Compositions and methods for treating defects in avascular cartilage tissue by directly administering one or more metabolites of simvastatin

Abstract

To provide methods for the treatment of patients suffering from injured or degenerating avascular cartilaginous tissue.SOLUTION: Provided is a method comprising the step for administering directly to the site of the avascular tissue of a subject controlled release hydrogel formulations of one or more simvastatin metabolites selected from 3'-hydroxy simvastatin (hSV), 6'-exomethylene simvastatin (eSV), 3',5'-dihydrodiol simvastatin, 3',5'-dihydrodiol simvastatin (dSV), and simvastatin-beta-hydroxy acid (SVA).SELECTED DRAWING: None

Description

Technical Field

[0001] Priority Claim This application claims priority to U.S. Provisional Patent Application No. 62 / 506,104, filed May 15, 2017, the entire disclosure of which is hereby incorporated by reference herein.

[0002] Embodiments of the present invention generally relate to therapeutic pharmacology, and in particular, effective methods and compositions for treating subjects suffering from diseases and conditions characterized by damaged or otherwise defective cartilage tissue, i.e., avascular cartilage tissue, by directly administering one or more metabolites of simvastatin to the avascular tissue.

Background Art

[0003] Simvastatin (SV) is a drug currently widely prescribed for treating cardiovascular disease / hypercholesterolemia, and its derivatives have recently been used in many other applications, including those for promoting chondrogenesis in intervertebral disc cells and improving intervertebral disc disease.

[0004] Based on an investigation of a library of over 30,000 natural compounds, Mundy and co-workers discovered that statins such as simvastatin (SV), which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are the only type of molecule that specifically increases BMP-2 mRNA in murine and human osteoblasts in vitro and induces subsequent bone formation in vivo. Statins are commonly prescribed cholesterol-lowering drugs that inhibit the cholesterol biosynthesis pathway. Since then, the discovery and underlying mechanism of this "side effect" of statins on bone anabolic action and bone protective effect have been the subject of intensive research, such as for regimens for osteoporosis. Further studies have also demonstrated that SV increases BMP-2 expression in various types of cells, such as non-transformed osteoblasts, bone marrow stromal cells, human vascular smooth muscle cells, and rat chondrocytes (see, for example, Zhang, H et al. (2008) Spine, 33(16), Zhang, H. et al. (2009) Arthritis Res Ther Arthritis Research & Therapy, 11(6), and Than, K.D. et al. (2014) The Spine Journal, 14(6), pp. 1017-1028, the entire disclosures of which are hereby incorporated by reference herein).

[0005] Degenerative disc disease (DDD) is considered a major cause of low back pain and is a common medical problem that imposes a significant socioeconomic burden. However, current clinical guidelines for the treatment of DDD are often associated with complications, especially when surgical intervention is involved. The biological repair or regeneration of the degenerative intervertebral disc (IVD) has been recommended with recent advances in recombinant therapeutic proteins, including recombinant human bone morphogenetic protein-2 (BMP-2). However, the required dosages of these recombinant human growth factors often exceed physiological dosages, increasing concerns regarding the potential for toxicity and other undesired complications.

[0006] As an alternative to current treatment regimens, tissue engineering and regenerative approaches have been the subject of intensive research efforts over the past decade. In particular, growth factors such as the bone morphogenetic protein (BMP) family have shown great promise in stimulating matrix regeneration within damaged intervertebral disc tissue. Although the initial results are encouraging , the clinical use of recombinant growth factors presents a number of concerns, including unwanted blood vessel growth within avascular intervertebral disc tissue, the need for supra-physiological concentrations for therapeutic efficacy which increases the risk of side effects, and the high costs associated with the manufacture of clinical grade recombinant proteins. As a result, there is a greater desire for regenerative pharmaceuticals without such problems.

[0007] The inventors have extensively studied the effects of SV over a period of more than 10 years and reported that SV stimulation promotes several phenotypic expressions of mammalian nucleus pulposus (NP) cells, including aggrecan, type II collagen, and glycosaminoglycans which help delay the progression of degeneration and promote the repair of degenerated IVDs. SV was found to be effective in promoting chondrogenesis by upregulating endogenous BMP-2 expression in treated NP cells and promoting in vivo repair of the affected IVD. A further advantage of SV treatment proposed for DDD is that the intradiscal injection procedure does not require open surgery, which also minimizes the risk of postoperative pain and recovery time, as well as excessive disc perturbation which ultimately results in unsightly deformities. The procedure is common and can be performed by many other clinical specialists in addition to spinal surgeons, making such an approach more affordable, practical, and adoptable within current healthcare systems. Therefore, SV is considered a promising alternative to protein-based regenerative pharmaceuticals for the treatment of DDD.

[0008] Based on the similarities between the compositions of the intervertebral disc and the meniscus, direct administration of SV is also being considered for use in improving healing by treating meniscal tissue defects and stimulating chondrogenesis in a manner similar to the intervertebral disc model. Meniscal tears are one of the most common knee injuries, causing substantial loss of productivity and a decline in quality of life for a large portion of the population, even among younger people. Physicians have reported that approximately one-third of the national population over the age of 50 has had at least one meniscal tear, and this group becomes more vulnerable to stability / falls and chronic pain as they age.

