Prophylactic skin treatment for radiation therapy

Abstract

To provide a method for manufacturing a medicament for reducing skin fibrosis by prophylactically treating skin prior to radiation therapy.SOLUTION: The present invention provides use of deferoxamine (DFO) for the manufacture of a medicament for decreasing radiation-induced fibrosis, wherein the medicament is prepared to be administered according to a dosage regimen comprising: administering an effective amount of DFO to a region of skin of a subject at a treatment site for a first period of time prior to a radiation treatment; administering an effective amount of DFO to the region of skin during a second period of time; administering radiation to the region of skin during the second period of time; and administering an effective amount of DFO to the region of skin for a third period of time subsequent to the radiation treatment.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Application No. 62 / 938,209, filed on November 20, 2019, which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference All publications and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0003] Statement Regarding Federally Sponsored Research This invention was made with government support under Contract DE026914 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] This invention relates to prophylactic skin treatment for radiation therapy.

Background Art

[0005] After heart disease, cancer is the leading cause of death in the United States. In 2017, an estimated 1.6 million new cancer cases were diagnosed and 600,000 cancer - related deaths were predicted. However, in recent years, substantial medical advances have been made in surgery, chemotherapy, and radiation therapy, increasing both the number of cancer survivors and the length of their survival. Due to this improvement, for example, long - term problems related to the treatment of cancer by radiation therapy have become increasingly apparent and have been shown to have a great impact on quality of life. Radiation - induced soft - tissue injury is one of the most common side effects of radiation therapy, affecting more than 90% of patients, and the resulting soft - tissue atrophy and fibrosis can lead to both severe cosmetic and long - term functional impairment.

[0006] Radiation therapy is a mainstay in the treatment of many malignancies. However, radiation therapy can cause secondary damage to surrounding tissues with attendant avascularity, fibrosis, and atrophy, and the damaged tissues may be difficult to reconstruct. Inevitably, radiation therapy (RT) is associated with pathologic-level progressive skin fibrosis.

[0007] In the United States, over 5.6 million soft tissue reconstructions are performed annually, with over half related to the sequelae of tumor resection and adjuvant radiation therapy. Even with an overlying intact epithelium, insufficient underlying soft tissue can result in visible asymmetry and contour abnormalities, contributing to unstable wounds and inadequate protection of vital organs and structures, including bone, implanted hardware, and major blood vessels. Although radiation therapy has been shown to be highly effective in reducing the local recurrence risk of various tumors, secondary damage to adjacent soft tissue with attendant microvascular occlusion and fibrosis can significantly complicate reconstruction strategies.

[0008] Chronic radiation injury is characterized by epidermal thinning, eosinophilic homogeneous amorphization of dermal collagen with sclerosis, scattered large and atypical fibroblasts, and fibrous thickening with occlusion of the lumen of deep blood vessels. The development of vascular injury and fibrosis is thought to result from radiation-induced cytokine expression, generation of reactive oxygen species, and apoptosis of cells, and soft tissue reconstruction of such affected sites is extremely difficult despite this. Autologous fat transplantation to address soft tissue deficiency after radiation has become increasingly popular, but fibroinflammatory changes and avascularity are associated with a worse fat graft outcome. In cell-assisted lipotransfer, improved retention has been noted, but the functional heterogeneity among stromal cells used to concentrate aspirated adipose tissue, combined with concerns about local recurrence in the post-tumor region, limits the widespread adoption of this strategy. Deferoxamine (DFO) is an iron chelating agent approved by the FDA for acute iron poisoning and chronic iron overload, and it has also been shown to increase angiogenesis. DFO has been demonstrated to increase hypoxia-inducible factor 1 alpha (HIF-1α) activity and improve the expression of angiogenic growth factors. Studies have also shown that local injection of DFO improves the survival of ischemic flaps in both mouse and pig models, with increased skin flap blood perfusion and capillary density noted in DFO-treated animals. Furthermore, in the context of irradiated bone, multiple reports have found that DFO promotes bone regeneration after distraction osteogenesis depending on improved vascular distribution.

[0009] The potential of angiogenesis agents and DFO as an antioxidant with the potential to improve fat graft survival in healthy subjects has also been studied, and its use to increase the survival rate of fat grafts in plastic surgery has been proposed. Importantly, recently, DFO has been suggested to promote fat graft survival in a rat model. However, in fat grafts injected with DFO, no changes in cell apoptosis were observed, but more inflammation and fibrosis were noted. The repeated manipulation of fat grafts with each injection may have contributed to this observation. In addition, it has been claimed that the adipogenic differentiation of endogenous stromal cells contributes to long-term fat graft retention, and direct exposure of fat grafts to DFO may be harmful to this process. Studies have shown that intracellular iron deficiency due to DFO administration severely blunts adipocyte differentiation. Therefore, these findings dampen the enthusiasm for direct injection of DFO into fat grafts.

[0010] Current treatment options for radiation-induced fibrosis (RIF) are limited. When injected into irradiated tissue prior to fat grafting, the iron chelator deferoxamine (DFO) has previously been shown to improve cutaneous angiogenesis. Applicants describe a method of topical delivery of DFO prior to and immediately after radiation exposure, which may mitigate the chronic effects of radiation damage to the skin.

Summary of the Invention

Means for Solving the Problems

[0011] A method for reducing radiation-induced fibrosis is provided, the method comprising: administering an effective amount of DFO to a region of the subject's skin at the treatment site over a first period prior to radiation treatment; administering an effective amount of DFO to the region of the skin during a second period; administering radiation to the region of the skin during the second period; and administering an effective amount of DFO to the region of the skin over a third period subsequent to the radiation treatment.

