On the Optimization of the Protocol for Automated Radiosyntheses of [68Ga]Ga-Pentixafor, [68Ga]Ga-FAPI-4 and [68Ga]Ga-DOTATATE in a Modular-Lab Standard.

Objectives: The present work describes the automated radiochemical synthesis of different PET tracers like [68Ga]Ga-Pentixafor, [68Ga]Ga-FAPI-4 and [68Ga]Ga-DOTATATE using optimized single protocol in the non-cassette based Eckert & Ziegler (EZ) Modular Lab (fixed tubing system) without any modification in the inbuilt human machine interface (HMI) software. Recently, PET agents viz. [68Ga]Ga-Pentixafor and [68Ga]Ga-FAPI-4 are gaining prominence for the diagnosis of overexpressed Chemokine Receptor-4 (CXCR4) and Fibroblast Activation Protein (FAP) receptor, respectively, in the microenvironment of numerous cancer types. The promising results observed with the clinical usage of [68Ga]Ga-DOTATATE produced using the automated protocol, provided impetus for the clinical translation of [68Ga]Ga-Pentixafor and [68Ga]Ga-FAPI-4 using the in-house developed automated radiolabeling protocol. Methods: Herein we report a single radiolabeling protocol for the automated preparation of [68Ga]Ga-Pentixafor and [68Ga]Ga-FAPI-4 in the non-cassette based EZ Modular-Lab Standard radiochemistry module, without any changes in schematic, graphical user interface (GUI) software and time list, from that used for routine production of [68Ga]Ga-DOTATATE in our centre, since 2015. Physico-chemical quality control and in-vitro stability analyses were carried out using radio-TLC and radio-HPLC. Results: The automated protocol yielded reliable and consistent non-decay corrected (ndc) radiochemical yield (RCY) of (84.4%±0.9%) and (85.5%±1.4%) respectively, for [68Ga]Ga-Pentixafor and [68Ga]Ga-FAPI-4, with RCP>98%, which are comparable to the RCY of (84.4%±1.2%) and RCP (99.1%±0.3%) for [68Ga]Ga-DOTATATE. The biological quality control studies confirmed the formulations to be of ready-to-use pharmaceutical grade. Conclusion: The consistent and reliable RCY and RCP of multiple 68Ga-labeled PET tracers by single automated radiochemistry protocol exhibits the versatility of the EZ Modular Lab.

Clinical Internal Dosimetry and Biodistribution of 177 Lu-DOTA-Trastuzumab in HER2-Positive Metastatic and Locally Advanced Breast Carcinoma.

OBJECTIVE: The aim of this study was to assess the biodistribution and dosimetry of 177 Lu-DOTA-trastuzumab in patients with HER2-positive breast carcinoma using whole-body (WB) planar imaging at multiple time points. PATIENTS AND METHODS: This study was a prospective evaluation of HER2-positive metastatic/locally advanced breast carcinoma patients who underwent gamma camera imaging for dosimetry and biodistribution studies by using 177 Lu-DOTA-trastuzumab. The standard diagnostic dosimetry protocol was followed, which included cold trastuzumab injection followed by in-house produced 177 Lu-DOTA-trastuzumab. Serial WB planar images (anterior and posterior) were obtained on gamma camera after the infusion of 177 Lu-DOTA-trastuzumab at multiple time points. Whole-body and organ regions of interest were drawn, and the numbers of disintegrations were obtained. The mean absorbed doses for the liver, spleen, kidneys, heart, red marrow, and tumor were obtained from OLINDA EXM v2.1.1 and ORIGIN software. RESULTS: The study included a cohort of 21 female breast carcinoma patients. Tracer activity ( 177 Lu-DOTA-trastuzumab) was noted in the physiological organs such as the liver, spleen, kidneys, heart, as well as in the tumors. On visual analysis of 177 Lu-DOTA-trastuzumab biodistribution, the liver activity showed gradual clearance over time, and although spleen was comparatively faintly visualized than liver and similarly, kidneys were faintly visualized suggestive of the alternate route of tracer excretion. The maximum number of patients (n = 12) showed 2 components of clearance, namely, fast and slow. The average effective half-life of all the patients (including single and 2 components of clearance) was 106.25 ± 22.14 hours (84.11-128.39 hours). The mean absorbed dose for the liver, spleen, kidneys, heart, whole body, and red marrow was 1.0702 ± 0.731, 1.4114 ± 0.462, 1.4232 ± 0.364, 1.4719 ± 0.602, 0.2412 ± 0.0295, and 0.1485 ± 0.0213 mGy/MBq, respectively, by OLINDA EXM and 0.5741 ± 0.333, 0.8096 ± 0.224, 0.7943 ± 0.235, 1.8971 ± 0.713, and 0.09619 ± 0.0144 for liver, spleen, kidneys, heart and whole body respectively by ORIGIN. The absorbed radiation dose for tumor was 1.94E+2 by OLINDA EXM software and 1.78E+2 by ORIGIN software. In this study, during and after infusion of 177 Lu-DOTA-trastuzumab, no major adverse effects were noted in any patient except 1 patient who had grade 1 nausea and managed conservatively by antiemetic drug. CONCLUSIONS: The results of our study demonstrated expected and favorable biodistribution and dosimetry with 177 Lu-DOTA-trastuzumab in HER2-positive breast carcinoma patients. We noticed the mean absorbed dose to the normal organs within the limits of maximum tolerable dose, and also tumor dose was higher than the normal liver dose. Therefore, we conclude that 177 Lu-DOTA-trastuzumab radioimmunotherapy is feasible and a safe treatment option for treating HER2-positive breast carcinoma patients.

