Biological and Pharmaceutical Bulletin Advance Publication by J-STAGE Advance Publication DOI:10.1248/bpb.b15-00728 December 25, 2015
Biol. Pharm. Bull. Regular Article
Comparison of Absorbents and Drugs for Internal
Decorporation of Radiocesium: Advances of Polyvinyl Alcohol
Hydrogel Microsphere Preparations Containing Magnetite and
Prussian Blue.
Izumi Tanaka, Hiroshi Ishihara*, Haruko Yakumaru, Mika Tanaka,
Kazuko Yokochi, Katsushi Tajima, Makoto Akashi
Internal Decorporation Research Team, Research Program for Radiation Medicine,
Research Center for Radiation Emergency Medicine, National Institute of Radiological
Sciences, 4-9-1, Anagawa, Inage-ku, Chiba-shi, Chiba 263-85555, Japan
*Corresponding author. Internal Decorporation Research Team, Research Program for
Radiation Medicine, Research Center for Radiation Emergency Medicine, National
Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba-shi, Chiba 263-85555,
Japan. Tel: +81-43-206-3162; Fax: +81-43-284-1769; Email: [email protected]
Ⓒ 2015 The Pharmaceutical Society of Japan Summary
Radiocesium nuclides, used as a gamma ray source in various types of industrial equipments
and found in nuclear waste, are strictly controlled to avoid their leakage into the
environment. When large amounts of radiocesium are accidentally incorporated into the
human body, decorporation therapy should be considered. Although standard
decorporation methods have been studied since the 1960s and were established in the 1970s
with the drug Radiogardase® (a Prussian blue preparation), application of recent advances
in pharmacokinetics and ethical standards could improve these methods. Here we designed
a modern dosage form of hydrogel containing cesium-absorbents to alleviate intestinal mucosa irritation due to the cesium-binding capacity of the absorbents. The effectiveness of the dosage form on fecal excretion was confirmed by quantitative mouse experiments.
The total cesium excretion rate of the crystal form (1.37 ± 0.09) was improved by the hydrogel form (1.52 ± 0.10) at the same dose of Prussian blue, with a longer gastrointestinal tract transit time. Using a mouse model, we compared the effects of several drugs on fecal and urinary excretion of internal cesium, without the use of absorbents. Only phenylephrine hydrochloride significantly enhanced cesium excretion (excretion rate of
1.17 ± 0.08) via the urinary pathway, whereas none of the diuretic drugs tested had this
Biological and Pharmaceutical Bulletin Advance Publication effect. These findings indicate that modifying the dosage form of cesium absorbents is important for the decorporation of internal radiocesium contamination.
Key Words: Radiocesium; Prussian blue; Zeolite; Diuretics; Phenylephrine
Biological and Pharmaceutical Bulletin Advance Publication
INTRODUCTION
Internal contamination with radiocesium generated by nuclear fission may occur
after nuclear weapon testing and serious nuclear plant accidents 1). In addition,
concentrated radiocesium is frequently used industrially as a source of gamma rays.
Accidental incorporation of radiocesium may also occur due to mishandling, inadequate
security, terrorism or complex disasters. 2) Studies of contamination countermeasures have been performed, but few studies have examined methods of internal contamination.
Cesium is an alkaline metal and the biokinetics of cesium have been studied in
detail. 3) Briefly, ingested cesium rapidly disperses throughout the whole-body in ion
form without sedimentation. Cesium in body fluids is incorporated into several cell types
by ionic pumps, and then leaked back to the fluid via potassium channels in the plasma
membrane. Cesium particularly accumulates in skeletal muscle, because of its large
influx/efflux rate, where it is retained for several months in human. Cesium in the body
fluid is mainly excreted in urine. Although large amounts of cesium are released to the
gastrointestinal (GI) tract, most of it is reabsorbed by the intestine and returned to the body
fluid. Thus, the final rate of excretion of cesium from the body through feces, urine and
sweat is estimated to be 13%, 85% and 2%, respectively, in human. 3)
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Medical treatment for radiocesium decorporation is recommended when the committed dose is predicted to be 30 to 300 mSv. 4) The effectiveness of oral administration of Prussian blue is established based on studies of various cesium absorbents. 5) In principle, ingested Prussian blue crystals capture radiocesium ions released in the GI tract, and are excreted with the feces while blocking intestinal reabsorption, thus enhancing the fecal radiocesium decorporation rate. 3) Since the 1970s,
Radiogardase®, a capsule containing Prussian blue crystals, has been sold on the market for treating serious accidental internal exposure to radiocesium. 6, 7) Although standard treatment using domestic stockpiles of Radiogardase® is established, 8) medical concepts are evolving to enhance the effectiveness and reduce patient discomfort. Recent findings and technologic advancements reveal room for improvement of the radiocesium decorporation method.
In this paper, we describe an improved radiocesium decorporation treatment based on modifications of the form of cesium absorbents, and confirmed by comparison with the present absorbents. The effects were quantified by decorporation tests in mice.
We also examined the effect of drugs that act to facilitate urinary and fecal excretion of body fluids.
