Abstract
Introduction:
This study examined the decorporation potential of lansoprazole (LNP) as radioactivity decorporation agent for radiothallium (201Tl) in internally contaminated BALB/c mice and New Zealand White rabbits using radiometry and gamma scintigraphy.
Methods:
Animals were divided into three groups, that is, control, pretreatment-1 (1X LNP), and pretreatment-2 (2X LNP). Mice received LNP intraperitoneally, while in rabbits LNP was given through oral route 0.5 and 1.5 h before radiothallium administration, respectively. Mice and rabbits used in the experiment were administered 1.48 and 11.1 MBq radiothallium (201TlCl) through intravenous and oral route, respectively. Once started as prophylactic, LNP was continued as therapeutic twice a day till the end of study period. Radiometry and gamma scintigraphy were used to monitor radiothallium retention and uptake patterns in animals. Gamma scintigraphic images of rabbits were taken at different time intervals up to 72 h and were analyzed for comparative uptake pattern of 201TlCl in all the groups.
Results:
LNP treatment significantly increased the 201Tl elimination over untreated control and considerably reduced the retention of 201Tl in various tissues and organs. Decrease in radiothallium uptake up to 40% was observed in LNP-treated mice as compared to untreated control. While in rabbits, whole-body radioactivity burden at 72 h was found to be 31.24%, 26%, and 18.54% in untreated control, 9 and 18 mg/kg LNP-treated groups, respectively.
Conclusion:
LNP exhibited dose-dependent decorporation potential to effectively enhance the elimination of 201Tl in mice and rabbits experimentally contaminated with 201TlCl.
Introduction
Radioactive contamination may occur due to a number of scenarios such as those related to terrorist attack using radioactive dirty devices, 1 accidents during radionuclide transport, 2 and even in case of incidents involving the use of radioactivity in medicine, industry, and research. 3 Graveness of such accidents involving spillage or dispersal of radioactive material depends on the physicochemical property of radionuclide and its behavior 4,5 in living organism once internalized through any route. Typically, after radioactive contamination or radiation exposures, there are no immediate acute health effects, but in the long run leukemia, genetic defects, malignancies, and teratogenic abnormality may arise as stochastic effects.
Besides inhalation, ingestion is a major route for entry of radionuclides into the human body in case of any radioactive fallout scenario. 6 Radiocesium (137Cs) is a fission product of various isotopes of plutonium, thorium, and uranium with a yield of about 6%. 7 Radiocesium that is abundantly found in spent nuclear fuels, used as fission byproduct after nuclear accidents, and usually present in nuclear fallouts 8 has an intermediate half-life, that is, more than 30 years. Biological half-life of radiocaesium is 70 days, which is a concern requiring exploration of approaches to eliminate it faster from biological system to minimize the risk from radiation. Radiocesium poses significant health hazard once absorbed in body and may lead to deterministic and stochastic effects depending on the amount incorporated.
Radiocesium (137Cs) and its chemical analog radiothallium (201Tl) mimic physiological functioning of potassium ion and therefore can get readily absorbed and easily incorporated into all living cells, particularly in muscle and cardiac cells. 9,10
Once radionuclides are internalized, they may reside in tissues and organs for an extended period of time depending on their respective half-lives. 137Cs and 201Tl ingested during any nuclear accident get absorbed through active process involving hydrogen/potassium adenosine triphosphatase (H+/K+ ATPase) and through passive diffusion as well. 11 Biokinetics of cesium has been studied in detail and it is well-known that ingested cesium rapidly disperses throughout the 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. 12 Cesium particularly accumulates in the skeletal muscle, because of its large influx/efflux rate.
Presently, oral administration of Prussian blue (PB) is the main treatment for decorporation of radiocesium from the body. 13,14 Radiogardase is the only Food and Drug Administration (FDA)-approved formulation of PB for treatment of radiocesium contamination or poisoning, availability of which is a major concern besides its high cost. Therefore, there is a need to look out for alternate readily available and inexpensive drug or approach to decorporate radiocesium and other similarly behaving monovalent ions.
