Metabolism of disodium ethane-1-hydroxy-1,1-diphosphonate (disodium etidronate) in the rat, rabbit, dog and monkey

TOXICOLOGY

AND APPLIED PHARMACOLOGY

Metabolism (Disodium

of Disodium Etidronate)

21, 503-515

(1972)

Ethane-I-Hydroxy-I,I-Diphosphonate in the Rat, Rabbit, Dog and

Monkey

WILLIAM R. MICHAEL, WILLIAM R. KING, AND J. M. WAKIM’ The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 39175, Cincinnati, Ohio 45239 Received June 4,197l

Metabolism of Disodium Ethane-l-Hydroxy-1,1-Diphosphonate (Disodium Etidronate) in the Rat, Rabbit, Dog and Monkey. MICHAEL, WILLIAM R., KING, WILLIAM R., and WAKIM, J. M. (1972). Toxicol Appl. Pharmacol. 21, 503-515. Absorption of disodium ethane-l-hydroxy-l,ldiphosphonate(disodiumetidronate) from the gastrointestinaltract of the rat, rabbit and monkey after po intubation was lO%. Absorption wasprimarily from the stomach.Weanling rats and young dogsabsorbedmoreof the compoundthan adultsof the samespecies.Disodiumetidronate wasnot metabolizedby the rat or dog, and there was virtually no enterohepaticcirculation of the compound in the rat. Approximately half of the absorbed disodium etidronate was excretedin the urine, while the remaininghalf wasdepositedin the skeleton. The half-life of disodiumetidronate in the skeletonof the rat was 12 days following po administration. INTRODUCTION Disodium ethane-I-hydroxy-l,l-diphosphonate (disodium etidronate)2 is 1 of a group of geminal diphosphonates that have a marked effect on calcium hydroxyapatite crystal growth and dissolution in vitro (Francis, 1969; Fleisch et al., 1968) and calciumphosphate metabolism in vitro and in oiuo (Fleisch et al., 1969a,b; Francis et al., 1969; Jowsey et al., 1970; King et al., 1971; Michael et al., 1971; Russellet al., 1969; Bassett et al., 1969). The importance of metabolic and biochemical studies as part of the overall safety evaluation of drugs and food additives has been emphasized by the Joint FAO/WHO Expert Committee on Food Additives (1967). The present metabolic study in several speciesis one part of the total safety evaluation of disodium etidronate in light of its intended usein a dentifrice as an anticalculus agent and as a drug to regulate calciumphosphate metabolism. METHODS Disodium ethane-1-hydroxy- 1,l -diphosphonate-1-i4C (disodium etidronate- 1-i4C) was prepared by treating phosphorus tetraoxide (P406) with acetic acid-I-i4C in a mixture of water and n-propyl sulfone (Quimby and Prentice, 1968). Specific activity of the compound was 4.5 mCi/g. Disodium etidronate-32P was prepared by refluxing a 1 Present address: Physical Science Section, University of Tennessee at Martin, Martin, Tennessee. * Disodium etidronate is the name disodium ethane-1-hydroxy-l,l-diphosphonate. 0 1972 by Academic

Press, Inc.

approved

by the United

503

States

Adopted

Names

Council

for

504

MICHAEL,

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AND

WAKIM

mixture of 32PC13,water and acetic acid at 120-l 30°C for 5 hr to yield a pure crystalline condensate, which was then refluxed in water for 24 hr. After crystallization (48 hr at room temperature), filtration and an aqueous wash, crystalline disodium etidronate32P (specific activity of 2.9 mCi/g) was obtained. Chemical Purity

Chemical purity of both the 14C- and 32P-disodium etidronate was shown to be greater than 99% by the following criteria: (a) Thin-layer chromatography using cellulose MN3003 coated plates and a solvent system of 1% (w/v) p-toluenesulfonic acid monohydrate dissolved in water, acetone, tetrahydrofuran and isobutanol (30, 5, 30, 40, v/v) revealed only 1 spot, which had an R, value of approximately 0.4 (the R, of pure disodium etidronate). The quantity of phosphorus as determined by X-ray emission and the total 14C and 32P in that spot were consistent with values calculated for a compound containing 2 phosphorus atoms per molecule and having the specific activities noted above. (b) No impurities were observed with reverse isotope dilution technique in either the i4C- or 32P-labeled compound. The procedure used for this assay is described below. (c) No differences were found between the nuclear magnetic resonance (NMR) spectrum and X-ray diffraction pattern of the 14C-labeled compound and standard disodium etidronate. Because of safety considerations, NMR and X-ray diffraction studies were not carried out on the 32P-labeled compound. Disposition

