Radioprotectors & mitigators in radiation therapy: Harnessing diverse pathways for optimizing clinical outcomes

(i) Blocking nuclear DNA damage

DNA is a critical radiation target. Even one unrepaired double-strand break can trigger apoptosis. Ionizing radiation acts directly or indirectly through water radiolysis, producing ROS like hydroxyl radicals, superoxide, hydrogen peroxide, and hydroperoxyl radicals¹¹. These ROS inflict oxidative damage to critical cellular components, including DNA, lipids, and proteins, contributing to genomic instability and cell death. In mitochondria, radiation disrupts the electron transport chain, causing electron leakage and more superoxide. Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide, which is detoxified by catalase or glutathione peroxidise (GPx). If unchecked, hydrogen peroxide reacts with iron, forming highly reactive hydroxyl radicals that tilt the balance toward damage¹².

Antioxidants that neutralise free radicals can prevent DNA damage. Genistein (from soy) scavenges radicals and enhances DNA repair. Curcumin, derived from turmeric, exhibits strong antioxidant properties by neutralizing hydroxyl radicals and reducing oxidative stress in normal tissues. Osmium compounds, including those derived from tulsi (Ocimum sanctum), are known for their ability to counteract radiation-induced damage. Additionally, glutathione and cysteine derivatives, as thiol-based compounds, protect DNA by donating electrons to neutralize ROS¹³. Enzyme mimics like SOD mimics (superoxide dismutase mimetics) and GPx mimics (selenium-based compounds) act by catalyzing the breakdown of ROS, such as superoxide anions and hydrogen peroxide, into less harmful molecules, thereby enhancing the radioprotection of normal cells. Alternatively, compounds that induce cell cycle arrest or delay can provide cells with more time to repair DNA damage before progressing to mitosis⁹. CHK1 (Checkpoint Kinase) inhibitors disrupt cell checkpoints, increasing vulnerability to radiation, while CDK (Cyclin Dependent Kinase) inhibitors like palbociclib induce G1 arrest, enhancing radioprotection. G2/M arrest can sensitize cells by trapping them during less repair-friendly phases. By refining radiation delivery and combining with such agents, normal tissue damage can be minimised¹⁴.

(ii) Mitochondrial stabilization

Mitochondria are vulnerable to oxidative damage induced by radiation-generated ROS. Radiation damages mitochondria, opening the mitochondrial permeability transition pore (MPTP), disrupting membrane potential, and leading to cell death¹⁵. MPTP inhibitors like cyclosporin A and sanglifehrin A prevent mitochondrial destabilization. Inhibitors of cyclophilin D and p53 can further stabilize mitochondria. Since excessive p53 activation leads to apoptosis, its regulation is key. Agents like sodium orthovanadate block p53 signalling, reducing cell death in sensitive tissues¹⁶. 3,3′-Diselenodipropionic acid (DSePA) a selenium-based compound, offers strong radioprotection. It preserves glutathione and SOD, reduces liver damage, and improves survival in irradiated mice. At 2 mg/kg, DSePA improves gastrointestinal and spleen integrity and shows high lung absorption, enhancing protection in chest radiation¹⁷. Pharmacokinetic studies indicate that DSePA is absorbed effectively in lung tissues, enhancing its protective effects during thoracic irradiation scenarios¹⁸.

Radioprotectors activate MAPK, PI3K/Akt, NF-κB, and STAT3 pathways, inducing DNA repair genes (XRCC1, ATM, DNAPKCS), blocking apoptosis, and promoting survival via Bcl2, BclxL, and XIAP. Also shown are SAP kinase inhibition and mitochondrial stabilization.