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MATERIALS AND METHODS
Cesium-absorbents and Reagents The ferrocyanides used as cesium absorbents in the
present study comprised water-soluble colloidal preparations of Prussian blue
III II [KFe Fe (CN)6], microcrystal preparations less than 1 μm in diameter of Prussian blue
III II II II II II [Fe 4Fe 3(CN)18-KCl-nH2O], K2Ni Fe (CN)6 and K2Co Fe (CN)6. The preparations
III II were made by mixing 0.25 mol/L solutions of Fe Cl3, CoCl2, NiCl2, or K4[Fe (CN)6]
(Wako Pure Chemical Industries, Ltd). Dialysis was performed to purify the preparations
or to substitue the alkali metals. Insoluble Prussian blue with 14 to 16 water molecules
corresponding to Radiogardase® was prepared by freeze-drying or dehydrating the
microcrystals at 80°C. The sizes of the microcrystals and colloid were measured using
the dynamic and static light-scattering method with a DelsaTM Max (Beckman-Coulter
Inc.), supported by Beckman Coulter K.K. Japan. Synthetic zeolite with a mean particle
size of 2 to 4 μm (Wako Pure Chemical Industries, Ltd.), and apple pectin (Wako) were also used.
Drugs for Mouse Experiments The following drugs were used in this study:
Radiogardase® (HEYL Chemisch-Pharmazeutische Fabrik GmbH und Co., KG), phenylephrine hydrochloride (Neo-Synesin Kowa, Kowa Pharmaceutical Co., Ltd.), adrenaline (Bosmin® injection, Dai-ichi Sankyo Co. Ltd), clonidine hydrochloride (Wako), phentolamine mesilate (Regitin® injection, Novartis Pharma), nisoldipine (Baymycard®,
Bayer HealthCare), and hydralazine hydrochloride (Apresoline®, Novartis Pharma). As a cathartic, D-sorbitol (Wako) and magnesium succinate (Magcorol®, Horii
Pharmaceutical Ind., Ltd.) were used. As diuretics, acetazolamide sodium (Diamox®,
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Sanwa Kagaku Kenkyusho Co., Ltd.), furosemide (Lasix®, Nichi-Iko Pharmaceutical Co.,
Ltd.), trichlormethiazide (Fluitran®, Shionogi & Co., Ltd.), indapamide (Natrix®, Kyoto
Pharmaceutical Ind., Ltd), potassium canrenoate (Soldactone®, Pfizer Japan Inc.),
eplerenone (Selara®, Pfizer Japan Inc.) and isosorbide (Isobide®, Kowa Pharmaceutical
Co., Ltd.) were used. Their administration doses (per kg-body weight) in mice
corresponded to the upper limit dose in humans.
Preparation of Polyvinyl Alcohol Hydrogel A mixture of 5% polyvinyl alcohol (PVA,
polymerization degree ~500, Wako) in 0.25 mol/L HCl containing magnetite silica
particles (final conc. = 20mg/mL of sicastarR-M plain, diameter of 350nm, Micromod
Partikeltechnologie GmbH) and Prussian blue or metal-ferrocyanide microcrystals of
II II II II K2Ni Fe (CN)6 and K2Co Fe (CN)6 (final conc. = 66.3 μmol/mL of ferrocyanide) was kept on ice. After adding 50% glutaraldehyde (Wako) to a final concentration of 1.5%,
the mixture was emulsified with a 4-fold volume of mineral oil (Sigma-Aldrich Co., LLC.)
containing 2.5% sorbitan sesquioleate (Sigma-Aldrich). Polymerization of PVA was
achieved by keeping the mixture at 50°C. The PVA gel was isolated, purified by
sequential extractions with ether, ethanol and water, and sieved. The microsphere of the
magnetic PVA preparations of cesium-absorbents with diameters ranging from 10 to 50 μm were freeze-dried to insolubilize the ferrocyanides and for storage. The freeze-dried preparations were hydrated before use.
Cesium Absorption Test in vitro To evaluate the absorption of cesium in vitro, solid absorbents (corresponding to 1 μmol ferrocyanide) were mixed with 2.5-fold molar amounts (2.5 μmol) of cesium chloride containing 440.5 Bq (1.0 pmole) of 137Cs (Eckert
& Ziegler Isotope Products). Radioactivity in the supernatant was measured to calculate
Biological and Pharmaceutical Bulletin Advance Publication
radiocesium binding in the solid. For water-soluble absorbents, the solution was put into a dialyzed membrane and immersed in solution containing 2.5-fold molar amounts of the expected absorbance capacity of cesium chloride with 137Cs. After incubation at 37°C
simulating in vivo treatment for the appropriate time, the radioactivity of the supernatant or
outer solution was measured. A coaxial HPGe gamma detector (ORTEC, P-type, model
GEM35P4-76), calibrated using a radioactivity standard gamma volume source (Japan
Radioisotope Association), was used for quantify the radioactivity.
Acclimatization of Mice Mice were treated in accordance with the Guidelines for
Proper Conduct of Animal Experiments (Science Council of Japan) and with minimum
stress, as previously reported. 9-11) As breeding mice in metabolic cages can induce stress,
we habituated young C3H/He inbred mice with a mild nature in metabolic cages. Thus,
C3H/He inbred mice 4 weeks of age obtained from Japan SLC Co. were acclimatized to
the metabolic cages for 4 weeks with one mouse per metabolic cage in animal room
maintained at 23°C with 55% humidity, and lights (10 to 50 lx) on from 07:00 to 19:00.
