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188Re-labeled bisphosphonates as potential bifunctional agents for therapy in patients with bone metastases
ARTICLE IN PRESS
Applied Radiation and Isotopes 62 (2005) 541–549 www.elsevier.com/locate/apradiso
188
Re-labeled bisphosphonates as potential bifunctional agents for therapy in patients with bone metastases Amal El-Mabhouha, John R. Mercera,b, a
Faculty of Pharmacy and Pharmaceutical Sciences, 3118 Dentistry Pharmacy Center, University of Alberta, Edmonton Alta, Canada T6G-2N8 b Faculty of Medicine, 3118 Dentistry Pharmacy Center, University of Alberta, Edmonton Alta, Canada T6G-2N8 Received 2 June 2004; received in revised form 30 September 2004; accepted 25 October 2004
Abstract Two new bisphosphonates have been examined for their ability to bind 188Re and deliver it selectively to bone. The bisphosphonates are prototype compounds with potential to deliver rhenium radionuclides and a second therapy modality to bone metastases. A conjugate between diethylenetriaminepentaacetic acid and bisphosphonate (DTPA/BP) and a conjugate between 5-fluorouracil and bisphosphonate (5-FU/BP) were prepared and labeled at high radiochemical purity with 188Re and biodistribution studies were carried out in normal Balb/C mice. The compounds showed rapid blood clearance and elimination from soft tissues with substantial retention of activity in the bone comparable to 188Re-hydroxyethylidine diphosphonate used as a control. At 8 h bone activity was 3.51% of injected dose for 188Re-DTPA/BP and 6.38% of injected dose for 188Re-5-FU/BP representing 69.6% and 80.6% of total body radioactivity, respectively. The two compounds show the potential for combination therapy of painful bone metastases. r 2004 Elsevier Ltd. All rights reserved. Keywords: Bone pain palliation; Bisphosphonates; Radiotherapy; Rhenium radioisotopes
1. Introduction The intractable and debilitating pain that may accompany cancer is often produced by bone metastases. While local external beam irradiation is the first choice for palliative treatment for patients with a limited number of lesions (Lin et al., 1997), systemic radiotherapy with radiopharmaceuticals is preferable with widespread bone metastases with multifocal sites of pain (Liepe et al., 2003). Systemic radionuclide therapy with b-emitting radiopharmaceutical has been used since 1942 for the management of painful bone metastases Corresponding author. Tel.: +1 780 492 5364; fax: +1 780 492 1217. E-mail address: [email protected] (J.R. Mercer).
(Pecher, 1942) and it remains a widely used and effective modality (for recent reviews see Roque et al. (2003) and Serafini (2001)) 188Re is an excellent candidate for bparticle therapy (Boschi et al., 2003; Du et al., 2000; Hsieh et al., 1999; Hsieh et al., 1996; Knapp et al., 1997; Lin et al., 1997; Wang et al., 1995, 1996;). Its energetic bparticles (2.1 MeV) have a maximum penetration in tissue of 10–11 mm making this radionuclide a suitable option for large tumor masses. Its g-ray emission (0.155 MeV) can be exploited for dosimetric purposes and to observe biological distribution during therapy (Chen et al., 2001; Hsieh et al., 1996; Hashimoto, 1998; Boschi et al., 2003). It also has a relatively short physical half-life of 16.9 h which allows the use of high doses and reduces the problem of radioactive waste handling and storage (Chen et al., 2001; Boschi et al., 2003; Lin et al.,
0969-8043/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.10.004
ARTICLE IN PRESS A. El-Mabhouh, J.R. Mercer / Applied Radiation and Isotopes 62 (2005) 541–549
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1997; Palmedo et al., 2003; Liepe et al., 2003). 188Re can be obtained by saline elution from a 188W/188Re generator system where the parent 188W has a 69-day half-life. Once the generator is installed 188Re is routinely available for clinical use (Knapp et al., 1997). Targeting of 188Re to bone has been accomplished effectively using the complex 188Re-hydroxyethylidine diphosphonate (188Re-HEDP, Fig. 1). This agent has shown therapeutic efficacy in the treatment of metastatic bone pain associated with cancer of the breast, prostate, lung and other locations (Maxon et al., 1998; Li et al., 2001; Liepe et al., 2003; Zhang et al., 2003). Bisphosphonates have been extensively studied due to their clinical effectiveness in treatment of a variety of pathological processes in the bone. For a systematic review of bisphosphonates in metastatic disease see Ross et al. (2004). Their present uses include treatment of hypercalcaemia, treatment of metastatic bone pain and as a prophylactic treatment for the prevention of pathological fractures and spinal cord compression (Coleman, 2001; Hortobagyi, 2002). The mechanism of the action of bisphosphonates has been extensively investigated (for comprehensive reviews see Reszka and
O O (OH)2P
N (OH)2P
2. Materials and methods OH
H C
N
N
O
2.1. Reagents and instruments OH
N
OH HO
O
O O
O
bisphosphonate DTPA conjugate (DTPA/BP) O
O (OH)2P
H N
(OH)2P
H N
C
O
N
O
O
F
bisphosphonate 5-fluorouracil conjugate (5-FU/BP) O (OH)2P
CH 3
(OH)2P
OH
Rodan (2003) and Fleisch (1998)). Bisphosphonates attach strongly and preferentially to bone with increased avidity for areas of osteoblastic and osteoclastic activity. Once associated with bone these compounds are potent inhibitors of osteoclastic bone resorption. Bearing in mind the attractive therapeutic features of 188 Re and the targeting potential of bisphosphonates, we have investigated the biodistribution and excretion of two 188Re-labeled novel bisphosphonate conjugates. The compounds are conjugates of the bisphosphonate functionality with diethylenetriaminepentaacetic acid and biphosphonate (DTPA/BP) and with 5-fluorouracil and biphosphonate (5-FU/BP) with the structures shown in Fig. 1. Studies were performed in Balb/C mice with particular attention to the accumulation of 188Re in bone. The bisphosphonate portion of these conjugates should allow binding of 188Re and targeting to bone lesions while the conjugated functionalities make these agents potential multimodality agents for therapy. We propose that the DTPA/BP chelate will permit therapeutic radionuclides to be targeted to the bone while the 5-fluorouracil bisphosphonate conjugate may be more selectively delivered to bone metastases without systemic toxicity.
O hydroxyethylidine diphosphonate (HEDP) Fig. 1. Structures for the bisphosphonate conjugates with DTPA and 5-fluorouracil, and for 1-hydroxyethane-1,1-diphosphonic acid (HEDP).