One of the possible strategies could be the use of proton pump inhibitors (PPIs), a class of drugs that are among the most frequently prescribed FDA-approved medicines (Table 1) with their widespread use in primary and secondary level health care. Among this list of PPIs, lansoprazole (LNP) is a primary drug of choice prescribed by physicians worldwide for management of gastroesophageal reflux disorders, peptic ulcers, and other related health disorders. 15 It is hypothesized that H+/K+ ATPase inhibitors may have some role in the metabolism of accidentally incorporated radionuclides, especially monovalent cations like 137Cs and 201Tl.
FDA-approved indications of the PPIs.
FDA: Food and Drug Administration; PPI: proton pump inhibitor; NSAID: Nonsteroidal anti-inflammatory drug.
LNP hampers gastric acid secretion by inhibiting proton pumps, that is, H+/K+-ATPase of gastric mucosa. 16 H+/K+ ATPase is an enzyme placed in the membrane of gastric parietal cell that executes the final step in acid secretion. LNP binds covalently to parietal cell H+/K(+)-ATPase and makes it nonfunctional resulting in the inhibition of gastric acid secretion, thereby impeding the exchange of H+ and K+. 17 LNP is more potent than omeprazole and other PPIs as it directly inhibits Cytochrome P450 2C19 (CYP2C19); however, LNP does not cause inhibition of CYP2C19 18 to the level which is clinically significant as in case of omeprazole. PPIs influence drug absorption and metabolism by interacting with adenosine triphosphate–dependent P-glycoprotein or with the cytochrome P450 enzyme system, thereby distressing both gastrointestinal (GI) first-pass metabolism and hepatic clearance. 19
This article studied the effect of oral LNP intervention on the uptake and retention patterns of radiothallium in experimental animals. Radioactive thallium (201Tl; half-life; 73.1 h) is a radiopharmaceutical commonly used for myocardial scintigraphy in nuclear medicine. Thallium shares the biodistribution profile and medical management strategies with cesium, a chemical analog. 20,21 Easy availability of radiothallium and its relatively short half-life makes it a radionuclide of choice to plan and conduct in vitro and in vivo animal studies as a representation for radioactive cesium (137Cs).
Material and methods
Material
Lansoprazole
LNP (Figure 1) was obtained as a gift sample from M/s Ranbaxy India Pvt. Ltd, Mumbai, Maharashtra, India, as a dry powder and was dissolved in 10% dimethyl sulfoxide (DMSO) and dose adjusted to appropriate concentration before intraperitoneal administration in mice models. LNP granules were collected from LAN-30 capsules (Intas Pharmaceuticals Ltd, Ahmedabad, Gujrat, India) purchased from market for oral administration in New Zealand White rabbits. All other chemicals used were of reagent grade.
Structure of lansoprazole (formula: C16H14F3N3O2 S; molar mass 369.363 g/mol).
Radiothallium
Radiothallium (201Tl) was obtained as thallous chloride (201TlCl) solution from Regional Centre for Radiopharmaceuticals, Board of Radiation and Isotope Technology (BRIT), India, at Institute of Nuclear Medicine & Allied Sciences, Delhi.
Equipment
Dose calibrator (CRC15 R; Capintec Inc., Florham Park, NJ, USA) for radiometry, gamma camera (Symbia TruePoint; Siemens, Munich, Germany) for gamma scintigraphy, and image processing software (Syngo; Siemens, Munich, Germany) were used for the study. A statistical software package (PASW Statistics 18) was used for analysis of the study data.
Method
Two animal species, that is, BALB/c mice and New Zealand White rabbits, were chosen to carry out the designed study. Radiothallium biodistribution study was performed in BALB/c mice, whereas the efficacy study of LNP for radiothallium decorporation was done in both mice and rabbit models using radiometry and gamma scintigraphy technique. Procedural details are as described below:
201TlCl biodistribution and efficacy study in BALB/c mice
Animal selection and preparation. Healthy male BALB/c mice with average weight 32 ± 2 g were taken for the study and were obtained from Central Animal House Facility of Institute of Nuclear Medicine & Allied Sciences (INMAS), DRDO, Delhi, India. The study was initiated after receiving approval from the duly constituted Institutional Animal Ethical Committee. Animals were acclimatized to laboratory conditions for 1 week prior to the study. They were housed in stainless steel cages to the normal diurnal and nocturnal rhythms. The test room was air-conditioned with temperature of 23 ± 2°C and humidity of 65 ± 5%. They were given free access to standard laboratory animal feed (Golden Feed, Delhi, India) and water ad libitum. All efforts were made to minimize the number of animals and their suffering during the conduct of experiments.
