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Nuclear Medicine and Biology xx (2009) xxx–xxxwww.elsevier.com/locate/nucmedbio

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Multispecies animal investigation on biodistribution, pharmacokineticsand toxicity of 177Lu-EDTMP, a potential bone pain palliation agent☆

Domokos Máthéa,⁎, Lajos Balogha, András Polyáka, Réka Királya, Teréz Máriánb,Dariusz Pawlakc, John J. Zaknund, Maroor R.A. Pillaid, Győző A. Jánokia

aDepartment of Applied Radioisotopes and Animal Experimentation, National ‘Frédéric Joliot-Curie’ Institute of Radiobiology andRadiohygiene, H-1221 Budapest, Hungary

bInstitute of Nuclear Medicine, Debrecen University, Debrecen, HungarycInstitute of Atomic Energy, Radioisotope Centre POLATOM, Swierk-Otwock, Poland

dInternational Atomic Energy Agency (IAEA), Vienna, Austria

Received 14 May 2009; received in revised form 15 August 2009; accepted 28 September 2009

Abstract

Introduction: Radionuclide therapy (RNT) is an effective method for bone pain palliation in patients suffering from bone metastasis. Due tothe long half-life, easy production and relatively low β− energy, 177Lu [T1/2=6.73 days, Eβmax=497 keV, Eγ=113 keV (6.4%), 208 keV(11%)]-based radiopharmaceuticals offer logistical advantage for wider use. This paper reports the results of a multispecies biodistributionand toxicity studies of 177Lu-EDTMP to collect preclinical data for starting human clinical trials.Methods: 177Lu-EDTMP with radiochemical purity greater than 99% was formulated by using a lyophilized kit of EDTMP (35 mg ofEDTMP, 5.72 g of CaO and 14.1 mg of NaOH). Biodistribution studies were conducted in mice and rabbits. Small animal imaging wasperformed using NanoSPECT/CT (Mediso, Ltd., Hungary) and digital autoradiography. Gamma camera imaging was done in rabbits anddogs. Four levels of activity (9.25 through 37 MBq/kg body weight) of 177Lu-EDTMP were injected in four groups of three dogs each tostudy the toxicological effects.Results: 177Lu-EDTMP accumulated almost exclusively in the skeletal system (peak ca. 41% of the injected activity in bone with terminalelimination half-life of 2130 and 1870 h in mice and rabbits, respectively) with a peak uptake during 1–3 h. Excretion of theradiopharmaceutical was through the urinary system. Imaging studies showed that all species (mouse, rat, rabbit and dog) take up thecompound in regions of remodeling bone, while kidney retention is not visible after 1 day postinjection (pi). In dogs, the highest appliedactivity (37 MBq/kg body weight) led to a moderate decrease in platelet concentration (mean, 160 g/L) at 1 week pi with no toxicity.Conclusion: The protracted effective half-life of 177Lu-EDTMP in bone supports that modifying the EDTMP molecule by introducing 177Ludoes not alter its biological behaviour as a specific bone-seeking tracer. Species-specific pharmacokinetic behavior differences wereobserved. Toxicity studies in dogs did not show any biological adverse effects. The studies demonstrate that 177Lu-EDTMP is a promisingradiopharmaceutical that can be further evaluated for establishing as a radiopharmaceutical for human use.© 2009 Elsevier Inc. All rights reserved.

Keywords: Radionuclide therapy; Bone seeking radiopharmaceuticals; In vivo biodistribution; Animal studies; Small animal imaging

☆ Part of this work was conducted under an International AtomicEnergy Agency (IAEA) Coordinated Research Project and a EuropeanCommission FP6 grant to the European Molecular Imaging Laboratories(LSH-CT 2004-503569).

⁎ Corresponding author. Tel.: +36 1 482 2012; fax: +36 1 482 2007.E-mail address: mdomokos@hp.osski.hu (D. Máthé).

0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.nucmedbio.2009.09.004

1. Introduction

Treatment of unresectable, often incurable multipleskeletal cancer metastases is one of the major challenges ofoncological practice. Most commonly used palliativetreatment modalities are external beam radiation therapy,morphine-derived analgesics and bisphosphonates [1–4].However, in a significant number of patients radiotherapy isnot an option due to the location or multiplicity of tumors,

Table 1Biodistribution of 177Lu-EDTMP in mice per organ and per gram of organ weight at various time points postinjection

Organs/tissues Time points

30 min 1 h 2 h

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

Blood 0.83 0.00 0.64 0.07 7.16 7.66 4.48 4.98 0.13 0.02 0.11 0.02Muscle 1.10 0.01 0.21 0.04 0.67 0.27 0.10 0.04 4.25 4.79 0.97 1.12Bone 38.46 2.62 18.00 2.23 40.82 3.98 19.22 1.81 38.38 3.17 19.55 3.28Bone marrow 0.00 0.01 3.69 2.32 0.00 0.00 1.66 0.50 0.00 0.00 0.00 0.00Thyroid 0.19 0.00 0.32 0.02 0.08 0.02 0.17 0.06 0.06 0.03 2.04 0.61Heart 0.04 0.00 0.65 0.04 0.02 0.01 0.39 0.13 0.01 0.00 0.13 0.03Lung 0.14 0.02 0.28 0.01 0.10 0.04 0.17 0.04 0.04 0.02 0.17 0.07Liver 0.33 0.01 1.44 0.22 0.21 0.05 0.85 0.28 0.18 0.02 0.15 0.02Kidneys 0.41 0.02 0.21 0.04 0.23 0.06 0.10 0.04 0.18 0.01 0.70 0.11

Organs/tissues Time points

3 days 5 days 1 week

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

Blood 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Muscle 0.10 0.11 0.02 0.02 0.38 0.40 0.08 0.08 0.02 0.00 0.00 0.00Bone 30.34 8.01 13.20 3.48 31.22 7.65 14.86 2.79 28.28 6.14 12.30 2.90Bone marrow 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Thyroid 0.02 0.01 0.56 0.54 0.03 0.01 0.75 0.32 0.02 0.02 0.48 0.34Heart 0.00 0.00 0.02 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.03 0.01Lung 0.01 0.00 0.05 0.02 0.01 0.00 0.03 0.01 0.01 0.00 0.04 0.01Liver 0.10 0.01 0.08 0.00 0.12 0.02 0.12 0.02 0.13 0.02 0.11 0.00Kidneys 0.04 0.00 0.18 0.02 0.02 0.01 0.11 0.02 0.02 0.00 0.06 0.01

Time points

3 h 6 h 24 h 48 h

%

Blood mass was calculated as 8% of body mass, bone mass was calculated as 10% of body mass, bone marrow mass was considered identical to blood mass. %IA: Percent of injected activity.

