The efficacy and safety of amifostine for the acute radiation syndrome

ABSTRACT

Introduction: A radiation countermeasure that can be used prior to radiation exposure to protect the population from the harmful effects of radiation exposure remains a major unmet medical need and is recognized as an important area for research. Despite substantial advances in the research and development for finding nontoxic, safe, and effective prophylactic countermeasures for the acute radiation syndrome (ARS), no such agent has been approved by the United States Food and Drug Administration (FDA).

Area covered: Despite the progress made to improve the effectiveness of amifostine as a radioprotector for ARS, none of the strategies have resolved the issue of its toxicity/side effects. Thus, the FDA has approved amifostine for limited clinical indications, but not for non-clinical uses. This article reviews recent strategies and progress that have been made to move forward this potentially useful countermeasure for ARS.

Expert opinion: Although the recent investigations have been promising for fielding safe and effective radiation countermeasures, additional work is needed to improve and advance drug design and delivery strategies to get FDA approval for broadened, non-clinical use of amifostine during a radiological/nuclear scenario.

KEYWORDS:

• Acute radiation syndrome
• amifostine
• aminothiols
• animal models
• FDA approval
• toxicity

1. Introduction

Radiation exposure from radiological accidents or other types of radiological/nuclear incidents (intentional or otherwise) can lead to various types of radiation injuries; all of which will require good diagnosis and treatment. Acute radiation syndrome (ARS) is an acute illness caused by radiation exposure of the whole or partial body by a high dose of penetrating ionizing radiation in a short time. In this age of terrorism and possible use of weapons of mass destruction, there is a compelling need to develop and establish federal, state and local capacities to counter these threats [1–4]. Such ‘medical threat-countering’ strategies are referred to as radiation countermeasures. Such agents are divided into three classes: radioprotectors (prophylaxis), radiomitigators (to be used soon after exposure), and therapeutics [5]. There are several radiation countermeasures under development that have been considered safe and efficacious to be used as radioprotective agents for radiological/nuclear events; however, they have not yet received FDA approval and need more studies [6–10]. All three currently FDA-approved radiation countermeasures (Neupogen/filgrastim/granulocyte colony-stimulating factor (G-CSF), Neulasta/pegfilgrastim/G-CSF, and Leukine/sargramostim/granulocyte-macrophage colony-stimulating factor (GM-CSF)) are defined as radiomitigators. These agents are approved specifically for a major ARS sub-syndrome, known as the hematopoietic sub-syndrome of ARS (H-ARS) [11–17]. It is important to note that at the present time no radioprotector for either H-ARS or for gastrointestinal-ARS (GI-ARS) has been approved by the FDA [18].

During the era of World War II, under the Manhattan Project, screening of more than 4,400 agents was carried out at the Walter Reed Army Research Institute of the US Army Drug Development Program to identify drugs to protect against injuries caused by ionizing radiation from nuclear/radiological detonation. This resulted in the selection of amifostine (WR-2721, WR implies Walter Reed, 2-[(3-aminopropyl)amino]ethanethiol dihydrogen phosphate) based on its excellent radioprotective efficacy and safety profile (Figure 1) [19]. Since then, amifostine has been pursued as the radioprotective agent of choice by diverse institutions, investigators, and government/funding agencies over the last six decades [20–24]. Though the brief message of this endeavor has been mixed [25–28], initial studies in animal models showed that amifostine could protect treated animals from high doses of ionizing radiation. Unlike other organ specific radioprotectants, amifostine is a broad-spectrum systemic cytoprotective agent. Preclinical studies in different animal models tend to encourage the belief that amifostine can protect almost all normal tissues from insults arising from irradiation or from particular types of chemotherapeutic agents. It is a strong, systemically effective radioprotector when used at high doses. However, it is toxic when administered at high doses required for radioprotection (survival benefits). The drug is hypotensive, causing both upper and lower GI disturbances [29,30]. These qualities lead to adverse behavioral responses and decreased performance [31–35]. As a result, this promising agent has been considered not suitable for use as a radioprotector for special operation forces, high-risk individuals, or for the general civilian population [36,37]. However, investigators have tried through various approaches to limit its side effects while retaining its robust radioprotective characteristics [38]: such efforts include, but not limited to threshold dosing regimens, modulating the drug delivery, and modifying the drug itself in order to retain its superior radioprotective activities.

Figure 1. The radioprotective domain of phosphorothioate.

Amifostine was referred to as Ethyol (trihydrate form of amifostine) after its purchase by MedImmune (Gaithersburg, MD, USA) in October 2001 from ALZA Corporation (Mountain View, CA, USA). In 2007, Astra Zeneca (Cambridge, UK) acquired MedImmune. Next, amifostine was acquired by the Clinigen Group (Staffordshire, UK) in August 2014. Clinigen and Cumberland Pharmaceuticals (Nashville, TN, USA) entered into an agreement for commercialization of amifostine in USA in May 2016. Generic version of amifostine is also marketed by Sun Pharmaceutical (Goregaon, Mumbai, India), Taj Pharmaceuticals (Goregaon West, Mumbai, India), Merro Pharmaceutical (Islamabad, Rawalpindi, Punjab, Pakistan), Luye Pharma (Shanghai, China), Mylan Pharmaceuticals (Canonsburg, PA, USA), and Kaifeng Mingren Pharmaceutical (Zhengzhou, Henan, China). It has obtained US FDA approval for two indications: (i) to reduce the accumulative renal toxicity associated with repeated administration of cisplatin in patients with ovarian cancer in 1996, (ii) to diminish the occurrence of xerostomia in patients undergoing post-operative radiotherapy for head and neck cancers (where the radiation port includes a considerable segment of the parotid glands, in 1999 (Table 1)) [39,40]. An enhanced uptake by the salivary gland and kidneys may be responsible for privileged protection of these tissues and organs. A recent study suggested that amifostine does not have a significant effect for preventing xerostomia and the benefits of amifostine should be weighed against its high cost and side effects [41].

Table 1. Indications for which amifostine has received full FDA approval.