Preparation of drug solution
LNP active pharmaceutical ingredient was dissolved in 10% DMSO and the concentration was adjusted to achieve 3 and 6 mg/200 μl, respectively, for intraperitoneal administration.
Procedure
Biodistribution of radiothallium was carried out in male BALB/c mice (n = 72). Animals were divided into three groups consisting of 24 mice in each, that is, groups I, II, and III. Each group was further subdivided into eight subgroups consisting of three mice each to be euthanized at different time points, that is, 15, 30, 45, 60, 90, 120, 150, and 180 min, respectively. Radiothallium solution was prepared and diluted using normal saline and pH was adjusted to 7.0. The concentration of radiothallium in the solution was adjusted to achieve the desired dose, that is, 1.48 MBq in 100 µl injected volume and injected via tail vein in all the three groups.
While group I animals served as control, groups II and III animals received 3 mg (1X) and 6 mg (2X) LNP intraperitoneally 0.5 h prior to intravenous radiothallium.
After intravenous administration of radiothallium, mice of each subgroup were euthanized at respective time points. Liver, heart, and muscles of hind limb were removed carefully and washed in chilled normal saline solution, weighed, and dried in a hot air oven for 5 min at 50°C to remove any extra water.
Radiometric analysis
Radioactive counts (kcps) in the removed organs/tissues were taken using gamma counter (Capintec Inc., Florham Park, NJ, USA. Radioactive counts in the tail were also taken for the purpose of quality control to validate the experimental procedure. Mice with more than 10% of total radioactivity count in their tail were rejected and replaced in the experiment. Results were expressed in terms of percentage of radiothallium incorporation in target tissues at different time points.
Gamma scintigraphic efficacy evaluation in New Zealand White rabbits
Animal selection and preparation
The experiments were conducted on female New Zealand White rabbits weighing approximately 3.5 ± 0.1 kg. The animals were obtained from the experimental animal facility of Institute of Nuclear Medicine & Allied Sciences (INMAS), Delhi. All animal experiments were approved by the Institutional Animal Ethical Committee and confirmed to the general national guidelines on the care and use of laboratory animals. The animals were maintained at controlled temperature and hygienic conditions and were provided with food and drinking water ad libitum. They were housed individually in cages and maintained on a 12-h day and night cycle. Animals were allowed to acclimatize for at least 1 week before starting the experiment.
Preparation of drug solution
LNP granules (9 mg equivalent) were suspended in 5 ml double-distilled water. The suspension was prepared freshly at the time of dosing to the animals of both treatment groups.
Procedure
To explore the gastric absorption of radiothallium and study the efficacy of LNP for the decorporation of radiothallium, New Zealand White rabbits (n = 18) were divided into three groups, that is, control, pretreatment-1, and pretreatment-2. The treatment given to each group is mentioned as below:
Control: Animals of the control group were left without any treatment and were given 11.1 MBq 201TlCl through oral gavage.
Pretreatment-1: Animals of the pretreatment-1 group were given 9 mg LNP (1X) in distilled water through oral gavage 90 min prior to oral administration of 11.1 MBq 201TlCl.
Pretreatment-2: Animals of the pretreatment-2 group were given 18 mg LNP (2X) in distilled water through oral gavage 90 min prior to oral administration of 11.1 MBq 201TlCl.
