11 Treatment of Degenerative Disc Disease and Disc Regeneration: Proteins and Genes
Daisuke Sakai and Jordy Schol
Treatment of degenerative disc disease (DDD) and disc regeneration by administration of proteins and genes has been studied for the past 2 decades. To facilitate treatment of DDD and disc regeneration, the agents need to possess anabolic effects, which promotes cell proliferation, enhance production of matrix, or inhibit catabolic pathways. Various peptides, proteins, and compounds derived synthetically or from natural herbs have been analyzed to possess an effect on IVD cell metabolism. Additionally, enhancements of specific genes by gene transduction have also been assessed to facilitate anabolic and anticatabolic responses in IVD cells. Despite abounding amounts of basic research published, very few proteins are found in the market as an actual medicinal product. One reason for this may be due to the character of the disease being affected by multiple factors that makes it difficult to prove the products’ efficacy in clinical trials.
Keywords: annulus fibrosus, gene therapy, intervertebral disc, nucleus pulposus, protein injections, regenerative medicine
11.1 Overview and Historical Perspective of Peptides, Proteins, and Compound Injection Therapy or Gene Therapy for Intervertebral Repair
Degeneration of the intervertebral disc (IVD) is the primary cause of degenerative disc disease (DDD) that is frequently associated with low back and neck pain.1,2 For the past 2 decades, there has been plenty of ongoing research to develop biological treatment strategies for IVD repair. The fundamental strategy behind biological IVD repair is to facilitate endogenous cell survival, viability, and function. The main functional role of IVD cells is to produce and remodel appropriate extracellular matrix (ECM). Among the biological treatment strategies, this chapter covers two major areas that have been reported in the literature: (1) to inject peptides, proteins, and compounds (excluding growth factors and growth factor-related proteins) and (2) IVD cell gene therapy. Numerous proteins and compounds have been shown to promote nucleus pulposus (NP) cell proliferation and stimulate ECM synthesis. Some assessed factors also exhibit anti-inflammatory effects or downregulate catabolic responses. Because the IVD consists of a compartment-like anatomy and is secluded from the vasculature, localized injections of agents are easier compared with other organs. Additionally, administration can be achieved by discography approaches, which makes it easier for the clinicians to deliver the agents compared with open surgery approaches often used in, for example, articular cartilage regeneration. However, it has been suggested that IVD puncture can induce degeneration.3 Systemic approaches to deliver agents to the IVD may not be optimal due to the avascular nature of the IVD, resulting in low efficiency and high dose requirements.
Another option to stimulate cells is by gene transfer, also known as gene therapy. (▶ Fig. 11.1) Gene therapy allows for the introduction of new or overexpressed genes into a cell population. Although very promising, gene therapy has for a long time been an undesirable field of research due to a failed gene therapy clinical trial in 1999 that resulted in the death of an 18-year-old patient with ornithine transcarbamylase deficiency. In the past few years, gene therapy has regained interest and has been investigated as a treatment option for a variety of musculoskeletal diseases. The genes tested include morphogens, growth factors, and anti-inflammatory agents. Proteins produced endogenously as a result of gene therapy are nascent molecules that have undergone posttranslational modification. Gene transfer also has the advantage that it can deliver products with an intracellular site of action, such as transcription factors, noncoding ribonucleic acid (RNA), and proteins that need to be inserted into a cell compartment or membrane.4 After an initial experimental feasibility study using adenovirus vector to deliver LacZ gene into rabbit IVDs, many genes have been transferred to IVD cells in in vitro and in vivo experimental models.5,6 However, gene therapy has not been performed in humans to treat DDD due to the fact that vectors with high gene transfection efficiency are primarily viral, which may induce serious side effects. Gene therapy may become more prominent with the development of safer, more efficient and controllable gene transfer methods.
Fig. 11.1 Schematic presentation of agent and gene therapy treatment mechanism. Injection of agents or viral vector to target cell in order to alter cell behavior or phenotype. (1) Receptor-pathway activation by injected agent. (2) Agent-receptor–mediated transcription activation of regenerative genes. (3) Translation of viral deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) either in cytosol or at the endoplasmic reticulum. (4) Incorporation of viral DNA or RNA into the host genome. (5) Activated transcription of regenerative genes by transcription vectors translated from transfected genetic material. (6) Introduced genetic material resulted in membrane receptors on cell membrane. (7) Secretion of proteins and compounds encoded by the introduced DNA or RNA.
11.2 Strategies Using Peptides, Proteins, and Compounds
Injection of peptides, proteins, and compounds is a common practice in the clinic. Proteins are biomolecules or macromolecules composed of long chain amino acid residues. Peptides on the other hand are also composed of amino acid chains, but are by definition up to 50 amino acids long. Both constitute the building blocks and machinery of the cells and are capable of stimulating cells by activating or inhibiting cell receptor activity or by resulting in ECM alterations (▶ Fig. 11.1; ▶ Table 11.1).
