Synthesis and 177Lu Labeling of the First Retro Analog of the HER2-Targeting A9 Peptide: A Superior Variant.

The retro analog of the HER2-targeting A9 peptide was synthesized by coupling amino acids in a reverse fashion and switching the N-terminal in the original sequence of the L-A9 peptide (QDVNTAVAW) to the C-terminal in rL-A9 (WAVATNVDQ). Modification in the backbone resulted in higher conformational stability of the retro peptide as evident from CD spectra. Molecular docking analysis revealed a higher HER2 binding affinity of [177Lu]Lu-DOTA-rL-A9 than the original radiopeptide [177Lu]Lu-DOTA-L-A9. Enormously enhanced metabolic stability of the retro analog led to significant elevation in tumor uptake and retention. SPECT imaging studies corroborated biodistribution results demonstrating a remarkably higher tumor signal for [177Lu]Lu-DOTA-rL-A9. The presently studied retro probe has promising efficiency for clinical screening.

Imaging of bacterial infection: Harnessing positron emission tomography and Cherenkov luminescence imaging with UBI-derived octapeptide.

Noninvasive imaging techniques for the early detection of infections are in high demand. In this study, we present the development of an infection imaging agent consisting of the antimicrobial peptide fragment UBI (31-38) conjugated to the chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which allows for labeling with the positron emitter Ga-68. The preclinical evaluation of [68 Ga]Ga-NODAGA-UBI (31-38) was conducted to investigate its potential for imaging bacterial infections caused by Staphylococcus aureus. The octapeptide derived from ubiquicidin, UBI (31-38), was synthesized and conjugated with the chelator NODAGA. The conjugate was then radiolabeled with Ga-68. The radiolabeling process and the stability of the radio formulation were confirmed through chromatography. The study included both in vitro evaluations using S. aureus and in vivo evaluations in an animal model of infection and inflammation. Positron emission tomography (PET) and Cherenkov luminescence imaging (CLI) were performed to visualize the targeted localization of the radio formulation at the site of infection. Ex vivo biodistribution studies were carried out to quantify the uptake of the radio formulation in different organs and tissues. Additionally, the uptake of [18 F]Fluorodeoxyglucose ([18 F] FDG) in the animal model was also studied for comparison. The [68 Ga]Ga-NODAGA-UBI (31-38) complex consistently exhibited high radiochemical purity (>90%) after formulation. The complex demonstrated stability in saline, phosphate-buffered saline, and human serum for up to 3 h. Notably, the complex displayed significantly higher uptake in S. aureus, which was inhibited in the presence of unconjugated UBI (29-41) peptide, confirming the specificity of the formulation for bacterial membranes. Bacterial imaging capability was also observed in PET and CLI images. Biodistribution results indicated a substantial target-to-nontarget ratio of approximately 4 at 1 h postinjection of the radio formulation. Conversely, the uptake of [18 F]FDG in the animal model did not allow for the discrimination of infected and inflamed sites. Our studies have demonstrated that [68 Ga]Ga-NODAGA-UBI (31-38) holds promise as a radiotracer for imaging bacterial infections caused by S. aureus.

Effect of ovarian follicular wave pattern and endocrine characteristics on pregnancy outcome in cows.