(iii) Blocking caspase activation and poly ADP-ribosyl-polymerase (PARP) cleavage

Targets for blocking apoptotic signalling pathways to prevent cell death include (i) Bcl-2 family proteins (anti-apoptotic: Bcl-2, Bcl-xL) and (ii) Caspases¹⁹. Bcl-2 and Bcl-xL prevent mitochondrial membrane damage, blocking cytochrome c release. Inhibiting pro-apoptotic proteins like Bax and Bak enhances survival. Caspase inhibitors, such as Z-VAD-FMK, can block caspase activation and prevent apoptotic cell death induced by radiation²⁰. ARP, a DNA repair enzyme, can worsen cell damage when overactivated by consuming too much NAD+ and ATP, leading to energy collapse and cell death²¹. Inhibiting Poly (ADP-Ribose) Polymerase (PARP) prevents this chain reaction, preserving energy and cell function. Blocking caspase activation and PARP cleavage helps maintain cell viability during radiation exposure.

(iv) Decreasing systemic cytokine-mediated cell death & immunomodulators

Inflammatory pathways activated by radiation involve NF-κB and TNF-α, which drive cytokine production. Suppressing these can reduce systemic damage. NF-κB activates genes for cytokines like TNF-α and interleukins. Blocking NF-κB reduces inflammation and limits chronic radiation effects like lung or heart injury. TNF-α, released by immune and blood vessel cells after radiation, promotes apoptosis and tissue damage. Inhibiting TNF-α or its receptors helps minimise these outcomes²². Additionally, chlorophyllin, a plant-derived antioxidant, has demonstrated radioprotective effects by reducing oxidative stress and modulating the inflammatory response, further limiting tissue injury and enhancing recovery.

Ionizing radiation (X, α, β, γ rays, neutrons, heavy ions) generates lipid peroxides, ceramides, and ROS, damaging DNA, membranes, and mitochondria, leading to cytochrome C-mediated apoptosis. Growth stimulating hormones (GSH) and antioxidants detoxify ROS. OH· forms via water radiolysis or Fenton’s reaction. Countermeasures target these pathways and activate pro-survival signals.

Radiation activates pro-survival transcription factors such as NF-κB, Akt, MAPK, and Nrf2, which promote genes tied to repair, growth, and detoxification. Their activation in normal tissue offers protective advantages. For example, in acute radiation injury, the agent CBLB502 activates NF-κB, triggering anti-apoptotic genes like c-IAP-1 and c-FLIP, helping cells survive. Importantly, this protection does not interfere with radiation’s tumour-killing effect. PI3K/Akt activation also protects cells by suppressing pro-apoptotic proteins like Bad and Bax and activating mTOR to promote DNA repair. Akt enhances survival under oxidative stress. STAT3 is another protective pathway. It regulates survival genes and works with NF-κB to amplify radioprotective responses. The TLR5 agonist CBLB502 activates both STAT3 and NF-κB, shielding normal cells while sparing tumour sensitivity. This dual-pathway support boosts cell defense and recovery. Small molecule activators like bardoxolone methyl and sulforaphane, along with Keap1 inhibitors, enhance Nrf2 activity, promoting DNA repair and reducing radiation-induced tissue injury. Nrf2 also synergizes with other pathways like NF-κB and MAPK to strengthen cellular defenses, maximizing radioprotection while maintaining redox balance²³.

Differential effects on tumour vis-a-vis normal tissue: An essential prerequisite for an ideal radioprotectors

The challenge for developing an ideal radioprotector is to protect normal tissues from radiation damage without protecting the tumour cells, which need to stay vulnerable for effective cancer treatment. By taking advantage of physiological differences such as enzyme activity, proliferation rate, oxygenation levels, and DNA repair capacities between normal and malignant tissues, selectivity can be achieved through various modes of action.

