Distribution of [14C]clodronate (dichloromethylene bisphosphonate) disodium in mice

TOXICOLOGY

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APPLIED

Distribution

Distribution

PHARMACOLOGY

(1987)

89,287-292

of [14C]Clodronate (Dichloromethylene Disodium in Mice

Bisphosphonate)

(Dichloromethylene Bisphosphonate) Disodium in Mice. H. A., AND AIRAKSINEN, M. M. (1987). Toxicol. Appl. Pharmocol. 89, 287-292. The distribution of 14C-labeled clodronate (dichloromethylene bisphosphonate), a new bisphosphonate for the treatment of osteolytic bone metastases and hypercalcemia, was studied in mice by whole-body autoradiogtaphy and by measuring the 14Cactivities in various tissues. [14C]Clodronatc was administered into the tail vein, and its distribution was followed from 5 min to 90 days after the injection. The drug disappeared promptly from the plasma and accumulated intensively in the bone and moderately in the spleen. In both tissues, relatively high radioactivities were measured as late as 90 days after the [‘4C]clodronate administration. Small amounts of 14Cactivity were also detected in the liver for 90 days. The results agree well with the previous observations that bisphosphonates deposit rapidly in the bone. Our findings indicate further that clodronate accumulates in the bone and the spleen for several months. Q 1987 Academic PBS, IIIC. M~NKK~NEN,

of [‘4C]Clodronate

J., YLITALO,

P., ELO,

The change of calcium metabolism in pathological conditions is a considerable problem in medicine, clearly shown by osteolytic bone metastases, hyperparathyroidism, and Paget’s disease. In the terminal stage of cancer with osteolytic bone metastases, hypercalcemia seems to be a major cause of death. Until recently, it has not been possible to treat hypercalcemia and bone resorption adequately, since there have been no suitable medications. Bisphosphonates, previously called diphosphonates, are a class of synthetic organic compounds effective in the treatment of the above-mentioned diseases (Fleisch, 1983). These compounds seem to block the growth and dissolution of hydroxyapatite crystals in the same way the pyrophosphates do in vitro. The bisphosphonates prevent or inhibit bone resorption either by binding to hydroxyapatite in such a way that the osteoclasts cannot split the crystals or by affecting the metabolism of osteoclasts (Fleisch and Felix, 1979). Most of the kinetic data on bisphosphonates have been collected with etidronate (hydroxyethylidene bisphosphonate, HEBP). The pharmacokinetics of clodronate (dichloromethylene bisphosphonate, ClzMBP), a

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newer bisphosphonate, is less well-known. Approximately half of the etidronate absorbed from the gastrointestinal tract enters bone, and the remaining half is excreted unchanged in the urine (Michael et al., 1972). Only minimal amounts of the drug enter the soft tissues (Michael et al., 1972; Fleisch, 1983). The half-life of etidronate in the bone of young rats is longer than 4 months (Francis and Martodam, 1983), while that of clodronate is unknown. In the present study, the distribution and deposition of [ “C]clodronate were studied in mice by whole-body autoradiography and by measuring the 14C activities in various tissues. METHODS Animals. Male and female NMRI mice (National Laboratory Animal Center, Kuopio, Finland), 90- 120 days of age and weighing 25-40 g at the time of drug administration, were used for whole-body autoradiography while only males were used for counting the radioactivities in various tissues. The animals were fed a standard pellet diet and had free accessto water. The temperature in the animal room was 20 + 0.5”C, and the relative humidity was 50%. Experiments. Whole-body autoradiography was performed according to the method described by Ullberg

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FIG. 1. Autoradiographs of mice (A) 5 min, (B) 8 h, (C) 7 days and (D) 2 1 days after the intravenous injection of [‘4C]clodronate (5 &i/4.7 17 mg/mouse). The abbreviations used are b, bone; g, gut; k, kidney; Ii, liver; lu, lung: r, rib; s, skin; sk, skull; sp, spleen; st, stomach; and v, vertebra. 1.6-fold enlargements are shown.

(1977). A single dose (5 &i/4.717 mg/mouse) of [‘4C]clodronate (sp act 1.06 pCi/mg; Oy Star Ab, Pharmaceutical Co., Tampem, Finland) was injected into the tail vein. Five and thirty minutes, 28, and 24 hr, and 3, 7, and 2 1 days after the dosage, four animals of each time group were frozen in liquid nitrogen, embedded in carboxymethyl cellulose, and cut into 20-pm-thick sections by a microtome (PVM, Cryo-Microtome, Type 45OP, LKB, Stockholm, Sweden). The tape-bearing sections were then freeze-dried for 1 day at -2o’C. About 10 sections from each animal were placed on the film (Kodak X-Omat AR Film, Eastman Kodak Co., Rochester, NV) and exposed for 12 weeks at -20°C. The fdms and photographs were developed by conventional dark-room procedures.