Indications	FDA approval	Dose	Treatment schedule	Caution
For reduction of cumulative renal toxicity with cisplatin chemotherapy of patients with ovarian cancer	1996	910 mg/m^2	Administered once daily as a 15-min iv infusion, starting 30 min prior to chemotherapy	Adequately hydrated prior to the infusion and kept in a supine position during the infusion, blood pressure should be monitored every five min during the infusion and thereafter as clinically indicated.
For reduction of moderate to severe xerostomia from irradiation of the head and neck	1999	200 mg/m^2	Administered once daily as a 3-min iv infusion, starting 15–30 min prior to standard fraction radiation therapy (1.8–2.0 Gy)	Adequately hydrated prior to the infusion, blood pressure be monitored at least before and immediately after the infusion, and thereafter as clinically indicated, antiemetic drug (5-HT3 receptor antagonists, alone or in combination with other antiemetics) be administered prior to and in conjunction with amifostine.

The above details are based on the amifostine label for injection [39]. 5-HT3; a serotonin receptor

2. Preclinical development of amifostine using various animal models

The development of radioprotectors has been dominated by the investigation of sulfhydryl compounds, specifically aminothiols and phosphorothioates. Several mechanisms have been suggested for radioprotection by the use of sulfhydryl compounds, such as radiation-induced repair of DNA damage, free radical scavenging, genomic stabilization, modulation of cellular metabolism, modification of cell-cycle progression, augmentation of selective DNA repair enzymes, and scavenging of metals (Figure 2) [42,43]. The most meticulously studied, efficient, and valuable radioprotective thiols produced are WR-2721 (amifostine) and other phosphorothioates, such as WR-3689 – S-2-(3-methylaminopropylamino) ethylphosphorothioic acid and WR-151,327 – S-2-(3-methylaminopropylamino) propylphosphorothioic acid [26,27]. These phosphorylated compounds are prodrugs for the active free aminothiols, such as WR-1065 – [2-(3-aminopropylamino)ethanethiol] from WR-2721, WR-151326 – [3-(3-methylaminopropylamino)propanethiol] from WR-151327, and their disulfides generated in vivo. These free thiols can form disulfides, and WR-2721 can also be metabolized to cysteamine [44,45]. All of these WR-associated phosphorothioates exhibit effective, but distinct degrees of radioprotectiveness and drug toxicity/side effects when assessed using small animal rodent models [46]. Furthermore, different routes of administration have been documented as well [47]. The inadequately studied WR-159243 is well-tolerated agent that can be used orally soon before acute radiation exposure, while still attaining high levels of radioprotection (30 days survival, dose reduction factor (DRF) of ~1.3 and therapeutic index of ~7.5).

Figure 2. Simplified diagram for the mechanism of action of amifostine. Amifostine protects against radiation/chemotherapeutic agent-induced DNA damage. It has anti-mutagenic, anti-carcinogenic characteristics, affect redox-sensitive transcription factors, gene expression, chromatin stability, enzymatic activity, affects growth and cell cycle progression, and prevents the up-regulation of inflammatory pathways.

In a preclinical study, WR-2721 demonstrated a DRF of 2.7 for H-ARS and 1.8 for GI-ARS, when administered intraperitoneally (ip) at a dose of 500 mg/kg to mice before irradiation (Table 2) [48–51]. The drug dose used was relatively high in terms of adverse side-effects, as LD₅₀ dose for the drug alone was 704 mg/kg [48]. In subsequent studies, several compounds were evaluated for the protection of the hematopoietic system, the GI system, the integumentary system, and pulmonary tissue. It was observed that the most effective aminothiol had the basic features of a free sulfhydryl group separated by no more than three carbon atoms from a nitrogen functional group (Table 3) [27,52]. In these reports, drugs were compared for dosing schedules, one-half of the maximum tolerated dose (MTD, the dose that is lethal to no more than 10% of mice tested). Administration of phosphorothioates offered a DRF of 1.7–2.3 for H-ARS and 1.2–1.6 for GI-ARS. Cysteamine, cystamine, and AET (2-aminoethylisothiouronium Br.HBr) demonstrated low therapeutic indexes compared to phosphorothioates. Earlier, a high DRF was attained at doses close to their MTD [26]. Not all phosphorothioates are effective as radioprotective agent when administered orally to rodents; furthermore, the phosphorothioate, namely amifostine, proved not to be effective in nonhuman primates (NHPs) when dispensed orally (Table 4) [26]. When amifostine was administered intramuscularly (im) at a dose of 300 mg/kg in mice 15 min prior or up to 2 h before irradiation, its DRF was 2.7 [53]. Phosphorothioates were useful against high-linear energy transfer (LET) exposures [54] indicating a protective mechanism. This implicates hydrogen donation to damaged DNA rather than solely scavenging reactive oxygen species (ROS).

Table 2. Radioprotective efficacy of amifostine (WR-2721) in mice.

Dose (mg/kg)	DRF		References
	H-ARS	GI-ARS
500	2.70	1.80	^[Citation48]
500	-	1.64	^[Citation49]
400	2.25	-	^[Citation50]
365	2.29	1.55	^[Citation51]

Table 3. Radioprotective efficacy of selected agents.

Name	Chemical Structure	Dose MTD/2 (mg/kg)	DRF
			Hematopoietic protection (30 min interval)	Gastrointestinal protection (30 min interval)	Pulmonary syndrome (180 days lethality)
WR-638	H2N-(CH2)2-S-PO3H2	450	1.91	1.30	1.29
WR-77,913	H2N-CH2-CHOH-CH2-S-PO3H2	900	1.86	1.24	1.29
WR-2721	H2N-(CH2)3-NH-(CH2)2-S-PO3H2	365	2.29	1.55	1.36
WR-3689	CH3-HN-(CH2)3-NH-(CH2)2-S-PO3H2	450	2.22	1.51	1.31
WR-44,923	H2N-(CH2)3-NH-(CH2)3-S-PO3H2	207	1.70	1.63	1.28
WR-151,327	CH3-HN-(CH2)3-NH-(CH2)3-S-PO3H2	315	1.93	1.40	1.27

Drugs were injected ip into BALB/c mice at half the maximum tolerated dose 30 min prior to irradiation [27]. These agents were also tested in some large animal models (canine, minipig, and NHP) [26]. WR3721 was found to be superior compared to other agents and its data with large animal models are presented in Table 4.

Table 4. Radioprotective efficacy of amifostine (WR-2721) in rodent, canine, and NHP.

