Introduction

Over 60,000 cases of head and neck cancer (HNC) are diagnosed in the United States each year (1). Radiation therapy (XRT) and chemotherapy serve an important role in HNC management, as such treatments significantly minimize recurrence rates in this patient population. While the effectiveness of these regimens continues to improve with time, the substantive detrimental effect of XRT to surrounding tissues remains a major challenge during the process of reconstruction (2,3,4,5). By decreasing osteocyte counts, increasing pathological vascular inflammation, and decreasing biomechanical strength, XRT significantly hampers the regenerative capabilities of bone and consequently results in pathological fractures and fracture site non-unions (6). Given the limited reconstructive options to remedy such complications after they occur, prophylaxis against XRT-induced tissue injury must form the cornerstone of care for HNC patients (7,8).

Amifostine (AMF) is one promising prophylactic therapeutic agent known to mitigate XRT-induced bony injury (7,8,9). The radio-cyto-protective benefits of this drug have been demonstrated by successfully reversing the deleterious effects of XRT on bone healing in murine models of mandibular fracture and distraction osteogenesis (10). In particular, our laboratory has repeatedly supported the ability of subcutaneous (SC) AMF formulations to improve fracture site cellularity, vascularity, mineralization, mechanical strength, and bony union rate (20% to 80%) (11,12,13,14,15,16,17,18). Despite these promising results, radiation oncologists remain reluctant to adopt this efficacious therapy in the clinical setting due to side effects such as nausea/vomiting and hypotension, and the onerous requirement of intravenous (IV) administration set forth by the FDA (7,19). The long-term clinical viability of AMF therefore depends on the discovery of alternative formulations and administration strategies that improve patient tolerance and compliance while preserving the radio-cyto-protective advantages.

Formed in vivo via an alkaline-phosphatase-mediated dephosphorylation reaction, WR-1065 is the pharmacologically-active metabolite of AMF and exerts its radio-protective properties by scavenging reactive free radicals formed by XRT (7,20). Interestingly, high concentrations of WR-1065 are known to cause hypotension, one of the most limiting side effects of AMF (21). Novel formulations and administration strategies of AMF may therefore relieve patient hypotension by limiting high initial peak exposure levels of WR-1065, which are associated with current IV administration. While our laboratory has repeatedly defended the bone-protecting efficacy SC AMF formulations in vivo, SC administration of AMF in humans is associated with injection site rashes, and only marginally remedies hypotension and nausea/vomiting (5,10,11,12,19).

Studies have demonstrated that oral (PO) AMF formulations provide adequate radio-protective efficacy against acute whole body XRT in rodent models, but it is not known whether a PO formulation of AMF can mitigate hypotension and other associated patient side effects associated with high systemic vascular levels of WR-1065 (22). The purpose of this study is to investigate the pharmacokinetic (PK) profiles of an enteric-coated AMF (EC-AMF) formulation utilizing in vitro and in vivo experimentation to determine peak and total exposure of WR-1065. We hypothesize that EC-AMF will result in more stable concentrations of WR-1065 and thus successfully minimize hypotension, nausea, and vomiting while maintaining efficacy as a radio-protectant therapy.

Materials and Methods

Materials

Analytical standards for WR-1065 measurement were prepared in plasma purchased from Lampire Biological Laboratories, Inc. (Everett, PA, USA). Mobile phase preparation for ultra performance liquid chromatography (UPLC) grade water and Liquid chromatography-mass spectrometry (LC-MS) grade acetonitrile was purchased from VWR (Radnor, PA, USA). Unless otherwise noted, all other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