[0012] In some variations, administering an effective amount of DFO to a skin area may include transdermally delivering the DFO. Administering an effective amount of DFO to a skin area may include applying a transdermal delivery device to the surface of the skin area at the treatment site. In some variations, the transdermal delivery system may include DFO encapsulated in reverse micelles.

[0013] In some variations, applying a transdermal delivery device to the surface of the skin area at the treatment site may further include applying a new transdermal delivery device at selected time intervals during each of the first, second, and third periods. The selected time interval may be from about 12 hours to about 36 hours. In some variations, the selected time interval is daily.

[0014] In some variations, the first period may be from about 3 days to about 21 days. In some variations, the second period may be from about 5 days to about 10 weeks. In some variations, the third period may be from about 2 weeks to about 8 weeks or more.

[0015] In some variations, administering radiation during the second period may further include administering radiation in a pattern of administering radiation across the first portion of the second period and then not administering radiation across the second portion of the second period. In some variations, the pattern of administering radiation and then not administering radiation may be repeated about 3 to 10 times during the second period. In some variations, the first portion of the time during the second period is from about 3 days to about 7 days, and the second portion of the time is from about 4 days to about 10 days.

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and from the appended drawings.

Brief Description of the Drawings

[0017]

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DETAILED DESCRIPTION OF THE INVENTION

[0018] By 2019, it is estimated that 2 million new patients will be diagnosed with cancer in the United States, and more than half of these patients will eventually receive radiation therapy (RT). Due to the increasing survival rate, the late effects of cancer treatment are becoming more apparent. Skin fibrosis damage is the most important dose-limiting factor in RT administration. The skin is extremely sensitive to RT, and more than 95% of patients experience acute skin reactions. Acute skin damage inevitably progresses over weeks to years to radiation-induced skin fibrosis (RIF), which is characterized by dermal sclerosis and microvascular hyperplasia leading to low perfusion and hypoxia. When RIF is severe, it can result in significant cosmetic and functional consequences that can substantially impact quality of life, including loss of range of motion and muscle strength. The pathogenesis of RIF is multifactorial and poorly understood. Key factors contributing to excessive soft tissue fibrosis include free radical generation, fibroblast activation, and damage to microvascular endothelial cells.

[0019] Current treatments for RIF are limited, showing significant benefits in only a few well-designed clinical trials, and there is no effective preventive regimen to alleviate this complication. Also, pentoxifylline, a methylxanthine derivative originally developed to improve blood flow in cardiovascular patients, reduces RIF and improves tissue function, especially when used in combination with vitamin E. The beneficial effects of pentoxifylline are thought to be largely driven by its ability to improve local blood flow, reduce blood viscosity and total peripheral vascular resistance, and thus reduce tissue hypoxia. Despite these reported benefits, a significant number of patients experience severe side effects, and poor tolerance and compliance clinically limit the use of pentoxifylline significantly.

[0020] The present invention provides a method for prophylactically minimizing the long-term development of skin fibrosis leading to scar formation and limitation of physical function in patients undergoing cancer radiotherapy (RT) following administration of deferoxamine. In some variations, the method can be performed in combination with autologous fat transplantation.

[0021] Subcutaneous injection of deferoxamine (DFO) prior to fat transplantation has been demonstrated to improve the soft tissue vascularization of previously irradiated sites and subsequent graft retention. DFO is an approved drug by the US Food and Drug Administration (FDA) and is commonly used to treat conditions related to iron overload. Depending on its iron chelation mechanism, DFO stabilizes hypoxia-inducible factor 1 alpha (HIF1α). This translocates to the nucleus and acts as a transcription factor for several potent pro-angiogenic genes including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase. The downstream result is improved tissue angiogenesis. Numerous studies have found that DFO treatment improves angiogenesis in hypoxic states of tissues including skin flaps, irradiated bone, and those related to diabetic foot ulcers. Radiation damage to the skin is a slow, progressive process that is particularly difficult to reverse completely when it is complete. Thus, an ideal treatment regimen would either be essentially prophylactic or target the earliest stages of this pathological process. Topical DFO treatment using a novel transdermal drug delivery system (TDDS) prior to and immediately after radiation exposure can improve tissue vascularization in the skin and mitigate the severity downstream of late chronic RIF.

[0022] In most patients treated according to RT, skin fibrosis and its long-term sequelae are frequent and often unavoidable side effects. In addition to aesthetic concerns, this significantly alters tissue morphology and function and can have a major impact on the patient's quality of life. RIF is a progressive disease that worsens months and years after radiation treatment. Therefore, preventing RIF prior to RT or targeting the earliest stages of its development can prevent more downstream amplification later and thus provide the most therapeutic benefit. The applicant has discovered that topical administration of DFO increases skin angiogenesis and perfusion and thereby can alleviate one key aspect of RIF. The most beneficial effects can be obtained by the methods described herein for prophylactic DFO treatment administered before, during, and after radiation exposure.

[0023] The main mechanism by which RIF appears in the skin is due to damage to irradiated microvessels. In the first 24 hours after RT, leukocytes infiltrate the vessels and fibrin plugs form. The endothelial cells lining the vessels then swell, undergo hyperplasia, leading to perivascular fibrosis, small vessel occlusion, low perfusion, and ultimately tissue hypoxia. Conditions of low oxygen partial pressure stimulate increased expression of collagen type 1 alpha 1 (COL1A1) and promote the development of tissue fibrosis. In addition to CD31 staining, laser Doppler imaging can be used as a surrogate measure of vascular density to show that radiation significantly reduces skin blood flow. It has been previously shown that subcutaneous injection of DFO into irradiated tissue prior to fat transplantation can increase perfusion of the overlying skin and thereby alleviate the fibrotic effects of RT. The applicant shows here for the first time that topical DFO administration by a novel TDDS can confer significant benefits when the skin is conditioned prior to RT.