Species	Radiation Dose (Gy)	Drug Dose (mg/kg)	Route of Administration	Time (h)	Percent Survival
Mice	8.25 (X-ray)	600	ip	0.25	100
		300			100
		150			100
		75			80
		50			10
		0			0
		600		0.5	86
				1	100
				1.5	93
				2	100
				3	93
		0		-	0
		700	po	1	90
				2	60
				3	50
		0		-	0
	9.5 (γ-photon)	700	po	2	87
				3	67
				4	13
				5	0
		0		-	0
Canine	4.5 (X-ray)	200	iv	0.5	100
		0		-	0
	6.5 (γ-photon)	150	iv	0.5	50
		0		-	0
NHP	10 (γ-photon)	250	iv	0.5	100
		0		-	0
		300	po	0.5	0
		0		-	0

The MTD and LD₅₀ with ip route in mice were 600 mg/kg and 950 mg/kg, respectively [26].

A low level of radioprotection was observed when phosphorothioates were dispensed to mice at a low dose, such as 75 mg/kg [26]. Although not fully authenticated, it is likely that the dose of amifostine suggested for use in head and neck cancer patients undergoing radiotherapy (200 mg/m² intravenous (iv)) [39] would be adequate to afford radioprotection, regardless of whether or not clinical support was used. There are several potentially radioprotective thiols, such as diethyldithiocarbamate (DDC), mercaptopropionylglycine (MPG), N-acetylcysteine (NAC), mesna, and cysteamine that have FDA approval for other indications and for which there is considerable clinical experience. Although these agents are less radioprotective for lethal doses of radiation, they may be far less toxic and well tolerated. Toxicity/side effect assessments of these agents indicate that NAC has the lowest toxicity, followed by amifostine, cysteamine, and DDC [34]. Researchers continued synthesizing and assessing newer derivatives of thiols, such as thiazolidine, prodrugs of cysteine, cysteamine, and WR-1065, for finding an optimal radioprotective agent for ARS [55].

The concept of dose conversion between various species of animal models and human is important for drug development [56]. Interspecies allometric scaling for dose conversion from animal to human (dose per kg to dose for body surface area) takes into the consideration the differences in body surface area, which is associated with animal weight. Allometric scaling is an approach where the conversion of drug dose is based on normalization of dose/kg to body surface area. This approach considers differences of attributes of anatomical, biochemical, and physiological processes among species, as well as differences in pharmacokinetics. Different equations described in a recently published article can be used as a reference for ‘dose extrapolation’ among species [56].

3. Mechanisms and toxicities of amifostine

Amifostine is an inactive phosphorylated aminothiol prodrug and it cannot protect until dephosphorylated to WR-1065, its active and free radical-scavenging sulfhydryl metabolite. It is dephosphorylated by alkaline phosphatase. Alkaline phosphatase is available in high concentrations in normal tissues and in relatively low concentrations in tumor/malignant cells. WR-1065 only provides protection to non-central nervous system (CNS) tissues, but not the CNS itself since it is not able to cross the blood–brain barrier. Once inside the cell, WR-1065 scavenges free radicals, allowing for the protection of intracellular constituents. Several studies demonstrate that there are additional mechanisms relative to radioprotective efficacy of amifostine [6,57,58]. Oxidation of WR-1065 to its polyamine-like disulfide metabolite (WR-33,278) is followed by a quick consumption of oxygen, suggesting that cellular anoxia may be a mechanism for radioprotection. This was supported by an increase in the oxygen saturation of the venous blood following iv administration of amifostine in the absence of hemoglobin-bound-oxygen. This promotes a decreased oxygen consumption by normal tissue; a process related to radioprotection by amifostine [59,60]. The latter effect has been referred to as the ‘Walberg-effect’ [61]. Furthermore, a high concentration of WR-33,278 condenses DNA and limits potential target sites for free-radical damage and for subsequent free radical scavenging [62]. Such event leads to a decrease in the number of DNA double-strand breaks. The drug-induced mitigation of a radiation-induced inhibition of cellular proliferation may be important to accelerate the recovery of endothelial and mucosal cells. Amifostine may upregulate the expression of proteins responsible for DNA repair and inhibition of apoptosis through Bcl-2 and hypoxia-inducible factor-1α (HIF-1α) [63–65].

In sum, amifostine and its immediate, dephosphorylated metabolite WR1065, exert radioprotective efficacy through various molecular and cellular processes [29]. Very often the efficacy of amifostine is attributed to free-radical scavenging, along with DNA protection and repair; all of which are coupled with the initial induction of cellular hypoxia [29,47,55,57–60]. At the cellular level, amifostine has significant effects on growth and cell cycle progression. In brief, amifostine protects against the DNA damage induced by ionizing radiation and chemotherapeutic agent associated free radicals. It has anti-mutagenic and anti-carcinogenic characteristics [29]. These latter endpoints for amifostine’s prophylactic efficacy have been demonstrated using small animal models [29], while (to our knowledge) the drug’s mitigative, anti-mutagenic actions have only been reported using in vitro testing systems [66,67]. At the molecular level, it has been demonstrated to affect redox-sensitive transcription factors, gene expression, chromatin stability, and enzymatic activity. At the cellular level, it has important effects on growth and cell cycle progression. Amifostine prevents the up-regulation of inflammatory pathways related to cancer therapy toxicity [23].

Tumor/malignant tissues are not well vascularized and demonstrate hypoxic microenvironments with low interstitial pH, and decreased alkaline phosphatase. Such microenvironments lead to low buildup of active drug within malignant tissues. Normal tissues may have as much as a 100 fold higher concentration of free thiol compared to malignant tissues [68]. Nonetheless, in some reports using animal models, it has been shown that amifostine does indeed diffuse into malignant tissues, perhaps slowly as compared to normal tissues. Still, it raises the question about its potential to protect malignant tissue from radiotherapy and chemotherapy [61,69].

The usual toxic effects of amifostine are hypotension, emesis, somnolence, and sneezing in patients who received single or multiple doses [46]. Other minor adverse effects are malaise, hypocalcemia, metallic taste, hiccups, chills, flushed feeling, idiosyncratic reactions, fever, and/or rash.