of Disodium Etidronate in the Rat, Rabbit, Dog, and Monkey

Rats. Three weanling (50 g) and 4 young adult (approximately 200 g) male SpragueDawley rats were fasted for 16 hr and then given disodium etidronate-i4C (50 mg/kg) by intragastric (ig) cannula. In addition, young adult rats were preconditioned to the compound by feeding a semi-purified diet (Table 1) containing 0.5% unlabeled disodium etidronate for 30 days. These animals were fasted for 16 hr and then administered the i4C-labeled material ig. The pH of the disodium etidronate-14C solution was 7.4, and the concentration was such that the weanling rats were given 0.5 ml while the young adult animals received 2 ml. Animals were housed individually in stainless steel cages which permitted separation and collection of urine, feces and expired CO*. Urine and feces were collected from all animals for 72 hr after dosing and, in addition, respiratory COZ was collected over the same time period from the young adult animals. Excreta, carcass and various organs in this experiment and subsequent studies described below were assayed for total i4C-content (Michael and Wakim, 1971). Nonfasted young adult rats were allowed 8 hr to consume a semi-purified diet containing 1% disodium etidronate-14C. Urine and feces collections were started when the diet was first offered, and the total collection time was 72 hr. An identical study was carried out on a group of rats that had been preconditioned to the semipurified diet containing 1% unlabeled disodium etidronate for 30 days prior to the 8 hr feeding of disodium etidronate-14C in the diet. The enterohepatic circulation of both po administered (stomach tube) and ip injected disodium etidronate- i4C was determined in fasted, young adult rats with cannulated bile ducts. Each rat received a total of 10 mg (approximately 50 mg/kg) of the labeled compound. 3 Brinkmann Instruments, Inc., Westbury, New York 11590.

METABOLISM OF DISODIUM ETIDRONATE

505

In a multiple po administration study, 10 mg of disodium etidronate-14C (2 ml of aqueous solution) was administered ig to fasted rats (13c-175 g) once per day for 5 consecutive days. Groups of 2 rats were sacrificed at 1 hr, and at 3, 5,7, 14,21 and 28 days after the last dose. Radioassay was performed to determine the length of retention of the compound in the carcass and bone. The mandibles and tibias were cleared of soft tissue by autoclaving. It was found that none of the compound was removed from the bone during autoclaving. TABLE 1 COMPOSITION OF SEMIPURIFIED DIET

Casein Sucrose

Fortified sucrose” Salt Mix USP No. 14b Celluflour” Fat soluble vitamin mix No. 7” Soybean oil

27% 46%

$s 32 1%

14%

’ Furnished the following in n&l00 g of ration: 0.3 menadione, 0.4 thiamine, 0.5 riboflavin, 2.0 niacin, 2.0 calcium pantothenate. 0.4 ovridoxine. 0.025 biotin. 300 choline chloride, 200 inositol, 10 ascorbic acid and 10 p-aminobenzoic acid. b Nutritional Biochemicals Corporation, Cleveland, Ohio. c Chicago Dietetic Supply House, Chicago, Illinois. d Furnished the following per 100 g of ration: 1200 IU vitamin A. 1200 IU vitamin D2 and 14.7 IU vitamin E ‘as D-a-tocopheryl acetate.

In a final study to determine what effect the quantity of disodium etidronate-14C had on absorption and disposition, groups of at least 3 fasted young adult male rats were administered the compound ig at dose levels of 0.1, 1, 2, 5, 10 and 200 mg (0.5 mg-1 .O g/kg). Rabbits. Three male New Zealand rabbits, weighing an average of 2.7 kg were fasted for 22 hr and then given 50 mg/kg of disodium etidronate-14C ig. Dogs. Dogs used in these studies were purebred beagle females obtained from the American Animal Industries of Indianapolis, Indiana. Three young dogs, 4-6 mo of age, weighing 4-7 kg, were fasted for 22 hr and then given a 50-mg/kg dose (4 ml of solution) of disodium etidronate-14C ig. In studies using disodium etidronate-32P, 8 young dogs (4-6 mo of age, weighing 4-7 kg) and 4 older dogs (5-8 yr of age, weighing approximately 1 l-l 5 kg) were used. In addition, 2 young dogs were employed to determine whether absorption of disodium etidronate-32P occurred from the stomach. Each dog was anesthetized using enough sodium thiamyla14 (usually 6-7 ml) to produce the desired level of anesthesia and was 4 Parke, Davis & Company, Detroit, Michigan.

