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A Walker 256 tumor-induced osteogenic small animal model for the evaluation of [99mTc] diphosphonate radiopharmaceuticals
In! J. Nuci. Med. Biol Vol 12. No. 3. pp. 197-208. 1985 Pnnted ,n Great Bntaln. All rqhts reserved
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0047-0740185 $3.00 + 0.00 c‘ 1985 Pergamon Press Ltd
A Walker 256 Tumor-Induced Osteogenic Small Animal Model for the Evaluation of [99mTc] Diphosphonate Radiopharmaceuticals K. T. CHENG,’ S. M. SHAW,’ T. C. PINKERTON,‘*
D. J. HOCH’ and D. C. VAN SICKLE3 ‘School of Pharmacy. Department of Medicinal Chemistry, ‘School of Science, Department of Chemistry and ‘School of Veterinary Medicine. Department of Veterinary Anatomy. Purdue University. West Lafayette. IN 47907. U.S.A. (Received 22 March 1985)
A mammalian model has been developed for the in viuo evaluation of bone imaging agents. The model is based upon the quantification of a discrete. initial secondary periosteal osteogenesis induced in cortical bone immediately adjacent lo an intramuscularly implanted Walker 256 tumor in Fisher 344 rats. Evaluation of the model consists of a histopathological examination of the periosteal bone formation. biodistribution studies on %“Tc-MDP and W”‘Tc-HMDP commercial kit preparations, and biodistribution studies on two “‘“‘Tc-HEDP component fractions isolated after anion exchange chromatographic separations from an investigative “carrier added” preparation. Reversed phase HPLC separations of the W”‘Tc-MDP and W”Tc-HMDP commercial kit preparations illustrate distinct differences in chemical composition between the two bone agents.
mineralized bone. +“’ Fogelman@) has suggested that the skeletal uptake in this region is more likely due to factors affecting mineralization, rather than the presence of immature collagen, with the technetium diphosphonates being adsorbed onto the surface of newly deposited hydroxyapatite crystals. Genant et a/.“*’ speculate that this “short term” uptake of bone imaging agents results from an ion exchange process on bone surfaces in contact with the circulating fluids, thus indicating periosteal, endosteal, trabecular and lacunar canalicular (osteonal) surfaces as predominate sites of rapid exchange. In the case of skeletal abnormalities involving osteolytic lesions the increased uptake of bone imaging agents parallels the increased rate of the compensatory osteogenic response. (“) Differences in biodistribution between various bone imaging agents appear to be dependent upon differences in (i) the diffusional processes across physiological barrierqt8) (ii) the degree of affinity of
Introduction Radiopharmaceutical bone imaging constitutes a sensitive means of detecting skeletal abnormalities involving discrete osteogenesis.“.” Bone imaging can detect bone metastases from six months to one year before visualization by radiography becomes possible.“-” Traumatic bone injuries, such as stress fractures. which are initially undetected radiographically, can also be detected by bone imaging.‘@ The “‘“Tc-labeled diphosphonates are currently the agents of choice for bone imaging”’ (Table 1). Although the exact mechanism for the localization of the bone imaging agents is not entirely known, the uptake of the ““Tc-diphosphonates on bone has been associated with regions of high metabolic bone turnover.‘*’ in particular. at the junction of osteoid and * Author to whom correspondence
should be addressed.
Table I. Chelating diohouhonate 4genr
kands
Ligand 0 R, 0 :I ~ HO-P-C-;-OH OH&
““‘Tc-MDP ““‘Tc-HMDP ““‘Tc-HEDP
OH
MDP. methylene diphosphonate (R, = H. RI = H) HMDP. hydroxymethylene diphosphonate (R, = H. R, = OH) HEDP. hydroxyethylidene diphosphonate (R, = CH,, R! = OH) 197
198
K. T.
CHENG et ui.
the agents for blood components (i.e. serum proteins)u4) and (iii) the ion exchange properties of the agents,‘2.12.‘5’
Research efforts aimed at improving the efficacy of bone imaging agents have been directed toward the production of technetium diphosphonates which exhibit a greater affinity for sites of osteogenesis”6.‘7’ and a more rapid clearance from the blood and soft tissue.“‘) In the search for bone imaging agents with higher selectivity for bone lesions, investigators must have a relatively inexpensive animal model which provides discrete sites of osteogenesis analogous to clinical abnormalities of interest. In this regard, the utilization of normal animals for the screening of potentially new bone’ imaging agents is not ideal, for a higher normal bone uptake does not imply an analogously higher lesion uptake”@ or faster soft tissue clearance. Three osteogenic animal models have been developed previously for the evaluation of bone imaging agents. These are (i) the pellet model, which involves the implantation of demineralized bone matrix;“3.‘9,20’ (ii) the drill hole model;u6) and (iii) the rachitogenic model.u2) Differences between the Walker 256 discrete periosteal osteogenic model and these previous models include the induction of excess callus osteogenesis with the pellet and drill hole models; the high cost of large animals (rabbits) in the case of the drill hole model; and the chronic pathophysiological state of the rachitogenic model. In a search for a low cost, rapid, and reproducible means of inducing osteogenesis, it was discovered that the intramuscular implantation of a Walker 256 tumor into Fisher 344 rats produced an increase in bone uptake of 99”Tc-HMDP on selected days during tumor growth, implying the presence of new bone.‘2’) Previous work by Hulth and Olerud(**) demonstrated that a VX2 carcinoma, when implanted in the proximity of bone, induced osteogenesis, even though the carcinoma had no natural tendency to grow in bone. In the work by Hulth and Olerud, discrete spiculae of new woven bone appeared at right angles to the bone surface while preosteoblasts had formed in the periosteum near areas of newly formed capillaries. The Walker 256 cells are easily propagated in the ascitic fluid of the host and/or readily stored in culture medium for prolonged periods, and tumor induction upon intramuscular implantation of the cell is successful in 100% of the animals; thus, the Walker 256 tumor in Fisher 344 rats provides a suitable, low cost small animal model for the rapid, reproducible production of discrete osteogenesis. The following study evaluates the potential utilization of the Walker 256 tumor model for the evaluation of bone imaging agents relative to their uptakes in this type of discrete osteogenesis. The investigation includes a histopathological study on the Walker 256 tumor and adjacent bone, multiple biodistribution experiments on two commercial bone imaging kit preparations (““Tc-MDP and “9’“Tc.-HMDP), and
multiple biodistribution experiments on two investigate 9y”‘Tc-HEDP component fractions isolated after an anion exchange high performance liquid chromatographic separation.
Experimental Tumor cell propagatiorr
The Walker 256 tumor cells were supplied initially by The Procter and Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio, U.S.A. The cells were stored in 10% dimethyl sulfoxide. 209~~ fetal calf serum and 70% media 199 at - 19OC in liquid nitrogen. The frozen tumor cells were thawed rapidly with agitation in a 37°C water bath. when needed for implantation. In order to maintain the cell line, 0.1-0.2 mL of cells were injected into the peritoneal cavity of Fisher 344 male rats (weighing 125-145 g. 6-7 weeks old). The tumor cells were allowed to propagate in the ascitic fluid in cico for 3-4 days. These animals were then euthanatized and the ascitic fluid was collected by rinsing the peritoneal cavity of the animals with 10-20 mL of Hanks Balanced Salt Solution (HMS0).(23) The solution was centrifuged at 1OOOrpm for lOmin, and the supernatant was discarded. The recovered cell pellet was treated with 0.83% ammonium chloride to lyse the erythrocytes. This solution was centrifuged at 1000 rpm for 10 min. The lysed erythrocytes were removed by decanting the supernatant. The tumor cells were resuspended in the appropriate volume of HBSO to obtain a final concentration of 10’ viable cells per tenth of a milliliter. The number of viable cells was determined with trypan blue and a hemocytometer. Tumor induction
Fisher 344 inbred male rats (weighing 125-145 g, 6-7 weeks old) were used throughout the study. A tumor was induced by injecting a suspension of 10’ viable Walker 256 tumor cells in 0. IO-O. 15 mL of HBSO intramuscularly into the inferior region of the gastroenemius of the left leg of a Fisher 344 rat. Animals were kept in individual cages with controlled temperature and light. Standard laboratory chow and tap water were allowed ad libitum. Radiopharmaceutical preparations
Sodium [%“Tc]pertechnetate was obtained from v9Mo/99Tc]generators and supplied in saline from Syncor International Inc., Indianapolis, IN, U.S.A. The 99mTc-diphosphonate kits, OsteoliteO 9”“Tc-MDP (Lot No. 6050, New England Nuclear, North Billerica, MA, U.S.A.) and Osteoscan@ 99”‘Tc-HMDP (Lot No. DL82065, Procter and Gamble, Co.. Cincinnati, OH. U.S.A.) were prepared in accordance to manufacturer’s instructions. Tests for aluminum content in each preparation were performed by standard procedures,‘Z4’ and all results were negative. Instant thin layer chromatography with acetone and normal saline’?” indicated that the
Evaluation
of bone imaging
preparations were greater than 90% radiochemically pure and contained less than 5% “hydrolyzed reduced” technetium. Liquid chromatography
qf commercial kits
In order to evaluate the compositional nature of the commercial tin-reduced technetium diphosphonate radiopharmaceuticals, the Osteolite@ 99”Tc-MDP and Osteoscan@ 99”‘Tc-HMDP kit preparations were subjected to reversed phase ion-pairing high performance liquid chromatography. In order to accommodate the possible presence of polymeric species a silica bonded reversed phase packing of large pore diameter was chosen (Vydac Cl 8, 4.6 mm i.d. x 25 cm 1. 5 pm particle diameter, pore size 33OA, Ranin Instrument Co., Wobum, Mass., U.S.A.). The reversed phase separations were conducted at ambient temperatures with an isocratic aqueous mobile phase containing 1.8 x 10-j M tetrabutyl ammonium hydroxide as the ion-pairing reagent, adjusted to pH 6 with acetic acid. In addition, the mobile phase contained ethyl acetate as an organic modifier (4;; by volume for the 99’“Tc-MDP separation and So/, by volume for the 99”Tc-HMDP separation). Constant flow rates were maintained with a LDC Constametric III@ dual piston pump (Laboratory Data Control, Riviera Beach, Fla., U.S.A.). Samples were injected via a Rheodyne Model 7125 injection valve equipped with a 2OpL loop (Rheodyne Inc., Cotati, Calif., U.S.A.). The y9mTc-diphosphonate separated components were detected by passage of the eluent flow into the well of a NaI(TI) well 7 counter via 0.25 mm i.d. stainless steel tubing.