Intricacies in the Preparation of Patient Doses of [177Lu]Lu-Rituximab and [177Lu]Lu-Trastuzumab Using Low Specific Activity [177Lu]LuCl3: Methodological Aspects.

The development of humanized monoclonal antibodies (MAbs) with Lutetium-177 ([177Lu]Lu3+) has brought a paradigm shift in the arena of targeted therapy of various cancers. [177Lu]Lu-DOTA-Rituximab and [177Lu]Lu-DOTA-Trastuzumab have gained prominence due to their improved therapeutic efficacy in the treatment of lymphoma and breast cancer. The clinical dose formulation of these radiolabeled MAbs, using low specific activity [177Lu]LuCl3, requires extensive optimization of the radiolabeling protocol. The present study merits the development of a single protocol which has been optimized for conjugation of Rituximab and Trastuzumab with p-NCS-benzyl-DOTA and further radiolabeling these immunoconjugates (ICs) with low specific activity [177Lu]LuCl3. Herein, we report a consistent and reproducible protocol for clinical dose formulations of [177Lu]Lu-DOTA-Rituximab and [177Lu]Lu-DOTA-Trastuzumab (~9.25 GBq each, equivalent to ~2 patient doses) with radiochemical yield (RCY) between 84 and 86% and radiochemical purities (RCP) >99%. The in vitro stabilities of both these radioimmunoconjugates (RICs) were retained up to 120 h post-radiolabeling, upon storage with L-ascorbic acid as stabilizer (concentration: ~ 220-240 µg/37MBq) at -20 °C. The ready-to-use formulation of clinical doses[177Lu]Lu-DOTA-Rituximab and [177Lu]Lu-DOTA-Trastuzumab has been successfully achieved by employing a single optimized protocol. While [177Lu]Lu-DOTA-Rituximab has exhibited a high degree of localization in retroperitoneal nodal mass of refractory lymphoma patient, high uptake of [177Lu]Lu-DOTA-Trastuzumab has been observed in metastatic breast carcinoma patient with multiple skeletal metastases.

Evaluation of Different Methods for the Detection of Anti- Thyroglobulin Autoantibody: Prevalence of Anti-Thyroglobulin Autoantibody and Anti-Microsomal Autoantibody in Thyroid Cancer Patients.

Four anti-thyroglobulin autoantibodies (TgAb) assays were evaluated for their reference interval, method agreement, concordance etc. Prevalence of TgAb and anti-thyroid peroxidase was studied in differentiated thyroid cancer (DTC) and control. Reference intervals for TgAb assays varied from method to method due to varied assay designs. For TgAb correlation coefficients ranged from 0.74 to 0.99 whereas concordance ranged from 81 to 96.1%. Prevalence of thyroid antibodies mainly TgAb was increased in DTC primarily in females. Use of sensitive immunoassays is recommended for thyroid autoantibody measurement. Diagnosis and follow-up are difficult in DTC with coexisting thyroid autoimmunity. Hence, careful monitoring with regular surveillance is suggested.

