Regulation of bone regeneration with approved small molecule compounds

MINI REVIEW

Regulation of bone regeneration with approved small molecule compounds

Erica J. Carbone1,2,3, Komal Rajpura1,2, Tao Jiang1,2,3, Cato T. Laurencin1,2,4,5,6 and Kevin W.-H. Lo1,2,3,4*

1Institute for Regenerative Engineering, School of Medicine, University of Connecticut Health Center, Farmington, CT, USA; 2The Raymond and Beverly Sackler Center for Biomedical, Biological, Physical and Engineering Sciences, School of Medicine, University of Connecticut Health Center, Farmington, CT, USA; 3Department of Medicine, Division of Endocrinology, School of Medicine, University of Connecticut Health Center, Farmington, CT, USA; 4Department of Biomedical Engineering, School of Engineering, University of Connecticut, Storrs, CT, USA; 5Department of Orthopaedic Surgery, School of Medicine, University of Connecticut Health Center, Farmington, CT, USA; 6Department of Chemical, Materials and Biomolecular Engineering, School of Engineering, University of Connecticut, Storrs, CT, USA

Received: 24 June 2014; Accepted in revised form: 17 August 2014; Published: 26 September 2014

Abstract

Stimulation of bone formation using recombinant growth factors has been a promising strategy for bone repair and regeneration. However, small molecules have been suggested as alternatives to recombinant protein-based treatments because of their unique advantages. To date, therapeutic methods for orthopaedic applications still heavily rely on therapeutic protein-based growth factors. This trend is likely to be reversed since innovative drug discovery strategies, such as broad-spectrum database analysis and high throughput functional screens, have led to the discovery of many novel small molecule compounds and the development of novel applications of existing approved small molecule compounds. It should be noted that some of these small molecules with osteoinductive potential have been approved for human use due to their efficacy in treating other health ailments. Thus, these approved small molecule compounds are highly translatable to orthopaedic applications. In this article, we review the literature, paying attention to the prospects of existing approved small molecule therapeutics with bone regenerative capacity. Future directions of bone repair and regeneration using these approved small molecule drugs will be discussed as well.

Keywords: small molecules; bone regeneration; drug discovery

In context

Skeletal diseases and injuries, such as bone fractures, affect a significant portion of the population. While several proteinbased medications are currently approved for the treatment of skeletal disorders via bone repair and regeneration, it is known that these protein-based therapeutics can cause significant side-effects. An alternative to protein-based pharmaceuticals is urgently needed in order for the therapeutics to be safer, more effective, and more affordable. Small moleculebased drugs are more appealing for treating bone diseases because they are more affordable, more stable, and require a smaller dosage to achieve a bone regenerative effect. Interestingly, some small molecule drugs that are already being prescribed to treat a variety of medical conditions have shown potential to be used as therapeutics for bone repair and regeneration. This review article provides a summary of recent findings of approved small molecule compounds with bone regenerative capability.

*Correspondence to: Kevin W.-H. Lo, School of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA, Email: wlo@uchc.edu

Advances in Regenerative Biology 2014. © 2014 Erica J. Carbone et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 Unported (CC BY 4.0) License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

Citation: Advances in Regenerative Biology 2014, 1: 25276 - http://dx.doi.org/10.3402/arb.v1.25276

 

Skeletal diseases and injuries, such as bone fractures, affect a significant portion of the population. While several protein-based medications are currently approved for the treatment of skeletal disorders via bone repair and regeneration, it is known that these protein-based therapeutics can cause significant side-effects. An alternative to protein-based pharmaceuticals is urgently needed in order for the therapeutics to be safer, more effective, and more affordable. Small molecule-based drugs are more appealing for treating bone diseases because they are more affordable, more stable, and require a lower dosage to achieve a bone regenerative effect. Interestingly, some small molecule drugs that are already being prescribed to treat a variety of medical conditions show potential to be used as therapeutics for bone repair and regeneration. This review article provides a summary of recent findings of approved small molecule compounds with bone regenerative capability.