199
agents
day for seven consecutive days. Each animal was administered 10 PCi of 99”‘T~-HMDP (0.02 mg/kg) .via the jugular vein while under ether anesthesia. Each animal was euthanatized by cardiac puncture at 3 h post injection. The tibia adjacent to the tumor, the contralateral tibia, the femur of the tumor leg and the contralateral femur were removed and isolated from soft tissue. The 99”?c activity in the bone was determined by means of a NaI(TI) scintillation well crystal detector. The bone samples were weighed and the data was expressed as percent of injected dose per gram of tissue. Histology
Based upon the significant by increased 99mTc-HMDP uptake in the tibia adjacent to the tumor, the eighth day after tumor implantation was selected for histological study. Ten rats were divided randomly into two groups. Tumor cells were implanted into five rats with an additional five rats receiving only HBSO. Eight days post-injection, all of the animals were euthanatized and the tibia of each animal removed. The tibias were decalcified in a mixture of formic acid and sodium citrate. processed for hard tissue histopathology in a standard fashion, and serial sections were stained with hematoxylineosin and safranin O-fast green. Biodisrrihution
studies
Three separate experimental trials were conducted during the biodistribution study of the 99”Tc-MDP and 99”‘Tc-HMDP commercial kit preparations. In each experiment, the Fisher 344 rats were randomly selected and divided into tumor bearing groups and normal control groups for each radiopharmaceutical lmestigatice preparation product. Each animal group contained 6 animals; An investigative technetium hydroxyethylidene therefore the overall biodistribution pooled means ““7~-HEDP complex solution was prepared by the for the 99’“Tc-MDP and 99”Tc-HMDP in the study were accumulated from 18 tumor-bearing animals reduction of [99”Tc]pertechnetate with sodium borohydride in the presence of [99mTc]pertechnetate and 18 normal animals. “carrier” by a procedure previously described.“5~26~27’ In each study, eight days after tumor implantation, The ““‘Tc-HEDP solution was subjected to anion 10 p Cl of the 99”Tc-diphosphonate preparation along exchange separation on Aminex-A29 (4.6 mm with 1 pCi of *%.rwas injected into ether-anesthetized i.d. x 25 cm 1. Bio-Rad Laboratories, Richmond, rats via the jugular vein. Three hours after injection Calif.. U.S.A.) with an aqueous mobile phase of each rat was euthanatized by cardiac puncture. The 0.85 M sodium acetate at pH -8.“‘) Two blood. heart, stomach, spleen, liver, kidney, and ‘““‘Tc-HEDP component fractions were isolated for in muscle in the tibia contralateral to the tumor were removed. The tibia adjacent to the tumor, the tibia riro evaluation from the chromatographic separation of approximately twelve 99mTc-HEDP complexes. The contralateral to the tumor bearing leg, the femur of isolated fractions containing the pure 99”Tc-HEDP the tumor bearing leg and the femur contralateral to the tumor bearing leg were excised from the tumor complexes were made isotonic in acetate and buffered animals, defleshed and blotted dry. All samples were to pH = 7.4 by dilution with 0.05 M orthophosphate. weighed and counted for 99mT~ and “Sr activity independently. Corresponding tissues were removed from normal control animals and analyzed m the “‘“‘Tc-HMDP was used as an indicator of osteoblastic activity induced on the inferior surface of the same manner. Activity was expressed as percent of tibia adjacent to the tumor. Tumor cells were iminjected dose per gram of tissue. The 99”Tc-HEDP planted in 24 Fisher 344 rats by methods just de- component fractions isolated by an HPLC separation. and designated as “YU” and “X”,“” were scribed. Starting from the second day after tumor implantation. three rats were chosen randomly each evaluated by means of two biodistribution experi-
CHENG et al.
K. T.
200
ments each in which groups consisted of 6 tumor bearing animals and 6 normal animals for each component fraction. The same experiment procedure as described above was employed, except the use of “Sr was found to be unnecessary. Strontium
correction
The 99mTcuptakes in the bone tissues were found to vary slightly with the inherent differences in age among the animals employed for each of the three experiments carried out during the study. The ages of the Fisher 344 ranged from 6 to 7 weeks. The slight variation in age between the animal experimental groups was compensated by utilizing %r as an internal standard. Speckman and Norris(28) have demonstrated the correlation between the age of rats and mice and the 85Srretention in normal bone. The ““Tc bone uptakes could be normalized from the inherent group variation through multiplication by a *%r correction factor, taken as the ratio of the mean 85Sr uptake in normal bone among the specific animal group to the mean *‘Sr uptake in normal bone for all non-diseased animals employed in the three experiments. The normalization improved the coefficient of variation in the pooled mean bone uptakes from 0 to 8%. Subramanian et al. (*‘) has employed a similar *‘Sr correction to normalize for the weight and age of New Zealand white rabbits used to evaluate the uptake of 99”‘Tc-diphosphonates in callus lesions with the drill hole model.
Results and Discussion Histology
of bone
In order to determine the New bone formation. potential for induced osteogenesis during the growth of the Walker 256 tumor, injection of 99”Tc-HMDP were administered to groups of animals during the
-
tibia
I ----
femur
??
0
growth period. The 9y”‘Tc-HMDP activity in the bone adjacent to the tumor was compared to that of the contralateral bone. The results expressed as ratios (i.e. bone adjacent to tumorcontralateral bone) for both the tibia and femur are illustrated in Fig. I. There appeared to be no significant difference in 99”‘Tc-HMDP uptake between the bone adjacent to the tumor and the contralateral bone from the second day to the sixth day after tumor cell implantation: increase of however, there was a significant *“‘Tc-HMDP uptake in the tibia adjacent to the tumor on the seventh and eighth day. This increase in uptake indicated the suspected presence of increased osteogenesis. A corresponding increase in 99mTcactivity was not observed in the femur. The properties of the Walker 256 tumor have been found previously to include rapid growth, malignancy, osseous invasiveness, hypercalcemia and osteolysis. (30-34)It is likely that this transient osteogenesis corresponds to an observed drop in serum calcium which immediately follows the tumor’s primary hypercalcemic response on day six.‘32’ Several days beyond day eight the osteogenesis is overcome by osteolysis, followed by death of the host.‘3@3J’ Microscopy. After eight days of tumor growth. all implanted animals had enlargements in the region of the gastroenemius (Fig. 2). The enlargements in the muscle consisted of packed round mononucleated cells measuring 15-30 pm in diameter. The tumors when stained with safranin-0 and fast green were variegated in color. The cell’s cytoplasm and their immediate matrix stained red while the green areas appeared to be a degenerating collaginous matrix. Tumor cells were found occasionally in the M-vessels of the proximal metaphysis of the tibia and in one instance there was a nidus of cells in the metaphyseal medullary cavity resembling the tumor. The tumor in the muscle produced a discrete secondary periosteal
,
1
I
I
I
I
1
I
1
2
3
4
5
6
7
6
DAYS AFTER TUMOR IMPLANTATION Fig. I. Ratios of *mTc-HMDP in the tibia and femur of the tumor bearing leg to the tibia and femur of the contralateral leg respectively vs days of tumor growth.
'01
Evaluation of bone imaging agents Table 2. Mean rissue distributions
for W”Tc-MDP
in normal
rats
Percent dose per gram of tissue at 3 h post dose TlSSUe
Expt.
Tibia Femur
I
2.34 + 0.19 2.14+0 II
Tibia muscle Blood Spleen Liver Kidney Stomach Heart
0.014 * 0 009 0.043 f 0.008 0.041 f 0.010 0.061 f 0.012 1.33*040 0.074 + 0.022 0.015 0.001
f_ i SD;
2.96 Z 0.30 2.84 f 0.36
3.14&O.ll 2.94 + 0.28
2.81 + 0.42 2.64 i 0.44
* 0.002 k 0.006 * 0.003 & 0.008 & 0.05 + 0.019
reaction in the adjacent tibia (Fig. 3). The same region in the control animals appeared to be normal (Figs 4 and 5). No mitosis was found either in the tumor or in the connective tissue surrounding the tumor. However. within the fibrous layer of the adjacent periosteum and the subadjacent cambrium layer, there were numerous mitotic figures as well as trabeculae of fiber bone forming perpendicularly to the long axis of the tibia1 diaphysis. Cells in this area resembled tumor cells and once the fibrous layer of the periosteum was breached, tumor cells were easily found in this area.
0.006 0 029 0.026 0.047
+ 0.001 + 0.003 + 0.004 + 0.30 1.09+ 0.20 0.03 I + 0.005 0.012 IO.002
0.009 0.034 0.030 0.048 0.97 0.048 0.014
Biodistribution of 99”Tc-HMDP and 99mTc-MDP. The biodistribution characteristics of the two commercial diphosphate kit preparations, 99mTc-HMDP and 99mTc-MDP were evaluated by means of the Walker 256 osteogenic model. The percent dose per gram uptakes of the “9”Tc-diphosphanates in the
Table 3. Mean tlssoe distributions
for *‘“Tc-HMDP
in normal
Percent dose per gram of tissue at 3 h post dose _.~~ ____~____~._ Expt. I Expt. 2 Expt. 3
1.97+
Tibia Femur
0.63 I .82 f 0.52
Tibia muscle Blood Spleen Liver Kldneh Stomach Heart
2.55 + 0.40 2.43 * 0.44
0.009 + 0.001 0.028 f 0.005 0.054 + 0.016 0.083 i 0.008 0.99t0.18 0.068 * 0.013 0.018 + 0.005
0.008 0.044 0.100 0.340 0.71 0.051 0.030
Table 4. Mean tissue distributions
Tibia (tumor bearing leg) Tibia (contralateral) Femur (tumor bearmg leg) Femur (contralateral) ;;$dmuscle Spleen LlVer Kldne! Stomach Heart
(contralateral)
Expt.
I
3.08 t 0.28 2.13 TO.15 2.32 z 0.15 1.82-0.18 0.049 0.012 0.043 0.078 1.05 0.080 0.015
I& 0.006 0.01 I 2 0.015 5 0.012 5 0.10 IO.010 i 0.008
0.004 0.008 0.010 0.012 0.43 0.23 0.002
r t * + t * i
rats -
Pooled
3.10 io.40 2.96 i 0.48
0.001 0.010 0.070 0.240 0.13 0.010 0.008
0.006 0.025 0.029 0.051 0.78 0.040 0.019
3.81 2.75 3.03 2.43 0.042 0.009 0.029 0.045 0.49 0.035 0.017
+ 0.59 t 0.16 : 0.36 _c 0.45 5 0.01 t_ 0.05 I t 0.006 I 0.006 _t 0.05 i_ 0.007 2 0.003
-
2.54 rt 0.57 2.40 t 0.57
i 0.001 * 0.002 i 0.005 +0.017 + 0.21 * 0.005 f 0.019
0.008 + 0.002 0.032 + 0.010 0.061 i 0.036 0.16iO.15 0.8350.15 0.053 * 0.014 0.022 _t 0.007
for 9Q”Tc-MDP m fumor bearing
Expt. 2
Mean
fp,isDi
rats
Percent dose per gram of tissue at 3 h post dose Tissue
+ F * + * + i
tumor bearing and the normal control animals at 3 h post dose for each experimental trial are listed in Tables 2, 3, 4 and 5. Fundamentally, the ““Tc-HMDP and 99’“Tc-MDP exhibited similar in uiuo characteristics. As expected, the ““Tc-activity was significantly higher in the lesioned tibias adjacent to the tumor than in the contralateral tibias or the tibias of the normal animals. The ratios of the uptakes in the lesioned tibias to the normal tibias of the control animals reveal a consistently higher uptake of the W’“Tc-HMDP compared 99’“Tc-MDP in this discrete secondary periosteal osteogenesis (Table 6). Although the uptake of the 99’“Tc-HMDP in this type of lesion appears to be favored over the et a1.,(29’has found the 99”Tc-MDP, Subramanian opposite to be the case for the uptake of these agents in callus lesions produced in New Zealand white rabbits. The uptakes in the contralateral tibias, normal control tibias, contralateral femurs and normal control femurs consistently indicated a greater nor-
Biodistribution of bone agents
Tissue
Mean
Expt. 3
0.007 0.030 0.022 0.037 0.50 0.040
z
Pooled
Expt. 2
Expt. 3 3.39 3.34 3.53 3.26
* 0.31 i 0.36 i 0.38 & 0.34
0.032 0.007 i* 0.004 0.002 0.029 If 0.005 0.040 + 0.006 1.04+0.13 0.037 + 0.006 0.014 * 0.004
Pooled
Mean
Rp+SDI 3.43 2.74 2.96 2.50 0.041 0.009 0.034 0.054 0.86 0.051 0.015
f + + *
0.37 0.61 0.61 0.72
* 0.009 + 0.003 + 0.008 _t 0.021 2 0.32 * 0.025 jr 0.002
-
202
K. T. CHENG er al. Table 5. Mean nssue distnbutions
for YPmTc-HMDP in wnor bearrng rats
Percent dose per gram of tissue at 3 h post dose
Tissue
Expt.