[0009] The inventors previously utilized a well-known meniscal injury model, created a circumferential lesion of sufficient thickness in the meniscus using a biopsy punch or k-wire, and used sustained drug delivery of SV with an FDA-approved biodegradable hydrogel, demonstrating new tissue growth within 4 weeks after injection (see Zhang & Lin, (2008) Spine, 33(16), and Zhang et al. (2016) The American Journal of Sports Medicine. doi:10.1177. The entire disclosure of which is incorporated herein by reference).

[0010] It is well known that systemically delivered SV undergoes extensive first-pass metabolism in the liver. As a result, the drug becomes rapidly hydrolyzed to several oxides, including 3'-hydroxy SV (hSV), 6'-exomethylene SV (eSV), 3',5'-dihydrodiol SV (dSV), and SV beta-hydroxy acid (SVA) (Figure 5). Some of these metabolite hydroxy acid forms, including SVA, have been found to be HMG-CoA reductase inhibitors, and subsequently, SVA has been recognized as a competitor to SV. As a result, it can be hypothesized that at least some of the therapeutic effects attributed to systemically administered SV are actually involved in one or more of the SV metabolites.

[0011] However, strictly speaking, even if SV is directly injected into the IVD or joint cavity, since both of them are avascular (without blood vessels that communicate or circulate blood or lymph), it would not be expected that SV metabolites would be present in the IVD or joint cavity. Direct injection bypasses liver metabolism, and thus it can be concluded that the regenerative effect of SV observed in avascular tissues is not involved with SV metabolites and is due to the pharmacophysiology of the SV activity itself.

[0012] As a result, prior to the investigations reported herein by the inventors, no studies have been conducted regarding which SV metabolites are similarly effective with respect to the increase in BMP-2 expression in avascular tissues, particularly with respect to eSV and SVA, which are metabolites known as competitive HMG-CoA reductase inhibitors. Furthermore, no studies have been conducted to verify whether SV metabolites that are non-HMG-CoA reductase inhibitors, namely hSV and dSV, can still control BMP-2 expression or regulate other cellular / molecular activators that have been observed. In particular, all potential advantages such as the increase in effectiveness accompanied by a decrease in injection volume, formulation advantages, and side effects associated with direct injection into the joint and intervertebral disc lead to further investigation of SV metabolites and strongly approach the discovery of an effective and relatively non-invasive method for regenerating defective avascular tissues. SUMMARY OF THE INVENTION

[0013] The inventors have surprisingly confirmed that SVA, a metabolite of SV, inhibits mevalonate conversion more efficiently than SV and, when administered directly, promotes assimilation 5- to 6-fold more than SV alone in the regeneration of avascular tissue. Furthermore, SVA has been found to contribute to an anti-catabolic effect, provide a synergistic effect, and promote chondrogenesis as observed with SV. Interestingly, it has also been found that administration of SV metabolites, which are non-HMG-CoA reductase inhibitors and include both dSV and hSV, promoted a certain degree of regeneration, suggesting that the mechanism associated with the effectiveness of the metabolites remains unclear. Thus, direct administration of a composition of one or more SV metabolites provides superior regenerative benefits to administration of SV alone for patients suffering from either DDD or meniscus injury.

[0014] Thus, one embodiment provides a method of repairing or delaying injury to substantially denatured or injured avascular cartilage tissue. The method includes directly administering to a site of avascular tissue in a subject in need thereof a composition comprising at least one oxidative metabolite of simvastatin (SV). In other embodiments, the method includes directly administering to a site of injured avascular tissue in a subject in need thereof at least one active that increases bone morphogenetic protein (BMP) expression without inhibiting HMG-CoA reductase. In certain embodiments, the at least one active is selected from hSV, dSV, and combinations thereof.

[0015] Another embodiment provides a controlled release composition formulated for injectable administration, the controlled release composition comprising at least one oxidative metabolite of simvastatin (SV).

[0016] Other embodiments are directed to methods for treating patients suffering from avascular cartilage tissue injury including, but not limited to, DDD and meniscus injury. These and other embodiments will be more fully described and made apparent by reference to the following drawings and detailed description.

[0017] The drawings are shown to illustrate specific embodiments and aspects of the present invention and should not be construed as limiting the full scope defined by the appended claims.

Brief Description of the Drawings

[0018]

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DETAILED DESCRIPTION OF THE INVENTION

[0019] Low back pain (LBP) is one of the most common medical problems in the United States, and about 80% of the US population is troubled at some point in their lives. It is also one of the most common reasons for missing work, and chronic LBP promotes opioid dependence and thus causes a huge socio-economic burden as well as public health problems. Among cases of LBP, whether specific (e.g., spinal tumors or infections) or nonspecific (without an obvious cause), degenerative disc disease (DDD) is considered a major contributor to LBP. Current clinical criteria for treating DDD are often associated with complications, especially when surgical intervention is involved. Therefore, the ability to biologically repair or regenerate abnormal discs in situ using therapeutic compounds is an attractive option for future treatment alternatives. Such a strategy is preferred not only to provide minimally invasive intervention but also to potentially promote the remodeling of the injured disc.