For the liquid scintillation counting, the mice received [‘4C]clodronate intravenously (iv), 1 &i/O.943 mg/animal. Five and thirty minutes, 2, 8, and 24 hr, and 3, 2 1, and 90 days after the label administration, the animals were anesthetized with ether, and blood samples for the separation of plasma were taken into heparinized syringes by cardiac puncture. There were four animals in each time group. The samples of various organs or tissues (skin, lung, liver, kidney, spleen, testis, muscle, femur + tibia, and brain) were removed and stored at -2o’C until their radioactivities were counted. The bone samples were treated according to Mahin and Lotberg (1966). Each sample was dissolved in 0.2 ml of 70% perchloric acid and 0.4 ml of 30% hydrogen peroxid at 75°C. The sample preparation was completed

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by addition of 6 ml of Cellosolve (E. Merck, Darmstadt, PRG) and 10 ml of 2,5diphenyloxaxole (PPO)-toluene (6 g/liter). Other tissues were prepared according to the routine procedure using a Lumasolve tissue solubilizer and Lipoluma scintillation solution (Lumac/3M B.V., Schaesberg, The Netherlands). The radioactivity was counted by a liquid scintillation counter (LKB Wallac, 12 15 Rackbeta, LKB, Stockholm, Sweden) using the external standardization. The counting efficiency varied from 70 to 90% for soft tissues and from 65 to 75% for bone samples.

RESULTS

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DISCUSSION

Whole-body autoradiographs showed that the distribution pattern of [ “C]clodronate

was similar both in male and in female mice in each time group. Five minutes after the iv injection, high levels of radioactivity were present in the bone and the kidney (Fig. 1A). In the lung, skin, and gut, 14C activities were also relatively high. At 30 min, autoradiographs still showed high radioactivities in the bone and the internal parts of the kidney; radioactivities in the cortical parts of the kidney and in the liver were lower. In other tissues, the radioactivity was undetectable. At 2, 8 (Fig. IB), and 24 hr and 3 days, the radioactivity was distributed into bone, kidney, liver, and spleen. The intestinal and stomach contents also showed some radioactivity. In the

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FIG. 2. The ‘%Z activities in various tissues of mice at given times after the intravenous injection of [ 14C]clodronate ( 1 &i/O.943 mg/mouse). The means k SD of four animals are given. SD is indicated when it exceeds the size of the symbols.

bone, the radioactivity was high at all these The radioactivities (dpm/g of tissue) of times. In the kidney, the radioactivity was [ 14C]clodronate in various organs at different lower compared to autoradiographs taken at times are illustrated in Fig. 2. After 5 min, 5 and 30 min. At 7 and 2 1 days (Figs. 1C and [‘4C]clodronate concentrated most intenlD), the radioactivity in the bone was still sively in the kidney. In the lung and the skin, very high. In the kidney it was hardly detect- the 14C activity was also relatively high at 5 able, while in the the spleen and the liver the min, but after 2 hr it had already decreased to labeling remained for at least 7-21 days. In almost undetectable levels. In the spleen, the the spleen, the radioactivity was somewhat 14C activity reached the top level more slowly higher than in the liver. In the spleen and the than in the other tissues, and it remained relaliver, the radioactivity showed no homogetively high until the end of the 90day obsernous distribution pattern. In the liver, the dis- vation period. In the liver, the radioactivity tribution was spot-like, and the organ con- also remained detectable for 90 days. Comtained small areas relatively free of radioacpared to plasma and the other tissues, the tivity. In the spleen, the radioactivity was bone (tibia + femur) showed very high radioconcentrated at the border between the white activities at all times. As soon as 5 min after and red pulps (Fig. 1C). the administration of [‘4C]clodronate, the