Gamma scintigraphic static images were taken after setting the camera parameters with respect to radioactivity used, that is, radiothallium (field size 256 × 256; 201Tl-NMG, Zoom-1.0, 300 s) to obtain scintigraphic images of higher resolution. Whole-body radiothallium retention: At different time points, whole-body imaging of control and test animal was done. In order to determine the whole-body retention of radiothallium, whole body of the animal was considered as region of interest (ROI) and comparative analysis of radiothallium retention was done after processing through inbuilt software “syngo” (Siemens, Erlangen, Germany). Bioavailability of radiothallium in soft tissue: Region of interests were drawn on the chosen muscle to determine radiothallium uptake at different time points in control and test groups. Analysis of data obtained was done using inbuilt software. Radiothallium retention in whole abdomen: Whole abdomen was taken as ROI to calculate the radioactive counts at various predefined time points in control and test animals. Radioactive counts were analyzed and recorded after processing. Radiothallium retention in stomach: Stomach of the animals was clearly identifiable and has been chosen as ROI to analyze radiothallium retention at different time points. Radioactive counts obtained were recorded and analyzed to calculate radiothallium retention in the stomach with respect to time. Radiothallium retention in lower abdomen part: Lower abdomen excluding kidneys was taken as ROI and radiothallium retention/increase in concentration was compared at different time intervals in control and test groups of animals.
In vitro blood radiometry
Rabbits were placed in individual restrainers. Ear was cleaned with 95% v/v ethanol. A polyethylene catheter was inserted into the marginal ear vein of left pinna of each rabbit for collection of blood samples. Blood samples (1 ml) were collected through indwelling vein catheter into heparinized vacutainer tubes at different time intervals, that is, before injecting radiothallium (201Tl), and postinjection at 10 min, 120 min, 360 min, 24 h, and 72 h. Radiometric counts were taken using gamma counter (Capintec) to determine the real-time radiothallium counts present in per milliliter blood volume.
In vitro urine radiometry
Rabbits were housed individually in stainless steel metabolic cages for collection of urine. Urine samples were collected at time intervals, that is, 24 and 72 h. Individual urine volumes were recorded at each collection. Radioactive counts in per milliliter collected urine were taken using gamma counter (Capintec) to calculate the cumulative radioactivity excreted out. Data obtained were compared to analyze the effect of LNP intervention on the concentration of radiothallium in urine.
Data analysis
Region of interests were drawn on the anticipated areas of animal body, that is, whole body, stomach, whole abdomen, and muscle count on thigh muscles, in order to obtain real-time radioactive counts per second. Data analysis was done using inbuilt software of the gamma camera (Syngope, Siemens).
Statistical analysis
Mean values were determined and error bars were calculated from the standard deviations. All data were presented as mean ± standard deviation. Data were statistically analyzed using one-way ANOVA and Student’s t-test applied for comparison between groups. Evaluations were made using the statistical software, and the results were considered statistically significant at 95% CI (p < 0.05).
Results
201TlCl biodistribution and efficacy study in BALB/c mice
Time–activity curve for 201TlCl (1.48 MBq) in each mouse was drawn for its uptake/retention in heart, liver, and limb muscles at different time points (Figure 2). In both control and treatment group animals, radiothallium uptake in the cardiac tissue started swiftly; however, differences in the uptake pattern were noted at different time points later. Similar pattern was followed by liver. In muscles, radiothallium uptake was higher at 30 min and thereafter started reducing. In LNP-treated groups 1X and 2X, a reduction in radiothallium uptake by 28% and 40%, respectively, was noted as compared to untreated control at the end of the study.
Radiothallium biodistribution in untreated control, 1X LNP, and 2X LNP–treated BALB/c mice.
Gamma scintigraphic efficacy evaluation in New Zealand White rabbits
Whole-body radiothallium retention
Gamma scintigraphic images (Figure 3) captured at different time intervals in control, test 1 (1X LNP), and test 2 (2X LNP) groups of animals up to 72 h were analyzed for radioactive counts depicting radiothallium uptake by considering whole body as ROI. Radioactive counts of the ROIs were compared to demonstrate the effect of LNP intervention (Figure 4). The radioactive counts per minute observed clearly depicted a reduction in whole-body radioactive burden with time in test groups as compared to control. Whole-body radioactivity burden in the control group of animals was found to be 31.24% at 72 h, whereas in animals of test 1 (1X LNP) and test 2 (2X LNP) groups, the radioactive body burden was observed to be 26.08% and 18.54%, respectively, in contrast to the radioactivity injected at the beginning of the experiment. LNP (2X) showed a significant reduction in whole-body radioactivity burden as compared to LNP (1X) and control groups.