To avoid generating high frequency sounds that are a source of stress for mice, we used the
metabolic cages made of methacrylate and polyethylene for housing and collection of
excreta, with stainless-steel mesh as the base-plate and cover, and glass tubes to supply
food and water (Natsume Seisakusho Co., Ltd).
Experiments to Measure GI Tract Passage of the Absorbents To measure the levels
of resorption in GI-tract of radiocesium from cesium absorbents, 5 mg of Prussian blue,
137 K2NiFe(CN)6 , K2CoFe(CN)6 or zeolite which bound to with 11 pmol of Cs at 37°C
overnight were orally administrated. After 24 h, each feces were collected and
radioactivity were measured.
Biological and Pharmaceutical Bulletin Advance Publication
The passing rate of the absorbents in the GI tract and intestinal absorption of
radiocesium released from the absorbents in the mice were measured. Absorbents were
labeled with 137Cs at 37°C overnight, then washed with saline. The absorbents were then orally administered to 4 mice per group at a.m. 10:00. The dose per mouse was 1.20kBq
137 II (2.72 pmol) of Cs in ferrocyanides [corresponding to 1.0 μmol of Fe (CN)6] or 1.25mg
of zeolite. The 137Cs levels in the feces and urine collected at regular intervals were
measured as described above. The amounts of the absorbents retained in the GI tract were determined by subtracting the sum of radioactivity in the feces and urine from the administered dose.
Experiments to Measure for Internal Decorporation of Radiocesium in Mice When the mice were 8 weeks old, we measured radiocesium decorporation rates in individual mice to exclude abnormal individuals. After subcutaneous injection of 5.0 kBq 137CsCl
into the interscapular space at a.m. 10:00, the radioactivity of all the excreted feces and
urine was measured daily. Twelve apparently healthy mice were divided into three
groups (two test groups and one control group) and used for the following experiments.
In each experiment, 5.0 kBq 137Cs was injected at Day 0. Test drugs and vehicle (water)
were administered for 3 times at 6-h intervals on Day 2. Urine and feces excreted on Day
2 to Day 3 were collected separatedly, and the radioactivity was measured for each of them as described above. Mean radioactivity in the Day 3 excreta (sum of urine plus feces) from 4 control mice was set at 1.00 as the standard. Relative rates of radioactivity in the urine and feces from the test mice were determined. The mice were reused for the next series of experiments after the internal radiocesium decreased below 500 Bq/mouse. The examinations were continued until the mice were 28 weeks old. The effects of each drug
Biological and Pharmaceutical Bulletin Advance Publication
on the excretion rate of radiocesium were calculated using data obtained from two
different batches of mice. The significances of the drug effects on the relative excretion
rates was determined based on Dunnett's test following one-way ANOVA.
Doses per kilogram body-weight for the examined drugs were determined based
on the upper limit of the daily dose for humans. Orally administered drugs were
administered through a stomach tube at the following daily doses per kilogram
body-weight: 150 mg Radiogardase®, 7.2 or 36 μmol of ferrocyanide in Prussian blue,
II II II II K2Ni Fe (CN)6 and K2Co Fe (CN)6 , 150 mg zeolite, 50 mg trichlormethiazide, 40 mg
indapamide, 10 mg eplerenone, 3.0 or 6.0 g isosorbide, 6.0 mg nisoldipine, 1mmol NaCl,
7.0 g magnesium succinate, or 6.5 g D-sorbitol. The volume was less than 20mL/kg
body-weight and the same volume of water was used as a negative control. Daily doses
per kilogram body-weight of drugs for subcutaneous injection were 500 mg acetazolamide
sodium, 100 mg furosemide, 160 mg potassium canrenoate, 3.0 g isosorbide, 1.0 mg or 2.0
mg phenylephrine hydrochloride, 0.1 mg adrenaline, 0.6 mg clonidine hydrochloride, 0.5
mg phentolamine mesilate, and 4.0 mg hydralazine hydrochloride.
Biological and Pharmaceutical Bulletin Advance Publication
RESULTS AND DISCUSSION
The present study aimed to improve the drugs currently used for decorporation of internally contaminating radiocesium. First, Prussian blue and related coordination complex were selected as cesium-absorbents, based on in vitro evaluation of the cesium binding capacities of various candidates (section 1). The absorbents were then modified to hydrogel preparation form for introduction to GI-tract (section 2), and the advantages of the hydrogel preparation form were compared based on in vivo experiments in mice
(section 3). The effects of commercial drugs on cesium excretion were also compared by the in vivo experiments (section 4).
1. Examination of Cesium-absorbents Studies of radiocesium absorbents performed since the 1960s have established the efficacy of porous minerals, 12) Prussian blue, 13) and related coordination crystals of ferrocyanides with transition metals 14) as absorbents. Transition metal ferrocyanides with modifications of the fine structure are currently under study. 15) First, we describe a part of a previous report for comparison with our results based on the usefulness of these methods for medical decorporation.