Acetic acid was purchased from Caledon Laboratories Ltd. and anhydrous sodium acetate was purchased from BDH Inc. Hydroxyethylidene diphosphonic acid (HEDP) was purchased from Fluka Chemie GmbH. All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). Double distilled deionized water was used for all the preparations. Silica gel instant thin-layer chromatography sheets (ITLC-SG) were purchased from Gelman Science Inc. A MINAXI g Auto-Gamma 5000 Series gamma counter was used to measure the activity in tissues and chromatography samples. Rhenium-188 was obtained by eluting a commercially available 37 GBq alumina-based 188 W/188Re generator (Oak Ridge National Laboratories) with 10–15 mL of normal saline, according to the manufacturer’s specifications, to provide solutions of no-carrier-added 188Re-sodium perrhenate (NaReO4). 2.2. Preparation of DTPA/BP and 5-FU/BP The bisphosphonate conjugates DTPA/BP and 5-FU/ BP shown in Fig. 1 were synthesized as described previously (El Mabhouh et al., 2004). Briefly, the key intermediate aminobisphosphonate tetraethyl ester was prepared according to the literature procedures (Kantoci et al., 1996). This intermediate was coupled with
ARTICLE IN PRESS A. El-Mabhouh, J.R. Mercer / Applied Radiation and Isotopes 62 (2005) 541–549
diethylenetriaminepentaacetic dianhydride in DMF at 60 1C. After stirring for 9 h, water was added dropwise to the hot reaction mixture and the mixture was then stirred for 5 h at room temperature. After removal of the solvent, the residue was purified by flash chromatography and the required DTPA/BP generated by reaction of the tetraethyl ester with bromotrimethylsilane at 0 1C. To prepare the 5-fluorouracil conjugate the aminobisphosphonate tetraethyl ester was first converted to the isocyanate by the action of triphosgene in acidified dry toluene. Reaction of this isocyanate with 5-fluorouracil was carried out at 90 1C in dry N,N-dimethylacetamide followed by deblocking of the isolated tetraethyl ester product with bromotrimethylsilane to produce the required 5-FU/BP. 2.3. Preparation of and 188Re-HEDP
188
Re-DTPA/BP,
188
Re-5-FU/BP
Reactions were generally carried out in sterilized, evacuated, sealed multidose vials. The vials were charged with 100 mL of 40 mg/mL SnCl2 in 10% aqueous acetic acid, 200 mL of 0.5 M sodium D-gluconate in 0.2 M sodium acetate pH 5 buffer solution and 40 mL of a 2.5 mg/mL aqueous solution of perrhenic acid. A solution of the bisphosphonate conjugate in sterilized DDD water was then added to the vials to produce a final concentration of 5 mg/mL (DTPA/BP or HEDP) or 2 mg/mL (5-FU/BP). Labeling reactions were initiated by the addition of 100–200 mL (nominally 2 MBq) of 188Re-sodium perrhenate to the vials. The reaction mixture was incubated at 90 1C for 3 h, cooled to room temperature and adjusted to pH 6.5–7.0 using 0.1 N HCl or 0.2 M NaOH. The radiochemical purity of 188ReDTPA/BP, 188Re-5-FU/BP or 188Re-HEDP was determined by using ITLC-SG strips (1 12 cm) developed with acetone or saline (0.9% NaCl). In all syntheses the radiolabeled compounds did not require further purification but were used directly in animal studies. 2.4. Preparation of 188Re-sodium perrhenate for injection Injection solutions of sodium perrhenate used in control studies were prepared by dilution of 100 mL (nominally 2 MBq) of 188Re-sodium perrhenate as eluted from the generator to 900 mL with normal saline. 2.5. Animal studies Female Balb/C mice aged between 6 and 8 weeks with weight range of 18–24 g were used in all the studies. Animals were obtained from the University of Alberta, Health Sciences Laboratory Services breeding program and were allowed to acclimatize for several days before being entered into the studies. All procedures are in accordance with the Canadian Council on Animal Care
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and were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. 2.6. Biodistribution studies Female Balb/C mice were injected via a lateral tail vein with the radioactive test agents (188Re-DTPA/BP, 188 Re-5-FU/BP, 188Re-HEDP and 188ReO 4 ) using volumes of approximately 0.1 mL and activities of approximately 200 KBq. Animal numbers are indicated in the Tables 1–5 and were generally 3 or 4 animals per time period. Drug mass was in the range of 500 mg per injection of DTPA/BP or HEDP and was reduced to 200 mg with 5-FU/BP due to potential toxicity with this agent. The mice were sacrificed using a CO2 chamber at times of 1, 2, 4 and 8 h after injection. Blood was collected by cardiac puncture at the time of sacrifice. Whole organs were collected for heart, liver, lungs, spleen, stomach, gastrointestinal tract (GI tract), kidneys, and brain. The thyroid was generally isolated with surrounding tissues of the neck and upper chest. Muscle was collected from the left hind leg, and the whole left tibia was isolated as a representative bone sample. The bone segment comprising the knee joint from the left femur was isolated in selected animals as a representative bone sample expected to exhibit increased osteoblastic activity due to terminal bone plate growth in these young animals. The whole tail was removed and counted separately. The tissues and residual carcass were weighed immediately upon dissection and activity for each sample was determined in a gamma counter. All radioactivity values were decay corrected to time of injection and efficiency corrected to provide true decays. Total blood weight was calculated as 7% of mouse body weight and total wet bone weight was estimated as 12% of mouse body weight.
3. Results All compounds were radiolabeled with high efficiency based on the ITLC analysis which identifies unbound 188 188 ReO ReO2 or other 4 and reduced hydrolyzed colloidal species if they are present. With acetone development free perrhenate moves with the solvent front and the 188Re-bisphosphonate conjugate stays at the origin on the strip, while using saline as eluent causes Re-188 colloidal impurity to remain at the origin while 188 Re-bisphosphonate conjugate and 188Re-sodium perrhenate migrate close to the solvent front. The expected labeled products accounted for 92% or more of total radioactivity using this analysis. No purification of the radiolabeled products was undertaken. Radiolabeled products were injected into test animals immediately following synthesis and quality control assays. DTPA and most bisphosphonates are of low toxicity and LD50
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Table 1 188 Re-DTPA/BP biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 3)
Table 4 188 ReO 4 biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 3)
Organ
Organ
Blood Liver Stomach Kidney Thyroid Bone Tail Joint
Time after injection (h) 1
2
4
8
0.5770.22 0.6470.25 1.2870.24 3.9871.72 0.8270.13 2.9370.69 3.3873.08 5.2570.93
0.3470.04 0.4370.10 0.8270.10 2.9370.39 0.7470.14 3.5870.30 1.1270.19 5.5970.34
0.1070.01 0.1270.00 0.3070.08 1.0270.14 0.2770.09 1.7470.26 0.6770.23 2.5770.29
0.0970.04 0.1370.06 0.2970.18 0.7570.22 0.2170.11 1.4070.31 1.4070.60 2.7870.59
Values are percent of injected dose per gram of wet tissue7standard deviation. Table 2 188 Re-5-FU/BP biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 4) Organ
Blood Liver Stomach Kidney Thyroid Bone Tail Joint
Time after injection (h) 1
2
4
8
0.5470.25 1.3670.62 1.1770.63 4.0471.18 0.6070.38 3.2171.28 3.1772.14 5.3172.40
0.1770.06 1.0670.55 0.6870.49 2.3870.55 0.4570.31 2.6571.05 1.9070.99 4.0971.38
0.1270.06 0.7870.81 0.8270.64 1.1770.31 0.4670.33 2.2671.18 1.8472.39 3.5372.21
0.0970.02 0.8470.27 0.7070.12 1.2670.26 0.3370.04 2.6770.13 1.7971.70 3.9170.20
Values are percent of injected dose per gram of wet tissue7standard deviation. Table 3 188 Re-HEDP biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 3) Organ
Blood Liver Stomach Kidney Thyroid Bone Tail Joint
Time after injection (h) 1a
2
4
8
0.7770.60 0.6070.31 1.5572.20 4.2271.07 0.8470.74 4.5271.44 3.3671.79 6.5771.05b
0.2870.32 0.2770.13 0.8271.31 2.0970.58 0.6370.75 2.6070.46 0.8770.06 5.10c
0.1270.00 0.2070.04 0.2270.05 1.8470.21 0.2770.05 2.9770.02 1.3070.62 5.13c
0.1470.06 0.2370.07 0.2970.15 1.3570.20 0.3770.19 3.2070.06 1.4470.97 5.12c
Values are percent of injected dose per gram of wet tissue7standard deviation. a n ¼ 4. b n ¼ 2. c n ¼ 1.
