Compositions and methods for treating defects in avascular cartilage tissue by direct administration of one or more metabolites of simvastatin
阅读说明：本技术 通过直接施用辛伐他汀的一种或多种代谢物来治疗无血管软骨组织中的缺损的组合物和方法 (Compositions and methods for treating defects in avascular cartilage tissue by direct administration of one or more metabolites of simvastatin ) 是由 C-Y·J·林 于 2018-05-15 设计创作，主要内容包括：一种或多种辛伐他汀代谢物3’-羟基辛伐他汀(hSV)、6’-外亚甲基辛伐他汀(eSV)、3’,5’-二氢二醇辛伐他汀、3’,5’-二氢二醇辛伐他汀(dSV)、辛伐他汀-β-羟基酸(SVA)的受控释放水凝胶制剂,以及用于治疗罹患受损的或退变的基本上无血管的软骨组织的患者的方法。(Controlled release hydrogel formulations of one or more simvastatin metabolites 3 '-hydroxysimvastatin (hSV), 6' -exomethylenesimvastatin (eSV), 3',5' -dihydrodiol simvastatin (dSV), simvastatin-beta-hydroxy acid (SVA), and methods for treating patients suffering from damaged or degenerated, substantially non-vascular cartilage tissue.)
1. A method of repairing or delaying damage to damaged, substantially non-vascular cartilage tissue, the method comprising directly administering to a site of non-vascular tissue of a subject in need thereof a composition comprising at least one oxidized metabolite of Simvastatin (SV).
2. The method of claim 1, wherein the step of administering comprises administering a controlled release formulation of the at least one oxidized metabolite of Simvastatin (SV) wherein the composition is released in the avascular cartilage tissue at a rate and in an amount effective to allow repair or delay damage.
3. The method of claim 1, wherein the at least one oxidized metabolite of SV is selected from the group consisting of 3 '-hydroxysimvastatin (hSV), 6' -exomethylene simvastatin (eSV), 3',5' -dihydrodiol simvastatin (dSV), simvastatin- β -hydroxyacid (SVA), and combinations thereof.
4. The method of claim 3, wherein the at least one oxidative metabolite of SV comprises SVA.
5. The method of claim 1, further comprising administering SV in combination with the at least one metabolite, wherein "combination" comprises simultaneous administration, tandem administration, or administration over a treatment time frame.
6. The method of claim 1, wherein the cartilage tissue is in an intervertebral disc.
7. The method of claim 1, wherein the cartilage tissue is in a joint.
8. The method of claim 7, wherein the cartilage tissue comprises meniscal cartilage.
9. The method of claim 6, wherein the subject suffers from degenerative disc disease and direct administration comprises intra-discal administration.
10. The method of claim 2, wherein the administering step comprises administering by injection.
11. The method of claim 10, wherein the injection is performed using fluoroscopy to guide a syringe carrying the controlled release formulation.
12. The method of claim 2, wherein the controlled release formulation comprises a hydrogel.
13. The method of claim 12, wherein the hydrogel comprises a hydrophobic polymer and a hydrophilic polymer.
14. The method of claim 13, wherein the polymer is a homopolymer or a copolymer.
15. The method of claim 2, wherein administering the controlled release composition promotes proliferation of chondrocytes or chondrocyte-like cells in a site of damaged cartilage.
16. The method of claim 2, wherein the subject is a mammal.
17. A method of repairing or delaying damage to damaged, substantially non-vascular cartilage tissue, the method comprising directly administering to a site of damaged, non-vascular tissue in a subject in need thereof at least one active that increases Bone Morphogenic Protein (BMP) expression without inhibiting HMG-CoA reductase.
18. The method of claim 17, wherein said at least one active is selected from the group consisting of hSV, dSV, and combinations thereof.
19. A controlled release composition formulated for injectable administration, said composition comprising at least one oxidized metabolite of Simvastatin (SV).
20. The controlled release composition of claim 19, further comprising a hydrogel, wherein an amount of the at least one metabolite is dispersed within the hydrogel.
