Open AccessAustin Journal of Nuclear Medicine andRadiotherapyResearch ArticleDevelopment of Radiolanthanide-Labeled-BisAlendronate Complexes for Bone Pain Palliation TherapyFakhari A1, Jalilian AR2*, Yousefnia H2, ShafieeArdestani M1, Johari-Daha F2, Mazidi M2 andKhalaj A11Faculty of Pharmacy, Tehran University of MedicalSciences, Tehran, Iran2Nuclear Science and Technology Research Institute(NSTRI), Tehran, Iran*Corresponding author: Amir R. Jalilian, NuclearScience and Technology Research Institute (NSTRI),Tehran, IranReceived: September 03, 2015; Accepted: September28, 2015; Published: September 30, 2015AbstractThe search for the development of new ligands with higher stability, betterpharmacokinetics and lower unwanted tissue uptakes (liver and GI) is stillongoing. In this work, synthesis, purification and structure characterization ofDTPA-bis-ALN conjugate is reported followed by evaluation in model animals.A DTPA-conjugated bis-alendronate analog (DTPA-bis-ALN) 3, was preparedfor possible bone pain palliation therapy after radiolabeling with Ho-166 andSm-153. Radiolanthanide-DTPA-bis-ALN complexes were prepared startingexcess amounts radiolanthanide chloride and DTPA-bis-ALN in 60-90 minat 50-60oC in phosphate buffer followed by solid phase purification on C18Sep Pak column. RTLC was used for radiochemical purity followed by log Pdetermination, stability studies and biodistribution studies in normal mice. Thepurified radiolabled complexes were prepared in high radiochemical purity( 98%, RTLC) and significant specific activity (7-10 GBq/mmol). The log P forthe complex was calculated as -0.594 and -0.43, consistent with water solublecomplexes. The complexes were stable in final solutions (25ºC) and presenceof human serum (37ºC). The biodistribution of the labeled compounds in normalmice demonstrated unwanted activity uptake in lungs, spleen and liver in case of166Ho-DTPA-bis-ALN and liver lung and kidney in case of 153Sm-DTPA-bis-ALN.Very limited bone uptake in both cases demonstrates complex instability or lossof bone avidity due to change of structure-activity relationship and/or anionicproperty of poly-dentate complex leading to renal excretion.Keywords:Alendronate; Sm-153; Ho-166; Solid phase extraction;biodistribution; DTPA-conjugateIntroductionBone pain arises in more than 50% of bone metastases usually anoutcome of various tumors metastases such as prostate (80%), breastand lung carcinoma (50%) [1,2]. A possible modality in bone painmanagement in these patients is the application of a systemic boneavid radiopharmaceutical for with potential benefits . Althoughnew alpha emitters have been introduced to the medical societyfor increasing the effectiveness of bone pain palliation, however,inaccessibility, high prices and limited vendors have negative effecton their vast applications .Radiolabeled bis-phosphonates such as 153Sm-EDTMP, 188/186ReHEDP, 177Lu-EDTMP and also 166Ho-EDTMP have been approvedby well-known pharmaceutical legal bodies, however, the researchand development of bone pain palliation compounds is an ongoing research area around the world. Recently many bone-seekingagent such as DO2A2P have been prepared and evaluated for theirstereoisomer studies , and still the application of radiolanthanidesis preferred  and they are entering faster and more efficient in theclinical trials in developing countries .including aliphatic chains as well as macrocycles have been reportedsuch as 166Ho-DOTMP [8,9], 166Ho-EDTMP [10,11], 166Ho-APDDMP, as well as 166Dy/166Ho-EDTMP  for management ofbone metastasis and pain prevention in breast cancer and multiplemyeloma .On the other hand, samarium-153 has favorable radiationcharacteristics, such as medium-energy beta particle emissions (Emax 810 keV, range of about 3.0 mm), medium-energy gamma photon(103 keV, 28%) in addition to particle emissions which make itsuitable for monitoring the therapy with imaging, widely used in painpalliation radiopharmaceutical in form of EDTMP complex .The search for the development of new ligands with higherstability, better pharmacokinetics and lower unwanted tissue uptakes(liver and GI) is still ongoing.The uni-elemental abundance of natural holmium, makesholmium-166 (Eβ- max 1.84 MeV, T1/2 26.8 h), an accessible,inexpensive radionuclide with enough specific activity forradiolabeling and targeted therapy modalities.Considering the inhibitory binding affinity constant (Ki) of bisphosphonates used in clinics alendronic acid and the idea of developingbone avid agents based on alendronic acid is of great interest. Inrecent studies, using simple bis-phosphonate –radiolanthanidecomplexes such as 177Lu-zoledronate , 166Ho-pamidronate ,177Lu-pamidronate; and 177Lu-alendronate  have not shown anyimproved properties in their bone avidity compared to their clinicalrivals. The resulting complexes were not stable in vivo and/or showedlower bone uptake compared to other therapeutic bisphosphonates.Possible therapeutic 166Ho bone-seeking bis-phosphonate agentsAn interesting novel approach that has been aimed in this work isAustin J Nucl Med Radiother - Volume 2 Issue 1 - 2015Submit your Manuscript www.austinpublishinggroup.comJalilian et al. All rights are reservedCitation: Fakhari A, Jalilian AR, Yousefnia H, Shafiee-Ardestani M, Johari-Daha F, et al. Development ofRadiolanthanide-Labeled-Bis-Alendronate Complexes for Bone Pain Palliation Therapy. Austin J Nucl MedRadiother. 2015; 2(1): 1012.
Jalilian ARAustin Publishing Groupirradiation of 100 µg of natural 165Ho(NO3)3 (165Ho, 99.99% fromISOTEC Inc.) and Sm2O3 (from ISOTEC Inc.) respectively accordingto reported procedures at the Tehran Research Reactor at a thermalneutron flux of 4 1013 n.cm-2.s-1.Preparation of fresh cyclic DTPA dianhydrideCyclic DTPA dianhydride was prepared according to thereported method with slight modifications . Pyridine (1.2mL) and acetonitrile (1.2 mL) warmed to 50 C were mixed with asolution of DTPA (490 mg, 1.37 mmol) in acetic anhydride (0.8 mL,8.60 mmol) and heated at this temperature for 24 h. The resultinganhydride was insoluble in pyridine and acetonitrile. It was collectedby filtration, purified by repeated washing with acetic anhydride, andwith anhydrous ether at the end. The solid was dried under vacuumto remove the last trace of solvent. The melting point of white solidwas 182oC-185oC and its 1H NMR and IR spectra were in accordancewith the literature.Conjugation of cyclic DTPA di-anhydride with alendronateFigure 1: Chemical structure for DTPA-bis-ALN.developing bis phosphonate ligands including metal chelating agentsincluding DTPA moiety. The incorporation of two bis-phosphonatemoiety in a single molecule can lead to better targeting of bonetissue. The multi dentate poly-amino carboxylic acid containingbis-phosphonate ligand, presumably to form stable chelates withmany metals including lanthanides was developed by conjugationof cyclic DTPA dianhydride and [4-amino-1-hydroxy-1-(hydroxyoxido-phosphoryl)- butyl]phosphonic acid (Alendronic acid), as apossible carrier moiety, for the development as beta emitter-basedradiopharmaceuticals for bone pain palliation (Figure 1).In this work, synthesis, purification and structure characterizationof DTPA-bis-ALN conjugate is reported followed by the preparation,quality control and preliminary evaluation of related Ho-166 and Sm153 complexes.ExperimentalProduction of 166Ho was performed at the Tehran Research Reactor(TRR) using 165Ho (n, γ) 166Ho nuclear reaction. Natural