Kusum Vats

Radiolabeling, Stability Studies, and Pharmacokinetic Evaluation of Thulium-170-Labeled Acyclic and Cyclic Polyaminopolyphosphonic Acids

OBJECTIVE Thulium-170 [T1/2=128.4 days, Eβ(max)=968 keV, and Eγ=84 keV (3.26%)] could be considered an easily producible and cost-effective alternative to (89)Sr for the preparation of radiopharmaceuticals for palliation of bone pain arising due to skeletal metastases. Multidentate aminomethylene polyphosphonic acids have already been proven to be effective as carrier moieties for developing radiolabeled bone pain palliation agents using lanthanide radionuclides. Therefore, an attempt was made to evaluate the potential of a series of (170)Tm-labeled acyclic (diethylenetriaminepentamethylene phosphonic acid and triethylenetetraminehexamethylene phosphonic acid) and cyclic polyaminopolyphosphonic acids (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene phosphonic acid [DOTMP] and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetramethylene phosphonic acid [CTMP]) toward their use as alternative bone pain palliation agents. EXPERIMENTAL Thulium-170 was produced by irradiating the natural Tm2O3 target at a thermal neutron flux of 7×10(13) n·cm(-2)·s(-1) for a period of 60 days. All the phosphonic acid ligands were synthesized and characterized in-house. The protocols for radiolabeling the phosphonic acids with (170)Tm were standardized. Biological evaluation of the (170)Tm-labeled phosphonic acids were carried out in normal Wistar rats by biodistribution as well as by scintigraphic studies. RESULTS Thulium-170 was produced with adequate specific activity (173 Ci/g, 6.41 TBq/g) and high radionuclidic purity (99.62%). All the (170)Tm-labeled phosphonic acids, except (170)Tm-CTMP, were prepared with very high radiochemical purity (>98%) under optimized reaction conditions and exhibited high stability. All the agents showed selective skeletal accumulation with insignificant uptake in other vital organs/tissues and major clearance through renal pathway. These findings were also substantiated by scintigraphic studies. CONCLUSIONS Although all the (170)Tm-labeled phosphonic acids showed significant and selective skeletal accumulation, radiochemical studies indicate that (170)Tm-DOTMP is the best choice for carrying out further evaluation toward its use for clinical applications.

Single vial cold kits optimized for preparation of gastrin releasing peptide receptor (GRPR)-radioantagonist 68Ga-RM2 using three different 68Ge/68Ga generators

&NA; 68Ga‐RM2 is a gastrin releasing peptide receptor (GRPR) antagonist PET (positron emission tomography) radiotracer which is being investigated in clinical trials as a potential prostate cancer imaging agent. Simple, one‐step kit formulation of 68Ga‐RM2 would facilitate multicentre trials and allow easier integration in hospital radiopharmacy. Herein we report development of three sets of single‐vial RM2 cold kits validated for formulation with three respective 68Ge/68Ga generators eluted in 0.6 M, 0.1 M and 0.05 M HCl (hydrochloric acid). Cold kits of varied pH (2, 3, 4 and 5) were prepared using 2 M sodium acetate for three different 68Ge/68Ga generators to determine influence of pH on the radiochemical yield of 68Ga‐RM2. Buffer content was optimized with respect to volume of 68GaCl3 eluate to be added (1 mL/2 mL/ 5 mL). Sterility, apyrogenicity and long term stability of cold kits; in vitro and serum stability of 68Ga‐RM2 were investigated. In vitro cellular uptake and inhibition studies were performed to demonstrate the specificity of kit‐formulated 68Ga‐RM2. The radiochemical yield of 68Ga‐RM2 formulated from three different generators was observed to be maximum at pH 3 (99 ± 0.5%). Cold kits stored for 6 months at 0 °C also resulted in high radiochemical yield. 68Ga‐RM2 exhibited excellent in vitro stability (1 h) and serum stability (1 h). In vitro cellular uptake of 5 ± 0.8% in PC3 cells with >85% inhibition was observed for the 68Ga‐RM2 radiotracer indicating its specificity towards GRPR expression. These simple, robust kits shall allow hospitals with different generators to participate in clinical studies of 68Ga‐RM2 for screening of GRPR‐expressing prostate tumors.

