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Blockade of the Ogf-Ogfr Axis Enhances Repair Processes In The Pennsylvania State University The Graduate School Department of Neural and Behavioral Sciences BLOCKADE OF THE OGF-OGFR AXIS ENHANCES REPAIR PROCESSES IN DIABETIC SKIN AND BONE A Dissertation in Anatomy by Michelle B. Titunick ©2018 Michelle B. Titunick Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2018 The dissertation of Michelle B. Titunick was reviewed and approved* by the following: Patricia J. McLaughlin Professor of Neural and Behavioral Sciences Director of the Graduate Program in Anatomy Dissertation Advisor Chair of Committee Ian S. Zagon Distinguished Professor of Neural and Behavioral Sciences Gregory S. Lewis Assistant Professor of Orthopaedics and Rehabilitation Christopher Niyibizi Associate Professor of Orthopaedics and Rehabilitation *Signatures are on file in the Graduate School. ii ABSTRACT Almost 9.5% of the U.S. population had diabetes in 2015 with 5-10% having type 1 diabetes. In addition to diabetic complications such as retinopathy, nephropathy, neuropathy, and cardiovascular disease, there is also poor wound healing and delayed fracture healing. Preclinical studies from our laboratory have demonstrated that topical naltrexone (NTX) is an effective and safe treatment for diabetic complications such as corneal keratopathy, dry eye, and non-healing ulcers. Naltrexone is an opioid receptor antagonist that, at an appropriate dosage, blocks the opioid growth factor (OGF)-opioid growth factor receptor (OGFr) axis resulting in increased cell proliferation. [Met5]- enkephalin, or OGF, is a highly conserved endogenous opioid peptide that tonically regulates cell proliferation by delaying the G0/G1 phase of the cell cycle. Both systemic and topical NTX have been shown to enhance corneal epithelial wound healing in normal and diabetic rats, diabetic rabbits, as well as the type 2 diabetic model, db/db. Topical NTX reverses dry eye in diabetic animal models, and increases collagen formation and angiogenesis in cutaneous wounds of diabetic rats and mice. Phase 1 clinical trials with topical NTX administered to the eye have reported tolerability and safety of the therapy; preclinical pathology studies confirm the lack of toxicity following sustained topical application. The underlying cause of delayed healing of bone fractures in diabetes is unclear. Both preclinical and clinical studies report elevated levels of enkephalins in diabetics suggesting that the increase in OGF, an inhibitory growth factor, may suppress cell replication and contribute to poor wound and fracture healing. There is limited iii information on the presence and role of the OGF-OGFr pathway in normal bone growth or in the process of fracture repair. Furthermore, the ability to manipulate the OGF-OGFr axis with naltrexone during the process of bone fracture repair is unknown. This dissertation hypothesizes that topical naltrexone to block the OGF-OGFr regulatory pathway is an effective modulator of diabetic complications related to delays cutaneous wound healing and bone fracture repair. The first aim tested the hypothesis that topical naltrexone enhances closure of full-thickness cutaneous wounds in type 1 diabetic rats at a rate comparable to standard of care- Regranex®. In aims 2 and 3, studies were conducted to investigate the role of the OGF-OGFr axis in diabetic bone. Studies were designed to determine whether diabetes is associated with a dysregulation of the OGF-OGFr pathway that subsequently changes bone composition in a type 1 diabetic rat model, and whether blockade of the pathway alters the rate of repair of diabetic bone. Lastly, studies were conducted to test the hypothesis that systemic naltrexone blockade of the OGF-OGFr pathway in diabetic animal models may protect against the complications related to bone fracture and repair. Male Sprague Dawley rats (Charles River Laboratories) were used throughout all the experiments and type 1 diabetes was induced by injections of streptozotocin. In aim 1 topical application of NTX accelerated the rate of closure of 6mm full thickness cutaneous wounds at a rate comparable to a daily application of the standard of care Regranex®. Analyses of the skin revealed that naltrexone treatment increased DNA synthesis, as well as expression of platelet-derived growth factor and vascular endothelial growth factor, required for the granulation tissue formation and angiogenesis. iv In the second series of experiments, OGF expression was detected at a comparable level in bones from normal rats aged 1 day to adult. OGFr expression was detected in the femur at all ages, and expression was elevated at days 1 and 21. OGF and OGFr expression were increased in type 1 diabetic rat bone relative to normal, non-diabetic bone. Serum levels