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and the effect of analgesia is insufficient. Radiation delivereddirectly and in a targeted manner to the tumor cells and theirmicroenvironment is a possible way to overcome theseobstacles. In this context, intravenous radionuclide therapy(RNT) aimed at pain palliation of incurable multiple skeletalmetastases with bone-seeking agents is a well-established butyet underexploited modality [4–15]. According to the clinicaltrials, RNT improves the quality of life in a higher percentageof patients than applying conventional treatment methods,also in cases where morphine derivatives or bisphosphonatesgive less satisfactory effect [16–19]. Several isotopes havebeen applied in the above-referenced studies. Currently, themost widely used and registered preparations make use of153Sm (153Sm-EDTMP) and 89Sr (89Sr-chloride). Otherisotopes coupled to bone-seeking molecules in clinical useinclude 186Re [9,20–22] and 188Re [22–24].

Systemic application of ethylene diamine-N,N,N′,N′-tetrakis(methylene phosphonic acid) (EDTMP) labeledwith 153Sm as a bone-seeking agent has been studied andapplied in clinical practice in a large number of patients formore than two decades. The clinical efficacy of thisradiopharmaceutical is well demonstrated [4,6,15–20].However, the short half-life of 153Sm (T1/2 47 h) has beenthe major impediment, making its transportation difficult andthereby limiting its wider use.

Lutetium-177 [T1/2=6.73 days, Eβmax=497 keV, Eγ=113keV (6.4%), 208 keV (11%)] has several advantages to be

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

explored as a therapeutic radionuclide for bone painpalliation [25]. First, its lower beta-particle energy andrelatively long physical half-life allow the deposition of anadequate tumor irradiation dose with a constant dose rate[26,27]. A model calculation published suggests that 177Luis the optimal radionuclide for full beta-particle energydeposition in small tumor volumes [26]. The high crosssection of the target radionuclide [176Lu(n,γ)177Lu σ=2100barns] allows large-scale production of 177Lu even whileusing medium flux research reactors. The relatively longhalf-life (6.73 days) of 177Lu allows easy transportation ofthe isotope thereby improving the accessibility of RNT tomore patients and in countries which have no reactoraccessibility. The low-energy, low-yield [Eγ=113 keV(6.4%), 208 keV (11%)] gamma photon emissions of177Lu allow scintigraphic detection of the tracer in vivo toestimate the dose in individual patients. Considering theabove advantages of 177Lu, the International AtomicEnergy Agency (IAEA) initiated a project towards theintroduction of 177Lu-based radiopharmaceuticals for bonepain palliation.

A variety of cyclic and acyclic polyamino-polypho-sphonates were evaluated as potential radiopharmaceuticals[28–32]. In comparative evaluation studies of the 177Lucomplexes of different acyclic and cyclic phosphonateligands, DOTMP and EDTMP were found to give superiorresults over other phosphonates [30,31]. As already

IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

Table 1 (continued)

Time points

3 h 6 h 24 h 48 h

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

0.11 0.02 0.08 0.01 0.07 0.02 0.05 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.000.24 0.09 0.04 0.02 1.57 1.23 0.25 0.19 0.59 0.65 0.12 0.13 0.06 0.05 0.01 0.0136.76 3.39 15.52 3.22 35.52 5.77 13.12 3.11 33.54 10.03 15.76 5.49 30.91 9.90 14.68 3.560.00 0.00 0.00 0.00 0.01 0.00 0.01 0.01 0.43 0.52 0.44 0.53 0.00 0.00 0.00 0.000.02 0.01 0.54 0.28 0.02 0.01 0.61 0.30 0.04 0.04 0.84 0.51 0.01 0.01 0.20 0.200.01 0.01 0.11 0.05 0.02 0.02 0.52 0.56 0.02 0.02 0.20 0.20 0.00 0.00 0.03 0.010.02 0.00 0.12 0.03 0.02 0.01 0.11 0.02 0.02 0.01 0.08 0.04 0.01 0.00 0.04 0.010.14 0.02 0.13 0.03 0.23 0.07 0.17 0.06 0.09 0.05 0.11 0.05 0.12 0.03 0.13 0.020.15 0.01 0.60 0.12 0.96 0.98 2.01 1.74 0.14 0.04 0.66 0.22 0.05 0.01 0.24 0.04

Time points

2 weeks 4 weeks 6 weeks 8 weeks

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.54 0.42 0.11 0.09 0.20 0.27 0.15 0.18 0.31 0.30 0.09 0.04 0.23 0.23 0.09 0.0929.21 3.44 12.44 1.74 28.63 6.28 14.66 2.95 28.20 2.78 11.84 1.72 21.33 2.10 10.13 2.120.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.04 0.01 1.31 0.38 0.05 0.03 1.00 0.56 0.01 0.03 0.23 0.52 0.00 0.00 0.04 0.020.00 0.00 0.03 0.00 0.00 0.00 0.03 0.01 0.00 0.00 0.04 0.01 0.00 0.00 0.05 0.030.01 0.01 0.06 0.03 0.01 0.00 0.03 0.01 0.01 0.00 0.05 0.01 0.01 0.01 0.04 0.010.14 0.03 0.13 0.02 0.14 0.02 0.14 0.03 0.11 0.02 0.09 0.03 0.11 0.11 0.08 0.030.03 0.01 0.10 0.03 0.01 0.00 0.05 0.01 0.00 0.00 0.06 0.01 0.01 0.01 0.02 0.01

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sufficient data are available on the biological tolerance of theligand EDTMP as well as the radiopharmaceutical 153Sm-EDTMP, EDTMP was selected as the carrier ligand for theIAEA-sponsored studies. It was felt that replacing theradionuclide 153Sm with 177Lu in the EDTMP complexwill pose minimal additional chemical or radiologicaltoxicity issues. Consequently, a detailed study on thebiodistribution and imaging of 177Lu-EDTMP in rats, rabbitsand beagle dogs was conducted [33] which showed highlypromising results. In the present studies, we aimed atobtaining detailed biodistribution and pharmacokineticparameters of 177Lu-EDTMP in several species to studythe pharmacological effects. Mouse, rats, rabbits and dogs

Table 2Standard pharmacokinetic parameters of 177Lu-EDTMP calculated on the basis of

Organs/tissues t1/2 (h) AUD (%⁎h) AUC∞ (%h/g) MR

Blood 7.20 7.35 7.35 3Muscle 1170 406.24 794.65 618Bone 2130 37666 103293 641Thyroid 923 36.11 36.11 517Heart 209 9.31 9.31 263Lungs 753 13.93 24.78 263Liver 2260 169.13 528.39 649Kidneys 304 38.97 43.36 375

t1/2 — terminal elimination half-life; AUC∞ — area under the curve extrapolated tMRT∞ — mean residence time extrapolated to t=∞; Vz — volume of distribution

were used for the biodistribution and imaging studies. Datafrom the biodistribution studies were used to calculatepharmacokinetic parameters based on a noncompartmentalmodel. Skeletal retention of the agent and its potentialradiotoxic effects were studied by injecting 177Lu-EDTMPactivity at different levels in beagle dogs.