Tibia (tumor bearing leg) Tibia (contralateral) Femur (tumor bearing leg) Femur (contralateral)
I
3.14 If:0.66 2.3 I + 0.49 2.41 + 0.57 2.15 F0.40
Tiba muscle (contralateral) Blood Spleen Liver Kidney Stomach Heart
0.007 _t 0.003 0.037 F 0.006 0.069 + 0.020 0.144 ; 0.050 1.13*0.17 0.097 & 0.008 0.029 k 0.003
ma1 bone uptake for 99”Tc-MDP than for 99”‘Tc-HMDP in the Fisher 344 rats (Tables 2, 3, 4 and 5). The ratios of lesioned tibia to normal tibia (Table 6) between the three experimental trials (which were conducted on separate dates with different animal groups) indicate a systematic variation in the degree of osteogenesis. It was discovered that the mean ratio (A) between the weight of the lesioned tibia and the weight of the contralateral tibias for an animal group was linearly related, throughout the study, to the mean ratio (0) of the percent dose per gram uptake in the lesioned tibias to the percent dose per gram uptake in the normal control tibias (correlation coefficient 0.92). This indicated that the mean ratio of weights (I?) between the lesioned tibia and the contralateral tibia could be used as a quantitative measure of average osteogenesis for a given experiment. The formation of new woven bone on the lesioned tibia adjacent to the tumor obviously added weight to
Table 6. Ratios of uptakes in lesioned tibia to selected tissues for VC-MDP and 99”Tc-HMDP
Exot. 1 Lesioned tibia/normal ‘+?c-MDP w’“Tc-HMDP
tibia I .32 1.59
Le~;siM~;/contralateral260 *=Tc-HMDP 450
Expt. 2
1.29 I .33
Exot. 3 1.08 1.16
mean ,?_+SD, 1.23 kO.13 1.36 + 0.22
tibia muscle 420 (tumor 480 bearing 390 rats)* 120 380 520 450 + 70
Lesioned tibia/blood (tumor bearine rats) ““%-MDP’ ‘63 91 W”%-HMDP 85 61
106 120
87 i 22 91 +27
EXDt.? 3.40 + 2.64 i 2.8s k 2.40 *
ExDt.3 3.6 I + 0.28
0.63 0.65 0.55 0.53
3.42 i_ 0.40 3.61 2 0.47 3.43 + 0.67
0.009 + 0.001 0.051 + 0.004 0.072 + 0.027 0.225 * 0.100 0.73 * 0.09 0.058 k 0.006 0.040+0.015
0.007 0.030 0.033 0.059 0.72 0.047 0.024
i: 0.001 i: 0.002 f 0.003 * 0.013 50.16 f 0.008 f 0.005
Pooled Mean .v. k SD; 3.38 2.79 2.98 2.66
t f + t
0.24 0.57 0.58 0.68
0.008 0.039 0.058 0.143 0.86 0.067 0.03 I
+ z + + i 2
0.001 0.0 I I 0.022 0.083 0.23 0.026 0.008
the tibia, so an approximate weight of an individual lesion could be obtained from the difference in weights between the lesioned tibia (IV,) and the contralateral tibia (WC). The percent dose per gram uptake in the new woven bone (7; dose/g “NWB”) could be calculated from equation (l), % dose/g “NWB” =
y/; x, - ;,; xc w, - WC
% dose/g “NWB” (corrected) = (% dose/g T) (R) - (% dose/g N) R-1
Percent dose per gram of tissue at 3 h post dose (normalized) -__ ““ToMDP WVe-HMDP
Tissue
Coefficient of variation = (SD,/?)
??
100.
(1)
where % X, and %, XCrepresent the percent dose in the lesioned tibia and the contralateral tibia respectively. The ‘?jr normalized pooled mean tibia lesion uptakes for the 99’“Tc-MDP and 99mTc-HMDP, determined by means of equation (l), are given in Table 7. The values appear inordinately high because the systematic variation in tumor growth between the experiments was not taken into account. In calculating the absolute new woven bone uptake, the differences in tumor growth between the experiments could be corrected by utilizing an expression which incorporates the mean weight ratio (I?) between the lesioned tibia and contralateral tibia as estimate of osteogenesis, and the mean per dose per gram uptake in the normal control animals (% dose/g fl) as the best estimate of uptake in the cortical bone for an experimental animal group. This relationship, represented by equation (2) is equivalent to equation (1)
Table 7. sJSr normalized pooled mean bone tissue distributions
Lesioned tibia Normal tibia Lesioned tibia/normal tibia New woven bone (uncorrected for variation in tumor growth, equation 1) New woven bone (corrrected for variation in tumor growth, equation 2)
i
3.42 t 0.33 (10%)’ 2.78 i_ 0.28 (10%) 1.24 + 0.1 I(9%)
3.38 f. 0.16 (5x)* 2.52 + 0.43 (17”/,, 1.37f0.22(16:/,)
9.21 f 0.44(5’4)
9.43 * 0.54 (60,)
7.28 + 0.08 (1%)
7.32 + 0.31 (45’ 0)
(2)
bone
Hcol
bone
Bone marrow
. d
Fig. 2. A sagittal section of tibia adjacent to tumor after eight days of tumor growth (SOFG x 4).
Fig. 3. Enlargements of square area in Fig. 2 (SOFG x 130). (A) Periosteal bone reaction, (B) cortical bone and (C) bone marrow.
203
Fig. 4. A sagittal section of the normal control tibia and gastroenemius
(SOFG x 4).
Fig. 5. Enlargement of square area hematopoietic in Fig. 4 (SOFG x 130). (A) adjacent skeletal muscle, (B) cortical bone and (C) bone marrow.
204
Evaluation of bone Imaging agents Table
8. Summary
of climcal imaging
comparisons performance
between *“‘Tc-HMDP of osteogemc lwons
(38.41.42.4547) (3740) (37.38.41,4446) (37.39.41.43.44.46) (37,3X.40,42.45.46)
Lesion counts: both agents comparable Lesion to normal bone: HMDP z MDP Bone to soft tissue: HMDP > MDP Blood clearance: HMDP z MDP “Image quality”: HMDP slightly better
??Rclerence (number of Patients in each study). 42(60). 43( 15). 44(34). 45(49). 46(15). 47(20).
“““‘Tc-MDP TOI
References*
Observation I. 2. 3. 4. 5.
and
37(1Ol, 38(102).
39(7). 40(20). 41(15).
selected mobile phase conditions and could not where 9, dose/g T is the mean percent dose per gram be distinguished in either preparation. The uptake in the lesioned tibia for an experimental 9ymTc-HMDP preparation yielded nine peaks (Fig. 6) animal group.‘3” The %r normalized pooled mean while the 99mTc-MDP preparation gave six peaks (Fig. new woven bone uptakes for 99”Tc-MDP and 99”‘Tc-HMDP are given in Table 7. These estimates of 7), indicating the complicated compositional nature of these solutions. the absolute lesion uptakes are lower than the unSrivastava et a1.‘48J have isolated tin reduced corrected values. In principle, if no variation in the production of the new woven bone exist between the 99mTc-MDP complex fractions after reversed phase HPLC and demonstrated differences in bioexperiments, then each method should yield comdistribution with normal rats. Other investigators parable values. This fact has been born out in subhave also purified 99”‘Tc-diphosphonates complexes sequent studies where lesion production was very by various chromatographic methods and illustrated reproducible.“” the difference in biodistribution among the pure 99mTc With respect to soft tissues, the 99”‘Tc-MDP yielded complexes within the mixtures.‘9.‘s.5’) No previous a slightly higher muscle and blood uptake over that of 99”Tc-HMDP: however. the 99”Tc-MDP exhibited a lower liver and heart uptake compared to the 99”Tc-HMDP (Tables 2-5). With the exception of the spleen, in the case of 99”‘Tc-HMDP. no appreciable differences in soft tissue uptakes were found between the tumor bearing and normal animals (Tables 2, 3, 4 and 5). The lesioned tibia to contralateral tibia muscle ratios were slightly higher for 99”Tc-HMDP than U9”Tc-MDP. while the ratios of lesioned tibia OSTEOSCAN to blood appeared comparable between the agents (Table 6). Tc-HMDP Clinical observations mvolving 11 studies with a total of 347 patients (summarized in Table 8) have demonstrated that 99”‘Tc-HMDP does indeed possess a slightly higher bone to soft tissue ratio, a more rapid blood clearance and a slightly better “image quality” when compared to 99”‘Tc_MDP,‘37A:)The results of this biodistribution study with the Walker 256 osteogenic model are consistent with these clinical observations. It should be noted that all ““Tc-diphosphonate preparations. commercial and investigative, have been found to contain multiple technetium diphosphonate complexes which can be separated by chroReversed phase iOn_ matographic means. 19.X.Z’.48.491 pairing high performance liquid chromatographic separations of the Osteoscan@ 99”Tc-HMDP and the Osteoliteo “‘“‘Tc-MDP commercial kits prepared under conditions identical to those employed during the animal studies (i.e. “no 99mTc carrier 0 4 8 12 16 20 added”) are illustrated in Figs 6 and 7 respectively. In TIME (MIN) these separations 98 and 96”, respectively. of the Fig. 6. Reversed phase ion-pairing HPLC of Osteoscan @ “““Tc activity injected onto the chromatographic col- %“‘Tc-HMDP. (Gamma response 1 x 104cps full scale. So< umn could be recovered. Free P’“‘Tc]pertechnetate (v’v) ethylacetate. ambient temperature. flow rate eluted at a retention time of -4 min under the 0.35 mL:min, pressure 950 psi.)
L b
1,
I
I,
I,
1,
1.
K. T.
206
CHEYG et
al
diphosphonate preparations accentuates the importance of performing multiple experimental trials when evaluating the performance of bone agents with animal models. It is likely on occasion that the
99”Tc-MDP and 99”‘Tc-HMDP kit preparations would yield identical biodistribution characteristics due to the overlapping physical nature of the component compositions: this explains why some studies have not observed differences in soft tissue uptake and differences in blood clearance between the agents.“O.‘O’
OSTEOLITE
Tc-MDP
Biodistribution of HPLC separated 99mTc-HEDP complexes. Preliminary biodistribution studies were
II
11/ 0
4 TIME
8
1;
(MINI
Fig. 7. Reversed phase ion-pairing HPLC of Osteolitea 99”Tc-MDP. (Gamma response 1 x 104cps full scale, 4% (v/v)
ambient temperature, ethylacetate, 0.60 mL/min, pressure 1400 psi).
flow
rate
studies, however, have attempted to evaluate the differences in uptake of HPLC separated 99mTccomplexes with an osteogenic lesion model. The biodistribution characteristics of the 99”‘Tc-MDP and *“‘Tc-HMDP commercial bone kits given in Tables 2 through 7 represent composite distributions which reflect the physical properties of the individual complexes and the relative abundance of particular complexes within a preparation. Five primary factors have been found to affect the chemical composition of technetium diphosphonate preparations; these factors include (i) the technetium concentration,‘26.49’(ii) the reductant concentration,(9) (iii) the presence of residual oxygen,‘*@(iv) temperature(48) and (v) PH.(~~’ The variations in muscle uptakes for 99’“Tc-MDP between the experiments (Tables 2 and 4) suggests that the component composition of the *‘“Tc-MDP preparations might have differed slightly during the study. This is consistent with the findings that the greatest differences in uptakes between the individual HPLC separated W”‘Tc-MDP complexes is among the soft tissues.‘4*’ This potential for compositional variation of the complexes within the technetium
conducted on two investigative 99”Tc-HEDP component fractions isolated after anion exchange HPLC (Fig. 8) from a sodium borohydride reduced “carrier preparation described elsewhere.““.z6.‘7’ added” Higher resolution anion exchange HPLC separations with reanalysis of the separated components by HPLC confirm that these technetium diphosphonate complexes are kinetically stable and substitution inert.‘*‘) By varying the HPLC anion exchange counterion concentration at constant pH, temperature and flow rate, and measuring corrected retention times, the average negative charges on 12 of these complexes at pH 8 have been determined to range from - 1.5 to -8.0.‘27) The early elution of highly charged complexes from the anion exchange HPLC and size exclusion chromatography indicate that some of the %“‘Tc-HEDP complexes are polymeric in nature.‘27’ The %“Tc-HEDP component fraction “YU” (Fig. 8), chosen for this preliminary biodistribution study with the Walker 256 osteogenic model, contains two complexes Y and U (assumably monomeric polyprotic species) with low average negative charges at pH 8 of - 1.5 k 0.1 and - 1.6 + 0.1, and small partial molar volumes of 510 mL/mol and 520 mL/mol respectively!*” The WmTc-HEDP component “X”, on the other hand, contains a single complex (assumed to be polymeric in nature) with a high negative charge at
0221
SY 405 nm delectmn X
Y =: 2
P QR
2
0
A LJGL 0
u
TM
v
Z
20
40
60
RETENTION
80
TIME
100
(mln)
Fig. 8. Anion exchange HPLC of sodium borohydide reduced “carrier added” W’“Tc-HEDP on Aminex A-29. (Spectrophotometric detection at 405 nm, isocratic elution with 0.75 M sodium acetate, temperature 28’C. Row rate 0. I1 mlimin. pressure 950 psi.)