506

MICHAEL,

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WAKIM

then provided with a jugular vein cannula5 and urinary catheter.6 These remained in place during the first 8 hr of the study while the animals were maintained in restraining slings. Approximately 30 min after the dogs had regained consciousness, zero-time blood and urine samples were collected. The animals were then given 20 mg/kg of disodium etidronate-32P ig (2% solution, pH 7.4). Blood was collected from the jugular cannula every 15 min for the first 2 hr, every 30 min for the next 2 hr, every 60 min for the next 4 hr and then from the cephalic vein at 24,30 and 48 hr after dosing. Approximately 2 ml of blood was taken at each sampling period, and the exact weight was determined for radioassay. Urine was collected from the urinary catheter of each animal every 30 min for the first 8 hr, and then from the metabolism cage at 24 and 48 hr after dosing. Saliva or emesis was collected in stainless steel pans during the period of time that the dogs were maintained in the restraining slings and was radioassayed to determine if any 32P was lost through salivation. The 2 dogs used to determine the site of absorption were anesthetized with sodium pentobarbital’ to provide deep anesthesia, and a jugular vein cannula and a urinary catheter were inserted as before. The pyloric sphincter was ligated through a 3-in longitudinal incision in the abdominal wall above the pyloric sphincter and slightly right of midline. The incision was closed with suture, and procaine (2 %) was injected into the wound to minimize discomfort. The dogs were placed in the restraining slings and allowed to regain consciousness prior to dosing with disodium etidronate-32P. With the exception of the 2 dogs surgically treated to determine the site of absorption, all animals were maintained for 48 hr after dosing. At the end of the 4%hr collection period, each animal was killed and a gross autopsy was performed. Tissues were not taken for microscopic examination. The gastrointestinal tract and its contents, selected organs (brain, heart, lungs, spleen, kidneys and liver), blood, urine, feces, saliva, a femur and its marrow, and cage washes from 4 of the 8 young dogs and from all 4 older dogs were radioassayed. The remaining carcass of each dog was autoclaved to facilitate recovery of the skeleton, which along with the carcass and autoclave juices, were radioassayed. The carcasses of the other 4 young dogs were not assayed; however, the liver and kidneys of 2 of these animals were removed, weighed and extracted with chloroform-methanol (2: 1, v/v). Both the lipid and nonlipid residue fractions were radioassayed. Brains from the same 2 dogs were removed and radioassayed to determine whether disodium etidronate-32P was able to penetrate the blood-brain barrier. The 2 dogs with the ligated pyloric sphincter were necropsied about 4.5 hr after dosing. Monkqs. The 3 rhesus monkeys (2 male and 1 female) used in this study were approximately 4 yr old and were obtained from Woodward Asiatic Corp., San Francisco, California. Each monkey weighed 3 kg and was fasted at least 18 hr prior to the start of the study. These animals were not provided with jugular vein cannula and urinary catheter because of the difficulty in restraining this species over a long period of time. Two of the animals (1 male and 1 female) received 20 mg/kg of disodium etidronate-32P ig (2% solution, pH 7.4). The monkeys were maintained in stainless steel metabolism cages suitable for urine and feces collection for the duration of the 5 PE 50 Intramedic Polyethylene Tubing, Clay-Adams, New York, New York. 6 Bardex self-retention urinary catheter, Bardex, Murray Hill, New Jersey. ’ W. A. Butler Co., Columbus, Ohio.

507

METABOLISMOFDISODIUMETIDRONATE

study. Blood samples were taken from either the saphenous or jugular vein at 30 min and 1,2,4,8,24 and 48 hr after dosing. Urine and feces were taken from the metabolism cage at 24 and 48 hr .The remaining male monkey was fasted for 24 hr and then administered 50 mg/kg of disodium etidronate-14C ig. Urine and feces were collected for 72 hr. Identification of Metabolites of Disodium Etidronate-14C

i/

In order to determine whether or not disodium etidronate was catabolized, radioactivity present in the urine, feces and bone was analyzed by the technique of reverse isotope dilution. Samples containing 14C were added to 40-50 ml of water already containing 1.0 g of pure unlabeled disodium etidronate. Ethanol was added with agitation to initiate crystallization of the disodium etidronate. The crystallization procedure was repeated until a constant specific activity was obtained; 3 recrystallizations were usually sufficient. RESULTS Disposition of Disodium Etidronate in the Rat

Absorption of po administered disodium etidronate-14C in young adult animals was less than 3% of that fed, while in the weanling rats absorption averaged 10.5% (Table 2). In both age groups very little 14C was found in the kidneys and livers 72 hr after dosing. The fact that only 0.2”/, of the fed 14C was converted to CO, by young adult animals indicates that the absorbed compound was not appreciably catabolized. Data from young adult animals which were fed the diet containing the unlabeled TABLE 2 ABSORPTION AND DISPOSITION OF DISODIUM ETIDRONATE-14C IN RATS 72 HR AFTER ORAL ADMINISTRATION Young adult

Number of rats Body weight (g) Dose level (mg/kg)

4 193 f 206 50

Young

adult”

4 234 f 26 50

Weanling 3 53 rt5 50

% of Administered 14C Absorbed

Urine Organsc Carcass co2

Total

1.3 0.015 1.3 0.2

i It f i

0.5 0.012 0.5 0.2

2.0 i2.2 0.006 + 0.003 1.6~112.1

d

3.9 i 0.1 0.04 f 0.005 6.6 * 1.9

d

--2.8

3.6

10.5

92.7 i 4.7 0.08 i 0.02

84 + 14.5 0.9 f 1.2

78.0 +c11.0 0.1 f 0.05

Not absorbed

Fecal matter Gastrointestinal contents Total Total recovery of 14C

92.8 95.6

84.9 88.5

78.1 88.6

a Fed disodium etidronate at a dietary level of 0.5% for 30 days prior to dosing with the labeled compound. b Mean I SD. c Kidneys and liver. d Not measured.