2. Materials and methods

2.1. Radiopharmaceutical preparation and quality control

Lutetium-177 as chloride in 0.5N HCl was obtainedfrom AEA GmbH (Germany) or POLATOM Ltd. (Poland).

mouse biodistribution results and the noncompartmental model

T (h) MRT∞ (h) Vz (ml) VSS (ml) TC (ml/min)

.74 3.74 141 50.9 0.231800 141 226 2.10×10−3

3040 2.98 2.95 1.61×10−3

517 3690 1430 4.62×10−3

263 3240 2820 1.79×10−1

1430 4380 577 6.72×10−2

3340 618 632 3.15×10−3

517 1010 1190 3.84×10−2

o t=∞; AUD — area under the data; MRT— mean residence time at t time;; VSS — volume of distribution steady state; TC — total clearance.

Fig. 1. Time–activity curves of blood, bone and whole body in mice and rabbits. The sum of activity measured in all the organs and carcass was considered aswhole-body radioactivity. Time–activity levels in mice blood (A), bone (B) and whole body (C); and rabbits' blood (D), bone (E) and whole body (F) during thefirst 24 h are presented in the figure. Bars represent standard deviation.

ig. 2. Maximum intensity projections acquired at 3 h pi from mice injectedith 30 MBq of 177Lu-EDTMP. SPECT and CT images were reconstructednd are presented as overlaid images. Projection image in (A) shows theertebral column; projection image in (B) shows the right knee joint.tructures with metabolically active bone clearly show intense tracer uptake.ctivity accumulation other than in bone is seen only in the bladder.

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The specific activity of 177Lu used in these studies was N5Ci/mg (185 GBq/mg). Lyophilized kits of the radiopharma-ceutical used in the studies were produced at RadioisotopeCentre, POLATOM (Poland), or at Medi-Radiopharma, Ltd.(Hungary). The kit consisted of 35 mg EDTMP (0.077 mM),5.72 mg CaO (0.102 mM), 14.1 mg NaOH (0.35 mM). Theradiopharmaceutical is formulated by the addition of 1 mlof water for injection, the required amount of 177LuCl3,followed by adjusting the final volume to 5ml by the additionof normal saline and incubation at room temperature for30 min. For a typical labeling study, 2 GBq of 177Lu activityis used. The radiochemical purity of 177Lu-EDTMP wasdetermined by instant thin layer chromatography using water/methanol/NH4OH (20:20:1 volumetric mixture) as solventsystem in which the 177Lu-EDTMP moved to the solventfront, while the uncomplexed 177Lu remained at the pointof spotting. 177Lu-EDTMP complex having N99% radio-chemical purity was used for animal experiments.

2.2. Biodistribution studies in mice

All biodistribution and imaging studies were performed ina dedicated laboratory after clearance from the relevantethical committees. A veterinarian supervised the healthstatus of the animals.

177Lu-EDTMP was injected in CD-1 mice intravenouslyvia the tail vein using a microsyringe (Hamilton Co.,Switzerland), and the mice were sacrificed at different time

points starting from 30 min and ending at 8 week pi of thetracer. Five animals were taken for each time point. For theearly time points (0.5 to 24 h), 8–10 MBq of 177Lu-EDTMPactivity in a volume of 30 μl was injected. For theintermediate time points (2 to 5 days), 26–30 MBq of

FwavSA

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activity in a final volume of 30–50 μl and, for late timepoints (1 to 8 weeks), 46–50 MBq in a volume of 60–120μl were applied. At the appropriate time points, mice wereeuthanized with intraperitoneal injection of a veterinaryeuthanasia agent, T-61 ad us. vet. (Bayer, Inc., Leverkusen,Germany). Organs of interest were dissected, washed, driedand weighed. These included femur bone, bone marrow,muscle, heart, lung, liver, kidneys and thyroid glands. Forthyroid, the larynx and the first two cartilages of the trachea

Fig. 3. Results of the autoradiography studies. Sagittal sections of a mouse at 3 dayside of the image (third and fourth panel) represent internal reference activities covertebrae and ischiadic bone with the acetabular joint are magnified in Parts (B) andthe remodeling region with a relative absence of uptake in the internal spongiosa

were included. A total of 0.2–0.5 ml of blood was collectedby cardiac puncture to measure the blood activity. Bonemarrow samples were obtained by rinsing a random femurhead, lumen and condyli with 0.5 ml of physiological salineand collecting the washing fluid together with the resultingmass of bone marrow. Radioactivity was measured in awell-type gamma counter (NZ-310, Gamma, Hungary)previously calibrated for 177Lu. In mice, percent organcontribution to body mass was assumed as bone mass 10%,

s pi of 5 MBq 177Lu-EDTMP are presented in (A). Circular spots at the rightntaining 0.18, 1.8 and 9.2 kBq in the order of darkness. Regions of thoracic(C), respectively, to show uptake with high fixation in the bone surfaces andof the bones.

Fig. 4. SPECT/CT images of a Wistar rat injected with 40 MBq of 177Lu-EDTMP 3 h pi. The images shown are taken at the level of the midline (A)and the right knee joint (B). Remodeling bone structures predominantlyaccumulate the radiopharmaceutical.

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blood 8%, bone marrow 8% and muscle 27% of body mass.Percentages of injected activity in whole organs werecalculated. The uptakes are expressed as percent of injectedactivity (%IA) per organ/tissue. Pharmacokinetic para-meters were derived from the mean values of biodistribu-tion data acquired at different time points by applyingTopfit v. 2.0 pharmacokinetic software (Fischer Verlag,

Table 3Biodistribution of 177Lu-EDTMP in rabbits per organ and per gram of organ weig

Organs/tissues Time points

30 min 1 h

%IA S.D. %IA/g S.D. %IA S

Blood 2.95 0.14 0.02 0.00 1.62 0Muscle 0.18 0.12 0.00 0.00 0.71 0Bone 36.57 5.52 0.33 0.04 40.99 4Bone marrow 0.01 0.01 0.00 0.00 0.01 0Thyroid 0.25 0.16 0.42 0.18 0.1 0Heart 0.05 0.12 0.01 0.00 0.03 0Lung 0.12 0.06 0.00 0.00 0.13 0Liver 0.82 0.91 0.00 0.00 0.92 0Kidneys 1.39 0.48 0.03 0.02 1.02 0

Organs/tissues Time points

5 days 1 week

%IA S.D. %IA/g S.D. %IA S

Blood 0.00 0.00 0.00 0.00 0.00 0Muscle 0.01 0.01 0.00 0.00 0.02 0Bone 23.09 2.62 0.23 0.03 25.92 1Bone marrow 0.01 0.01 0.00 0.00 0.00 0Thyroid 0.00 0.00 0.00 0.00 0.01 0Heart 0.01 0.00 0.00 0.00 0.00 0Lung 0.06 0.02 0.00 0.00 0.01 0Liver 0.03 0.01 0.00 0.00 0.13 0Kidneys 0.02 0.02 0.00 0.00 0.01 0

Time points

3 h 6 h 2

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %

Blood mass was calculated as 8% of body mass, bone mass was calculated as 5%

Jena, Germany) using the noncompartmental model. Time–activity curves were drawn using Prism v. 5.0 software(GraphPad, Inc., USA).