Evaluation Table 9. Mean tissue distributions
of bone imaging agents
207
for P9’“Tc-HEDP. HPLC separated fraction YU and X in Fig. 8
Percent dose per gram of tissue at 3 h-post dose Expt. 4
Expt. 5
Pooled ~~__~_ mean RkSD,
Normal rats Tlbla Tibia muscle Blood
2.19 f 0.36 0.006 f 0.001 0.066 t 0.01 I
2.92 I 0.22 0.008 f 0.002 0.046 t 0.012
2.86 + 0.09 0.007 + 0.001 0.056 t 0.014
Tumor bearine rats Lesioned tibia Tibia muscle Blood
3.81 IO.25 0.016 + 0.005 0.073 + 0.017
3.98 i 0.36 0.018 f 0.003 0.064 + 0.004
3.90 f 0.12 0.017 f 0.001 0.069 + 0.006
Normal rats Tibia Tlbla muscle Blood
2.8010.17 0.011 * 0.004 0.017 * 0.004
0.010rt_0.004
0.011+ 0.001
0.027 I 0.007
0.022 f 0.007
Tumor bearing rats Lesioned tibia Tibia muscle Blood
3.61 + 0.24 0.010 * 0.001 0.024 f 0.004
3.63 iO.14 0.010 * 0.002 0.029 + 0.006
3.62 i 0.01 0.010 + 0.001 0.027 f 0.004
Fraction
YU
Fractron X
pH 8 of - 8.0 _t 0.3 and a large partial molar volume of - 1600 mL/mol.“” The biodistribution characteristics of the two 99”Tc-HEDP component fractions were determined with the Walker 256 osteogenic model by means of two experimental trials (Table 9). The consistency in the lesioned tibia to normal tibia ratios (Table 10) between the two experiments indicated that no variation in tumor growth existed between the experiments. This consistency was achieved through an improvement in techniques of tumor induction gained as a result of experience from previous experiments. The remarkable degree of reproducibility among all of the tissue uptakes between the two experiments (Table 9), result in part from the fact that pure 99”Tc-HEDP complexes were being evaluated as opposed to reaction mixtures. The most striking result is that the uptakes between the two types of 99”Tc-HEDP complexes in normal bone are virtually identical, while the uptakes in the lesioned tibia vary significantly (Table 9), leading to a difference between the lesion to normal bone ratios (Table 10). Although detailed subsequent biodistribution studies, involving six of the 99mT~-HEDP complexes from this separation (Fig. 8), indicate that the normal bone uptakes range from 0.41 to 3.41% dose/g, the osteogenic lesion uptakes do not parallel the normal bone uptakes.(35’ The large, high charges 99”‘Tc-HEDP complex “X” exhibited similar muscle and blood uptakes between the normal and tumor bearing rats; whereas, the small. low charged 99”‘Tc-HEDP complexed “YU” showed consistently higher blood uptakes in both the animal groups, compared with the 99”Tc-HEDP complex “X”. but lower uptakes in both the muscle and the blood for normal rats than in the tumor bearing rats (Table 9). These results indicate that higher lesion to normal bone ratios can be achieved with low
2.92 _+0.12
2.86 k 0.09
charged, monomeric 99’“Tc-diphosphonate complexes compared to high charged, polymeric complexes; however, at the expense of soft tissue uptakes (Table 10). The findings also implicate the mechanism behind the enhanced uptake of the *‘“Tc-HEDP complexes “YU”, in this discrete secondary periosteal osteogenesis, as involving more the interaction of the 99”‘Tc-HEDP complexes with the blood pool constituents than a likely difference in extraction efficiency by the bone tissue. In either case, the differences in physical properties between the complexes, play a critical role in determining the uptake of the 99”‘Tc-diphosphonate in osseous lesions.
Conclusions The Walker 256 tumor when implanted intramuscularly in Fisher 344 rats induces a discrete secondary periosteal osteogenesis in adjacent cortical bone. Biodistribution studies with the Walker 256 model on 99’“Tc-MDP and 99”‘Tc-HMDP commercial kits, and on two HPLC 99’“Tc-HEDP separated fractions demonstrate ihat the model can provide valuable information concerning the uptake characteristics of %‘“Tc-diphosphonates in this type of bone lesion. Advantages of the model include the relative Table 10. Ratios of uptakes in lesioned tibia to selected tissues for HPLC separated ““Tc-HEDP fractions YU and X in Fig. 8 Expt. 4 Lesioned tibia/normal 99’“Tc-HEDP, YU 99”Tc-HEDP, X
Expt. 5
Pooled mean
tibia
Lesioned tibiajcontralateral *“Tc-HEDP, YU W’“Tc-HEDP, X
1.37 1.29
1.36 1.24
1,37 + 0.01 1.27& 0.04
tibia muscle (tumor bearing rats) 240 210 225 + 20 361 362 + 1 363
Lesioned tibia/blood (tumor bearing rats) 52 ““‘Tc-HEDP, YU 62 150 120 ‘++“Tc-HEDP, X
57 + 7 135 i 20
K. T. CHENGet al.
208
low cost, the ease of maintaining the tumor line, either in vivo or in culture media, and the high incidence of tumor induction following intramuscular implantation of cell. Acknowledgemenn-This research has been funded in part by contributions from the American Cancer Society, the Indiana Elks Association and the Society of Nuclear Medicine. The authors wish to express their appreciation to Bruce Wigley, Margaret Hushek, John Ortman, Bonnie Lawson, Carla Powell, Martin Mikelsons and G. Michael Wilson for laboratory assistance in carrying out the animal studies.
Co, Miami Valley Laboratories. Cincinnati. Ohio. U.S.A. (Unnublished work). 22. Hulth A. and Olerud S. Acta Orthop. &and. 36, 230 (1965).
23. Baltimore J. P. In Cell and Tissue Culture, pp. 79-109 (Williams and Wilkins, Baltimore, U.S.A., 1965). 24. Gopel S. B. In Fundamentals of Nuclear Pharmacy, 2nd edn, p. 63 (Springer-Verlag, Berlin). 25. Vivian A., Ice R. D., Shen V. and Hetzel K. R. Radiochemical Purity of Radiopharmaceuticals. using Gelman Sprachrom (ITLC) Chromatograph, p. I-40.
(Gelman Instrument Co.) 26. Pinkerton T. C., Heineman W. R. and Deutsch E. .4nai. Chem. 52, 1106 (1980). 27. Wilson G. M. and Pinkerton T. C. Anal. Chem. 57( 1). 246 (1985).
References 1. Kirchner P. T. In Nuclear Medicine Review Syllabus pp. 540-586 (The Society of Nuclear Medicine, U.S.A., 1980). 2. Sy W. M. In Gamma Images in Benign and Metabolic Bone Diseases, Vol. 1, pp. 2-34 (CRC Press Inc., Boca Raton, U.S.A., 1980). 3. Citrin D. L., Bessent R. G. and Greig W. R. Clin. Radiol. 28, 107 (1977). 4. Tofe A. J., Francis M. D. and Harvey W. J. J. Nucl. Med. 16, 986 (1975). 5. Pauwels E. K. J., Blom J., Camps J. A. J., Hemans J. and Rijke A. M. Eur. J. Nucl. Med. B, 118 (1983). 6. Matin P. Semin. Nucl. Med. 13, 104 (1983). 7. Pauwels E. K. J., Schutte H. E. and Conis W. K. In Bone Scintigraphy, pp. 8-10 (Leiden University Press, Boston, U.S.A., 1981). 8. Fogelman F. Eur. J. Nucl. Med. 5, 473 (1980). 9. Van den Brand J. A. G. M., Das H. A., Dekker B. G., De Ligny, C. L. and Van den Hamer C. J. A. Int. J. Appl. Radiat. Isot. 33, 917 (1982).
10. Tilden R. L., Jackson J., Enneking W. F., Deland F. H. and McVey T., J. J. Nucl. Med. 14, 576 (1973). II. Christensen S. B. and Krogsgaard 0. W. J. Nucl. Med. 22, 237 (1981). 12. Genant H. K., Bautovich G. J., Singh M., Lathrop L. A. and Harner P. V. Radioloav. 113. 373 (1974). 13. Garcia D. A., Tow D. E., Kapur K. K: and Wells H. J. Nucl. Med. 17, 93 (1975). 14. Schumichen C., Koch K., Kraus A., Kuhliche G., Weiler K., Wenn A. and Hoffman G. J. Nucl. Med. 21, 1080 (1980). 15. Pinkerton T. C., Ferguson D. L., Deutsch E., Heineman W. R. and Libson K. Int. J. Appl. Radiat. 33,907 (1982). 16. Subramanian G., Proc. Third World Congress of Nucl. Med. & Biol., Paris, France, Aug. 29-Sept. 2, 1982, p. 1204. 17. Rosenthal1 L., Stem J. and Arzoumanian A. Clin. Nucf. Med. 7, 403 (1982).
18. Ekelman W. C. and Volkert W. A. Int. J. Appl. Radiat. Isot. 33, 945 (1982).
19. Van Deputte K. L. and Urist M. R. Clin. Orthop. 43, 257 (1965). 20. Francis M. D., Ferguson D. L., Tofe A. J., Bevan J. A. and Michaels S. E. J. Nucl. Med. 21, 1185 (1980). 21. Johnson K. and Martodam R. The Procter and Gamble
28. Speckman T. W. and Norris W. P. Radiat. Res. 23, 461 (1964).
29. Subramanian
G., McAfee J. G.. Thomas F. D. et al.
Radiology 149, 823 (1983).
30. Brammer I., Zywietz F. and Jung H. Eur. J. Cancer 15, 1329 (1979).
31. Schafer A., Raue F., Minne H. and Zieglar R. Calc$ Tissue Res. 22, 368 (1977).
32. Minnie H., Raue F., Bellwinkel S. and Ziegler R. Acta Endocr. 78, 613 (1976).
33. Earle W. R. Am. J. Cancer 24, 566 (1935). 34. Fisher E. R. and Fisher B. Cancer Res. 21, 527 (1961). 35. Cheng K. T. PhD Thesis, Purdue University, West Lafayette, Indiana (1985). 36. Robinson C. D. Drugs of Today, M(5), 235 (1982). 37. Littlefield J. L. and Rudd T. G. J. Nucl. Med. 24, 463 (1983). 38. Domstad P. A., Coupal J. J., Kim E. E. et al. Radiology 136, 209 (1980). 39. Muranaka A., Yasuhiko I., Nobuaki 0. et al. Jap. J. Nucf. Med. M(2), 273 (1981).
40. Rosenthal1 L., Arzoumanian A., Damiew B. and Tremblay J. Clin. Nucl. Med. 6, 353 (1981). 41. Nakano S., Hasegawa Y., Shiomura et al. Jap. J. Nucl. Med. 18(10), 1485 (1981). 42. Okahasi S., Ishimaru T., Maeda H. ef al. Radioisotopes 30, 458 (1981).
43. Ishii C., Mori Y., Hata Y. et al. Radioisotopes 30, 401 (1981).
44. Kosuda S., Sanmiya T., Kitagawa I. ef al. Radioisotopes 30, 404 (1981).
45. Honda H., Oshiumi Y., Kamoi I. et al. Jap. J. Nucl. Med. M(9), 1315 (1981). 46. Oshiro N., Bito S., Ito H. et al. Jap. J. Nucl. Med. 305 (1982). 47. Silberstein E. B. Radiology 136, 747 (1980). 48. Srivastava S. C., Meinken G. E., Richards P., L. A. and Benson W. R. Presented to Third Congress of Nuclear Medicine and Biology,
19(2),
Ford World
Paris, France, August 29-September 1982. 49. Tanabe S., Zodda J. P., Deutsch E. and He&man W. R. Int. J. Appl. Radiat. Isot. 34, 1577 (1983). 50. Pauwels E. K. J., Born J., Camps J. A. J., Hermans J. and Rijke A. M. Eur. J. Nucl. med. 8, 118 (1983). 51. Tanabe S.. Zodda J. P.. Libson K.. Deutsch E. and Heineman ‘W. R. Int. J: Appl. Radiat. Isot. 34, 1585 (1983).