Time after injection (h) 1
2
4
8
Blood 7.3870.45 4.1370.84 1.1670.38 0.3570.12 Liver 3.9370.83 2.1270.42 0.4570.18 0.1670.07 Stomach 30.8977.14 18.6871.87 5.3471.29 1.6070.91 Kidney 4.1170.55 2.2970.39 0.6270.13 0.2070.08 Thyroid 26.2473.92 18.3575.10 12.4971.47 3.2571.25 Bone 2.7170.25 1.6470.41 0.4170.15 0.2070.06 Tail 4.2270.03 4.8373.98 1.1270.44 1.1371.17 Values are percent of injected dose per gram of wet tissue7standard deviation.
values for 5-fluorouracil are quoted in various sources as being several hundreds of milligrams per kilogram (e.g. Burns and Beland, 1984). No physiological effects or toxicity was observed at the dose levels used in these studies. The bisphosphonate conjugates are all rapidly removed from the blood within the first hour after intravenous injection as indicated by the blood concentrations of activity shown in Tables 1–3. Total blood activity was calculated using the value of 7% of body mass as blood mass and is shown in Table 5. By 1 h 1% or less of the injected activity remains in the blood and this value continues to decline up to 8 h. In contrast 188 Re activity is high and persistent in the blood following injection of 188Re-perrhenate, representing 9.92% of the injected dose at 1 h and 1.66% at 4 h. The whole body activity was determined by summing the activities from all dissected tissues and the residual carcass (Table 5, Fig. 2). The two test compounds and 188 Re-HEDP show similar behavior with rapid initial excretion over the first hour following injection followed by a slow decrease in the residual activity up to 8 h. The biodistribution data in selected tissues for 188Re-DTPA/ BP, 188Re-5-FU/BP, 188Re-HEDP and 188ReO 4 are shown in Tables 1–4, respectively. The radioactivity in the heart, skeletal muscle and brain was low at all time points with all tested compounds. Whole carcass activity for 188Re-HEDP, 188 Re-DTPA/BP and 188Re-5-FU/BP after organ dissection is predominantly due to activity in the bone rather than significant tissue retention. Perrhenate (188ReO 4 ), evaluated here as a control agent, shows high blood levels and higher whole body retention but low bone uptake when compared with the other tested compounds. At early time periods following IV injection, activity from the three 188Re-labelled bisphosphonates were found mainly in bone and kidney. Renal excretion accounted for the rapid early blood activity clearance and explains the kidney activity levels
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Table 5 Activity retained in whole body, total blood and total wet bone at various times after intravenous injection of test compounds Time (h) Injected compound
Activity location
1
2
4
8
188
Whole body Total blood Total bone Bone/body 100%
15.5474.38 0.8270.25 7.2171.22 46.4
10.8472.10 0.4570.06 8.2570.75 76.1
5.1370.29 0.1470.01 4.4470.58 86.5
5.0470.46 0.1370.05 3.5170.65 69.6
188
Whole body Total blood Total bone Bone/body 100%
17.8176.71 0.8070.38 7.8872.75 44.2
11.9172.39 0.2770.08 7.1171.99 59.7
9.6076.42 0.1770.08 5.3072.08 55.2
7.9270.78 0.1370.03 6.3870.84 80.6
188
Whole body Total blood Total bone Bone/body 100%
15.4574.14 1.0370.59 10.4072.60 67.3
11.1172.29 0.5770.39 9.0271.21 81.2
7.6370.48 0.1870.02 7.5670.79 99.1
7.1871.19 0.1970.07 7.8570.61 109
188
Whole body Total blood Total bone
61.8177.79 9.9270.58 6.2470.53
42.9673.10 5.6170.74 3.8370.74
14.3372.73 1.6670.45 1.0170.30
5.0372.24 0.4870.17 0.4770.16
Re-DTPA/BP
Re-5-FU/BP
Re-HEDP
Re-O 4
Values represent the mean percent of total injected radioactive dose7standard deviation (n ¼ 3). Whole bone to whole body ratios are calculated as (whole bone activity/whole body activity) 100%.
whole body activity (%dose)
80
perrhenate Re-188-HEDP Re-188-5-FU/BP Re-188-DTPA
70 60 50 40 30 20 10 0 1h
2h
4h
8h
time Fig. 2. Percent of injected 188Re activity retained in whole body for the bisphosphonate conjugates and for perrhenate over 8 h in Balb/C mice (n ¼ 3).
at 1 h. For 188Re-HEDP the kidney activity had declined to 2.09% injected dose per gram at 2 h while for the test compounds 188Re-DTPA/BP and 188Re-5-FU/BP there was a relatively high activity in the kidneys up to 8 h. Liver activity was generally unremarkable for 188ReHEDP and 188Re-DTPA/BP, being highest at 1 h and then significantly decreased over the 8 h of the study. The absence of liver activity is reassuring as an
indication that labeled colloidal impurities, often produced when high tin concentrations are used in reduction radiolabeling with rhenium, were not present in our preparations. The activity in the liver for 188Re-5FU/BP was somewhat higher and more persistent representing 1.36% of the injected dose at 1 h and 0.84% of the injected dose at 8 h. Tail activities are significantly higher for the bisphosphonates than for the perrhenate and we relate this to the bone content of this structure while the high variability of activity in the tail in some cases indicates some retention of activity at the injection site. The bone activity for the control and test compounds is expressed as percent of injected dose per gram of tissue in Tables 1–4 and as whole body bone activity, using 12% of body mass to represent wet bone, in Table 5 and Fig. 3. As expected, 188Re-perrhenate does not accumulate to any extent in the bone. HEDP, as a positive control known to bind 188Re and deliver it to the bone, had 10.40% of the injected dose in the bone at 1 h and this value decreased only minimally to 7.85% at 8 h. The two test compounds showed similar though somewhat reduced bone binding with values for 188ReDTPA/BP and 188Re-5-FU/BP of 7.21% and 7.88% at 1 h, which declined to 3.51% and 6.38% by 8 h, respectively. Table 5 also reports the percent of total body activity at each time period that is due to bound radioactivity in the bone. These values show that the whole body activity at the later time periods is
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14
perrhenate Re-188-HEDP Re-188-5-FU/BP Re-188-DTPA/BP
bone activity (%dose)
12 10 8 6 4 2 0 1h
2h
4h
8h
time Fig. 3. Percent of injected 188Re activity retained in total bone for the bisphosphonate conjugates and for perrhenate over 8 h in Balb/C mice (n ¼ 3).
predominantly bone activity for 188Re-HEDP as well as the two test compounds. Tables 1–3 indicate significantly higher uptake of the bisphosphonate conjugates in the bone segment comprising the knee joint relative to the bone shaft. In young mice the joints are areas of increased osteoblastic activity due to bone growth with a bone remodeling activity similar to that at the sites of osteoblastic bone metastases. The joint activity for 188Re-HEDP at 1 h was 6.57% ID/g and this decreased only minimally to 5.12% ID/g by 8 h. The test conjugates 188Re-DTPA and 188Re5-FU/BP showed 5.25% ID/g and 5.31% ID/g respectively, at 1 h. Although some of this initially bound activity was lost by 8 h, the value of 2.78% for 188ReDTPA and 3.91% ID/g for 188Re-5-FU/BP still represent significant concentration of activity in this tissue. These results are promising as they indicate effective targeting of the bisphosphonate analogs to the bone and increased uptake at sites of active bone growth.