The effect of two (2W) versus three (3W) wave patterns of follicular dynamics and concurrent endocrine milieu of follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestradiol 17-ß (E2) and progesterone (P4) were investigated during one interoestrous interval (IEI) before insemination, on ensuing pregnancy, in 70 lactating Jersey crossbred cows. The findings were evaluated for between [included all (overall) 2W-O and 3W-O cows] and within [after separating pregnant (P) and non-pregnant (NP) cows in 2W and 3W] wave patterns. The propensity of two (58.6%, n = 41) and three (41.4%, n = 29) wave patterns was similar (p = .15). The IEI, shorter by 2.6 days for 2W-O versus 3W-O (p < .0009), predicted wave pattern as 100% 2W-O cows had IEI of ≤21 days, present only in 27.6% 3W-O cows (p < .0001). The ovulatory follicle persisted for a significantly shorter duration for 3W-O versus 2W-O cows. The average FSH, LH, E2 and P4 concentrations during the IEI did not differ for between and within the wave patterns. Pregnancy rate (%) of 58.6 versus 41.4 (p = .15) for 2W-O versus 3W-O and 56.1-P versus 43.9-NP (p = .44) for within 2W was similar, but tended to differ for within the 3W pattern (69.0-P versus 31.0-NP, p = .06). The pregnancy outcome was influenced by the age of ovulatory follicle for between the wave patterns and by follicular count as well as FSH surge concentration for within the wave patterns. A shorter luteal phase reduced the pregnancy outcome, a novel finding of the present study.

68 Ga-labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators.

The dual interaction with integrins and neuropilin-1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with 68 GaCl3 . Negatively charged 68 Ga-DOTAGA-iRGD and neutral 68 Ga-NODAGA-iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral 68 Ga-NODAGA-iRGD, but 68 Ga-DOTAGA-iRGD exhibited better tumor-to-blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.

Design, synthesis, and comparative evaluation of 99m Tc(CO)3 -labeled N-terminal and C-terminal modified asparagine-glycine-arginine peptide constructs.

The present study describes modification of asparagine-glycine-arginine (NGR) peptide at N-terminally and C-terminally by introduction of a tridentate chelating scaffold via click chemistry reaction. The N-terminal and C-terminal modified peptides were radiometalated with [99m Tc(CO)3 ]+ precursor. The influence of these moieties at the two termini on the targeting properties of NGR peptide was determined by in vitro cell uptake studies and in vivo biodistribution studies. The two radiolabeled constructs did not exhibit any significant variation in uptake in murine melanoma B16F10 cells during in vitro studies. In vivo studies revealed nearly similar tumor uptake of N-terminally modified peptide construct 5 and C-terminally construct 6 at 2 h p.i. (1.9 ± 0.1 vs 2.4 ± 0.2% ID/g, respectively). The tumor-to-blood (T/B) and tumor-to-liver (T/L) ratios of the two radiometalated peptides were also quite similar. The two constructs cleared from all the major organs (heart, lungs, spleen, stomach, and blood) at 4 h p.i. (<1% ID/g). Blocking studies carried out by coinjection of cCNGRC peptide led to approximately 50% reduction in the tumor uptake at 2 h p.i. This work thus illustrates the possibility of convenient modification/radiometalation of NGR peptide at either N- or C-terminus without hampering tumor targeting and pharmacokinetics.

Preparation and preliminary bioevaluation studies of 68 Ga-NOTA-rituximab fragments as radioimmunoscintigraphic agents for non-Hodgkin lymphoma.

Rituximab is used for the treatment of non-Hodgkin lymphoma (NHL). This study focuses on development of 68 Ga-labeled rituximab fragments, (68 Ga-NOTA-F (ab')-rituximab and 68 Ga-NOTA-F (ab')2 -rituximab, as PET-imaging agents for NHL. Rituximab was digested with immobilized pepsin and papain to yield F (ab')2 and Fab fragments respectively that were characterized by size exclusion HPLC (SE-HPLC) and SDS-PAGE. They were conjugated with p-SCN-Bn-NOTA, labeled with 68 Ga and characterized by SE-HPLC. Intact rituximab was labeled with gallium-68 for comparison. Specificity of 68 Ga-labeled immunoconjugates was ascertained by immunoreactivity and cell binding studies in Raji cells, while biodistribution studies were performed in normal Swiss mice. Gradient SDS-PAGE under nonreducing condition showed molecular weights of F (ab')2 -rituximab and F (ab')-rituximab as approximately 100 and 40 Kd, respectively. Radiochemical purity (RCP) of 68 Ga-NOTA-F (ab')2 -rituximab and 68 Ga-NOTA-F (ab')-rituximab were 98.2 ± 0.5% and 98.8 ± 0.2% respectively with retention times of 17.1 ± 0.1 min and 19.3 ± 0.1 min in SE-HPLC. 68 Ga-labeled rituximab fragments were stable in saline and serum up to 2-hour post preparation and exhibited specificity to CD20 antigen. Immunoreactivity of 68 Ga-labeled immunoconjugates was greater than 80%. Clearance of the fragmented radioimmunoconjugates was predominantly through renal route. Preliminary results from this study demonstrate the potential of 68 Ga- NOTA-F (ab')2 -rituximab and 68 Ga-NOTA-F (ab')-rituximab as PET imaging agents for NHL.