2.3. Imaging studies in mouse

2.3.1. SPECT-CT studiesThree CD-1 mice were injected with 50 μl (30 MBq) of

177Lu-EDTMP. Three hours postinjection, mice were anaes-thetized by intraperitoneal injection of ketamine [4 mg/kgbody weight (bw)] (SBH-Ketamin, SBH Ltd.) and xylazine(1 mg/kg bw) (Xylavet, Alfasan, Holland). A dedicatedsmall animal imaging system, NanoSPECT/CT (Mediso)equipped with a multiplexed multi-pinhole (nine pinholes,aperture 1.4 mm) collimator was used to acquire whole-bodySPECT/CT images. Energy window was centered at 208keV with ±10% width for all imaging studies. Acquisitiontimes were defined to obtain 100 000 counts for eachprojection with 24 projections. Images and maximumintensity projections (MIPs) were reconstructed using thededicated software Invivoscope (Bioscan, Inc., Washington,USA) and Mediso InterViewXP (Mediso).

2.4. Autoradiography in mice

For whole-body autoradiography, three mice wereinjected intravenously with 5 MBq of 177Lu-EDTMP. Theanimals were euthanized at 3 day pi and frozen in liquidnitrogen. Fifty-micrometer-thin cryostat sections were pre-

ht at various time points postinjection

2 h

.D. %IA/g S.D. %IA S.D. %IA/g S.D

.46 0.01 0.00 0.51 0.3 0.00 0.00

.24 0.00 0.00 1.11 0.88 0.00 0.00

.54 0.29 0.05 38.83 4.71 0.27 0.04

.02 0.00 0.00 0.03 0.08 0.00 0.00

.05 0.17 0.00 0.06 0.02 0.10 0.11

.01 0.00 0.00 0.41 0.53 0.00 0.00

.07 0.00 0.00 0.45 0.38 0.02 0.00

.26 0.01 0.00 0.54 0.31 0.01 0.01

.16 0.02 0.00 0.98 0.19 0.02 0.00

2 weeks

.D. %IA/g S.D. %IA S.D. %IA/g S.D

.00 0.00 0.00 0.00 0.00 0.00 0.00

.00 0.00 0.00 0.32 0.25 0.00 0.00

.97 0.26 0.02 24.96 2.57 0.25 0.03

.00 0.00 0.00 0.00 0.00 0.00 0.00

.01 0.00 0.00 0.01 0.01 0.00 0.00

.00 0.00 0.00 0.00 0.00 0.00 0.00

.00 0.00 0.00 0.01 0.01 0.00 0.00

.04 0.00 0.00 0.08 0.06 0.00 0.00

.00 0.00 0.00 0.02 0.02 0.00 0.00

4 h 48 h

IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D

of body mass, bone marrow mass was considered identical to blood mass

.

.

.

.

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pared using Leica CM 3600 instrument (Leica Co., Germany).The sections together with activity standards containing 0.18,1.8 and 9.2 kBq were exposed to phosphor imaging plates(MolecularDynamics Phosphor Imager,GEHealthcare,USA).

2.5. Imaging studies in rats

Forty megabecquerel of 177Lu-EDTMP was injected intothe tail vein of three rats and imaged after 3 h using theNanoSPECT/CT system. The anaesthesia and imagingparameters used were the same as described above exceptthat a pinhole aperture with 2.0 mm in diameter was applied.

2.6. Biodistribution studies in rabbits

Three New Zealand white rabbits (Biofarm, Ltd.,Hungary) were used for each time point varying from30 min to 8 weeks. 177Lu-EDTMP tracer was injected via theauricular vein. Rabbits used for early time points (30 min to1 week) received 100 MBq (in 0.3–0.4 ml), while the onesused for later time point studies received 200 MBq (in 0.6–0.7 ml) of activity. Animals were anaesthetized withintramuscular injection of ketamin (dose 100 mg/kg bw)and xylazine (dose 10 mg/kg bw). One millilitre of bloodwas drawn from the heart and then the animals wereeuthanized with intracardial injection of 1 ml T-61 ad us. vet.Bone (tibia was taken as sample), bone marrow, muscle,heart, lung, liver, kidneys and thyroid glands were collected.Organs were cleaned, dried and weighed. Radioactivity

Table 3 (continued)

Time points

3 h 6 h 2

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %

3.17 0.05 0.02 0.00 0.08 0.01 0.00 0.00 00.65 0.37 0.00 0.00 0.51 0.56 0.00 0.00 036.91 8.92 0.26 0.06 33.42 2.00 0.27 0.04 20.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00.03 0.01 0.04 0.00 0.03 0.02 0.04 0.02 00.22 0.30 0.03 0.00 0.07 0.05 0.00 0.00 00.04 0.04 0.00 0.00 0.06 0.05 0.00 0.00 00.35 0.40 0.01 0.00 0.29 0.08 0.00 0.00 00.55 0.30 0.01 0.02 0.72 0.50 0.01 0.00 0

Time points

3 weeks 4 weeks 6

%IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D. %

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00.06 0.06 0.00 0.00 0.03 0.03 0.00 0.00 025.02 1.22 0.25 0.01 24.05 1.98 0.24 0.02 20.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00.10 0.08 0.00 0.00 0.02 0.02 0.00 0.00 00.01 0.01 0.00 0.00 0.01 0.01 0.00 0.00 00.01 0.01 0.00 0.00 0.01 0.00 0.00 0.00 00.09 0.06 0.00 0.00 0.01 0.00 0.00 0.00 00.03 0.03 0.00 0.00 0.01 0.01 0.00 0.00 0

in the organ was measured in a well-type gamma counter(NZ-310, Gamma, Hungary). Bone marrow was obtainedby physical removal of it from the tibial plateau, epicondyliand tibial lumen. In rabbits, percent organ contribution tobody weight was assumed as bone mass 5%, blood 8%, bonemarrow 8% and muscle 50% of body mass. Pharmacokineticparameters were calculated as described above.