Background

Bone repair and reconstruction using therapeutic biological factors is a very common orthopaedic procedure (1, 2) (Fig. 1). This is because certain biological factors are able to promote bone regeneration via osteoblastic differentiation and maturation. Traditionally, these compounds have been protein polypeptides. Over the past several decades, bone morphogenetic proteins (BMPs) have been the most widely used biologicals in the treatment of skeletal disorders (1, 3, 4). Unfortunately, BMPs used for current orthopaedic applications suffer from a number of limitations (5, 6). Osteoinductive small molecules have the potential to overcome many limitations, including those of growth factors, such as poor availability, contamination with bacterial cells or toxic debris, and adverse immunogenic reaction (Table 1). In fact, small molecules with regenerative potential have been suggested as a viable option for tissue engineering (7). A number of small molecules have been identified that can be employed as molecular tools to direct stem cell differentiation, or to reprogram somatic cells to a more naive state (8). In addition, multiple research groups have reported hundreds of small molecules that can promote bone repair and regeneration [please refer to (5, 6, 911) for the list]. It is worth noting that some of these small molecule compounds have been approved by the Food and Drug Administration (FDA) as remedies for other health ailments (5, 12). Since the clinical data regarding bioavailability and toxicity of these drugs in humans are well-documented, they could translate very quickly into orthopaedic applications once further research establishes their osteoinductive abilities in a larger, pre-clinical animal model. This review focuses on the approved small molecule compounds and their potential with regard to bone repair and regeneration, as well as perspectives concerning future directions.

Fig 1

Fig. 1.   The role of biological factors in the bone formation process. (a) Small molecule-based biological factors travel into the cytoplasm to stimulate the signalling cascade associated with osteoblastogenesis and bone formation. (b) Protein-based biological factors bind with a membrane receptor, activating the signalling cascade through the intracellular receptor domain. In both cases, the activated signalling cascade will modify the transcription factors that play a vital role in transcription of genes associated with osteoblast-specific protein expression. These proteins are important for osteogenesis, as they will promote the differentiation of mesenchymal stem cells (MSCs) or osteoprogenitor cells into mature osteoblasts, ultimately inducing bone formation. Figure was modified from (5).


Table 1.  Comparison of therapeutic proteins and small molecules. Table was adapted from (5).
  Advantages Disadvantages
Protein Specific Unstable Impurities
    High cost
    Immunogenic
    Given by injection
Small molecule Ease of manufacturing Low cost Non-specific, off target side-effects
  Stable  
  Non-immunogenic  
  Orally available  
  Ease of delivery  

Approved small molecule compounds with regenerative capacity of skeletal tissue

In this section, we focus on a number of FDA-approved small molecule compounds: simvastatin, alendronate, retinoic acid, rapamycin, doxycycline, dexamethasone, vitamin C, and vitamin D3. The background information and experimental evidences of osteoinductive capabilities for each drug are summarised in Table 2.