holmiumnitrate with purity of 99.99% was obtained from ISOTEC Inc.Samarium-152 Oxide with purity of 98% was obtained from ISOTECInc., USA and used in radionuclide production. Whatman No. 2 wasobtained from Whatman (Maidstone, UK). Radio-chromatographywas performed by using a Bioscan AR-2000 radio TLC scannerinstrument (Bioscan, Paris, France). A high purity Germanium(HPGe) detector coupled with a Canberra (model GC1020-7500SL)multichannel analyzer and a dose calibrator ISOMED 1010 (Dresden,Germany) were used for counting distributed activity in mice organs.All other chemical reagents were purchased from Merck (Darmstadt,Germany). Calculations were based on the 80.6 keV peak for 166Hoand 103 keV for 153Sm. The approval of NSTRI Ethics Committee wasobtained for conducting this research. The wild-type mice (NMRI)were purchased from Pasteur Institute of Iran, Karaj, and all weighing20-25 g and were acclimatized at proper rodent diet and 12h/12h day/night lofradiolanthanideHolmium-166 and samarium-153 were produced by neutronSubmit your Manuscript www.austinpublishinggroup.comThe DTPA conjugation to alendronate was performed basedon reported similar methods with slight modifications . To amagnetically stirred solution of cyclic DTPA dianhydride (108 mg,0.3 mmol) in anhydrous DMF (2 mL) was added triethylamine (0.16mL, 1.2 mmol) under nitrogen (final pH. 8). After 15 min a solutionof alendronic acid (32.7 mg, 0.08 mmol) in DMF (1 mL) was addeddropwise to the reaction mixture at room temperature for 30 min.The reaction mixture was then allowed to stir for 24h at 60ºC. A fewdrops of water were added to quench the reaction. To the reactionmixture diethyl ether was added and the precipitate was recoveredand further purified using flash chromatography. Yield: 48% (32.7mg). The structure of compound was determined using spectroscopicmethods.Radiolabeling of DTPA-bis-ALN with radiolanthanidesA stock solution of DTPA-bis-ALN in pure ethanol wasprepared (20mg.mL-1). For labeling, an appropriate amount of theradiolanthanide solution containing the required activity (0.1 mL, 4mCi) was added to the desired amount of DTPA-bis-ALN solution(60µL). The pH adjusted using phosphate buffer to 5. The complexsolutions were kept at room temperature for 1-6 h. Also anotherset of experiment was performed at 60ºC warm bath for 1-6 h. Theradiochemical purity was determined using ITLC and HPLC. The80-88% radiochemical purity was obtained. For further purificationthe reaction mixture was passed through a freshly activated C18Sep-Pak column (pre-washed by ethanol and water) and fractionswere collected by washing with water: methanol mixture. Thefinal solution was passed through a 0.22-µm membrane filter andpH was adjusted to 7-8.5 with 0.05 molL-1 phosphate buffer (pH5.5). For Radiochemical purity of the complexes, instant thin layerchromatography was used. A 5µl sample of the final fraction wasspotted on a chromatography Whatman No. 3, paper, and developedin Whatman 3 MM chromatography paper or ITLC-SG eluted withNH4OH (56%): MeOH (%100): H2O (%100) (0.2:2:4; v/v/v) as mobilephase mixture.Sterility and apyrogenicity of the radiopharmaceuticalSterility was controlled on a random sampling following decay ofradioactivity. The Limulus Amoebocyte Lysate (LAL) test was usedAustin J Nucl Med Radiother 2(1): id1012 (2015) - Page - 02