4. Discussion Bisphosphonates are known to chelate rhenium radioisotopes producing stable complexes (Koudelkova and Jedinakova-Krizova, 2003; Hashimoto and Yoshihara, 1996; Hashimoto, 1998; Maxon et al., 1990) a characteristic which was demonstrated to extend to the present ligands. The radiolabeling of bisphosphonates conjugates (DTPA/BP and 5-FU/BP) with 188Re was achieved by using gluconate as a transchelator in 0.2 M sodium acetate buffer at pH 5.0 (Du et al., 2000). The facile reoxidation of reduced 188Re was prevented by using an evacuated vial and a high
concentration of SnCl2 solution (4 mg/mL). The excess of stannous ions has been found to be necessary to reduce the carrier-added perrhenate and to drive the reduction reaction to completion as far as possible (Deutsch et al., 1986). In the radiolabeling reaction to produce 188Re-DTPA/ BP, 188Re-5-FU/BP and 188Re-HEDP, carrier perrhenic acid was added to the essentially carrier-free radionuclide eluted from the 188W/188Re generator. The presence of macroscopic amounts of stable rhenium is known to increase the labeling yield and the in vivo stability of 188Re-labeled bisphosphonate conjugates (Palmedo et al., 2000; Hashimoto, 1998; Arteaga de Murphy et al., 2001; Hsieh et al., 1999; Lin et al., 1999). The concentration of the perrhenic acid was a critical factor affecting both the synthesis and the in vivo biodistribution of the 188Re-labeled bisphosphonate conjugates. The optimum concentration of carrier was determined by evaluation of the radiolabeled conjugate produced. At a perrhenate concentration lower than 100 mg/mL in the reaction mixture reduced bone uptake of the conjugates was observed, while at a concentration higher than 100 mg/mL colored solutions resulted making the product unsuitable for in vivo use. The addition of small amounts of carrier rhenium will not be a clinical concern since levels can be kept below a toxic threshold for the metal and the concentration of the complexes in vivo will be well below a level that would saturate bisphosphonate binding sites on the bone. Successful labeling also required incubation times of several hours at a temperature of 90 1C and pH 5. Balb/C mice were used in all our studies. This strain is widely used in animal research studies and was readily available to us from the local breeding program. As the present study was a preliminary investigation, and in consideration of animal ethics we restricted our numbers of mice to 3 or 4 per time point in each study. Although this allows distribution and pharmacokinetic data to be generated and general conclusions to be made, the statistical significance of some of the observations would need to be confirmed with a more extensive study. Injected 188Re-perrhenate does not bind to bone but distributes to the gastric mucosa, gastrointestinal tract and thyroid (Hsieh et al., 1999) as observed in our control study (Table 4). These organs do not show significant accumulation of activity with our test compounds or 188Re-HEDP as an indication that there is no perrhenate present in the injected formulations and that this species is not produced following in vivo breakdown of the complexes. 188Re-hydroxyethylidene diphosphonate (188Re-HEDP) was used as a positive control in this study (Table 3). 188Re-HEDP showed the expected distribution, as described by others (Maxon et al., 1998; Lin et al., 1997, 1999; Lisic et al., 2001), with
ARTICLE IN PRESS A. El-Mabhouh, J.R. Mercer / Applied Radiation and Isotopes 62 (2005) 541–549
the kidney and the bone having the highest concentration of activity at all time points investigated. The persistent activity in the tail up to 8 h is partially due to the bony structure of this tissue. The predominate urinary excretion, of bisphosphonates accounts for the early activity in the kidney as has been observed in previous animal and human studies (Liepe et al., 2003; Lisic et al., 2001; Lin et al., 1997). After IV injection the two test compounds show distribution and excretion patterns similar to 188ReHEDP, with a substantial portion of the injected activity accumulating in the bone. Bone binding in the tail may account for some of the activity noted in this structure although the presence of injection site activity contributes to a high variability in this measurement. Kidney activity for the test compounds remained relatively high up to 8 h while all other soft tissue levels declined rapidly. While early kidney activities would represent blood clearance and urinary excretion these later activities indicate some delay in clearance of activity from the kidney. The nature of this delay has not yet been investigated. Tables 1 and 2 show that, following intravenous administration of the test compounds, the majority of bone binding is complete within the first hour. The blood levels of circulating activity are very low, there is little soft tissue uptake and most of the remaining activity has been cleared from the body. The bone remains the major repository of the whole body retained radioactivity up to 8 h for 188Re-HEDP and the two test compounds as shown in Table 5 and Fig. 3. Although we see relatively high standard deviations in the measured activity levels for bone, which will contribute some uncertainty to these values, the substantial uptake and retention of radioactivity in bone relative to other tissues is encouraging bearing in mind that our study is performed with normal bone. It is recognized that the distribution of bisphosphonates to the bone in normal mice is not uniform but rather will be concentrated in the ends of the bone where bone growth and consequently osteoblastic activity is more pronounced. Joint bone surface will provide a normal animal model for the activity present at sites of osteoblastic bone metastases. In the present study joint samples showed higher radioactivity concentration than whole bone throughout the 8 h of the study. Bisphosphonates such as 188Re-HEDP, used for palliation of bone pain, and 99mTc-methylene diphosphonate, used for imaging bone lesions show binding to normal bone as well as enhanced binding at bone metastases. This provides a precedent to suggest that our bisphosphonate conjugates will exhibit similar targeting toward metastatic bone lesions. The two compounds investigated in this study present the possibility of multimodality treatment of bone metastatic disease. The DTPA/BP compound is a
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prototype for chelate complexes that could deliver a variety of therapeutic radionuclides to bone lesions in combination with or in place of the rhenium radionuclides. Bouchet et al. (2000) provided an analysis of available radionuclides used in palliation of bone pain by observing the capacity of these agents to deliver a high radiation dose to the bone while minimizing the marrow dose. Low-energy b or electron emitters such as 117m Sn and 33P were concluded to be more likely to deliver a therapeutic dose to the bone while sparing bone marrow. DTPA has been demonstrated to be a strong chelator for 117mSn (Swailem et al., 1998), while chelates other than DTPA may be more appropriate for optimizing binding stability for specific radiometals. Conjugates between bisphosphonates and chemotherapy agents have been investigated previously (e.g. Hosain et al., 1996; Wingen et al., 1986), but this appears to be a relatively unexploited approach to treatment of bone metastases. Bisphosphonates tolerate a variety of modifications to the basic structural backbone while retaining bone binding capability. Therefore, a wide spectrum of chemotherapy agents could be envisioned with labile linkers that would permit their release at the site of bone metastatic involvement following rapid delivery and binding mediated by the bisphosphonate moiety. The addition of 188Re to these compounds would provide the capacity for combined radiotherapy and chemotherapy.
5. Conclusion In this study 188Re-DTPA/BP and 188Re-5-FU/BP were prepared with high radiochemical yield and stability. The biodistribution results for these compounds in Balb/C mice showed a high bone uptake especially in the joints, rapid plasma clearance and low soft tissue uptake. This raises the possibility of selective therapy to bone metastatic lesions while avoiding systemic toxicity. The dual nature of the two test compounds also provides the possibility for therapy with radionuclide mixtures or with combinations of rhenium radionuclides and chemotherapy agents. Bisphosphonate conjugates may have an unexploited potential in the treatment of metastatic bone cancer and further studies in animal models of metastatic bone disease are warranted to fully elaborate the tumoricidal behavior of both drug and radionuclide complexes prior to their consideration in human clinical trials.
Acknowledgements Financial assistance in the form of an Alberta Cancer Board Research Initiatives Grant is gratefully acknowledged.
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diphosphonates for the treatment of bone metastases. Nucl. Med. Biol. 26, 973–976. Kantoci, D., Denike, J.K., Wechter, W.J., 1996. Synthesis of aminobisphosphonates. Synth. Comm. 26, 2037–2039. Knapp Jr, F.F., Beets, A.L., Guhlke, S., Zamora, P.O., Bender, H., Palmedo, H., Biersack, H.J., 1997. Availability of rhenium-188 from the alumina-based tungsten-188/rhenium-188 generator for preparation of rhenium-188-labeled radiopharmaceuticals for cancer treatment. Anticancer Res 17, 1783–1795. Koudelkova, M., Jedinakova-Krizova, V., 2003. Capillary electrophoretic and thin-layer chromatographic characterization of rhenium complexation with 1-hydroxyethylidenediphosphonic acid. J. Chromatogr. A. 990, 317–323. Li, S., Liu, J., Zhang, H., Tian, M., Wang, J., Zheng, X., 2001. Rhenium-188 HEDP to treat painful bone metastases. Clin. Nucl. Med. 26, 919–922. Liepe, K., Kropp, J., Runge, R., Kotzerke, J., 2003. Therapeutic efficiency of rhenium-188-HEDP in human prostate cancer skeletal metastases. Br. J. Cancer 89, 625–629. Lin, W.Y., Lin, C.P., Yeh, S.J., Hsieh, B.T., Tsai, Z.T., Ting, G., Yen, T.C., Wang, S.J., Knapp, F.F., Stabin, M.G., 1997. Rhenium-188 hydroxyethylidene diphosphonate: a new generator-produced radiotherapeutic drug of potential value for the treatment of bone metastases. Eur. J. Nucl. Med. 24, 590–595. Lin, W.Y., Hsieh, J.F., Lin, C.P., Hsieh, B.T., Ting, G., Wang, S.J., Knapp Jr., F.F., 1999. Effect of reaction conditions on preparations of rhenium-188 hydroxyethylidene diphosphonate complexes. Nucl. Med. Biol. 26, 455–459. Lisic, E.C., Phillips, M., Ensor, D., Nash, K.L., Beets, A., Knapp, F.F., 2001. Synthesis of a new bisphosphonic acid ligand (SEDP) and preparation of a (188)Re-(Sn)SEDP bone seeking radiotracer. Nucl. Med. Biol. 28, 419–424. Maxon III., H.R., Schroder, L.E., Thomas, S.R., Hertzberg, V.S., Deutsch, E.A., Scher, H.I., Samaratunga, R.C., Libson, K.F., Williams, C.C., Moulton, J.S., et al., 1990. Re-186(Sn) HEDP for treatment of painful osseous metastases: initial clinical experience in 20 patients with hormone-resistant prostate cancer. Radiology 176, 155–159. Maxon III., H.R., Schroder, L.E., Washburn, L.C., Thomas, S.R., Samaratunga, R.C., Biniakiewicz, D., Moulton, J.S., Cummings, D., Ehrhardt, G.J., Morris, V., 1998. Rhenium188(Sn)HEDP for treatment of osseous metastases. J. Nucl. Med. 39, 659–663. Palmedo, H., Guhlke, S., Bender, H., Sartor, J., Schoeneich, G., Risse, J., Grunwald, F., Knapp Jr., F.F., Biersack, H.J., 2000. Dose escalation study with rhenium-188 hydroxyethylidene diphosphonate in prostate cancer patients with osseous metastases. Eur. J. Nucl. Med. 27, 123–130. Palmedo, H., Manka-Waluch, A., Albers, P., Schmidt-Wolf, I.G., Reinhardt, M., Ezziddin, S., Joe, A., Roedel, R., Fimmers, R., Knapp Jr., F.F., Guhlke, S., Biersack, H.J., 2003. Repeated bone-targeted therapy for hormone-refractory prostate carcinoma: tandomized phase II trial with the new, high-energy radiopharmaceutical rhenium-188 hydroxyethylidenediphosphonate. J. Clin. Oncol. 21, 2869–2875. Pecher, P., 1942. Biological investigation with radioactive calcium and strontium. Preliminary report on the use of radioactive strontium in the treatment of metastatic bone cancer. Univ. Calif. Publ. Pharmacol. 2, 117–149.