Comparison of the efficacy of 177Lu-EDTMP, 177Lu-DOTMP and 188Re-HEDP towards bone osteosarcoma: an in vitro study

Among the radiopharmaceuticals harnessed for palliation of bone pain in osseous metastases patients, 177Lu labeled phosphonates such as EDTMP (Ethylene diamine tetramethylene phosphonic acid) as well as DOTMP (1,4,7,10-tetraaza-cyclo-dodecane-1,4,7,10 tetraethylene phosphonic acid) and 188Re-labeled HEDP (1,1-hydroxyethylene diphosphonic acid) seem to be the most favorable for treatment of small and medium/large size bone lesions, respectively. A comparative assessment of 177Lu-EDTMP, 177Lu-DOTMP and 188Re-HEDP in osteosarcoma tumor cell line was carried out to evaluate their relative efficacy. It was found that 188Re-HEDP is more potent in induction of cell toxicity and apoptosis compared to the 177Lu-EDTMP and 177Lu-DOTMP, thus 188Re-HEDP might have great clinical significance.

177Lu-labeled cyclic Asn-Gly-Arg peptide tagged carbon nanospheres as tumor targeting radio-nanoprobes

&NA; This study explores the potential of 177Lu‐labeled carbon nanospheres as radio‐nanoprobes for molecular imaging and therapy. The carboxyl functionalized surface of carbon nanospheres (CNS) was conjugated with [Gly‐Gly‐Gly‐c(Asn‐Gly‐Arg)], G3‐cNGR peptide through amide bond for targeting tumor vasculature and with [2‐(4‐Aminobenzyl)‐1,4,7,10‐tetraazacyclododecane tetraacetic acid], p‐NH2‐Bz‐DOTA for chelation with 177Lu. The nanosphere‐peptide conjugate, DOTA‐CNS‐cNGR, was characterized by dynamic light scattering and zeta potential measurements, IR and UV experiments and did not show any in vitro cytotoxicity. The pharmacokinetics and biodistribution of 177Lu‐labeled nanosphere‐peptide conjugate, 177Lu‐DOTA‐CNS‐cNGR was compared with 177Lu‐DOTA‐CNS (without the peptide) as well as with 177Lu‐DOTA‐cNGR (without carbon nanospheres). The radiolabeled nanosphere‐peptide conjugate exhibited higher tumor accumulation than nanosphere‐free radiolabeled peptide. The accumulation of the two radiolabeled probes in the tumor reduced to half during blocking studies with unlabeled G3‐cNGR peptide. 177Lu‐DOTA‐CNS exhibited higher tumor uptake than 177Lu‐DOTA‐CNS‐cNGR but rapid clearance of the latter nanoprobe from non‐target organs resulted in significantly higher (p < 0.05) tumor‐to‐blood and tumor‐to‐muscle ratios at 24 and 48 h p.i. It is evident from this study that carbon nanospheres conjugated to specific vectors shall form an important part of targeted radionanomedicine in future.

68Ga-labeled HBED-CC variant of uPAR targeting peptide AE105 compared with 68Ga-NODAGA-AE105

AIMS The urokinase Plasminogen Activator Receptors (uPAR) over-expressed on tumor cells and their invasive microenvironment are clinically significant molecular targets for cancer research. uPARexpressing cancerous lesions can be suitably identified and their progression can be monitored with radiolabeled uPAR targeted imaging probes. Hence this study aimed at preparing and evaluating two 68Ga-labeled AE105 peptide conjugates, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 as uPAR PET-probes. METHOD The peptide conjugates, HBED-CC-AE105-NH2 and NODAGA-AE105-NH2 were manually synthesized by standard Fmoc solid phase strategy and subsequently radiolabeled with 68Ga eluted from a commercial 68Ge/68Ga generator. In vitro cell studies for the two radiotracers were performed with uPAR positive U87MG cells. Biodistribution studies were carried out in mouse xenografts with the subcutaneously induced U87MG tumor. RESULTS The two radiotracers, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 that were prepared in >95% radiochemical yield and >96% radiochemical purity, exhibited excellent in vitro stability. In vivo evaluation studies revealed higher uptake of 68Ga-HBED-CC-AE105 in U87MG tumor as compared to 68Ga-NODAGAAE105; however, increased lipophilicity of 68Ga-HBED-CC-AE105 resulted in slower clearance from blood and other non-target organs. The uPAR specificity of the two radiotracers was ascertained by significant (p<0.05) reduction in the tumor uptake with a co-injected blocking dose of unlabeled AE-105 peptide. CONCLUSION Amongst the two radiotracers studied, the neutral 68Ga-NODAGA-AE105 with more hydrophilic chelator exhibited faster clearance from non-target organs. The conjugation of HBED-CC chelator (less hydrophilic) resulted in negatively charged 68Ga-HBED-CC-AE105 which was observed to have high retention in blood that decreased target to non-target ratios.