[0020] Currently available remodeling regimens involve the use of recombinant growth factors, which not only have very high manufacturing costs but also raise concerns regarding toxicity and other unwanted complications associated with exceeding the required physiological dosages. Thus, unfortunately, current standard treatments for DDD focus more on pain control, spinal stabilization, and slowing disease progression than on disc repair.

[0021] The inventors have previously shown that simvastatin (SV), a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor commonly prescribed as a cholesterol-lowering drug, promotes phenotypic expression of mammalian nucleus pulposus (NP) cells when drug-treated in vitro. In vivo, when the intervertebral disc (IVD) affected in a DDD rat model was injected with a controlled-release formulation of SV, the compound delayed the progression of degeneration and most significantly promoted the repair of the deteriorated IVD (anabolic effect). Furthermore, the known pleiotropic effects of simvastatin in anti-inflammation were also observed, where the expression of enzymes that degrade the extracellular matrix was suppressed (catabolic effect). These matrix metalloproteinases (MMPs) are typically stimulated by pro-inflammatory cytokines in the diseased intervertebral disc. The results provide the first evidence that SV is an excellent alternative to recombinant proteins for treating DDD. Nevertheless, the hydrophobicity of the SV prodrug significantly limits its local delivery using currently available / approved hydrogel vehicles.

[0022] Interestingly, in a recent study conducted by the inventors, simvastatin beta-hydroxy acid (SVA), an active hydrolytic metabolite of SV, was observed to be actually more effective in the upregulation of endogenous bone morphogenetic protein-2 (BMP-2), a mediator of intervertebral disc repair resulting from the effects observed with the prodrug SV, suggesting that at least one of the SV metabolites may dictate the effectiveness of SV observed in the treatment of IVDs.

[0023] NP cells are commonly referred to as "chondrocyte-like" cells. This is because these cells are initially notochordal but are gradually replaced during childhood by round cells that resemble chondrocytes of articular cartilage. NP cells maintain a chondrogenic phenotype for constructing the IVD tissue matrix, and when NP cells are exposed to BMP-2, the expression of the chondrogenic phenotype is promoted. Furthermore, recent findings have also shown that endogenously produced BMPs, including BMP-2, interfere with the effects of pro-inflammatory cytokines. Both phenomena are consistent with the above results. This experiment is designed to show that BMP-2 expression stimulated in avascular tissues is enhanced by administering the active metabolite SVA, which is also a potent competitor in the inhibition of HMG-CoA reductase, and by a composition containing SVA and at least one additional SV metabolite, as well as by a composition containing at least one active SV metabolite containing SV and metabolites other than HMG-CoA reductase inhibitors.

[0024] Statins are potent inhibitors of cholesterol biosynthesis. However, ongoing studies also indicate that some of the cholesterol-independent or "pleiotropic" effects of statins are more beneficial than can be expected from changes in lipid levels alone. Statins containing SV affect the enzymatic activity of protein prenylation, which is important for the function of downstream low-molecular-weight G-proteins. These G-proteins are regulators of many physiological responses and intracellular signaling pathways, including polarity, gene transcription, and intracellular vesicular transport. Therefore, there is interest in the effects of SV metabolites on Rho family and its subfamily Rac G-proteins. SV confers an anti-inflammatory effect by inactivating Rho, which is related to what was observed in the suppression of MMP, while Rac reduces oxidative stress when inhibited by SV. Recent studies have reported that oxidative stress induced in NP cells is associated with intervertebral disc degeneration. Therefore, it is important to elucidate the role of SV metabolites in the downregulation of the two G-proteins observed with the SV prodrug.

[0025] In systemic delivery, SV undergoes hepatic metabolism and generates various metabolites including several hydroxy acids such as SVA (see Figure 5). These acidic metabolites may compete with the prodrug SV in terms of rate-limiting enzyme activity and potentially have a significant impact on certain biological activities. However, with respect to delivery to avascular tissues, neither systemic administration nor direct administration of SV is expected to confer active metabolites.

[0026] As described above, the discovery of the pleiotropic effects of statins on BMP-2 upregulation has generated intense investigation, particularly regarding their effects on bone anabolism and bone protection. To elucidate the basic mechanisms underlying upregulation, extended studies have also been conducted, and the results indicate that statins increase the expression of BMP-2 through the Ras / PI3K / Akt / MAPK (mitogen-activated protein kinase) / BMP-2 pathway (Ghosh-Choudhury N et al., J Biol Chem. 2007;282(7)). Chen et al. (Chen P.Y. et al., Nutr Res. 2010;30(3):191-199) confirmed these results and further reported that the PI3K / Akt pathway involved in statin-induced bone formation is dependent on the activation of the small molecular weight GTPase, Ras, which is promoted by localizing proteins on the intracellular membrane. In addition to bone, BMP has also been suggested as a potential therapeutic agent for IVD degeneration in studies focusing on the use of BMP2, 4, 7, and 14. All of these growth factors act on and function through the same receptor that requires the presence of BMPRII. However, only one study has investigated the expression of this receptor in human IVD tissue. Wang H. et al. (J Mol Med-Jmm. 2004;82(2):126-134) used reverse transcriptase PCR to demonstrate the expression of BMP receptors in six human idiopathic scoliosis intervertebral discs (IVD discs) and showed that mRNA for three receptors was expressed.