Synthesis, radiolabeling, and evaluation of gastrin releasing peptide receptor antagonist 68 Ga-HBED-CC-RM26.

The acyclic chelator HBED-CC has attained huge clinical significance owing to high thermodynamic and kinetic stability of 68 Ga-HBED-CC chelate. It provides an excellent platform for quick preparation of 68 Ga-based radiotracers in high yield. Thus, the present study aimed at conjugation of gastrin releasing peptide receptor (GRPr) antagonist, RM26, with HBED-CC chelator for 68 Ga-labeling. In vitro and vivo behavior of the peptide tracer, 68 Ga-HBED-CC-PEG2 -RM26, was assessed and compared with 68 Ga-NODAGA-PEG2 -RM26. The peptide tracers, 68 Ga-HBED-CC-PEG2 -RM26 and 68 Ga-NODAGA-PEG2 -RM26, prepared either by wet chemistry or formulated using freeze-dried kits exhibited excellent radiochemical yield and in vitro stability. The two peptide tracers cleared rapidly from the blood. Biodistribution studies in normal mice demonstrated slightly higher or comparable uptake of 68 Ga-HBED-CC-PEG2 -RM26 in GRPr-expressing organs pancreas, stomach, and intestine. The preliminary studies suggest high potential of 68 Ga-HBED-CC-PEG2 -RM26 for further investigation as a GRPr imaging agent and the wide scope of HBED-CC chelator in development of 68 Ga-based peptide tracers.

Ce-141-labeled DOTMP: A theranostic option in management of pain due to skeletal metastases.

Owing to its favorable radioactive decay characteristics (T1/2  = 32.51 d, Eß [max] = 434.6 keV [70.5%] and 580.0 keV [29.5%], Eγ  = 145.4 keV [48.5%]), 141 Ce could be envisaged as a theranostic radionuclide for use in nuclear medicine. The present article reports synthesis and evaluation of 141 Ce complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP) as a potent theranostic agent targeting metastatic skeletal lesions. Ce-141 was produced with 314 ± 29 MBq/mg (n = 6) specific activity and >99.9% radionuclidic purity (n = 6). Around 185 MBq dose of [141 Ce]Ce-DOTMP was synthesized with 98.6 ± 0.5% (n = 4) radiochemical yield under optimized conditions of reaction, and the preparation showed adequately high in vitro stability. Biodistribution studies in normal Wistar rats demonstrated significant skeletal localization and retention of injected activity (2.73 ± 0.28% and 2.63 ± 0.22% of injected activity per gram in femur at 3 hours and 14 days post-injection, respectively) with rapid clearance from non-target organs. The results of biodistribution studies were corroborated by serial scintigraphic imaging studies. These results demonstrate the potential utility of 141 Ce-DOTMP as a theranostic molecule for personalized patient care of cancer patients suffering from painful metastatic skeletal lesions.