Biological and Pharmaceutical Bulletin Advance Publication
III II Prussian blue is a coordination complex of Fe and Fe (CN)6 with water
crystals, alkaline metal, and anions. The binding affinity of alkaline metal is Cs> Rb>
13) III II K> Na. An equimolar complex of Fe and Fe (CN)6 is soluble (Fig. 1a) and a 4:3
III II 16) complex of Fe and Fe (CN)6 with excess water forms microcrystals (Fig. 1b).
Removal of the water crystals by drying under 100°C leads to the formation of stable
III II water-insoluble crystals of Fe 4[Fe (CN)6]3-(14-16)H2O with irregular shapes and sizes
(Fig. 1c). 17) Excess dehydration and heating at more than 550°C generates degradative
products of iron oxides by the removal of cyanogen. For safe use as a drug, release of
II cyanogen in the GI tract should be avoided, even though the Fe (CN)6 ion is chemically
stable. Ultimately, an aqueous complex with 14 to 16 water molecules was determined to
be the safest form of Prussian blue, 14, 18) and the capsule formulation is used as the
medical decorporation drug called Radiogardase®. Although the surface area of the 14
to 16 water crystals is extraordinarily smaller than that of microcrystals or the colloidal
form, large differences in the binding velocity of radiocesium were not observed (Fig. 2a),
indicating that the coordination structure of the water-insoluble crystals is not rigid and
alkaline metal ions readily move though the lattice. 17)
A certain type of coordination complex of ferricyanide and non-iron divalent
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II III x- period-4 transition metals, such as Co [Fe (CN)6] , also binds cesium similar to Prussian
blue. 19) This type of crystal, however, cannot be used as a drug as it easily releases the
transition metal ion, which is toxic to humans, due to the instability of the lattice created
by non-stoichiometric coordination. 20, 21) Another type of coordination complex of
ferrocyanide and divalent period-4 transition metals having a more stable coordination has
been used as a conventional cesium absorbent. 14, 22) Examples of such compounds,
II II II II K2Ni Fe (CN)6 and K2Co Fe (CN)6, are shown in Fig. 1d and 1e. Their maximal
absorption capacities of cesium in physiologic saline in vitro are 0.9 and 1.3 mol Cs per
II mole of Fe (CN)6, respectively, and greater than that of Prussian blue [0.7 mol per mole
II Fe (CN)6] (Fig. 2b and 2c). The 24 h-cesium binding capacity of Radiogardase®
(water-insoluble crystal of Prussian blue) is maximal at pH7.5 and 85 to 90 % of maximum at pH9.0. 23) The microcrystal form of Prussian blue, however, exhibits maximal absorption capacity only at pH7 (Fig. 2b) as like previous report. 22) The
II II II II microcrystal forms of K2Ni Fe (CN)6 and K2Co Fe (CN)6 maintain their capacity among
a wide range of pH levels, between 1.0 to 9.0 in saline (Fig. 2b). After binding with the
carrier to prevent dissolution of the constituent in the GI tract, 24) these ferrocyanides were
examined for decorporation of radiocesium in livestocks in the 1970s. 25, 26) Currently,
Biological and Pharmaceutical Bulletin Advance Publication
ferrocyanide is applied for the removal of radiocesium from animals 27) or the environment
28, 29) by binding with insoluble carriers. Unfortunately, the recent trends of emphasizing
the adverse effects 30, 31) or allergenic effects 32) of the transition metals have hampered
their medical application.
Ore, such as zeolite, which is also used to bind radiocesium, and its application
for decorporation in livestock have been examined since the 1950s. 33) Zeolite has some
cesium-binding capacity of ~ 0.4 μmol/mg at pH 1.0 and pH 7.0 (Fig 2c, lanes 10 and 11).
Orally administered zeolite that held radiocesium (2.2 pmol/mg) loses the bound cesium
during passage through the GI tract, while ferrocyanides hold on to the bound cesium in
vivo (Fig. 3a). This indicates that cesium would be released from zeolite in the GI tract
with its complicated chemical environment and resorbed by the mice body. On the other
hand, zeolite is useful for environmental decontamination of radionuclides due to its
thermal- and chemical- stability, compared with ferrocyanides, which are degraded at
temperatures higher than 550°C, at an alkaline pH > 12, or with chelates. Incineration at
800°C of zeolite with excreta containing ferrocyanides with radiocesium created a form of
waste suitable for long-term storage and reduced the volume (Figs. 1i, 2c).
Few organic substances that capture radiocesium, like pectin, are known. We
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tested the effect of apple pectin on cesium incorporation. Dialysis at 37°C of 2.5% pectin
in saline solution placed in 133-fold volume of 0.015 or 0.15 mmol/L cesium in saline
solution increased the concentration of cesium in the pectin solution to 0.85 mmol/L
(57-fold of the original concentration) after 24 h (the data not shown). Reducing the pectin solution to 0.25% pectin reduced the concentration rate to 30-fold with either 0.015
or 0.15 mmol/L cesium solution. These findings indicate that the ability of pectin to
capture cesium was in equilibrium and could be changed by the environment. Even if
some dosage form of pectin that maintained a concentration of 2.5% was orally administered to humans or animals, the effect on the fecal cesium excretion would not be obvious because of large individual variations. This is consistent with a previous report that the effect of pectin is not significant for decorporation in animals. 34) Thus, Prussian
blue is a much better base material for the preparation of cesium absorbents for medical
use.