[0027] In Example 1, BMPRII localizes to NP and inner annulus fibrosus (AF) cells of 30 human intervertebral discs. Slight immunoreactivity was observed in cells from the outer AF. Cells in the NP showed immunoreactivity at a higher rate than those in the inner AF. This suggests that BMP applied to the human IVD will exhibit the most excellent effects within the NP and inner AF. These results are different from those observed in mice, in which receptor expression is only observed in the endplate cartilage and AF. Interestingly, changes in the expression level were not observed along with the degree of degeneration.

[0028] Findings from studies investigating SVs that stimulate endogenous BMP-2 expression in treated NP cells and increase chondrogenic phenotype expression (aggrecan and type II collagen mRNA expression and sGAG content) are consistent with the above observations, demonstrating that the small molecule is as effective as osteogenesis in promoting chondrogenesis. However, surprisingly, when these cells were treated with SVA, an active hydrolytic metabolite of SV, the upregulation of BMP-2 was stimulated more strongly. SV is widely prescribed to treat hypercholesterolemia and hypertriglyceridemia. In humans, it undergoes rapid metabolism to form four major oxidative NADPH-dependent metabolites, 3'-hydroxy SV (hSV), 6'-exomethylene SV (eSV), 3',5'-dihydrodiol SV (dSV), and SVA (Figure 5). Among these, SVA is the most potent competitor of SV in HMG-CoA reductase inhibition. This raised questions about whether SVA is dominant in the overall scheme of BMP-2 upregulation. To test how different the levels of BMP-2 induced by both SV and SVA would be, the inventors conducted tests using an in vitro model system developed in previous studies. Rat NP cells harvested from the caudal intervertebral disc were first cultured in monolayers and then in alginate beads (Zhang H. et al., Spine, 2008; 33(16), the entire disclosure of which is incorporated herein by reference). The cells were treated with DMSO (vehicle), 1 or 3 μM of SV or SVA. The cells were then collected at predetermined time points and RNA was extracted. Gene expression was analyzed by RT-qPCR. The results showed that at 1 μM, the mRNA expression of BMP-2 was the same or two-fold in cells treated with SVA compared to those treated with SV from day 1 to day 3. However, by day 7, the difference increased dramatically. The levels of BMP-2 induced in the SVA group were 5-6 times higher than those in the SV group (Figure 1A). When the treatment concentration was increased to 3 μM, the difference further increased (Figure 1B).

[0029] The results showed superior efficacy of SVA over SV for BMP-2 upregulation. The event of BMP-2 upregulation is a result of HMG-CoA reductase inhibition and can be achieved by these as SV, SVA, and eSV are all inhibitors. Further surprisingly, some regenerative abilities are established by administration of hSV and dSV, which are non-HMG-CoA reductase inhibitors, but the pathway is unknown.

[0030] SV can block the synthesis of either isoprenoid intermediate, farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP), and it is known that these inhibit the functions of downstream low molecular weight G-proteins such as the Ras, Rho, Rab families. The question of the mechanism pathway regarding the effects of hSV and dSV, which are non-HMG-CoA reductase inhibitors (whether they prenylate proteins or directly affect G-proteins) remains unresolved. Since Ras and Rho control BMP-2 expression through the Ras / PI3K / Akt / MAPK / BMP-2 pathway and both Rho and Rac can be associated with the observed anabolic and catabolic effects, a detailed analysis of the mechanism by which the metabolites contribute to the efficacy obtained with SV could lead to additional therapeutic strategies. A scheme explaining this is shown in Figure 2.

[0031] The classical mechanism understood for cholesterol lowering by statins is that statins act by competitively inhibiting HMG-CoA reductase, an enzyme that is involved in the first step of the mevalonate (MVA) pathway. This competition reduces the rate at which HMG-CoA reductase can produce MVA and the next molecule in the cascade for synthesizing the prenylation enzyme substrates FPP and GGPP, which ultimately helps to produce cholesterol. As shown in previous studies by the inventors (Zhang H.N., Lin C.Y. Spine. 2008;33(16), which is hereby incorporated by reference in its entirety), when SV was present, BMP-2 mRNA expression in NP cells always responded in a time- and dose-dependent manner. Furthermore, the stimulation in NP treated with 3 μM SV was independent of the presence of cholesterol (Figure 3A) as well as FPP (Figure 3B). Instead, when the cells were pretreated with MVA, the stimulation was completely reversed As was shown by observation, the stimulation was actually involved in the MVA pathway. Interestingly, reversal was also achieved when the downstream substrate GGPP was replenished (Figure 3B). Next, when either of the GGTase inhibitors GGTI-286 and POH was given to mimic the inhibition of GGPP enzyme activation by SV, both of them were able to increase BMP-2 mRNA expression, but the levels were much lower than those observed with SV (Figure 3C). When IVD cells were co-treated with GGTI286 and POH, the BMP-2 upregulation was significantly higher than with each individual treatment (Figure 3D). However, the BMP-2 upregulation by co-treatment still did not reach the level corresponding to that with SV treatment, suggesting that a mechanism distinct from the inhibition of HMG-CoA reductase can synergistically promote BMP-2 expression. Without being bound by theory, the inventors have discovered that SV metabolites other than the HMG-CoA reductase inhibitor, namely hSV and dSV, also affect BMP-2 upregulation.