Jalilian ARAustin Publishing Groupfor validation of radiopharmaceutical production according to theEuropean protocol .Stability of radiolanthanide-DTPA-bis-ALN complexes infinal formulationFor serum stability studies, 300µL of freshly prepared healthyhuman serum was added to 7.4MBq (200 µCi, 100 µL) of radiolabeledcomplex final solution and the resulting mixture was incubated at37ºCfor 48 h. Every 12 h to a portion of the 50 µL of the mixture,trichloroacetic acid (10%, 100 µL) was added and the mixturewas centrifuged at 3000 rpm for 5min followed by decanting thesupernatant from the debris. The stability was determined by paperchromatography analysis of supernatant using Whatman 3 MMchromatography paper or ITLC-SG eluted with NH4OH (56%):MeOH (%100): H2O (%100) (0.2:2:4; v/v/v).In vitro protein binding of radiolanthanide-DTPA- bis-ALN in presence of human serum: In vitro protein binding ofradiolanthanide-DTPA- bis -ALN was carried out in human bloodby protein precipitation according to published procedure . To3 mL fresh human plasma, 1mL of the labeled complex was mixedand incubated for 1h at 37ºC. Contents of the tube were centrifugedat 3000 rpm for 10 min for separation of serum and blood cells. Aftermixing approximately equal volume of 10% Trichloroacetic Acid(TCA), the mixture was centrifuged at 3000 rpm for 10 min. Residuewas separated from supernatant and both layers were counted forradioactivity in a well type gamma counter. Protein binding of thecomplex was expressed as the fraction of radioactivity bound toprotein, in percentage of the total radioactivity.In vitro stability of radiolanthanide-DTPA- bis -ALN inpresence of human serumFinal solution (200 µCi, 50 µL) was incubated in the presence offreshly prepared human serum (300 µL) (Purchased from IranianBlood Transfusion Organization, Tehran) and kept at 37ºC for 2 days.Every 30 min to a portion of the mixture (50 µl), trichloroacetic acid(10%, 100µl) was added and the mixture was centrifuged at 3000 rpmfor 5 min followed by decanting the supernatant from the debris. Thestability was determined by performing frequent ITLC analysis ofsupernatant using above mentioned ITLC system.Hydroxyapatite binding assayThe hydroxyapatite binding assay was performed accordingto the procedure described previously , with only a slightmodification. In brief, to vials containing 1.0, 2.0, 5.0, 10.0, 20.0 and50.0 mg of solid hydroxyapatite, 2ml of saline solution of pH 7.4were added and the mixtures were shaken for 1h. Then, 50 ml of theradioactive preparation was added and the mixtures were shakenfor 24 h at room temperature. The suspensions were centrifuged,and two aliquots of the supernatant liquid were taken from each vialand the radioactivity was measured with a well-type counter. Testexperiments were performed using a similar procedure, but in theabsence of hydroxyapatite. The percentage binding of radiolanthanideto Hydroxyapatite (HA) was calculated according to HB 1-A/B 100,where A is the mean radioactivity value of the supernatant sampleunder study and B is the mean total value of whole activity used.OH OOOO OOHO OHHONNNaOHOOOHOONNNOOFigure 2: Schematic diagram of the synthesis of DTPA cyclic di-anhydride.radiolanthanide-DTPA- bis -ALN were determined in normal mice.For each compound, 100 µL (150µCi) of radioactive solution wasinjected directly to normal mice through caudal vein. The animals(n 3) were sacrificed at selected times after injection and percentageof injected dose in the tissues was determined with a γ -ray scintillationor a dose calibrator.Results and DiscussionPreparation and structure confirmation of DTPA cyclic dianhydrideIn order to prepare the bi-functional ligand, DTPA cyclic dianhydride, which was not cost effective, we tried the general procedurefor its preparation . The reaction was performed in pyridinecontaining DTPA acid form and acetic anhydride. The filtered masswas washed with cold acetic anhydride to remove the residues of thereactant. The solid was dried in oven for a couple of hours and finallyre-crystallized to get a high purity product, suitable for spectroscopicand radiolabeling steps (Figure 