508

MICHAEL, KING, AND WAKIM

compound for 30 days prior to ig administration of disodium etidronate-14C were indistinguishable from those of rats exposed only once to the compound (Table 2). It is concluded that repeated exposure to disodium etidronate did not alter the extent of absorption or disposition of the labeled compound in the rat. Table 3 shows the absorption and disposition of disodium etidronate-14C in nonfasted young adult rats 72 hr following consumption of a diet containing the labeled compound. Again it is apparent that previous exposure to the compound did not appreciably alter the extent of absorption or distribution. The rather large amount of radioactivity found in the contents of the gastrointestinal tract of animals fed disodium etidronate-14C as part of the diet reflects the slow passage of the solid diet through the gastrointestinal tract. TABLE 3 ABSORPTION

AND DISPOSITION OF DISODIUM ETIDRONATE-14C 72 HR AFTER BEING FED IN THE DIET TO NONFASTED RATS”

__-~

Single meal following 30 days of control diet

Number of rats Body weight (g) Total disodium etidronate-14C consumed (mg)

3 278 zt 15’ 93

---

Single meal following 30 days of disodium etidronate dieP 3 179*8 97

% of Administered 14C --~-.

Absorbed

Urine Organsd Carcass Total Not absorbed Feces

Gastrointestinal contents Total Total recovery of “C

0.9 It 0.3 0.03 zt 0.006 1.4 & 1.4 -2.3 91.6 + 5.3 17.7 i 6.5 109.3 ~111.6 i 1

0.6 i: 0.4 0.02 * 0 1.8 zk 2.5 2.4 90.8 iz 13.5 16.0 rk 2.1 106.8 109.2 zt 8

n Animals allowed access to disodium etidronate-14C diet for 8 hr. b Animals fed diet containing unlabeled disodium etidronate at a level of 1% for 30 days prior to feeding the disodium etidronate-14C diet. Approximately 146 mg of disodium etidronate was consumed per day. Food consumption during the 30-day feeding period was approximately 3 less than that of the control group. c Mean k SD. d Kidneys and liver.

To further verify that disodium etidronate-14C was not appreciably absorbed and did not enter the enterohepatic circulation for excretion, the compound was administered both po and ip to rats with cannulated bile ducts. The absorption of disodium etidronate-14C in these rats (Table 4) was greater than that found in the intact animal (Table 2). The increased absorption may have resulted from an increased residence time for the compound in the stomach as the consequence of surgery. The 72-hr

METABOLISM

OF DISODIUM

ETIDRONATE

509

collection of bile from fasted rats fed disodium etidronateJ4C by stomach tube contained less than 0.01 y0 of the administered dose; thus, i4C in the feces after po administration must represent unabsorbed material. When the disodium etidronate was administered ip, only 0.1 y0 of the dose was recovered in the bile after 72 hr. In these animals 33 ‘A of the dose was excreted in the urine, and 4 % was found in the feces; the latter very likely was due to contamination of the feces with urine in the metabolism cage. TABLE 4 ABSORPTION AND DISPOSITION OF DISODIUM ETIDRONATEJ~C IN BILE-DUCT-CANNULATED RATS 72 HR. AFTER ORAL OR IP ADMINISTRATION OF 10 MG OF THE COMPOUND (-50 MG/KG)

% of Administered 14CG Fraction

Oral dose Intraperitoneal dose -___47 i lb 414

Fecal matter Urine 612 33 i 19 Bile co.01 0.1 * 0.04 11 f3 Carcass 51 * 14 Total recovery 90 f 8 88 i 26 n Four rats (averageweight 200 g) wereused in each group. The po dosewasadministered by stomach tube. b Mean & SD. Dynamics of Disodium EtidronateJ4C in the rat