2.7. Imaging studies in rabbits

Three rabbits were injected as above with 40 MBq (in0.2 ml) of activity. Planar whole-body images in dorsoven-tral (DV), ventrodorsal (VD) and left lateral view wereacquired in a 256×256×16 matrix at different time pointsstarting from 30 min and ending at 4 weeks using a digitalgamma camera (Nucline X-Ring/R, Mediso). At the time ofimaging, 1-ml blood samples were drawn and countedtogether with a standard of injected activity.

2.8. Dose escalation studies in healthy beagle dogs

Four activity levels (9.25, 18.5, 27.75 and 37 MBq/kg)were studied. Three randomized healthy male beagle dogsweighing between 13.2 and 23.4 kg were used for eachactivity level. The tracer was injected through the cephalicvein. Venous blood samples (4 ml, cephalic or jugular vein)were drawn from each of the injected animals, to determinethe biochemical parameters. Serum levels of alanineaminotransferase, alkaline phosphatase, gamma-glutamyl

4 h 48 h

IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

.03 0.02 0.00 0.00 0.01 0.01 0.00 0.00

.26 0.29 0.00 0.00 0.04 0.02 0.00 0.008.68 3.18 0.24 0.02 25.87 3.91 0.23 0.07.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00.05 0.03 0.05 0.04 0.05 0.05 0.00 0.00.08 0.04 0.00 0.00 0.02 0.02 0.00 0.00.06 0.03 0.00 0.00 0.03 0.04 0.00 0.00.39 0.24 0.01 0.00 0.10 0.02 0.00 0.00.21 0.08 0.01 0.00 0.05 0.03 0.00 0.00

weeks 8 weeks

IA S.D. %IA/g S.D. %IA S.D. %IA/g S.D.

.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

.01 0.00 0.00 0.00 0.01 0.00 0.00 0.001.95 1.05 0.22 0.01 20.43 2.53 0.20 0.02.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00.01 0.00 0.00 0.00 0.01 0.00 0.00 0.00.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

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transferase, urea and creatinine were measured. Urea/creatinine ratio was calculated to verify possible renaldamage. Automated complete blood counts performedincluded white blood cells (WBC), red blood cells andplatelets. All measurements were done by a dedicatedveterinary laboratory (VetMedLabor, Ltd., Budapest, Hun-gary). Toxicity to bone marrow was defined as reduction inplatelet concentration below 100 g/L (103/μl) or WBC levelbelow 4 g/L.

Cumulative urine up to 72 h was collected in fractionsduring the time intervals 0–6 h, 6–24 h, 1–2 days and 2–3days via an indwelling Foley catheter. At each time pointthe urinary bladder was rinsed with saline and the finalvolume was documented. Activity concentration in thecorresponding pooled fractions was determined in 1-mlsamples using the NZ-310 gamma counter.

Imaging studies in dogs consisted of VD and DV viewsby using a Nucline XRing/R (Mediso) gamma cameraequipped with a high-resolution, general-purpose collimator.Whole-body (512×1024) and spot images (256×256) wereacquired at different time points starting at 30 min and up to4 weeks pi. Prior to imaging, animals were anaesthetized byintravenous injection of ketamin and xylazine at a dose of100 and 5 mg/kg bw, respectively. Images were processedwith the Pmod 2.7 software package (Pmod TechnologiesCo., Zürich, Switzerland).

3. Results

3.1. Biodistribution studies in mice

The results of the biodistribution studies in mice at differenttime points studied are summarized in Table 1. Accumulationof activity in the bone is fast and peaks at 2 h pi. (40.82±3.98%IA in bone). Activity accumulation was primarily observed inthe bones with fast excretion via the urinary system. No organsother than the bone showed retention of activity in significantamount at any time point. The pharmacokinetic parameterscalculated from the biodistribution data are presented inTable 2. Time–activity curves of 177Lu-EDTMP in blood,bone and whole body are given in Fig. 1A–C.

Table 4Standard pharmacokinetic parameters of 177Lu-EDTMP calculated on the basis of

Organs/tissue t1/2 (h) AUD (%⁎h) AUC∞ (%h/g) MR

Blood 8.91 30.97 30.97 2Muscle 250 115.99 119.60 348Bone 1870 31806.8 87063.5 639Thyroid 297 14.53 14.53 397Heart 215 7.15 7.15 325Lungs 181 11.10 11.10 310Liver 219 65.54 68.69 276Kidneys 102 29.19 29.19 160

t1/2 — terminal elimination half-life; AUC∞ — area under the curve extrapolatedMRT— mean residence time at t time; MRT∞ — mean residence time extrapolatestate; TC — total clearance.

3.2. Imaging studies in mouse and rat

Fig. 2 shows maximal intensity projection (MIP) SPECTimages in a mouse overlaid on CT images. Results of thedigital autoradiography study in mouse are presented in Fig. 3,clearly illustrating the distribution of 177Lu-EDTMP withpredominant accumulation in the skeleton. Internal referenceactivity sources are also shown in the figure (black round spot).Fig. 4 showsMIP images of overlaid SPECT and CT studies ina rat at two different lateral projections.

3.3. Biodistribution and imaging studies in rabbits

Results of the biodistribution studies in rabbits are shownin Table 3 and the pharmacokinetic parameters derived fromthe data are summarized in Table 4. Time–activity curves ofthe tracer in blood, bone and whole body are given in Fig.1D–F. Ventro-dorsal images of rabbits at different timepoints (1 h, 1 day, 1 week and 4 weeks) pi are presented inFig. 5. The results of the biodistribution studies arecomparable to those obtained in mice. Three hourspostinjection, the bone is the only organ having activityretention higher than 1 %IA.

3.4. Dog imaging studies

Fig. 6 shows the ventrodorsal images of two dogs at1 h and 1 week pi of 177Lu-EDTMP. The upper panel (Aand B) represents the dog which received 9.25 MBq/kgbw, and the lower panel (C and D) is the dog whichreceived 37MBq/kg bw. Results of the activity excreted inthe urine during different time intervals of the fourdifferent dosing groups of dogs are presented in Table 5.Most of the nonbone accumulated activity was excretedbetween 0 and 6 h. Activity excretion during 48–72 h wasrelatively insignificant.

3.5. Toxicity studies

Table 6 summarizes the results of the biochemicalanalyses of the group of three dogs which received thehighest injected activity (37 MBq/kg). All the biochemicalparameters studied are within the normal range. The ratioof serum urea to creatinine was below 0.1 thereby

rabbit biodistribution results and the noncompartmental model

T (h) MRT∞ (h) Vz (ml) VSS (ml) TC (ml/min)

.83 2.83 41.5 9.12 5. 38×10−2

389 302 326 13.9×10−2

2800 3.11 3.22 1.91×10−5

397 2950 2740 1.15×10−2

325 4350 4550 2.33×10−1

310 2350 2790 1.50×10−1

340 460 495 2.43×10−2

160 505 546 5.71×10−2

to t=∞; AUD — area under the data.d to t=∞; Vz — volume of distribution; VSS — volume of distribution steady

Fig. 5. Planar ventrodorsal scintigraphic images of rabbits 1 h (A), 1 day (B), 1 week (C) and 4 weeks (D) postinjection of 40 MBq of 177Lu-EDTMP.

