0.21, Figure 6A), but the difference did not reach statistical significance. In collagenase-treated human discs, the collagen content in the treated upper CEPs (327.75 ± 188.65 μg / mg dry weight) tended to be lower than in the untreated CEPs of their pairs (334.7186 ± 208.01 μg / mg dry weight, p = 0.07, Figure 6B).

[0125] Effect of enzyme treatment on NP tissue composition To understand how the enzyme treatment delivered to the CEP affected the composition of NP tissue, we measured sGAG content in human NP tissue samples assayed for fluorescein (Table 3). Collagenase treatment had a small local effect on the sGAG content of adjacent NP tissue. Specifically, the mean sGAG of NP samples adjacent to a CEP treated with collagenase at location A was 15.2% lower than that of NP tissue samples adjacent to an untreated CEP at location D within the same disc (p = 0.027) and 13.8% lower than that of tissue NP samples at location B within the same disc (p = 0.026). Comparisons of mean sGAG between NP tissue samples at other locations were not significant.

[0126] Effect of enzymatic treatment on the biomechanical behavior of whole intervertebral discs To determine the biomechanical effects of CEP matrix modification at the whole disc level, we compared the compressive modulus and total creep strain between groups by analysis of variance. In bovine discs, the total creep strain and compressive modulus were similar between discs with collagenase-treated, buffer-treated, and untreated CEPs (Figure 7A). Similarly, in human discs, the total creep strain and compressive modulus were similar between discs with collagenase-treated and buffer-treated CEPs (Figure 7B).

[0127] Consideration Here, we investigated intradiscal treatment by modifying the matrix of CEPs to enhance the transport of small solutes throughout the intervertebral disc. Collagenase enzyme was loaded into a degradable hydrogel carrier and then delivered to the superior CEP via a needle under fluoroscopic guidance. Results showed that collagenase treatment (0.15 U) significantly reduced the amount of sGAG in the treated CEPs. Enzyme treatment of human lumbar CEPs and bovine coccygeal CEPs reduced sGAG by 33.5% and 40%, respectively (Figure 5). Enzyme treatment also resulted in a reduction in collagen content, but the treatment effect was heterogeneous, and the difference in CEP collagen content between treated and untreated CEPs from the same disc was not statistically significant. Importantly, enzyme treatment of CEPs was consistent with greater transport of 376 Da fluorescein to the nucleus pulposus under static and cyclic compressive loading. Specifically, NP tissue adjacent to bovine and human CEPs treated with collagenase had 10.8-fold (bovine) and 14-fold (human) higher fluorescein concentrations compared to NP tissue adjacent to CEPs treated with buffer after loading (Figure 4). The similar pattern of findings in bovine discs with relatively healthy CEP and NP tissues and human discs with more degenerated CEP and NP tissues lends confidence to the generalizability of the treatment mechanism and the overall magnitude of the treatment effect. In a previous study of human CEPs, we observed that in-house treatment of CEPs with cleaved human recombinant MMP-8 dose-dependently reduced sGAG content, increased fluorescein uptake into unloaded CEPs, and improved nutrient transport and NP cell viability (Dolor et al., 2019). The new findings demonstrate for the first time the concept of CEP matrix modification in compressively loaded whole discs, and together, these findings support the potential of CEP matrix modification to improve the transport of small solutes in the size range of essential nutrients, such as glucose (180 Da).

[0128] The goal of CEP matrix modification is to reduce solute-blocking matrix components, improve CEP permeability to nutrients, and enhance disc cell viability / function (Dolor et al., 2019, Wong et al., 2019). In a previous study, a 6–22% reduction in the amount of sGAG in CEPs was consistent with a 16–24% increase in passive fluorescein uptake (Dolor et al., 2019). In vivo, intervertebral discs are subjected to a combination of static and cyclic loading, which improves solute transport through CEPs by inducing convection (Sampson et al., 2019). The effect of fluid flow on solute convection may explain why similar enzyme doses used in the current study resulted in a greater increase in fluorescein transport through CEPs than that observed with passive diffusion.