4. Strategies for use and regulatory approval of amifostine as a prophylaxis agent in limiting ARS and associated delayed pathological sequelae

Substantial effort has been made by several investigators to make amifostine more user-friendly by trying to reduce its toxicity and side effects by developing novel chemical analogs, drug formulations, and drug delivery systems. The overall goal of this endeavor is to maintain the efficacy and to extend the time window for effective drug administration. Specifically, such efforts have comprised of (i) the chemical synthesis of better tolerated analogs [26,27]; (ii) the radioprotective synergy by combining lower doses of amifostine with lower doses of another pharmacological agent with less or fewer side effects (such as α-tocopherol, γ-tocotrienol (GT3), selenomethionine); i.e., a poly-pharmacy approach [70,71]; (iii) minimize side effects of amifostine by additional use of supplemental pharmacological agent designed to minimize emetic effects of amifostine (or use of anti-emetics) [6]; (iv) use of low doses of amifostine to protect against carcinogenesis and/or mutagenesis, while forgoing some of inherently toxic cytoprotective attributes of amifostine [72,73]; and (v) improve the approaches for ‘slow-release’ drug delivery [74–76]. Generally, these approaches have proven to be successful in lowering drug toxicity/side effects, but have not been successful in totally eliminating it. Considering current rigorous regulatory environment that controls drug use in humans, such an effort to reduce toxicity of amifostine to a minimum will be critical if amifostine, phosphonol or any other phosphorothioates are to be approved by the FDA or other international drug regulatory agencies (such as The European Medicines Agency, Pharmaceuticals and Medical Devices Agency of Japan) for use as a radioprotector. Some important and ongoing approaches for improving amifostine use are discussed below:

4.1. Chemically re-engineering better tolerated analogs of amifostine

Attempts to improve the radioprotective efficacy, improve the drug pharmacology, and reduce the side effects of amifostine by re-engineering a phosphorothioate analog have been pursued for the past several decades [26,27,76]. The radioprotective domain of this class of drugs seems to be its hydrophilic chemical core, containing a sulfhydryl group, protected by a phosphate, and linked by two or three carbons to at least one or two amino groups (Figure 1).

The domain responsible for side effects is attributed to the length and terminal nature of the aminoalkyl group and its hydrophobicity. The pharmacology of phosphorothioate species seems to be an amalgamation of both these functional groups, the aminoalkyl and sulfhydryl [26,27]. The chemical modifications within the two functional domains producing greater hydrophilicity, along with increased coverage of sulfur group, resulted in improved therapeutic indices [26]. It is vital to note, that such improvement did not generate ideal pharmacologic profiles. The time window for radioprotection and the poor bioavailability remained important concerns.

Phosphonol (WR-3689) is close to amifostine in its radioprotective efficacy (polyamine like affinities and targeted action on DNA) but suffers from side effects comparable to amifostine although to somewhat lesser extent. Its bioavailability is marginally higher than amifostine when administered orally. In brief, phosphonol may be considered an improved drug compared with amifostine due to is higher protective effects [77,78].

A new and quite promising radioprotective aminothiol analog, PrC-210, has been developed and evaluated in preclinical animal models. PrC-210 is a five carbon, single thiol alkylamine (aminothiol derived organosulphur compound). Important considerations in the designing this aminothiol were (i) a small molecular size for efficient transmembrane diffusion, (ii) positive charged amines on an alkyl backbone for strong ionic interaction with DNA, and (iii) a perpendicular, alkyl side-chain with a terminal thiol projecting away from the DNA backbone to enable scavenging of ROS around DNA [79]. PrC-210, when administered prior to irradiation (prophylaxix) via ip injection, provided 100% protection in ICR mice against total-body irradiation which was 100% lethal to untreated control mice (8.63 Gy). In this study, PrC-210 dose was 252 mg/kg and it was administered 30 min prior to irradiation, similar to the amifostine. Furthermore, PrC-210 was effective when administered anywhere between 7.5 and 60 min before irradiation to mice [80]. When administered topically (370 mM in ethanol:propylene glycol:water solution) to Sprague-Dawley female rats prior to cutaneous irradiation (17.3 Gy to skin), it provided 100% prevention against grade 2–3 radiation-induced dermatitis. The DRF for PrC-210 was 1.6 in prophylaxed, acutely irradiated (i.e., at a dose of 252 mg/kg ip 30 min prior to 8.75 Gy ¹³⁷Cs γ-irradiation) ICR female mice [79]. Furthermore, PrC-210 also protected mice and rats against total-body irradiation, 100% survival in rat and mouse models against 100% lethal dose of ¹³⁷Cs γ-radiation. This drug appeared to lack retching and emesis-producing properties when tested in a ferret model [73]. In addition, there was no evidence of drug-induced hypotension in arterial cannulated rats treated with PrC-210 [81]. The comparison of PrC-210 and amifostine side effects was striking because PrC-210 was free of all side effects investigated.

A variety of N-(aminoalkanoyl)-S-acylcysteamine and N,N’-is(aminoalkanoyl)cystamine salt derivatives have been synthesized and such agents have been tested for their toxicity/side effects and radioprotective efficacy using murine model, and also compared with amifostine and S-acetylcysteamine hydrochloride. One of the most attractive compounds of this series was N-glycyl-S-acetylcysteamine trifluoroacetate (16, I 102) [82]. Side effects and radioprotective efficacy were investigated in murine model and found to be promising when compared to amifostine and S-acetylcysteamine hydrochloride. The structure–activity relationships of these agents have been well studied.

Several analogs of amifostine (such as DRDE-07: (S-2(2-aminoethylamino) ethyl phenyl sulfide), DRDE-30: (S-2(2-aminoethyl amino) ethyl propyl sulfide), and DRDE-35: (S-2(2-aminoethyl amino) ethyl butyl sulfide)) have been synthesized by Defense Research and Development Establishment, Gwalior, India (Government of India, Ministry of Defense Institution), and tested for their radioprotective efficacy using C57BL/6 mice model of total-body irradiation. These agents were reported to be promising as radioprotectors when administered 30 min before irradiation [83]. Several parameters such as animal survival, body weight, hematological parameters, GI histology (structural integrity, vilus height, regenerating crypts and mitotic figures), and colony forming unit – spleen (CFU-S) were examined. These analogs significantly improved all above listed parameters in a dose dependent manner. All three analogs were effective orally as well as through ip route of administration. Earlier, these agents were reported to be effective against Sulfur Mustard [84,85]. Recently, DRDE-30 has also demonstrated to attenuate bleomycin-induced pulmonary fibrosis in murine model [86].