CopyrIght
0047-0740185 $3.00 + 0.00 c‘ 1985 Pergamon Press Ltd
A Walker 256 Tumor-Induced Osteogenic Small Animal Model for the Evaluation of [99mTc] Diphosphonate Radiopharmaceuticals K. T. CHENG,’ S. M. SHAW,’ T. C. PINKERTON,‘*
D. J. HOCH’ and D. C. VAN SICKLE3 ‘School of Pharmacy. Department of Medicinal Chemistry, ‘School of Science, Department of Chemistry and ‘School of Veterinary Medicine. Department of Veterinary Anatomy. Purdue University. West Lafayette. IN 47907. U.S.A. (Received 22 March 1985)
A mammalian model has been developed for the in viuo evaluation of bone imaging agents. The model is based upon the quantification of a discrete. initial secondary periosteal osteogenesis induced in cortical bone immediately adjacent lo an intramuscularly implanted Walker 256 tumor in Fisher 344 rats. Evaluation of the model consists of a histopathological examination of the periosteal bone formation. biodistribution studies on %“Tc-MDP and W”‘Tc-HMDP commercial kit preparations, and biodistribution studies on two “‘“‘Tc-HEDP component fractions isolated after anion exchange chromatographic separations from an investigative “carrier added” preparation. Reversed phase HPLC separations of the W”‘Tc-MDP and W”Tc-HMDP commercial kit preparations illustrate distinct differences in chemical composition between the two bone agents.
mineralized bone. +“’ Fogelman@) has suggested that the skeletal uptake in this region is more likely due to factors affecting mineralization, rather than the presence of immature collagen, with the technetium diphosphonates being adsorbed onto the surface of newly deposited hydroxyapatite crystals. Genant et a/.“*’ speculate that this “short term” uptake of bone imaging agents results from an ion exchange process on bone surfaces in contact with the circulating fluids, thus indicating periosteal, endosteal, trabecular and lacunar canalicular (osteonal) surfaces as predominate sites of rapid exchange. In the case of skeletal abnormalities involving osteolytic lesions the increased uptake of bone imaging agents parallels the increased rate of the compensatory osteogenic response. (“) Differences in biodistribution between various bone imaging agents appear to be dependent upon differences in (i) the diffusional processes across physiological barrierqt8) (ii) the degree of affinity of
Introduction Radiopharmaceutical bone imaging constitutes a sensitive means of detecting skeletal abnormalities involving discrete osteogenesis.“.” Bone imaging can detect bone metastases from six months to one year before visualization by radiography becomes possible.“-” Traumatic bone injuries, such as stress fractures. which are initially undetected radiographically, can also be detected by bone imaging.‘@ The “‘“Tc-labeled diphosphonates are currently the agents of choice for bone imaging”’ (Table 1). Although the exact mechanism for the localization of the bone imaging agents is not entirely known, the uptake of the ““Tc-diphosphonates on bone has been associated with regions of high metabolic bone turnover.‘*’ in particular. at the junction of osteoid and * Author to whom correspondence
should be addressed.
Table I. Chelating diohouhonate 4genr
kands
Ligand 0 R, 0 :I ~ HO-P-C-;-OH OH&
““‘Tc-MDP ““‘Tc-HMDP ““‘Tc-HEDP
OH
MDP. methylene diphosphonate (R, = H. RI = H) HMDP. hydroxymethylene diphosphonate (R, = H. R, = OH) HEDP. hydroxyethylidene diphosphonate (R, = CH,, R! = OH) 197
198
K. T.
CHENG et ui.
the agents for blood components (i.e. serum proteins)u4) and (iii) the ion exchange properties of the agents,‘2.12.‘5’
Research efforts aimed at improving the efficacy of bone imaging agents have been directed toward the production of technetium diphosphonates which exhibit a greater affinity for sites of osteogenesis”6.‘7’ and a more rapid clearance from the blood and soft tissue.“‘) In the search for bone imaging agents with higher selectivity for bone lesions, investigators must have a relatively inexpensive animal model which provides discrete sites of osteogenesis analogous to clinical abnormalities of interest. In this regard, the utilization of normal animals for the screening of potentially new bone’ imaging agents is not ideal, for a higher normal bone uptake does not imply an analogously higher lesion uptake”@ or faster soft tissue clearance. Three osteogenic animal models have been developed previously for the evaluation of bone imaging agents. These are (i) the pellet model, which involves the implantation of demineralized bone matrix;“3.‘9,20’ (ii) the drill hole model;u6) and (iii) the rachitogenic model.u2) Differences between the Walker 256 discrete periosteal osteogenic model and these previous models include the induction of excess callus osteogenesis with the pellet and drill hole models; the high cost of large animals (rabbits) in the case of the drill hole model; and the chronic pathophysiological state of the rachitogenic model. In a search for a low cost, rapid, and reproducible means of inducing osteogenesis, it was discovered that the intramuscular implantation of a Walker 256 tumor into Fisher 344 rats produced an increase in bone uptake of 99”Tc-HMDP on selected days during tumor growth, implying the presence of new bone.‘2’) Previous work by Hulth and Olerud(**) demonstrated that a VX2 carcinoma, when implanted in the proximity of bone, induced osteogenesis, even though the carcinoma had no natural tendency to grow in bone. In the work by Hulth and Olerud, discrete spiculae of new woven bone appeared at right angles to the bone surface while preosteoblasts had formed in the periosteum near areas of newly formed capillaries. The Walker 256 cells are easily propagated in the ascitic fluid of the host and/or readily stored in culture medium for prolonged periods, and tumor induction upon intramuscular implantation of the cell is successful in 100% of the animals; thus, the Walker 256 tumor in Fisher 344 rats provides a suitable, low cost small animal model for the rapid, reproducible production of discrete osteogenesis. The following study evaluates the potential utilization of the Walker 256 tumor model for the evaluation of bone imaging agents relative to their uptakes in this type of discrete osteogenesis. The investigation includes a histopathological study on the Walker 256 tumor and adjacent bone, multiple biodistribution experiments on two commercial bone imaging kit preparations (““Tc-MDP and “9’“Tc.-HMDP), and
multiple biodistribution experiments on two investigate 9y”‘Tc-HEDP component fractions isolated after an anion exchange high performance liquid chromatographic separation.
Experimental Tumor cell propagatiorr
The Walker 256 tumor cells were supplied initially by The Procter and Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio, U.S.A. The cells were stored in 10% dimethyl sulfoxide. 209~~ fetal calf serum and 70% media 199 at - 19OC in liquid nitrogen. The frozen tumor cells were thawed rapidly with agitation in a 37°C water bath. when needed for implantation. In order to maintain the cell line, 0.1-0.2 mL of cells were injected into the peritoneal cavity of Fisher 344 male rats (weighing 125-145 g. 6-7 weeks old). The tumor cells were allowed to propagate in the ascitic fluid in cico for 3-4 days. These animals were then euthanatized and the ascitic fluid was collected by rinsing the peritoneal cavity of the animals with 10-20 mL of Hanks Balanced Salt Solution (HMS0).(23) The solution was centrifuged at 1OOOrpm for lOmin, and the supernatant was discarded. The recovered cell pellet was treated with 0.83% ammonium chloride to lyse the erythrocytes. This solution was centrifuged at 1000 rpm for 10 min. The lysed erythrocytes were removed by decanting the supernatant. The tumor cells were resuspended in the appropriate volume of HBSO to obtain a final concentration of 10’ viable cells per tenth of a milliliter. The number of viable cells was determined with trypan blue and a hemocytometer. Tumor induction
Fisher 344 inbred male rats (weighing 125-145 g, 6-7 weeks old) were used throughout the study. A tumor was induced by injecting a suspension of 10’ viable Walker 256 tumor cells in 0. IO-O. 15 mL of HBSO intramuscularly into the inferior region of the gastroenemius of the left leg of a Fisher 344 rat. Animals were kept in individual cages with controlled temperature and light. Standard laboratory chow and tap water were allowed ad libitum. Radiopharmaceutical preparations
Sodium [%“Tc]pertechnetate was obtained from v9Mo/99Tc]generators and supplied in saline from Syncor International Inc., Indianapolis, IN, U.S.A. The 99mTc-diphosphonate kits, OsteoliteO 9”“Tc-MDP (Lot No. 6050, New England Nuclear, North Billerica, MA, U.S.A.) and Osteoscan@ 99”‘Tc-HMDP (Lot No. DL82065, Procter and Gamble, Co.. Cincinnati, OH. U.S.A.) were prepared in accordance to manufacturer’s instructions. Tests for aluminum content in each preparation were performed by standard procedures,‘Z4’ and all results were negative. Instant thin layer chromatography with acetone and normal saline’?” indicated that the
Evaluation
of bone imaging
preparations were greater than 90% radiochemically pure and contained less than 5% “hydrolyzed reduced” technetium. Liquid chromatography
qf commercial kits
In order to evaluate the compositional nature of the commercial tin-reduced technetium diphosphonate radiopharmaceuticals, the Osteolite@ 99”Tc-MDP and Osteoscan@ 99”‘Tc-HMDP kit preparations were subjected to reversed phase ion-pairing high performance liquid chromatography. In order to accommodate the possible presence of polymeric species a silica bonded reversed phase packing of large pore diameter was chosen (Vydac Cl 8, 4.6 mm i.d. x 25 cm 1. 5 pm particle diameter, pore size 33OA, Ranin Instrument Co., Wobum, Mass., U.S.A.). The reversed phase separations were conducted at ambient temperatures with an isocratic aqueous mobile phase containing 1.8 x 10-j M tetrabutyl ammonium hydroxide as the ion-pairing reagent, adjusted to pH 6 with acetic acid. In addition, the mobile phase contained ethyl acetate as an organic modifier (4;; by volume for the 99’“Tc-MDP separation and So/, by volume for the 99”Tc-HMDP separation). Constant flow rates were maintained with a LDC Constametric III@ dual piston pump (Laboratory Data Control, Riviera Beach, Fla., U.S.A.). Samples were injected via a Rheodyne Model 7125 injection valve equipped with a 2OpL loop (Rheodyne Inc., Cotati, Calif., U.S.A.). The y9mTc-diphosphonate separated components were detected by passage of the eluent flow into the well of a NaI(TI) well 7 counter via 0.25 mm i.d. stainless steel tubing.