[0032] One embodiment is a method of repairing or delaying damage to a substantially avascular cartilage tissue that has been injured, the method comprising administering a composition comprising at least one oxidative metabolite of simvastatin (SV) directly to a site of avascular tissue in a subject in need thereof. According to further specific embodiments, the administering step comprises administering a controlled release formulation of at least one oxidative metabolite of simvastatin (SV), and the composition is released at a rate and in an amount effective to repair or delay damage in the avascular cartilage tissue. At least one oxidative metabolite of SV is selected from the group consisting of 3'-hydroxysimvastatin (hSV), 6'-exomethylene simvastatin (eSV), 3',5'-dihydrodiol simvastatin, 3',5'-dihydrodiol simvastatin (dSV), simvastatin-beta-hydroxy acid (SVA), and combinations thereof. According to highly specific embodiments, at least one oxidative metabolite of SV comprises SVA.

[0033] In some embodiments, it is contemplated that SV may be administered together with at least one metabolite, where "together with" includes co-administration, tandem administration, or administration within a treatment time frame. If the administration is co-administration, it may be in one dosage unit or multiple units. The treatment time frame may be any time frame during which the patient is undergoing treatment for an injured or degenerated cartilage tissue. The treatment regimen may include a single administration or multiple administrations over the treatment time frame. According to highly specific embodiments, the cartilage tissue includes intervertebral disc cartilage / fibrocartilage, and in other specific embodiments, the cartilage tissue is in a joint. According to further specific embodiments, the cartilage tissue includes meniscal cartilage.

[0034] According to some embodiments, when the subject suffers from degenerative disc disease, administration includes, for example, direct administration into the disc lumen by injection or by means of a guide catheter. The injection may be performed using a fluoroscope to guide a syringe carrying a controlled release composition of one or more of the metabolites, with or without a formulation, for example, SV. Administration of the controlled release composition promotes the proliferation of chondrocytes or chondrocyte-like cells at the damaged cartilage site. According to certain embodiments, the subject is a mammal, and in highly specific embodiments, the mammal is a human.

[0035] According to certain embodiments, a controlled release composition is provided that includes one or more hydrogels containing an active agent. Exemplary hydrogels suitable for drug delivery formulations include chitosan (CT), cyclodextrin (CD), p-dioxanone (DX), ethylene glycol (EG), ethylene glycol dimethacrylate (EGDMA), hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide (MBAAm), poly(acrylic acid), polyacrylamide, polycaprolactone, poly( ethylene glycol), poly(ethyleneimine), poly(ethylene oxide), poly(ethyl methacrylate), poly(hydroxyethyl methacrylate), poly(hydroxypropyl methacrylamide), poly(lactic acid) (PLA), poly(lactic acid-co-glycolic acid) (PLGA), poly(methyl methacrylate) (PMMA), poly(propylene oxide), poly(vinyl alcohol) (PVA), poly(vinyl acetate), poly(vinylamine), and combinations thereof.

[0036] According to some embodiments, the hydrogel comprises a hydrophobic polymer and a hydrophilic polymer, and in some embodiments, the polymer is a homopolymer or a copolymer. In the hydrogel, the hydrophilic polymer may be included in the range of about 10% to 50%, about 20% to 40%, or about 20% to 30%, and the hydrophobic polymer may be included in the range of 40% to 90%, about 60% to 80%, or about 70% - 80%. According to very specific embodiments, the hydrophilic polymer is included. In other very specific embodiments, the hydrophobic polymer comprises CT. More specifically, HA comprises HA-Na polyanion, and CT comprises CT-NH3 + comprises a polycation, and the mass ratio of CT to HA (CT to HA) is about 60:40. In this paragraph, "about" means + / -2%. At least one metabolite (with or without including SV) of a certain amount is dispersed within the hydrogel matrix. The metabolite is selected from the group consisting of 3'-hydroxysimvastatin (hSV), 6'-exomethylene simvastatin (eSV), 3',5'-dihydrodiol simvastatin, 3',5'-dihydrodiol simvastatin (dSV), simvastatin-beta-hydroxy acid (SVA), and combinations thereof. According to specific embodiments, the active agent comprises SVA. According to other specific embodiments, the active agent is selected from hSV, dSV, and combinations thereof. According to some embodiments, the amount of the active agent dispersed in the controlled release hydrogel is included in the range of 1 to 50 mg / ml, including all ranges and numerical amounts therebetween.

[0037] Another embodiment provides a method for repairing or delaying the injury of a substantially avascular cartilage tissue that has been damaged. The method includes the step of directly administering at least one active agent that increases bone morphogenetic protein (BMP) expression without inhibiting HMG-CoA reductase to the site of the damaged avascular tissue of a subject in need thereof. According to specific embodiments, the active agent is selected from hSV, dSV, and combinations thereof.