Overall, only a small number of rationally designed analogs of amifostine have been synthesized and investigated for side effects/toxicity, pharmacology, and radioprotective efficacy using various preclinical animal models. Further effort is warranted and might prove fruitful from a radiation countermeasure development perspective.

4.2. Nanoparticle preparation, PEGylation and molecular conjugates designed to extend amifostine (or it’s active metabolite, WR 1065) circulating half-life

Although amifostine is an efficacious and potentially useful radiation countermeasure, its limitations due to short systemic circulation time and fast clearance from the tissues hamper its sustained bioactivity. With the development of nanotechnology in the area of drug development, the nanoparticles have proved to be powerful drug delivery systems for improving the biological activity of the drug [87]. Two oral formulations of amifostine have been prepared through nanotechnology with median particle sizes of 257 and 240 nm. After oral administration to mice, tissue distribution study demonstrated presence of its active metabolite, WR-1065, in various organs with some degree of selectivity in bone marrow, jejunum, and kidneys [76]. Radioprotective efficacy evaluation orally demonstrated enhanced mouse survival, hematopoietic cell survival, and surviving crypt cell counts in animals irradiated with ¹³⁷Cs γ-radiation source [88]. The nanoparticles of WR1065 prepared by spray drying using polylactide-co-glycolide (PLGA) resulted in significant radioprotection (mice survival, bone marrow recovery, and intestinal injury mitigation) when administered orally 1 h before irradiation at a dose of 500 mg/kg [89]. These nanoparticles were of 206 nm in diameter and having 21.7% w/w WR1065. There are reports for dual targeting functionality and dual drug delivery using nanoparticle technology [90].

PEGylation, covalently conjugating drug with polyethylene glycol (PEG), has demonstrated enormous potential in improving drug safety, pharmacokinetics, and pharmacodynamics. Since PEG is FDA approved for clinical use, PEGylation is used for the development of drugs [91]. PEGylated amifostine was prepared by its conjugation with the 4-arm PEG (5,000 Da). The large PEG-amifostine conjugated molecules could be internalized into cells and translocated to acidic organelles and demonstrate prolonged hydrolysis time. Results demonstrated sustained transformation of amifostine to WR1065 with higher efficiency in the elimination of ROS, prevention of cisplatin-induced nephrotoxicity, and impeded the interaction between free sulfhydryls and functional biomolecules providing improved safety profile [92].

4.3. Use of low doses of amifostine for cytoprotection

It is of great interest to know whether low doses of amifostine can be used to radioprotect humans at the risk to ionizing radiation exposures. Preclinical studies using rodent model have demonstrated that low, nontoxic doses of amifostine can protect hematopoietic progenitors within blood-forming tissues of irradiated mice. Although the radioprotection may not be enough to provide an overall survival benefit, it might however be enough to partially protect vital tissues. It is reasonable to assume that fractional protection by a pre-exposure drug can be successfully leveraged by the use of another agent used post-exposure [6,37,93].

As mentioned above, it has been demonstrated in a murine model that radioprotective doses for amifostine to protect hematopoietic organs of mice appear to be between 25 and 50 mg/kg. The mature and lineage-restricted progenitors are more responsive to the low doses of amifostine than the more primitive, multipotential progenitors [73]. Amifostine had a marginal effect on the bipotential and multipotential progenitors of bone marrow under a steady-state. Prophylactic doses of >50 mg/kg of amifostine resulted in moderate regeneration of multipotential progenitors and the significant regeneration of bipotential progenitors in irradiated mice. There was no regeneration of more primitive progenitors. Amifostine dose of 25 mg/kg failed to stimulate radioprotective effects on any of the progenitor subtypes. In brief, radioprotective doses for amifostine appear to lie between 25 and 50 mg/kg. Furthermore, the protection afforded by low doses of amifostine can be enhanced by the combined administration of other nontoxic agents [70,71,94].

4.4. Toxicity mitigation with anti-emetic agents

The upper and lower GI tract disturbances associated with the use of high doses of amifostine can be reduced with the use of 5-HT₃-receptor antagonist-based antiemetic drugs, such as granisetron or ondansetron [38,95]. Canines treated with increasing doses of amifostine (12.5–100 mg/kg) demonstrated a dose-dependent emesis. The use of granisetron at a dose of 80 mg/kg shortly before amifostine administration diminished the emesis and also the percentage of emetic animals [6]. A few canines remained sensitive to amifostine irrespective of the relatively low dose used.

4.5. Poly-pharmacy approach – combination of lower doses of amifostine and another radiation countermeasure

There are several reports for the poly-pharmacy approach in the area of radiation countermeasures [96–98]. The combination of three cytokines/growth factors, PEGylated G-CSF, PEGylated GM-CSF, and PEGylated interleukin-11 (IL-11) has been shown to enhance radiomitigation in the murine model demonstrating enhanced recovery in complete blood counts [99,100]. This study supports and extends earlier findings of the beneficial effects of combined cytokine therapy that serves to enhance hematopoietic recovery of vital trilineal bone marrow compartments of acutely irradiated mice [101]. Another example of the utility of the ‘poly-pharmacy’ approach to mitigate ARS includes the combination of angiotensin-converting enzyme (ACE) inhibitor, Captopril, and EUK-207, selenomethionine complexes in limiting the extent of radiation-induced lung damage in Sprague-Dawley rats. This rodent model has been shown to be a well-established animal model for radiation-induced lung damage [96,102]. It is important to note that captopril is effective, well tolerated, and has widespread clinical use.

Based on existing information with other radiation countermeasures, some of which are mentioned above, it is a logical step to investigate whether amifostine could enhance the radioprotective efficacy of another radiation countermeasure that acts through a different pathway, or if the efficacy of amifostine can be improved by another unrelated agent. Such poly-pharmacy approaches need to be tried with amifostine as its adverse, toxic side-effects clearly limited its non-clinical use as a sole radioprotective agent [71,103,104]. The advantageous therapeutic usefulness of amifostine is well known and the combined therapy with amifostine with other agents for the potential prevention and treatment of individuals at high risk of developing ARS is becoming an increasingly important strategy.