Statins or 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are highly effective cholesterol-lowering drugs that are believed to reduce the morbidity associated with coronary artery disease. In 1999, through an examination of 30,000 compounds in a search for a bone morphogenetic (BMP)-2 inducing stimulant, investigators found that HMG-CoA reductase inhibitor statin was the only compound that specifically increased BMP-2 messenger RNA (mRNA) levels in osteoblasts.7 This finding confirmed BMP’s capacity to enhance matrix production by IVD cells.8 These findings led to the work of Zhang and Lin in 2008, which tested the stimulatory effect of simvastatin on IVD cell metabolism.9 The results of this study showed that simvastatin significantly upregulated BMP-2 mRNA expression, followed by aggrecan and type II collagen gene expression and proteoglycan deposition by IVD cells. This effect was mediated through the inhibition of the mevalonate pathway. The same group further reported an in vivo study using a rattail IVD puncture model to test whether simvastatin can facilitate IVD regeneration. In this study, simvastatin was loaded in poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) gel and injected in the injured IVD. Results showed that this treatment increased aggrecan expression and sulfated glycosaminoglycan (sGAG) content, and significantly increased mRNA levels of BMP-2 and type II collagen in the IVD. Magnetic resonance imaging (MRI) findings and histology also demonstrated milder degeneration in the IVDs receiving treatment, which suggested that single injection of simvastatin possesses the potential to retard or regenerate the degenerative IVD. Other research groups looked into lovastatin, the first statin in the market. It was tested in vitro and showed upregulation of genes encoding type II collagen and transcription factor SRY (sex determining region Y) box 9 (SOX9) in human degenerative NP cells, while suppressing the expression of type I collagen.10 Subsequently, by in vivo examination, it was confirmed that intradiscal administration of lovastatin solution upregulated the expressions of BMP-2 and SOX9 and promoted chondrogenesis of rat caudal discs after needle puncture and substance injection.11 These results demonstrate potential regenerative effects of statins in IVD degeneration. Where we are now with the application for IVD regeneration or associated back pain still remains to be clarified. Although a large-scale animal study has been reported to determine dose and effect, no clinical trials or study in humans has been found to date and this potential treatment awaits investigation.12
Bisphosphonates are drugs reported to be effective in conditions with bone fragility, and commonly are prescribed to elderly patients for the treatment of osteoporosis. A previous study on correlation of bone mineral density and lumbar IVD degeneration has shown that bone mineral density of not only the lumbar vertebrae but also the calcaneus and radius was mutually related to lumbar IVD degeneration from an early stage of degeneration.13 A laboratory investigation using ovariectomized (OVX) rats has shown strong association between osteopenia and disc degeneration.14 These facts led the experiment to test whether alendronate, one of the most common bisphosphonates in the market, has any effect on the progression of IVD degeneration. Results demonstrated that subcutaneous injections of alendronate in OVX rats helped in preserving the anatomical structure and function of the adjacent vertebrae and motion segments. Also retarded progression of IVD degeneration and maintained matrix metabolism was observed compared with the saline control.15 Up until now no clinical investigation has been reported.
Lactoferricin is an iron- and heparin-binding glycoprotein from the transferrin family, which is synthesized by epithelial cells and is present in most mammalian exocrine secretions. Lactoferricin has been isolated from bovine milk by pepsin digestion as LfcinB16 and has been shown to possess anti-inflammatory, antioxidative, anticatabolic, and anabolic effects on bovine disc cells in vitro.17,18 Furthermore, a recent report indicates that LfcinB synergistically stimulates anabolic effect in IVD cells with BMP-7. This effect results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (1) phosphorylation of SMAD1/5/8; (2) down-regulation of SMAD inhibitory factors (i.e., noggin [BMP receptor antagonist] and SMAD6 [inhibitory SMAD]); and (3) upregulation of SMAD4 (universal co-SMAD). (▶ Fig. 11.2) These data indicate that LfcinB suppression of noggin may eliminate the negative feedback of BMP-7, thereby maximizing biological activity of BMP-7 and ultimately shifting homeostasis to a proanabolic state in disc cells.19 No clinical investigations are available.
Fig. 11.2 ERK/MAPK and SMAD mediated pathway by lactoferricin activation. LfcinB stimulates ERK/MAPK pathway resulting activation of R-SMAD1/5/8 allowing for SMAD4-SMAD1/5/8 combination for matrix component and SOX9 expression. Moreover, noggin and SMAD6 are inhibited by ERK/MAPK activation. (Adapted with permission from Ellman et al 2013.19) Abbreviations: BMP, bone morphogenetic protein; NP, nucleus pulposus; Sp-1, specificity protein 1; Lfin-B, lactoferricin.
11.2.4 Peniel 2000
A novel peptide named Peniel 2000 (P2K) with a binding activity to transforming growth factor (TGF)-β1 was discovered using a knowledge-based in silico drug discovery strategy from biglycan, an ECM component of the IVD. TGF-β1 stimulation can demonstrate an anabolic effect in IVD cells. TGF-β1 is able to signal through type I receptors ALK1 and ALK5 with opposing effects (▶ Fig. 11.3). ALK1 dependent pathway activates through phosphorylation of SMAD1/5/8 and ALK5 dependent pathway activates through SMAD2/3 phosphorylation. A study has shown that regulation of TGF-β signaling through these two independent pathways is a key mechanism in the degenerative process of IVDs. TGF-β1 could stimulate the synthesis of ECM components by the SMAD2 pathway via ALK5 and inhibit the regeneration of IVD tissue by the SMAD1/5/8 pathway via ALK1. It was reported that TGF-β1 activates both anabolic SMAD2 and antianabolic SMAD1/5/8 pathways, and a tight balance between the two pathways inhibits the regeneration of degenerative IVDs. P2K treatment blocks the antianabolic pathway by SMAD1/5/8, but allows a minimal activation of anabolic pathway by SMAD2, thus induces the expression of ECM in IVDs and stimulates regeneration of IVDs.20 No clinical application of P2K has been reported.
Fig. 11.3 Mechanism of TGFβ1-ALK1 activation inhibited by P2K. P2K inhibits binding of TGFβ1 to ALK1, resulting in decreased inhibition of the ALK5-mediated pathway. (Adapted with permission from Kwon et al 2013.20) Abbreviations: ECM, extracellular matrix; TGF, transforming growth factor.