21. The controlled release composition of claim 20, wherein the at least one metabolite is selected from the group consisting of 3 '-hydroxysimvastatin (hSV), 6' -exomethylene simvastatin (eSV), 3',5' -dihydrodiol simvastatin (dSV), simvastatin- β -hydroxy acid (SVA), and combinations thereof.
22. The controlled release composition of claim 21, wherein the at least one metabolite comprises SVA.
23. The controlled release composition of claim 21, wherein the at least one metabolite comprises hSV, dSV, or both.
24. The controlled release composition of claim 20, wherein the hydrogel comprises a hydrophobic polymer and a hydrophilic polymer.
25. The controlled release composition of claim 24, wherein the hydrophilic polymer in the hydrogel ranges from about 10% to 50%, from about 20% to 40%, or from about 20% to 30%.
26. The controlled release composition of claim 24, wherein the hydrophobic polymer in the hydrogel ranges from 40% to 90%, from about 60% to 80%, or from about 70% -80%.
27. The controlled release composition according to claim 20, wherein the amount of dispersed metabolite ranges from 1 to 50 mg/ml.
Technical Field
Embodiments of the present invention relate generally to therapeutic pharmacology, and specifically to methods and compositions effective for treating subjects suffering from diseases and conditions characterized by damage or otherwise impairment of cartilage tissue (i.e., avascular cartilage tissue) by direct administration of one or more metabolites of simvastatin (simvastatin) to avascular tissue.
Background
Currently, Simvastatin (SV) is a widely prescribed drug for the treatment of cardiovascular disease/hypercholesterolemia, and its derivatives are also used in many other applications, including the recent use in promoting disc cell chondrogenesis and ameliorating disc disease.
Based on examination of a library of more than 30000 native compounds, Mundy and colleagues found that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, a statin, including Simvastatin (SV) is the only molecule that specifically increases BMP-2mRNA in mouse and human bone cells in vitro and induces subsequent bone formation in vivo. Statins are commonly used cholesterol lowering prescription drugs that inhibit the cholesterol biosynthesis pathway. Since the discovery of this "side effect" on bone anabolism, the skeletal protection and underlying mechanisms of statins have been the subject of considerable research including osteoporosis treatment programs. Further studies have also shown that SV increases BMP-2 expression in various cell types, such as untransformed osteoblasts, myeloid stromal cells, human vascular smooth muscle cells and rat chondrocytes (see, e.g., Zhang, h. et al, (2008) Spine,33(16), Zhang, h. et al, (2009) Arthritis Res The Arthritis research & Therapy,11(6), and Than, k.d. et al, (2014) The Spine Journal,14(6), 1017-.
Degenerative Disc Disease (DDD) is considered to be the leading cause of lower back pain, a common medical problem, which also imposes a significant socio-economic burden. However, current clinical criteria for DDD treatment are often associated with complications, particularly when surgical intervention is involved. In recent years, the bioremediation or regeneration of degenerative disc (IVD) has been advocated with the development of recombinant therapeutic proteins including recombinant human bone morphogenetic protein-2 (BMP-2). However, the required doses of these recombinant human growth factors are often supraphysiological, which raises concerns about the possibility of toxicity and other undesirable complications.
As an alternative to current treatment regimens, tissue engineering and regenerative approaches have been the focus of research work in the past decade. In particular, growth factors such as the Bone Morphogenetic Protein (BMP) family have shown great potential in stimulating matrix regeneration in damaged disc tissue. Although the initial results are encouraging, clinical use of recombinant growth factors raises a number of concerns, including: undesired vascular growth within avascular disc tissue, the supraphysiological concentrations required for therapeutic effectiveness, which increase the risk of side effects, and the high costs associated with clinical grade recombinant protein production. Therefore, regenerative drugs without such problems are more desirable.
Over a decade, the present investigators have extensively studied the role of SV and reported that SV stimulation promotes several phenotypic expressions of mammalian Nucleus Pulposus (NP) cells, including aggrecan, type II collagen, and sulfated glycosaminoglycans, which in turn help to delay the development of degeneration and help repair of degenerative IVD. SV has been found to effectively promote chondrogenesis by upregulating the expression of endogenous BMP-2 in treated NP cells, which in turn contributes to the repair of affected IVD in vivo. Other benefits of the proposed SV treatment for DDD include: the intra-discal injection procedure does not require open surgery, which can minimize post-operative pain and recovery time as well as the risk of over-involvement of the disc and ultimately leading to deformed degeneration. The procedure is common and can be performed by many other clinical specialists besides the spinal surgeon, making this approach more economical, practical and applicable in current healthcare systems. Therefore, SV is considered a promising alternative to protein-based regenerative medicine for the treatment of DDD.