[0129] CEPs are rich in type II collagen (Moore, 2006; Nosikova et al., 2012; Schollmeier et al., 2000; Roberts, 1991) and aggrecan. For matrix modification, we used a commercially available form of collagenase P from Clostridium histolyticum (C. histolyticum) because this enzyme can cleave peptide bonds in the four types of collagen (I, II, III, and IV) (Alipour et al., 2016) and has proteolytic activity on proteoglycans. Matrix metalloproteinases (MMPs) also have high specificity for type II collagen and aggrecan (Dolor et al., 2019; Hideaki and Kashiwagi, 2002). In the current study, we chose a general collagenase enzyme rather than a specific human recombinant MMP because we treated both human and bovine CEPs. Nevertheless, the effect of collagenase P on human CEP composition was similar to that observed with cleaved human MMP-8 (Dolor et al., 2019). At the same time, our findings suggest that both enzymes reduce sGAG content in CEPs and have more subtle effects on collagen. Although collagen content in collagenase-treated CEPs was typically lower than that in buffer-treated CEPs, these differences were not statistically significant (Figure 6). One possible explanation is that cleaved collagen fragments remain intertwined within the CEP matrix, requiring longer exposure times to remove these fragments (Jiang et al., 2020).

[0130] Targeted delivery of matrix-modifying enzymes to CEPs presents several technical challenges. A 50 μL injection volume was selected based on preliminary intradiscal pressure testing. Injecting larger volumes increased backpressure, making it difficult to prevent leakage. The hydrogel carrier was selected to control the release of the enzyme treatment (auxiliary material). Intradiscal delivery was performed using a 17G Tuohy epidural needle with a curved end at the needle tip, which allowed the hydrogel to exit laterally at a 45-degree angle. In combination with the oblique needle trajectory, it was possible to perform the treatment adjacent to the target CEP (Figure 1). While these factors assisted in localizing the enzyme and minimizing its impact on the spinal cord tissue, we found that NP tissue adjacent to CEPs treated with these enzymes had a 15% lower sGAG content compared to NP tissue adjacent to untreated CEPs from the same intervertebral disc (Table 3). This suggests that other strategies may be necessary to further reduce off-target treatment effects. For example, attaching enzymes to large liposomal nanoparticles can help restrict enzyme mobility and reduce unwanted enzyme migration in NPs ( Dolor et al., 2019 ).

[0131] Proper CEP function requires balancing conflicting biotransport and biomechanical demands; therefore, successful matrix modification strategies to improve biotransport must be sensitive to any biomechanical effects. Specifically, excessive severing and removal of collagen fibrils or proteoglycans within the CEP may inadvertently reduce disc elasticity and risk clinically significant disc damage. The collagen network helps resist lateral CEP elongation that occurs during disc compression (Fields et al., 2010; DeLucca et al., 2016; Fields et al., 2014), and proteoglycans in the CEP are thought to help prevent the loss of proteoglycan aggregates from the nucleus pulposus (Roberts et al., 1996). The discs with enzyme-treated CEP had similar whole-disc elastic modulus and creep strain to those treated with buffer solution (Figure 7), suggesting that the moderate dose used here was sufficient to improve solute transport without affecting whole-disc behavior under static and cyclic compressive loading. Additional mechanical testing using more complex loading protocols is required to further understand the biomechanical effects of CEP matrix modification.

[0132] conclusion We found that intradiscal delivery of matrix-modifying enzymes to CEPs significantly reduced the amount of solute-blocking matrix components, particularly sGAGs. Treating CEPs in this way dramatically improved small solute transport into the disc, an important step toward in vivo preclinical testing.