199
agents
day for seven consecutive days. Each animal was administered 10 PCi of 99”‘T~-HMDP (0.02 mg/kg) .via the jugular vein while under ether anesthesia. Each animal was euthanatized by cardiac puncture at 3 h post injection. The tibia adjacent to the tumor, the contralateral tibia, the femur of the tumor leg and the contralateral femur were removed and isolated from soft tissue. The 99”?c activity in the bone was determined by means of a NaI(TI) scintillation well crystal detector. The bone samples were weighed and the data was expressed as percent of injected dose per gram of tissue. Histology
Based upon the significant by increased 99mTc-HMDP uptake in the tibia adjacent to the tumor, the eighth day after tumor implantation was selected for histological study. Ten rats were divided randomly into two groups. Tumor cells were implanted into five rats with an additional five rats receiving only HBSO. Eight days post-injection, all of the animals were euthanatized and the tibia of each animal removed. The tibias were decalcified in a mixture of formic acid and sodium citrate. processed for hard tissue histopathology in a standard fashion, and serial sections were stained with hematoxylineosin and safranin O-fast green. Biodisrrihution
studies
Three separate experimental trials were conducted during the biodistribution study of the 99”Tc-MDP and 99”‘Tc-HMDP commercial kit preparations. In each experiment, the Fisher 344 rats were randomly selected and divided into tumor bearing groups and normal control groups for each radiopharmaceutical lmestigatice preparation product. Each animal group contained 6 animals; An investigative technetium hydroxyethylidene therefore the overall biodistribution pooled means ““7~-HEDP complex solution was prepared by the for the 99’“Tc-MDP and 99”Tc-HMDP in the study were accumulated from 18 tumor-bearing animals reduction of [99”Tc]pertechnetate with sodium borohydride in the presence of [99mTc]pertechnetate and 18 normal animals. “carrier” by a procedure previously described.“5~26~27’ In each study, eight days after tumor implantation, The ““‘Tc-HEDP solution was subjected to anion 10 p Cl of the 99”Tc-diphosphonate preparation along exchange separation on Aminex-A29 (4.6 mm with 1 pCi of *%.rwas injected into ether-anesthetized i.d. x 25 cm 1. Bio-Rad Laboratories, Richmond, rats via the jugular vein. Three hours after injection Calif.. U.S.A.) with an aqueous mobile phase of each rat was euthanatized by cardiac puncture. The 0.85 M sodium acetate at pH -8.“‘) Two blood. heart, stomach, spleen, liver, kidney, and ‘““‘Tc-HEDP component fractions were isolated for in muscle in the tibia contralateral to the tumor were removed. The tibia adjacent to the tumor, the tibia riro evaluation from the chromatographic separation of approximately twelve 99mTc-HEDP complexes. The contralateral to the tumor bearing leg, the femur of isolated fractions containing the pure 99”Tc-HEDP the tumor bearing leg and the femur contralateral to the tumor bearing leg were excised from the tumor complexes were made isotonic in acetate and buffered animals, defleshed and blotted dry. All samples were to pH = 7.4 by dilution with 0.05 M orthophosphate. weighed and counted for 99mT~ and “Sr activity independently. Corresponding tissues were removed from normal control animals and analyzed m the “‘“‘Tc-HMDP was used as an indicator of osteoblastic activity induced on the inferior surface of the same manner. Activity was expressed as percent of tibia adjacent to the tumor. Tumor cells were iminjected dose per gram of tissue. The 99”Tc-HEDP planted in 24 Fisher 344 rats by methods just de- component fractions isolated by an HPLC separation. and designated as “YU” and “X”,“” were scribed. Starting from the second day after tumor implantation. three rats were chosen randomly each evaluated by means of two biodistribution experi-
CHENG et al.
K. T.
200
ments each in which groups consisted of 6 tumor bearing animals and 6 normal animals for each component fraction. The same experiment procedure as described above was employed, except the use of “Sr was found to be unnecessary. Strontium
correction
The 99mTcuptakes in the bone tissues were found to vary slightly with the inherent differences in age among the animals employed for each of the three experiments carried out during the study. The ages of the Fisher 344 ranged from 6 to 7 weeks. The slight variation in age between the animal experimental groups was compensated by utilizing %r as an internal standard. Speckman and Norris(28) have demonstrated the correlation between the age of rats and mice and the 85Srretention in normal bone. The ““Tc bone uptakes could be normalized from the inherent group variation through multiplication by a *%r correction factor, taken as the ratio of the mean 85Sr uptake in normal bone among the specific animal group to the mean *‘Sr uptake in normal bone for all non-diseased animals employed in the three experiments. The normalization improved the coefficient of variation in the pooled mean bone uptakes from 0 to 8%. Subramanian et al. (*‘) has employed a similar *‘Sr correction to normalize for the weight and age of New Zealand white rabbits used to evaluate the uptake of 99”‘Tc-diphosphonates in callus lesions with the drill hole model.
Results and Discussion Histology
of bone
In order to determine the New bone formation. potential for induced osteogenesis during the growth of the Walker 256 tumor, injection of 99”Tc-HMDP were administered to groups of animals during the
-
tibia
I ----
femur
??
0
growth period. The 9y”‘Tc-HMDP activity in the bone adjacent to the tumor was compared to that of the contralateral bone. The results expressed as ratios (i.e. bone adjacent to tumorcontralateral bone) for both the tibia and femur are illustrated in Fig. I. There appeared to be no significant difference in 99”‘Tc-HMDP uptake between the bone adjacent to the tumor and the contralateral bone from the second day to the sixth day after tumor cell implantation: increase of however, there was a significant *“‘Tc-HMDP uptake in the tibia adjacent to the tumor on the seventh and eighth day. This increase in uptake indicated the suspected presence of increased osteogenesis. A corresponding increase in 99mTcactivity was not observed in the femur. The properties of the Walker 256 tumor have been found previously to include rapid growth, malignancy, osseous invasiveness, hypercalcemia and osteolysis. (30-34)It is likely that this transient osteogenesis corresponds to an observed drop in serum calcium which immediately follows the tumor’s primary hypercalcemic response on day six.‘32’ Several days beyond day eight the osteogenesis is overcome by osteolysis, followed by death of the host.‘3@3J’ Microscopy. After eight days of tumor growth. all implanted animals had enlargements in the region of the gastroenemius (Fig. 2). The enlargements in the muscle consisted of packed round mononucleated cells measuring 15-30 pm in diameter. The tumors when stained with safranin-0 and fast green were variegated in color. The cell’s cytoplasm and their immediate matrix stained red while the green areas appeared to be a degenerating collaginous matrix. Tumor cells were found occasionally in the M-vessels of the proximal metaphysis of the tibia and in one instance there was a nidus of cells in the metaphyseal medullary cavity resembling the tumor. The tumor in the muscle produced a discrete secondary periosteal
,
1
I
I
I
I
1
I
1
2
3
4
5
6
7
6
DAYS AFTER TUMOR IMPLANTATION Fig. I. Ratios of *mTc-HMDP in the tibia and femur of the tumor bearing leg to the tibia and femur of the contralateral leg respectively vs days of tumor growth.
'01
Evaluation of bone imaging agents Table 2. Mean rissue distributions
for W”Tc-MDP
in normal
rats
Percent dose per gram of tissue at 3 h post dose TlSSUe
Expt.
Tibia Femur
I
2.34 + 0.19 2.14+0 II
Tibia muscle Blood Spleen Liver Kidney Stomach Heart
0.014 * 0 009 0.043 f 0.008 0.041 f 0.010 0.061 f 0.012 1.33*040 0.074 + 0.022 0.015 0.001
f_ i SD;
2.96 Z 0.30 2.84 f 0.36
3.14&O.ll 2.94 + 0.28
2.81 + 0.42 2.64 i 0.44
* 0.002 k 0.006 * 0.003 & 0.008 & 0.05 + 0.019
reaction in the adjacent tibia (Fig. 3). The same region in the control animals appeared to be normal (Figs 4 and 5). No mitosis was found either in the tumor or in the connective tissue surrounding the tumor. However. within the fibrous layer of the adjacent periosteum and the subadjacent cambrium layer, there were numerous mitotic figures as well as trabeculae of fiber bone forming perpendicularly to the long axis of the tibia1 diaphysis. Cells in this area resembled tumor cells and once the fibrous layer of the periosteum was breached, tumor cells were easily found in this area.
0.006 0 029 0.026 0.047
+ 0.001 + 0.003 + 0.004 + 0.30 1.09+ 0.20 0.03 I + 0.005 0.012 IO.002
0.009 0.034 0.030 0.048 0.97 0.048 0.014
Biodistribution of 99”Tc-HMDP and 99mTc-MDP. The biodistribution characteristics of the two commercial diphosphate kit preparations, 99mTc-HMDP and 99mTc-MDP were evaluated by means of the Walker 256 osteogenic model. The percent dose per gram uptakes of the “9”Tc-diphosphanates in the
Table 3. Mean tlssoe distributions
for *‘“Tc-HMDP
in normal
Percent dose per gram of tissue at 3 h post dose _.~~ ____~____~._ Expt. I Expt. 2 Expt. 3
1.97+
Tibia Femur
0.63 I .82 f 0.52
Tibia muscle Blood Spleen Liver Kldneh Stomach Heart
2.55 + 0.40 2.43 * 0.44
0.009 + 0.001 0.028 f 0.005 0.054 + 0.016 0.083 i 0.008 0.99t0.18 0.068 * 0.013 0.018 + 0.005
0.008 0.044 0.100 0.340 0.71 0.051 0.030
Table 4. Mean tissue distributions
Tibia (tumor bearing leg) Tibia (contralateral) Femur (tumor bearmg leg) Femur (contralateral) ;;$dmuscle Spleen LlVer Kldne! Stomach Heart
(contralateral)
Expt.
I
3.08 t 0.28 2.13 TO.15 2.32 z 0.15 1.82-0.18 0.049 0.012 0.043 0.078 1.05 0.080 0.015
I& 0.006 0.01 I 2 0.015 5 0.012 5 0.10 IO.010 i 0.008
0.004 0.008 0.010 0.012 0.43 0.23 0.002
r t * + t * i
rats -
Pooled
3.10 io.40 2.96 i 0.48
0.001 0.010 0.070 0.240 0.13 0.010 0.008
0.006 0.025 0.029 0.051 0.78 0.040 0.019
3.81 2.75 3.03 2.43 0.042 0.009 0.029 0.045 0.49 0.035 0.017
+ 0.59 t 0.16 : 0.36 _c 0.45 5 0.01 t_ 0.05 I t 0.006 I 0.006 _t 0.05 i_ 0.007 2 0.003
-
2.54 rt 0.57 2.40 t 0.57
i 0.001 * 0.002 i 0.005 +0.017 + 0.21 * 0.005 f 0.019
0.008 + 0.002 0.032 + 0.010 0.061 i 0.036 0.16iO.15 0.8350.15 0.053 * 0.014 0.022 _t 0.007
for 9Q”Tc-MDP m fumor bearing
Expt. 2
Mean
fp,isDi
rats
Percent dose per gram of tissue at 3 h post dose Tissue
+ F * + * + i
tumor bearing and the normal control animals at 3 h post dose for each experimental trial are listed in Tables 2, 3, 4 and 5. Fundamentally, the ““Tc-HMDP and 99’“Tc-MDP exhibited similar in uiuo characteristics. As expected, the ““Tc-activity was significantly higher in the lesioned tibias adjacent to the tumor than in the contralateral tibias or the tibias of the normal animals. The ratios of the uptakes in the lesioned tibias to the normal tibias of the control animals reveal a consistently higher uptake of the W’“Tc-HMDP compared 99’“Tc-MDP in this discrete secondary periosteal osteogenesis (Table 6). Although the uptake of the 99’“Tc-HMDP in this type of lesion appears to be favored over the et a1.,(29’has found the 99”Tc-MDP, Subramanian opposite to be the case for the uptake of these agents in callus lesions produced in New Zealand white rabbits. The uptakes in the contralateral tibias, normal control tibias, contralateral femurs and normal control femurs consistently indicated a greater nor-
Biodistribution of bone agents
Tissue
Mean
Expt. 3
0.007 0.030 0.022 0.037 0.50 0.040
z
Pooled
Expt. 2
Expt. 3 3.39 3.34 3.53 3.26
* 0.31 i 0.36 i 0.38 & 0.34
0.032 0.007 i* 0.004 0.002 0.029 If 0.005 0.040 + 0.006 1.04+0.13 0.037 + 0.006 0.014 * 0.004
Pooled
Mean
Rp+SDI 3.43 2.74 2.96 2.50 0.041 0.009 0.034 0.054 0.86 0.051 0.015
f + + *
0.37 0.61 0.61 0.72
* 0.009 + 0.003 + 0.008 _t 0.021 2 0.32 * 0.025 jr 0.002
-
202
K. T. CHENG er al. Table 5. Mean nssue distnbutions
for YPmTc-HMDP in wnor bearrng rats
Percent dose per gram of tissue at 3 h post dose
Tissue
Expt.