Table 5 shows the absorption, excretion and retention in young adult rats at various time intervals after administration of disodium etidronate-i4C ig for 5 consecutive days. One hour after the last dose, i4C was detected in the feces, and most of the remaining radioactivity was recovered in the gastrointestinal wash. The carcass contained 1.2% of the total administered 14C, and absorption averaged just over 2 y0 in these animals. Three days after the last dose, more than 90 % of the material had been excreted in the feces, while the urine and carcass contained about 1% of the dose. These values remained relatively constant until the end of the study at 4 wk. Radioactivity remaining in the carcass 72 hr after 1 (Table 2) or 5 daily doses (Table 5) of sodium etidronate was found to be deposited primarily in the skeleton. Loss of i4C from the tibia with time during the 4-wk test period after 5 daily doses (Table 5) was found to follow first-order kinetics. The half-life of disodium etidronate-i4C in the tibia calculated from the first-order rate constant was approximately 12 days. The quantity of disodium etidronate- 14C fed did not affect the absorption or disposition patterns in fasted rats when the total dose was in the range of 0.1-10 mg (0.5-60 mg/kg) (Table 6). At the highest dose level (200 mg) the carcass contained 5 % and the urine 11 0/0of the administered i4C, indicating that absorption was greatly increased. Ulceration of the gastrointestinal tract was observed in earlier studies conducted in this laboratory in rats that received LD50 (1.35 g/kg) and near-LDSO doses. If similar damage occurred in rats dosed at the 200-mg level (1 .Og/kg), increased absorption by these animals would very likely occur. The concentration of 14C in the bone (Table 6) was severalfold higher than that in the carcass. Only trace amounts of i4C were detected in the soft tissues.

510

MICHAEL,

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TABLE 5 ABSORPTION

AND DISPOSITION OF DISODIUM AFTER ORAL ADMINISTRATION OF

1 Hr

ETIDRONATE-lJC IN THE YOUNG ADULT FOR 5 CONSECUTIVE DAYS~

RAT”

10 MC/DAY

3Days

5Days

7Days

14Days

21 Days 28 Days

% of Administered “‘C

Absorbed

Urine Organsd Carcass

0.9 10.02 1.2 ~2.1

Total

0.4 eo.02 0.7 ~~I.1

0.3 0.5 CO.04 10.02 0.4 0.7

93.0 0.3

91.0 91.0 CO.02 X0.04

91.0 91.0 CO.02 co.02

91.o CO.02

93.3 94.4

91.0 91.7

91.0 91.7

91.0 91.7

0.7

0.3 co.02 0.4

0.4 co.02 0.4

0.3 co.02 0.4

0.7

0.8

0.7

1.T

Not absorbed

Feces 85.5 Gastrointestinalcontents 20.0 -~ Total 105.5 Total Recovery 107.6

91.0 92.2

91.0 91.8

pg Equivalentsof disodiumetidronate-W/g Long bone (tibia) Mandible

61 24

31 12

25 10

23

11

10

5

7 4

5 4

a130-175g. b Disodium etidronate-14C given by intragastric cannula following overnight (16 hr) fast. c Time of sacrifice after administration of last dose. Two rats per group sacrificed at each time period. d Kidneys and liver.

TABLE 6 EFFECT

OF DOSE

ON DISODIUM

ETIDRONATE-14C

Disodium etidronate-t4C dose(mg) 0.1 Number of rats 3 Body weight (g) 204+ 17” ~ ~.___

1 3 202*4 -

ABSORPTION

2 3 188zk28

AND

5 3 165ztl5

DISPOSITION

IN THE RAT

10 3 1621t24

2co 4 2OOzk24

% of Administered 14C ____-~ - ~..~~~ -

-

Absorbed

Urine Organsb

l.Odc1.4 0.4*0.1 2.7*1.1 2.3kO.6 2.541.5 ll.Oi8.0 co.01 co.02 CO.02 1.1 I- 1.0 Carcass <3 0.7 kO.7 1.4& 0.9 2.8 ho.4 2.6 ~2.0 5.0 zt 2.5 ____ -__ -___-__Total 4.0 1.1 4.1 5.1 5.1 16.1 Not absorbed(feces) 101.2& 17 98.1 * 8 97.0It 3 89.1zk6 90.3 i 17.4 59.4zk15 Total recovery 105.2 99.2 101.1 94.2 95.4 75.5 pg Equivalents of disodiumetidronate-t4C/g Long bone(tibia) Mandible Carcass ’ Mean rt SD. b Kidneys and liver.

0.02 co.01 co.01

0.61 0.21 0.03

2.3 1.0 0.23

16.7 7.4 1.15

30.2 3.5 2.18

580 350 89

METABOLISM

Disposition

OF DISODIUh4

511

ETIDRONATE

of D&odium Etidronate in the Rabbit

Absorption and disposition patterns of disodium etidronate-14C in the rabbit may be seen in Table 7. Total absorption of the compound was not appreciably different from that observed in the rat. The large quantity of 14Cfound in the intestinal contents reflects a slow passage of materials through the gastrointestinal tracts of the rabbit. TABLE 7 ABSORPTIONAND DISPOSITIONOF DISODIUM ETIDRONATE-l"C IN THE RABBIT 72 HR AFTERORALADMINISTRATION

Number of rabbits Body weight (kg) Disodium etidronateJ4C dose (mg/kg) Absorbed

Urine Organsb Carcass Total

3 2.7 zt 0.17"

50 okof AdministeredJ4C 3.3 i 3.1 co.03 * 0.01 0.50 * 0.5 3.8

Not absorbed

Feces Gastrointestinal contents Total Total recovery

78.0f 8.3 25.8 xtl1.2 ___-

103.8 107.6

a Mean f SD. b Liver and kidneys.