For instance, amifostine, an FDA-approved radioprotector, is selectively targeted by the enzyme alkaline phosphatase, which is abundantly present in healthy tissues than in cancerous cells. Here, the active form of amifostine scavenges radiation-induced free radicals and also supports DNA repair. On the other hand, tumour cells frequently lack the enzyme activity required to change amifostine into its active form, which leaves tumour tissues with little to no protection. Compared to normal tissues that receive adequate oxygen, tumour tissues are less vulnerable to ROS-induced oxidative damage because they frequently reside in hypoxic environments. Amifostine and other radioprotectors work mainly by scavenging ROS, which are more prevalent in situations with higher oxygen levels. As a result, tumour cells, which are already less dependent on ROS-induced oxidative damage, continue to be vulnerable, whereas normal oxygenated tissues gain from the reduction of ROS-induced damage by the radioprotector. Tumour cells are especially susceptible to radiation-induced DNA damage because they frequently have compromised DNA repair mechanisms. Normal cells, on the other hand, usually have efficient repair systems. Radiation can efficiently target and kill cancer cells because of this differential response. Moreover, without improving the tumour’s ability to repair DNA damage, radioprotectors can increase the ability of healthy cells to repair radiation-induced damage by strengthening cellular antioxidant defences or DNA repair pathways. Apart from the aforementioned differential effects, HemaMax (Recombinant Human Interleukin 12), a promising radioprotective drug, reduces radiation damage to healthy tissues by utilizing the body’s immunological response²⁴. IL-12 is essential for both haematopoiesis and immunological control. Especially in the hematopoietic system, i.e., bone marrow, which is extremely vulnerable to radiation damage, HemaMax boosts the immune system and aids in regenerating damaged tissues. The preclinical studies have shown that HemaMax treatment leads to a considerable increase in the survival rates of animals irradiated with high absorbed doses by reducing the gastrointestinal and haematological syndromes. However, IL-12 does not directly protect malignant tissues, therefore tumour cells are not protected by this immune-mediated defence, which keeps the tumour vulnerable to radiation-induced damage²⁴.

Amifostine

Amifostine, an inactive prodrug, is converted by alkaline phosphatase in normal endothelial cells into an active thiol metabolite that neutralizes reactive metabolites from platinum and alkylating agents. Its selective cytoprotection results from higher concentrations in healthy cells (⁓100-fold greater than in tumour cells) due to the tumour microenvironment and differential enzyme expression. Amifostine’s mechanisms include scavenging secondary radicals, donating electrons for chemical repair, stabilizing DNA repair, inhibiting cell-cycle progression, and inducing hypoxia, protecting normal tissues while preserving cancer treatment efficacy⁵⁹. This multi-faceted mechanism of action allows amifostine to protect normal tissues from the damaging effects of radiation and chemotherapy while still allowing these treatments to target cancer cells.

The U.S. FDA approved intravenous amifostine in 1996 to reduce renal toxicity from repeated cisplatin in advanced ovarian cancer and in 1999 to decrease moderate to severe xerostomia in head and neck cancer patients undergoing postoperative radiotherapy⁵⁹. Indeed, amifostine holds promise for various oncologic contexts beyond its current FDA-approved indications. Ongoing research explores new administration schedules and routes to reduce side effects like nausea, vomiting, and hypotension, and to expand applications beyond current indications. However, toxicity and side effects limit its use in non-clinical radiological or nuclear scenarios, requiring further research for FDA approval in these contexts.

Palifermin

Palifermin, a recombinant human keratinocyte growth factor (KGF) developed by Amgen, is a 140-residue protein produced using E. coli. Initial preclinical data demonstrated that Palifermin could reduce gastrointestinal injury and mortality resulting from a variety of toxic exposures⁶⁰. These studies also showed that palifermin has protective effects on many epithelial tissues, particularly when administered prior to the toxic radiation exposure. Toxicology studies were conducted using animal models, including mice, rats, and monkeys. The endogenous form of palifermin is expressed in the kidneys of rats. The FDA approved palifermin in 2004 for reducing severe oral mucositis in haematological malignancy patients undergoing chemoradiotherapy. However, the gain in radioprotection remains hampered with a low margin of tolerance⁶¹. In a phase II trial for head and neck cancer, palifermin reduced the incidence, severity, and duration of oral mucositis during concurrent chemotherapy and hyperfractionated radiotherapy⁶². Extensive preclinical research, coupled with a burgeoning field of clinical investigation, has been dedicated to the development of agents like palifermin aimed at safeguarding normal tissues from the deleterious effects of irradiation.