Table 2.  FDA-approved small molecule compounds (<1,000 Da) that have recently shown promise as bone-regenerating therapeutics in vitro and in vivo
Approved small molecules Molecular formula Mechanisms of action In vitro cell models In vivo animal models References
Alendronate C4H13NO7P2 25276_ILF0001.jpg Deprotonated O and P-C-P backbone target hydroxyapatite; inhibits protein-tyrosine phosphatase and farnesyl diphosphate synthase Human adipose tissue-derived stem cells (hADSCs); monocytic macrophages Rat-calvarial defect model (19, 4244)
Retinoic acid C20H28O2 25276_ILF0002.jpg Binds to retinoic acid receptors; might inhibit Runx2 and up-regulate bone morphogenic protein and hedgehog signalling Murine adipose-derived adult stromal cells (ADASs); primary rat calvarial osteoblasts; bone marrow stromal cells; cranial suture–derived mesenchymal cells Mouse – calvarial implantation of ADASs ‘primed’ with retinoic acid (2729, 45)
Sirolimus (rapamycin) C51H79NO13 25276_ILF0003.jpg Inhibits mTOR pathway; stimulates BMP/Smad signalling pathway Human embryonic stem cells; ROS 17/2.8; pre-osteoblastic cells; primary mouse bone marrow stromal cells (BMSCs) Mouse – closed femur fracture model (31, 32)
Simvastatin (statin) C25H38O5 25276_ILF0004.jpg Targets HMG-CoA-reductase; likely stimulates BMP-2 expression Mouse osteoblast-like MC3T3-E1 cells; Human osteoblast MG63 cells; mouse embryonic stem cells; rat and human bone marrow stromal cells; murine embryonic stem cells (mESCs) Mouse – subcutaneous injection over the calvaria; rat – systemic treatment; Mouse – gap fracture bridging (1416, 4648)
FK506 (analogue of rapamycin) C43H67NO12 25276_ILF0005.jpg Stimulates BMP/Smad signalling pathway; might activate BMP receptors C17; mouse myoblast C2C12; MC3T3-E1; rat bone marrow cells; rat mesenchymal stem cells (MSCs); stromal cell line ST-2 Rat – subcutaneous implantation (33, 37, 4951)
Doxycycline (tetracycline) C22H24N2O8 25276_ILF0006.jpg Inhibits matrix metalloproteinases, specifically collagenase MC3T3-E1, Osteoprogenitor cells from human femoral cancellous bone Dog – periodontal implantation; Rabbit – implantation of Titanium zirconium coated with doxycycline Human – bilateral infrabony defect repair (3941, 52)
Dexamethasone C22H29FO5 25276_ILF0007.jpg Regulates chemokine (C-C motif) ligand 5 (CCL5) expression during osteogenesis; induces Cbfa1 and Osx gene expression; induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation Human mesenchymal stem cells; primary rat osteoblasts; Rat – calvarial defect model (5357)
Ascorbic Acid (vitamin C) C6H8O6 25276_ILF0008.jpg Increases expression of osteopontin, osteonectin, and RUNX2; promotes increased Col1/α2β1 integrin-mediated intracellular signalling Human suspension mononuclear cells (MNCs) Ovariectomised mouse – oral administration (5759)
Vitamin D3 C27H44O 25276_ILF0009.jpg Increases expression of type 1 collagen proteins, osteopontin, osteocalcin, and bone sialoprotein Human adipose-derived mesenchymal stromal cells Human osteocalcin enhancer/promoter-luciferase transgenic mouse – oral administration (60, 61)
All molecular structures were adapted from PubChem Compound database.

The statins compose a family of medications used to lower cholesterol by inhibiting the activity of HMG-CoA reductase, an enzyme that plays an important role in the production of cholesterol in the liver (13). Over the past decade, statins have drawn attention as bone regenerative therapies due to their anabolic effects on bone metabolism (14). Specifically, the small molecule simvastatin has been shown to provide an essential signal to induce osteogenic differentiation of stem cells. Pagkalos and colleagues showed that treatment with simvastatin induced differentiation of murine embryonic stem cells (mESCs) into osteoblasts, as indicated by increased osteogenic gene expression and matrix mineralisation (15). Moreover, numerous in vivo studies have demonstrated the anabolic effect of simvastatin when used in conjugation with appropriate delivery systems (16). Specifically, simvastatin has been shown to enhance cancellous bone density and cancellous bone compressive strength through local application in various animal models (16). Further research using statin analogues in animal models could provide more data supporting the quick translation of statin pharmaceuticals as bone regenerative therapies.

