Peptide-drug conjugate designated for targeted delivery to HER2-expressing cancer cells.

Targeted therapy of the highest globally incident breast cancer shall resolve the issue of off-target toxicity concurring with augmented killing of specific diseased cells. Thus, the goal of this study was to prepare a peptide-drug conjugate targeting elevated expression of HER2 receptors in breast cancer. Towards this, the rL-A9 peptide was conjugated with the chemotherapeutic drug doxorubicin (DOX) through a N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. The synthesized peptide-drug conjugate, rL-A9-DOX, was characterized by mass spectrometry. Molecular docking studies, based on binding energy data, suggested a stronger interaction of rL-A9-DOX with the HER2 receptor in comparison to the unconjugated peptide, rL-A9. The cytotoxic effect of the rL-A9-DOX conjugate was observed to be higher in HER2-positive SKOV3 cells compared to HER2-negative MDA-MB-231 cells, indicating selective cell killing. Cellular internalization of the rL-A9-DOX conjugate was evident from the flow cytometry analysis, where a noticeable shift in mean fluorescent intensity (MFI) was observed for the conjugate compared to the control group. This data was further validated by confocal microscopy, where the fluorescent signal ascertained nuclear accumulation of rL-A9-DOX. The present studies highlight the promising potential of rL-A9-DOX for targeted delivery of the drug into a defined group of cancer cells.

Nuclear Localization Signal Enhances the Targeting and Therapeutic Efficacy of a Porphyrin-Based Molecular Cargo: A Systemic In Vitro and Ex Vivo Evaluation.

The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with 177Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 µCi (9.25 MBq) and 500 µCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.

Design, synthesis and evaluation of 177Lu-labeled inverso and retro-inverso A9 peptide variants targeting HER2-overexpression.

Several HER2-specific peptides are being continuously explored to find a candidate with suitable pharmacokinetic properties for development of effective radiopharmaceutical that can find applications for clinical screening of breast cancer patients. In the present work with an aim of preparing a radiopeptide with improved metabolic stability and in vivo pharmacokinetic performance we modified our previously reported [177Lu]DOTA-L-A9 peptide. Here we designed an 'inverso' peptide with all d-amino acids and a 'retro-inverso' peptide where sequence of d-amino acids was reversed. Higher secondary structure stabilization of retro- inverso A9 variant compared to inverso A9 peptide was evident by circular dichroism studies. The two radiopeptides [177Lu]DOTA-D-A9 and [177Lu]DOTA-rD-A9 exhibited significantly improved in vivo metabolic stability over the original l-peptide. The retro-inverso variant, [177Lu]DOTA-rD-A9 demonstrated better pharmacokinetic behavior with significantly higher tumor uptake than the inverso peptide, [177Lu]DOTA-D-A9 and the original peptide, [177Lu]DOTA-L-A9. In the present case of A9 peptide, reversal of the peptide sequence of d-amino acids boosted the uptake and retention of radioactivity in HER2-positive tumor. The present study can thus guide the design and development of newer and improved versions of peptides.

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.

Targeting HER2-receptors with 177Lu-labeled triazole stapled cyclic peptidomimetic.

In this study on-resin Cu(I)-catalyzed click reaction was performed to synthesize triazole-stapled cyclic peptidomimetic, DOTA-c[TZ]A9 targeting HER2 receptor expression in breast cancers. Spectroscopic (circular dichroism) and docking analysis provided evidence of enhanced helicity and secondary structure stabilization along with improved HER2 affinity in comparison to the corresponding linear peptide, DOTA-[Pra1, Aza7]A9. 177Lu-labeled cyclic peptide, 177Lu-DOTA-c[TZ]A9 displayed higher in vitro serum stability and in vivo metabolic stability and better HER2 binding affinity {Kd of 16.93 ± 3.02 nM} than the linear counterpart, [177Lu]DOTA-[Pra1, Aza7]A9 {Kd of 26.28 ± 2.87 nM}. Biodistribution profile in SKBR3 tumor bearing SCID mice demonstrated elevated radioactivity levels and prolonged retention of cyclic peptide in the tumor compared to the linear peptide. Thus, solid phase click cyclization technique can be extended towards preparation of triazole-stapled peptides targeting different receptors with improved stability and efficacy.

[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.

Evaluation of the effect of a cell penetrating peptide (TAT) towards tailoring the targeting efficacy and tumor uptake of porphyrin.