[0133] References Alipour, H., Raz, A., Zakeri, S., Dinparast Djadid, N., 2016. Therapeutic applications of collagenase(metalloproteases): A review. 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Matta,A.,Karim,M.Z.,Isenman,D.E.,Erwin,W.M.,2017.Molecular Therapy for Degenerative Disc Disease:Clues from Secretome Analysis of the Notochordal Cell-Rich Nucleus Pulposus.Scientific Reports 7.doi.org / 10.1038 / srep45623 Moore,R.J.,2006.The vertebral endplate:Disc degeneration,disc regeneration, in:European Spine Journal.doi.org / 10.1007 / s00586-006-0170-4 Nachemson,A.M.D..The Load on Lumbar Disks in Different Positions of the Body.Clinical Orthopaedics and Related Research:March 1966-Volume 45-Issue-p 107-122 Nachemson,A.,Lewin,T.,Maroudas,A.,Freeman,M.A.R.,1970.In vitro diffusion of DYE through the end-plates and the annulus fibrosus of human lumbar inter-vertebral discs.Acta Orthopaedica 41,589-607.doi.org / 10.3109 / 17453677008991550 Nosikova,Y.S.,Santerre,J.P.,Grynpas,M.,Gibson,G.,Kandel,R.A.,2012.Characterization of the annulus fibrosus-vertebral body interface:Identification of new structural features.Journal of Anatomy 221,577-589.doi.org / 10.1111 / j.1469-7580.2012.01537.x Roberts S,Urban JP,Evans H,Eisenstein SM.Transport properties of the human cartilage endplate in relation to its composition and calcification.Spine.1996 Feb;21(4):415-420.DOI:10.1097 / 00007632-199602150-00003.PMID:8658243 Roberts S,M.J.D.V.W.S.A.S.,1991.1948394.Spine(Phila Pa 1976)16,1030-1038. Rodriguez,A.G.,Slichter,C.K.,Acosta,F.L.,Rodriguez-Soto,A.E.,Burghardt,A.J.,Majumdar,S.,Lotz,J.C.,2011.Human disc nucleus properties and vertebral endplate permeability.Spine(Phila Pa 1976)36,512-520.doi.org / 10.1097 / BRS.0b013e3181f72b94 Sampson,S.L.,Sylvia,M.,Fields,A.J.,2019.Effects of dynamic loading on solute transport through the human cartilage endplate.Journal of Biomechanics 83,273-279.doi.org / 10.1016 / j.jbiomech.2018.12.004 Schollmeier G,Lahr-Eigen R,Lewandrowski KU.Observations on fiber-forming collagens in the anulus fibrosus.Spine(Phila Pa 1976).2000 Nov 1;25(21):2736-41.doi:10.1097 / 00007632-200011010-00004.PMID:11064517 Vassilaki,M.,Hurwitz Dc,E.L.,2014.Insights in public health:perspectives on pain in the low back and neck:global burden,epidemiology,and management.Hawai’i journal of medicine & public health:a journal of Asia Pacific Medicine & Public Health,73(4),122-126. Wilke HJ, Neef P, Caimi M, Hoogland T, Claes LE. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine(Phila Pa 1976). 1999 Apr 15;24(8):755 - 62. doi:10.1097 / 00007632 - 199904150 - 00005. PMID:10222525 Wong, J., Sampson, S.L., Bell - Briones, H., Ouyang, A., Lazar, A.A., Lotz, J.C., Fields, A.J., 2019. Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy. Osteoarthritis and Cartilage 27, 956 - 964. doi.org / 10.1016 / j.joca.2019.01.013 [Table 1] [Table 2] [Table 3]

[0134] Example 2 Treatment of CEP with full-length MMP-8 reduces collagen content and increases small solute transport To test for enzymes with collagen-binding ability, we used full-length recombinant human MMP-8 (MMP8-1132H, Creative BioMart). Human CEPs from different donors were treated with equivalent doses of each form of the enzyme (2 U / mL in 0.1 mL of reaction buffer) for 18 h at 37 °C. Results showed that collagen content was 25.8% lower in site-matched CEP tissues treated with full-length but not cleaved MMP-8 (Figure 11). Conversely, sGAG was 14.8% lower in CEPs treated with cleaved, but not full-length, MMP-8 (p = 0.89). These data suggest that full-length MMP-8 primarily reduces collagen, whereas cleaved MMP-8 primarily reduces aggrecan or sGAG in CEPs. Treatment with full-length MMP-8 also improved small solute transport. Briefly, the CEP was placed in a custom permeameter whose upstream inlet was connected to a pressurized solute reservoir (fluorescein, 376 Da). Fluid and solutes passing through the CEP were collected downstream for 80 min (steady state) and the solute concentration (C 下流 ) and upstream values ​​(C = C 下流 / C 上流 ) was normalized to . This validated approach allows for comparison of solutes and treatments [3]. In a pilot study, the fluorescein concentration ratio C from one patient was approximately two-fold higher in site-matched biopsies treated with full-length MMP-8 (2.0 U / mL) than in biopsies treated with control buffer (Figure 12).