Alendronate is in a class of medications called bisphosphonates, which are used for treating osteoporosis in women who have experienced menopause (17). It is a potent inhibitor of osteoclastic bone resorption (18). Recent studies have demonstrated that alendronate enhances alkaline phosphatase (ALP) activity and matrix calcification through endogenous bone morphogenic protein 2 (BMP-2) production in human adipose-derived stem cells (hADSCs) (19). The effect of alendronate on in vivo osteogenesis was evidenced in a critical-sized calvarial defect rat model, implanted with a hADSC-loaded poly(lactic-co-glycolic acid) (PLGA)-based scaffold. Their results revealed that local administration of alendronate on hADSC-seeded scaffolds had a significant effect on bone repair and regeneration at week 12 (19). These results would be beneficial in a clinical setting, since bone regenerative therapies using adipose-derived stem cells would be a less invasive procedure than using bone marrow–derived stem cells. More recently, Bobyn et al. demonstrated that alendronate increased peri-implant bone formation in a dog model, where alendronate-coated cylindrical rods were implanted into the femoral intramedullary canals (20). These in vivo results using animal models confirm that bisphosphonates, specifically alendronate, have the potential to induce localised bone regeneration in humans as well. However, the use of alendronate for regenerating bone is associated with concerns. As alendronate functions to inhibit bone turnover and induce substantial increase in bone mass, the natural bone remodelling process is affected. Clinical studies have shown that alendronate, when systematically administered, leads to positive bone balance and the effect is sustained even long after the therapy is suspended (21, 22). Therefore, localised delivery of alendronate should be more beneficial and desired than systemic administration for bone regeneration. In addition, the dose of alendronate and duration of treatment should be carefully determined.

Retinoids are metabolites of vitamin A and are currently being used to treat various cancers, acne, and psoriasis (2326). All-trans-retinoic acid (ATRA) has been suggested as a therapeutic for bone regeneration. For instance, ATRA has been shown to increase ALP activity in a variety of stem cells including adipose-derived adult stromal (ADAS) cells (27), bone marrow–derived stromal cells (BMSC) (28), and cranial suture–derived mesenchymal cells (29). Wan et al. pre-cultured ADAS cells with retinoic acid for 15 days, and then seeded them onto PLGA-based scaffolds. Their results indicated that in vivo bone formation was significantly enhanced 2 weeks after implantation, without retinoic acid-mediated osteoclastogenic resorption (27). Additional research should be conducted in order to confirm the positive effects that ATRA has on bone health, specifically stimulation of bone formation, without subsequent bone resorption.

Immunosuppressants have also been investigated for regenerating skeletal tissue. Rapamycin is currently prescribed for preventing graft rejection and treating autoimmune diseases (30). Lee et al. demonstrated that continuous treatment with rapamycin stimulated differentiation of human embryonic stem cells (hESCs) into an osteoblastic lineage, evidenced by mineralised bone nodules and upregulation of both early and late stage osteogenic markers (31). Intriguingly, Singha et al. observed that rapamycin inhibited cell proliferation and differentiation in osteoblast-like MC3T3-E1 cells, as well as in primary mouse BMSCs (32). FK-506, an analogue of rapamycin, has been shown to increase ALP activity, expression of osteoblastic genes, and bone nodule formation in rat mesenchymal stem cells (33). Nevertheless, the effects of FK-506 on bone formation are likely to be dependent on the drug administration method and dose. It has been suggested that systematical administration of FK-506 causes osteopenia in rodents and humans (34, 35); while local administration of FK-506 promotes osteogenic differentiation (36, 37). In addition, studies have shown that FK-506 at the concentrations appropriate for immunosuppression mainly inhibits osteogenic differentiation; however, at higher concentrations, it stimulates osteogenic differentiation by upregulating BMP/Smad signalling (38). These results suggest that the local use of FK-506 at sufficiently high doses may be beneficial for bone formation, provided that any systemic adverse effect can be avoided. Overall, the utilisation of rapamycin should be looked at further for bone repair and regeneration since the combined effects of its immunosuppressive and bone regenerative properties would be beneficial for use with stem cells therapies and allograft transplants.