Tibia (tumor bearing leg) Tibia (contralateral) Femur (tumor bearing leg) Femur (contralateral)
I
3.14 If:0.66 2.3 I + 0.49 2.41 + 0.57 2.15 F0.40
Tiba muscle (contralateral) Blood Spleen Liver Kidney Stomach Heart
0.007 _t 0.003 0.037 F 0.006 0.069 + 0.020 0.144 ; 0.050 1.13*0.17 0.097 & 0.008 0.029 k 0.003
ma1 bone uptake for 99”Tc-MDP than for 99”‘Tc-HMDP in the Fisher 344 rats (Tables 2, 3, 4 and 5). The ratios of lesioned tibia to normal tibia (Table 6) between the three experimental trials (which were conducted on separate dates with different animal groups) indicate a systematic variation in the degree of osteogenesis. It was discovered that the mean ratio (A) between the weight of the lesioned tibia and the weight of the contralateral tibias for an animal group was linearly related, throughout the study, to the mean ratio (0) of the percent dose per gram uptake in the lesioned tibias to the percent dose per gram uptake in the normal control tibias (correlation coefficient 0.92). This indicated that the mean ratio of weights (I?) between the lesioned tibia and the contralateral tibia could be used as a quantitative measure of average osteogenesis for a given experiment. The formation of new woven bone on the lesioned tibia adjacent to the tumor obviously added weight to
Table 6. Ratios of uptakes in lesioned tibia to selected tissues for VC-MDP and 99”Tc-HMDP
Exot. 1 Lesioned tibia/normal ‘+?c-MDP w’“Tc-HMDP
tibia I .32 1.59
Le~;siM~;/contralateral260 *=Tc-HMDP 450
Expt. 2
1.29 I .33
Exot. 3 1.08 1.16
mean ,?_+SD, 1.23 kO.13 1.36 + 0.22
tibia muscle 420 (tumor 480 bearing 390 rats)* 120 380 520 450 + 70
Lesioned tibia/blood (tumor bearine rats) ““%-MDP’ ‘63 91 W”%-HMDP 85 61
106 120
87 i 22 91 +27
EXDt.? 3.40 + 2.64 i 2.8s k 2.40 *
ExDt.3 3.6 I + 0.28
0.63 0.65 0.55 0.53
3.42 i_ 0.40 3.61 2 0.47 3.43 + 0.67
0.009 + 0.001 0.051 + 0.004 0.072 + 0.027 0.225 * 0.100 0.73 * 0.09 0.058 k 0.006 0.040+0.015
0.007 0.030 0.033 0.059 0.72 0.047 0.024
i: 0.001 i: 0.002 f 0.003 * 0.013 50.16 f 0.008 f 0.005
Pooled Mean .v. k SD; 3.38 2.79 2.98 2.66
t f + t
0.24 0.57 0.58 0.68
0.008 0.039 0.058 0.143 0.86 0.067 0.03 I
+ z + + i 2
0.001 0.0 I I 0.022 0.083 0.23 0.026 0.008
the tibia, so an approximate weight of an individual lesion could be obtained from the difference in weights between the lesioned tibia (IV,) and the contralateral tibia (WC). The percent dose per gram uptake in the new woven bone (7; dose/g “NWB”) could be calculated from equation (l), % dose/g “NWB” =
y/; x, - ;,; xc w, - WC
% dose/g “NWB” (corrected) = (% dose/g T) (R) - (% dose/g N) R-1
Percent dose per gram of tissue at 3 h post dose (normalized) -__ ““ToMDP WVe-HMDP
Tissue
Coefficient of variation = (SD,/?)
??
100.
(1)
where % X, and %, XCrepresent the percent dose in the lesioned tibia and the contralateral tibia respectively. The ‘?jr normalized pooled mean tibia lesion uptakes for the 99’“Tc-MDP and 99mTc-HMDP, determined by means of equation (l), are given in Table 7. The values appear inordinately high because the systematic variation in tumor growth between the experiments was not taken into account. In calculating the absolute new woven bone uptake, the differences in tumor growth between the experiments could be corrected by utilizing an expression which incorporates the mean weight ratio (I?) between the lesioned tibia and contralateral tibia as estimate of osteogenesis, and the mean per dose per gram uptake in the normal control animals (% dose/g fl) as the best estimate of uptake in the cortical bone for an experimental animal group. This relationship, represented by equation (2) is equivalent to equation (1)
Table 7. sJSr normalized pooled mean bone tissue distributions
Lesioned tibia Normal tibia Lesioned tibia/normal tibia New woven bone (uncorrected for variation in tumor growth, equation 1) New woven bone (corrrected for variation in tumor growth, equation 2)
i
3.42 t 0.33 (10%)’ 2.78 i_ 0.28 (10%) 1.24 + 0.1 I(9%)
3.38 f. 0.16 (5x)* 2.52 + 0.43 (17”/,, 1.37f0.22(16:/,)
9.21 f 0.44(5’4)
9.43 * 0.54 (60,)
7.28 + 0.08 (1%)
7.32 + 0.31 (45’ 0)
(2)
bone
Hcol
bone
Bone marrow
. d
Fig. 2. A sagittal section of tibia adjacent to tumor after eight days of tumor growth (SOFG x 4).
Fig. 3. Enlargements of square area in Fig. 2 (SOFG x 130). (A) Periosteal bone reaction, (B) cortical bone and (C) bone marrow.
203
Fig. 4. A sagittal section of the normal control tibia and gastroenemius
(SOFG x 4).
Fig. 5. Enlargement of square area hematopoietic in Fig. 4 (SOFG x 130). (A) adjacent skeletal muscle, (B) cortical bone and (C) bone marrow.
204
Evaluation of bone Imaging agents Table
8. Summary
of climcal imaging
comparisons performance
between *“‘Tc-HMDP of osteogemc lwons
(38.41.42.4547) (3740) (37.38.41,4446) (37.39.41.43.44.46) (37,3X.40,42.45.46)
Lesion counts: both agents comparable Lesion to normal bone: HMDP z MDP Bone to soft tissue: HMDP > MDP Blood clearance: HMDP z MDP “Image quality”: HMDP slightly better
??Rclerence (number of Patients in each study). 42(60). 43( 15). 44(34). 45(49). 46(15). 47(20).
“““‘Tc-MDP TOI
References*
Observation I. 2. 3. 4. 5.
and
37(1Ol, 38(102).
39(7). 40(20). 41(15).
selected mobile phase conditions and could not where 9, dose/g T is the mean percent dose per gram be distinguished in either preparation. The uptake in the lesioned tibia for an experimental 9ymTc-HMDP preparation yielded nine peaks (Fig. 6) animal group.‘3” The %r normalized pooled mean while the 99mTc-MDP preparation gave six peaks (Fig. new woven bone uptakes for 99”Tc-MDP and 99”‘Tc-HMDP are given in Table 7. These estimates of 7), indicating the complicated compositional nature of these solutions. the absolute lesion uptakes are lower than the unSrivastava et a1.‘48J have isolated tin reduced corrected values. In principle, if no variation in the production of the new woven bone exist between the 99mTc-MDP complex fractions after reversed phase HPLC and demonstrated differences in bioexperiments, then each method should yield comdistribution with normal rats. Other investigators parable values. This fact has been born out in subhave also purified 99”‘Tc-diphosphonates complexes sequent studies where lesion production was very by various chromatographic methods and illustrated reproducible.“” the difference in biodistribution among the pure 99mTc With respect to soft tissues, the 99”‘Tc-MDP yielded complexes within the mixtures.‘9.‘s.5’) No previous a slightly higher muscle and blood uptake over that of 99”Tc-HMDP: however. the 99”Tc-MDP exhibited a lower liver and heart uptake compared to the 99”Tc-HMDP (Tables 2-5). With the exception of the spleen, in the case of 99”‘Tc-HMDP. no appreciable differences in soft tissue uptakes were found between the tumor bearing and normal animals (Tables 2, 3, 4 and 5). The lesioned tibia to contralateral tibia muscle ratios were slightly higher for 99”Tc-HMDP than U9”Tc-MDP. while the ratios of lesioned tibia OSTEOSCAN to blood appeared comparable between the agents (Table 6). Tc-HMDP Clinical observations mvolving 11 studies with a total of 347 patients (summarized in Table 8) have demonstrated that 99”‘Tc-HMDP does indeed possess a slightly higher bone to soft tissue ratio, a more rapid blood clearance and a slightly better “image quality” when compared to 99”‘Tc_MDP,‘37A:)The results of this biodistribution study with the Walker 256 osteogenic model are consistent with these clinical observations. It should be noted that all ““Tc-diphosphonate preparations. commercial and investigative, have been found to contain multiple technetium diphosphonate complexes which can be separated by chroReversed phase iOn_ matographic means. 19.X.Z’.48.491 pairing high performance liquid chromatographic separations of the Osteoscan@ 99”Tc-HMDP and the Osteoliteo “‘“‘Tc-MDP commercial kits prepared under conditions identical to those employed during the animal studies (i.e. “no 99mTc carrier 0 4 8 12 16 20 added”) are illustrated in Figs 6 and 7 respectively. In TIME (MIN) these separations 98 and 96”, respectively. of the Fig. 6. Reversed phase ion-pairing HPLC of Osteoscan @ “““Tc activity injected onto the chromatographic col- %“‘Tc-HMDP. (Gamma response 1 x 104cps full scale. So< umn could be recovered. Free P’“‘Tc]pertechnetate (v’v) ethylacetate. ambient temperature. flow rate eluted at a retention time of -4 min under the 0.35 mL:min, pressure 950 psi.)
L b
1,
I
I,
I,
1,
1.
K. T.
206
CHEYG et
al
diphosphonate preparations accentuates the importance of performing multiple experimental trials when evaluating the performance of bone agents with animal models. It is likely on occasion that the
99”Tc-MDP and 99”‘Tc-HMDP kit preparations would yield identical biodistribution characteristics due to the overlapping physical nature of the component compositions: this explains why some studies have not observed differences in soft tissue uptake and differences in blood clearance between the agents.“O.‘O’
OSTEOLITE
Tc-MDP
Biodistribution of HPLC separated 99mTc-HEDP complexes. Preliminary biodistribution studies were
II
11/ 0
4 TIME
8
1;
(MINI
Fig. 7. Reversed phase ion-pairing HPLC of Osteolitea 99”Tc-MDP. (Gamma response 1 x 104cps full scale, 4% (v/v)
ambient temperature, ethylacetate, 0.60 mL/min, pressure 1400 psi).
flow
rate
studies, however, have attempted to evaluate the differences in uptake of HPLC separated 99mTccomplexes with an osteogenic lesion model. The biodistribution characteristics of the 99”‘Tc-MDP and *“‘Tc-HMDP commercial bone kits given in Tables 2 through 7 represent composite distributions which reflect the physical properties of the individual complexes and the relative abundance of particular complexes within a preparation. Five primary factors have been found to affect the chemical composition of technetium diphosphonate preparations; these factors include (i) the technetium concentration,‘26.49’(ii) the reductant concentration,(9) (iii) the presence of residual oxygen,‘*@(iv) temperature(48) and (v) PH.(~~’ The variations in muscle uptakes for 99’“Tc-MDP between the experiments (Tables 2 and 4) suggests that the component composition of the *‘“Tc-MDP preparations might have differed slightly during the study. This is consistent with the findings that the greatest differences in uptakes between the individual HPLC separated W”‘Tc-MDP complexes is among the soft tissues.‘4*’ This potential for compositional variation of the complexes within the technetium
conducted on two investigative 99”Tc-HEDP component fractions isolated after anion exchange HPLC (Fig. 8) from a sodium borohydride reduced “carrier preparation described elsewhere.““.z6.‘7’ added” Higher resolution anion exchange HPLC separations with reanalysis of the separated components by HPLC confirm that these technetium diphosphonate complexes are kinetically stable and substitution inert.‘*‘) By varying the HPLC anion exchange counterion concentration at constant pH, temperature and flow rate, and measuring corrected retention times, the average negative charges on 12 of these complexes at pH 8 have been determined to range from - 1.5 to -8.0.‘27) The early elution of highly charged complexes from the anion exchange HPLC and size exclusion chromatography indicate that some of the %“‘Tc-HEDP complexes are polymeric in nature.‘27’ The %“Tc-HEDP component fraction “YU” (Fig. 8), chosen for this preliminary biodistribution study with the Walker 256 osteogenic model, contains two complexes Y and U (assumably monomeric polyprotic species) with low average negative charges at pH 8 of - 1.5 k 0.1 and - 1.6 + 0.1, and small partial molar volumes of 510 mL/mol and 520 mL/mol respectively!*” The WmTc-HEDP component “X”, on the other hand, contains a single complex (assumed to be polymeric in nature) with a high negative charge at
0221
SY 405 nm delectmn X
Y =: 2
P QR
2
0
A LJGL 0
u
TM
v
Z
20
40
60
RETENTION
80
TIME
100
(mln)
Fig. 8. Anion exchange HPLC of sodium borohydide reduced “carrier added” W’“Tc-HEDP on Aminex A-29. (Spectrophotometric detection at 405 nm, isocratic elution with 0.75 M sodium acetate, temperature 28’C. Row rate 0. I1 mlimin. pressure 950 psi.)