Palifermin prevents and treats oral mucositis by binding to KGF receptors on buccal cells, stimulating epithelial cell proliferation, differentiation, and migration in the tongue and mouth. The KGF receptor is widely distributed across various tissues, with notable concentrations observed in regions such as the tongue, oesophagus, salivary gland, and other gastrointestinal tract organs.

Table II^62-75 shows clinical trials investigating the role of amifostine and palifermin for mitigating radiation-induced toxicities in cancer patients and stem cell transplantation, particularly for hematologic conditions. Their synergistic potential is under study, but further research is needed to clarify benefits and risks. Current evidence should guide their use. Radioprotection is essential in radiation therapy to minimise normal tissue damage. Amifostine and palifermin are FDA-approved radioprotectors used in clinical settings.

The use of amifostine in preventing chemotherapy-associated neutropenia has been inconsistent across studies. However, a notable large randomized trial published since 2002 demonstrated a significantly lower frequency of grades 3 to 4 neutropenia in the amifostine arm. A large randomized trial since 2002 showed reduced grade 3-4 neutropenia with amifostine, but no benefit for thrombocytopenia, leading to recommendations against its use for thrombocytopenia prevention in chemotherapy⁷⁶. Amifostine also reduces cisplatin-associated nephrotoxicity in ovarian cancer and acute/late xerostomia in head and neck cancer patients undergoing fractionated radiotherapy.

Palifermin effectively prevents severe oral mucositis in hematopoietic stem-cell transplant (HSCT) patients with hematologic malignancies, reducing its incidence, severity, and duration. Updated guidelines support palifermin’s use for mucositis prevention in HSCT. Some studies have also shown that palifermin is associated with a decrease in the use of patient-controlled analgesia (PCA) and a subsequent reduction in severe mucositis. Additionally, it contributes to a decrease in the number of days requiring analgesics and antibiotics, further highlighting its prophylactic benefits. When comparing amifostine and palifermin, as portrayed in supplementary table I^77-99, it’s important to note that they are used to prevent different types of toxicities.

Radioprotectors in clinical oncology, specifically amifostine and palifermin, have shown promising results as both agents have demonstrated significant potential in radiation protection, with their therapeutic roles being well-documented and supported by clinical trials. The comparative analysis of the effectiveness of amifostine and palifermin further underscores their potential as lead agents in clinical studies. However, it is important to note that while these findings are encouraging, further research is necessary to fully understand the long-term effects and potential side effects of these agents. As we continue to advance in the field of oncology, the role of the above radioprotectors will undoubtedly become more crucial in improving patient outcomes and quality of life.

Final destination: Challenges in adoption in clinics and opportunities to overcome the challenges in bringing these agents to clinics

Incorporating new therapeutic approaches, such as radioprotectors, into clinical practice presents a host of challenges that necessitate strategic navigation. While radioprotectors hold promise in mitigating the adverse effects of radiation therapy and improving treatment outcomes, their adoption in clinics faces numerous hurdles, from concerns surrounding safety and efficacy to regulatory complexities and market dynamics; the path to integrating radioprotective agents into routine clinical care is fraught with obstacles. To ensure the successful translation of research innovations into real-world clinical applications, healthcare providers and researchers can work together to address various challenges in effectively utilizing these agents along with advanced and precision-based radiation therapies.

The figure shows how combining advanced techniques (proton therapy, CIRT, IMRT, SRS) with radioprotectors (amifostine, BIO 300, entolimod) improves tumour targeting, reduces toxicity, enhances radiosensitivity, and minimises side effects for better patient-centred care.