2). Washing/drying steps were veryimportant in that more repetition of these steps afforded high-purityproduct with rather long shelf-life. Such samples can be stored atroom temperature under a blanket of N2 for up to one year.DTPA cyclic anhydride was characterized by IR spectroscopy.The formation of 1730 cm-1 peak indicated anhydride carbonyl groupformation which is accompanied by a weaker 1695 cm-1 carboxylicacid peak of the untouched COOH. The IR spectrum of cyclic DTPAdianhydride is shown in (Figure 3).1H NMR spectrum of the above compound was recorded inDMSO at 25µC. The chemical shifts of CH2CO groups have the lowestfield are very close so that a major singlet is observed around 3.76ppm (a). The NCH2CH2N groups are more shielded and because oftheir similarity, a broad multiplet is observed at 2.6-2.56 ppm (b,c).The DMSO peak is observed at 2.5 ppm as a multiplet (d). The 1HNMR spectrum of cyclic DTPA-di-anhydride was in accordance withthe literature.Biodistribution studiesThe biodistribution of free radiolanthanide cations as well as ofSubmit your Manuscript www.austinpublishinggroup.comFigure 3: FT-IR spectrum of DTPA cyclic di-anhydride prepared in this study.Austin J Nucl Med Radiother 2(1): id1012 (2015) - Page - 03
Jalilian AR2Austin Publishing GroupOHOPO OHOHP OHO OONH2 OHOHOONNN1OHOPO2O OHOHP OHOHOHNHO O OOHOONNNHN3O OHHOPOHOHO PHO O OHFigure 4: Reaction steps for preparation of DTPA-bis-ALN conjugate.Production of the precursorVarious reaction conditions were tried for DTPA- bis -ALNconjugation. The conjugation was performed in DMF, DMSO as wellas aqueous mixtures as the best reaction solvent showed to be DMFsince the reaction work-up seemed feasible due to the precipitationof the final compound followed by solid washing using varioussolvents. For better yields the alendronate free base (1) was addedto the ccDTPA (2) and not vice versa. Nitrogen atmosphere was alsomandatory for the conjugation reaction since the presence of watercan reduce the conjugation yields. The reaction scheme is shown in(Figure 4).The 1H NMR study of the conjugate demonstrated the 1:2conjugation ratio for DTPA: alendronate with the presence of allexpected peaks for both alendronate and DTPA moieties (FigureFigure 5: 1H NMR spectrum of DTPA-bis-ALN conjugate.Submit your Manuscript www.austinpublishinggroup.comFigure 6: ITLC chromatograms of radiolanthanide solutions in 10 mM DTPAsolution (pH 4) (left) and in 10% ammonium acetate: methanol (1:1) (right)on Whatman No. 1 Paper.5). Due to superimposition of many signals at close chemical shifts,the exact peak determination for all CH2 groups for alendronate andDTPA was not possible.IR data for the conjugate demonstrated 3470 cm-1 consistent withcarboxylic acid O-H and N-H stretch, 1634 cm-1 related to amide C Ostretch due to successful amide bond formation, also the presence ofP-O bond (at 1180 cm-1), P-O-H functional group at 2362 cm-1 asdescribed for phosphoric acid derivatives as reported .The radiochemical purity in 10 mmol.L-1 DTPA aq. solution(solvent 1), free radiolanthanide cation is complexed to morelipophilic radiolanthanide -DTPA form and migrates to higher Rf.Small radioactive fraction remaining at the origin could be relatedto other La ionic species, not forming radiolanthanide-DTPAcomplex, such as LaCl4-, etc. and/or colloids. On the other hand, 10%ammonium acetate: methanol mixture (1:1) (solvent 2) was also usedfor the determination of radiochemical purity. The fast eluting specieswas radiolanthanide cation. Other ionic forms of radiolanthanidesuch as LaCl4- as well as colloids remained at the origin (Rf 0) (Figure6).Figure 7: ITLC chromatograms of 153Sm-DTPA-bis-ALN solutions priorto solid phase purification (right) and after solid phase purification (left) inNH4OH: MeOH: H2O (0.2:2:4) as mobile phase on Whatman No. 1 Paper.Austin J Nucl Med Radiother 2(1): id1012 (2015) - Page - 04