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indicating no renal toxicity. Serial measurements of whiteblood cells and platelets for all the activity groups arepresented in Fig. 7. The normal range for white blood cellsand the lower cut of limit for platelets are shown withhorizontal bars. Neither WBC nor platelet counts reducedbelow toxic levels in any of the activity group studied.Reduction of platelet counts below the normal range wasseen at 1 week and persisted till the fourth week in thecase of dogs administered with the highest activity. Thecounts increased to normal range 5 weeks postadministra-tion of activity. The follow-up on the dogs by theveterinary physician did not report any changes ordeterioration on the overall health status of the animals.In three dogs, mild, transient urethritis attributed tocatheterization was observed.

4. Discussion

The composition of the freeze-dried EDTMP kits used inthe present studies was matched to the registered product of153Sm-EDTMP. Detailed quality control of the kits includingsterility, apyrogenicity and long-term stability was estab-lished. The radiochemical purity as well as the stability of thecomplex over a period of time was studied. The 177Lu-

EDTMP complexation yield was greater than 99% to startwith and there was no significant reduction in theradiochemical purity over several days of storage. Injectionof the labeled product in this study did not result in systemicreaction in any of the species studied.

Biodistribution studies were done both in mice and inrabbits, and the percent injected activity (%IA) associatedwith each organ was calculated based on the activitymeasured on the whole organ or part of the tissues. Thereis significant difference in the body composition betweendifferent species such as mice and rabbits. Body compositionof CD-1 mouse (n=10) and New Zealand white rabbit (n=6)strains was measured preceding the present studies. Based onour previous experience and the present studies, we took10% for bone and 27% for muscle in the case of mice and 5%for bone and 50% for muscle in case of rabbits.

The results of the biodistribution studies in mice andrabbits summarized in Tables 1 and 3 show that 177Lu-EDTMP concentrates in the bone; very high uptake is seen inearly time points and persists till the last time point studied.Activity not bound to bone is excreted via the kidneys andbladder. The activity from blood is cleared very fast.

Pharmacokinetic parameters in mice and rabbits summa-rized in Tables 2 and 4, respectively, give importantinformation about the biokinetics of 177Lu-EDTMP. We

Fig. 6. Planar ventrodorsal scintigraphic images of beagle dogs at differentactivities and different time points. (A) and (B) represent the images of a doginjected with 9.25 MBq/kg bw of 177Lu-EDTMP at 1 h and 1 week,respectively. (C) and (D) represent the images of a dog injected with 37MBq/ kg bw at 1 h and 1 week pi, respectively.

able 5esults of urine collection in the dog dose groups

og dosingroup

Radioactivity cumulated in urine in % of IA in theindicated period postinjection

0–6 h 6–24 h 24–48 h 48–72 h Total 0–72 h

7 MBq/kg bw. 34.8 (9.1) 3.0 (0.6) 1.5 (0.5) 0.4 (0.2) 39.0 (8.4)9.8 MBq/kg bw 41.9 (7.9) 2.2 (1.3) 1.1 (0.3) 0.2 (0.1) 46.4 (7.7)8.6 MBq/kg bw 36.2 (9.5) 5.0 (1.3) 2.1 (1.3) 0.3 (0.1) 42.5 (7.3).3 MBq/kg bw 38.1 (7.8) 4.2 (2.1) 1.1 (0.3) 0.3 (0.1) 44.2 (6.0)

alues are shown as mean (S.D.) Percent of urinary excretion over timeeriod in groups of three dogs treatedwith different activities of 177Lu-EDTP.

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chose the noncompartmental model for calculation of thepharmacokinetic parameters as one of the reported studiesfound seven compartments for 99mTc-methylydene dipho-sphonate which were not realistically handled in thecalculations [34]. Data obtained in our study were relevantto the noncompartmental model which has minimal numberof presumptions and restrictions. Results of the pharmaco-kinetic modeling studies support stable and long-lastingbinding of 177Lu-EDTMP to bone. The terminal half-life inbone was 2130 and 1870 h, whereas in blood was 7.3 and 8.9h in mice and rabbits, respectively. All organs other thanbone having long terminal elimination half-lives containedminuscule fraction of radioactivity as reflected by very smallvalues of area under the curve (AUC∞). The bone-to-muscle

ratio of AUC∞ was 130, while the bone-to-blood ratio ofAUC∞ was 14 053 in mice. In rabbits, the bone-to-muscleratio of AUC∞was 732 and the bone-to-blood ratio of AUC∞was 2811. A higher bone mass index and faster bonemetabolism with higher mineralization rate might explain thefaster tracer uptake and slightly higher whole-body uptake inmice [35,36]. The difference in the body composition ofrabbits and mice will lead to varied accumulated radioactiv-ity in the skeleton of those two species. In turn, this couldresult in different radiation doses. Both the species studiedhave an important role in extrapolating the results to humans.Mice provide a basis for extrapolation to the adult human,while rabbits offer a model for physiologic processes andbiodistribution in the growing bone (nutrition of epiphysealplates is the same in the rabbit as in man) [37,38].

Autoradiography studies show uptake of the activity byboth long and spongeous bones in the remodeling regionsand bone surfaces (Fig. 3). Joints and surfaces of thevertebrae and the ischiadic bone accumulate more radioac-tivity than the internal spongiosa of the same bones (Fig. 3,blown-up Sections B and C).

Imaging studies in rabbits and dogs showed protractedbone localization (Figs. 5 and 6). Rapid bone uptake togetherwith fast blood clearance of the tracer results in good qualityimages at all time points. By using imaging studies and theactivity measured in urine of dogs, it is reasonable to statethat the pharmacokinetic behavior of 177Lu-EDTMP in dogsis similar to mice and rabbits.