Based on the similarities between disc and meniscus composition, it is also contemplated to administer SV directly for treatment of a meniscal tissue defect, thereby improving healing by stimulating cartilage formation in a similar manner as in the disc model. Meniscal tears are one of the most common injuries to the knee joint, leading to severe loss of productivity and reduced quality of life in most populations, even among young people. Physicians report that approximately one third of the population over the age of 50 have at least one meniscal tear, which makes the population more vulnerable to instability/falls and chronic pain near advanced age.
The present investigators previously used a well-known meniscus lesion model in which a circular full thickness lesion was formed in The meniscus using a biopsy punch or k-wire, and in combination with FDA approved biodegradable hydrogels, using SV sustained drug delivery, which demonstrated new tissue growth within four weeks post injection (see Zhang & Lin, (2008) Spine,33(16), and Zhang et al, (2016) The American Journal of Sports.
It is well known that systemically delivered SVs undergo extensive first pass metabolism in the liver. As a result, the drug rapidly hydrolyzes into several oxidation products (fig. 5), including 3 '-hydroxy SV (hsv), 6' -exomethylene (exomethylene) SV (esv), 3',5' -dihydrodiol SV (dsv), and SV beta-hydroxy acid (SVA). Some of the hydroxyacid forms of these metabolites, including SVA, have also been found to be HMG-CoA reductase inhibitors, after which SVA has been found to be a competitor of SV. Thus, it can be postulated that at least some actually involve one or more SV metabolites due to the therapeutic effects of systemically administered SV.
However, it is crucial that direct injection of SV into the IVD or joint space is not expected to result in the presence of SV metabolites in the IVD or joint space, both avascular (blood or lymphatic vessels that do not conduct or circulate blood). Direct injection bypasses liver metabolism and it can therefore be concluded that the observed regenerative effects of SV in avascular tissue do not involve SV metabolites, but rather are due to the physiopharmacology of the SV active itself.
Therefore, prior to the studies reported herein by the present inventors, no studies have been conducted to determine whether any metabolite of SV has a similar effect on increasing BMP-2 expression in avascular tissues, particularly metabolite-eSV and SVA, which are known to be competitive HMG-CoA reductase inhibitors. Furthermore, no studies have been conducted to confirm whether SV metabolites, i.e., hSV and dSV, that are non-HMG-CoA reductase inhibitors, are still capable of modulating BMP-2 expression or modulating other cellular/molecular activities observed. Especially when injected directly into joints and intervertebral discs, potential benefits such as reduced injection volume, formulation advantages, and increased efficacy and reduced side effects, all make further studies of SV metabolites an attractive approach to finding effective, relatively non-invasive methods for regenerating defective avascular tissue.
Summary of The Invention
The present investigators unexpectedly determined that the SV metabolite SVA inhibits mevalonate conversion more effectively than SV and, after direct administration, is 5-6 fold higher in regeneration of avascular tissue than the anabolism of SV alone. Furthermore, SVA has been found to contribute to anti-catabolism which synergistically promotes cartilage formation as observed by SV. Interestingly, it was also found that administration of SV metabolites other than HMG-CoA reductase inhibitors (including dSV and hSV) both promoted regeneration to some extent, suggesting that there is a mechanism that has not been elucidated so far with respect to the efficacy of the metabolite. Thus, direct administration of a composition of one or more SV metabolites provides greater regenerative benefit to patients suffering from DDD or meniscal damage than SV alone.
Accordingly, one embodiment provides a method of repairing or delaying damage to degenerated or damaged, substantially non-vascular cartilage tissue. The method comprises directly administering to an avascular tissue site of a subject in need thereof a composition comprising at least one oxidized metabolite of Simvastatin (SV). In other embodiments, the methods comprise directly administering to a site of damaged avascular tissue of a subject in need thereof at least one active that increases Bone Morphogenetic Protein (BMP) expression without inhibiting HMG-CoA reductase. In particular embodiments, the at least one active is selected from the group consisting of hSV, dSV, and combinations thereof.