Gamma scintigraphic images of New Zealand White rabbits showing the effect of lansoprazole administration on radiothallium uptake in untreated controls and treatment groups (11.1 MBq 201TlCl was given orally in all groups and gamma scintigraphic images were taken at different time intervals, that is, 0.5, 2, 6, 24, and 72 h).
Whole-body radiothallium (201TlCl) retention.
Bioavailability of radiothallium in soft tissue
A small ROI was drawn on the thigh muscle of the hind limb on the scintigraphic images of animals taken at different time intervals ranging from 0.5 h to 72 h. Radioactive count of that particular ROI was taken into consideration and was compared at different time points among control and test groups (Figure 5). A significant reduction in radiothallium uptake was observed in the test group of animals when compared with control. Radiothallium bioavailability in terms of radioactive count in ROI was found to be 0.58% in control, whereas in test 1 (1X LNP) and test 2 (2X LNP) groups it was noted to be 0.32% and 0.15% only. As visualized in the graph, we can see the pattern of slower uptake of radiothallium in soft tissues, that is, muscles of animals of the treated group when compared with control.
Radiothallium (201TlCl) uptake in soft tissues at different time intervals.
Radiothallium retention in whole abdomen
Identical ROIs on the whole abdomen including the stomach, liver, gallbladder, spleen, pancreas, kidneys, and upper intestinal parts were drawn on control, test 1, and test 2 groups of animals (Figure 6). Radioactivity burden in the whole abdomen was observed over a period of different time intervals.
Whole abdomen radiothallium (201TlCl) retention.
Radiothallium retention in stomach
ROIs were drawn on the stomach, which was visible throughout the gamma scintigraphic study and radioactive counts were noted from each group. Data obtained were analyzed and compared (Figure 7) for the radiothallium uptake/retention pattern in the stomach. In the control group of animals, radiothallium count was high up to 30 min and thereafter started decreasing at 6 h and only residual amount of radioactivity remained at 24 h, which clearly depicted fair absorption of radiothallium in the control group, resulting in high radioactivity body burden. However, in pretreatment groups 1 and 2, radiothallium radioactive counts were found to be stable up to 24 h, indicating entrapment of radiothallium in H+/K ATPases due to their inhibition by LNP resulting in less whole-body absorption. At 72 h, radioactivity in the stomach was minimal in both the treatment groups (1X and 2X LNP) with low radioactivity body burden suggestive of removal of excess radiothallium with time from the stomach through food transiting in the GI system.
Radiothallium (201TlCl) retention in the stomach.
In vitro blood radiometry
At 24 and 72 h, a notable difference in radiothallium concentration between both treated groups can be observed in comparison with control and among themselves (Figure 8). 2X LNP–treated animals were left with one-third of radiothallium concentration in 1X LNP–treated animals at 24 h. Radiothallium concentrations at 72 h were found to be 4.97%, 3.75%, and 1.86% in control, 1X, and 2X LNP–treated groups, respectively.
Radiothallium (201TlCl) in blood at different time intervals.
In vitro urine radiometry
Noteworthy distinction was observed in radioactivity counts per milliliter of urine at 24 and 72 h in radiothallium concentration between control and treatment groups. Radioactive counts were found to be 0.44, 0.67, and 1.16 MBq per ml of excreted urine at 72 h in control, 1X, and 2X LNP–treated groups, respectively. The data obtained are indicative of faster renal excretion of radiothallium in LNP-treated groups as compared to control (Figure 9).
Radiothallium (201TlCl) in urine at different time intervals.
Discussion
This study was undertaken to evaluate the potential of using LNP, a H+/K+ ATPase inhibitor, as decorporation agent for prevention and inhibition of radiothallium absorption in vivo using gamma scintigraphy and radiometry (Pictorial summary of study; appendix ‘I’). Gamma scintigraphy is a proven nuclear medicine technique for evaluation of various pharmaceutical preparations and now a widely accepted modality being considered in drug development. 22,23 LNP inhibits absorption of radiothallium by inhibiting H+/K+ ATPase and interrupting H+/K+ transport mechanism, and thereby not allowing radiothallium, an analog of potassium ion, to get absorbed from gastric lumen. 24
Radiothallium being a chemical analog of potassium ion absorbs fairly through the GI system using both active and passive routes. 16,17 Active absorption is mediated by H+/K+ ATPases, which are biodistributed throughout the GI system including the stomach and lower parts of the intestine. Radiothallium absorption is >95% when administered orally without any intervention, which is clearly evident from our results. However, LNP intervention resulted in poor absorption of radiothallium in test group of animals, probably due to obstruction in H+/K+ ATPase pathway, although no attempt was made in this study to examine the molecular mechanisms behind this observation.