In Vitro Comparison of AMF and WR-1065

To assess the effectiveness of AMF as a radio-protectant of osteogenesis and to determine the radio-protective effects of AMF Trihydrate (LKT Laboratories; Product ID A4933) versus its active metabolite WR-1065 (LKT Laboratories), irradiated calvarial osteoblasts (MC3T3 cells) were pretreated with AMF or WR-1065. First, MC3T3 cells were cultured and plated at a density of 100,000 cells per well in 6-well plates. After 24 h, 8 mM of either AMF or WR-1065 was applied to each well, the plate was radiated at a dose of 10 Gy, and the drug was immediately removed (n = 3/group). Upon completion of XRT, the plates were washed with phosphate buffered saline (PBS) and alpha-MEM was applied to each well to allow for cell maintenance and growth. Osteogenic differentiation medium (ODM) was administered to induce osteoblast differentiation three days after XRT. To understand the differences in osteogenic potential between the groups treated with AMF Trihydrate and WR-1065, we performed an alkaline phosphatase (ALP) stain 8 days after ODM application as previously described (Sigma Kit #245) (23). Proliferating osteoblasts exhibit high ALP activity, which makes the enzyme a suitable marker for quantifying the level of differentiation and osteogenic potential (24).

In Vivo Determination of Pharmacokinetics

Experimental Design

Two PK experiments were performed in vivo using male CD rats (9 months old) to determine the ability of PO AMF to both deliver efficacious levels of WR-1065 and alleviate the observed side effects associated with IV and/or SC administration.

In the first experiment, we compared the PK profiles of AMF administered at 10 mg/kg via IV (currently-approved route) versus the corresponding parameters after direct injection of AMF to the jejunum of the upper gastrointestinal tract. The basis of this experiment was twofold: to establish a relative level of conversion of AMF to WR-1065 in the intestines and to delineate the level of systemic WR-1065 absorption compared to IV administration. Direct jejunal injection of AMF simulates the PO delivery of the active therapeutic to the upper GI tract with minimal degradation in the stomach, which represent potentially desirable features of an effective PO formulation. Insight into the potential efficacy afforded by and toxicity resulting from, an oral delivery strategy designed for conversion and absorption in the upper GI tract can be obtained from comparison to the corresponding PK profile acquired after IV administration, which provides a reference point for both efficacy as well as toxicity.

In the second experiment, we compared the PK profiles of rats that received 100 mg/kg AMF as an enteric-coated PO formulation versus SC-administered AMF formulated in saline (i.e. non-coated) at the same dose. Enteric-coating is designed to protect active pharmaceutical ingredients from gastric stomach decomposition, thus facilitating release in the upper GI tract. In this experiment, AMF was coated with Eudragit L100–55, an enteric-coating designed for dissolution at pH 5.5. EC-AMF was prepared by a CRO (Pharma Spray Drying, LLC, Bedford Hills, NY) exactly as described by Gula and colleagues at the Beijing Institute of Radiation Medicine in 2013 (Supplementary Material 1) (22). Briefly, AMF was dissolved in deionized water at a concentration of 250 mg/ml. Separately, Eudragit L100–55 was dissolved in ethanol at a concentration of 20 mg/ml. The aqueous and ethanol solutions were mixed together at a ratio of 1:25 and homogenized to form an emulsion. The emulsion was spray-dried using a Buchi B-290/295 spray dryer into a collection package (Supplementary Material 2). The formulation properties and performance characteristics, including drug load and in vitro dissolution rate, for EC-AMF prepared by this procedure have been determined and described previously (Supplementary Material 3) (22). The SC-administered AMF was injected into the flank (100 mg/kg; Ethyol, MedImmune, Gaithersburg, MD).

This corresponds to the typical pH environment in the duodenal or jejunal regions of the intestines. We have previously demonstrated the efficacy of SC administered AMF at 100 mg/kg in rat models of bone healing and repair; this provided a pharmacokinetic reference for evaluating the corresponding enteric-coated delivery of AMF.

Amifostine Dosing Procedure and Plasma Collection

All pharmacokinetic studies were performed using rats with pre-implanted jugular access catheters (Charles River Laboratories). All AMF formulations were prepared fresh on the day of administration as a solution in 0.9% normal saline.