[0038] Examples are presented to illustrate and prove specific embodiments and should not be construed as limiting the full scope of the invention as defined by the appended claims.

Example

[0039] This example demonstrates the effectiveness of a model system of modified human NP cells and tests SV and its metabolites. The prior literature was all based on in vitro and in vivo investigations using rodents and thus worked towards establishing a model system with degenerated human NP cells. Interestingly, the results show different patterns regarding the human cell response to drugs compared to other studies using rat and pig cells. When IVD cells collected from human patients presenting with DDD were exposed to SV, these cells were stimulated and maintained or even increased in a dose-dependent manner their chondrogenic phenotype. However, there were different expression patterns compared to those in rat IVD cells (see Zhang HN, Lin CY, Spine. 2008; 33(16)). SV upregulated BMP-2 mRNA expression in both human NP cells and annulus fibrosus (AF) cells, as observed in rat cells. Furthermore, both NP cells and AF cells expressed the BMP-2 receptor, BMPRII, and both types of cells were sensitive to the upregulation of BMP-2 induced by SV and mediated the expected pathway (data not shown). However, when human NP cells were treated with the same doses of SV (0.3 to 3 μM) that were also used to treat rat cells, the mRNA expression of aggrecan and type II collagen was not affected. Alternatively, SV suppressed type I collagen mRNA expression in a dose-dependent manner and thus significantly increased the ratio of type II to type I collagen (Figure 4). This phenomenon was only observed in human NP cells compared to those from other species used (rat (Zang et al., 2008), rabbit and pig, data not shown). The mRNA expression of aggrecan, type II collagen and type I collagen in human AF cells did not change even after treatment. The result that the ratio of Col II / Col I (also referred to as the "differentiation index") increased suggests that SV would have suppressed the dedifferentiation of human NP cells in degenerated intervertebral discs and assisted in maintaining their chondrogenic phenotype.

[0040] Based on this discovery, in this study, human NP cells are used to further facilitate the strategies proposed to repair human IVD. However, to obtain a high degree of consistency, especially regarding experiments using CRISPR genome editing technology, a human NP cell line derived from Taipei Medical University and Hospital, Taipei, Taiwan, by Dr. Win-Ping Deng (Liu M.C. et al., Tissue Eng Part C-Me. 2014; 20(1): 1-10, which is hereby incorporated by reference in its entirety) was used.

[0041] SV metabolites All compounds in this study, including SVA, eSV, hSV, and dSV, were synthesized and characterized (e.g., chemical structure, solubility, particle size, impurities, and polymorphism) by AAPharmaSyn, LLC, a global chemistry contract research organization founded and operated by former Pfizer chemists with extensive knowledge and experience in statins, including the success in the development of LIPITOR (trademark) (atorvastatin). This company also developed hSV and dSV, two SV metabolites that are non-HMG-CoA reductase inhibitors, for research.

[0042] Treatment design As described above, immortalized human NP (ihNP) cells are grown, subsequently encapsulated in alginate beads, and their phenotypes are maintained in a three-dimensional environment (Zang et al., 2008). Newly formed alginate beads are cultured in each well of a 6-well plate and placed in DMEM / F12 medium containing 10% FBS medium + 2 mM L-glutamine + 50 μg / mL vitamin C. After 3 days, the medium is changed, and the cells are treated as described above for both SV and SVA with 0.3, 1, and 3 μM of the SV prodrug and each SV metabolite, respectively. The cells are removed from the alginate beads on days 1, 2, 3, and 7 after treatment. The cells are rinsed with 0.15 M NaCl and then incubated in lysis buffer (55 mmol / L sodium citrate and 0.15 M NaCl, pH 6.0) at 37°C for 15 minutes. The cells are pelleted by centrifugation, and the lysate is collected for evaluation. Total RNA is extracted using Trizol reagent, followed by DNase digestion using the RNeasy Mini Kit and RNase-free DNase set. The concentration of total RNA is determined at 260 nm using a spectrophotometer. Reverse transcription is performed using the SuperScript First-Strand Synthesis System. Real-time polymerase chain reaction is used, and the gene expression level of BMP-2 is quantified using the TaqMan real-time PCR Kit, which is performed using the Gene Amp 7700 Sequence Detection System. The amount of gene expression of BMP-2 is calculated using a standard sample and normalized using the GAPDH internal control. Furthermore, combined treatments using all metabolites at each of the three concentrations are also performed to examine whether the overall contribution from these compounds is synergistic or antagonistic.