Another embodiment provides a controlled release composition formulated for injection administration comprising at least one oxidized metabolite of Simvastatin (SV).
Other embodiments relate to methods of treating patients suffering from avascular cartilage tissue damage, including but not limited to DDD and meniscus damage.
These and other embodiments will be described and illustrated more fully with reference to the following drawings and detailed description.
Brief Description of Drawings
The drawings are intended to illustrate particular embodiments and aspects of the invention, and should not be construed as limiting the full scope as defined by the appended claims.
FIG. 1A
FIG. 2 gives a schematic representation of the effect of SV metabolites on the IVD repair observed for SV.
FIG. 3A demonstrates that the up-regulation of BMP-2mRNA expression in rat NP cells is independent of the presence of cholesterol when treated with 3 μ M (effective in vitro dose) SV; fig. 3B shows that stimulation is not associated with inhibition of FPP, but is highly influenced by inhibition of the downstream substrate GGPP along the MVA pathway; FIG. 3C shows that inhibition of a single GGT enzyme achieved BMP-2 stimulation, but at a level that was not comparable to that directly stimulated by simvastatin; figure 3D shows that when both inhibition occurred simultaneously, the stimulation was significantly increased compared to each inhibition alone, although the levels were still below the expression promoted by simvastatin.
FIG. 4 illustrates the effect of simvastatin on aggrecan, type II collagen, type I collagen mRNA expression and "differentiation index" type II/type I collagen ratio in human NP and AF cells. Data were normalized to GAPDH and expressed as a ratio to vehicle (vs vehicle, P <0.05, P < 0.01).
FIG. 5 shows the structures and pathways of simvastatin and its metabolites.
FIG. 6A is a photograph showing an intact 1.4mm defect in the right medial meniscus; the photograph of fig. 6B shows the injection of the hydrogel composition into the left medial meniscus.
FIG. 7A is a 2X hematoxylin & eosin histological stain (H & E) 8 weeks after injury of only the injured control group, showing no repair tissue; figure 7B is 2XH & E treated 8 weeks post injury, showing the presence of repaired tissue.
FIG. 8A is 40X H & E treated 8 weeks after injury, showing repaired tissue; FIG. 8B is 40X BMP-II immunohistochemistry treated 8 weeks after injury, showing repaired tissue.
FIG. 9A is a 40X (right medial meniscus) Safranon-O stain of the treated 8 weeks post-injury showing repaired tissue at the repair site; fig. 9B is the same subject as fig. 9A, showing 40X positive SAFO staining of tissue cells located in
FIG. 10A is 40X (right medial meniscus) COL-I immunohistochemistry treated 8 weeks after injury, showing repaired tissue at the repair site; FIG. 10B is the same as the subject of FIG. 10A, showing COL-I positive staining of tissue cells located in the
FIG. 11A is 40X (right medial meniscus) COL-II immunohistochemistry treated 8 weeks after injury, showing repaired tissue at the repair site; FIG. 11B is the same as the subject of FIG. 11A, showing COL-II positive staining of tissue cells located in the
Detailed Description
Lower Back Pain (LBP) is one of the most common medical problems in the united states, afflicting approximately 80% of the us population at some point in their life. This is also one of the most common causes of absenteeism, and chronic LBP exacerbates dependence on narcotics; thereby causing a huge socio-economic burden and public health problems. In cases of LBP, either specific (e.g., spinal tumors or infections) or non-specific (no apparent cause), Degenerative Disc Disease (DDD) is considered to be the major cause of LBP. Current clinical criteria for treating DDD are often associated with complications, particularly when surgical intervention is involved. Thus, the ability to bioremediate or regenerate an abnormal disc in situ with a therapeutic compound is a very attractive option for future treatment options. Such a strategy is attractive not only because it provides the least invasive intervention, but also because it potentially facilitates reconstruction of the damaged disc.
