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.

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.

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.

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.

Biochemical separation of Cetuximab-Fab from papain-digested antibody fragments and radiolabeling with 64Cu for potential use in radioimmunotheranostics.

Engineered Fab fragments of monoclonal antibodies (mAbs) after radiolabeling with suitable radiometals have the potential to play a key role in personalized radioimmunotheranostics of cancer patients. In this study, we have generated Fab fragment of Cetuximab, a mAb targeting epidermal growth factor receptor (EGFR) expression and purified from the Fc and other fragments by ultrafiltration and affinity chromatography. The Cetuximab-Fab was conjugated with a suitable bifunctional chelator and radiolabeled with no-carrier-added (NCA) 64Cu produced via 64Zn (n, p) 64Cu reaction in a nuclear reactor. The radioimmunoconjugate obtained after size exclusion chromatographic separation possessed >95% radiochemical purity and it retained its integrity over at least three half-lives of the radiometal. Biodistribution studies was performed in fibrosarcoma tumor bearing Swiss mice, which demonstrated the explicit need for purification of the Cetuximab-Fab from Fc fragments. Enhanced and rapid tumor uptake with decent tumor-to-background ratio with prolonged retention was observed when radiolabeled purified Cetuximab-Fab was intravenously administered in animal models. Overall, this preclinical study established the pivotal role of separation science and technology to obtain the radioimmunoconjugate with requisite purity in order to demonstrate optimal pharmacokinetics and maximized treatment 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.

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.

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.

Synthesis and evaluation of [177 Lu]Lu-labeled porphyrin loaded PAMAM dendrimer: Impact on tumor uptake and pharmacokinetics.

The objective of the present work is to evaluate the ability of the radiolabeled PAMAM dendrimers (polyamidoamine) towards facilitating the delivery of an in-house synthesized porphyrin derivative in the tumorous lesions to evaluate their candidature for possible application in endo-radionuclide therapy. For this, PAMAM particles were conjugated with a porphyrin derivative namely, 5,10,15,20-tetrakis-(4-carboxymethyleneoxyphenyl)porphyrin (STAP), synthesized in-house following a two-step reaction. The average number of porphyrin molecules loaded per PAMAM particle was evaluated using ultraviolet-visible spectrophotometry and was found to be approximately 2. STAP conjugated PAMAM particles were further conjugated with p-NCS-benzyl-DOTA (subsequently referred as DOTA) to facilitate radiolabeling with 177 Lu. On an average, two p-NCS-benzyl-DOTA molecules were observed to be attached per PAMAM-STAP particle. DOTA-PAMAM-STAP conjugate was radiolabeled with 177 Lu with a final radiochemical purity of >95%, which was determined by paper chromatography using two different mobile phases viz. 0.1 M sodium citrate buffer (pH 5.0) and 10 mM DTPA. Biological behavior of [177 Lu]Lu-DOTA-PAMAM-STAP conjugate was investigated in fibrosarcoma bearing Swiss mice model wherein accumulation of radiolabeled particles was observed in liver, GIT, spleen, and kidneys at 3 h post-administration. However, accumulated activity exhibited rapid clearance from majority of the organs at 24 h post-administration. [177 Lu]Lu-DOTA-PAMAM-STAP conjugate exhibited an appreciable uptake in tumor mass [6.09 ± 1.22 percentage injected activity/organ (% IA/organ)] at 3 h post-administration (p.i.) which was found to reduce to 1.05 ± 0.13 % IA/organ at 24 h post-administration. The results obtained in biodistribution studies were further corroborated through scintigraphic imaging performed in the same animal model. Despite of an appreciable accumulation in tumor mass, the lower retention of the [177 Lu]Lu-DOTA-PAMAM-STAP conjugate therein, at longer time point (24 h p.i.) may limit its possible potential as a radio-therapeutic agent and indicates towards need for further structural manoeuvring to attain favorable in vivo performance.

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.

A solvent extraction-based procedure for removal of 46Sc impurity from reactor produced [45Ca]CaCl2 for its potential use in bone pain palliation.

Calcium-45 [T½ = 163 d, Eß (max) = 0.3 MeV] is a pure ß- emitting radioisotope which can be envisaged for potential use in palliative care of pain due to skeletal metastases of primary cancer. During production of 45Ca in nuclear reactor via 44Ca (n,γ) 45Ca route, 46Sc is co-produced as a radionuclidic impurity. In this study, we have optimized a single-step solvent extraction procedure for complete removal of 46Sc impurity from [45Ca]CaCl2. The purified radiotracer was administered intravenously in normal Wistar rats and preferential bone uptake could be demonstrated by ex vivo biodistribution studies.