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colloid as a radiation synovectomy agent. Eur. J. Nucl. Med. 22, 505–507. Wang, S.J., Lin, W.Y., Chen, M.N., Hsieh, B.T., Shen, L.H., Tsai, Z.T., Ting, G., Knapp Jr., F.F., 1996. Biodistribution of rhenium-188 lipiodol infused via the hepatic artery of rats with hepatic tumours. Eur. J. Nucl. Med. 23, 13–17. Wingen, F., Sterz, H., Blum, H., Moller, H., Pittermann, W., Pool, B.L., Sinn, H.J., Spring, H., Schmahl, D., 1986. Synthesis, antitumor activity, distribution and toxicity of 4-[4-[bis(2-chloroethyl)amino]phenyl]-1-hydroxybutane-1 1-bisphosphonic acid (BAD), a new lost derivative with increased accumulation in rat osteosarcoma. J. Cancer Res. Clin. Oncol. 111, 209–219. Zhang, H., Tian, M., Li, S., Liu, J., Tanada, S., Endo, K., 2003. Rhenium-188-HEDP therapy for the palliation of pain due to osseous metastases in lung cancer patients. Cancer Biother. Radiopharm. 18, 719–726.
Applied Radiation and Isotopes 62 (2005) 541–549 www.elsevier.com/locate/apradiso
188
Re-labeled bisphosphonates as potential bifunctional agents for therapy in patients with bone metastases Amal El-Mabhouha, John R. Mercera,b, a
Faculty of Pharmacy and Pharmaceutical Sciences, 3118 Dentistry Pharmacy Center, University of Alberta, Edmonton Alta, Canada T6G-2N8 b Faculty of Medicine, 3118 Dentistry Pharmacy Center, University of Alberta, Edmonton Alta, Canada T6G-2N8 Received 2 June 2004; received in revised form 30 September 2004; accepted 25 October 2004
Abstract Two new bisphosphonates have been examined for their ability to bind 188Re and deliver it selectively to bone. The bisphosphonates are prototype compounds with potential to deliver rhenium radionuclides and a second therapy modality to bone metastases. A conjugate between diethylenetriaminepentaacetic acid and bisphosphonate (DTPA/BP) and a conjugate between 5-fluorouracil and bisphosphonate (5-FU/BP) were prepared and labeled at high radiochemical purity with 188Re and biodistribution studies were carried out in normal Balb/C mice. The compounds showed rapid blood clearance and elimination from soft tissues with substantial retention of activity in the bone comparable to 188Re-hydroxyethylidine diphosphonate used as a control. At 8 h bone activity was 3.51% of injected dose for 188Re-DTPA/BP and 6.38% of injected dose for 188Re-5-FU/BP representing 69.6% and 80.6% of total body radioactivity, respectively. The two compounds show the potential for combination therapy of painful bone metastases. r 2004 Elsevier Ltd. All rights reserved. Keywords: Bone pain palliation; Bisphosphonates; Radiotherapy; Rhenium radioisotopes
1. Introduction The intractable and debilitating pain that may accompany cancer is often produced by bone metastases. While local external beam irradiation is the first choice for palliative treatment for patients with a limited number of lesions (Lin et al., 1997), systemic radiotherapy with radiopharmaceuticals is preferable with widespread bone metastases with multifocal sites of pain (Liepe et al., 2003). Systemic radionuclide therapy with b-emitting radiopharmaceutical has been used since 1942 for the management of painful bone metastases Corresponding author. Tel.: +1 780 492 5364; fax: +1 780 492 1217. E-mail address: [email protected] (J.R. Mercer).
(Pecher, 1942) and it remains a widely used and effective modality (for recent reviews see Roque et al. (2003) and Serafini (2001)) 188Re is an excellent candidate for bparticle therapy (Boschi et al., 2003; Du et al., 2000; Hsieh et al., 1999; Hsieh et al., 1996; Knapp et al., 1997; Lin et al., 1997; Wang et al., 1995, 1996;). Its energetic bparticles (2.1 MeV) have a maximum penetration in tissue of 10–11 mm making this radionuclide a suitable option for large tumor masses. Its g-ray emission (0.155 MeV) can be exploited for dosimetric purposes and to observe biological distribution during therapy (Chen et al., 2001; Hsieh et al., 1996; Hashimoto, 1998; Boschi et al., 2003). It also has a relatively short physical half-life of 16.9 h which allows the use of high doses and reduces the problem of radioactive waste handling and storage (Chen et al., 2001; Boschi et al., 2003; Lin et al.,
0969-8043/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.10.004
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1997; Palmedo et al., 2003; Liepe et al., 2003). 188Re can be obtained by saline elution from a 188W/188Re generator system where the parent 188W has a 69-day half-life. Once the generator is installed 188Re is routinely available for clinical use (Knapp et al., 1997). Targeting of 188Re to bone has been accomplished effectively using the complex 188Re-hydroxyethylidine diphosphonate (188Re-HEDP, Fig. 1). This agent has shown therapeutic efficacy in the treatment of metastatic bone pain associated with cancer of the breast, prostate, lung and other locations (Maxon et al., 1998; Li et al., 2001; Liepe et al., 2003; Zhang et al., 2003). Bisphosphonates have been extensively studied due to their clinical effectiveness in treatment of a variety of pathological processes in the bone. For a systematic review of bisphosphonates in metastatic disease see Ross et al. (2004). Their present uses include treatment of hypercalcaemia, treatment of metastatic bone pain and as a prophylactic treatment for the prevention of pathological fractures and spinal cord compression (Coleman, 2001; Hortobagyi, 2002). The mechanism of the action of bisphosphonates has been extensively investigated (for comprehensive reviews see Reszka and
O O (OH)2P
N (OH)2P
2. Materials and methods OH
H C
N
N
O
2.1. Reagents and instruments OH
N
OH HO
O
O O
O
bisphosphonate DTPA conjugate (DTPA/BP) O
O (OH)2P
H N
(OH)2P
H N
C
O
N
O
O
F
bisphosphonate 5-fluorouracil conjugate (5-FU/BP) O (OH)2P
CH 3
(OH)2P
OH
Rodan (2003) and Fleisch (1998)). Bisphosphonates attach strongly and preferentially to bone with increased avidity for areas of osteoblastic and osteoclastic activity. Once associated with bone these compounds are potent inhibitors of osteoclastic bone resorption. Bearing in mind the attractive therapeutic features of 188 Re and the targeting potential of bisphosphonates, we have investigated the biodistribution and excretion of two 188Re-labeled novel bisphosphonate conjugates. The compounds are conjugates of the bisphosphonate functionality with diethylenetriaminepentaacetic acid and biphosphonate (DTPA/BP) and with 5-fluorouracil and biphosphonate (5-FU/BP) with the structures shown in Fig. 1. Studies were performed in Balb/C mice with particular attention to the accumulation of 188Re in bone. The bisphosphonate portion of these conjugates should allow binding of 188Re and targeting to bone lesions while the conjugated functionalities make these agents potential multimodality agents for therapy. We propose that the DTPA/BP chelate will permit therapeutic radionuclides to be targeted to the bone while the 5-fluorouracil bisphosphonate conjugate may be more selectively delivered to bone metastases without systemic toxicity.