of OGF were also increased in diabetic rats. Histological analyses of femurs from normal and type 1 diabetic rats revealed no significant differences in the number of osteoclasts, but did indicate with safranin O staining a decrease in calcified cartilage in diabetic rats. In comparison to normal bone composition, Ki67 staining showed a decrease in proliferative cells, and VEGF staining revealed a decrease in vascularity in diabetic bone. Seven days following fracture, the callus was examined by radiography and histology. Calluses in normal femurs had more cartilage than in diabetic bones, and more granulation tissue was evident in diabetic bones in comparison to diabetic bones treated with naltrexone. Bone tissue treated with naltrexone from diabetic rats displayed elevated levels of Ki67 staining relative to tissue treated with vehicle suggesting that topical application of naltrexone may accelerate early phases of bone repair. Serum OGF levels increased in DB rats with fractures treated with vehicle compared to the serum of DB rats without a fracture, suggesting fracture or fracture repair may increase serum enkephalin levels. NTX-treated rats had decreased serum OGF levels relative to that of diabetic vehicle-treated rats. Assessment of diabetic bone composition in animals treated systemically with either saline or naltrexone revealed that naltrexone-treated bones appeared to be stronger and absorbed more energy than vehicle-treated diabetic bones. No significant differences v between the 3 groups were seen in trabecular μCT measurements. Diabetic bone treated with either vehicle or naltrexone had significantly lower cross-sectional areas and cortical areas. No gross morphological differences were detected after 21 days of naltrexone therapy. In summary, naltrexone is effective in enhancing full-thickness cutaneous wound healing and early phases of fracture repair, and is able to change mechanical properties of diabetic bone. Naltrexone is comparable to the current standard of care for non-healing wounds, Regranex®, and has been proven to be safe in multiple tolerability studies. Local NTX can decrease granulation tissue in diabetic fracture calluses and increase cartilage and bone volume. Systemic naltrexone can increase diabetic bone strength and reduce OGFr expression. vi TABLE OF CONTENTS LIST OF FIGURES ………………………………..……………………………………………………………. xi LIST OF TABLES …………………………………..………………………………………………………….. xii ABBREVIATIONS .………………………………..………………………………………………………….. xiii ACKNOWLEDGEMENTS .……………………….………………………………………………………… xvi CHAPTER 1: INTRODUCTION …………………………………………………………………………. 1 1.1. Diabetes ……………….………………………………………………………………………… 2 1.2. Animal Models ……………………………………………………………………………….. 2 1.3. Cutaneous Wound Healing ……………………….……………………………………. 5 1.3.1. Cellular and Molecular Basis of Full-Thickness Wound Healing ……… 5 1.3.1.1. Hemostasis ....………………………………………………………………. 5 1.3.1.2. Inflammation ..……………….…………………………………………….. 6 1.3.1.3. Proliferation ....……………………………………………………………… 7 1.3.1.4. Remodeling ….……………………………………………………………… 8 1.3.2. Cutaneous Healing Complications, Amputation, and Diabetes ……… 8 1.3.3. Current Treatments in Wound Healing …………………………………………. 9 1.4. Bone Growth and Remodeling ……………………………………………………….. 12 1.4.1. Cellular and Molecular Basis of Fracture Healing …………………………… 12 1.4.1.1. Inflammation ……………………………………………………………….. 12 1.4.1.2. Soft Callus Formation …………………………………………………… 13 1.4.1.3. Hard Callus Formation …………….…………………………………… 14 1.4.1.4. Bone Remodeling ……………………………………….………………… 15 1.4.2. Bone Healing Complications and Fractures in the Diabetic Population ………………………………………………………………………………………………. 15 1.4.3. Current Treatments in Fracture Repair ………………………………………….. 18 1.5. Opioids …………………………………………..………………………………………………. 19 1.6. The OGF-OGFr Axis ..………………………………………..……………………………… 21 1.7. Opioid Receptor Antagonists ..……………………………………………………..… 21 1.7.1. Naltrexone …………………………………………………………………………………….. 21 1.7.1.1. Function of Naltrexone Blockade Actions ……………………… 22 1.7.2. Naloxone ……………………………………………………………………………………….. 24 1.8. Current Gap in Knowledge ..……………………………………………………………. 26 1.9. Hypothesis and Specific Aims .………………………………………………………… 26 1.10. References ………..………………………..…………………………………………………… 28 vii CHAPTER 2: TOPICAL NALTREXONE IS A SAFE AND EFFECTIVE ALTERNATIVE TO STANDARD TREATMENT OF DIABETIC WOUNDS ……………………………………… 53 2.1. Abstract ……………………………………………………………………………………………….. 54 2.1.1. Objective ……………………………………………………………………………………….. 54 2.1.2. Approach ………………………………………………………………………………………. 54 2.1.3. Results …………………………………………………………………………………………… 54 2.1.4. Innovation and Conclusion ……..…………………………………………………….. 55 2.2. Introduction ………………………………………………………………..……………………… 55 2.3. Clinical Problem Addressed ……………………………………………………..…………. 57 2.4. Materials
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