2. Hydrogel Formulation of Cesium Absorbents. Currently, only one
absorbent for radiocesium decorporation is available on the market, Radiogardase®, which is a capsule preparation of Prussian blue 14-16 water crystals with sizes between 1 μm to a few millimeters (Fig. 1c). The crystal structure has a large advantage to minimize
Biological and Pharmaceutical Bulletin Advance Publication
absorbed dose by radiocesium in patients because the water-insoluble crystal firmly bind
radiocesium in GI-tract and does not release radiocesium before the excretion. 16) The
dose regimen recommended by the Health Protection Agency of the UK, 4) is 1 g orally 3
times per day. Based on the current pharmaceutical view, passage of such crystals
through the digestive tract is associated with the risk of physical damage. In fact, it has been reported that a maximal daily dose of 10 g leads to dysphoria in the digestive tract. 35)
Additionally, it is difficult to control the transit time of Prussian blue with radiocesium in the digestive tract. A longer retention time leads to an increase in the effect/dose rate, which allows for minimization of the dose. Thus, there is room for improvement of radiocesium absorbents as decorporating molecules.
We describe here how Prussian blue preparations for radiocesium decorporation are improved by the use of hydrogel. Hydrated microcrystals less than 1 μm in size of
II II II II Prussian blue, K2Ni Fe (CN)6 or K2Co Fe (CN)6 (Fig. 1b, 1d, 1e) were suspended with siliconized magnetite in PVA gel. They were solidified into spherical shapes with diameters of 10 to 50 μm (microspheres, Fig. 1f, 1g, 1h). The ferrocyanide crystals were dehydrated by lyophilization of the hydrogel to prevent decomposition of the crystal.
The lyophilized preparations could be stored and rehydrated before use. The absorption
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II capacity of radiocesium per mole of Fe (CN)6 was not changed in the hydrogel dosage form (Fig. 2c).
Hydrogel preparations of drugs cause mucoadhesiveness with minimal physical and chemical stress to the GI mucosa in mammals. 36, 37) The retention time of the hydrogel form in the murine GI tract tended to be longer than that of the Prussian blue
14-16 hydrous crystals (Fig. 3b). Since elongation of the retention time of Prussian blue binding radiocesium increases exposure time in the GI-tract, Radiogardase® minimizes the effective dose in the intestine. Large crystals of Radiogardase® with a spicular shape, however, physically irritate the digestive tract mucosa and restrict upper limit of dose, as described above. 35) When using the hydrogel dosage form of cesium-absorbents, the required dose is expected to increase because of the mild effect on mucosa, similar to a microsphere preparation for the GI-tract. 38)
3. Excretion of Radiocesium by Hydrogel Preparations of Cesium
Absorbents in Mice. Some factors that cause physiologic and psychologic variations among individuals reduce reproducibility in animal experiments. For example, handling-induced stress increases blood glucocorticoid level, 39) which drastically affect the transition and excretion of body fluids. Although housing a mouse in a metabolic
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cage sometimes leads to agitated behaviour, suggesting psychological stress, calm
behavior was observed in most of the C3H/He inbred mice that were acclimatized to
metabolic cages beginning at 4 weeks of age. When the body-burden of 12 mice injected
with radiocesium was measured, similar decreases with small variations among 12 mice
were observed from Day1 to Day5 (Fig. 3c upper panel). Comparison of the daily
decrease rate (Fig. 3c) indicated that individual differences were lower on Days 2, 3, and 5.
A similar excretion rate profile was observed in different batches of 12 mice (data not
shown). Thus, we administered drugs on Day 2 and measured the daily excretion on Day
3 to compare the effects of the drugs on the daily excretion rate.
The urinary and fecal excretion rate was 0.65 ± 0.03 (95% confidence intervals) and 0.35 ± 0.05, respectively, in C3H/He inbred mice (Fig. 3d, lane 1). To reveal the drug effects on these different excretion pathways, we measured urinary and fecal
excretion separately in mice.
The effects of dosage forms of cesium absorbents on decorporation were
examined. Oral administration of the standard dose of Prussian blue (Radiogardase®) increased the total excretion rate to 1.37 ± 0.09 (Fig. 3d, lane 2), although the urinary excretion rate was reduced to 0.53 ± 0.10 from 0.65 ± 0.03. A similar excretion rate was
Biological and Pharmaceutical Bulletin Advance Publication
observed following administration of a 20% dose of the microcrystal form (Fig. 1b) of
Prussian blue with a longer retention time in the GI tract (Fig. 3b, Fig. 3d lane 4). At the
standard dose, further increase of the total excretion rate to 1.58 ± 0.09 was observed (Fig.
3d, lane 3). The dose-effect relationship was maintained when the PVA dosage form (Fig.