Dispositiotz of Disodium Etidronate in the Dog

Total absorption of fed disodium etidronate- r4C over 72 hr, or disodium etidronate32P over the 4%hr collection period, averaged about 21% in young dogs, while about 14 % of the administered disodium etidronate -32P was absorbed by old dogs (Table 8). Radioactivity not absorbed was found in the feces, intestinal contents and vomitus. Disposition of the i4C- and 32P-labeled compounds was virtually identical, even though different dose levels of the labeled compounds were administered. It is apparent that animal-to-animal variation was extreme. For example, in the 8 young dogs, total absorption ranged from 8 to 27 % of the fed 32P, In old dogs, 2 animals had very limited absorption (2-5 %) while the other 2 revealed appreciable absorption (22-24 %). An emetic response was noted in only 2 dogs (both young) following administration of disodium etidronate-)*P. In each case, the vomitus was collected and radioassayed. It may be seen that most of the radioactivity absorbed by the dog was found either in the urine or in the skeleton, and that there was very little (~2% of the fed) in the internal organs and carcass (Table 8). In young dogs where bone growth and remodeling were quite active, about 45% of the absorbed disodium etidronate-32P was in the skeleton, and approximately 50% was excreted in the urine. In old dogs where bone turnover was decreased, approximately 25% of the absorbed 32P was found in the skeleton, while approximately 65 % was excreted in the urine. This greater uptake of 32P by the bone in young dogs was also reflected in the increased concentration of

512

MICHAEL, KlNG,AND

WAKIM

disodium etidronate 32Pin the femur. In both young and old dogs, the concentration of 32Pin the bone was found to be about 3 times as great as in the marrow and about 5 times asgreat as the peak blood level. TABLE

8

ABSORPTION OF DISODIUM ETIDRONATE-14C (50 MG/KG) AND DISODILJM ETIDRONATE-32P (~OMG/KG) INTHEDOGAND MONKEYFOLLOWINGORALADMINISTRATION Young dogs

~~.~~

14C No. of animals

3

Old dogs

32P

32P Adminztered

8

% of

Monkeys 14C

32P

1 dose

2

Absorbed

Urine Organsc Skeleton Carcass Total

8.7 * 4.7” 0.63 x+z0.05

11.8*

10.3 0.3 9.2 0.8

i 6.58 ic 0.06 $4.32 i 0.26

8.9 1.4 3.1 0.2

i It It &

8.61 0.75 2.22 0.06

21.1

20.6

13.6

60.4 i 18.43

81.3 + 11.34

0.1 co.02 0.5 0.6

0.4 0.5 3.6 1.7

(0.3; 0.4Y (0.2; 0.7)

(0.1; 7.0) (0.1; 3.3)

6.2 (0.7; 11.4)

Not absorbed

Feces 80.9 Gastrointestinal 2.4 content Vomitus Total

97

0.1 zt 0.23

0

0.05

6.8 f 15.00’

0

-

91.5 (102; 81) 0.3 (0.1; 0.5)

0

83.3

67.3

81.3

97.1

91.8 (102.1; 81.5)

104.4

0.4 88.3

0 94.9

0 97.7

0.1 (0; 0.2) 97.9 (102.8; 92.9)

Miscellaneous

Cagewash Total recovery

Femur Bone marrow Peak blood level

---

pg Equivalentsof 14C-or 32P-labeled disodiumetidronate 29.7 zk 15.7 10.5f 6.4 0.94 -

67 zt 47.2

-

9.4 xt 6.3 5.9 i 3.8

3.6i4.1 2.1h1.8

-

1.1 (0; 2.1) 0.8 (0; 1.5)

a Mean + SD. b Values of each animal. c Combined values representing brain, heart, lungs, spleen, kidneys and liver.

dOnly 1 carcass assayed. e Two dogs had an emetic response.

In young dogs, the amount of radioactivity found in the internal organs (exclusive of the gastrointestinal tract) was insignificant. The kidneys contained lessthan 0.06 % of the fed 32Pwhile lessthan 0.02% was found in the liver. The radioactive material found in the kidney and liver could not be extracted into chloroform-methanol. Radioactivity from disodium etidronate- 32Pdid not readily penetrate the blood-brain “barrier” of these animals, as less than 0.001% of the total dose was found in the brain. The 2 old dogs that absorbed greater than 20 % of the fed compound had peak levels of 3.5 and 3.8 pg of disodium etidronate-32P/g of blood within 30-45 min after dosing. Total absorption in thesedogs was about the sameas that observed in the young dogs