Jalilian ARAustin Publishing GroupLabeling optimization studiesIn order to obtain maximum complexation yields, severalexperiments were carried out varying different reaction parameterssuch as ligand concentration, pH, reaction time and temperature.Ligand concentration was varied between a wide range startingfrom 10 to 50mg/ml for DTPA-bis- ALN. It was observed that atroom temperature 99% complexation was achieved with 15 mg/mlof DTPA-bis-ALN. The best ITLC mobile phase was considered bywhatman No.2 paper using NH4OH: MeOH: H2O (0.2:2:4) as shownin (Figure 7).In order to achieve higher specific activities for the final labeledcompounds, excess amounts of lanthanides were used in theradiolabeling procedure; however a solid phase purification step usingC18 SepPak was designed and applied to the final mixtures in orderto get higher radiochemical purities by removing excess amountsof unlabeled cations. By fraction eluting of the loaded cartridgesapplying the same solvent system used in the radio chromatographyof the labeling procedure, the radiolabeled complex was eluted inthe first 1-3 ml fractions. (Figure 8). Demonstrates the elution of theunlabeled cations from the column.Labeling yield increased with increasing molar ratio Ho: DTPAbis-ALN (from 1:5 to 1:50) and reached more than 99% in 60 minutesfollowed by C18 Sep Pak purification. The stability of prepared166Ho/153Sm-DTPA-bis-ALN complex was checked up to 24 hoursafter preparation. The complex was stable in final pharmaceuticalsample and its radiochemical purity was above 99% even 24 hoursafter preparation using Whatman 2 MM eluted with NH4OH: MeOH:H2O (0.2:2:4).Stability test was developed for the complex in presence of humanserum at 37ºC using ITLC as mentioned above and all data within 48were above 89% at all time intervals.For determination of protein binding the data showed the 57%protein binding using ITLC of the serum-radiopharmaceuticalmixture, while 43% is found in free form in the circulation. Theprotein binding for the DTPA-bis-ALN ligand has been reported indifferent references from 54% in free form ; however no proteinbiding for metal DTPA-bis-ALN complexes were available in theliterature.HA assay demonstrated low capacity binding forHo/153Sm-166Figure 8: Cationic radiolanthanide content of solid phase elution’s for 153SmDTPA-bis-ALN solution (right) and 166Ho-DTPA-bis-ALN solution eluted byNH4OH: MeOH: H2O (0.2:2:4) mixture.Figure 9: Biodistribution of 166HoCl3 (up) and 153SmCl3 (below) (1.85 MBq,50µCi) in normal mice at various time intervals after iv injection via tail vein(ID/g%: percentage of injected dose per gram of tissue) (n 3).DTPA-bis-ALN complexes to hydroxy apatite. Even at 50 mg amountof HA, less than 30% binding was observed (Table 1).BiodistributionFor better comparison biodistribution study was performed forfree Ho3 and 153Sm3 as well. The %ID/g data for free radiolanthanidesare summarized in (Figure 9).Holmium-166 cation: The liver radioactivity uptake of the cationis comparable to other radio-lanthanides such as Yb and Tb .About 1 % of the cation radioactivity accumulates in the liver in 48h. Low blood radioactivity content demonstrates the rapid removalof 166Ho from the circulation after injection. Lung, muscle and skindo not demonstrate significant 166Ho uptake while it is in accordancewith other cations accumulation. A low bone uptake is observed for166Ho which remains almost constant after 48 h (0.7%). Spleen alsohas significant 166Ho uptake possibly related to reticuloendothelialsystem. The free cation is soluble in water and it can be excreted viathe urinary tract .Samarium-153 cation: The liver uptake of the cation isTable 1: Hydroxy apatite binding assay for complexes at 37ºC in 24 hr.Ligand / Hydroxyapatite1535 mg10 mg15 mg20 mg25 mg50 mgHo-DTPA-bis-ALN18 0.2%21 0.3%23 0.92%26 0.09%28 0.1%29 0.03%Sm-DTPA- bis -ALN15 0.93%22 0.23%25 0.39%26 0.32%29 0.53%30 0.32%166Submit your Manuscript www.austinpublishinggroup.comAustin J Nucl Med Radiother 2(1): id1012 (2015) - Page - 05