4.5.1. Combination of amifostine with growth factor/cytokine

The combination of amifostine and G-CSF has been attempted successfully for mitigating ARS in murine model where amifostine was used as a radioprotector and G-CSF as a radiomitigator [105]. The study was conducted to determine whether amifostine can be used to protect hematopoietic stem cells against irradiation, and G-CSF can be used to stimulate the amifostine-protected cells to proliferate and reconstitute the hematopoietic system [105,106]. C3H/HeN mice received treatment of 200 mg/kg amifostine ip 30 min before ⁶⁰Co γ-irradiation and 125 µg/kg G-CSF sc from day 1–16 post-irradiation. The LD_50/30 values for mice administered saline, G-CSF, amifostine, and amifostine plus G-CSF were 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. Based on above LD_50/30 values, it is obvious that the DRF of the combined treatment of amifostine and G-CSF was higher than the DRFs for either amifostine or G-CSF treatment. Accelerated recovery of bone marrow and splenic multipotential stem cells (CFU-s), granulocyte-macrophage progenitor cells, and peripheral blood cells, was detected in mice treated with amifostine and G-CSF. Furthermore, the G-CSF administered post-irradiation accelerated hematopoietic reconstitution with amifostine-protected stem and progenitor cells and leading to an unambiguous survival-enhancing effect of the pre-irradiation administered amifostine [106]. Such combination treatment approach using amifostine and cytokine has also been discussed in relation to oncology patients with chemotherapy and radiotherapy [107,108].

4.5.2. Combination with vitamin E

Vitamin E is a less effective radioprotector compared to amifostine but it has a wider window of protection against radiation injury, as well as it is less toxic. Amifostine or phosphorothioate WR-3689 (S-2([3-methylaminopropyl]amino)ethylphosphorothioic acid) have been combined with α-tocopherol (a component of vitamin E), and also selenomethionine with positive, survival promoting outcomes [70,109,110]. A prophylactically administered combination of amifostine and vitamin E in acutely irradiated rats has been shown to be beneficial, in terms of protecting and maintaining organ-specific (liver) functions as well [111].

GT3 is currently under advanced development as a radiation countermeasure [112]. Optimal radioprotective doses of GT3 for acutely, potentially lethally irradiated mice have been estimated to be 200 mg/kg [113]. However, the radioprotective efficacy of much lower doses of GT3 (50 mg/kg) can be enhanced significantly by complementary prophylaxis with low, but nontoxic doses of amifostine (30 and 50 mg/kg) [71]. This study using one fourth of the optimal dose of GT3 for murine H-ARS and a very low dose of amifostine suggests that both agents can be used at lower doses which may be free of any side effects of either agent and can still achieve optimal radioprotection.

4.5.3. Combination with metformin

Metformin (1,1-dimethylbiguanide hydrochloride, a biguanide derivate) is the most prescribed agent for the treatment of type 2 diabetes. It has been reported to have radiomitigative potential for ARS [114,115]. When administered 24 h after irradiation as a single dose, metformin significantly potentiated the radioprotective efficacy of amifostine and other sulfhydryl agents in several cell types [114]. It has been proposed that metformin reduces endogenous ROS, cell-cycle progression of radiation-injured cells and increases time for repair. Metformin administered 24 h post-irradiation and in combination with sulfhydryl agents had comparable radioprotective efficacy to that observed for amifostine administered 30 min prior to irradiation. As such, metformin might well be considered as a potentially useful radiation countermeasure for combination with other radioprotective agents.

4.5.4. Combined effects of amifostine and antioxidative salts

The radioprotective efficacy of the metal-containing compounds has been shown to be relatively low, but they have been found to enhance the radioprotective effects of amifostine and other thiols, while reducing toxicity of thiols [27,110]. Several theories (relative to selenium-based enhancements) have been suggested: these include but not limited to: (a) dampening of alkaline phosphatase activity in bone marrow and (b) the stimulation of glutathione peroxidase activity [116]. These observations have resulted in clinical efforts to combine selenium (Se) and amifostine to attenuate side effects of radiotherapy and chemotherapy of malignancies [103,117]. Preclinical studies, using rodent models, have been performed in order to better understand the nature of the adjuvant effect of selenium on radioprotection. For example, the effect of Se pretreatment on the toxicity and radioprotective efficacy of amifostine was studied in male CD2F1 mice [110]. Administration of 1.6 mg/kg Se 24 h before ip injection of amifostine (800–1,200 mg/kg) decreased the toxicity of amifostine. Se alone (1.6 mg/kg) administered 24 h prior to ⁶⁰Co γ-irradiation increased the survival with a DRF of 1.1. When Se was injected 24 h before ip administration of amifostine (200–600 mg/kg 30 min before irradiation), a synergistic, radioprotective effect was observed. The DRF with 400 mg/kg of amifostine and Se was 2.6, a surprisingly high value as compared to a DRF of 2.2 without Se pretreatment.

4.5.5. Combination of amifostine with other agents

Amifostine has been tested in combination with prostaglandin E2 (PGE2) or its synthetic analog, misoprostol. The DRF values of amifostine alone, PGE2 alone, or PGE2 plus amifostine have been reported to be 1.9, 1.45, and 2.15, respectively [118]. Both PGE2 and misoprostol used singly have been found to be effective in mitigating GI-ARS [119]. The protective role of amifostine in mitigating GI-ARS by the prostaglandin synthesis inhibitor indomethacin has substantiated a positive role of amifostine on the prostaglandin metabolism [120]. Interestingly, prostaglandins have been shown to have a hematopoietic effect as well. The prostaglandins have been reported to enhance erythroid and multilineage progenitors, homing of hematopoietic stem cells, and hematopoietic stem cell survival [121,122].

β-glucan, a polyglucose administered post-irradiation, has been shown to enhance the radioprotective efficacy of amifostine administered before irradiation. The DRF values for mice survival were shown to be 1.37, 1.08, and 1.52 for amifostine, β-glucan, and amifostine plus β-glucan, respectively [123]. The combination of amifostine (200 mg/kg, ip, 30 min prior to irradiation), Se (0.8 mg/kg, ip, 20 h before irradiation), and β-glucan (75 mg/kg iv, 20 h before irradiation) to C3H/HeN mice has been reported as a successful poly-pharmacy approach [124]. The additive effect of amifostine administered pre-irradiation and glucan administered post-irradiation has also been reported by another group [125].