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bone radioactivity was high, and it reached the peak value at 30 min decreasing only very slowly during the course of the 9Oday experiment. In the muscle, testis, and brain, the 14C activity was minimal at all times. These results indicate that [ 14C]clodronate disappears rapidly from the plasma, is incorporated rapidly and strongly into the bone, and accumulates also in the spleen and to a lesser extent in the liver. Since clodronate is not metabolized in mammals (Fleisch, 1983), the radioactivities are considered to indicate the unchanged drug. Our results agree well with previous knowledge about the high alhnity of bisphosphonates to deposit onto ossified tissue, resulting in their rapid clearance from the blood and incorporation into the bone. In young rats, the half-life of etidronate in the bone is longer than 4 months (Francis and Martodam, 1983) and in older animals it is correlated with the metabolic activity of the bone tissue (Fleisch, 1980). In the present study, the halflife of clodronate in the bone could not be exactly determined, but it seems to be at least 3 months. The peak level of etidronate in the bone is reached within 1 hr (Bisaz et al., 1978). According to our results, the peak level of clodronate is reached within 30 min. The high concentration of [‘4C]clodronate in the kidney immediately after administration and the disappearance of radioactivity from there agree well with the fact that unincorporated bisphosphonates are excreted rapidly and unchanged via the kidneys (see Fleisch, 1983). The strong accumulation of [‘4C]clodronate in the spleen for several months is a new and interesting discovery. No corresponding findings have been published for any of the bisphosphonates. Rohlin (1976) and Rohlin and Nosslin ( 1977) have reported on the up take of 9g”Tc-labeled etidronate into the spleen and liver of rats. They suggested that the accumulation was due to the presence of 99mTc-Sn colloid which is hydrolyzed from ‘“Tc-etidronate i;? vivo. However, the col-

loid formation cannot be the reason for the accumulation of [ “C]clodronate. It has been established that local high concentrations of extracellular iron will result in abnormal uptake and retention of 99mTc-labeled etidronate in soft tissues in the absence of elevated calcium levels, and the affinity of the pure carrier molecule to iron may be even higher than that of 99”Tc-labeled compound (Jones et al., 1976). We are now studying whether the interaction of clodronate with iron is involved in the accumulation of the drug in the spleen. ACKNOWLEDGMENTS The study was financially supported by Oy Star Ab, Pharmaceutical Co., Tampere, Finland. Our thanks are also due to Dr. Eeva-Liisa Sainio for her valuable advice in preparing autoradiographs.

REFERENCES BISAZ, S., JUNG, A., AND FLEISCH, H. (1978). Uptake by bone of pyrophosphate, diphosphonates and their technetium derivatives. Clin. Sci. Mol. Med. 54,265272. FLEISCH, H. (1980). Experimental basis for the clinical use of diphosphonates in Paget’s disease of bone. Arthritis Rheum. 23,1162- 117 1. FLEIXH, H. (1983). Bisphosphonates: Mechanisms of action and clinical application. In Bone and Mineral Research. (W. A. Peck, Ed.), Annual 1. Excerpta Medica, Amsterdam. ~EISCH, H., AND FELIX, R. (1979). Diphosphonates. Calcif: Tissue Int. 27,9 l-94. FRANCIS, M.D., ANDMARTODAM, R.R.(1983).Chemical, biochemical, and medicinal properties of the diphosphonates. In The Role of Phosphonates in Living Systems (R. L. Hilderbrand, Ed.), p. 68. CRC Press, Boca Raton, FL. JONES,A. G., FRANCIS,M. D., AND DAVIS, M. A. ( 1976). Bone scanning: Radionuclidic reaction mechanisms. Semin. Nucl. Med. 6,3- 18. IMAHIN, D. T., AND LOFBERG, R. T. (1966). A simplified method of sample preparation for determination of tritium, carbon-14 or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. B&hem. 16, 500509. MICHAEL, W. R., KING, W. R., AND WAKIM J. M. ( 1972). Metabolism of disodium ethane- 1-hydroxy-

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l,l-diphosphonate (disodium etidronate) in the rat, rabbit, dog, and monkey. Toxicol. Appl. Pharmacol. 21,503-515. ROHLIN, M.

(1976). Whole-body autoradiography of 99mTc-labeled ethylene- 1-hydroxy- I, 1-diphosphonate (EHDP) in young rats. Acta Radio/. Ther. Phys. Biol. 15,410-416. ROHLIN, M., AND NOSSLIN, B. (1977). Quantitative analysis ofthe distribution of 99mTc-labelled ethylene hydroxy diphosphonate in young rats. Eur. J. Nucl. &fed.2,113-115. ULLBERG, S. (1977). The technique of whole-body autoradiography. Cryosectioning of large specimens. Sci-

ence Tools, The LM3 Instrument Journal, Special Issue on Whole-Body Autoradiography-1977,2-29. PAULI YLITALO HEIRRI A. ELO MAUNO M. AIRARSINEN Department of Pharmacology and Toxicology University of Kuopio P.O.B. 6 SF-70211 Kuopio Finand Received August 6, 1986