Prophylactic administration of Amifostine protects vessel thickness in the setting of irradiated bone

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Summary

Although often beneficial in the treatment of head and neck cancer (HNC), radiation therapy (XRT) leads to the depletion of vascular supply and eventually decreased perfusion of the tissue. Specifically, previous studies have demonstrated the depletion of vessel volume fraction (VVF) and vessel thickness (VT) associated with XRT. Amifostine (AMF) provides protection from the detrimental effects of radiation damage, allowing for reliable post-irradiation fracture healing in the murine mandible. The purpose of this study is to investigate the prophylactic ability of AMF to protect the vascular network in an irradiated field. Sprague–Dawley rats (n = 17) were divided into 3 groups: control (C, n = 5), radiated (XRT, n = 7), and radiated mandibles treated with Amifostine (AMF XRT, n = 5). Both groups receiving radiation underwent a previously established, human equivalent dose of XRT totaling 35 Gy, equally fractionated over 5 days. The AMF XRT group received a weight dependent (0.5 mg AMF/5 g body weight) subcutaneous injection of AMF 45 min prior to XRT. Following a 56-day recovery period, mandibles were perfused, dissected, and imaged with μCT. ANOVA was used for comparisons between groups and p < 0.05 was considered statistically significant. Stereologic analysis demonstrated a significant and quantifiable restoration of VT in AMF treated mandibles as compared to those treated with radiation alone (0.061 ± 0.011 mm versus 0.042 ± 0.004 mm, p = 0.027). Interestingly, further analysis demonstrated no significant difference in VT between control mandibles and those treated with AMF (0.067 ± 0.016 mm versus 0.061 ± 0.011 mm, p = 0.633). AMF treatment also showed an increase in VVF, however those results were not statistically significant from VVF values demonstrated by the XRT group. Our data support the contention that AMF therapy acts prophylactically to protect vessel thickness. Based on these findings, we support the continued investigation of this treatment paradigm in its potential translation for the prevention of vascular depletion after radiotherapy.

Introduction

Head and neck cancer (HNC) and its associated complications will account for 53,000 new cases and 13,000 deaths in 2013.1 The current standard of care for cancer of this nature often includes operative resection, subsequent reconstruction, and adjuvant radiotherapy. Although often beneficial in the clinical management of HNC, a number of previous studies demonstrated that radiotherapy leads to decreased regenerate vascularity and cellularity, resulting in unacceptably high rates of reconstructive sequelae.2, 3, 4, 5 Additionally, ischemia and hypovascularity as a result of radiation therapy6 predisposes patients to the devastating complications of osteoradionecrosis, pathologic fracture, and non-union.7, 8 Thus, a means to prevent these pathologies would be highly desirable.

Amifostine (AMF) is an organic thiophosphate prodrug under investigation for its cytoprotective capabilities in the setting of XRT. As a result of dephosphorylation in vivo, AMF becomes active as WR-1065. AMF acts as a cellular antioxidant, scavenging radiation-induced free oxygen species that damage local vascularity and tissue.9, 10 Moreover, activated AMF neutralizes reactive metabolites of platinum and alkylating agents and functions as a stabilizer of the global tumor suppressor, p53. In a recent phase III trial, AMF reduced the incidence of xerostomia in patients undergoing radiotherapy for head and neck cancer.9 Our model seeks to build upon these results in soft tissue by further investigating the effects of AMF in bone.

In previous studies, AMF demonstrated selective protection of normal, non-cancerous tissue, which is attributed to higher alkaline phosphatase activity, higher pH, and vascular permeation of normal tissues. Additionally, when treated with AMF prior to radiation, no changes in cancer reoccurrence rates are seen.8, 9, 10 This characteristic is of utmost importance in regards to the clinical applicability of AMF to protect and preserve local vascular supply in an irradiated field. Previous work in our laboratory demonstrated this effect as it relates to bone regeneration in both our irradiated models of distraction osteogenesis and pathologic fracture.11, 12, 13, 14, 15, 16, 17, 18, 19 In this study, we sought to investigate the radio-protective potential of AMF by examining the extent to which it protects the vascular supply in the murine mandible in normal, un-injured bone. The specific aim of this report is to determine the detailed pattern of vascular damage engendered by radiation in the mandible and in so doing help to better understand the pathologic mechanism of radiation injury to the vascular system. Furthermore we aim to determine the efficacy of AMF to protect the bony vasculature from the harmful effects of radiation. We hypothesize that AMF will act prophylactically to protect vascularity thereby potentially reduce the costly occurrence of osteoradionecrosis, pathologic fracture, and the other consequences of hypovascularity associated with radiation therapy.

Section snippets

Materials and methods

Animal experimentation was conducted in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals: Eighth Edition. Animal protocol # PRO00001267 was approved by the University of Michigan's Committee for the Utilization and Care of Animals (UCUCA) prior to implementation.

Results

AMF appeared to ameliorate the incidence of radiation-induced alopecia and oral mucositis. All animals underwent successful perfusions, were dissected, decalcified and subjected to subsequent micro-CT scanning. Stereologic analysis and ANOVA demonstrated a significant and quantifiable protection of VT in AMF-treated mandibles as compared to those treated with radiation alone (0.061 ± 0.011 mm versus 0.042 ± 0.004 mm, p = 0.027). Interestingly, further analysis demonstrated no significant

Discussion

Pathologic fractures and osteoradionecrosis continue to manifest themselves as damaging consequences of often life-saving radiotherapy. These sequelae can have devastating and life-altering consequences for patients stricken with these conditions, leaving them with the inability to eat, drink, or socialize normally. Here we demonstrated the clinically relevant capability of AMF to protect an essential metric of vascularity, vessel thickness, in the setting of irradiation. Radiation is known to

Disclosure/financial support

All authors state that they have no conflicts of interest.

Funding support

NIH T32-GM008616 to C.L. Marcelo, for A. Donneys.

NIH R01 CA 12587-01 to S. R. Buchman.

NIH R01 CA 12587-06 to S. R. Buchman.

Acknowledgments

The authors thank Mary Davis and David Karnack for assistance with the delivery of radiotherapy. The authors do not have a conflict of interest to declare. Supported by grants from the National Institutes of Health (CA12587-01 and CA12587-06) to S.R.B and (T32-GM008616) to C.L.M. for A.D.

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