Broncho-Vaxom, a bacterial lysate, has been investigated in combination with amifostine and shown to potentiate its efficacy as radioprotector as well as radiomitigator when amifostine was administered as a radioprotector [126,127]. When both the agents were administered prior to irradiation, the DRF values in mouse survival study have been reported as 1.92, 1.17, and 2.07 for amifostine, Broncho-Vaxom, and amifostine plus Broncho-Vaxom, respectively [126].

The polysaccharide from Sipunculus nudus (a species of unsegmented marine worms) administered post-irradiation along with IL-11 and G-CSF, has been shown to enhance the radioprotective efficacy of amifostine administered as a radioprotector in irradiated mice [128]. Amifostine administered prior to irradiation and peptidoglycan (a polymer from the bacterial cell wall consisting of sugars and amino acids) administered post-irradiation have enhanced the survival of irradiated mice [129].

5. Recent developments with amifostine

Regardless of the lack of FDA approval to use amifostine non-clinically as a radioprotector for ARS in humans associated with nuclear/radiological incidents, we strongly believe this agent, with its several encouraging attributes, merits additional consideration in order to get FDA approval for additional generalized use during radiation exposure emergencies. However, few laboratories have continued to investigate different aspects of its radioprotective efficacies, as well as pursuing work molecular targets, pharmacology, ways to limit its toxicity, and developing modern strategies for drug delivery and extending drug action. Notwithstanding these investigative deficits, a recent investigation demonstrated differential gene expression in lymphohematopoietic tissues of mice administered 100 mg/kg (minimally toxic doses) and exposed to sublethal doses of ⁶⁰Co γ-radiation. Irradiation elicited both early and late-arising gene responses that were altered by amifostine prophylaxes, with such treatments leading to wide-spread dampening of irradiation-related gene response [130]. A select group of proto-oncogenes reacted in much the same manner as did entire group of arrayed genes. Disparities in gene expression caused by sublethal dose of radiation were observed and amifostine prophylaxis considerably changed gene expression. This investigation suggests that amifostine may have additional useful attributes which we are not aware of. At present, we are conducting metabolomics and lipidomic studies using blood and various tissue samples collected from amifostine-treated and irradiated/unirradiated animals. In these studies, comparisons are being made between animals treated with survival-optimal doses of amifostine but associated with toxic side effects, and animals treated with sub-optimal doses of amifostine that are devoid of any side effects. The goal of such investigations is to identify the downstream metabolic products in animals treated with different doses of amifostine to identify biomarkers which may help to identify the dose of amifostine that is devoid of any side effects. Our initial observations are encouraging and incoming data suggest that the extent to which amifostine can correct radiation-induced perturbations of given metabolic pathways are better with lower dose 50 mg/kg compared to higher dose of 200 mg/kg (unpublished observations). In brief, there is a need for further investigations to optimize the drug dose, administration route, and treatment schedule to reduce the undesired side effects to broaden its clinical application.

6. Conclusion

The pursuit of an ideal radioprotective agent to be used under various radiological/nuclear scenarios has continued for more than 60 years. However to date, we do not have any FDA-approved radioprotective agents that can be administered to individuals at high risk to injurious radiation exposures resulting from radiological/nuclear accidents or from intentional terrorism. There are several agents that have demonstrated potential, but these agents need to be thoroughly investigated and further developed for FDA approval following Animal Rule.

The production of ROS and reactive nitrogen species (RNS) largely underpin cell and tissue injuries induced by ionizing radiation. Efficient defensive mechanisms have evolved within cells in order to cope with such oxidative stresses generated by radiation exposure. As a result of an understanding of such critical radiobiological processes, earlier pharmacologic work concentrated on sulfhydryl drugs and other antioxidants to counter ionizing radiation-induced harmful effects. A primary goal of such investigations was to recognize radioprotective sulfhydryl drugs that generated higher DRF values compared to agents with marginal radioprotective efficacy and low toxicity. Initially, amifostine (WR 2721) was recognized as an encouraging radioprotector among the vast collection of potential radiation-countering pharmacologic agents. It was demonstrated that the drug successfully quenched DNA damage by scavenging free radicals induced by irradiation. However, as in the case of amifostine and several other aminothiols and phosphorothioates, toxicity limits its effectiveness. As the radioprotective efficacy of amifostine increases with higher doses of the drug, so does its detrimental, toxic side-effects. Amifostine has several side effects, such as upper and lower GI disturbances as well as hypotension, making it incompatible for its use as a prophylactic drug administered to population during emergencies. Further R&D investigations for its use outside clinical settings are required to exploit its positive attributes while minimizing its toxic side effects. By either modifying drug dose, reworking its structure, altering its route of administration, or blending it with other potent radioprotective agents at lower doses, amifostine may retain its optimal efficacy and minimize the side effects. This agent might also be tested and used in combination with other FDA-approved drugs for other indications following the poly-pharmacy approach since there is a good possibility that additive or synergistic effects can be achieved [42].

Due to the scientific community’s continued interest and the increased funding in the US post 9/11 for terrorism-associated radiation countermeasure research, significant progress has been and continues to be made to identify, develop, and to receive FDA-approval for countermeasures that can protect population at large from acute radiation injuries. Even with all of its positive characteristics, clinical use of amifostine remains limited. It is difficult to discard amifostine as a viable radioprotector for human use only because of side effects that are quite manageable. Amifostine might well be dispensed safely, with significant benefit, to populations under risk to the radiation injury that are not tasked to execute emergency duties. In brief, amifostine is an effective, promising, and systemically active drug that provide not only cytoprotection to various vital tissues, but also to promote survival in fatally exposed individuals. Despite the reasonable advancements in the usefulness of amifostine as a radioprotector for ARS, none of the strategies described to date completely remove amifostine’s toxic side effects. Although the work-effort on amifostine and other aminothiols has been assuring for developing a safe and effective radioprotector, additional work is definitely required to advance the current drug design and delivery approaches.