METABOLISM

OF DISODIUM

513

ETIDRONATE

which absorbed 20 % or more of the administered dose and where the peak blood level of disodium etidronate-32P averaged 6.8 pg/g of blood. The differences in peak blood levels found in the 2 age groups probably reflect a difference in blood volume, since the quantity of disodium etidronate-32P in the blood at the time of the peak level was found to be directly proportional to the quantity of the compound deposited in the skeleton of both age groups. Radioassay data from the 2 dogs fed disodium etidronate-32P following ligation of the pyloric sphincter are shown in Table 9. No attempt was made to determine disposition of the administered 32P among skeleton, internal organs and soft carcass; a total recovery of the fed radioactivity was not obtained. It is assumed in this study that any 32P not recovered in the gastrointestinal contents, vomitus and/or saliva and stomach was absorbed. On this basis, absorption was about 33 % in one dog and about 59 7; in the other. Blood analyses confirm that absorption was extensive in the latter animal, as a peak level of 6.8 pg of disodium etidronate-32P/g of blood was reached 1.5 hr after dosing. In contrast to intact or unaltered animals, the level of 32P in the blood of this animal did not diminish once a peak was reached, but rather remained relatively constant until the time of death, which may have resulted from surgical shock. Even though these dogs were surgically altered, the peak blood level, absorption and urine excretion data (Table 9) confirm that appreciable absorption of disodium etidronate did take place from the stomach. These data help to explain the very early appearance of the labeled compound in the blood of dogs after po administration. TABLE 9 DISPOSITION OF ORALLY ADMINISTERED DISODIUM ETIDRONATE-~~P IN THE DOG FOLLOWING LIGATION OF THE PYLORIC SPHINCTER (DOSE LEVEL, 20 MG/KG)

y0of Administered 32Pa 31b 32b Fraction

Urine Stomach contents Stomach Saliva Peak blood level

0.8 34.6 6.7

2.1 63.0 3.7

0

CO.1

6.8

1.2

(pg of disodium etidronate-32P/g) a 48 Hr after dosing. b Animal number. Disposition of Disodium Etidronate in the Monkey

Absorption of disodium etidronate- 32P averaged approximately 6 ‘A in the monkey as shown in Table 8. Again animal variation was extreme as 1 animal absorbed ~1% of the dose while absorption in the other was about 12% of the administered dose. Approximately 58 % of the absorbed 32P was in the skeleton, while urine excretion was very limited (6.5 ‘A of absorbed 32P). Because all long bones were included in radioassay of the skeleton, concentration of disodium etidronate-32P in the femur was not determined in the monkey. However, it appears that the level would have been approximately 3-4 pg/g based upon the peak blood level and marrow concentration, assuming the same ratios as those experienced in the dog. The 1 monkey that absorbed appreciable

514

MICHAEL,

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AND

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disodium etidronate-‘*P had a peak level of 1.4 pg/g of blood, and this occurred about 1 hr after dosing. Absorption of disodium etidronate-32P by the second monkey was so limited that there was no detectable radioactivity in the blood. Carcass and organ radioactivity in the monkey accounted for approximately 35% of the total absorbed; however, it should be noted that this was only approximately 2 % of the fed 32P (Table 8). A similar comparison would be meaningless for the monkey that received disodium etidronate-i4C because of the very small amount of radioactivity absorbed (< 1 %). Identljication

of Metabolites of Disodium Etidronate-14C

Reverse isotopic dilution analysis of disodium etidronate was performed on different biological samples obtained from rats and dogs given disodium etidronate-i4C ig. Analyses were performed on urine from the rat and on urine, feces, serum and bone from dogs. An impurity of l-2% may be missed by this technique. However, in all cases, 95-100’~ of the i4C was precipitated with standard disodium etidronate. DISCUSSION

These studies have shown that absorption of disodium etidronate was very limited from the gastrointestinal tract of the rat, rabbit and monkey. The dog, however, was able to absorb the compound more readily than the other species. An age effect on absorption was also demonstrated; weanling rats and young dogs (tl yr of age) were found to adsorb about twice as much of a single po dose as adults of the respective species. These studies also suggest that absorption of disodium etidronate from the diet is extremely poor, probably reflecting the inability of animals to absorb the heavy metal salts of disodium etidronate which likely form in the gastrointestinal tract from metals in the diet. Reasons for species and age differences in absorption are not clear. Disodium etidronate was found to be absorbed primarily from the stomach, suggested by rapid appearance of the compound in the blood of all species and confirmed by ligation of the pyloric sphincter in the dog. The skeleton was the target site for deposition of disodium etidronate in all species. Although there were some species variations, about half of the absorbed po dose was deposited in the skeleton, while half was excreted in the urine. There was very little retention of the compound in the soft tissues or organs. The peak blood level of disodium etidronate-32P in young dogs after po administration was found to average 5.9 pg/g of blood (Table 8). In 2 of these animals where absorption of disodium etidronate-)*P was very limited, the peak blood levels (at approximately 3 hr post dose) were less than 1 pg of disodium etidronate-32P/g of blood. In the other 6 dogs where absorption was approximately 20 % or more, a peak blood level of 6.8 pg/g was obtained 30 min after dosing. The concentration of 32P fell to undetectable levels by 4 hr after dosing. These studies also showed that there was little or no enterohepatic circulation of the absorbed compound. It appears from the data that disodium etidronate was not catabolized appreciably, since only 0.2”! of the administered dose was detected in the expired CO, of the rat Reverse isotopic dilution experiments were unable to detect metabolites, indicating no appreciable conjugation or metabolic alteration of the compound. The in vitro activity of disodium etidronate in preventing hydroxyapatite crystal growth and slowing crystal dissolution reflects the ability of the compound to chemisorb