Example

[0043] This example tests and demonstrates the effect of the SV metabolite on small molecular weight GTP-binding proteins (G-proteins). Members of the Ras and Rho GTPase families are major substrates for post-translational modification by prenylation. Both Ras and Rho are low molecular weight GTP-binding proteins that cycle between an inactive GDP-bound state and an active GTP-bound state. In endothelial cells, the translocation of Ras from the cytoplasm to the plasma membrane is dependent on farnesylation, while Rho translocation is dependent on geranylgeranylation. Statins inhibit the isoprenylation of both Ras and Rho, resulting in the accumulation of inactive Ras and Rho in the cytoplasm. Since Rho is the major target of geranylgeranylation, the inhibition of Rho and its downstream target Rho kinase is a promising mechanism mediating some of the pleiotropic effects of statins on the vascular wall, leukocytes, and bone. Various studies have suggested that the inactivation of Rho is involved in statin-induced BMP-2 expression, which is consistent with preliminary findings showing that inhibition of GGPP by SV governs BMP-2 upregulation in treated IVD cells. Furthermore, Luan et al. reported that statins inhibit the secretion of MMP-1, -2, -3, and -9 from vascular smooth muscle cells and macrophages, and since the secretion of MMPs was restored by reapplying GGPP, inhibition of GGPP-mediated prenylation was suggested to be the mechanism underlying this phenomenon. On the other hand, Rac, a subfamily of Rho, has been recognized to be associated with an increased production of reactive oxygen species (ROS), which causes vascular dysfunction in hypertension through the activation of NADPH oxidase. However, in IVD, in a hypoxic environment and obtaining energy mainly through glycolysis, NP cells still generated ROS through oxidative metabolism with angiogenesis, especially in aged or degenerated intervertebral discs. Chen et al., Cell Physiol Biochem. 2014;34(4):1175-1189 hypothesized that the increased apoptosis of NP cells under oxidative stress should be involved in the development of IVD degeneration. For this aspect, the inhibited Rac-1 activity could be considered as a potential mechanism contributing to the anabolic effects observed herein using NP cells treated with SV.Based on this study, it is important to understand how SV metabolites affect the Rho family, which includes its subfamily Rac, and to use the results observed in the SV treatment.

[0044] Study design The CRISPR / Cas technology is used to efficiently disrupt low molecular weight G-protein genes in ihNP cells. According to web tools (www.benchling.com and CRISPRscan.org), single-guide (sg) RNAs with both high predictive activity and high specificity for the target Rho or Rac genes are designed. To generate the sgRNA and Cas9 double-expression vector, a pair of complementary DNA oligos with compatible overhangs are annealed and cloned into a modified pX458 vector carrying a U6 promoter to drive sgRNA expression and a ubiquitously expressed promoter to drive the expression of high-fidelity eSpCas9(1.1)-2A-GFP (modified from Addgene plasmids #43138 and #71814). The sgRNA editing activity is evaluated in human 293T cells by the T7E1 assay (New England Biolabs) and compared with the EMX1 sgRNA, which has been shown to efficiently modify the genome (Ran et al., Nat Protoc. 2013;8(11):2281-308, which is hereby incorporated by reference in its entirety). The effective sgRNA / Cas9 vector is transfected into ihNP cells, and 48 hours later, based on GFP expression, the transfected cells are sorted into 96-well plates at 1 cell per well. Cell clones are cultured, genotyped, and biallelic nonsense mutations are confirmed by Sanger sequencing. At least two independent targeted clones and non-targeted wild-type clones are used for the study.

[0045] Treatment design Grow Rho- or Rac1-deficient ihNP cells and wild-type control cells, culture them, and treat them with SV metabolites respectively. Extract total RNA on days 1, 2, 3, and 7 after treatment, perform RT-qPCR, and quantify the gene expression level of BMP-2.

Example

[0046] The following example demonstrates meniscus repair and characterizes the torn meniscus repaired according to an embodiment of the present invention. In particular, this example shows that when treating an injured meniscus using the injection of a hydrogel formulation of SVA, chondrogenesis is stimulated, verifiable meniscus repair is obtained, and SVA is established as a long-term viable therapeutic intervention for avascular meniscus tears that are currently considered non-renewable.

[0047] Meniscus tears are a common knee injury, and approximately one million corrective surgeries are performed annually in the United States. The meniscus is an essential component in the knee, designed to transmit loads across the tibiofemoral joint and reduce the amount of stress applied to the articular cartilage. Previously, the standard surgical procedure for non-renewable meniscus tears that cause discomfort to patients was partial or total meniscectomy due to low healing potential and poor angiogenesis. However, studies have shown that removing the meniscus can sometimes lead to early osteoarthritis because significant stress is applied to the articular cartilage. The purpose of this experiment / study is to characterize the meniscus tissue repaired by partial injection of a hydrogel composition containing SVA.

[0048] Method Bilateral lesions in female white New Zealand rabbits (8 - 9 weeks old) were created in the avascular anterior portion of the medial meniscus using a 1.4 mm diameter k-wire. An SVA-hydrogel mixture was inserted into the defect to effect sustained drug delivery at the injury site. The animals healed 8 weeks after injury. Hematoxylin and eosin (H&E) and Safranin O (Safran-O) histological staining were utilized to analyze the morphological changes of the repaired tissue at the defect site. Collagen I, II, and BMP-II immunohistochemistry was performed to determine the composition of the repaired tissue. The study groups included an uninjured control group, a control group with injury only, and a treatment group with injury + SVA hydrogel repair treatment.

[0049] Results Previous studies conducted by the inventors have demonstrated that SVA can be utilized as a therapeutic agent to stimulate chondrogenesis via upregulation of the bone morphogenetic protein 2 (BMP-2) pathway and improve the degenerative changes associated with intervertebral disc disease in a rat model. This pathway was also utilized in this study and led to the morphology observed in the meniscal repair tissue (Figures 7B through 11B). The repair tissue was still fragile at 8 weeks post-repair, but these figures show that the tissue contained structured nucleated cells at the repair site. This is in contrast to Figure 7A, which shows no repaired tissue in the injury-only group 8 weeks after injury.