Clinical Dose Preparation of [177Lu]Lu-DOTA-Pertuzumab Using Medium Specific Activity [177Lu]LuCl3 for Radioimmunotherapy of Breast and Epithelial Ovarian Cancers, with HER2 Receptor Overexpression.

Background: The overexpression of human epidermal growth factor receptor 2 (HER2) is commonly associated with metastatic breast cancer and epithelial ovarian cancer. The U.S. Food and Drug Administration (FDA) has approved Trastuzumab as an anti-HER2 agent for the metastatic breast and epithelial ovarian cancer. However, Trastuzumab has severe limitations in the treatment of metastatic breast cancer associated with ligand-dependent dimerization of HER2 receptor at the extracellular domain-II (ECD-II) region. The therapeutic approach in combination of pertuzumab and trastuzumab is found to be effective in preventing HER2 dimerization at the ECD-II region. The radioimmunotherapeutic approach, utilizing both these anti-HER2 agents (trastuzumab/pertuzumab), radiolabeled with [177Lu]Lu3+, has proved to be clinically efficacious with promising potential. Toward this, the formulation for clinical doses of [177Lu]Lu-DOTA-pertuzumab has been optimized using medium specific activity (0.81 GBq/µg) [177Lu]LuCl3. Materials and Methods: Preconcentrated pertuzumab was conjugated with p-NCS-benzyl-DOTA. Purified DOTA-benzyl-pertuzumab conjugate was radiolabeled with carrier-added [177Lu]LuCl3. Quality control parameters were evaluated for the [177Lu]Lu-DOTA-pertuzumab. In vivo biodistribution was carried out at different time points postadministration. Specific cell binding, immunoreactivity, and internalization were investigated by using SKOV3 and SKBR3 cells. Results: In this study, the authors reported a consistent and reproducible protocol for clinical dose formulations of [177Lu]Lu-DOTA-pertuzumab, with a radiochemical yield of 86.67% ± 1.03% and radiochemical purity (RCP) of 99.36% ± 0.36% (n = 10). Preclinical cell binding studies of [177Lu]Lu-DOTA-pertuzumab revealed specific binding with SKOV3 and SKBR3 cells up to 24.4% ± 1.4% and 23.2% ± 0.8%, respectively. The uptakes in SKOV3- and SKBR3-xenografted tumor in severe combined immunodeficiency mice were observed to be 25.9% ± 0.8% and 25.2% ± 1.2% ID/g at 48 and 120 h postinjection, respectively. Conclusions: A protocol was optimized for the preparation of ready-to-use clinical dose of [177Lu]Lu-DOTA-pertuzumab, in hospital radiopharmacy settings. The retention of RCP of the radiopharmaceutical, on storage in saline and serum, at -20°C, up to 120 h postradiolabeling, confirmed its in vitro stability.

Automated radiochemical synthesis of pharmaceutical grade [18 F]FLT using 3-N-Boc-5'-O-dimethoxytrityl-3'-O-nosyl-thymidine precursor and its Sep-Pak® purification employing selective elution from reversed phase.

Pharmaceutical grade 3'-deoxy-3'-[18 F]fluorothymidine [18 F]FLT was synthesized using 3-N-Boc-5'-O-dimethoxytrityl-3'-O-nosyl-thymidine (BOC-Nosyl) precursor, in the general purpose TRACERlab FX modules. Purification of [18 F]FLT, via solid phase extraction (SPE) after radiosynthesis, using a combination of different SPE cartridges, yielded satisfactory results, with radiochemical and chemical purity >99%. While the non-decay corrected radiochemical yield (RCY) with 20 mg (24 µmole) of BOC-Nosyl precursor was found to be 6.80 ± 0.16%, the decay corrected radiochemical yield (RCY) was 9.95 ± 0.24%. Residual acetone, acetonitrile, and ethanol levels were found to be 22.97 ± 0.76, 109.08 ± 0.93, and 7,666.45 ± 3.7 ppm, respectively. A simplified method for solid-phase purification of [18 F]FLT was developed, circumventing the need for HPLC purification. Biodistribution in C57BL/6 mice with B16F10 cell line-induced melanoma showed tumor to blood ratio of ~3.8 at 90 min. PET/CT imaging of normal rabbit injected with [18 F]FLT shows selective uptake in the bone marrow and small intestine. [18 F]FLT was found to be excreted through the kidneys and get collected in the urinary bladder, 120 min post injection. PET/CT imaging performed in rabbit model at 30, 60, 90, and 120 min post [18 F]FLT injections showed concordance with tissue distribution kinetics of mice tumor model.