An improved kit formulation for one-pot synthesis of [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 for routine clinical use in cancer imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide derivatives have immense potential for non-invasive monitoring of malignancies overexpressing integrin αv ß3 receptors. Easy availability of suitable radiotracers would augment the utility of this class of molecular imaging agents. Towards this, the present article describes the development of an improved lyophilized kit for the routine clinical formulation of [99m Tc]Tc complex of HYNIC-conjugated dimeric cyclic RGD peptide derivative E-[c(RGDfK)]2 (E = glutamic acid, f = phenyl alanine, K = lysine) without using Sn2+ and systematic evaluation of its efficacy. Five batches of the kits were prepared, and [99m Tc]Tc-HYNIC-E[c(RGDfK)]2 radiotracer was synthesized with high radiochemical purity (98.6 ± 0.5%) and specific activity (124.8 GBq/µmol) using the kits. Biodistribution studies in C57BL/6 mice bearing melanoma tumor exhibited significant accumulation of the radiotracer in tumor (5.32 ± 0.56 %ID/g at 60 min p.i.), and this uptake was also found to be receptor-specific by blocking studies. Preliminary human clinical investigations carried out in 10 breast cancer patients revealed high radiotracer uptake in the tumor along with good tumor-to-background contrast. The developed kit formulation showed an exceptionally high shelf-life of at least 18 months. These results demonstrated promising attributes of the developed kit formulation and warrant more extensive clinical investigations.

Preparation and comparative evaluation of 99m Tc-HYNIC-cNGR and 99m Tc-HYNIC-PEG2 -cNGR as tumor-targeting molecular imaging probes.

The tripeptide sequence asparagine-glycine-arginine (NGR) specifically recognizes aminopeptidase N (APN or CD13) receptors highly expressed on tumor cells and vasculature. Thus, NGR peptides can precisely deliver therapeutic and diagnostic compounds to CD13 expressing cancer sites. In this regard, 2 NGR peptide ligands, HYNIC-c(NGR) and HYNIC-PEG2 -c(NGR), were synthesized, radiolabeled with 99m Tc, and evaluated in CD13-positive human fibrosarcoma HT-1080 tumor xenografts. The radiotracers, 99m Tc-HYNIC-c(NGR) and 99m Tc-HYNIC-PEG2 -c(NGR), could be prepared in approximately 95% radiochemical purity and exhibited excellent in vitro and in vivo stability. The radiotracers were hydrophilic in nature with log P values being -2.33 ± 0.05 and -2.61 ± 0.08. The uptake of 2 radiotracers 99m Tc-HYNIC-c(NGR) and 99m Tc-HYNIC-PEG2 -c(NGR) was similar in nude mice bearing human fibrosarcoma HT-1080 tumor xenografts, which was significantly reduced (P < .05) during blocking studies. The 2 radiotracers being hydrophilic cleared rapidly from blood, liver, and intestine and were excreted through renal pathway. The pharmacokinetics of 99m Tc-labeled HYNIC peptide could not be modulated through introduction of PEG2 unit, thus posing a challenge for studies with other linkers towards enhanced tumor uptake and retention.

Synthesis and biodistribution studies of 99m Tc labeled fatty acid derivatives prepared via "Click approach" for potential use in cardiac imaging.

123 I-Iodophenylpentadecanoic acid (IPPA) is a metabolic agent used in nuclear medicine for diagnosis of myocardial defects. Efforts are underway worldwide to develop a 99m Tc substitute of the above radiopharmaceutical for the aforementioned application. Herein, we report synthesis and biodistribution studies of 99m Tc labeled fatty acids (8, 11, and 15 carbons) obtained via "click chemistry" for its potential use in myocardial imaging. ω-Bromo fatty acids (8C/11C/15C) were synthetically modified at bromo terminal to introduce a heterocyclic triazole with glycine sidearm in a five step procedure. Modified fatty acids were subsequently radiolabeled with preformed [99m Tc(CO)3 ]+ synthon to yield the desired fatty acid complexes which were evaluated in Swiss mice. All the radiolabeled complexes were obtained with radiochemical purities >80%, as characterized by HPLC. Biodistribution studies of all three complexes in Swiss mice showed myocardial uptake of ~6-9% ID/g at 2 minutes post-injection, close to* I-IPPA (~9% ID/g). Complexes exhibited significant retention in the myocardium up to 30 minutes (~1% ID/g) but were lower to the standard agent (~7% ID/g). Similar uptake of activity in myocardium for the newly synthesized complexes in comparison to 125 I-IPPA along with favorable in vivo pharmacokinetics merits potential for the present "click" design of complexes for myocardial imaging.