There was no difference among the four different activitygroups (9.25, 18.5, 27.75 and 37 MBq/kg bw) in terms of theactivity excreted or excretion rate in urine (Table 5). As thespecific activity of the tracer used was identical and only theactivity injected varied, it can be inferred that there is nomass effect of the tracer on body localization. The nonboneaccumulated activity is excreted preferentially through therenal route. By pooling the results of the animals of allgroups, it can be seen that 96.6% of the urinary excretionoccurs in the first 24 h. The results provide strong support tothe inference that there will not be any significant radiation-related adverse effects to any organs other than the bone dueto activity accumulation. Activity uptake in bone marrow isalso insignificant. The energy deposition of 177Lu betaparticles is almost totally achieved within the volume of asmall-diameter (maximally 5 mm) sphere [26,27]. From the

TR

Dg

3119

Vp

Table 6Biochemical parameters of the dogs treated with 37 MBq/kg bw of 177Lu-EDTMP

Parameters Normal range Pretreatment 2 days 1 week 2 weeks 3 weeks 4 weeks 5 weeks

ALT 10–60 IU/L 52.6 (25.7) 38.5 (38.5) 46.6 (36.7) 106 (13.3) 48.6 (43.6) 45.3 (12.4) 56.0 (5.5)ALKP 40–300 IU/L 87 (41.9) 96 (49.5) 64 (13.4) 256 (40.4) 78 (11.1) 217 (33.4) 87 (26.3)GGT 0–10 IU/L 0.0 (0.0) 0.5 (0.4) 0.9 (0.4) 2.1 (1.2) 1.3 (0.4) 3.7 (0.5) 0.0 (0.0)Urea 4.5–9.0 mmol/L 4.5 (0.4) 4.5 (0.4) 5.8 (0.5) 3.6 (0.4) 4.5 (0.5) 5.6 (1.1) 4.8 (1.0)Serum creatinine 40–140 μmol/L 75 (12.4) 56.2 (12.3) 70.6 (22.2) 58.5 (15.4) 59.5 (17.8) 68.6 (19.3) 62.9 (13.2)Serum urea/creatinine ratio Below 0.10 0.06 (0.01) 0.08 (0.03) 0.09 (0.04) 0.06 (0.01) 0.08 (0.03) 0.08 (0.01) 0.08 (0.03)

Values are shown as mean (S.D.).ALT — Alanine transaminase; ALKP — alkaline phosphatase; GGT — gamma-glutamyl transferase.

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radioactivity accumulated in the bones, the radiation damagecould only reach the bone marrow.

Biochemical parameters to assess the liver and kidneyfunction determined in the four activity groups eachconsisting of three dogs did not reveal any abnormalitiesfor the length of the follow-up period of 5 weeks.Biochemical parameters of the highest activity group (37MBq/ kg bw) alone are presented in Table 4. The reason foran above normal alanine transaminase value in the secondweek is not understood as it is not part of any definite trendand hence no inference is drawn. The tracer being excretedmainly through the renal pathway, the kidney and bladder arethe critical organs [39,40] in this regard. Measured

Fig. 7. White blood cells (top) and platelet counts (bottom) at different timepoints of rabbits injected with four different levels (9.25–37 MBq/kg bw)of 177Lu-EDTMP activity. Bars represent standard deviation (n=3). Thelines parallel to the x-axis represent the normal range (top) and lower cutoff limit (bottom).

biochemical parameters and serum urea-to-creatinine ratiodo not indicate any damage to the renal system, even at thehighest level of activity injected (Table 6).

A drop below normal values for platelet and WBC countswas seen in the highest activity group (Fig. 7). As opposed toplatelets, the trend in the WBC counts was not unidirection-al. This could probably be due to a reactive leukocytosis toiatrogenic urethritis caused by catheterization. This changein WBC counts indicates that the proliferative capability ofthe bone marrow in dogs is not hindered even at the highestlevel of injected activity. Neither WBC nor platelet countsreached the toxicity levels. The results clearly suggest thatthere are no adverse effects or toxicity up to 37 MBq/kg bwof injected activity.

5. Conclusion

We examined the biodistribution, pharmacokinetic,biochemical and hematologic parameters of 177Lu-EDTMPin different species in order to collect data to support ahuman clinical trial. The biodistribution, autoradiographyand imaging studies of 177Lu-EDTMP clearly demonstratethat the tracer is taken up almost exclusively by the skeletalsystem, with minimal activity accumulation in any otherorgan. Multidose studies up to 37 MBq/kg bw in dogs didnot result in any adverse effects as seen from the biochemicalparameters and hematological measurements. Based on theresults of the present studies, we consider 177Lu-EDTMP asuitable radiopharmaceutical for human clinical trials as ametastatic bone pain palliation agent. Data collected in thepresent studies formed the basis for initiating a multicentreclinical trial of 177Lu-EDTMP carried out under an IAEACoordinated Research Project.

Acknowledgments

The protocol for the reported studies was finalized duringa consultants' meeting organized by IAEA during 2006 andthe authors are thankful to Dr. W.A. Volkert, Dr. RenataMikolajczak and Dr. Meera Venkatesh who participated inthe consultants' meeting and gave valuable suggestions.Károly Haller and Norbert Fésüs are acknowledged for theirkind help in performing the experiments. Radioisotope

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Centre POLATOM and Medi-Radiopharma Ltd. are grate-fully acknowledged for their support in providing theEDTMP kits.

References

[1] Mercadante S, Fulfaro F. Management of painful bone metastases.Curr Opin Oncol 2007;19:308–14.

[2] Cicek M, Oursler MJ. Breast cancer bone metastasis and current smalltherapeutics. Cancer Metastasis Rev 2006;25:635–44.

[3] Berenson JR, Rajdev L, Broder M. Managing bone complications ofsolid tumors. Cancer Biol Ther 2006;9:1086–9.

[4] Macedo A, Araujo A, Carvalho Melo F, et al. Cost-effectiveness ofsamarium-153-EDTMP for the treatment of pain due to multiple bonemetastases in hormone-refractory prostate cancer versus conventionalpain therapy, in Portugal. Acta Med Port 2006;19:421–6.

[5] Knapp FF, Mirzadeh S, Beets AL, et al. Reactor produced radio-isotopes from ORNL for bone pain palliation. Appl Radiat Isot1998;49:309–15.

[6] Anderson MP, Wiseman AG, Dispenzieri A, et al. High-dosesamarium-153 ethylene diamine tetramethylene phosphonate: lowtoxicity of systemic irradiation in patients with osteosarcoma andskeletal metastases. J Clin Onc 2002;20:189–96.

[7] Bauman G, Charette M, Reid R, Sathya J. Radiopharmaceuticals forthe palliation of painful bone metastasis—a systemic review. Radio-ther Oncol 2005;75:258–70.

[8] Liepe K, Runge R, Kotzerke J. The benefit of bone-seekingradiopharmaceuticals in the treatment of metastatic bone pain. JCancer Res Clin Oncol 2005;131:60–6.

[9] Minutoli F, Herberg A, Spadaro P, et al. [186Re]HEDP in the palliationof painful bone metastases from cancers other than prostate and breast.Q J Nucl Med Mol Imaging 2006;50:355–62.

[10] Silberstein EB. Dosage and response in the radiopharmaceuticaltherapy of painful osseous metastases. J Nucl Med 1996;37:249–52.

