Nucl. Med. Biol. Vol. 16, No. 3, pp. 255-259, 1989

hr. J. Rodiar. Appl. Instrum. Part B

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The Biodistribution of [75Se]bis-

[/I -(NJ&N-Trimethylamino)ethyl]selenide

Diiodide in Adult Guinea Pigs

W. K. SIDNEY YU, S. M. SHAW*, J. M. BARTLETT?, D. C. VAN SICKLES and B. H. MOCK6

School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana, U.S.A.

(Receiued 5 July 1988)

The biodistribution of [“Se]BISTAES was studied in guinea pigs. A higher concentration of radioactivity was observed in articular cartilage than in other tissues or organs. A minimal amount of radioactivity was found in the blood, muscle and bone. The compound was excreted rapidly in urine. The target to background ratios were encouraging. [‘%e]BISTAES has potential as an articular cartilage imaging agent and further studies in osteoarthritic animals are merited.

Introduction

Primary and secondary osteoarthritis are common

joint diseases involving the destruction of articular

cartilage. Although radiography is commonly used in the diagnosis of osteoarthritis, it does not directly reveal damage to articular cartilage. Radiography reveals secondary structural changes such as joint space narrowing, subchondral bone sclerosis, sub- chondral cyst formation and osteophyte formation. Therefore, radiography is not sufficiently sensitive for early diagnosis of osteoarthritis, but is utilized to confirm the above described structural changes that occur in the advanced stage of osteoarthritis.

Arthroscopy is more sensitive than radiography and allows the physician to directly examine the articular cartilage surface. However, the procedures are relatively complicated. Magnetic resonance imaging is highly sensitive in assessing osteoarthritis. Due to the high cost of instruments and the long data acquisition time, the utilization of magnetic resonance imaging is still very limited.

Radionuclide scintigraphy is another technique for the detection of osteoarthritis. The current radio- pharmaceuticals used in osteoarthritis imaging are bone scanning agents such as *Tc-methylene diph-

*Author for correspondence. tPresent address: The Dow Chemical Company, Centr,aI

Research, Biotechnology Laboratory, 1701 Building, Midland, MI 48674, U.S.A.

SSchool of Veterinary Medicine, Purdue University, West Lafayette, Indiana, U.S.A.

gIndiana University Medical Center, Indianapolis, Indiana, U.S.A.

osphonate (%“Tc-MDP). Although osteoarthritis imaging using *Tc-MDP is more sensitive than radiography, the specificity is unsatisfactory. It is difficult to differentiate osteoarthritis from various bone abnormalities. The availability of an agent that localizes directly in the articular cartilage would greatly enhance the usefulness of radionuclide scintigraphy.

The “Se labeled bisquatemary ammonium com- pound, [75Se]bis[B-(iV,N,N-trimethylamino)ethyl]sel- enide diiodide (r’Se]BISTAES), has been synthesized and the biodistribution studied in normal rabbits (Yu et al., 1988). The concentration of the compound was significantly higher in the articular cartilage than in background tissues such as the femur, tibia and the blood. The favorable target tissue to background tissue ratios indicate that (‘%e]BISTAES may have merit as an articular cartilage imaging agent. Since articular cartilage is the initial site of destruction of osteoarthritis (Sissons, 1983; Sokoloff, 1983), it would appear that [“Se)BISTAES would improve the sensitivity and specificity of radionuclide scintigraphy in the diagnosis of osteoarthritis. Based on the posi- tive evidence gained from the rabbit study, the agent was evaluated further in normal mature guinea pigs. The results of the investigation are reported herein.