[0024] It is likely that the protective role of DFO may be related to its downstream angiogenesis-promoting effect. DFO is an FDA-approved iron chelator, which has shown recent benefits in the context of ischemic and irradiated tissues. DFO chelates iron, which leads to increased HIF1α and, consequently, increased expression of several potent angiogenesis-promoting genes such as VEGF. Preventively treating patients with DFO can confer significant benefits to scalp perfusion during the period immediately after RT. Moreover, preventive treatment can be significantly more effective than radiation post-treatment alone. RT is a scheduled treatment, often planned weeks or months in advance, so targeting the skin during this early time window is clinically feasible and can have a great bridging benefit to cancer patients even with a moderate alleviation of skin fibrosis.

[0025] Accordingly, a method for reducing radiation-induced fibrosis is provided herein, the method comprising: administering an effective amount of DFO to an area of the subject's skin at the treatment site over a first period prior to radiation treatment; administering an effective amount of DFO to the area of the skin during a second period; administering radiation to the area of the skin during the second period; and administering an effective amount of DFO to the area of the skin over a third period subsequent to the radiation treatment.

[0026] Administering an effective amount of DFO to the area of the skin can include transdermally delivering DFO. Administering an effective amount of DFO to the area of the skin can include applying a transdermal delivery device to the surface of the area of the skin at the treatment site. In some variations, administering an effective amount of (if) DFO can include the area of the skin surrounding the area of the skin at the treatment site. The area of the surrounding area of the skin can be about 5%, 10%, 25%, 50%, or more of the area of the area of the skin at the treatment site. In some variations, the transdermal delivery system can include DFO encapsulated in reverse micelles.

[0027] Applying a transdermal delivery device to the surface of the skin region at the treatment site may further include applying a new transdermal delivery device at selected time intervals during each of the first, second, and third periods. The selected time intervals can be about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 48 hours, or any number of hours in between. In some variations, the selected time intervals are daily.

[0028] In some variations, the first period can be any number of days between about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 18 days, about 21 days, or in between. In some variations, the second period can be from about 5 days to about 10 weeks. In some variations, the third period can be about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, or more.

[0029] In some variations, administering radiation during the second period further includes administering radiation in a pattern of administering radiation across the first portion of the second period and then not administering radiation across the second portion of the second period. In some variations, the pattern of administering radiation and then not administering radiation can be repeated about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 times, or more during the second period. In some variations, the first portion of the time during the second period is about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, and the second portion of the time is about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more.

[0030] Another aspect of the present invention provides a method of preconditioning a soft tissue site to be irradiated with radiation with DFO to improve vascularization prior to transplantation of a fat graft. Typically, HIF-1α is degraded by prolyl hydroxylase domain-containing protein 2 (PHD2). DFO has been shown to stabilize HIF-1α by chelating the iron cofactor of PHD2 activity, leading to an increase in downstream angiogenic factors and mobilization of endothelial progenitor cells. This is thought to be the mechanism by which DFO promotes angiogenesis in ischemic skin flaps, improves wound healing in diabetic mice, and enhances callus size, mineralization, and mechanical strength at irradiated bone injury sites. Furthermore, radiation-induced avascular regression has also been observed with DFO treatment during distraction osteogenesis of the mandibular defect. All of these findings support the potential of DFO to precondition irradiated recipient sites for subsequent fat transplantation by stabilizing HIF-1α and increasing angiogenic gene expression.

[0031] Preconditioning irradiated tissue at the fat graft site with DFO prior to transplantation of the fat graft facilitates earlier angiogenesis of the fat graft. Histological analysis of the treated skin following this method revealed increased vascularization when the fat graft was placed in the recipient site preconditioned with DFO. This is a bridge to improved volume retention. Interestingly, the effects related to DFO can reach a plateau after four treatments, but the addition of a fat graft to DFO-treated irradiated tissue leads to further improvement in vascularization. This suggests that alternative mechanisms by the transplanted adipocytes and associated stromal cells can also be used to improve vascularization after fat transplantation. Finally, the effect of DFO treatment on skin vascularization is not related to significant changes in dermal thickness and collagen content compared to the decreased dermal thickness and collagen content after fat transplantation. Architectural changes observed in the dermis with decreased collagen secondary to fat transplantation may not necessarily be the result of improved vascularization alone.

[0032] In patients with radiation fibrosis and soft tissue atrophy, pre-conditioning the tissue by serial DFO injections prior to fat grafting can be logistically difficult and may not be well tolerated by the patient. Percutaneous delivery of DFO to irradiated tissue before and / or after fat graft implantation can be used as an alternative to delivery of DFO by direct injection. Also, such an approach may potentially be effective in pre-conditioning irradiated tissue for fat grafting and will likely be better tolerated by the patient. Alternatively, nanoparticle formulations of DFO have also been developed and their controlled release of DFO can similarly be used to improve the vascularization of irradiated skin. Also, these nanoparticles can be directly injected with the fat graft to promote earlier angiogenesis.

[0033] Since DFO promotes the expression of multiple angiogenic factors by stabilizing HIF-1α, concerns can also be raised regarding its use at the site after resection of irradiated tumors. To our knowledge, studies have not demonstrated an increased risk of cancer recurrence after local administration of DFO, although several reports have suggested an anti-tumor effect. Iron is required for oxygen transport, cell metabolism, and growth and is particularly important in cells with active growth. Not surprisingly, iron chelators such as DFO have been found to reduce liver fibrosis and their effect on iron metabolism has been shown to clinically reduce the progression of hepatocellular carcinoma. Iron dependence has also been reported in human epidermal growth factor receptor 2 positive breast cancer cells and multiple breast cancer cell lines have been shown to be sensitive to iron chelation. Therefore, these reports suggest that local application of DFO may not be associated with an increased risk of cancer recurrence.