II II 1h) was administered (Fig. 3d, lanes 5 and 6). PVA gel containing K2Ni Fe (CN)6
II II (nickel ferrocyanide) or K2Co Fe (CN)6 (cobalt ferrocyanide) had a similar excretion rate
as Prussian blue (Fig. 3d, lane 7 or 8), even though they had the cesium binding capacity in
vitro of 1.8-fold or 1.2-fold, respectively (Fig. 2c). In all dosage forms, the fecal
excretion rate increased 2 to 3 times and the urinary excretion rate was 0.8 to 1.0 times lower than that of the control (Fig. 3d). The results indicated that dosage forms that
control transition time in the GI tract are more important for decorporation of radiocesium
than the cesium-absorbance capacity of the base substance. The hydrogel dosage form of
the cesium- absorbent reported in this paper is also expected to facilitate waste-disposal
procedures. Radiocesium with Prussian blue in a hydrogel can be collected from feces
using a magnet, and stably stored for a long time by transferring to a stable carrier, such as
zeolite, by co-incineration (Fig. 1i).
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4. Confirmation of the Drug Effects on Radiocesium Excretion in the Mouse
Model. The effects of drugs on decorporation of radiocesium without absorbents have
been studied since the 1960s. For example, acetazolamide sodium, a diuretic drug that
inhibits carbonate dehydratase enhances cesium excretion. 40) On the other hand, the diuretic drug thiazide, which affects reabsorption of alkaline metals and hydrogen ions in the kidney tubules, has no significant effect on the cesium excretion rate. 41) We
examined the effects on urinary and fecal excretion of internal radiocesium using typical
and common diuretic drugs. As shown in Fig. 3e (lanes 1 to 8), none of the diuretic
drugs significantly enhanced the total excretion rates of radiocesium. Although the
urinary excretion rates tended to be enhanced with furosemide, indapamide, and isosorbide,
the increase was not significant (Fig. 3e, lanes 3, 5, and 8). In contrast, acetazolamide
sodium, furosemide, and trichlormethiazide significantly decreased fecal excretion rates
(Fig. 3e, lanes 2, 3, and 4). In particular, trichlormethiazide significantly reduced the
total excretion rate (Fig. 3e, lane 4). These findings suggest that forced urination by
drugs acting on the kidney may actually decrease the fecal and total excretion rate of
internal radiocesium.
Of the drugs examined, only phenylephrine hydrochloride, an α1 adrenergic
Biological and Pharmaceutical Bulletin Advance Publication agonist, significantly increased both the urinary and total excretion rates (Fig. 3e, lane 9).
The absence of a significant increase in the total or urinary excretion rate with other adrenergic agonists, such as adrenaline and clonidine hydrochloride (Fig. 3e, lane 11, 12); an antagonist, such as phentolamine mesilate (Fig. 3e, lane 13); or vasodilators, such as nisoldipine and hydralazine hydrochloride (Fig. 3e, lanes 14, 15), suggests that changes in regional blood flow do not markedly affect cesium excretion. Because phenylephrine is used to induce frequent urination in experimental animals, 42) the urinary excretion of cesium is probably enhanced by a decrease in the bladder volume. A significant increase in the urinary excretion rate was observed following NaCl administration (Fig. 3e, lane 16), which stimulates water intake in animals. 43) The fecal excretion rate was significantly increased by the administration of cathartics, magnesium succinate and D-sorbitol (Fig. 3e, lanes 17, 18). No significant increase in the total excretion rate, however, was observed with theses drugs. Therefore, there is no rationale for using diuretic drugs in addition to cesium absorbents for decorporation.
5. Conclusion Regarding Decorporation of Radiocesium. Oral administration of Prussian blue is the standard treatment method for the decorporation of radiocesium in the body. Modification of the dosage form, such as the use of a hydrogel,
Biological and Pharmaceutical Bulletin Advance Publication improves the safety and efficacy by controlling the transit time in the GI tract, and is a more effective approach compared to changing the base absorbent.
Conflict of Interest The authors declare no conflict of interest.