Multimodal Applications of Zinc Gallate-Based Persistent Luminescent Nanoparticles in Cancer Treatment: Tumor Margining, Diagnosis, and Boron Neutron Capture Therapy.

On the basis of the boron neutron capture therapy (BNCT) modality, we have designed and synthesized a zinc gallate (ZnGa2O4)-based nanoformulation for developing an innovative theranostic approach for cancer treatment. Initially, the (ZnGa1.995Cr0.005O4 or ZnGa2O4:(0.5%)Cr persistent luminescence nanoparticles (PLNPs) embedded on silica matrix were synthesized. Their surface functionalization was performed using organic synthesis strategies to attach the amine functional moieties which were further coupled with poly(vicinal diol). These diols were helpful for conjugation with 10B(OH)3, which subsequently served to couple with an in-house-synthesized variant of pH-(low)-insertion peptide (pHLIP) finally giving a tumor-targeting nanoformulation. Most importantly, the polymeric diols helped in conjugation of a substantial number of 10B to provide the therapeutic dose required for effective BNCT. This nanoformulation internalized substantially (∼80%) to WEHI-164 cancer cells within 6 h. Tumor homing studies indicated that the accumulation of this formulation at the acidic tumor site was within 2 h. The in vitro evaluation of the formulation against WEHI-164 cancer cells followed by neutron irradiation revealed its potent cytotoxicity with IC50 ∼ 25 µM. In the case of studies on animal models, the melanoma-induced C57BL/6 and fibrosarcoma-induced BALB/c mice were treated with formulations through intratumoral and intravenous injections, respectively, followed by neutron irradiation, leading to a significant killing of the cancer cells, which was evidenced by a reduction in tumor volume (75-80%) as compared with a control tumor. Furthermore, the histopathological studies confirmed a damaging effect only on tumor cells, while there was no sign of damage to the vital organs in treated mice as well as in controls.

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.

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.

Preparation of 177Lu-PSMA-617 in Hospital Radiopharmacy: Convenient Formulation of a Clinical Dose Using a Single-Vial Freeze-Dried PSMA-617 Kit Developed In-House.

OBJECTIVE: In the recent time, endoradionuclide therapy for metastatic castration-resistant prostate carcinoma employing 177Lu-PSMA-617 has yielded encouraging results and several clinical trials with the agent are currently ongoing. Routine preparation of 177Lu-PSMA-617 patient doses can be made simpler and convenient, if the ingredients essential for radiolabeling are made available in a ready-to-use lyophilized form. METHODS: PSMA-617 freeze-dried kit was formulated and used for the preparation of 177Lu-PSMA-617 clinical dose with high radiochemical purity using low/medium specific activity 177Lu. Detailed radiochemical studies were performed to determine the maximum activity and volume of 177LuCl3, which can be added in the kit for the formulation of 177Lu-PSMA-617. Studies were also performed to determine the shelf life of the kit to ensure its long-term usage. Studies were performed in buffer as well as human serum medium to determine the stability of the 177Lu-PSMA-617 complex after storing in respective media up to 7 days postpreparation. About ten patient doses of 177Lu-PSMA-617 were administered, and posttherapy scans were acquired. RESULTS: The formulated freeze-dried kit of PSMA-617 could be radiolabeled with an average percentage radiochemical purity > 98.53 ± 0.38. The freeze-dried kit was found suitable for tolerating up to 0.5 mL of 177LuCl3 (in 0.01 N HCl) and specific activity of 555 MBq/µg (15 mCi/µg) for the preparation of the patient dose of 177Lu-PSMA-617. The 177Lu-PSMA-617 complex prepared using the freeze-dried kit of PSMA-617 was observed to maintain % radiochemical purity (RCP) of 96.74 ± 0.87 and 94.81 ± 2.66, respectively, even after storing up to 7 days in buffer and human serum, respectively. 177Lu-PSMA-617 prepared using the in-house formulated freeze-dried kit of PSMA-617 exhibited accumulation in metastatic lesions picked up in a pretherapy PET scan. Reduction in number as well as size of lesions was observed in posttherapy scans acquired after two months of administering the first therapeutic dose of 177Lu-PSMA-617. CONCLUSIONS: The freeze-dried kit of PSMA-617 could be used for the preparation of 177Lu-PSMA-617 with high radiochemical purity (>98%) in a reproducible manner. 177Lu-PSMA-617 prepared using the developed kit was successfully evaluated in patients suffering from metastatic prostate cancer.