For the first experiment described above, the AMF formulation was administered at 10 mg/kg (final dose) for both the IV (n = 2) and direct jejunal routes of administration (n = 3). For IV administration, doses were administered directly through the jugular cannula as a bolus dose. For direct jejunal administration, the jejunum of the small intestine was isolated (using the Ligament of Trietz as a reference point) and remained exposed for direct dose injection into the jejunum using a standard needle and syringe. A small drop of VetClose® surgical adhesive was used to seal the site of injection. Following injection, the intestine was gently placed back into the abdominal cavity and the site draped with moistened gauze pads and Parafilm™. Blood samples (0.5 ml per time point) were collected from the cannulae at 0, 5, 15, and 30 min and 1, 2 and 4 h post-dose. Whole blood was deposited into K3 EDTA tubes, placed on wet ice, centrifuged at 4°C, and plasma frozen at −80°C within 1 h of collection. Blood volume removed at each time point was replaced with 0.9% saline. Animals were euthanized by IV injection of pentobarbital (Fatal-Plus®) through the jugular cannulae and death was confirmed by performing a bilateral pneumothorax.

For the second experiment described above, EC-AMF and SC administered AMF were given at 100 mg/kg (final dose) for both routes of administration (n = 4 rats each). EC-AMF was administered via oral gavage and SC doses were administered in the scapular region using a syringe and 26 gauge needle. Blood and plasma were collected as described for the first experiment.

Plasma Preparation Protocol for WR-1065 Measurement

A chemical derivatization protocol developed previously in our laboratory was employed to stabilize the reactive thiol of WR-1065 before LC-MS/mass spectrometry (MS) analysis. Briefly, frozen plasma samples (−80°C) were thawed at room temperature and 100 μl of each was diluted with 300 μl of 25 mM ammonium bicarbonate. Disulfide bonds were reduced to free thiols via the addition of 10 μL of 200 mM dithiothreitol. After brief vortexing, the mixture was incubated at 60°C for 30 min. The mixture was then diluted further with 65 μl of 25 mM ammonium bicarbonate. Free thiols were then alkylated to prevent disulfide reformation by adding 35 μl of 200 mM N-ethylmaleimide (prepared in 50% methanol (v/v)) to yield the modified WR-1065 (WR-1065-EMI). The mixture was incubated at room temperature in the dark for 30 min. To quench remaining alkylation reagent, 13 μl of 200 mM dithiothreitol was added and the mixture was vortexed briefly. The mixture was stored at room temperature in the dark for 15 min. Proteins were precipitated via addition of 209 μl of cold (4°C) 10% trichloroacetic acid. After vortexing briefly, the proteins were pelleted via centrifugation at 14,000 rpm for 10 min at 4°C. 150 μl of supernatant was filtered (0.22 μm) into a clean micro-centrifuge tube. 40 μl of the filtered supernatant was diluted with 120 μl of internal standard spiking solution (spiked WR-1065-MMI concentration 125 nM) and placed in an autosampler (4°C) for LC-MS/MS analysis. The internal standard WR-1065-MMI corresponds to the N-methymaleimide analog of WR-1065 prepared in ammonium bicarbonate buffer (pH 7.8) using the same procedure described above for plasma.

LC-MS/MS Analysis of Derivatized WR-1065 (WR-1065-EMI)

Chromatographic separation was performed with a Nexera X2 UPLC system (Shimadzu Corp., Kyoto, Japan). Processed plasma samples were incubated in the autosampler at 4°C until analysis. 5 μl of sample was injected onto a Kinetex reversed-phase C8 column (1.7 μm, 2.1 mm × 150 mm) from Phenomenex (Torrance, CA, USA) installed in a column heating unit set at 40°C. A binary gradient program was used to separate the analyte and internal standard using a flow rate of 0.4 ml/min. The mobile phases were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The initial mobile phase composition was 3% B. This was held for the first 1.5 min of each run followed by a 2 min gradient to 90% B. The composition was held at 90% B for 0.5 min. The composition returned to 3% B over a 0.5 min gradient and remained at 3% B for the duration of the run. The total run time was 6 min.