[0034] DFO treatment can improve radiation-induced avascularity and this improved perfusion can improve the quality of the recipient site for fat grafting. After DFO treatment, the long-term retention of fat grafts injected into the irradiated site was significantly improved.

[0035] Reconstruction of irradiated tissue is difficult due to radiation-induced alterations in the recipient bed. Fibrotic inflammatory changes and avascularity have been shown to impact fat graft retention, and cell-based strategies have been shown to improve outcomes, but to date, regulatory and safety concerns have limited their translational potential. As an alternative approach, preconditioning irradiated tissue with deferoxamine improves local perfusion, which is associated with improved radiographic and histological fat graft outcomes. Thus, deferoxamine-based preconditioning prior to fat grafting holds promise for improving the reconstruction outcomes of irradiated tissue.

Example

[0036] Experiment Example 1 Adult 60-day-old male Crl:NU-Foxl Nu Immunocompromised mice were used in this study's experiments. Twelve mice were treated with a total of 30 Gy of external beam radiation delivered as six divided doses of 5 Gy each over 12 days, followed by a 5-week recovery. An additional six non-irradiated mice were used as healthy controls for laser Doppler analysis (LDA) and skin analysis. The irradiated mice were divided into two treatment groups: the DFO experimental group and the saline control group. After recovery, the mice underwent a total of seven treatments with either an injection of DFO (1 mg in 100 μl saline) or 100 μl of saline alone, just under the dermis every other day. Figure 1 shows an overview of the treatment of this irradiated scalp.

[0037] After radiation exposure, fat grafting was performed on the irradiated mice. After obtaining informed consent, under the approved IRB protocol #2188, aspirated adipose tissue was obtained from three healthy female donors aged 45, 49, and 51 without other medical comorbidities. Allowing the aspirated adipose tissue to sediment over 15 minutes, the layers were separated by gravity sedimentation, and then the oil and blood layers were removed by vacuum aspiration. The remaining adipose layer was centrifuged at 1300 rcf for 3 minutes at 4°C. Any remaining oil and blood were removed again, and the remaining fat was transferred from a 14-gauge needle into a 1-cc syringe for injection. Fat grafting was performed under the scalp by creating subcutaneous tunnels with the needle and then injecting 200 μl of aspirated adipose tissue in a retrograde fashion while withdrawing the needle.

[0038] Laser Doppler analysis ("LDA") was performed to measure perfusion at the radiation-exposed site using a Perimed PIM3 laser Doppler perfusion imager (Dataveg, Sweden). The laser Doppler perfusion index (LDPI) of the signal generated by LDA was used for comparison purposes. LDPI is the product of blood cell velocity and concentration and is represented by a color spectrum, with black / dark blue representing low perfusion and red representing high perfusion. LDA was performed prior to radiation exposure, after completion of radiation exposure and recovery, and then 24 hours after each treatment with DFO or saline. LDA was also performed every two weeks after fat grafting.

[0039] Five images were taken from each mouse, and the average LDPI of the five images was recorded. Figure 2A shows representative photographs of heat maps of mouse scalp before radiation, after radiation, and after treatment with either saline or DFO. Darker areas represent lower perfusion, and brighter areas represent higher perfusion. Figure 2B shows that quantification of the laser Doppler perfusion index demonstrates a significant decrease in perfusion after radiation. Laser Doppler analysis shows improved perfusion of irradiated tissue depending on DFO treatment. Laser Doppler analysis enables an estimate of in vivo local blood perfusion in microcirculation depending on the frequency shift of light scattered by moving red blood cells. This facilitated longitudinal measurements in the same animals after each treatment with DFO. DFO treatment (T) (upper line in Figure 2B) caused a significant increase in perfusion after four treatments (T4) compared to saline injection (lower line in Figure 2B), and reached a plateau after five treatments (T5) (*p<0.05).

[0040] Also, mice were imaged using a MicroCAT-II in vivo X-ray microCT scanner (Imtek; Knoxville, TN) two days after fat graft injection for baseline volume measurements. Thereafter, fat graft volume retention was analyzed a total of eight weeks every two weeks using microtomography, and the images were reconstructed as three-dimensional surfaces depending on cubic spline interpolation. All reconstructions were performed by a single researcher to avoid inter-observer variability.

[0041] For skin analysis, scalp skin biopsies were harvested from both treatment groups after completion of radiation and 8 weeks after fat grafting therefrom. For sectioning, specimens were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. For dermis thickness measurement, sections were stained with hematoxylin and eosin (H&E) and imaged using a Leica DM5000B optical microscope (Leica Microsystems; Buffalo Grove, IL) at 20× magnification. Dermis measurements were performed on 10 stained sections from each sample. Also, picrosirius red staining was performed for collagen content. Vascular distribution was determined by CD31 immunofluorescence staining (1:100 Ab28364; Abcam; Cambridge, MA and 1:200 AF547; Thermo Fisher Scientific; Waltham, MA) and DAPI counterstaining to visualize cell nuclei. Fluorescent images were obtained at 20× magnification using an X-Cite120 fluorescent illumination system (LumenDynamicsGroup; Ontario, Canada). Quantification of CD31 staining was performed using ImageJ (National Institutes of Health; Bethesda, MD), and the area of pixels positive per high-power field was measured to determine vascular density (11). Also, a comparison of both dermis thickness and CD31 immunofluorescence staining was made against non-irradiated skin.

[0042] After completion of radiation and 5 weeks of recovery, it was noted that scalp perfusion significantly dropped from 265.23 ± 7.01 LDPI (pre-radiation baseline) to 176.70 ± 2.59 LDPI (Figure 2B). However, treatment of the scalp with 1 mg of DFO every other day after radiation recovery resulted in increased LDPI, which was significant after 4 treatments (205.08 ± 2.30 LDPI) (*p < 0.05). However, 3 additional treatments with DFO did not result in any significant increase in perfusion, so no increase in LDPI measurement was noted after 4 treatments. In contrast, as shown by the lower line in Figure 2B, injection of saline alone did not result in a change in LDPI measurement throughout the treatment course.