Evaluation Table 9. Mean tissue distributions
of bone imaging agents
207
for P9’“Tc-HEDP. HPLC separated fraction YU and X in Fig. 8
Percent dose per gram of tissue at 3 h-post dose Expt. 4
Expt. 5
Pooled ~~__~_ mean RkSD,
Normal rats Tlbla Tibia muscle Blood
2.19 f 0.36 0.006 f 0.001 0.066 t 0.01 I
2.92 I 0.22 0.008 f 0.002 0.046 t 0.012
2.86 + 0.09 0.007 + 0.001 0.056 t 0.014
Tumor bearine rats Lesioned tibia Tibia muscle Blood
3.81 IO.25 0.016 + 0.005 0.073 + 0.017
3.98 i 0.36 0.018 f 0.003 0.064 + 0.004
3.90 f 0.12 0.017 f 0.001 0.069 + 0.006
Normal rats Tibia Tlbla muscle Blood
2.8010.17 0.011 * 0.004 0.017 * 0.004
0.010rt_0.004
0.011+ 0.001
0.027 I 0.007
0.022 f 0.007
Tumor bearing rats Lesioned tibia Tibia muscle Blood
3.61 + 0.24 0.010 * 0.001 0.024 f 0.004
3.63 iO.14 0.010 * 0.002 0.029 + 0.006
3.62 i 0.01 0.010 + 0.001 0.027 f 0.004
Fraction
YU
Fractron X
pH 8 of - 8.0 _t 0.3 and a large partial molar volume of - 1600 mL/mol.“” The biodistribution characteristics of the two 99”Tc-HEDP component fractions were determined with the Walker 256 osteogenic model by means of two experimental trials (Table 9). The consistency in the lesioned tibia to normal tibia ratios (Table 10) between the two experiments indicated that no variation in tumor growth existed between the experiments. This consistency was achieved through an improvement in techniques of tumor induction gained as a result of experience from previous experiments. The remarkable degree of reproducibility among all of the tissue uptakes between the two experiments (Table 9), result in part from the fact that pure 99”Tc-HEDP complexes were being evaluated as opposed to reaction mixtures. The most striking result is that the uptakes between the two types of 99”Tc-HEDP complexes in normal bone are virtually identical, while the uptakes in the lesioned tibia vary significantly (Table 9), leading to a difference between the lesion to normal bone ratios (Table 10). Although detailed subsequent biodistribution studies, involving six of the 99mT~-HEDP complexes from this separation (Fig. 8), indicate that the normal bone uptakes range from 0.41 to 3.41% dose/g, the osteogenic lesion uptakes do not parallel the normal bone uptakes.(35’ The large, high charges 99”‘Tc-HEDP complex “X” exhibited similar muscle and blood uptakes between the normal and tumor bearing rats; whereas, the small. low charged 99”‘Tc-HEDP complexed “YU” showed consistently higher blood uptakes in both the animal groups, compared with the 99”Tc-HEDP complex “X”. but lower uptakes in both the muscle and the blood for normal rats than in the tumor bearing rats (Table 9). These results indicate that higher lesion to normal bone ratios can be achieved with low
2.92 _+0.12
2.86 k 0.09
charged, monomeric 99’“Tc-diphosphonate complexes compared to high charged, polymeric complexes; however, at the expense of soft tissue uptakes (Table 10). The findings also implicate the mechanism behind the enhanced uptake of the *‘“Tc-HEDP complexes “YU”, in this discrete secondary periosteal osteogenesis, as involving more the interaction of the 99”‘Tc-HEDP complexes with the blood pool constituents than a likely difference in extraction efficiency by the bone tissue. In either case, the differences in physical properties between the complexes, play a critical role in determining the uptake of the 99”‘Tc-diphosphonate in osseous lesions.
Conclusions The Walker 256 tumor when implanted intramuscularly in Fisher 344 rats induces a discrete secondary periosteal osteogenesis in adjacent cortical bone. Biodistribution studies with the Walker 256 model on 99’“Tc-MDP and 99”‘Tc-HMDP commercial kits, and on two HPLC 99’“Tc-HEDP separated fractions demonstrate ihat the model can provide valuable information concerning the uptake characteristics of %‘“Tc-diphosphonates in this type of bone lesion. Advantages of the model include the relative Table 10. Ratios of uptakes in lesioned tibia to selected tissues for HPLC separated ““Tc-HEDP fractions YU and X in Fig. 8 Expt. 4 Lesioned tibia/normal 99’“Tc-HEDP, YU 99”Tc-HEDP, X
Expt. 5
Pooled mean
tibia
Lesioned tibiajcontralateral *“Tc-HEDP, YU W’“Tc-HEDP, X
1.37 1.29
1.36 1.24
1,37 + 0.01 1.27& 0.04
tibia muscle (tumor bearing rats) 240 210 225 + 20 361 362 + 1 363
Lesioned tibia/blood (tumor bearing rats) 52 ““‘Tc-HEDP, YU 62 150 120 ‘++“Tc-HEDP, X
57 + 7 135 i 20
K. T. CHENGet al.
208
low cost, the ease of maintaining the tumor line, either in vivo or in culture media, and the high incidence of tumor induction following intramuscular implantation of cell. Acknowledgemenn-This research has been funded in part by contributions from the American Cancer Society, the Indiana Elks Association and the Society of Nuclear Medicine. The authors wish to express their appreciation to Bruce Wigley, Margaret Hushek, John Ortman, Bonnie Lawson, Carla Powell, Martin Mikelsons and G. Michael Wilson for laboratory assistance in carrying out the animal studies.
Co, Miami Valley Laboratories. Cincinnati. Ohio. U.S.A. (Unnublished work). 22. Hulth A. and Olerud S. Acta Orthop. &and. 36, 230 (1965).
23. Baltimore J. P. In Cell and Tissue Culture, pp. 79-109 (Williams and Wilkins, Baltimore, U.S.A., 1965). 24. Gopel S. B. In Fundamentals of Nuclear Pharmacy, 2nd edn, p. 63 (Springer-Verlag, Berlin). 25. Vivian A., Ice R. D., Shen V. and Hetzel K. R. Radiochemical Purity of Radiopharmaceuticals. using Gelman Sprachrom (ITLC) Chromatograph, p. I-40.
(Gelman Instrument Co.) 26. Pinkerton T. C., Heineman W. R. and Deutsch E. .4nai. Chem. 52, 1106 (1980). 27. Wilson G. M. and Pinkerton T. C. Anal. Chem. 57( 1). 246 (1985).
References 1. Kirchner P. T. In Nuclear Medicine Review Syllabus pp. 540-586 (The Society of Nuclear Medicine, U.S.A., 1980). 2. Sy W. M. In Gamma Images in Benign and Metabolic Bone Diseases, Vol. 1, pp. 2-34 (CRC Press Inc., Boca Raton, U.S.A., 1980). 3. Citrin D. L., Bessent R. G. and Greig W. R. Clin. Radiol. 28, 107 (1977). 4. Tofe A. J., Francis M. D. and Harvey W. J. J. Nucl. Med. 16, 986 (1975). 5. Pauwels E. K. J., Blom J., Camps J. A. J., Hemans J. and Rijke A. M. Eur. J. Nucl. Med. B, 118 (1983). 6. Matin P. Semin. Nucl. Med. 13, 104 (1983). 7. Pauwels E. K. J., Schutte H. E. and Conis W. K. In Bone Scintigraphy, pp. 8-10 (Leiden University Press, Boston, U.S.A., 1981). 8. Fogelman F. Eur. J. Nucl. Med. 5, 473 (1980). 9. Van den Brand J. A. G. M., Das H. A., Dekker B. G., De Ligny, C. L. and Van den Hamer C. J. A. Int. J. Appl. Radiat. Isot. 33, 917 (1982).
10. Tilden R. L., Jackson J., Enneking W. F., Deland F. H. and McVey T., J. J. Nucl. Med. 14, 576 (1973). II. Christensen S. B. and Krogsgaard 0. W. J. Nucl. Med. 22, 237 (1981). 12. Genant H. K., Bautovich G. J., Singh M., Lathrop L. A. and Harner P. V. Radioloav. 113. 373 (1974). 13. Garcia D. A., Tow D. E., Kapur K. K: and Wells H. J. Nucl. Med. 17, 93 (1975). 14. Schumichen C., Koch K., Kraus A., Kuhliche G., Weiler K., Wenn A. and Hoffman G. J. Nucl. Med. 21, 1080 (1980). 15. Pinkerton T. C., Ferguson D. L., Deutsch E., Heineman W. R. and Libson K. Int. J. Appl. Radiat. 33,907 (1982). 16. Subramanian G., Proc. Third World Congress of Nucl. Med. & Biol., Paris, France, Aug. 29-Sept. 2, 1982, p. 1204. 17. Rosenthal1 L., Stem J. and Arzoumanian A. Clin. Nucf. Med. 7, 403 (1982).
18. Ekelman W. C. and Volkert W. A. Int. J. Appl. Radiat. Isot. 33, 945 (1982).
19. Van Deputte K. L. and Urist M. R. Clin. Orthop. 43, 257 (1965). 20. Francis M. D., Ferguson D. L., Tofe A. J., Bevan J. A. and Michaels S. E. J. Nucl. Med. 21, 1185 (1980). 21. Johnson K. and Martodam R. The Procter and Gamble
28. Speckman T. W. and Norris W. P. Radiat. Res. 23, 461 (1964).
29. Subramanian
G., McAfee J. G.. Thomas F. D. et al.
Radiology 149, 823 (1983).
30. Brammer I., Zywietz F. and Jung H. Eur. J. Cancer 15, 1329 (1979).
31. Schafer A., Raue F., Minne H. and Zieglar R. Calc$ Tissue Res. 22, 368 (1977).
32. Minnie H., Raue F., Bellwinkel S. and Ziegler R. Acta Endocr. 78, 613 (1976).
33. Earle W. R. Am. J. Cancer 24, 566 (1935). 34. Fisher E. R. and Fisher B. Cancer Res. 21, 527 (1961). 35. Cheng K. T. PhD Thesis, Purdue University, West Lafayette, Indiana (1985). 36. Robinson C. D. Drugs of Today, M(5), 235 (1982). 37. Littlefield J. L. and Rudd T. G. J. Nucl. Med. 24, 463 (1983). 38. Domstad P. A., Coupal J. J., Kim E. E. et al. Radiology 136, 209 (1980). 39. Muranaka A., Yasuhiko I., Nobuaki 0. et al. Jap. J. Nucf. Med. M(2), 273 (1981).
40. Rosenthal1 L., Arzoumanian A., Damiew B. and Tremblay J. Clin. Nucl. Med. 6, 353 (1981). 41. Nakano S., Hasegawa Y., Shiomura et al. Jap. J. Nucl. Med. 18(10), 1485 (1981). 42. Okahasi S., Ishimaru T., Maeda H. ef al. Radioisotopes 30, 458 (1981).
43. Ishii C., Mori Y., Hata Y. et al. Radioisotopes 30, 401 (1981).
44. Kosuda S., Sanmiya T., Kitagawa I. ef al. Radioisotopes 30, 404 (1981).
45. Honda H., Oshiumi Y., Kamoi I. et al. Jap. J. Nucl. Med. M(9), 1315 (1981). 46. Oshiro N., Bito S., Ito H. et al. Jap. J. Nucl. Med. 305 (1982). 47. Silberstein E. B. Radiology 136, 747 (1980). 48. Srivastava S. C., Meinken G. E., Richards P., L. A. and Benson W. R. Presented to Third Congress of Nuclear Medicine and Biology,
19(2),
Ford World
Paris, France, August 29-September 1982. 49. Tanabe S., Zodda J. P., Deutsch E. and He&man W. R. Int. J. Appl. Radiat. Isot. 34, 1577 (1983). 50. Pauwels E. K. J., Born J., Camps J. A. J., Hermans J. and Rijke A. M. Eur. J. Nucl. med. 8, 118 (1983). 51. Tanabe S.. Zodda J. P.. Libson K.. Deutsch E. and Heineman ‘W. R. Int. J: Appl. Radiat. Isot. 34, 1585 (1983).