[0135] Example 3 CEP treatment with full-length MMP-8 has few direct and indirect off-target matrix effects Mass spectrometry was used to profile peptides, including "release products" from CEP proteolysis by MMP-8 (n = 4 patients), in untreated (n = 4 patients) and MMP-8-treated (n = 2 patients) human CEP biopsies. We identified 8,664 peptides corresponding to 250 unique proteins in the untreated CEP samples and 5,653 peptides belonging to 201 unique proteins in the treated CEP samples. Exogenous MMP-8 was identified in all treated samples but not in any of the untreated samples, suggesting good penetration of the enzyme into the tissue. Among the proteins discovered were known targets of full-length MMP-8, including collagen II (COL2A1). In release products from full-length MMP-8-treated CEPs, we identified 288 peptides belonging to 24 unique proteins, with collagen II being the most abundant (more than three times more abundant than the next most abundant peptide). Two collagen subtypes (COL3A1 and COL5A2) not identified in treated or untreated CEPs were also identified in the releasate. This collagen enrichment suggests the productive collagenolytic activity of MMP-8. Finally, of the proteins identified in untreated CEPs, only 16 (6.4%) were found in the treated releasate, supporting the specificity of full-length MMP-8 activity with little direct or indirect off-target effects on the matrix. To our knowledge, this is the first proteomic analysis of human CEPs.

[0136] Example 4 CEP cells show reduced sensitivity to matrix fragments derived from MMP-8 proteolysis. We used cell culture to test the response of CEP cells to matrix fragments resulting from proteolysis by MMP-8. Briefly, CEP cells were isolated from surgical waste tissue collected from five patients (3 / 2 females / 2 males; 64.6 ± 4.1 years old), expanded for three passages, and then cultured for 72 hours in four conditions: basal growth medium (negative control); medium supplemented with 100 ng / mL Pam2CSK4 (a TLR2 / 6 agonist, positive control); and medium supplemented with matrix fragments ("releasate") resulting from 12 hours of treatment of patient-matched CEP tissue with 0.2 or 2.0 U / mL full-length MMP-8. After culture, the medium was assayed by ELISA. Whereas a TLR2 / 6 agonist (Pam2CSK4) significantly increased the production of inflammatory cytokines (IL-6) and chemokines (CCL5) in CEP cells, we found almost minimal effects of MMP-8-derived release (Figure 13).

[0137] Example 5 Intradiscal treatment of CEPs with full-length MMP-8 increases NP cell viability We also tested CEP treatment in a bovine organ culture model. Superior CEPs from intervertebral discs from two fresh bovine tails (3 discs / tail) were intradiscally treated with full-length MMP-8 (3.0 U / mL) in an alginate carrier (50 μL) or control buffer (0 U / mL, HBSS) as described above. Discs were then cultured through a porous platen in growth medium (low-glucose DMEM containing 5% FBS and NaCl) for 2 weeks at 21% / 5% O2 / CO2 and 37°C, with daily compressive loading (0.2 MPa, 8 h / day). After culture, cell viability was measured using confocal microscopy after staining with the Live / Dead assay (1 disc / group), and fluorescein transport was measured after biomechanical testing as described above [2] (2 discs / group). In discs with MMP-8-treated CEPs, cell viability in the central NP was 13% higher (89% vs. 76%, Figure 14A), consistent with up to threefold higher fluorescein concentrations near the treated CEPs (Figure 14B). Although confirmation in additional discs is required, these results are consistent with our findings in diffusion chambers

[10] and suggest that the zone of improved nutrient transport induced by CEP treatment is sufficient to improve cell viability in the central NP. More generally, these results have important translational implications, as they suggest that a one-time treatment to reduce the impairment of solute transport through CEPs can have an impact on long-term improvements in NP cell viability.

Claims

1. A method for treating degenerative disc disease in a subject in need thereof, comprising locally administering a therapeutically effective amount of one or more matrix-modifying enzymes to the cartilage endplate (CEP).

2. 10. The method of claim 1, wherein the treatment increases the permeability of the CEP and intervertebral disc nutrient transport.

3. 3. The method of claim 1 or 2, wherein the one or more matrix-modifying enzymes have proteolytic activity against collagen, proteoglycans, or a combination thereof.

4. The method of claim 3 , wherein the one or more matrix-modifying enzymes comprises collagenase.

5. 5. The method of claim 4, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof.

6. 6. The method of claim 5, wherein the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof.

7. The method of claim 6, wherein the MMP8 is a full-length MMP8.

8. The method of any one of claims 1 to 7, wherein the one or more matrix-modifying enzymes are encapsulated in a hydrogel.

9. The method of claim 8 , wherein the hydrogel comprises alginate.

10. 10. The method of claim 9, wherein the concentration of the alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%).