[0043] In statistical analysis, data are presented as mean ± SE. Student's two-tailed t-test was used for comparison between two groups, and analysis of variance with Tukey's post hoc test was used for multiple-group comparison. All analyses were performed using StatPlus software (Analyst-Soft, Alexandria, Va.). Values of *p < 0.05 were considered significant.

[0044] In vivo radiographic analysis of fat grafts showed that DFO-preconditioned irradiated mice retained more fat volume (89.24% ± 1.69) at 2 weeks compared to saline-injected control mice (74.03 + 7.91) (Figs. 3A - C). Fat graft volume retention was consistently greater in DFO-treated mice (upper line in Fig. 3C) compared to saline control mice (lower line in Fig. 3C), and at 6 and 8 weeks, these differences were statistically significant (week 6: 73.17% ± 4.26 DFO vs. 52.40% ± 4.83 saline treatment; week 8: 71.75% ± 3.70 DFO vs. 49.47% + 4.62 saline treatment; *p < 0.05).

[0045] After irradiation and saline control treatment, the vascular distribution of skin biopsies demonstrated by CD31 staining was found to be significantly lower than that of non-irradiated healthy skin (*p < 0.05) (Figs. 4A - E and Fig. 5). However, as shown in Fig. 4A, treatment of irradiated skin with DFO resulted in increased CD31 staining, but this did not reach healthy skin levels. Also, as expected, skin biopsies obtained 8 weeks after fat grafting demonstrated increased CD31 staining compared to irradiated skin injected with saline control. Interestingly, slightly more CD31 staining was also noted after fat grafting in DFO-preconditioned mice relative to saline control fat grafted mice, but this difference was not significant.

[0046] In addition, skin perfusion after fat transplantation was measured by LDA, and the LDPI values were found to be lower than immediately after the completion of DFO or saline preconditioning due to changes in the three-dimensional architecture of the target area after fat engraftment. However, two weeks after injection of the fat grafts, significantly more perfusion was still noted in DFO-preconditioned mice (86.33 ± 2.00 vs. 65.72 ± 2.02 LDPI for saline controls; *p < 0.05) (Figure 5). Also, perfusion continued to increase in DFO-preconditioned mice after fat transplantation (upper line in Figure 6B), but perfusion also increased similarly in saline-injected control mice after fat transplantation (lower line in Figure 6B), and no significant difference in LDA was noted between the two groups two weeks later (127.78 ± 2.29 vs. 119.18 ± 4.09 LDPI for DFO and saline-treated mice 8 weeks after transplantation; p > 0.05) (Figures 6A - B).

[0047] Finally, the dermal thickness of irradiated skin after saline treatment was significantly greater than that of healthy non-irradiated skin (*p < 0.05) (Figures 7 - 8). Treatment of irradiated skin with DFO resulted in a slight decrease in dermal thickness (242.09 ± 7.22 µm) compared to saline-injected mice (256.71 ± 16.76 µm), but this was not significantly less. However, fat transplantation was found to significantly decrease dermal thickness regardless of whether the site was preconditioned with saline or DFO, but there was no significant difference when comparing these two groups (p > 0.05). In contrast to these findings, picrosirius red staining revealed a significantly increased collagen content after irradiation and saline treatment (*p < 0.05). Treatment of irradiated skin with DFO resulted in a slight decrease in collagen content, which was not statistically significant. And, similar to our observations of dermal thickness, fat transplantation was found to significantly reduce collagen content regardless of whether the site was preconditioned with saline or DFO (*p < 0.05) (Figures 9 - 10).

[0048] Therefore, local injection of DFO into irradiated avascular skin improved perfusion measured by laser Doppler analysis. Laser Doppler analysis enabled an in vivo estimate of local blood perfusion in the microcirculation based on the frequency shift of light scattered by moving erythrocytes. This facilitated longitudinal measurements in the same animals after each treatment with DFO. Histological analysis of the treated skin also revealed an increased vascular distribution as determined by CD31 staining after DFO treatment. At this time, the fat grafts were placed at the DFO-preconditioned recipient sites. This was a bridge to improved volume retention. Interestingly, the effects related to DFO were seen to reach a plateau after four treatments, but the addition of fat grafts to DFO-treated irradiated tissue led to a further improvement in vascular distribution. This suggests that alternative mechanisms by the transplanted adipocytes and associated stromal cells can also be used to improve the vascular distribution after fat grafting. Finally, no significant changes in dermal thickness and collagen content were found to be associated with the effect of DFO treatment on skin vascular distribution.

[0049] Example 2 Adult 60-day-old male Crl:NU-FoxlNU immunocompromised mice were used in this study. Twelve mice were treated with a total of 30 Gy of external beam radiation delivered as six split doses of 5 Gy every other day for 12 days, followed by a one-month recovery. An additional six non-irradiated mice were used as healthy controls for laser Doppler analysis (LDA) and skin analysis. The irradiated mice were divided into two treatment groups: the DFO experimental group and the control group. After recovery, we applied a transdermal delivery system to the irradiated scalp skin of the DFO experimental group. It contained a dry film with 13.4% weight / weight% of DFO encapsulated in inverse micelles by a nonionic surfactant stabilized by polyvinylpyrrolidone (PVP) in an ethyl cellulose matrix, cut into 5 / 8-inch circles, and covered with a silicon sheet of the same size. An identical transdermal delivery device without DFO was applied to the irradiated scalp skin of the control group mice. The transdermal delivery system was left in place for two days and then replaced with a new device. After treatment by radiation and seven exchanges of the transdermal delivery device, fat grafting was performed on the irradiated mice as described in Example 1 above.