Currently available reconstitution protocols involve the use of recombinant growth factors that are not only prohibitively expensive to manufacture, but also raise concerns about toxicity and other adverse complications associated with the required supraphysiological doses. Thus, unfortunately, current standards of care for DDD focus on pain control, spinal stabilization, and slowing of disease progression, rather than disc repair.
The present inventors have previously disclosed that Simvastatin (SV) (3-hydroxy-3-methylglutaryl coenzyme a (HMG-CoA) reductase inhibitor, a commonly used cholesterol lowering prescription drug) and drugs promote phenotypic expression of mammalian Nucleus Pulposus (NP) cells upon in vitro treatment. In vivo, when injected into the affected intervertebral disc (IVD) in a rat model of DDD with a controlled release formulation of SV, the compounds retard the progression of degeneration, most notably also promote repair (anabolism) of degenerated IVD. In addition, the known pleiotropic effects of simvastatin in anti-inflammation were also observed, in which the expression of enzymes that degrade the extracellular matrix (anti-catabolism) was inhibited. These Matrix Metalloproteinases (MMPs) are usually stimulated by pro-inflammatory cytokines in pathological intervertebral discs. The results provide preliminary evidence that SV is a better alternative to recombinant proteins for the treatment of DDD. Nonetheless, the hydrophobicity of SV prodrugs also greatly limits their local delivery with currently available/approved hydrogel vehicles.
Encouraging, in the recent studies conducted by the present inventors, it was observed that the active hydrolysis metabolite simvastatin β -hydroxy acid (SVA) of SV is actually more effective in up-regulating endogenous bone morphogenetic protein-2 (BMP-2), a mediator of disc repair caused by the prodrug SV, suggesting that at least one SV metabolite may determine the efficacy of SV observed in IVD treatment.
NP cells are commonly referred to as "chondrocyte-like" cells because these cells are initially notochordal cells but are gradually replaced in childhood by round cells resembling the chondrocytes of articular cartilage. The NP cells maintain the chondrogenic phenotype for the composition of the IVD tissue matrix, and exposure of the NP cells to BMP-2 promotes expression of the chondrogenic phenotype. In addition, recent findings have also shown that endogenously produced BMPs, including BMP-2, interfere with the actions of proinflammatory cytokines. Both phenomena are consistent with the results described above. This experiment is intended to show that the stimulated expression of BMP-2 in avascular tissues is enhanced by administering the active metabolite SVA (SVA is also a potent competitor for inhibiting HMG-CoA reductase) as well as compositions comprising SVA and at least one other SV metabolite, and compositions comprising SV and at least one active SV metabolite, including metabolites that are not HMG-CoA reductase inhibitors.
Statins are potent inhibitors of cholesterol biosynthesis. However, ongoing research also indicates that some of the cholesterol-independent or "pleiotropic" effects of statins are more beneficial than would be expected by changes in lipid levels alone. Statins (including SV) affect the enzymatic activity of protein prenylation, which is critical for the function of downstream small G proteins. These G proteins are modulators of many physiological responses and intracellular signaling pathways, including polarity, gene transcription, and intracellular vesicle transport. Thus, particular attention has been given to the effects of SV metabolites on Rac, G proteins of the Rho family and subfamilies thereof. SV exerts an anti-inflammatory effect by inactivating Rho, which is associated with the observation in inhibition of MMPs. On the other hand, Rac, when inhibited by SV, reduces oxidative stress. Recent studies have reported that oxidative stress induced in NP cells is associated with disc degeneration. Therefore, it is important to elucidate the role of SV metabolites in the down-regulation of two G proteins observed with SV prodrugs.
In systemic delivery, SVs undergo hepatic metabolism to produce a variety of metabolites, including several hydroxy acids, such as SVA (see fig. 5). These acid metabolites may compete with the prodrug SV in rate-limiting enzyme activity and may have an effect on certain biological activities. However, systemic administration of SV or direct administration of SV to provide active metabolites would not be expected for delivery to avascular tissue.