The liquid chromatography system was configured to an LC-MS 8050 triple quadrupole mass spectrometer (Shimadzu Corp., Kyoto, Japan) for online detection and measurement of derivatized WR-1065. It was operated using the software package LabSolutions version 5.75 SP2 (Shimadzu Corp., Kyoto, Japan). The mass spectrometer was equipped with a heated electrospray ionization source set in positive ion mode with the following temperature, voltage and gas flow settings: interface temperature 400°C; desolvation temperature 250°C; heat block temperature 400°C; interface voltage 4 kV; nebulizing gas flow 3 L/min.; drying gas flow 10 L/min; and heating gas flow 10 L/min. The in vivo-derivatized metabolite WR-1065-EMI and the spiked internal standard WR-1065-MMI were measured in MRM mode. For WR-1065-EMI, the target transition 260.2 > 186.1 was measured for quantitation with an offset collision energy setting of −20.0 eV. A qualitative reference ion transition (260.2 > 243.1) was also monitored for specificity (offset collision energy −16.0 eV). The relative peak area for the reference ion transition compared to the target transition was within the range of 35% ± 10% to be considered a specific signal of WR-1065-EMI. For the internal standard WR-1065-MMI, the target transition 246.2 > 215.1 was measured for quantitation with an offset collision energy setting of −16.0 eV. The reference ion transition of WR-1065-MMI was (246.2 > 229.2) (offset collision energy −16.0 eV). The relative peak area for the reference ion transition compared to the target transition was within the range of 65% ± 10% to be considered a specific signal of WR-1065-MMI. The retention times for WR-1065-EMI and WR-1065-MMI were 1.96 and 2.37 mins respectively. Concentrations for all standard and unknown samples were determined from extrapolation of the measured WR-1065-EMI: WR-1065-MMI ratio onto a calibration curve acquired in each batch.

Calculation of Pharmacokinetic Parameters

Non-compartmental analysis of the concentration-time profiles of the levels of WR-1065 measured in plasma was performed with the Phoenix 64 WinNonlin® (Version 6.4) software package to obtain PK parameters and to calculate bioavailability.

Results

In Vitro

WR-1065 Protects the Ability of MC3T3 Cells to Differentiate into Functionally Active Osteoblasts in the Presence of XRT, while AMF Does Not

When comparing the efficacy of AMF and WR-1065 for the ability to protect osteogenic differentiation potential after XRT, ALP stain was performed as described above. AMF did not confer any increase in protection, while WR-1065 restored differentiation to a level similar to that of control samples without XRT (Fig. 1a). Figure 1b illustrates the ineffectiveness of AMF in protecting the osteogenic potential of MC3T3 cells upon XRT exposure (right). Extensive staining after WR-1065 treatment demonstrates the efficacy of protection of MC3T3 cells by this formulation in the preservation of osteogenic potential compared to control and AMF (middle). Without any treatment or with AMF alone, XRT-exposed cells are completely inhibited from differentiation. These results demonstrate that AMF is not active, and is required to be converted to WR-1065 in order to exert its effect on tissues. These results also imply that this treatment can effectively maintain bone-forming cells during XRT if sufficient levels of the metabolite WR-1065 reach the target craniofacial tissues.

Fig. 1
figure 1

(a) Control, WR-1065 and AMF plates stained via Alkaline Phosphatase assay early in cell life (day 6). The spotted red color of the WR-1065 plate indicates strong osteogenic differentiation potential and thus supports the radioprotective efficacy of WR-1065 compared to control and AMF (b) Control, WR-1065, and AMF plates stained via Alizarin Red assay later in cell life (day 18). The dark red color of the WR-1065 plate indicates the presence of calcified deposition and matrix mineralization, and support the radioprotective ability of WR-1065. The diameter of each plate is 3.5 cm. In sum, WR-1065 is an effective radioprotectant of MC3T3 cells, while AMF requires ALP to break down into its active metabolite (WR-1065).

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In Vivo

High Systemic Levels of WR-1065 Are Observed Following Direct Injection of AMF into the Upper GI Tract

An ideal PO formulation would allow for effective delivery of AMF to the upper GI tract, where it could then be metabolically converted by intestinal ALP to WR-1065 and absorbed systemically for delivery to target tissues. To evaluate the feasibility of intestinal conversion of AMF and subsequent systemic release of WR-1065, we surgically injected AMF to the jejunal region in rats and compared the resulting PK profile against an equivalent IV dose (10 mg/kg) in another rat cohort. The PK profile of the IV cohort serves as a reference for comparison since it displays features indicative of both the efficacy of, and side effects associated with, this route of administration. Figure 2 displays the time-concentration profiles of WR-1065 measured in rat plasma from both routes. The IV profile exhibits expected features, including a high initial concentration of WR-1065 followed by gradual clearance over a few hours. The high initial systemic levels of WR-1065 speak to the rapid in vivo conversion of AMF, and indicate the role WR-1065 plays in the onset of hypotension and nausea/vomiting.