Cell penetrating peptides (CPPs) are known to possess a unique capacity to penetrate biological membranes and translocate various molecules into the cells. Therefore, porphyrin-CPP conjugates could be envisaged to boost the intracellular delivery of porphyrins thereby providing an improved tool for the development of agents for multi-modal applications for cancer management. Working in this direction, an unsymmetrically substituted porphyrin derivative was conjugated with a transactivating transcriptional activator peptide (TAT) and various in vitro and in vivo studies were carried out in order to study the effect of adding a CPP to the porphyrin derivative. MTT assay revealed the preferential light dependent toxicity of the porphyrin derivative which was further enhanced upon peptide conjugation. Fluorescence and flow cytometry studies revealed the relatively higher cellular internalization of the porphyrin-TAT conjugate in comparison with the porphyrin derivative. The elevated light dependent cell toxicity of the porphyrin-TAT conjugate along with its capability of generating cytotoxic singlet oxygen indicated the advantages of using the porphyrin-TAT conjugate for PDT applications. Also, porphyrin and the porphyrin-peptide conjugate were radiolabelled with 68Ga to investigate their possible potential as PET agents. In vivo biodistribution studies revealed a higher tumor uptake for the 68Ga-porphyrin-TAT conjugate (6.32 ± 1.24% IA per g) than for 68Ga-porphyrin (2.45 ± 0.88% IA per g) at 60 min post-administration. However, the observation of a higher non-target retention of the radiolabelled agents during in vivo studies might pose a limitation on their possible application in PET imaging.

Preparation of radiolabeled erlotinib analogues and analysis of the effect of linkers.

Erlotinib is a first generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) which was granted Food and Drug administration (FDA) approval for treatment of patients with locally advanced or metastatic NSCLC. The present study aimed at development of radiolabeled erlotinib variants as tyrosine kinase inhibitors. Three DOTA-erlotinib conjugates were prepared for radiolabeling with 177Lu. The terminal alkyne group of erlotinib was modified by performing Cu-catalyzed click chemistry and three different linkers were introduced which were then conjugated to the chelator, DOTA. The DOTA-erlotinib conjugates were characterized by 1H NMR and ESI-MS. 177Lu-DOTA-erlotinib complexes were characterized using natLu-DOTA-erlotinib conjugates. The 177Lu-complexes exhibited high in vitro stability in human serum up to 48 h. They were highly hydrophilic in nature as observed from their log Po/w values (177Lu-DOTA-propyl-Er: -2.5 ± 0.1; 177Lu-DOTA-PEG3-Er: -3.0 ± 0.1; 177Lu-DOTA-PEG6-Er: -3.3 ± 0.1). The MTT assay in A431 human epidermoid carcinoma cell lines indicates that the chemical modification at the terminal alkyne group of the erlotinib molecule does not have significant effect on its TKI property. Biodistribution studies in normal Swiss mice demonstrated fast clearance and excretion of 177Lu-labeled erlotinib complexes. These studies indicate that erlotinib variants with hydrophobic pharmacokinetic modifiers/chelators may enhance the retention of 177Lu-labeled complexes in blood thereby increasing the probability to reach EGFR-expressing tumor.

Synthesis and comparative evaluation of 177Lu-labeled PEG and non-PEG variant peptides as HER2-targeting probes.

Highest global cancer incidence of female breast cancer is a matter of great concern. HER2-positive breast cancers have high mortality rate hence detection at an early stage is vital for successful treatment, improved cancer care and survival rate. Radiolabeled peptides have emerged as new alternatives to radiolabeled antibodies to overcome the limitations of slow clearance and uptake in non-target tissues. Herein, DOTA-A9 peptide and its pegylated variant were constructed on solid phase and radiolabeled with [177Lu]LuCl3. [177Lu]DOTA-A9 and [177Lu]DOTA-PEG4-A9 displayed high binding affinity (Kd = 48.4 ± 1.4 and 55.7 ± 12.3 nM respectively) in human breast carcinoma SKBR3 cells. Two radiopeptides exhibited renal excretion and rapid clearance from normal organs. Uptake in SKBR3 tumor and tumor-to-background ratios were significantly higher (p < 0.05) for [177Lu]DOTA-PEG4-A9 at the three time points investigated. Xenografts could be clearly visualized by [177Lu]DOTA-PEG4-A9 in SPECT images at 3, 24 and 48 h p.i. indicating the potential for further exploration as HER2-targeting probe. The encouraging in vivo profile of PEG construct, [177Lu]DOTA-PEG4-A9 incentivizes future studies for clinical applications.

Assessment of 177 Lu-labeled carboxyl-terminated polyamidoamine (PAMAM) dendrimer-RGD peptide conjugate.

Structurally unique polyamidoamine (PAMAM) dendrimers implanted with targeting biological moiety along with complexed radiometal constitute a favorable nano-system for diagnosis and therapy of targeted tumor sites. In the present study, carboxyl functionalities of PAMAM- generation 4 dendrimer (PAMAM-G4-COOH) were conjugated with ε-amino group of lysine of cRGDfK peptide to impart integrin αv ß3 targeting capability. Reaction of p-NH2 -Bn-DOTA with PAMAM was accomplished via acid-amine coupling using EDC/NHS for 177 Lu-complexation. 177 Lu-labeled nano-system, 177 Lu-PAMAM-DOTA-cRGDfK demonstrated receptor-mediated uptake in murine melanoma B16F10 cells during in vitro cell uptake studies. In vivo biodistribution studies demonstrated low tumor uptake and retention of 177 Lu-PAMAM-DOTA-cRGDfK which may be attributed to rapid blood clearance. However, fast clearance from non-target organs resulted in higher target to background ratio. Tumor uptake of targeted nano-system, 177 Lu-PAMAM-DOTA-cRGDfK was observed to be significantly (p < 0.05) higher in comparison to 177 Lu-PAMAM-DOTA without the targeting peptide. Inhibition studies with unlabeled cRGDfK resulted in 60% reduction in tumor uptake of 177 Lu-PAMAM-DOTA-cRGDfK, indicating specificity of the developed nano-system towards integrin αv ß3 receptors.