Jalilian ARAustin Publishing Groupformation of larger complex structures due to phosphate groups asobserved for 99mTc-MDP.In case of 153Sm-DTPA-bis-ALN complex however the activitycontent was much lower in reticuloendothelial system compared to166Ho-DTPA-bis-ALN. Instead, the main site of accumulation andexcretion was shown to be kidneys. This might be a result of watersolubility as well as m9ore anionic nature of complex compared tothat of 166Ho-DTPA-bis-ALN.Figure10: Percentage of injected dose per gram of 166Ho-DTPA-bis-ALN inwild-type mice tissues at 2, 4, 24 and 48 h post injection (n 3).comparable with many other radio-lanthanides mimicking calciumcation accumulation [22 ]; about 3-4% of the activity accumulates inthe liver after 4 h and remains constant for 48 h. The blood content islow at all time intervals and this shows the rapid removal of activityfrom the circulation.The lung, muscle and also skin do not demonstrate significantuptake which is in accordance with other cations accumulation .A 1% bone uptake is observed for the cation which remains almostconstant after 48 h. The spleen also has significant uptake possiblyrelated to reticuloendothelial uptake. The kidney plays an importantrole in 153Sm cation excretion during 48 h (Figure 10).The distribution of injected dose in rat organs up to 48 h afterinjection of 166Ho-DTPA-bis-ALN (200 µCi/ 150µl) solution wasdetermined. Based on these results, it was concluded that the majorportion of the injected activity of 166Ho-DTPA- bis -ALN wasextracted from blood circulation into other tissues (Figure 11).The liver radioactivity uptake of the Ho-DTPA-bis-ALN wasincomparable to other radio-lanthanide complexes. About 20 % of theradioactivity accumulates in the liver in 48 h. On the other hand highlung radioactivity was also demonstrates the unwanted accumulationin reticuloendothelial tissues. One other possible mechanism is the166Both complexes demonstrated poor/unacceptable behavior fora possible bone avid radiopharmaceutical compared to even theirdirectly complexed La-alendronate complexes. Increased molecularsize as well as presence of many chelating functional groups in thestructure possibly led to the formation of non bone avid complexeswith lower complex stability constants compared to La-bisphosphonates.ConclusionA comparative accumulation study for the complex was obtainedin high radiochemical purity ITLC ( 99%) and HPLC ( 99.9%)and satisfactory stability in presence of human serum and finalformulations were obtained. HA binding assay demonstrated 95%binding from 5-20 mg of HA in 24h at room temperature. Thecomplex protein binding was about 55-58%. The biodistributionof the labeled compound in wild-rodents demonstrated unwantedactivity uptake in lungs, spleen and liver in case of 166Ho-DTPA-bisALN and liver lung and kidney in case of 153Sm-DTPA-bis-ALN. Verylimited bone uptake in both cases demonstrates complex instabilityor loss of bone avidity due to change of structure-activity relationshipand/or anionic property of polydentate complex leading to renalexcretion.AcknowledgmentWe acknowledge the financial support of Deputy of Research,Tehran University of Medical Sciences for conducting this researchproject. The authors wish to also to thank Pars Isotope Co., Tehran,Iran for providing animal facility