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.

68Ga labeled Erlotinib: A novel PET probe for imaging EGFR over-expressing tumors.

Molecular imaging using radiolabeled Tyrosine Kinase Inhibitors (TKI) is a promising strategy for detection and staging of EGFR-positive cancers. A novel analogue of one such TKI, Erlotinib has been developed for PET imaging by derivatizing the parent Erlotinib molecule for conjugation with the bifunctional chelator p-SCN-Bn-NOTA towards radiolabeling with 68Ga. NOTA-Erlotinib conjugate was synthesized and characterized by NMR and ESI-MS techniques. The conjugate was radiolabeled with 68Ga in 95±2% yield, as evidenced by HPLC characterization. The logP value of 68Ga-NOTA-Erlotinib was - (0.6±0.1). The 68Ga-NOTA-Erlotinib conjugate was characterized using its natGa-NOTA-Erlotinib surrogate. Cell viability studies showed that the NOTA-Erlotinib conjugate retained the biological efficacy of the parent Erlotinib molecule. Further, 68Ga-NOTA-Erlotinib exhibited an uptake of 9.8±0.4% in A431 cells which was inhibited by 55.1±0.2% on addition of cold Erlotinib (10µg) confirming the specificity of the radioconjugate for EGFR expressing cells. In the biodistribution studies carried out in tumor bearing SCID mice, 68Ga-NOTA-Erlotinib conjugate showed moderate tumor accumulation (1.5±0.1% ID/g at 30minp.i.; 0.7±0.2% ID/g at 1hp.i.). Hepatobiliary clearance of the radioconjugate was observed. The 68Ga-NOTA-Erlotinib conjugate was found to have high in vivo stability as determined by the metabolite analysis study using urine sample of the Swiss mice injected with the preparation. The overall properties of 68Ga-NOTA-Erlotinib are promising and merit further exploration. To the best of our knowledge, this is the first report on the design of a 68Ga labeled Erlotinib for PET imaging of EGFR and opens avenues for the successful development of 68Ga labeled TKI for imaging of EGFR over-expressing tumors.

68Ga labeled fatty acids for cardiac metabolic imaging: Influence of different bifunctional chelators.

Development of 68Ga labeled fatty acids is of immense interest due to the availability of 68Ga through a generator and its superiority over SPECT based tracers in carrying out dynamic imaging on a PET scanner. Our present work explores the influence of different chelators on the cardiac uptake and pharmacokinetics of the 68Ga-labeled fatty acids. Two new 68Ga labeled fatty acids were synthesized by conjugation of 11-aminoundecanoic acid with the bifunctional chelators (BFCs) viz. p-SCN-Bn-DTPA (S-2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid) and p-SCN-Bn-NODAGA (S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid) and their comparison was carried out with the previously reported 68Ga-NOTA-undecanoic acid. Both the conjugates were radiolabeled with 68Ga in high yields and purities (>95%). Their formation was established by preparation and characterization of their inactive analogs with natGa at macroscopic levels. Biodistribution studies of the complexes in Swiss mice showed lower initial myocardial uptake for 68Ga-NODAGA-undecanoic acid (3.8±0.6%ID/g) and 68Ga-DTPA-undecanoic acid (1.3±0.5%ID/g) complexes in comparison to previously reported 68Ga-NOTA-undecanoic acid complex (7.4±2.8%ID/g) at 2min p.i. However, significant retention of the tracer in the myocardium was observed in the case of 68Ga-NODAGA-undecanoic complex, which led to improved heart/non-target ratios of the complex over time in comparison to the other 68Ga complexes. Similarly, the DTPA complex exhibited increased washout from the liver in comparison to other 68Ga derivatives. The ß oxidation mechanism in myocytes was investigated by isolating the myocardial extract post intravenous injection of the respective 68Ga complexes and analyzing them by radio-HPLC, which showed metabolic transformation of the parent fatty acid complex peak in all the three complexes. This study has provided an insight into the design characteristics of 68Ga labeled fatty acids to achieve the desired myocardial imaging characteristics.