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REFERENCES
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exchange of caesium of ferrocyanides. J. Inorg. Nucl. Chem., 26, 1111-1115 (1964). 15) Sangvanich T, Sukwarotwat V, Wiacek RJ, Grudzien RM, Fryxell GE, Addleman RS, Timchalk C, Yantasee W. Selective capture of cesium and thallium from natural waters and simulated wastes with copper ferrocyanide functionalized mesoporous silica. J. Hazard. Mater., 182, 225-231 (2010). 16) Verzijl JM, Joore HC, van Dijk A, Wierckx FC, Savelkoul TJ, Glerum JH. In vitro cyanide release of four Prussian blue salts used for the treatment of cesium contaminated persons. J. Toxicol. Clin. Toxicol., 31, 553-562 (1993). 17) Buser HJ, Schwarzenbach D, Petter W, Ludi, A. The crystal structure of Prussian
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26) Matsusaka N, Takeyama S, Matsuda Y, Kobayashi H, Yuyama A, Watari K, Imai K. Efficacy of nickel ferrocyanide-anion exchange resin for reducing egg contamination with 137Cs in laying Japanese quails. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med., 38, 217-221 (1980). 27) Timchalk C, Creim JA, Sukwarotwat V, Wiacek R, Addleman RS, Fryxell GE, Yantasee W. In vitro and in vivo evaluation of a novel ferrocyanide functionalized nanoporous silica decorporation agent for cesium in rats. Health Phys., 99, 420-429 (2010). 28) Sangvanich T, Sukwarotwat V, Wiacek RJ, Grudzien RM, Fryxell GE, Addleman RS, Timchalk C, Yantasee W. Selective capture of cesium and thallium from natural waters and simulated wastes with copper ferrocyanide functionalized mesoporous silica. J. Hazard. Mater., 182, 225-231 (2010). 29) Parab H, Sudersanan M. Engineering a lignocellulosic biosorbent--coir pith for removal of cesium from aqueous solutions: equilibrium and kinetic studies. Water Res., 44, 854-860 (2010). 30) Costa M, Davidson TL, Chen H, Ke Q, Zhang P, Yan Y, Huang C, Kluz T. Nickel carcinogenesis: epigenetics and hypoxia signaling. Mutat. Res., 592, 79-88 (2005). 31) De Boeck M, Kirsch-Volders M, Lison D. Cobalt and antimony: genotoxicity and carcinogenicity. Mutat. Res., 533, 135-152 (2003). 32) Bakula A, Lugović-Mihić L, Situm M, Turcin J, Sinković A. Contact allergy in the mouth: diversity of clinical presentations and diagnosis of common allergens relevant to dental practice. Acta Clin. Croat., 50, 553-561 (2011). 33) Mraz FR, Patrick H. Some factors influencing the excretory pattern of cesium-134 in rats. Arch. Biochem. Biophys., 71, 121-125 (1957). 34) Le-Gall B, Taran F, Renault D, Wilk JC, Ansoborlo E. Comparison of Prussian blue and apple-pectin efficacy on 137Cs decorporation in rats. Biochimie, 88, 1837-1841 (2006). 35) Melo DR, Lipsztein JL, de Oliveira CA, Bertelli L. 137Cs internal contamination involving a Brazilian accident, and the efficacy of Prussian Blue treatment. Health Phys., 66, 245-252 (1994). 36) Goto T, Morishita M, Kavimandan NJ, Takayama K, Peppas NA. Gastrointestinal transit and mucoadhesive characteristics of complexation hydrogels in rats. J. Pharm. Sci., 95, 462-469 (2006). 37) Varum FJ, McConnell EL, Sousa JJ, Veiga F, Basit AW. Mucoadhesion and the gastrointestinal tract. Crit. Rev. Ther. Drug Carrier Syst., 25, 207-258 (2008).
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38) Dhaliwal S, Jain S, Singh HP, Tiwary AK. Mucoadhesive microspheres for gastroretentive delivery of acyclovir: in vitro and in vivo evaluation. AAPS J., 10, 322-330 (2008). 39) Cutolo M, Sulli A, Pizzorni C, Secchi ME, Soldano S, Seriolo B, Straub RH, Otsa K, Maestroni GJ. Circadian rhythms: glucocorticoids and arthritis. Ann. N. Y. Acad. Sci., 1069, 289–299 (2006). 40) Sastry BV, Bush MT. Enhancement of cesium-137 excretion by rats treated with acetazolamide. Science, 136, 257-258 (1962). 41) Harrison J, McNeill KG. Effect of chlorothiazide on cesium-137 excretion in human subjects. Can. Med. Assoc. J., 89, 1266-1269 (1963). 42) Akiyama K, Hora M, Tatemichi S, Masuda N, Nakamura S, Yamagishi R, Kitazawa M. KMD-3213, a uroselective and long-acting alpha(1a)-adrenoceptor antagonist, tested in a novel rat model. J. Pharmacol. Exp .Ther., 291, 81-91 (1999). 43) Wasserman RH, Comar CL, Tapper DN. Influence of dietary potassium and sodium on cesium-137 retention in rats. Proc. Soc. Exp. Biol. Med., 113, 305-307 (1963).
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Legend of Figures
Fig. 1. Preparations of Cesium Absorbents
Prussian blue in the colloidal form (a), microcrystals of Prussian blue >100 H2O
II II II II (b), crystals of Prussian blue⋅(14-16) H2O (c), K2Ni Fe (CN)6 (d) and K2Co Fe (CN)6
(e) used in this study are shown. Mean diameters are indicated at the bottom. The
dosage form of the PVA hydrogels containing magnetite and Prussian blue (f),
II II II II K2Ni Fe (CN)6 (g), and K2Co Fe (CN)6 (h), before (left) and after (right) dehydration plus rehydration are shown. Bars indicate 10 μm. As an example of the disposal process, radiocesium-bound PVA-Prussian blue was magnetically collected from feces
(i-left, magnetic field is the upper), then the waste was covered with zeolite (i-middle), and incinerated (i-right).