Formulation and clinical translation of [177Lu]Lu-trastuzumab for radioimmunotheranostics of metastatic breast cancer.

Trastuzumab (Herceptin®) is an approved immunotherapeutic agent used for the treatment of metastatic breast cancer over-expressing HER2 antigen receptors. The aim of the present work is to standardize the formulation protocol of [177Lu]Lu-trastuzumab addressing various reaction parameters, evaluating the efficacy of the radiolabeled product by in vitro investigations, scaling-up the preparation for administration in patients and performing preliminary clinical studies in patients suffering from metastatic breast cancer. Trastuzumab was conjugated with a suitable bi-functional chelating agent namely, p-NCS-benzyl-DOTA. On average 6.15 ± 0.92 p-NCS-benzyl-DOTA molecules were observed to be attached to each trastuzumab moiety. [177Lu]Lu-trastuzumab could be prepared with >95% radiochemical purity (% RCP) employing the optimized radiolabeling procedure. In vitro studies revealed the affinity of [177Lu]Lu-trastuzumab towards HER2 +ve cancer cell lines as well as against HER2 protein (K d = 13.61 nM and 11.36 nM, respectively). The value for percentage immunoreactive fraction (% IRF) for [177Lu]Lu-trastuzumab was observed to be 76.92 ± 2.80. Bio-distribution studies in Swiss mice revealed non-specific uptake in the blood, liver, lungs and heart followed by gradual clearance of activity predominantly through the hepatobiliary route. Preliminary clinical studies carried out in 8 cancer patients with immunohistochemically proven HER2 positive metastatic breast cancer revealed preferential localization of [177Lu]Lu-trastuzumab in breast cancer lesions, which was in concordance with [18F]FDG-PET scans recorded earlier in the same patient indicating the potential of the agent towards radioimmunotheranostic applications.

68Ga-labeled PET tracers for targeting tumor hypoxia: Role of bifunctional chelators on pharmacokinetics.

INTRODUCTION: By virtue of their oxygen dependant accumulation in hypoxic cells, radiolabeled nitroimidazole analogues have been widely used for detecting tumor hypoxia. Present study evaluates two 2-nitroimidazole (2-NIM) based 68Ga-labeled radiotracers, [68Ga]Ga-DOTAGA-2-NIM and [68Ga]Ga-NODAGA-2-NIM, for hypoxia targeting applications. METHODS: Bifunctional chelating agents suitable for radiolabeling with 68Ga, viz. 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) and 1,4,7-triazacyclododececane,1-(glutaric acid)-4,7-diacetic acid (NODAGA), were coupled to appropriately modified 2-nitroimidazole to obtain 2-NIM-DOTAGA and 2-NIM-NODAGA, respectively. These ligands were radiolabeled using [68Ga]GaCl3 obtained from a commercial 68Ge/68Ga-generator to obtain corresponding 68Ga-complexes. Both the radiotracers were tested for their hypoxia selectivity in CHO cells under hypoxic and normoxic conditions. Biodistribution studies in fibrosarcoma tumor bearing Swiss mice were carried out to evaluate the radiotracer in vivo. RESULTS: The 68Ga complexes of 2-NIM-DOTAGA and 2-NIM-NODAGA could be prepared in ~82% and ~90% yield, respectively. In vitro studies of the complexes in CHO cells showed significant accumulation of [68Ga]Ga-NODAGA-2-NIM complex under hypoxic conditions with hypoxic to normoxic ratio of 2.88 ± 0.36 at 180 min post incubation. The [68Ga]Ga-DOTAGA-2-NIM complex also showed hypoxia selectivity albeit to a lesser extent. Biodistribution studies of the complexes in Swiss mice bearing fibrosarcoma tumor showed significant tumor uptake by both radiolabeled complexes. [68Ga]Ga-NODAGA-2-NIM showed a more favorable pharmacokinetics with respect to [68Ga]Ga-DOTAGA-2-NIM. CONCLUSION: The nitroimidazole radiotracer with NODAGA chelator displayed more favorable pharmacokinetics and good hypoxia selectivity, making it a promising candidate for further investigation. The present study also provides an insight into the possible role of bifunctional chelator on overall pharmacokinetics of small molecule radiotracers.