As mentioned previously, the discovery of the pleiotropic effects of statins on BMP-2 upregulation has led to extensive research, particularly their role in bone anabolism and bone protection. Extensive studies have also been carried out to elucidate the underlying mechanism of such upregulation, and the results indicate that statins increase BMP-2 expression via 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):191-9) confirmed these results and further reported that the statin-induced osteogenesis PI3K/Akt pathway is dependent on activation of small GTPase Ras, which promotes activation of small GTPase Ras by localizing proteins on the intracellular membrane. In addition to bone, BMP is also considered a potential therapeutic agent for IVD degeneration, and studies focused on the use of
In example 1, BMPRII was localized in the NP and inner Annulus Fibrosis (AF) cells of 30 human intervertebral discs. Little immunopositivity was seen in cells of external AF. Cells in NP show a higher immune positive rate than cells in internal AF. This suggests that BMP applied in human IVD will show the greatest effect in NP and internal AF. These results are different from those observed in mice where receptor expression was observed only in the cartilage endplate and AF. Interestingly, no changes in expression levels with the degree of degeneration were observed.
The results of the study by SV stimulating endogenous BMP-2 expression in treated NP cells to increase chondrogenic phenotype expression (aggrecan and type II collagen mRNA expression and sGAG content) are consistent with the above observations, confirming that this small molecule is as effective in promoting cartilage formation as bone formation. However, surprisingly, the degree of stimulation of BMP-2 up-regulation is greatly increased when these cells are treated with SVA, an active hydrolysis metabolite of SV. SV has been widely prescribed for the treatment of hypercholesterolemia and hypertriglyceridemia. In humans, SV undergoes rapid metabolism, forming four major oxidative, NADPH-dependent metabolites: 3 '-hydroxy SV (hSV), 6' -exomethylene SV (eSV), 3',5' -dihydrodiol SV (dSV), and SVA (FIG. 5). Among them, SVA is the most potent competitor of SV in HMG-CoA reductase inhibition. This presents a problem as to whether SVA dominates the overall BMP-2 upregulation scheme. To test whether there were any differences in the levels of BMP-2 induced by SV and SVA, respectively, the present investigators performed tests using in vitro model systems developed in previous studies. Rat NP cells harvested from the tail disc were first cultured in a monolayer and then in alginate beads (Zhang H. et al, Spine, 2008; 33(16), the entire disclosure of which is incorporated herein by reference). Cells were treated with 1 μ M or 3 μ M DMSO (vehicle), SV or SVA. Cells were then collected at predetermined time points and RNA was extracted. Gene expression was analyzed by RT-qPCR. The results showed that from
The results show that SVA has better effect than SV in BMP-2 up-regulation. The BMP-2 up-regulation event is the result of HMG-CoA reductase inhibition, which can be achieved by SV, SVA and eSV because they are inhibitors. It is further surprising that some regenerative potential was established by administration of non-HMG-CoA reductase inhibitors hSV and dSV, although the pathway is not clear.
SV is known to block the synthesis of either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP), an isoprenoid intermediate, and thereby inhibit the function of downstream small G-proteins such as the Ras, Rho, Rab families. The question of the mechanistic pathways of action of the non-HMG-CoA reductase inhibitors hSV and dSV, whether they directly affect protein prenylation or G-protein, remains open. Since both Ras and Rho regulate BMP-2 expression via the Ras/PI3K/Akt/MAPK/BMP-2 pathway and both Rho and Rac can be correlated with observed anabolic and anti-catabolic effects, the mechanism by which the profiling of how metabolites contribute to SV gain-of-effect provides additional therapeutic strategies. The scheme of these is illustrated in fig. 2.