[0050] As described in Example 1 above, laser Doppler analysis ("LDA") was performed before and after fat transplantation to measure perfusion at the radiation-irradiated site. Figures 11 and 12 show that mice with the DFO transdermal delivery patch (upper line in Figure 11) showed a significant improvement in blood flow compared to mice treated with a transdermal delivery device without DFO (*p<0.05). Figure 11 shows that the scalp (upper line) pretreated with the DFO transdermal delivery system had significantly higher perfusion than the scalp (lower line) pretreated with a transdermal delivery system lacking DFO one week after fat transplantation, as demonstrated by quantification of the laser Doppler perfusion index (*p<0.05). Figure 12 shows a representative LDA image of the mouse scalp. Perfusion is shown in mice prior to radiotherapy (leftmost image), in the experimental group (upper two images) pretreated with DFO delivered by a transdermal delivery device, and in the control group (lower two images) pretreated with a transdermal delivery device without DFO, immediately after and one week after the end of radiotherapy. Darker areas represent lower perfusion and brighter areas represent higher perfusion.

[0051] In vivo radiography analysis of fat grafts showed that irradiated mice preconditioned with DFO retained more fat volume two weeks later compared to control mice (Figures 13-14). Fat graft volume retention was consistently greater in mice treated with transdermal DFO (upper line in Figure 13) compared to control mice whose transdermal delivery device lacked DFO (lower line in Figure 13). Figure 14 shows a representative 3D reconstruction of fat grafts two weeks after transplantation in irradiated scalp preconditioned with either DFO (upper) or control (lower).

[0052] For skin analysis, scalp skin biopsies were harvested from both treatment groups at the time of fat graft placement after completion of radiation, by excising a skin patch at the fat graft incision site. Scalp skin was also harvested from healthy mice that were not irradiated. Skin stiffness was measured using an MTS-Bionix 200 with an interface SM-19 force transducer. Stress-strain curves were generated as shown in the figure, and then the Young's modulus (slope) was calculated to determine stiffness. Figure 15A shows the stress-strain curve of a healthy, non-irradiated mouse, Figure 15B shows the stress-strain curve of an irradiated mouse treated with a transdermal delivery device without DFO, and Figure 15C shows the stress-strain curve of the experimental group of irradiated mice treated with DFO via a transdermal delivery system. Figure 16 summarizes the Young's modulus data for the three groups. These data indicate that treatment of the skin with DFO after radiation therapy results in reduced skin stiffness.

[0053] Example 3 Adult female 60-day-old CD-1 nude immunocompromised mice (Crl:CD1-Foxn1nu, Charles River) were used for the experimental work (total n = 16). The mice were maintained in sterile microisolators at the Stanford University Research Animal Facility (4 mice / cage) and given free access to water and rodent chow. Stanford University guidelines were followed. All experiments were conducted in accordance with Stanford University Animal Care and Use Committee guidelines under an approved APLAC protocol (APLAC#31212).

[0054] DFO-TDDS Delivery: DFO was topically delivered by a monolithic matrix-type TDDS containing DFO dispersed in a biodegradable polymer, as described in Duscher, et al., "Transdermal deferoxamine prevents pressure-induced diabetic ulcers," Proceedings of the National Academy of Sciences 2015; Vol. 112(1): p. 94-99, which is hereby incorporated by reference in its entirety. The patch provided sustained release of the active ingredient. DFO is hydrophilic and was complexed with polyvinylpyrrolidone (PVP) at a concentration of 1 mg in 100 microliters to stabilize its amorphous form and promote 24-hour skin penetration (Figure 17A). Mice were divided into four experimental groups (n = 4 / group): 1) radiation alone (IR without DFO), 2) radiation followed by DFO treatment (IRtxDFO), 3) radiation preceded and followed by DFO treatment (IRppxDFO), and 4) no radiation and no DFO (no IR) (Figure 17B). Therapeutic DFO treatment began 4 weeks after completion of a 12-day 30 Gy radiation period and continued for 2 weeks. Preventive DFO delivery began 2 weeks prior to the start of radiation and continued until 6 weeks after completion. The DFO-TDDS was attached to mouse skin overlapping the cranial crown with leucotape for reinforcement. Super glue at both ends was used with three anchor sutures to secure the leucotape band under the mouse's chin (Figure 17C). Each DFO-TDDS was replaced every 24 hours.

[0055] Irradiation: Mouse scalp was irradiated with 30 Gy. Delivered in a total of 12 days in six 5 Gy doses given every other day. The rest of the body was protected using a lead shield. The dosing and fractionation protocol was selected based on a previous protocol that generates RIF.

[0056] Laser Doppler analysis (LDA): To measure the perfusion of the irradiated site, laser Doppler analysis (LDA) was performed. A Perimed PIM3 laser Doppler perfusion imager ( Järfälla (J.rf.IIa), Sweden) was used. The signal generated by LDA (laser Doppler perfusion index) was used for comparison purposes. This index is the product of blood cell velocity and concentration and is represented by a color spectrum, where black / dark blue represents low perfusion and red represents high perfusion. LDA was performed immediately after radiation exposure and 6 weeks after radiation exposure. Before measurements were taken of the region of interest (ROI) on the mouse scalp, the mice were anesthetized (isoflurane; 2–3% induction, 1–2% maintenance) and placed on a heat pad for 5 minutes (Figure 18A). Five images were taken from each mouse, and the mean laser Doppler perfusion index of the five images was recorded to give a single mean value per mouse.