To expedite the translation of radioprotectors and mitigators, the Radiation Research Program and SBIR Development Center within the National Cancer Institute issued four requests for proposals (RFPs) from 2010 to 2013¹⁰⁰. Through the SBIR programme, four contracts and 11 grants were funded for the development of novel radioprotectors. Overall, 50 per cent of the companies (6 out of 12) successfully advanced their investigational drugs into prospective clinical trials in cancer patients¹⁰⁰. Therefore, their development and application in clinical settings are of great importance and continue to be the subject of ongoing research.

Many radioprotectors require intravenous administration, which is less convenient and more invasive compared to oral administration. This limits patient compliance and ease of use in outpatient settings. The interaction between radioprotectors and the tumour microenvironment, as well as the immune niche, is not well understood. Without robust data, clinicians are hesitant to adopt these agents widely. Additionally, contemporary radiotherapy practice involves using altered fractionation strategies like extreme hypofractionation (SRS and SBRT). These approaches are at a higher risk of causing severe toxicities. The role of radioprotectors in these settings is crucial, but their effectiveness in reducing toxicity in these high-risk scenarios is not well established. This uncertainty hampers their adoption in clinical protocols involving extreme fractionation schedules. Radioprotectors have not shown effectiveness in protecting against the most severe radiation-induced toxicities, particularly in serial organs such as the brain and spinal cord. These organs are highly sensitive to radiation, and damage can lead to serious and often irreversible complications. The inability of radioprotectors to mitigate these severe toxicities limits their perceived value in clinical practice. A few challenges are briefly described below for better understanding.

The integration of radioprotectors into clinical radiotherapy offers significant potential to mitigate normal tissue damage and enhance patient outcomes, yet their adoption faces substantial challenges, including safety concerns, limited efficacy in modern radiotherapy techniques, clinician skepticism, and regulatory and commercial barriers. These hurdles necessitate strategic approaches to facilitate the translation of radioprotective agents into routine clinical practice. Collaborative efforts among researchers, clinicians, regulatory agencies, and industry partners, supported by increased research funding, public-private partnerships, robust clinical trials, regulatory guidance, education, and market incentives, can address these challenges. Supplementary table II^101-111 summarizes key challenges in radioprotector use in cancer radiotherapy and corresponding strategies to overcome them, providing a framework for advancing their clinical integration.

Radioprotective agents hold immense promise in improving the therapeutic ratio of radiotherapy by minimizing normal tissue toxicity without compromising tumour control. By implementing these strategies and fostering collaboration among radiobiologists and clinical radiation oncologists, it is possible to overcome the challenges associated with translating radioprotectors and mitigators to clinics. Through collective efforts and commitment to advancing radiation safety and patient care, radioprotectors can become valuable platforms in enhancing the effectiveness and safety of radiation therapy for cancer patients. A key concern among clinicians is the potential for radioprotectors to inadvertently shield tumour tissue from radiation, thereby compromising treatment efficacy. However, clinical evidence supports that the use of radioprotectors such as amifostine does not compromise tumour control in patients undergoing radiotherapy. For example, a pivotal phase III randomized trial in head and neck cancer demonstrated that while amifostine significantly reduced acute and chronic xerostomia, there was no significant difference in local-regional control, disease-free survival, or overall survival between patients receiving amifostine and those who did not, indicating preservation of antitumourefficacy¹⁰⁴. Meta-analyses of randomized controlled trials further confirm that amifostine does not confer a tumour protective effect, as no significant differences were observed in complete or partial tumour response rates when compared to placebo or observation groups. Additionally, multiple clinical studies have concluded that amifostine does not reduce the efficacy of radiotherapy against tumours, although the statistical power of individual studies may limit the ability to detect very small differences in tumour control. These findings collectively provide reassurance that, when used appropriately, amifostine selectively protects normal tissues without shielding tumour cells from the effects of radiation.

Radioprotective agents through ongoing research, clinical validation, and responsible integration hold immense promise in improving the therapeutic ratio of radiotherapy. By minimising normal tissue toxicity without compromising tumour control, they represent a critical step towards more effective, personalized, and patient centred cancer care.