O hydroxyethylidine diphosphonate (HEDP) Fig. 1. Structures for the bisphosphonate conjugates with DTPA and 5-fluorouracil, and for 1-hydroxyethane-1,1-diphosphonic acid (HEDP).
Acetic acid was purchased from Caledon Laboratories Ltd. and anhydrous sodium acetate was purchased from BDH Inc. Hydroxyethylidene diphosphonic acid (HEDP) was purchased from Fluka Chemie GmbH. All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). Double distilled deionized water was used for all the preparations. Silica gel instant thin-layer chromatography sheets (ITLC-SG) were purchased from Gelman Science Inc. A MINAXI g Auto-Gamma 5000 Series gamma counter was used to measure the activity in tissues and chromatography samples. Rhenium-188 was obtained by eluting a commercially available 37 GBq alumina-based 188 W/188Re generator (Oak Ridge National Laboratories) with 10–15 mL of normal saline, according to the manufacturer’s specifications, to provide solutions of no-carrier-added 188Re-sodium perrhenate (NaReO4). 2.2. Preparation of DTPA/BP and 5-FU/BP The bisphosphonate conjugates DTPA/BP and 5-FU/ BP shown in Fig. 1 were synthesized as described previously (El Mabhouh et al., 2004). Briefly, the key intermediate aminobisphosphonate tetraethyl ester was prepared according to the literature procedures (Kantoci et al., 1996). This intermediate was coupled with
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diethylenetriaminepentaacetic dianhydride in DMF at 60 1C. After stirring for 9 h, water was added dropwise to the hot reaction mixture and the mixture was then stirred for 5 h at room temperature. After removal of the solvent, the residue was purified by flash chromatography and the required DTPA/BP generated by reaction of the tetraethyl ester with bromotrimethylsilane at 0 1C. To prepare the 5-fluorouracil conjugate the aminobisphosphonate tetraethyl ester was first converted to the isocyanate by the action of triphosgene in acidified dry toluene. Reaction of this isocyanate with 5-fluorouracil was carried out at 90 1C in dry N,N-dimethylacetamide followed by deblocking of the isolated tetraethyl ester product with bromotrimethylsilane to produce the required 5-FU/BP. 2.3. Preparation of and 188Re-HEDP
188
Re-DTPA/BP,
188
Re-5-FU/BP
Reactions were generally carried out in sterilized, evacuated, sealed multidose vials. The vials were charged with 100 mL of 40 mg/mL SnCl2 in 10% aqueous acetic acid, 200 mL of 0.5 M sodium D-gluconate in 0.2 M sodium acetate pH 5 buffer solution and 40 mL of a 2.5 mg/mL aqueous solution of perrhenic acid. A solution of the bisphosphonate conjugate in sterilized DDD water was then added to the vials to produce a final concentration of 5 mg/mL (DTPA/BP or HEDP) or 2 mg/mL (5-FU/BP). Labeling reactions were initiated by the addition of 100–200 mL (nominally 2 MBq) of 188Re-sodium perrhenate to the vials. The reaction mixture was incubated at 90 1C for 3 h, cooled to room temperature and adjusted to pH 6.5–7.0 using 0.1 N HCl or 0.2 M NaOH. The radiochemical purity of 188ReDTPA/BP, 188Re-5-FU/BP or 188Re-HEDP was determined by using ITLC-SG strips (1 12 cm) developed with acetone or saline (0.9% NaCl). In all syntheses the radiolabeled compounds did not require further purification but were used directly in animal studies. 2.4. Preparation of 188Re-sodium perrhenate for injection Injection solutions of sodium perrhenate used in control studies were prepared by dilution of 100 mL (nominally 2 MBq) of 188Re-sodium perrhenate as eluted from the generator to 900 mL with normal saline. 2.5. Animal studies Female Balb/C mice aged between 6 and 8 weeks with weight range of 18–24 g were used in all the studies. Animals were obtained from the University of Alberta, Health Sciences Laboratory Services breeding program and were allowed to acclimatize for several days before being entered into the studies. All procedures are in accordance with the Canadian Council on Animal Care
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and were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. 2.6. Biodistribution studies Female Balb/C mice were injected via a lateral tail vein with the radioactive test agents (188Re-DTPA/BP, 188 Re-5-FU/BP, 188Re-HEDP and 188ReO 4 ) using volumes of approximately 0.1 mL and activities of approximately 200 KBq. Animal numbers are indicated in the Tables 1–5 and were generally 3 or 4 animals per time period. Drug mass was in the range of 500 mg per injection of DTPA/BP or HEDP and was reduced to 200 mg with 5-FU/BP due to potential toxicity with this agent. The mice were sacrificed using a CO2 chamber at times of 1, 2, 4 and 8 h after injection. Blood was collected by cardiac puncture at the time of sacrifice. Whole organs were collected for heart, liver, lungs, spleen, stomach, gastrointestinal tract (GI tract), kidneys, and brain. The thyroid was generally isolated with surrounding tissues of the neck and upper chest. Muscle was collected from the left hind leg, and the whole left tibia was isolated as a representative bone sample. The bone segment comprising the knee joint from the left femur was isolated in selected animals as a representative bone sample expected to exhibit increased osteoblastic activity due to terminal bone plate growth in these young animals. The whole tail was removed and counted separately. The tissues and residual carcass were weighed immediately upon dissection and activity for each sample was determined in a gamma counter. All radioactivity values were decay corrected to time of injection and efficiency corrected to provide true decays. Total blood weight was calculated as 7% of mouse body weight and total wet bone weight was estimated as 12% of mouse body weight.
3. Results All compounds were radiolabeled with high efficiency based on the ITLC analysis which identifies unbound 188 188 ReO ReO2 or other 4 and reduced hydrolyzed colloidal species if they are present. With acetone development free perrhenate moves with the solvent front and the 188Re-bisphosphonate conjugate stays at the origin on the strip, while using saline as eluent causes Re-188 colloidal impurity to remain at the origin while 188 Re-bisphosphonate conjugate and 188Re-sodium perrhenate migrate close to the solvent front. The expected labeled products accounted for 92% or more of total radioactivity using this analysis. No purification of the radiolabeled products was undertaken. Radiolabeled products were injected into test animals immediately following synthesis and quality control assays. DTPA and most bisphosphonates are of low toxicity and LD50
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Table 1 188 Re-DTPA/BP biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 3)
Table 4 188 ReO 4 biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 3)
Organ
Organ
Blood Liver Stomach Kidney Thyroid Bone Tail Joint
Time after injection (h) 1
2
4
8
0.5770.22 0.6470.25 1.2870.24 3.9871.72 0.8270.13 2.9370.69 3.3873.08 5.2570.93
0.3470.04 0.4370.10 0.8270.10 2.9370.39 0.7470.14 3.5870.30 1.1270.19 5.5970.34
0.1070.01 0.1270.00 0.3070.08 1.0270.14 0.2770.09 1.7470.26 0.6770.23 2.5770.29
0.0970.04 0.1370.06 0.2970.18 0.7570.22 0.2170.11 1.4070.31 1.4070.60 2.7870.59
Values are percent of injected dose per gram of wet tissue7standard deviation. Table 2 188 Re-5-FU/BP biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 4) Organ
Blood Liver Stomach Kidney Thyroid Bone Tail Joint
Time after injection (h) 1
2
4
8
0.5470.25 1.3670.62 1.1770.63 4.0471.18 0.6070.38 3.2171.28 3.1772.14 5.3172.40
0.1770.06 1.0670.55 0.6870.49 2.3870.55 0.4570.31 2.6571.05 1.9070.99 4.0971.38
0.1270.06 0.7870.81 0.8270.64 1.1770.31 0.4670.33 2.2671.18 1.8472.39 3.5372.21
0.0970.02 0.8470.27 0.7070.12 1.2670.26 0.3370.04 2.6770.13 1.7971.70 3.9170.20
Values are percent of injected dose per gram of wet tissue7standard deviation. Table 3 188 Re-HEDP biodistribution in normal Balb/C mice as determined by radioactive distribution (n ¼ 3) Organ
Blood Liver Stomach Kidney Thyroid Bone Tail Joint
Time after injection (h) 1a
2
4
8
0.7770.60 0.6070.31 1.5572.20 4.2271.07 0.8470.74 4.5271.44 3.3671.79 6.5771.05b
0.2870.32 0.2770.13 0.8271.31 2.0970.58 0.6370.75 2.6070.46 0.8770.06 5.10c
0.1270.00 0.2070.04 0.2270.05 1.8470.21 0.2770.05 2.9770.02 1.3070.62 5.13c
0.1470.06 0.2370.07 0.2970.15 1.3570.20 0.3770.19 3.2070.06 1.4470.97 5.12c
Values are percent of injected dose per gram of wet tissue7standard deviation. a n ¼ 4. b n ¼ 2. c n ¼ 1.