[0057] Histology: Six weeks after radiation exposure, the mice were sacrificed and the scalp skin was processed for histological analysis. For sectioning, the specimens were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Cat#l5710) at 4°C for 18 hours, processed, and embedded in paraffin. For evaluation of dermal thickness, sections were stained with hematoxylin and eosin (H&E, Abcam, Cambridge, Mass., ab245880), and for evaluation of the collagen fiber network, sections were stained with picrosirius red (Abcam, ab150681). Standard protocols were used. The dermis was defined as the vertical distance from the basal layer of the epidermis down to the subcutaneous tissue overlapping it, and was measured for 10 randomly selected sections per mouse at 20× magnification. For evaluation of the collagen fiber network, picrosirius-stained skin was imaged (lower row in Fig. 20A). Polarization and 40× magnification were used (a total of 100 images per condition, 25 images per mouse). Slides were imaged using a Leica DM5000B optical microscope (Leica Microsystems, Buffalo Grove, Ill.). For evaluation of vascular distribution, immunostaining of mouse endothelial cells was performed. Paraffin slides were blocked with 1× Powerblock (Biogenex, HK083-50K), incubated with a 1:100 dilution of unconjugated anti-mouse CD31 (PECAM, Abcam, Ab28364) in 0.1× Powerblock at 37°C for 1 hour. The specimens were washed with phosphate-buffered saline (PBS, Gibco®, 10010023), incubated with an AlexaFluor647-conjugated secondary antibody (Abcam, Ab10079) at 37°C for 1 hour, washed with PBS, and then mounted on glass slides with DAPI FluoroMount G (SouthernBiotech, 0100-20). Fluorescent images were taken using an LSM880 inverted confocal (Airyscan, GaAsP detector, 880, Beckman). Standard fields of view (1024×1024) were used for all images.

[0058] Statistical analysis: Data are presented as mean and standard error of the mean (SEM) for parametric and median and range for non-parametric. Images of picrosirius red-stained slides were color deconvolved, converted to grayscale, binarized, and skeletonized. A novel algorithm running in MATLAB® (R2018b, MathWorks, Natick, MA) was used. Thirteen parameters of the collagen fibers were extracted from the skeletonized images (including length, width, branch points, and luminance), and dimensionality reduction was performed to generate a two-dimensional t-distributed stochastic neighbor embedding (TSNE) plot to visualize the collective differences in the collagen fiber network patterns between groups. Quantification of CD31 staining was performed using ImageJ (National Institutes of Health, Bethesda, MD) on three images per mouse per condition, measuring the area of pixels positive per high-power field in the dermis to determine the vascular density (Figure 20C). The Mann-Whitney test (non-parametric) was used to compare the means between two groups, and the Kruskal-Wallis test (non-parametric) was used to compare the means between three or more groups, using PRISM (Graphpad) software. Values of *p < 0.05 were considered statistically significant. The TSNE plot indicated that the collagen fibers in the skin of mice receiving continuous DFO treatment were most clearly visible and may represent more remodeling after radiation exposure.

[0059] Pre-treatment with DFO improves tissue perfusion after RT: Tissue perfusion measurements obtained by LDA immediately after RT showed that prophylactic treatment with TDDS-DFO significantly alleviated the harmful effects of RT on skin perfusion (**p < 0.01) (Figures 18B and 18C). Six weeks after RT, both prophylactic DFO and therapeutic DFO tended to have improved perfusion (Figures 18B and 18D). As expected, irradiated mice that did not receive DFO had significantly worse scalp perfusion than non-irradiated mice (**p < 0.01).

[0060] DFO improves neovascularization: As expected, the skin of non-irradiated mice formed significantly more blood vessels than the skin of untreated irradiated mice (****p < 0.0001). Similarly, the skin from mice receiving prophylactic DFO formed blood vessels well compared to that of non-irradiated mice and significantly more than the skin of irradiated mice that did not receive any DFO (*p < 0.05). In contrast, the skin of mice receiving DFO only after irradiation had significantly less angiogenesis than the skin of non-irradiated mice (***p < 0.001) (Figures 19A and 19B).

[0061] DFO improves dermal thickness and reduces total collagen content: To evaluate whether DFO-TDDS treatment could alleviate skin RIF, mouse scalp skin for histological evaluation of dermal thickness and collagen fiber network was harvested 6 weeks after irradiation. Analysis of hematoxylin-stained skin revealed that irradiation significantly increased dermal thickness (all ****p < 0.0001), and the dermis of irradiated but DFO-treated skin was more similar to that of non-irradiated skin, and mice receiving continuous DFO treatment showed the greatest benefit (Figures 20A [upper row] & 20B).

[0062] DFO treatment induces remodeling of the collagen fiber network: The collagen fiber network of mouse scalp skin was stained with picrosirius red (lower row of Figure 20A), modeled using a new computational algorithm, and represented in two-dimensional space using t-distributed stochastic neighbor embedding (TSNE plot) (Figure 20C). The TSNE plot indicated that the collagen fibers of mouse skin receiving continuous DFO treatment were most clearly visible, suggesting perhaps more post-irradiation remodeling.

[0063] Preventively treating mice with DFO conferred a significant benefit on scalp perfusion during the period immediately after RT. Moreover, preventive treatment was significantly more effective than treatment after radiation alone.

[0064] When a feature or element is said to be "on" another feature or element in this application, it can be directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is said to be "directly on" another feature or element, there are no intervening features or elements. When a feature or element is said to be "connected to", "attached to", or "associated with" another feature or element, it will be understood that it can be directly connected, attached, or associated with the other feature or element, or intervening features or elements may be present. In contrast, when a feature or element is said to be "directly connected to", "directly attached to", or "directly associated with" another feature or element, there are no intervening features or elements. Although described or shown for one embodiment, the features and elements so described or shown may be applicable to other embodiments. It will also be recognized by those skilled in the art that referring to a structure or feature "adjacent to" another feature may have portions that overlap or underlie the adjacent feature.