Materials and Methods

Synthesis of [73Se]BISTAES

Nonradioactive BISTAES was synthesized and identified according to the procedures described pre- viously (Yu ef al., 1988; Kung and Biau, 1980). The ‘?!4e labeled BISTAES was synthesized in an identical

255

256 W. K. SIDNEY Yv et al.

Table 1. Percent radiochemical purity of [“Se]BISTAES’ at various times of storage

Temperature Storage time (weeks)

Solvent (“C) 0 1 2 3 4 5 6 8

Ab 4 95.c 94.7 97.1 94.9 96.8 93.9 93.7 95.2 : 4 4 96.9 96.7 97.9 94.8 96.6 96.7 97.3 93.9 95.8 93.6 93.7 97.7

98.6 95.8 98.7 99.3

A ::

95.4 94.9 97.0 94.6 93.7 94.3 95.3 95.1 B 96.7 96.8 96.8 93.8 94.1 93.9 94.3 95.9 C 25 96.9 96.9 97.1 97.6 97.2 98.5 97.4 99.0

A 40 95.4 95.9 96.5 92.3 87.2 92.8 90.1 90.9 40 96.7 96.1 97.1 95.1 90.7 92.3 90.4 93.4 40 96.9 97.5 96.9 97.1 93.4 90.5 96.3 97.6

‘1.5 mCi/mg/mL. blO% sodium iodide in ethanol. ‘18% sodium iodide in ethanol. dAcetonitrile:normal saline (2: 1). ‘Mean of two samples.

manner. The yield of the synthesis was 40% and the specific activity was 1.5 mCi/mg. The product was identified by conducting TLC using the non- radioactive BISTAES as the reference standard and three different solvent systems: 18% sodium in eth- anol, 10% sodium iodide in ethanol and acetonitrile: normal saline (2: 1). The Rf values were 0.5, 0.3, and 0.1 respectively.

Stability of [“Se]BISTAES

In order to establish its stability, [7sSe]BISTAES was stored in normal saline (1.5 mCi/mg/mL) at 4,25 and 40°C for 8 weeks. The identity and radiochemical purity of the [‘?Ge]BISTAES were determined weekly by TLC.

Biodistribution study

Prior to the biodistribution study, the r’Se]BISTAES was dissolved in normal saline and filtered through a sterile 0.22~ filter. Six bio- distribution replicates were conducted. In each repli- cate, six male guinea pigs weighing 600-700 g were injected intraperitoneally with the [“Se]BISTAES at a dose of 15 pg/kg (9-12 &i/animal). Immediately before euthanization of each animal, the pupillary response to light was observed. One animal each was euthanized with carbon dioxide at 15 and 30 min and at 1,2,4 and 6 h post-injection. The organs/tissues of interest were removed, rinsed with normal saline, blotted dry and weighed. A blood sample was ob- tained by cardiac puncture immediately after the animal was euthanized. The plasma and the blood cells were separated and weighed. The amount of radioactivity in the tissues/organs was determined with a NaI(T1) scintillation detector. The concen- tration of [7JSe]BISTAES was expressed as the percentage of injected dose per gram of sample.

Excretion of [ “Se]BISTAES

Five guinea pigs weighing 600-700 g were injected as described previously. Each animal was maintained in a metabolism cage for 10 h. The urine and feces from each animal were collected separately, volume

or weight determined, and radioactivity measured. The percentage of parent compound in the urine was determined by TLC using the same solvent systems as described previously.

Results and Discussion

The radiochemical purity of the [75Se]BISTAES compound stored at different temperatures is presented in Table 1. The radiochemical purity of the [75Se]BISTAES stored at 4 and 25°C remained rela- tively constant throughout the entire period of study, while the radiochemical purity of the compound stored at 40°C decreased slightly after the third week of the study. However, the radiochemical purity was higher than 90% in all but one circumstance. The results indicate that the [“Se]BISTAES was relatively stable when stored at an ambient temperature or below and could be used for a reasonable time period after preparation.

Bisquatemary ammonium compounds are well known for their ganglionic blocking actions. Mclsaac found that the inhibition of pupillary response to light was the first observable pharmacologic action of hexamethonium and occurred at a plasma concen- tration of 1 pg/mL (McIsaac, 1962). When the plasma hexamethonium concentration was reduced to 0.3 pg/mL, no pharmacological action was ob- served. In the present study, no inhibition of pupil- lary response to light was observed, thus suggesting that no pharmacological action occurred in the guinea pigs receiving the [‘$e]BISTAES at a dose of

15 /us/b. The results of the biodistribution study are

presented in Table 2. The articular cartilage of the knee and shoulder contained a higher level of radio- activity than did the other organs and/or tissues except for the kidney and bladder. The highest concentration of radioactivity in articular cartilage occurred at 60 min post-injection.