Time after injection (h) 1
2
4
8
Blood 7.3870.45 4.1370.84 1.1670.38 0.3570.12 Liver 3.9370.83 2.1270.42 0.4570.18 0.1670.07 Stomach 30.8977.14 18.6871.87 5.3471.29 1.6070.91 Kidney 4.1170.55 2.2970.39 0.6270.13 0.2070.08 Thyroid 26.2473.92 18.3575.10 12.4971.47 3.2571.25 Bone 2.7170.25 1.6470.41 0.4170.15 0.2070.06 Tail 4.2270.03 4.8373.98 1.1270.44 1.1371.17 Values are percent of injected dose per gram of wet tissue7standard deviation.
values for 5-fluorouracil are quoted in various sources as being several hundreds of milligrams per kilogram (e.g. Burns and Beland, 1984). No physiological effects or toxicity was observed at the dose levels used in these studies. The bisphosphonate conjugates are all rapidly removed from the blood within the first hour after intravenous injection as indicated by the blood concentrations of activity shown in Tables 1–3. Total blood activity was calculated using the value of 7% of body mass as blood mass and is shown in Table 5. By 1 h 1% or less of the injected activity remains in the blood and this value continues to decline up to 8 h. In contrast 188 Re activity is high and persistent in the blood following injection of 188Re-perrhenate, representing 9.92% of the injected dose at 1 h and 1.66% at 4 h. The whole body activity was determined by summing the activities from all dissected tissues and the residual carcass (Table 5, Fig. 2). The two test compounds and 188 Re-HEDP show similar behavior with rapid initial excretion over the first hour following injection followed by a slow decrease in the residual activity up to 8 h. The biodistribution data in selected tissues for 188Re-DTPA/ BP, 188Re-5-FU/BP, 188Re-HEDP and 188ReO 4 are shown in Tables 1–4, respectively. The radioactivity in the heart, skeletal muscle and brain was low at all time points with all tested compounds. Whole carcass activity for 188Re-HEDP, 188 Re-DTPA/BP and 188Re-5-FU/BP after organ dissection is predominantly due to activity in the bone rather than significant tissue retention. Perrhenate (188ReO 4 ), evaluated here as a control agent, shows high blood levels and higher whole body retention but low bone uptake when compared with the other tested compounds. At early time periods following IV injection, activity from the three 188Re-labelled bisphosphonates were found mainly in bone and kidney. Renal excretion accounted for the rapid early blood activity clearance and explains the kidney activity levels
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Table 5 Activity retained in whole body, total blood and total wet bone at various times after intravenous injection of test compounds Time (h) Injected compound
Activity location
1
2
4
8
188
Whole body Total blood Total bone Bone/body 100%
15.5474.38 0.8270.25 7.2171.22 46.4
10.8472.10 0.4570.06 8.2570.75 76.1
5.1370.29 0.1470.01 4.4470.58 86.5
5.0470.46 0.1370.05 3.5170.65 69.6
188
Whole body Total blood Total bone Bone/body 100%
17.8176.71 0.8070.38 7.8872.75 44.2
11.9172.39 0.2770.08 7.1171.99 59.7
9.6076.42 0.1770.08 5.3072.08 55.2
7.9270.78 0.1370.03 6.3870.84 80.6
188
Whole body Total blood Total bone Bone/body 100%
15.4574.14 1.0370.59 10.4072.60 67.3
11.1172.29 0.5770.39 9.0271.21 81.2
7.6370.48 0.1870.02 7.5670.79 99.1
7.1871.19 0.1970.07 7.8570.61 109
188
Whole body Total blood Total bone
61.8177.79 9.9270.58 6.2470.53
42.9673.10 5.6170.74 3.8370.74
14.3372.73 1.6670.45 1.0170.30
5.0372.24 0.4870.17 0.4770.16
Re-DTPA/BP
Re-5-FU/BP
Re-HEDP
Re-O 4
Values represent the mean percent of total injected radioactive dose7standard deviation (n ¼ 3). Whole bone to whole body ratios are calculated as (whole bone activity/whole body activity) 100%.
whole body activity (%dose)
80
perrhenate Re-188-HEDP Re-188-5-FU/BP Re-188-DTPA
70 60 50 40 30 20 10 0 1h
2h
4h
8h
time Fig. 2. Percent of injected 188Re activity retained in whole body for the bisphosphonate conjugates and for perrhenate over 8 h in Balb/C mice (n ¼ 3).
at 1 h. For 188Re-HEDP the kidney activity had declined to 2.09% injected dose per gram at 2 h while for the test compounds 188Re-DTPA/BP and 188Re-5-FU/BP there was a relatively high activity in the kidneys up to 8 h. Liver activity was generally unremarkable for 188ReHEDP and 188Re-DTPA/BP, being highest at 1 h and then significantly decreased over the 8 h of the study. The absence of liver activity is reassuring as an
indication that labeled colloidal impurities, often produced when high tin concentrations are used in reduction radiolabeling with rhenium, were not present in our preparations. The activity in the liver for 188Re-5FU/BP was somewhat higher and more persistent representing 1.36% of the injected dose at 1 h and 0.84% of the injected dose at 8 h. Tail activities are significantly higher for the bisphosphonates than for the perrhenate and we relate this to the bone content of this structure while the high variability of activity in the tail in some cases indicates some retention of activity at the injection site. The bone activity for the control and test compounds is expressed as percent of injected dose per gram of tissue in Tables 1–4 and as whole body bone activity, using 12% of body mass to represent wet bone, in Table 5 and Fig. 3. As expected, 188Re-perrhenate does not accumulate to any extent in the bone. HEDP, as a positive control known to bind 188Re and deliver it to the bone, had 10.40% of the injected dose in the bone at 1 h and this value decreased only minimally to 7.85% at 8 h. The two test compounds showed similar though somewhat reduced bone binding with values for 188ReDTPA/BP and 188Re-5-FU/BP of 7.21% and 7.88% at 1 h, which declined to 3.51% and 6.38% by 8 h, respectively. Table 5 also reports the percent of total body activity at each time period that is due to bound radioactivity in the bone. These values show that the whole body activity at the later time periods is
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14
perrhenate Re-188-HEDP Re-188-5-FU/BP Re-188-DTPA/BP
bone activity (%dose)
12 10 8 6 4 2 0 1h
2h
4h
8h
time Fig. 3. Percent of injected 188Re activity retained in total bone for the bisphosphonate conjugates and for perrhenate over 8 h in Balb/C mice (n ¼ 3).
predominantly bone activity for 188Re-HEDP as well as the two test compounds. Tables 1–3 indicate significantly higher uptake of the bisphosphonate conjugates in the bone segment comprising the knee joint relative to the bone shaft. In young mice the joints are areas of increased osteoblastic activity due to bone growth with a bone remodeling activity similar to that at the sites of osteoblastic bone metastases. The joint activity for 188Re-HEDP at 1 h was 6.57% ID/g and this decreased only minimally to 5.12% ID/g by 8 h. The test conjugates 188Re-DTPA and 188Re5-FU/BP showed 5.25% ID/g and 5.31% ID/g respectively, at 1 h. Although some of this initially bound activity was lost by 8 h, the value of 2.78% for 188ReDTPA and 3.91% ID/g for 188Re-5-FU/BP still represent significant concentration of activity in this tissue. These results are promising as they indicate effective targeting of the bisphosphonate analogs to the bone and increased uptake at sites of active bone growth.