Fig. 2
figure 2

Time-concentration profile of WR-1065 (minutes, ng/ml) in rat blood plasma after IV and direct jejunal (DJ) AMF administration. The IV curve demonstrates a high initial concentration of WR-1065 followed by gradual clearance over a few hours, while the DJ curve is comparably stable. The high initial systemic levels of WR-1065 demonstrate the role WR-1065 plays in causing patient side effects.

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In contrast, the profile representing direct jejunal injection of AMF exhibits low initial exposure levels of WR-1065. Its maximum concentration (cmax) was observed at 30 min before its gradual clearance over a few hours. As shown in Table I, the areas under the plasma drug concentration-time curves (AUC) for direct jejunal versus IV administration were 1940 and 3155 ng*hr/ml, respectively. This corresponds to a systemic bioavailability (%F) of WR-1065 after direct jejunal administration of 61.5%. The achievement of sufficient absorption and proper bioavailability of amifostine within the jejunum first requires conversion of AMF to WR-1065 in the intestinal tract. In contrast to AMF, which exhibits low cellular and tissue permeability, WR-1065 actively penetrates cellular membranes by passive transport (25). As supported by the pharmacokinetic data in Table I, this implies that a PO administration strategy designed for site directed release of an equivalent dose of AMF to the upper GI tract may facilitate high conversion to WR-1065 by intestinal ALP, followed by systemic absorption and total exposure levels that are comparable to direct exposure through the IV route. This assumes that AMF is formulated for adequate protection from gastric decomposition while transported through the stomach (e.g. enteric coating). Furthermore, the profile suggests that high systemic exposure may be achieved while significantly reducing the peak exposure (i.e. cmax) that leads to the side effects. However, it should be stated that systemic levels of WR-1065 cannot directly be correlated with tissue distribution and radio-protective efficacy.

Table I Pharmacokinetic Profile of WR-1065 after Administration of Amifostine Directly into the Jejunum and Intravenously
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EC-AMF Formulated for PO Administration Delivers Similar Systemic Exposure Levels of WR-1065 Compared to an Equivalent Radio-Protective Dose Administered Subcutaneously

We prepared an EC-AMF (Eudragit L100–55) designed for stable stomach transport and subsequent pH-dependent release of the drug in the intestinal tract. To assess the potential of this PO formulation for delivering efficacious levels of AMF, we compared its PK profile to an equivalent dose administered subcutaneously. The dose level administered through both routes was 100 mg/kg, which we’ve demonstrated to be an efficacious, radio-protective dose in rats when administered subcutaneously. As shown in Fig. 3, the SC route of administration yields higher systemic levels of WR-1065 along with a more rapid peak exposure level. As summarized in Table II, peak levels are reached in the blood stream in under 30 min, while peak levels are reached in almost twice as long after administration of EC-AMF (PO). From their corresponding AUCs (Table II), the total systemic exposure of WR-1065 from SC administration of AMF was almost 10-fold greater than the exposure achieved from the PO administration of EC-AMF.

Fig. 3
figure 3

Time-concentration profile of WR-1065 (hours, ng/ml) in rat blood plasma after SC and PO AMF administration. The SC curve shows higher systemic levels of WR-1065 and a more rapid peak exposure level compared to the PO curve. Peak levels are reached in the blood stream in under 30 min after SC administration, while peak levels are reached in almost twice as long after oral administration. The total systemic exposure of WR-1065 from SC administration of AMF was almost 10-fold greater than the exposure achieved from PO administration.