[11] Srivastava S, Dadachova E. Recent advances in radionuclide therapy.Semin Nucl Med 2001;31:330–41.

[12] Finlay IG, Mason MD, Shelley M. Radioisotopes for the palliation ofmetastatic bone cancer: a systematic review. Lancet Oncol2005;6:392–400.

[13] Hosain F, Spencer RP. Radiopharmaceuticals for palliation ofmetastatic osseous lesions: Biologic and physical background. SeminNucl Med 1992;22:11–6.

[14] Pandit-Taskar N, Batraki M, Divgi CR. Radiopharmaceutical therapyfor palliation of bone pain from osseous metastases. J Nucl Med2004;45:1358–65.

[15] Sartor O. Overview of samarium Sm-153 lexidronam in the treatmentof painful metastatic bone disease. Rev Urol 2004;6(Suppl 10):S3–S12.

[16] Sartor O, Reid RH, Bushnell DL, Quick DP, Ell PJ. Safety and efficacyof repeat administration of samarium Sm-153 lexidronam to patientswith metastatic bone pain. Cancer 2007;109:637–43.

[17] Coronado M, Redondo A, Coya J, et al. Clinical role of Sm-153-EDTMP in the treatment of painful bone metastatic disease. Clin NuclMed 2006;31:605–10.

[18] Wang RF, Zhang CL, Zhu SL, Zhu M. A comparative study ofsamarium-153-ethylenediaminetetramethylene phosphonic acid withpamidronate disodium in the treatment of patients with painfulmetastatic bone cancer. Med Princ Pract 2003;12:97–101.

[19] Sartor O, Reid RH, Hoskin PJ, et al. Samarium-153-lexidronamcomplex for treatment of painful bone metastases in hormone-refractory prostate cancer. Urology 2004;63:940–5.

[20] Brenner W, Kampen WU, Kampen AM, Henze E. Skeletal uptake andsoft-tissue retention of 186Re-HEDP and 153Sm-EDTMP in patientswith metastatic bone disease. J Nucl Med 2001;42:230–6.

[21] Ogawa K, Mukai T, Asano D, et al. Therapeutic effects of a 186Re-complex-conjugated bisphosphonate for the palliation of metastaticbone pain in an animal model. J Nucl Med 2007;48:122–7.

[22] Oh SJ, Won KS, Moon DH, et al. Preparation and biological evaluationof 188Re-ethylenediamine-N,N,N′,N′tetrakis(methylene phosphonicacid) as a potential agent for bone pain palliation. Nucl Med Commun2002;23:75–83.

[23] Liepe K, Franke WG, Kropp J, Koch R, Runge R, Hliscs R.Comparison of rhenium-188, rhenium-186-HEDP and strontium-89 inpalliation of painful bone metastases. Nuklearmedizin 2000;39:146–51.

[24] Li S, Liu J, Zhang H, Tian M, Wang J, Zheng X. Rhenium-188 HEDPto treat painful bone metastases. Clin Nucl Med 2001 Nov;26(11):919–22.

[25] Pillai MRA, Chakraborty S, Das T, Venkatesh M, Ramamoorthy N.Production logistics of 177Lu for radionuclide therapy. Appl Radiat Isot2003;59:109–18.

[26] Bernhardt P, Benjegård SA, Kölby L, et al. Dosimetric comparison ofradionuclides for therapy of somatostatin receptor-expressing tumors.Int J Rad Onc Biol Phys 2001;51:514–24.

[27] Syme AM, Kirkby C, Riauka TA, Fallone BG, McQuarrie SA. MonteCarlo investigation of single cell beta dosimetry for intraperitonealradionuclide therapy. Phys Med Biol 2004;49:1959–72.

[28] Ando A, Ando I, Tonami N, et al. 177Lu-EDTMP: a potentialtherapeutic bone agent. Nucl Med Comm 1998;19:587–91.

[29] Rutty Solá AG, Arguelles MG, Botazzini D, et al. Lutetium-177EDTMP for bone pain palliation. Preparation, biodistribution and pre-clinical studies. Radiochim Acta 2000;88:157–61.

[30] Chakraborty S, Das T, Unni PR, et al. 177Lu-labeled polyaminopho-sphonates as potential agents for bone pain palliation. Nucl MedCommun 2002;23:67–74.

[31] Das T, Chakraborty S, Unni PR, Sarma HD, Samuel G, Banerjee S,Venkatesh M, Ramamoorthy N, Pillai MRA. 177Lu labeled cyclicpolyaminophosphonates as potential agents for bone pain palliation.Appl. Radiat. Isot 2002;57:177–84.

[32] Das T, Chakraborty S, Sarma HD, Banerjee S. 177Lu-DOTMP: a viableagent for palliative radiotherapy of painful bone metastasis. RadiochimActa 2008;96:55–61.

[33] Chakraborty S, Das T, Banerjee S, Balogh L, Chaudhari PR, SarmaHD, et al. 177Lu-EDTMP: a viable bone pain palliative in skeletalmetastasis. Cancer Biother Radiopharm 2008;23:202–13.

[34] Sagar VV, Piccone JM, Charkes ND. Studies of skeletal tracer kinetics:III. Tc-99m(Sn)methylenediphosphonate uptake in the canine tibia as afunction of blood flow. J Nucl Med 1979;20:1257–61.

[35] Kumar V, Kumar D, Howman-Giles RB, Little DG. Is 99mTc-labelledpamidronate a better agent than 99mTc-medronate for bone imaging?Nucl Med Commun 2007;28:101–7.

[36] Toegel S, Hoffmann O, Wadsak W, et al. Uptake of bone-seekers issolely associated with mineralisation! A study with 99mTc-MDP,153Sm-EDTMP and 18F-fluoride on osteoblasts. Eur J Nucl Med MolImaging 2006;33:491–4.

[37] Essman SC, Lewis MR, Miller WH. Intraorgan biodistribution anddosimetry of 153Sm-ethylenediaminetetramethylene phosphonate injuvenile rabbit tibia: implications for targeted radiotherapy ofosteosarcoma. J Nucl Med 2005;46:2076–82.

[38] Essman SC, Lattimer J, Cook JL, Turnquist S, Kuroki K. Effectsof 153Sm-ethylenediaminetetramethylene phosphonate on physealand articular cartilage in juvenile rabbits. J Nucl Med 2003;44:1510–5.

[39] Eary JF, Collins C, Stabin M, et al. Samarium-153-EDTMPbiodistribution and dosimetry estimation. J Nucl Med 1993;34:1031–6.

[40] Brenner W, Kampen WU, Brummer C, von Forstner C, Zuhayra M,Czech N, Muhle C, Henze E. Bone uptake studies in rabbits before andafter high-dose treatment with 153Sm-EDTMP or 186Re-HEDP. J NuclMed 2003;44:247–51.