(90) Y/(177) Lu-labelled Cetuximab immunoconjugates: radiochemistry optimization to clinical dose formulation.

Radiolabelled monoclonal antibodies (mAbs) are increasingly being utilized in cancer theranostics, which is a significant move toward tailored treatment for individual patients. Cetuximab is a recombinant, human-mouse chimeric IgG1 mAb that binds to the epidermal growth factor receptor with high affinity. We have optimized a protocol for formulation of clinically relevant doses (~2.22 GBq) of (90) Y-labelled Cetuximab and (177) Lu-labelled Cetuximab by conjugation of the mAb with a suitable bifunctional chelator, N-[(R)-2-amino-3-(paraisothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N″,N″-pentaacetic acid (CHX-A″-DTPA). The radioimmunoconjugates demonstrated reasonably high specific activity (1.26 ± 0.27 GBq/mg for (90) Y-CHX-A″-DTPA-Cetuximab and 1.14 ± 0.15 GBq/mg for (177) Lu-CHX-A″-DTPA-Cetuximab), high radiochemical purity (>95%) and appreciable in vitro stability under physiological conditions. Preliminary biodistribution studies with both (90) Y-CHX-A″-DTPA-Cetuximab and (177) Lu-CHX-A″-DTPA-Cetuximab in Swiss mice bearing fibrosarcoma tumours demonstrated significant tumour uptake at 24-h post-injection (p.i.) (~16%ID/g) with good tumour-to-background contrast. The results of the biodistribution studies were further corroborated by ex vivo Cerenkov luminescence imaging after administration of (90) Y-CHX-A″-DTPA-Cetuximab in tumour-bearing mice. The tumour uptake at 24 h p.i. was significantly reduced with excess unlabelled Cetuximab, suggesting that the uptake was receptor mediated. The results of this study hold promise, and this strategy should be further explored for clinical translation.

Single vial kit formulation of DOTATATE for preparation of (177) Lu-labeled therapeutic radiopharmaceutical at hospital radiopharmacy.

The clinical applications of radiolabeled somatostatin analogue (177) Lu-DOTA-Tyr(3) -Thr(8) -Octreotide ((177) Lu-DOTATATE) constitute a promising treatment option for patients with disseminated and inoperable neuroendocrine (NET) tumors. Formulation of (177) Lu-DOTATATE in hospital radiopharmacy under aseptic conditions in a safe and reliable manner is a major constraint for its extensive use. The present work was intended to develop a kit for the safe preparation of the therapeutic radiopharmaceutical, viz. (177) Lu-DOTATATE of high quality that can be easily adapted at conventional hospital radiopharmacies. Single vial kits of DOTATATE were formulated and evaluated for suitability for radiolabeling as well as stability on its storage. Patient dose of (177) Lu-DOTATATE (7.4 GBq) could be successfully prepared using semi-automated in-house setup that assures safe handling and high yields of product of pharmaceutical purity suitable for clinical use. Fast clearance of activity via renal route was observed in preclinical biodistribution studies of (177) Lu-DOTATATE carried out in normal Swiss mice. Deployment of in-house produced (177) LuCl3 , cold kits and easy adaptability of synthesis setup at hospital radiopharmacy for preparation is likely to expand applications of peptide receptor radionuclide therapy.