Synthesis and comparative in vivo evaluation of 99mTc(CO)3-labeled PEGylated and non-PEGylated cRGDfK peptide monomers

This work aimed at studying the effect of insertion of medium PEG (PEG7) on the pharmacokinetic behavior of cRGDfK peptide in comparison with the non‐PEGylated analogue. The cRGDfK peptide has thus been derivatized at ε‐amino group of lysine by conjugation with N3–PEG7–COOH/N3–CH2–COOH to prepare a PEGylated and a non‐PEGylated analogue of cRGDfK. A tridentate chelator was then incorporated by click chemistry conjugation of the two peptide azides for radiolabeling with [99mTc(CO)3(H2O)3]+ precursor. Comparative in vivo evaluation of the two 99mTc(CO)3‐labeled radiotracers, 99mTc(CO)3–Pra–Tz–CH2–cRGDfK 5 and 99mTc(CO)3–Pra–Tz–PEG7–cRGDfK 6, was carried out in C57BL/6 mice bearing αvβ3‐positive melanoma tumors to determine their potential toward targeting integrin αvβ3 receptors. The radiotracers exhibited excellent stability in saline as well as in serum. Maximum tumor uptake for the two radiotracers was observed at 30 min p.i. (5: 3.0 ± 0.7% ID/g; 6: 4.1 ± 0.5% ID/g). The two neutral 99mTc(CO)3 radiotracers prepared exhibited receptor‐mediated uptake in melanoma tumor. The increase in the tumor uptake on introduction of PEG7 unit was accompanied by slower clearance from other organs which resulted in decreased target‐to‐background ratios. The in vivo kinetics of 99mTc(CO)3‐labeled radiotracer, 99mTc(CO)3–Pra–Tz–CH2–cRGDfK 5 with only methylene unit as the spacer, was found to be more favorable due to higher tumor/blood, tumor/liver, tumor/kidney, and tumor/lung ratios.

Radiosynthesis and evaluation of a 99mTc-folic acid radiotracer prepared using [99mTcN(PNP)]2+ metal fragment

Folate receptors (FR) are over-expressed on a wide variety of tumor cells and are a potential molecular target for radiolabeled folates. In this respect, several SPECT and PET based radiofolates have been evaluated in the past albeit with their high renal uptake posing limitation towards their clinical use. To overcome this, a new 99mTc labeled folic acid was synthesized via the use of [99mTcN(PNP)]2+ metal fragment, where the presence of the latter pharmacophore redirects in vivo clearance via the hepatobiliary pathway. In this respect, folic acid was derivatized at the γ-acid group with a cysteine BFCA (bifunctional chelating agent) and subsequently reacted with the preformed [99mTcN]2+ intermediate in presence of PNP2 (bisphosphine) ligand, to yield the final complex. While preliminary, in vivo distribution of the complex exhibited high association of activity with liver and intestines and provided support to the rationality of the present design as clearance of labeled folic acid could be effected via the hepatic route, the in vitro studies of the folic acid-cysteine conjugate carried out in KB-31 cells, did not show much promise with reduction in receptor affinity in comparison with the native folic acid. The route followed herein to prepare a folic-acid based radiotracer constitutes the first report of radiolabeling folic acid using the [99mTcN(PNP)]2+ as a radiosynthon. Modification in the structure of conjugate by linking the BFCA through a long-chain linker can be envisaged to improve the affinity of [99mTcN(PNP)]-folic acid complex towards FRs.