Fig. 2. Absorbance of Cesium in Physiologic Saline in vitro
(a). Velocity of cesium absorbance of Prussian blue 14-16 hydrous crystals
(black) and microcrystals (blue). (b). 24 h-binding rates of cesium-absorbent Prussian
II II II II blue microcrystals (blue), K2Ni Fe (CN)6 (cyan), and K2Co Fe (CN)6 (green) at various
II 2- pH levels. (c). 24 h-binding (moles-cesium per mol of Fe (CN)6 in the absorbents) of the microcrystals (1, 4, 6) or PVA-hydrogel dosage form (2, 3, 5, 7) before lyophilization
(2) or rehydration following lyophilization (3, 5, 7) of Prussian blue (1, 2, 3),
II II II II K2Co Fe (CN)6 (4, 5), or K2Ni Fe (CN)6 (6, 7). 24 h binding (μ mole-cesium/mg dried
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absorbents) of Prussian blue 14-16 hydrous crystals (PBcr) at pH 7.0 (8) or pH 1.0 (9), and
zeolite (Z) at pH 7.0 (10) or pH 1.0 (11) is also shown.
Fig. 3. Mouse Excretion of Radiocesium
(a). Retention rate of radiocesium bound with microcrystals of cesium-absorbents of Prussian blue (1, PB), K2CoFe(CN)6 (2, Co), K2NiFe(CN)6 (3, Ni),
or zeolite (4, Z) in feces after passage through the GI tract. The mean and 95%
confidence intervals (CI) of four mice [mean ± standard deviation (s.d.) of body weight in
each group of mice was as follows: 1=31.5±1.1g, 2=30.3±1.3g, 3=31.2±0.7g,
4=30.9±1.3g) are shown. (b). Transit of cesium-absorbents in the GI tract. After oral
administration of radiocesium bound with the Prussian blue 14-16 hydrous crystals (1,
black), Prussian blue microcrystals (2, blue), or hydrogel containing Prussian blue (3,
green) to 4 mice, feces and urine were collected at the indicated times. Mean ± s.d. of
body weight in each group of mice was as follows: 1=31.4±1.2g, 2=30.4±1.1g,
3=30.7±0.8g. The retention rates of the absorbents were then calculated. The mean and
stanard deviation of four mice are shown. (c). Daily excretion rate of internal
radiocesium among 12 mice (mean and s.d. of body weight were 22.9g and 1.5g,
respectively) after subcutaneous injection of 137CsCl. The daily decrease in the
body-burden is shown in the upper right. The levels of internal cesium in each mouse
were calculated by the measuring daily excretion. Only standard deviation bars are
shown to emphasize individual differences. (d). Effect of the dosage forms of cesium absorbents on excretion rates of internal radiocesium. Mice were injected with radiocesium on Day 0, then absorbents of Prussian blue 14-16 hydrous crystals (cr, 2),
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Prussian blue microcrystals (mc, 3, 4), PVA with Prussian blue (pv, 5, 6), PVA with
II II II II K2Co Fe (CN)6 (Co, pv, 7), or PVA with K2Ni Fe (CN)6 (Ni, pv, 8) in the doses of 36
μmol (h, 2, 3, 5, 7, 8) or 7.2 μmol (l, 4, 6) of FeII(CN)6 per kilogram body-weight were orally administered to the mice three times on Day2. As a control, water was administered as described above (W, 1). All the urine and feces excreted on Day 2 to
Day 3 from each individual were separately collected and measured. Mean ± s.d. of body weight in each group was as follows: 1=29.5±1.6g, 2=28.8±0.7g, 3=29.3±0.8g,
4=27.5±0.4g, 5=29.4±0.7g, 6=29.7±1.1g, 7=30.6±0.7g, 8=30.1±0.8g. Means and 95%
CI of urinary (the lower cyan parts of bars) and fecal (the upper brown) excretion rates of
4 mice are indicated. The 95% CI of the total excretion rate are indicated at the bottom.
Significant data are indicated as red (fecal excretion), blue (urinal excretion), and black
(total excretion) asterisks (*: P <0.05, ** P <0.01). (e). Effect of drugs on excretion rate
of internal radiocesium. After radiocesium injection on Day 0, the following drugs were
administered subcutaneously (sc) or orally (po) to mice 3 times on Day2: water po (1,
water), acetazolamide sodium sc (2, AZA), furosemide sc (3, FCM), trichlormethiazide po
(4, TMZ), indapamide po (5, IDP), potassium canrenoate sc (6, CRN), eplerenone po (7,
ELN), isosorbide sc (8, ISB), phenylephrine sc at lower (9, PLPl) or higher (10, PLPh)
doses, adrenaline sc (11, ADR), chlonidine sc (12, CND), phentolamine hydrochloride sc
(13, PTL), nisoldipine po (14, NSD), hydralazine hydrochloride sc (15, HRZ), isotonic
sodium po (16, PSS), magnesium succinate po (17, MGS), and D-sorbitol po (18, SOR).
Mean ± s.d. of body weight in each group was as follows: 1=26.2±1.4g, 2=26.0±0.4g,
3=26.5±1.1g, 4=24.3±1.2g, 5=24.4±0.3g, 6=24.7±1.5g, 7=28.3±2.2g, 8=27.5±0.5g,
9=26.6±0.6g, 10=27.0±1.1g, 11=26.8±0.6g, 12=29.2±0.9g, 13=27.5±0.9g, 14=29.1±2.2g,
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15=29.4±3.1g, 16=26.2±0.8g, 17=25.8±0.6g, 18=26.0±0.6g. Means, 95% CI and the statistical notation are the same as in 3d.
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Fig. 1
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Fig. 2
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Fig. 3
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