Tetracyclines are antibiotics used to treat bacterial infections. Apart from their anti-microbial activity, Eglence et al. observed that human osteoprogenitor cells exposed to low concentrations of a common tetracycline, doxycycline, showed osteoblast differentiation to the same extent as when the cells were exposed to BMP-2 (39). Interestingly, in vivo studies have shown that the anti-microbial and osteogenic properties of doxycycline make it a valuable drug to simultaneously combat infection and facilitate healing of oral infrabony defects (40). Recently, the benefits of using doxycycline as an osteogenic agent on the surface of bone implants have been explored by Walter and colleagues using an in vivo rabbit model (41). In short, their results indicated that titanium zirconium (TiZr) implants coated with doxycycline showed an increase in various bone-forming markers after 8 weeks of implantation, as evidenced by osteoblastic differentiation, bone remodelling, and bone matrix formation (41). Overall, the small molecule doxycycline, due to its enhanced osteogenic effect and anti-microbial activities, is a very promising candidate for orthopaedic applications.

Additional small molecule compounds such as dexamethasone, vitamin C, and vitamin D3 that have been reported in the context of osteogenesis are summarised in Table 2. These molecules have all been shown to increase expression of genes associated with bone formation in vitro and in vivo. In fact, dexamethasone and vitamin C are routinely used as supplements to induce osteogenic differentiation of cells for laboratory studies. In addition, vitamin D3 has been well-established as an essential dietary component to help the body absorb calcium, subsequently lessening the chance of bone loss and bone fracture later in life. Further investigations can determine how these widely available small molecules can be used as bone regenerative pharmaceuticals in the clinic.

Future perspective and conclusion

Hundreds of small molecules have demonstrated the ability to promote osteogenesis, a few of which are already approved for other indications, making them safe for human use. The in vitro and in vivo studies reviewed here have indicated that local administration of many FDA-approved small molecules can induce and accelerate bone fracture healing. Next, various pre-clinical animal model studies can facilitate the translation of the research results into clinical applications including treatments for osteoporosis, long bone fractures and non-unions, and spinal fusion. However, biological activities, structure–activity relationships, and modes of action of these small molecules for bone regeneration have been only primarily investigated and need to be studied in further detail. Moreover, an effective targeted drug delivery strategy is required to limit the adverse effects of small molecule drugs without changing their efficacy in bone regeneration. Biodegradable polymer and ceramic scaffolds have been established as viable strategies for sustained-release drug delivery to target sites (9). Future studies should focus on combining osteoinductive small molecules identified by advanced screening technologies with biocompatible drug delivery devices to allow these approved pharmaceuticals, with minimal side-effects, to transition quickly into orthopaedic applications.

Acknowledgements

This work was supported by the funding from the State of Connecticut Stem Cell Research Foundation (13-SCA-UCHC-01), NIH-R21-AR060480 and NSF-EFRI# 1332329. We thank the Raymond and Beverly Sackler Center for Biomedical, Biological, Physical and Engineering Sciences Foundation for supporting our Institute. Dr. Cato Laurencin was the recipient of the Presidential Faculty Fellowship Award from President William Clinton and the Presidential Award for Excellence in Science, Mathematics, and Engineering Mentorship from President Barack Obama. Finally, we thank all members of the IRE, past and present, and numerous colleagues and friends for their helpful discussions.

Conflict of interest and funding

The authors have not received funding nor benefits from industry nor elsewhere to conduct this study.

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About The Authors

Erica J. Carbone
University of Connecticut Health Center
United States

Komal Rajpura
University of Connecticut
United States

Tao Jiang

United States

Cato T. Laurencin
University of Connecticut
United States

Kevin W.-H. Lo
University of Connecticut Health Center
United States

Department of Medicine, Assistant Professor