11. The method of claim 10, wherein the concentration of the alginate in the hydrogel is about 1% by weight.

12. The method according to any one of claims 9 to 11, wherein the alginate is at least partially oxidized.

13. The method of claim 12, wherein about 4% to about 10% of the alginate is oxidized.

14. 14. The method of any one of claims 8 to 13, wherein the hydrogel maintains delivery of the matrix-modifying enzyme for at least 2 days.

15. 8. The method of any one of claims 1 to 7, wherein the one or more matrix-modifying enzymes are encapsulated in liposomes or conjugated to the surface of liposomes.

16. 16. The method of claim 15, wherein the liposome comprises a phospholipid.

17. 17. The method of claim 16, wherein the phospholipid is phosphatidylcholine or phosphatidylethanolamine.

18. The method of any one of claims 15 to 17, wherein the liposome further comprises cholesterol.

19. The method of any one of claims 15 to 18, wherein the liposome further comprises polyethylene glycol.

20. 20. The method of any one of claims 1 to 19, wherein the one or more matrix modifying enzymes are administered in a volume small enough so that intradiscal pressure does not increase substantially.

21. 21. The method of claim 20, wherein the one or more matrix-modifying enzymes are administered in a volume of 50 μL or less.

22. 22. The method of any one of claims 1 to 21, wherein the one or more matrix modifying enzymes are administered within or adjacent to the CEP.

23. 23. The method of any one of claims 1 to 22, wherein the one or more matrix-modifying enzymes are administered with an epidural needle.

24. 24. The method of claim 23, wherein the epidural needle has a curved end such that the matrix-modifying enzyme exits the epidural needle laterally at an angle of about 45 degrees.

25. 25. The method of any one of claims 1 to 24, further comprising using medical imaging to guide local administration of the one or more matrix-modifying enzymes to the CEP or to select which intervertebral discs may benefit from treatment with the one or more matrix-modifying enzymes.

26. 26. The method of claim 25, wherein the locally administering of the one or more matrix-modifying enzymes to the CEP is performed under fluoroscopic guidance, computed tomography (CT) guidance, combined CT fluoroscopic guidance, or ultrasound guidance.

27. 27. The method of any one of claims 1 to 26, wherein the one or more matrix-modifying enzymes are administered locally to the CEP without exposing the nucleus pulposus to the one or more matrix-modifying enzymes.

28. The method of any one of claims 1 to 27, further comprising administering a growth factor.

29. 29. The method of any one of claims 1 to 28, further comprising administering an analgesic or anti-inflammatory agent.

30. 30. The method of any one of claims 1 to 29, wherein multiple cycles of treatment are administered to the subject.

31. A composition comprising one or more matrix modifying enzymes for use in a method for treating degenerative disc disease.

32. 32. The composition of claim 31, further comprising a pharmaceutically acceptable excipient.

33. 33. The composition of claim 31 or 32, wherein the one or more matrix-modifying enzymes comprises collagenase.

34. 34. The composition of claim 33, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof.

35. 35. The composition of claim 34, wherein the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof.

36. 36. The composition of claim 35, wherein the MMP8 is full-length MMP8.

37. The composition of any one of claims 31 to 36, wherein the one or more matrix-modifying enzymes are encapsulated in a hydrogel.

38. 38. The composition of claim 37, wherein the hydrogel comprises an alginate.

39. 39. The composition of claim 38, wherein the concentration of the alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%).

40. 40. The composition of claim 39, wherein the concentration of the alginate in the hydrogel is about 1% by weight.

41. 41. The composition of any one of claims 38 to 40, wherein the alginate is at least partially oxidized.

42. 42. The composition of claim 41, wherein about 4% to about 10% of the alginate is oxidized.

43. 43. The composition of any one of claims 37 to 42, wherein the hydrogel maintains delivery of the matrix-modifying enzyme for at least 2 days.

44. 37. The composition of any one of claims 31 to 36, wherein the one or more matrix-modifying enzymes are encapsulated in or conjugated to the surface of a liposome.

45. 45. The composition of claim 44, wherein the liposome comprises a phospholipid.

46. 46. ​​The composition of claim 45, wherein the phospholipid is phosphatidylcholine or phosphatidylethanolamine.