[0065] The terms used in this application are for the purpose of describing particular embodiments only and are not intended to limit the invention. For example, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" used in this application are intended to include the plural forms. Further, the terms "comprises" and / or "comprising", as used herein, specify the presence of the recited features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. The term "and / or" used in this application includes any and all combinations of one or more of the associated listed items and may be omitted as " / ".

[0066] Spatially relative terms, such as "below", "beneath", "lower", "vertically above", "upper", and the like, may be used in this application for ease of description to describe the relationship of one element or feature to another element or feature (singular or plural) illustrated in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. For example, if the device in the figures is turned upside down, an element described as "below" or "vertically below" another element or feature will be oriented "vertically above" the other element or feature. Thus, the exemplary term "below" can encompass both an orientation of directly above and directly below. The device may be otherwise oriented (rotated 90 degrees or at some other orientation), and the spatially relative descriptors used in this application may be interpreted accordingly. Similarly, unless specifically stated otherwise, the terms "above", "below", "vertical", "horizontal", and the like are used in this application for descriptive purposes only.

[0067] The terms "first" and "second" may be used in this application to describe various features / elements (including steps), but unless the context otherwise indicates, these features / elements should not be limited by these terms. These terms may be used to distinguish one feature / element from another. Thus, without departing from the teachings of the present invention, the first feature / element discussed below could be termed the second feature / element, and similarly, the second feature / element discussed below could be termed the first feature / element.

[0068] In this specification and the following claims, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", are meant to imply that various components can be used together in a method and product (e.g., a composition, an apparatus including a device, and a method). For example, the term "comprise" will be understood to imply the inclusion of any recited element or step, without the exclusion of any other element or step.

[0069] As used in this application, including its use in the examples, and unless otherwise expressly specified, all numbers used in the specification and claims are to be read as if prefaced by the word "about" or "approximately" even if the term does not explicitly appear. The phrase "about" or "approximately" may be used when describing magnitude and / or position to indicate that the value and / or position being described is within a reasonable expected range of values and / or positions. For example, a numerical value can have a value that is + / −0.1%, + / −1%, + / −2%, + / −5%, + / −10%, etc. of the stated value (or range of values). Also, unless the context indicates otherwise, any numerical value given in this application is to be understood as including about or approximately that value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited in this application is intended to include all subranges subsumed therein. As will be appropriately understood by those skilled in the art, it is also understood that when a value is disclosed (that when), values of "less than or equal to", "greater than or equal to", and the possible ranges between the values are also disclosed. For example, if the value "X" is disclosed, then "less than or equal to X" and "greater than or equal to X" (where, for example, X is a numerical value) are also disclosed. In this application, it is also understood that the data is provided in several different formats and that this data represents ranges of any combination of endpoints and starting points, as well as data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that values greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as the range between 10 and 15 are considered disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0070] Although various illustrative embodiments have been described above, various changes can be made to the various embodiments without departing from the scope of the invention as claimed. For example, the order in which the various described method steps are performed can often be changed in alternative embodiments, and in other alternative embodiments, one or more method steps can be skipped entirely. Any feature of the various device and system embodiments can be included in some embodiments and not included in others. Accordingly, the above description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention set forth in the claims.

[0071] The examples and illustrations included in this application show, as illustration and not limitation, specific embodiments in which the subject matter can be practiced. As noted, other embodiments can be utilized and derived therefrom, with structural and logical substitutions and changes being made, without departing from the scope of this disclosure. Such embodiments of the subject matter of the invention can, in this application, be referred to individually or collectively by the term "invention" for convenience only and without any intention of self-limiting the scope of this application to any single invention or inventive concept when more than one is actually disclosed. Thus, although specific embodiments have been illustrated and described in this application, any configuration calculated to achieve the same purpose can be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of the various embodiments. Combinations of the above embodiments and other embodiments not specifically described in this application will be apparent to those skilled in the art upon review of the above description.

Claims

1. Here's how: administering an effective amount of DFO to an area of ​​the subject's skin at a treatment site for a first period of time prior to radiation treatment; administering an effective amount of DFO to the area of ​​skin during a second period of time; administering radiation to the area of ​​skin during a second period of time; administering an effective amount of DFO to the area of ​​skin for a third period of time following the radiation treatment; 23. A method for reducing radiation-induced fibrosis comprising:

2. The method of claim 1 , wherein administering an effective amount of DFO to an area of ​​skin comprises delivering DFO transdermally.

3. 3. The method of claim 1 or 2, wherein administering an effective amount of DFO to an area of ​​skin comprises applying a transdermal delivery device to the surface of the area of ​​skin at the treatment site.

4. The method of claim 3 , wherein the transdermal delivery system comprises DFO encapsulated in reverse micelles.

5. 4. The method of claim 3, wherein applying a transdermal delivery device to the surface of the area of ​​skin at the treatment site further comprises applying a new transdermal delivery device at selected time intervals during each of the first, second, and third time periods.

6. 6. The method of claim 5, wherein the selected time interval is from 12 hours to 36 hours.

7. The method of claim 5 , wherein the selected time interval is daily.

8. 8. The method of any one of claims 1 to 7, wherein the first period of time is from 3 to 21 days.

9. 9. The method of any one of claims 1 to 8, wherein the second period of time is from 5 days to 10 weeks.

10. 10. The method of claim 9, wherein administering radiation during the second time period further comprises administering radiation in a pattern of administering radiation for a first portion of the second time period and then not administering radiation for a second portion of the second time period.

11. 11. The method of claim 10, wherein the pattern of administering radiation followed by no radiation is repeated 3 to 10 times during the second period of time.

12. 12. The method of claim 11, wherein the first portion of time in the second period of time is from 3 to 7 days and the second portion of time is from 4 to 10 days.

13. 13. The method of any one of claims 1 to 12, wherein the third period of time is from 2 weeks to 8 weeks.

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