7. Expert opinion

Amifostine is a promising, well-studied, effective radioprotector (an agent administered prior to radiation exposure) for ARS. However, to date, no radioprotector for ARS has received FDA approval for non-clinical use in humans. There are several reasons for such failure in fielding of radioprotectors compared to radiomitigators. The use of radioprotectors presents a higher level of risk. They would require greater scrutiny by FDA since such countermeasures would be dispensed to healthy individuals under threat of radiation exposure. This situation is quite different than for the radiomitigators that would be dispensed to actually exposed persons. Furthermore, all three radiomitigators approved by the FDA for radiological/nuclear exposure contingencies and for the unwanted radiation exposure-associated H-ARS, were already approved for other clinical indications [11–17] and were in clinic for several decades. These agents were approved following the FDA Animal Rule established for such purpose.

Despite the recent progress in optimizing amifostine’s radioprotective use, none of the approaches taken to date (for using amifostine as a radioprotector for H-ARS) have reduced side effects to sufficiently low levels that would satisfy the regulatory requirements of the FDA. Although the preclinical work with amifostine and related aminothiols have indeed been promising in terms of improving drug efficacy and safety profiles, still further work is needed for better drug design and delivery strategies. We consider that a realistic, practical ‘game-plan’ to get amifostine approved for H-ARS would be to use a ‘poly-pharmacy approach’ where more than one agent, particularly with different modes of action, are combined at lower doses to attain the synergistic or additive outcomes. Under such situation, low doses of few agents without any side effects can be combined. Several poly-pharmacy approaches with amifostine have been tried over last 25 years, with some reported accomplishments using preclinical, murine animal models. One such approach has been to combine low doses of amifostine with lower doses of GT3, a component of vitamin E. GT3 is under advanced development following the FDA Animal Rule and provided consistent efficacy in irradiated murine and NHP models. Recent studies using combined treatment with these two agents demonstrated promising results in the murine model of H-ARS using total-body ⁶⁰Co γ–radiation [71]. This approach of combined treatment was based on earlier reports of combining amifostine with another component of vitamin E, α-tocopherol, and few other thiols [70].

In addition to the side effects, other constraints of amifostine are route of administration (i.e., iv route/sc) and the narrow time-window (~30 min prior to radiation exposure) for optimal efficacy [131]. The utility of amifostine as a radiation countermeasure is unlikely to be completely appreciated if the mode of administration is limited to iv dispensation. Efforts have been made to develop noninvasive options of delivery (e.g. transdermal patches, pulmonary inhalers and microspheres for oral sustained-release) [132]. Such endeavors need to be continued for ultimate success. Furthermore, the window of amifostine administration needs to be improved to make amifostine effective during a radiological or nuclear situation. Though significant effort has been made to increase the treatment window of amifostine, such attempts need to be continued until we get complete success [26,42,133].

Regardless of the current lack of FDA approval to use amifostine non-clinically as a radioprotector for H-ARS in humans associated with nuclear/radiological incidents, we strongly believe that this agent, with its many positive, radioprotective attributes, merits additional consideration in order to secure full regulatory approval for generalized use during radiation exposure emergencies. However, few laboratories have continued to investigate different aspects of its radioprotective efficacies, including investigations of molecular targets, pharmacology, ways to limit its toxicity, and developing modern strategies for drug delivery and extending drug action. To achieve this goal, a recent investigation show differential gene expression in lymphohematopoietic tissues of mice administered 100 mg/kg (minimally toxic doses) and exposed to sublethal doses of ⁶⁰Co γ-radiation. Irradiation introduced both early and late-arising gene responses that were altered by amifostine prophylactic use and such treatment led to wide-spread dampening of irradiation-related gene response [130]. A group of proto-oncogenes reacted to the entire group of arrayed genes. Disparities in gene expression caused by sublethal dose of radiation were observed and amifostine prophylaxis considerably changed gene expression. This investigation suggests that amifostine may have additional useful attributes which we are not aware of. At present, we are conducting metabolomics and lipidomic studies using blood and various tissue samples collected from amifostine-treated and irradiated/unirradiated animals. In these studies, comparisons are being made between animals treated with the optimal dose of amifostine, where a significant survival benefit was observed but may be associated with side effects, and animals treated with the sub-optimal dose of amifostine which may be devoid of any side effects. The goal of such investigations is to identify the downstream metabolic products in animals treated with different doses of amifostine to identify biomarkers which may help to identify the dose of amifostine that is devoid of any side effects. Our initial observations are encouraging and incoming data suggest that pathway corrections are better with lower dose 50 mg/kg compared to higher dose of 200 mg/kg (unpublished observations). In brief, there is a need for further investigations to simplify the drug dose, administration route, and treatment schedule to reduce the undesired side effects to broaden its clinical application.

Article highlights

• Amifostine is a promising radioprotector but it has not received FDA approval for the indication of ARS due to its side effects at doses required for radioprotection against ARS, and other clinically relevant limitations.

• It has received FDA approval for limited indications; to reduce the renal toxicity as a result of cisplatin treatment of individuals with ovarian cancer and to reduce the xerostomia in patients undergoing radiotherapy treatment for head and neck cancer.

• The radioprotective doses for amifostine to protect hematopoietic tissues of mice are significantly lower; lie between 25 and 50 mg/kg, compared to its dose used in survival studies.

• One important approach is poly-pharmacy with the use of small doses of two or more radiation countermeasures with different modes of action to improve the treatment outcome.

• Several approaches are being tried to get amifostine or its improved analogs approved by FDA for ARS.

• Amifostine modulates several genes in irradiated mice and appears to reverse the radiation-induced changes on some genes.

This box summarizes key points contained in the article.

Author contributions

VK Singh and TM Seed drafted the manuscript, reviewed critically for intellectual content, and approved the final version to be published. Both authors agree to be accountable for all aspects of the work.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Acknowledgments

The opinions or assertions contained herein are the private views of the authors and are not necessarily those of the Armed Forces Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, or the Department of Defense. Mention of specific therapeutic agents does not constitute endorsement by the U.S. Department of Defense, and trade names are used only for the purpose of clarification. We apologize to those having contributed substantially to the topics discussed herein that we were unable to cite because of space constraints.

Additional information

Funding

This paper was funded by the Armed Forces Radiobiology Research Institute intramural project [# BB291731].