The degree of localization of [“Se]BISTAES in the bone and the muscle was very minimal when com- pared to other tissues and/or organs. Throughout the

[‘%e]BISTAES in guinea pigs 257

Table 2. Biodistribution of [7sSc]BISTAES’ in minea uigs

Percentage in&ted dose per gram tissue

15min 30 min Organ (N = 5) (N = 5)

Menisci 0.09 * 0.02s 0.17 +0.03 Knee’ 0.28 * 0.05 0.43 * 0.13 ShouldeP 0.41 * 0.10 0.47 +0.16 Trachea 0.11 *0.01 0.21 + 0.06 Bile 0.42 + 0.34 0.39 * 0.34 Liver 0.18 kO.03 0.17 f0.03 Kidney 0.36 ?r 0.14 0.41 k 0.08 Bone’ 0.04 i 0.00 0.06 + 0.01 Boner 0.04 * 0.01 0.06 f 0.01 Muscle’ 0.03 * 0.01 0.04 f 0.01 Muscle’ 0.03 f 0.00 0.04 0.01 * Bladder 0.40+0.15 0.57 0.37 * Testis 0.10 kO.02 0.06 f 0.01 Heart 0.07 + 0.02 0.08 + 0.02 Blood 0.14 + 0.02 0.15 f0.03 Plasma 0.29 + 0.07 0.29 0.08 f Blood cells 0.07 * 0.03 0.10 5 0.04 G-I 0.12 +0.06 0.06 0.02 k

‘r%e]BISTAES and/or metabolites. ‘Mean f 1 SD. ‘Pooled articular cartilage from both knees. dPooled articular cartilage from both shoulders. ‘Front limbs. ‘Hind limbs.

(NI_h6) (N’_h5) (N”=” 6) (N6: 4)

0.22 * 0.03 0.13 io.03 0.04 * 0.00 0.01 f 0.00 0.73 f 0.18 0.34 f 0.05 0.14 +0.02 0.01 f 0.01 0.69 + 0.21 0.48 + 0.21 0.19 kO.07 0.01 * 0.01 0.31 + 0.07 0.28 + 0.09 0.11 f 0.03 0.02 * 0.01 0.27 + 0.27 0.05 f 0.04 0.05 * 0.03 0.02 f 0.01 0.17 +0.03 0.13 f 0.02 0.11 * 0.03 0.08 + 0.01 0.47 + 0.06 0.36 f 0.10 0.33 f 0.13 0.18 + 0.07 0.06 + 0.01 0.03 + 0.00 0.02 + 0.01 0.01 + 0.08 0.06 + 0.01 0.03 * 0.01 0.02 i 0.00 0.01 * 0.00 0.04 f 0.01 0.02 f 0.00 0.01 + 0.08 0.01 * 0.08 0.04 * 0.01 0.02 f 0.01 0.01 f 0.00 0.01 f 0.01 0.68 * 0.22 0.40 + 0.23 0.40 + 0.13 0.39 * 0.21 0.06 f 0.01 0.02 f 0.01 0.02 f 0.01 0.02 * 0.01 0.07 + 0.01 0.03 f 0.01 0.03 f 0.01 0.02 f 0.01 0.14 * 0.01 0.08 f 0.01 0.02 * 0.02 0.01 + 0.00 0.26 F 0.04 0.10 + 0.03 0.03 f 0.01 0.01 f 0.00

0.07 * 0.01 0.04 + 0.02 0.01 + 0.00 0.01 i 0.00 0.05 f 0.01 0.03 f 0.01 0.02 * 0.01 0.03 f 0.01

entire period of the study, there was only a very small amount of radioactivity in these tissues. Since the articular cartilage is adjacent to bone and muscle, the ratio of activity in articular cartilage to bone and to muscle were of particular interest in determining the usefulness of the [75Se]BISTAES for imaging carti- lage. As presented in Table 3, the maximum target to background ratio occurred at 1 and 2 h post- injection. At that time, the articular cartilage (knee) to bone and to muscle ratios were 12 and 21 respectively, while the articular cartilage (shoulder) to bone and to muscle ratios were 12 and 20 respectively.