47. The composition of any one of claims 44 to 46, wherein the liposome further comprises cholesterol.

48. 48. The composition of any one of claims 44 to 47, wherein the liposome further comprises polyethylene glycol.

49. The composition of any one of claims 31 to 48, further comprising a growth factor.

50. 50. The composition of any one of claims 31 to 49, further comprising an analgesic or anti-inflammatory agent.

51. A method for increasing intervertebral disc solute transport in a subject in need thereof, comprising locally administering to the cartilage endplate (CEP) an effective amount of one or more matrix-modifying enzymes.

52. 52. The method of claim 51, wherein said administering increases the permeability of said CEP and intervertebral disc nutrient transport.

53. 53. The method of claim 51 or 52, wherein the one or more matrix-modifying enzymes have proteolytic activity against collagen, proteoglycans, or a combination thereof.

54. 54. The method of claim 53, wherein the one or more matrix-modifying enzymes comprises collagenase.

55. 55. The method of claim 54, wherein the collagenase has proteolytic activity against collagen I, collagen II, collagen III, collagen IV, or any combination thereof.

56. 56. The method of claim 55, wherein the collagenase is collagenase P, matrix metalloproteinase 8 (MMP8), or a combination thereof.

57. 57. The method of claim 56, wherein the MMP8 is full-length MMP8.

58. 58. The method of any one of claims 51 to 57, wherein the one or more matrix-modifying enzymes are encapsulated in a hydrogel.

59. 59. The method of claim 58, wherein the hydrogel comprises alginate.

60. 60. The method of claim 59, wherein the concentration of the alginate in the hydrogel ranges from 0.5 to 10 weight percent (wt%).

61. 61. The method of claim 60, wherein the concentration of the alginate in the hydrogel is about 1% by weight.

62. 62. The method of any one of claims 59 to 61, wherein the alginate is at least partially oxidized.

63. 63. The method of claim 62, wherein about 4% to about 10% of the alginate is oxidized.

64. 64. The method of any one of claims 58 to 63, wherein the hydrogel maintains delivery of the matrix-modifying enzyme for at least 2 days.

65. 58. The method of any one of claims 51 to 57, wherein the one or more matrix-modifying enzymes are encapsulated in or conjugated to the surface of a liposome.

66. 66. The method of claim 65, wherein the liposome comprises a phospholipid.

67. 67. The method of claim 66, wherein the phospholipid is phosphatidylcholine or phosphatidylethanolamine.

68. 68. The method of any one of claims 65 to 67, wherein the liposome further comprises cholesterol.

69. 69. The method of any one of claims 65 to 68, wherein the liposome further comprises polyethylene glycol.

70. 70. The method of any one of claims 51 to 69, wherein the one or more matrix modifying enzymes are administered in a volume small enough so that intradiscal pressure does not increase substantially.

71. 71. The method of claim 70, wherein the one or more matrix-modifying enzymes are administered in a volume of 50 μL or less.

72. 72. The method of any one of claims 51 to 71, wherein the one or more matrix modifying enzymes are administered within or adjacent to the CEP.

73. 73. The method of any one of claims 51 to 72, wherein the one or more matrix modifying enzymes are administered with an epidural needle.

74. 74. The method of claim 73, wherein the epidural needle has a curved end such that the one or more matrix-modifying enzymes exit the epidural needle laterally at an angle of about 45 degrees.

75. 75. The method of any one of claims 51 to 74, further comprising using medical imaging to guide local administration of the one or more matrix-modifying enzymes to the CEP or to select which intervertebral discs may benefit from treatment with the one or more matrix-modifying enzymes.

76. 76. The method of claim 75, wherein the locally administering of the one or more matrix-modifying enzymes to the CEP is performed under fluoroscopic guidance, computed tomography (CT) guidance, combined CT fluoroscopic guidance, or ultrasound guidance.

77. 77. The method of any one of claims 51-76, wherein the one or more matrix modifying enzymes are administered locally to the CEP without exposing the nucleus pulposus to the one or more matrix modifying enzymes.

78. 78. The method of any one of claims 51 to 77, further comprising administering a growth factor.

79. 79. The method of any one of claims 51 to 78, further comprising administering an analgesic or anti-inflammatory agent.

80. 80. The method of any one of claims 51 to 79, further comprising detecting whether intervertebral disc solute transport is inhibited in the CEP prior to locally administering the one or more matrix modifying enzymes to the CEP to determine whether the subject will benefit from the treatment.

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