services and Mr. M. Mazidi forperforming animal tests.References1. Campa JA, Rayne R. The management of intractable bone pain: a clinician’sperspective. Semin Nucl Med.1992; 22: 3-10.2. Serafini AN, Therapy of metastatic bone pain. J Nucl Med. 2001; 42: 895-906.3. Eary JF, Collin C, Stabin M, Vernon C, Petersdorf S, Baker M, et al.Samarium-153-EDTMP biodistribution and dosimetry estimation. J Nucl Med.1993; 34: 1031-1036.4. Bagheri R, Jalilian AR, Bahrami-Samani A, Mazidi M, Ghannadi-Maragheh.M. Production of Holmium-166 DOTMP: A promising agent for bone marrowablation in hematologic malignancies. Iran J Nucl Med. 2011; 19: 12-20.5. Breitz HB, Wendt III RE, Stabin MS, Shen S, Erwin WD, Rajendrann JG,et al. 166Ho-DOTMP Radiation-Absorbed Dose Estimation for SkeletalTargeted Radiotherapy, J Nucl Med. 2006; 47: 534–542.6. Bahrami-Samani A, Bagheri R, Jalilian AR, Shirvani-Arani S, GhannadiMaragheh M, et al. M. Production, Quality Control and PharmacokineticStudies of 166Ho-EDTMP for Therapeutic Applications. Sci Pharm. 2010; 78:423-433.Figure 11: Percentage of injected dose per gram of 153Sm-DTPA-bis-ALN inwild-type mice tissues at 2, 4, 24 and 48 h post injection (n 3).Submit your Manuscript www.austinpublishinggroup.com7. Louw WK. Dormehl IC, van Rensburg AJ, Hugo N, Alberts AS, Forsyth OE,et al. A. Evaluation of samarium-153 and holmium-166-EDTMP in the normalbaboon model. Nucl Med Biol.1996; 23: 935-40.Austin J Nucl Med Radiother 2(1): id1012 (2015) - Page - 06
Jalilian ARAustin Publishing Group8. Zeevaart JR, Jarvis NV, Louw WK, Jackson, GE. Metal-ion speciation in bloodplasma incorporating the xy-4-aminopropilydenediphosphonate(APDDMP), in therapeuticradiopharmaceuticals. J Inorg Biochem. 2001; 83: 57-65.9. Pedraza-López M, Ferro-Flores G, de Murphy CA, Tendilla JI, VillanuevaSánchez O. Preparation of (166)Dy/(166)Ho-EDTMP: a potential in vivo generatorsystem for bone marrow ablation. Nucl Med Commun. 2004; 25: 615-21.10. Gano L, Marques F, Campello MP, Balbina M, Lacerda S, Santos I.et al. Radiolanthanide complexes with tetraazamacrocycles bearingmethylphosphonate pendant arms as bone seeking agents. Q J Nucl MedMol Imaging. 2007; 51: 6-15.11. Mirsaeed Nikzad, Amir R Jalilian, Simindokht Shirvani-Arani, Ali BahramiSamani, Hamid Golchoubian. Production, quality control and pharmacokineticstudies of 177Lu-zoledronate for bone pain palliation therapy, J Radioanal NuclChem. 2013; 298: 1273–128112. Asma Fakhari, Amir R Jalilian, Hassan Yousefnia, Fariba Johari-Daha,Mohammad Mazidi, Ali Khalaj. et al. Development of 166Ho-pamidronate forbone pain palliation therapy, J Radioanal Nucl Chem. 2014.15. Hnatowich DJ, Layne WW, Child RL. Radioactive labeling of antibody: Asimple and efficient method. Science 1983; 220: 613-619.16. Wang S, Luo J, Lantrip DA, Waters DJ, Mathias CJ, Green MA, et al†Design and Synthesis of [111In]DTPA-Folate for Use as a Tumor-TargetedRadiopharmaceutical. Bioconjugate Chem. 1997; 8: 673-679.17. CGRPP-guidelines, version 2 March 2007, EANM RadiopharmacyCommittee, guidelines on current good radiopharmacy, practice (cgrpp) inthe preparation of radiopharmaceuticals.18. Dar UK, Khan IU, Javed M, Ahmad F, Ali M, Hyder SW,et al. Preparation andbiodistribution in mice of a new radiopharmaceutical technetium-99m labeledmethotrexate, as a tumor diagnostic agent. Hell J Nucl Med. 2012; 15: 120124.19. Neves M, Gano L, Pereira N, Costa MC, Costa MR, Chandia M, et al.Synthesis, characterization an
(HPGe) detector coupled with a Canberra (model GC1020-7500SL) multichannel analyzer and a dose calibrator ISOMED 1010 (Dresden, Germany) were used for counting distributed activity in mice organs. All other chemical reagents were purchased from Merck (Darmstadt, Germany). Calculations were