Bioevaluation of (125) I Ocu-Prosta seeds for application in prostate cancer brachytherapy.

BACKGROUND & OBJECTIVES: In recent years, brachytherapy involving permanent radioactive seed implantation has emerged as an effective modality for the management of cancer of prostate. 125 I-Ocu-Prosta seeds were indigenously developed and studies were carried out to assess the safety of the indigenously developed 125 I-Ocu-Prosta seeds for treatment of prostate cancer. METHODS: Animal experiments were performed to assess the likelihood of in vivo release of 125 I from radioactive seeds and migration of seeds implanted in the prostate gland of the rabbit. In vivo release of 125 I activity was monitored by serial blood sampling from the auricular vein and subsequent measurement of 125 I activity. Serial computed tomography (CT) scans were done at regular intervals till 6 months post implant to assess the physical migration of the seeds. RESULTS: The laser welded seeds maintained their hermeticity and prevented the in vivo release of 125 I activity into the blood as no radioactivity was detected during follow up blood measurements. Our study showed that the miniature 125 I seeds were clearly resolved in CT images. Seeds remained within the prostate gland during the entire study period. Moreover, the seed displacement was minimal even within the prostate gland. INTERPRETATION & CONCLUSIONS: Our findings have demonstrated that indigenously developed 125 I-Ocu-Prosta seeds may be suitable for application in treatment of prostate cancer.

Evaluation of 177Lu-Labeled Pertuzumab F(ab')2 Fragments for HER2-Positive Cancer Targeting: A Comparative In Vitro and In Vivo Study.

Background: Radiolabeled antibody fragments present a promising opportunity as theranostic agents, offering distinct advantages over whole antibodies. In this study, the authors investigate the potential of [177Lu]Lu-DTPA-F(ab')2-pertuzumab as a theranostic agent for precise targeting of human epidermal growth factor receptor 2 (HER2)-positive cancers. Additionally, the authors aim to quantitatively assess the binding synergism in the presence of cold trastuzumab. Materials and Methods: F(ab')2-pertuzumab was prepared by pepsin digestion and conjugated with a bifunctional chelator. The immunoconjugate was radiolabeled with 177Lu and characterized by chromatography techniques. Binding parameters (affinity, specificity, and immunoreactivity) and cellular binding enhancement studies were evaluated in HER2-overexpressing and triple-negative cell lines. The in vivo enhancement in tumor uptake of the radiolabeled immunoformulation was assessed in severe combined immunodeficient (SCID) mice bearing tumors, both in the presence and absence of unlabeled trastuzumab. Results: The formulation of [177Lu]Lu-DTPA-F(ab')2-pertuzumab could be prepared in high yields and with consistent radiochemical purity, ensuring reproducibility. Comprehensive in vitro and in vivo evaluation studies confirmed high specificity and immunoreactivity of the formulation toward HER2 receptors. Binding synergism of radiolabeled pertuzumab fragments in the presence of trastuzumab to HER2 receptors was observed. Conclusions: The radioformulation of [177Lu]Lu-DTPA-F(ab')2-pertuzumab holds great promise as a targeted approach for addressing HER2-positive cancers. A potentially effective strategy to amplify therapeutic efficacy involves dual epitope targeting by combining radiolabeled pertuzumab with cold trastuzumab.

68Ga-Labeled Trastuzumab Fragments for ImmunoPET Imaging of Human Epidermal Growth Factor Receptor 2 Expression in Solid Cancers.