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Table II Pharmacokinetic Profile of WR-1065 after Administration of Amifostine Subcutaneously and Orally
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Using the PK profile for AMF administrated subcutaneously as a reference, the corresponding profile for the orally administered EC-AMF could be interpreted as potentially efficacious, although the reduced systemic exposure may also indicate a reduced distribution of WR-1065 to the target tissue. If the latter, then it would likely mean that there is reduced radio-protection of the irradiated tissue. On the other hand, the side effects caused from high WR-1065 exposure, characteristic of both SC and IV administration, may be mitigated by an orally administered EC-AMF formulation that limits the peak exposure of WR-1065 (cmax) to roughly one-tenth of the corresponding peak levels observed from SC administration (Table II). Based on this, it is possible that PO AMF formulations could have similar radioprotective effects as SC or IV formulations, but with reduced side effects as a result of lower peak exposure levels.

Discussion

AMF was first approved by the Food and Drug Administration for its demonstrated protection of tissues against XRT induced injury (7,8). Numerous studies subsequently established the ability of AMF to remediate vascular injury and cellular depletion in multiple settings, including in HNC and breast cancer (26). In this manner, our lab has demonstrated that AMF has a transformative ability to encourage bony union by preventing XRT injury in cases where this was not previously possible (11,12,13,14,15,16,17,18). While AMF offers selective radio-protection of normal tissue to facilitate bony and soft tissue healing after XRT, its current method of delivery and related side effect profile have limited its clinical acceptance. The current route of administration is either IV or SC, with both requiring painful needle injections for proper dose delivery. These methods are also associated with rapid and high peak exposure levels of WR-1065, which results in hypotension, nausea, vomiting, and injection site reactions. Consequently, most patients do not commonly receive AMF due to complex dosing strategies, the associated side effects, costs associated with administration, and inconvenience. Without further cultivation of this therapeutic option, the application and clinical adoption of this validated and efficacious therapy may never fully occur (9,27,28,29,30). Nonetheless, the growing incidence of HNC with over 75% of these patients requiring radiotherapy mandates the development of effective and more tolerated formulations of AMF or other radio-protectants.

In this study, we identify that AMF directly protects the cellularity associated with bone healing and regeneration from XRT injury once it has been metabolically converted to its active radio-protective metabolite WR-1065. We show that delivery of AMF safely to the upper GI tract (specifically the jejunum) yields 61.5% bioavailability of WR-1065 compared to an equivalent IV dose. Furthermore, the peak levels (cmax) associated with jejunal release are less than 30% compared to the corresponding peak levels observed from IV administration. Taken together, this suggests that a PO formulation designed for stable gastric transport and subsequent release in the upper intestinal tract may provide radio-protective efficacy with a reduction in side effects (reflected by reduced cmax relative to IV administration) when administered in this manner. While previous in vitro and in vivo studies have supported the radio-protective capacity of a PO formulation of AMF, this present study adds to the literature by demonstrating that orally administered AMF delivers a reduced peak level of WR-1065 compared to subcutaneously-administered AMF, which suggests an improved toxicity profile through the PO route (22,31,32).

The impact of pharmacotherapy depends not only on the quality of the product, but also on the feasibility of its use. Although AMF has proven to be effective for prophylaxis against XRT injury, its use has been limited because of side effects and inconvenient methods of administration. Here, we identify a potential target with which to innovate more convenient formulations in the future, and hope to increase the number of patients able to tolerate and benefit from this efficacious therapy. By minimizing the effects of XRT in a consistent, reliable, and feasible manner, we can prevent complications including osteoradionecrosis and XRT-induced fractures that mandate surgical management and significantly impair the quality of life among patients with HNC.

Conclusion

We first confirmed WR-1065 as the radio-protective species responsible for protecting and preserving the functionality of osteoblasts against XRT. We then evaluated a PO strategy designed to protect AMF against decomposition in the stomach, enabling subsequent dissolution and metabolic conversion to WR-1065 in the upper GI tract where there is an abundance of intestinal AP. Next, we confirmed that significant absorption of WR-1065 can be achieved from a PO route designed for targeted release of AMF in the upper GI tract. Finally, we prepared EC-AMF for PO delivery and compared it against SC AMF formulation at equivalent doses to assess its potential in providing radio-protective benefits while mitigating side-effects associated with high systemic levels of WR-1065. Together, these findings enhance our ability to create a clinically viable formulation of AMF that maintains efficacy against XRT injury among patients with HNC in the future.