Throughout the entire study, the concentration of radioactivity in the articular cartilage from the knee was about the same as that observed in the articular cartilage from the shoulder. This is to be expected since the articular cartilage from the knee and shoul- der are both hyaline cartilage, have similar functions, and are corresponding articulations.

The concentration of radioactivity in the menisci and trachea was much lower than that found in the

articular cartilage at corresponding time intervals up to 4 h (Table 2). A smaller amount of [“Se]BISTAES would be expected in the men&i. The menisci is classified as a fibrocartilage and contains a smaller amount of negatively charged glycosaminoglycans than articular cartilage. The localization of bis- quaternary ammonium compounds such as hexa- methonium and [“Se]BISTAES in the articular cartilage has been hypothesized to occur through the electrostatic interaction between the compound and the negatively charged matrical glycosaminoglycan such as the chondroitin sulfate (Yu et al., 1988; Asghar and Roth, 197la, b; Shindo et al., 1971). The lower concentration of radioactivity in the trachea may be attributed to a lesser degree of vascularity in the perichondrium and the lack of biomechanical motions as compared to the knee or shoulder. A more detailed discussion has been presented previously (Yu et al., 1988).

The concentration of radioactivity found in the blood cells ranged from 0.01 to 0.07% of the injected dose per gram of blood cells. Since the density of the

Table 3. Target to background tissue ratio

Target to background ratio

Target/background tissues

Articuiar cartilage (knee)/ bone (hind limbs) Articular cartilage (knee)/ muscle (hind limbs) Articular cartilage (shoulder)/ bone (front limbs) Articular cartilage (shoulder)/ muscle (front limbs)

1Smin 30 min (N = 5) (N = 5) (N’J6) (,‘=” 5) (N4_h6)

6.56 6.72 11.8 10.2 8.08

9.09 12.0 20.5 14.5 13.5

9.80 8.06 11.9 15.6 12.9

15.9 11.4 19.7 20.5 18.3

258 W. K. SIDNEY Yu et al.

blood is approximately one, the blood cells would account for 45% of the blood by weight. Thus, the concentration of radioactivity found in the blood cells can be calculated as 0.004-0.03% of the injected dose per gram of blood. Based upon the concen- tration of radioactivity found in the whole blood (0.02-0.14%), the amount of radioactivity in the blood cells was calculated to account for 20-21% of the total activity in the blood. The result agrees with Wasserman’s data (Wasserman, 1972). He found that 20% of 3H-hexamethonium was bound to erythro- cytes to guinea pigs. According to Wasserman, the 3H-hexamethonium was bound to the negatively charged glycosaminoglycan in the cell membrane on the external surface of erythrocytes.

The concentration of radioactivity in kidney and bladder was high and remained constant throughout the period of the biodistribution study (Table 2). As would be expected, the results of the excretion study (Tables 4 and 5) showed that 75-103% of the injected radioactivity was found in urine, while only O-l .37% of the injected dose was found in the feces. These observations agree well with those reported for hexa- methonium (Levine, 1960; Christensen and Holm, 1969; Taylor, 1985). The rapid excretion of the r5Se]BISTAES and hexamethonium in the urine can be attributed to the positive charge and the polarity of the compounds. The short residence time of [75Se]BISTAES in the animal body is both advan- tageous and disadvantageous. The rapid excretion allows a minimal time for patient positioning or instrument adjustment. However, the short residence time is advantageous from the view point of radiation dose to a patient. The short residence time and rapid excretion minimize the dose by shortening the effective half-life of the [75Se]BISTAES (Yu et al., 1988).