Recent Breakthrough in 68Ga-Radiopharmaceuticals Cold Kits for Convenient PET Radiopharmacy.

68Ga-PET has emerged as an important diagnostic tool for precise detection and monitoring of oncological situations. Availability, cost, and radiosynthesis procedure are determining steps for success of a radioisotope/radiopharmaceutical in nuclear medicine. Availability of 68Ga from a 68Ge/68Ga generator containing a long-lived parent radioisotope (68Ge: t1/2 = 271 days) and an inexpensive, simplified production of 68Ga-radiopharmaceuticals through kit methodology has allowed smooth accommodation of 68Ga-PET in clinics. The uncomplicated formulation of 68Ga-radiopharmaceuticals from a lyophilized, cold kit is an impending breakthrough in clinical PET. The huge success of 68Ga in neuroendocrine tumor and prostate cancer imaging along with the regulatory approval of respective cold kits has opened a pathway for development of kits for other evolving radiotracers. There is a definite scope for increased participation of commercial manufacturers and distributors of cold kits to spread the potential of 68Ga worldwide across all the geographical locations and satellite centers.

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.

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.

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 clinical translation of 99mTc-PSMA-11 for SPECT imaging of prostate cancer.

This study explores the feasibility of radiolabeling the HBED-CC-PSMA (PSMA-11) ligand with Tc-99m for SPECT imaging of prostate cancer patients. 68Ga-HBED-CC-PSMA (PSMA-11) is used clinically for PET/CT imaging of prostate cancer (PCa) patients. However, a PET/CT facility may not be affordable and/or accessible to remotely located health centers. Thus, economic considerations require development of a SPECT-based tracer to provide low cost effective health care to the entire global population. Hence, radiochemical parameters were varied and optimized to obtain the maximum radiochemical yield of 99mTc-PSMA-11. 99mTc-PSMA-11 could be prepared in 60 ± 5% radiochemical yield and >98% radiochemical purity with a specific activity of 15 ± 5 GBq µmol-1. The radiotracer exhibited high stability in vitro in human serum after 24 h. A cell uptake of 15.2 ± 1.2% was observed for 99mTc-PSMA-11 in PSMA-positive prostate carcinoma LNCaP cells. Rapid clearance from blood, liver, intestine, lungs and other major organs was observed during normal biodistribution studies. The radiotracer, 99mTc-PSMA-11, exhibited physiological distribution in salivary and lacrimal glands similar to that of 68Ga-PSMA-11 in mice and successfully identified primary tumors as well as metastatic lesions in human patients. This study thus highlights successful radiolabeling of HBED-CC-PSMA with Tc-99m and the potential of 99mTc-PSMA-11 as a SPECT imaging agent for PCa.

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

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.

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.

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

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.

Comparative evaluation of 68 Ga-labeled NODAGA, DOTAGA, and HBED-CC-conjugated cNGR peptide chelates as tumor-targeted molecular imaging probes.

The biological behavior of 68 Ga-based radiopharmaceuticals can be significantly affected by the chelators' attributes (size, charge, lipophilicity). Thus, this study aimed at examining the influence of three different chelators, DOTAGA, NODAGA, and HBED-CC on the distribution pattern of 68 Ga-labeled NGR peptides targeting CD13 receptors. 68 Ga-DOTAGA-c(NGR), 68 Ga-NODAGA-c(NGR), and 68 Ga-HBED-CC-c(NGR) were observed to be hydrophilic with respective log p values being -3.5 ± 0.2, -3.3 ± 0.08, and -2.8 ± 0.14. The three radiotracers exhibited nearly similar uptake in human fibrosarcoma HT-1080 tumor cells with 86%, 63%, and 33% reduction during blocking studies with unlabeled cNGR peptide for 68 Ga-DOTAGA-c(NGR), 68 Ga-NODAGA-c(NGR), and 68 Ga-HBED-CC-c(NGR), respectively, indicating higher receptor specificity of the first two radiotracers. The neutral radiotracer 68 Ga-NODAGA-c(NGR) demonstrated better target-to-non-target ratios during in vivo studies compared to its negatively charged counterparts, 68 Ga-DOTAGA-c(NGR) and 68 Ga-HBED-CC-c(NGR). The three radiotracers had similar HT-1080 tumor uptake and being hydrophilic exhibited renal excretion with minimal uptake in non-target organs. Significant reduction (p < .005) in HT-1080 tumor uptake of the radiotracers was observed during blocking studies. It may be inferred from these studies that the three radiotracers are promising probes for in vivo imaging of CD13 receptor expressing cancer sites; however, 68 Ga-NODAGA-c(NGR) is a better candidate.