4. Discussion Bisphosphonates are known to chelate rhenium radioisotopes producing stable complexes (Koudelkova and Jedinakova-Krizova, 2003; Hashimoto and Yoshihara, 1996; Hashimoto, 1998; Maxon et al., 1990) a characteristic which was demonstrated to extend to the present ligands. The radiolabeling of bisphosphonates conjugates (DTPA/BP and 5-FU/BP) with 188Re was achieved by using gluconate as a transchelator in 0.2 M sodium acetate buffer at pH 5.0 (Du et al., 2000). The facile reoxidation of reduced 188Re was prevented by using an evacuated vial and a high
concentration of SnCl2 solution (4 mg/mL). The excess of stannous ions has been found to be necessary to reduce the carrier-added perrhenate and to drive the reduction reaction to completion as far as possible (Deutsch et al., 1986). In the radiolabeling reaction to produce 188Re-DTPA/ BP, 188Re-5-FU/BP and 188Re-HEDP, carrier perrhenic acid was added to the essentially carrier-free radionuclide eluted from the 188W/188Re generator. The presence of macroscopic amounts of stable rhenium is known to increase the labeling yield and the in vivo stability of 188Re-labeled bisphosphonate conjugates (Palmedo et al., 2000; Hashimoto, 1998; Arteaga de Murphy et al., 2001; Hsieh et al., 1999; Lin et al., 1999). The concentration of the perrhenic acid was a critical factor affecting both the synthesis and the in vivo biodistribution of the 188Re-labeled bisphosphonate conjugates. The optimum concentration of carrier was determined by evaluation of the radiolabeled conjugate produced. At a perrhenate concentration lower than 100 mg/mL in the reaction mixture reduced bone uptake of the conjugates was observed, while at a concentration higher than 100 mg/mL colored solutions resulted making the product unsuitable for in vivo use. The addition of small amounts of carrier rhenium will not be a clinical concern since levels can be kept below a toxic threshold for the metal and the concentration of the complexes in vivo will be well below a level that would saturate bisphosphonate binding sites on the bone. Successful labeling also required incubation times of several hours at a temperature of 90 1C and pH 5. Balb/C mice were used in all our studies. This strain is widely used in animal research studies and was readily available to us from the local breeding program. As the present study was a preliminary investigation, and in consideration of animal ethics we restricted our numbers of mice to 3 or 4 per time point in each study. Although this allows distribution and pharmacokinetic data to be generated and general conclusions to be made, the statistical significance of some of the observations would need to be confirmed with a more extensive study. Injected 188Re-perrhenate does not bind to bone but distributes to the gastric mucosa, gastrointestinal tract and thyroid (Hsieh et al., 1999) as observed in our control study (Table 4). These organs do not show significant accumulation of activity with our test compounds or 188Re-HEDP as an indication that there is no perrhenate present in the injected formulations and that this species is not produced following in vivo breakdown of the complexes. 188Re-hydroxyethylidene diphosphonate (188Re-HEDP) was used as a positive control in this study (Table 3). 188Re-HEDP showed the expected distribution, as described by others (Maxon et al., 1998; Lin et al., 1997, 1999; Lisic et al., 2001), with
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the kidney and the bone having the highest concentration of activity at all time points investigated. The persistent activity in the tail up to 8 h is partially due to the bony structure of this tissue. The predominate urinary excretion, of bisphosphonates accounts for the early activity in the kidney as has been observed in previous animal and human studies (Liepe et al., 2003; Lisic et al., 2001; Lin et al., 1997). After IV injection the two test compounds show distribution and excretion patterns similar to 188ReHEDP, with a substantial portion of the injected activity accumulating in the bone. Bone binding in the tail may account for some of the activity noted in this structure although the presence of injection site activity contributes to a high variability in this measurement. Kidney activity for the test compounds remained relatively high up to 8 h while all other soft tissue levels declined rapidly. While early kidney activities would represent blood clearance and urinary excretion these later activities indicate some delay in clearance of activity from the kidney. The nature of this delay has not yet been investigated. Tables 1 and 2 show that, following intravenous administration of the test compounds, the majority of bone binding is complete within the first hour. The blood levels of circulating activity are very low, there is little soft tissue uptake and most of the remaining activity has been cleared from the body. The bone remains the major repository of the whole body retained radioactivity up to 8 h for 188Re-HEDP and the two test compounds as shown in Table 5 and Fig. 3. Although we see relatively high standard deviations in the measured activity levels for bone, which will contribute some uncertainty to these values, the substantial uptake and retention of radioactivity in bone relative to other tissues is encouraging bearing in mind that our study is performed with normal bone. It is recognized that the distribution of bisphosphonates to the bone in normal mice is not uniform but rather will be concentrated in the ends of the bone where bone growth and consequently osteoblastic activity is more pronounced. Joint bone surface will provide a normal animal model for the activity present at sites of osteoblastic bone metastases. In the present study joint samples showed higher radioactivity concentration than whole bone throughout the 8 h of the study. Bisphosphonates such as 188Re-HEDP, used for palliation of bone pain, and 99mTc-methylene diphosphonate, used for imaging bone lesions show binding to normal bone as well as enhanced binding at bone metastases. This provides a precedent to suggest that our bisphosphonate conjugates will exhibit similar targeting toward metastatic bone lesions. The two compounds investigated in this study present the possibility of multimodality treatment of bone metastatic disease. The DTPA/BP compound is a
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prototype for chelate complexes that could deliver a variety of therapeutic radionuclides to bone lesions in combination with or in place of the rhenium radionuclides. Bouchet et al. (2000) provided an analysis of available radionuclides used in palliation of bone pain by observing the capacity of these agents to deliver a high radiation dose to the bone while minimizing the marrow dose. Low-energy b or electron emitters such as 117m Sn and 33P were concluded to be more likely to deliver a therapeutic dose to the bone while sparing bone marrow. DTPA has been demonstrated to be a strong chelator for 117mSn (Swailem et al., 1998), while chelates other than DTPA may be more appropriate for optimizing binding stability for specific radiometals. Conjugates between bisphosphonates and chemotherapy agents have been investigated previously (e.g. Hosain et al., 1996; Wingen et al., 1986), but this appears to be a relatively unexploited approach to treatment of bone metastases. Bisphosphonates tolerate a variety of modifications to the basic structural backbone while retaining bone binding capability. Therefore, a wide spectrum of chemotherapy agents could be envisioned with labile linkers that would permit their release at the site of bone metastatic involvement following rapid delivery and binding mediated by the bisphosphonate moiety. The addition of 188Re to these compounds would provide the capacity for combined radiotherapy and chemotherapy.
5. Conclusion In this study 188Re-DTPA/BP and 188Re-5-FU/BP were prepared with high radiochemical yield and stability. The biodistribution results for these compounds in Balb/C mice showed a high bone uptake especially in the joints, rapid plasma clearance and low soft tissue uptake. This raises the possibility of selective therapy to bone metastatic lesions while avoiding systemic toxicity. The dual nature of the two test compounds also provides the possibility for therapy with radionuclide mixtures or with combinations of rhenium radionuclides and chemotherapy agents. Bisphosphonate conjugates may have an unexploited potential in the treatment of metastatic bone cancer and further studies in animal models of metastatic bone disease are warranted to fully elaborate the tumoricidal behavior of both drug and radionuclide complexes prior to their consideration in human clinical trials.
Acknowledgements Financial assistance in the form of an Alberta Cancer Board Research Initiatives Grant is gratefully acknowledged.
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