Background: Trastuzumab, the first humanized antibody approved for therapeutic use has shown promising results for the treatment of patients with human epidermal growth factor receptor 2 (HER2) positive cancers. The aim of this study was to formulate immunoPET agents based on trastuzumab fragments and demonstrate their potential for early diagnosis of HER2-positive tumors. Materials and Methods: F(ab')2 and F(ab') fragments of trastuzumab were prepared by enzymatic digestion and conjugated with chelator NOTA for labeling with 68Ga. For comparison, intact trastuzumab was also radiolabeled. In vitro stability, immunoreactivity, and binding affinity of radio formulations toward HER2 receptors were evaluated by performing in vitro studies in cancer cell lines. Biodistribution and PET imaging studies were performed in animal model bearing tumors. Results: 68Ga-NOTA-F(ab')-trastuzumab, 68Ga-NOTA-F(ab')2-trastuzumab, and 68Ga-NOTA-trastuzumab could be prepared with >98% radiochemical purity (% RCP) and were found to be stable when studied up to 4 h. In vitro binding studies revealed high affinity and specificity of formulations toward HER2 receptors. Specific tumor uptake of 68Ga-NOTA-F(ab')-trastuzumab and 68Ga-NOTA-F(ab')2-trastuzumab in HER2-positive tumors was observed in biodistribution and PET imaging studies. Conclusions: This study describes optimization of protocol for the formulation of 68Ga-NOTA-F(ab')-trastuzumab and 68Ga-NOTA-F(ab')2-trastuzumab for targeting HER2-overexpressing tumors. Further studies with these radioformulations are warranted to confirm their potential as immunoPET agents for management of HER2-positive breast and other solid tumors.

[99mTc]Tc-HYNIC-RM2: A potential SPECT probe targeting GRPR expression in prostate cancers.

INTRODUCTION: Elevated density of gastrin releasing peptide receptors (GRPR) in prostate cancer has led to exploration of several radiolabeled peptides for imaging and staging of the disease. The GRPR antagonist peptide RM2 has been successfully conjugated with several chelators and radiolabeled with gallium-68. The goal of this study was to synthesize a 99mTc-labeled probe and investigate its potential for SPECT imaging of prostate cancer. Towards this HYNIC-RM2 peptide conjugate was synthesized, radiolabeled with 99mTc and evaluated in GRPR-positive PC3 tumor xenografts. METHODS: HYNIC-RM2 was manually synthesized by standard Fmoc solid phase strategy and radiolabeled with 99mTc. In vitro cell studies were performed in GRPR-positive human prostate carcinoma (PC3) cells. Metabolic stability studies of [99mTc]Tc-HYNIC-RM2 were performed in normal mice in the presence as well as absence of neutral endopeptidase (NEP) inhibitor, phosphoramidon (PA). Biodistribution and imaging studies of [99mTc]Tc-HYNIC-RM2 were performed in SCID mice bearing PC3-xenograft. RESULTS: [99mTc]Tc-HYNIC-RM2 exhibited high binding affinity in low nanomolar range (Kd = 1.83 ± 0.31 nM). Metabolic stability studies in mice indicated that in the absence of PA, radiolabeled peptide was about 65 % intact in the blood at 15 min p.i., whereas proportion of intact radiolabeled peptide was enhanced to 90 % on co-administration of PA. Biodistribution studies in PC3 tumor bearing mice demonstrated high tumor uptake (8.02 ± 0.9%ID/g and 6.13 ± 0.44%ID/g at 1 h and 3 h p.i.). Co-administration of PA with the radiolabeled peptide resulted in further enhancement of tumor uptake (14.24 ± 0.76 % ID/g and 11.71 ± 0.59%ID/g at 1 h and 3 h p.i.). SPECT/CT images of [99mTc]Tc-HYNIC-RM2 could clearly visualize the tumor. Significant (p < 0.001) reduction in the tumor uptake with a co-injected blocking dose of unlabeled peptide ascertained the GRPR specificity of [99mTc]Tc-HYNIC-RM2. CONCLUSION: Encouraging results obtained in biodistribution and imaging studies indicate the potential of [99mTc]Tc-HYNIC-RM2 for further exploration as GRPR targeting agent.