As presented in Table 6, about 70% of the injected r5Se]BISTAES appeared to be excreted in the origi- nal form with either 10 or 18% sodium iodide in ethanol as the solvent system, while about 90% of the injected [75Se]BISTAES was found in the original form when acetonitrile : normal saline (2: 1) was used as the solvent. The migration of [?le]BISTAES in acetonitrile:normal saline (2: 1) was less than in the other solvent systems indicating a lower resolution capability for the acetonitrile: normal saline (2: 1) system. It is possible that the f5Se]BISTAES and any metabolites were not completely separated. While

Table 4. Recovery of radioactivity in urine over a 10-h interval

Animal Urine volume Percentage of number W) injected dose

I 21 85.2 2 5 75.5 3 82 97.3 4 96 103 5 20 90.9

Average: 90.7 SD: 9.59

Table 5. Recovery of radioactivity in feces over a 10-h interval

Animal Feces weight Percentage of number (g) injected dose

1 3.22 1.37 2 0.27 0.28 3 0 0 4 0.27 0.11 5 1.46 0.22

Mean: 0.39 SD: 0.49

[75Se]BISTAES appears to be metabolized, hexa- methonium has been shown to be excreted virtually unchanged (Taylor, 1985).

The biodistribution pattern of [‘?Je]BISTAES was similar to that observed for hexamethonium (Kom et al., 1979), but [“Se]BISTAES localized in articular cartilage to a greater extent when compared to “C-hexamethonium. The concentration of 14C radio- activity found in articular cartilage at 0.5 and 2 h was 0.29 and 0.043% injected dose/g respectively. The concentration of 75Se radioactivity observed in artic- ular cartilage was 0.43 and 0.34% injected dose/g at 0.5 and 2 h respectively. In addition, the target to background ratio observed for [“Se]BISTAES was higher than that observed for “C-hexamethonium. Based on the data provided in the literature (Kom et al., 1979), the articular cartilage to muscle radio- activity ratio for “C-hexamethonium was calculated to be 3.1 at both 0.5 and 2 h post-injection. As indicated in Table 3, the articular cartilage (knee) to muscle (hind limbs) radioactivity ratios for [7SSe]BISTAES at 0.5 and 2 h were 12 and 14.5, respectively.

There were some differences between the results of the biodistribution study in guinea pigs and in rabbits (Yu et al., 1988). These may be attributed to the difference in animal models and in the route of administration. The [“Se]BISTAES was injected in- travenously into the rabbits and intraperitoneally into the guinea pigs. As would be expected, the i.p. route of administration resulted in some difference in the pharmacokinetics. The maximum concentration of radioactivity in the articular cartilage was reached 60min post-injection in the guinea pigs, while the maximum concentration of radioactivity in the artic-

Table 6. Percentage of [‘&]BISTAES excreted in the urine over a IO-h interval

Solvent system’

Animal no. A B C

1 73.5 69.0 89.3 2 79.5 58.9 97.6 3 63.5 70.2 87.4 4 63.3 69.5 87.6 5 77.9 72.5 95.7

Mean: 12.8 68.1 91.5 SD: 5.8 4.7 4.1

‘Solvent systems: A = 18% sodium iodide in ethanol; B = 10% so-

dium iodide in ethanol; C = Acetonitrile:normal saline (2: I).

[%e]BISTAES in guinea pigs 259

ular cartilage occurred 15 min post-injection in the rabbits. There was a higher level of radioactivity in the blood of the guinea pigs as compared to that in the blood of the rabbits. The level of radioactivity in the organs or tissues of the rabbits decreased at a rate faster than in the organs or tissues from the guinea pigs. Finally, the target to background ratios for the rabbits were higher than those observed for the guinea pigs. In the rabbits, the articular cartilage to adjacent bone ratio was approximately 30 at 15 min post-injection. In the guinea pigs, the articular cartilage (knee) to bone (hind limbs) ratio was ap- proximately 12 at 60 min post-injection. The uptake of radioactivity in the articular cartilage and the target to background ratios found in this study are encouraging and indicate that further study of r’Se]BISTAES in an osteoarthritic guinea pigs model would be merited.

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