The classical mechanism by which statins lower cholesterol is that statins act by competitively inhibiting HMG-CoA reductase, the first committed enzyme of the Mevalonate (MVA) pathway. This competition reduces the rate of HMG-CoA reductase production of MVA, the next molecule in the cascade for synthesis of the prenylase substrates FPP and GGPP, which ultimately help produce cholesterol. As previously demonstrated by the present inventors (Zhang H.N., LinC.Y.spine.2008; 33(16), incorporated herein by reference in its entirety), BMP-2mRNA expression by NP cells always responds in a time-dependent and dose-dependent manner in the presence of SV. Furthermore, stimulation in NPs treated with 3 μ M SV was independent of the presence of cholesterol (fig. 3A) and FPP (fig. 3B). In contrast, stimulation actually involves the MVA pathway, and observations indicate that stimulation is completely reversed when cells are pretreated with MVA. Interestingly, when the downstream substrate, GGPP, was supplemented, a reversal was also achieved (fig. 3B). Next, when GGT enzyme inhibitors GGTI-286 and POH were administered to mimic the SV's inhibition of GGPP enzymatic activation, they were both able to increase BMP-2mRNA expression, but at levels much lower than those found with SV, respectively (FIG. 3C). BMP-2 upregulation was significantly higher when IVD cells were co-treated with GGTI286 and POH than each treatment alone (fig. 3D). However, the upregulation of BMP-2 by co-treatment has not reached comparable levels to SV treatment, suggesting that a mechanism other than the inhibition of HMG-CoA reductase may synergistically promote BMP-2 expression. Without being bound by mechanism, the inventors found that SV metabolites that are not HMG-CoA reductase inhibitors, i.e., hSV and dSV, also affect BMP-2 up-regulation.
One embodiment relates to a method of repairing or delaying damage to damaged, substantially non-vascular cartilage tissue, the method comprising directly administering to a site of non-vascular tissue of a subject in need thereof a composition comprising at least one oxidized metabolite of Simvastatin (SV). According to a more specific embodiment, the administering step comprises administering a controlled release formulation of at least one oxidized metabolite of Simvastatin (SV) wherein said composition is released in said avascular cartilage tissue at an effective rate and amount to allow repair or delay damage. At least one oxidized metabolite of SV is selected from the group consisting of 3 '-hydroxysimvastatin (hSV), 6' -exomethylene simvastatin (eSV), 3',5' -dihydrodiol simvastatin (dSV), simvastatin-beta-hydroxy acid (SVA), and combinations thereof. According to a very specific embodiment, the at least one oxidative metabolite of SV comprises SVA.
It is contemplated that in some embodiments, the SV may be administered in combination with at least one metabolite, wherein "combination" includes simultaneous administration, tandem administration, or administration over a treatment time frame. In the case of simultaneous administration, it may be presented as a single dosage unit or as multiple units. The treatment time frame may be any time frame during which the patient is receiving treatment for damaged or degenerated cartilage tissue. The treatment regimen may comprise a single administration or multiple administrations over the treatment time frame. According to very particular embodiments, the cartilage tissue comprises disc cartilage/fibrocartilage, and in other particular embodiments, the cartilage tissue is in a joint. According to an even more specific embodiment, the cartilage tissue comprises meniscal cartilage.
According to some aspects, where the subject suffers from a degenerative disc disease, administration includes direct administration to the intradiscal space, for example by injection or by a guide catheter. Fluoroscopy may be used to guide the injection from a syringe carrying the formulation (e.g., the controlled release composition of one or more metabolites with or without SV). Administration of the controlled release composition promotes proliferation of chondrocytes or chondrocyte-like cells at the site of damaged cartilage. According to a particular embodiment, the subject is a mammal, and in a very particular embodiment, the mammal is a human.
According to a particular embodiment, a controlled release composition comprising one or more hydrogels comprising an active is provided. Exemplary hydrogels suitable for use in 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 bisacrylamide (MBAAm), polyacrylic acid, polyacrylamide, polycaprolactone, polyethylene glycol, polyethyleneimine, polyethylene oxide, polyethyl methacrylate, polyhydroxyethyl methacrylate, polyhydroxypropylmethacrylamide, polylactic acid (PLA), polylactic-glycolic acid copolymer (PLGA), polymethyl methacrylate (PMMA), polypropylene oxide, polyvinyl alcohol (PVA), polyvinyl acetate, polyvinylamine, and combinations thereof.