Since animals of all groups were given a fixed dose of radiotracer, that is, radiothallium (201TlCl), counts of radioactivity considering same decay factors are comparable between control and treatment groups. In animals of the control group, it was evident from gamma scintigraphic images that radiothallium absorption from the initial parts of the GI system, that is, the stomach, was faster in comparison with LNP-treated groups. This could be due to the fact that in case of LNP-treated animals, proton pumps (H+/K+ ATPases) were blocked because of LNP intervention with some amount of the radiotracer still entrapped in inhibited proton pumps. On the other hand, in case of control animals, there was no such blocking of the proton pumps and radiothallium could easily get absorbed through the stomach and other GI regions.
It is notable while observing the GI retention of radiothallium that in case of control animals, radiothallium was fairly distributed throughout the GI system at 2 h, whereas in case of 1X LNP and 2X LNP groups, similar distribution of radiothallium in the GI system was visible at 6 h and 24 h, respectively. This shows that LNP treatment could limit the absorption of radiothallium in the GI tract at the designated doses (9 mg and 18 mg) in a concentration-dependent manner as compared to control at the same time points. The reason behind this phenomenon could possibly be the inhibition of H+/K+ ATPases present in parietal cells located in the inner cell linings of the stomach. It may be noted that the gastric mucosa in particular is abundantly lined with the epithelial cells responsible for H+ and K+ exchange 25 to facilitate gastric juice production.
It was also evident from gamma scintigraphy images that LNP treatment resulted in delayed uptake of radiothallium in other organs and tissues as compared to that in control animals. Radiothallium is known to absorb quickly and get distributed in the body with particularly more affinity to muscle cells including cardiac tissues. As seen from scintigraphy images, radiotracer uptake in blood pool and muscles was found to be slow as compared to control animals at similar time points.
Blood radiometry at different time points of the study also depicts the effect of LNP intervention in the treatment group as compared to control up to 72 h. Poor absorption of radiothallium and enhanced excretion through the renal system resulted in reduced systemic absorption of radiothallium in treatment group animals. Moreover, low concentration of radiothallium in blood circulation further led to reduced radionuclide uptake in the tissues of LNP-treated animals.
LNP also acts on H+/K+ ATPases situated on the renal medulla, which are responsible for reabsorption of potassium and thallium. Renal H+/K+-ATPase secretes H+ and recycles K+ via apical K+ channels and reabsorbs K+ via basolateral K+ channels during K+ depletion. Our results indicate that inhibition of renal H+/K+ ATPases probably hindered reabsorption of radiothallium ions leading to excess secretion in urine, as seen in the animals of LNP-treated groups. LNP may prove to be an effective and economic alternate to “radiogardase” (PB), the only proven and globally acclaimed radiocesium antidote. Moreover, easy availability, cost-effectiveness, and already established use of LNP as an approved drug for another indication are few of the other reasons, which may encourage further research in developing LNP as an alternative or adjunctive therapy to currently available treatment options for radiocesium poisoning.
Conclusion
LNP exhibited dose-dependent decorporation potential to effectively enhance the elimination of 201Tl in mice and rabbits experimentally contaminated with 201TlCl.
LNP, by inhibiting H+/K+ ATPases and interfering with absorption, secretion, and/or reabsorption of radiothallium in the body system, has showed a significant potential for enhanced elimination of radiothallium from the body. LNP, therefore, could be considered as a potential decorporation agent against radiothallium (201Tl) and more importantly by implication for its chemical analog radiocesium (137Cs).
Footnotes
Acknowledgement
The authors of the manuscript are thankful to the Director of Institute of Nuclear Medicine & Allied Sciences, Delhi, for their continuous support and guidance throughout the study.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Defence Research and Development Organization, Delhi, India (grant no. NBC 1.30, 2010).