[0050] Proteoglycan content, collagen I, and collagen II are essential components of native meniscal tissue. Proteoglycans in the meniscus allow the tissue to maintain the high water content involved in its ability to absorb compressive loads across the knee joint. Collagen I and II are involved in the meniscus's ability to resist tensile loads. Demonstration of proteoglycan, collagen I, and collagen II in the meniscal tissue repaired by the SVA hydrogel method (Figures 7B - 11B) shows similarities between the repaired meniscal tissue and native tissue.

[0051] It has already been hypothesized that SVA hydrogels would act through upregulation of the BMP-2 pathway. However, Figure 8B shows that at 8 weeks after injury, the tissue was not positive for staining with respect to BMP-2. A possible explanation for this finding is that the BMP-2 pathway is upregulated in the short term before repair to stimulate new tissue growth and is no longer active after 8 weeks.

[0052] The entire disclosure content of all the documents cited herein shall be incorporated herein by reference in its entirety.

Claims

1. A controlled release composition formulated for injectable administration to a joint cavity, comprising: Simvastatin-beta-hydroxy acid (SVA); and At least one hydrogel selected from the group consisting of chitosan (CT), cyclodextrin (CD), p-dioxanone (DX), ethylene glycol (EG), ethylene glycol dimethacrylate (EGDMA), hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide (MBAAm), poly(acrylic acid), polyacrylamide, polycaprolactone, poly(ethylene glycol), poly(ethyleneimine), poly(ethylene oxide), poly(ethyl methacrylate), poly(hydroxyethyl methacrylate), poly(hydroxypropyl methacrylamide), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(methyl methacrylate) (PMMA), poly(propylene oxide), poly(vinyl alcohol) (PVA), poly(vinyl acetate), poly(vinylamine), and combinations thereof The controlled release composition comprising the same.

2. The controlled release composition according to claim 1, wherein a certain amount of SVA is dispersed in the hydrogel.

3. The controlled release composition according to claim 2, wherein the amount of dispersed SVA ranges from 1 to 50 mg / ml.

4. The controlled release composition according to claim 2, wherein the hydrogel comprises chitosan and / or hyaluronic acid.

5. The controlled release composition according to claim 4, wherein the mass ratio of chitosan to hyaluronic acid is about 60:

40.

6. A controlled release composition for repairing an injury to substantially avascular cartilage tissue or delaying the progression of its degeneration, comprising: A hydrogel; and At least one oxidative metabolite of simvastatin (SV) dispersed in the hydrogel The controlled release composition, wherein The oxidative metabolite of SV is selected from the group consisting of simvastatin-beta-hydroxy acid (SVA), 3'-hydroxysimvastatin (hSV), 6'-exomethylene simvastatin (eSV), 3',5'-dihydrodihydroxy simvastatin, 3',5'-dihydrodihydroxy simvastatin (dSV), and combinations thereof, The controlled release composition is directly administered by injection into the joint cavity of a subject in need thereof. The SVA is released in an effective rate and amount to repair the damage to the substantially avascular cartilage tissue damaged in the avascular cartilage tissue or delay the progression of its degeneration. The controlled release composition. **Claim 7** The hydrogel is selected from the group consisting of chitosan (CT), cyclodextrin (CD), p-dioxanone (DX), ethylene glycol (EG), ethylene glycol dimethacrylate (EGDMA), hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide (MBAAm), poly(acrylic acid), polyacrylamide, polycaprolactone, poly(ethylene glycol), poly(ethylene imine), poly(ethylene oxide), poly(ethyl methacrylate), poly(hydroxyethyl methacrylate), poly(hydroxypropyl methacrylamide), poly(lactic acid) (PLA), poly(lactic acid-co-glycolic acid) (PLGA), poly(methyl methacrylate) (PMMA), poly(propylene oxide), poly(vinyl alcohol) (PVA), poly(vinyl acetate), poly(vinyl amine), and combinations thereof. The controlled release composition according to claim 6. **Claim 8** At least one oxidative metabolite of SV is SVA. The controlled release composition according to claim 6. **Claim 9** The cartilage tissue is in the intervertebral disc. The controlled release composition according to claim 6. **Claim 10** The cartilage tissue is in the joint. The controlled release composition according to claim 6. **Claim 11** The cartilage tissue includes meniscus cartilage. The controlled release composition according to claim 10. **Claim 12** The subject suffers from degenerative disc disease and the direct administration includes intradiscal administration. The controlled release composition according to claim 9. **Claim 13** The injection is performed using a fluoroscope to guide a syringe carrying the controlled release formulation. The controlled release composition according to claim 6. **Claim 14** The administration of the controlled release composition promotes the proliferation of chondrocytes or chondrocyte-like cells at the damaged cartilage site. The controlled release composition according to claim 6. **Claim 15** The subject is a mammal. The controlled release composition according to claim 6. **Claim 16** The mammal is a human. The controlled release composition according to claim 15.

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