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. The hydrophilic polymer may be included in the hydrogel in a range of about 10% to 50%, about 20% to 40%, or about 20% to 30%, and the hydrophobic polymer may be included in a range of 40% to 90%, about 60% to 80%, or about 70% -80%. According to a very specific embodiment, the hydrophilic polymer comprises. In other very specific embodiments, the hydrophobic polymer comprises CT. Even more specifically, HA comprises the HA-Na polyanion and CT comprises CT-NH3 +Polycation, and a mass ratio of CT to HA of about 60: 40. In this paragraph, "about" means +/-2%. An amount of the at least one metabolite (with or without SV) is dispersed in the hydrogel matrix. The metabolite is selected from the group consisting of 3 '-hydroxysimvastatin (hSV), 6' -exomethylenesimvastatin (eSV), 3',5' -dihydrodiol simvastatin (dSV), simvastatin-beta-hydroxy acid (SVA), and combinations thereof. According to a particular embodiment, theActives include SVA. According to other particular embodiments, the active is selected from the group consisting of hSV, dSV, and combinations thereof. According to some embodiments, the amount of active dispersed in the controlled release hydrogel is from 1 to 50mg/ml, including all ranges and values therebetween.
Another embodiment provides a method of repairing or delaying damage to damaged, substantially non-vascular cartilage tissue. The methods comprise directly administering to a site of damaged avascular tissue of a subject in need thereof at least one active that increases Bone Morphogenetic Protein (BMP) expression without inhibiting HMG-CoA reductase. According to a particular embodiment, the active is selected from the group consisting of hSV, dSV and combinations thereof.
The examples are presented to illustrate and support specific embodiments and should not be construed as limiting the full scope of the invention as defined by the appended claims.
Example 1
This example demonstrates the effectiveness of a degenerated human NP cell model system and tests for SV and its metabolites.
Previous publications were based on in vitro and in vivo studies using rodents, thus creating model systems with degenerated human NP cells. Interestingly, the results showed that human cells responded differently to the drug than other studies on rat and pig cells. When IVD cells harvested from a human patient with DDD are exposed to SV, these cells stimulate the maintenance or even increase of the chondrogenic phenotype in a dose-dependent manner. However, there were differences in expression patterns from rat IVD cells (see Zhang HN, Lin CYSpine.2008; 33 (16)). As observed in rat cells, SV upregulates BMP-2mRNA expression in human NP and Annulus Fibrosus (AF) cells. In addition, both NP and AF cells expressed the BMP-2 receptor BMPRII, suggesting that both cell types are susceptible to SV-induced BMP-2 upregulation to mediate defined pathways (data not shown). However, when human NP cells were treated with the same dose (0.3 to 3. mu.M) of SV as used for treating rat cells, mRNA expression of aggrecan and type II collagen was not affected.
Alternatively, SV inhibited expression of type I collagen mRNA in a dose-dependent manner, thus significantly increasing the ratio of type II to type I collagen (fig. 4). This phenomenon was observed only in human NP cells compared to the other species used (rats (Zang et al, 2008), rabbits and pigs, data not shown). The treatment did not alter the mRNA expression of aggrecan, type II collagen and type I collagen in human AF cells. The results of the increase in the Col II/Col I ratio (also referred to as the "differentiation index") indicate that SV may inhibit de-differentiation of human NP cells in degenerated intervertebral discs, which will help maintain their chondrogenic phenotype.
Based on this finding, human NP cells were used to better facilitate the proposed human IVD repair strategy in this study. However, to obtain higher order consistency, particularly for experiments using CRISPR genome editing techniques, a human NP cell line from Win-Ping Deng doctor (Liu M.C. et al, tissue Eng Part C-Me.2014; 20(1):1-10, incorporated herein by reference in its entirety) obtained from the subsidiary Hospital of the North medical university (Taipei, Taiwan) was employed.
SV metabolites
All compounds in this study, including SVA, eSV, hSV, and dSV, were created and operated by AAPharmaSyn, LLC (Global contract for chemistry research organization, created and operated by former Pfizer chemists, who possessed extensive knowledge and experience in statins, including the successful development of statins
Process design
Immortalized human np (ihnp) cells were expanded as previously described (Zang et al, 2008) and then encapsulated with alginate beads to maintain their phenotype in a three-dimensional environment. The newly formed alginate beads were cultured in each well of a 6-well plate and placed in DMEM/F12 medium containing 10% FBS medium +2mM L-glutamine + 50. mu.g/mL vitamin C. After three days, the medium was changed and the cells were treated with 0.3, 1 and 3 μ M of the SV prodrug and each SV metabolite, respectively, as previously described with SV and SVA. On
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