METABOLISM

OF DISODIUM

ETIDRONATE

515

onto the apatite crystal surface (Francis, 1969). This affinity for apatite (bone) was further demonstrated in the present metabolic study by the deposition and relatively long half-life (12 days) of disodium etidronate in the skeleton following po administration. In separate studies the half-life of disodium etidronate in bone was reported to be approximately 30 days after SC administration (King et aE., 1971). In these studies the dose level was 50 mg/kg/day for 5 days. The longer half-life probably reflects a more extensive diffusion of disodium etidronate into the bone due to a higher blood level of the compound because of SCrather than po administration. The effect of various systemic doses of disodium etidronate on bone development, and the relationship of disodium etidronate interaction with bone to calcium-phosphate metabolism, are discussedin considerable detail elsewhere(King et al., 1971). ACKNOWLEDGMENTS The authors are grateful for the expert technical assistanceof Mrs.Cynthia Mark and Messrs.D. D. Dearwester,L. Flora, W. J. Harvey, M. B. Hester and C. L. Varns and to Messrs.L. McCreary and H. Lampefor synthesisof the radioactive compounds,radioisotope analysesand reverse isotopic dilution studies.They would also like to thank Drs. R. H. Coots, M. D. Francisand W. B. Geho for encouragementduring the courseof this study. REFERENCES A. L., DONATH, A., MACAGNO, F., PREISIG, R., FLEISCH, H., and FRANCIS, M. D. (1969).Diphosphonatesin the treatment of myositisossificans.Lancet7625, 845. FLEISCH, H., RUSSELL, R. G. G., BISAZ, S., CASEY, P. A., and MUHLBAUER, R. C. (1968).The influenceof pyrophosphateanalogues(diphosphonates)on the precipitation and dissolution of calciumphosphatein vitro and in vivo. Calc. Tiss. Res. 2, Suppl., lO-10A. FLEISCH, H., RUSSELL, R. G. G., and FRANCIS, M. D. (1969a).Diphosphonatesinhibit hydroxyapatite dissolutionin vitro and bone resorption in tissueculture and in vivo. Science BASSETT,

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165, 1262-1264. H., RUSSELL, R. G. G., SIMPSON, B., and MUHLBAUER, R. C. (1969b).Prevention by a diphosphonateof immobilization “osteoporosis”in rats. Nature (London) 223,211-212. FRANCIS, M. D. (1969). The inhibition of calcium hydroxyapatite crystal growth by polyphosphonatesand polyphosphates.Calc. Tim. Res. 3, 151-162. FRANCIS, M. D., RUSSELL, R. G. G., and FLEISCH, H. (1969).Diphosphonates inhibit formation of calcium phosphatecrystals in vitro and pathological calcification in vivo. Science 165, 1264-1266. JOINT FAO/WHO EXPERT COMMI~EE ON Foot ADDITIVES. (1967). Specificationsfor the identity and purity of food additives and their toxicological evaluation: someemulsifiers and stabilizersand certain other substances.World Health Organ. Tech. Rep. Ser. 373, p. 12. JOWSEY, J., HOLLY, H. E., and LINMAN, J. W. (1970).The effect of sodiumetidronate in adult cats. J. Lab. Clin. Med. 76, 126-133. KING, W. R., FRANCIS, M. D., and MICHAEL, W. R. (1971).The effect of disodiumethane-lhydroxy-1,1-diphosphonate on bone formation in animals. Clin. Orthop. Rel. Res., in press. MICHAEL, W. R., andWAKIM, J. M. (1971).Metabolismof nitrilotriacetic acid (NTA). Toxicol. FLEISCH,

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W. R., KING, W. R., and FRANCIS, M. D. (1971).The effectiveness of diphosphonates in preventing “osteoporosis”of disusein the rat. Clin. Orthop. Rel. Res., in press. QUIMBY, 0. T., and PRENTICE, J. B. (1968).U.S. Pat. 3,400,149,Sept. 3. RUSSELL, R. G. G., BISAZ, S., and FLEISCH, H. (1969). Pyrophosphateand diphosphonates in calcium metabolismand their possiblerole in renal failure. Arch. Intern. Med. 124, 571-577. MICHAEL,