Estimation of Absorbed Doses of Indigenously Produced "Direct-route" Lutetium-177-[177Lu]Lu-DOTA-TATE PRRT in Normal Organs and Tumor Lesions in Patients of Metastatic Neuroendocrine Tumors: Comparison with No-Carrier-Added [177Lu]Lu-DOTA-TATE and the Trend with Multiple Cycles.

Background: Lutetium-177-labeled somatostatin analogue, [177Lu]Lu-DOTA-TATE is most commonly used across the world for peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors (NETs). The primary objective of this study was to estimate the absorbed doses in organs and tumor lesions in NET patients treated with indigenously produced "direct-route" [177Lu]Lu-labeled DOTA-TATE and impact of multiple treatment cycles on absorbed doses, and compare with those treated with no-carrier-added [177Lu]Lu-labeled DOTA-TATE. Materials and Methods: Sixty patients of NET were enrolled in this prospective study. These patients received up to 6 cycles of PRRT with [177Lu]Lu-DOTA-TATE (total 232 cycles) at 10- to 12-week intervals between the two successive therapy cycles. The patients were administered 5.55-7.4 GBq (150-200 mCi) of [177Lu]Lu-DOTA-TATE in 100 mL of normal saline over a period of 30 min. Postadministration whole-body planar scintigraphy were acquired at five time points 0.5 (prevoid), 2, 12, 24, and 72 h (postvoid) and one SPECT scan at 24 h (postvoid). Number of disintegrations was determined from time-activity curves generated by drawing regions of interests (ROIs) on the images. Tumor masses were derived from computed tomography (CT) data. The absorbed doses for normal organs and tumor lesions were calculated using OLINDA 2.1.1 software. The same were also estimated in a group of 22 patients who were treated with no-carrier-added [177Lu]Lu-labeled DOTA-TATE. Results: The mean absorbed organ doses (mean ± SD) in Gy/GBq received by normal organs were as follows: kidneys 0.64 ± 0.21, liver 0.10 ± 0.05, spleen 0.88 ± 0.35, bone marrow 0.04 ± 0.02, urinary bladder 0.26 ± 0.06, heart wall 0.04 ± 0.02, and whole-body 0.06 ± 0.02. Tumor dosimetry was performed in a total of 410 tumor lesions, the mean absorbed dose to the tumor lesions was 4.79 ± 4.23 Gy/GBq. Large variations were observed in absorbed doses received by these lesions (range: 0.15-21.26 Gy/GBq). With no-carrier-added [177Lu]Lu-DOTA-TATE, the mean absorbed organ doses (mean ± SD) in Gy/GBq received by normal organs were as follows: kidneys 0.76 ± 0.16, liver 0.10 ± 0.05, spleen 1.14 ± 0.31, bone marrow 0.05 ± 0.02, urinary bladder 0.27 ± 0.05, heart wall 0.06 ± 0.02, whole-body 0.07 ± 0.02, and tumor dose 5.87 ± 5.74. Conclusions: There was no statistically significant difference in the dosimetry data of patients treated with no-carrier-added (indirect route) [177Lu]Lu-labeled DOTA-TATE and the dosimetry data of patients treated with [177Lu]Lu-labeled with DOTA-TATE formulated using 177Lu produced through "Direct-route" and were comparable with the data reported.

Prospective evaluation of organ-specific dose and lesional doses following therapeutic [177Lu]Lu-EDTMP administration in patients with multiple skeletal metastases and its correlation with clinical hematological toxicity.

AIM: In patients with multiple skeletal metastases, accurate estimation of absorbed doses to radiosensitive bone marrow in bone-directed systemic radionuclide therapies (RNT) is critically important from clinical dose determination standpoint. The primary aim of the present study was to estimate the radiation absorbed doses of therapeutic [177Lu]Lu-EDTMP to bone marrow by two methods viz. Medical Internal Radiation Dose (MIRD) schema and using OLINDA software and correlate with hematological toxicity. METHODS: A total of 15 patients diagnosed to have multiple painful skeletal metastases being treated with [177Lu]Lu-EDTMP for palliation of pain, were enrolled for this prospective study. For all patients, urine was collected immediately after infusion of [177Lu]Lu-EDTMP up to 24 h post-administration and cumulative activity excreted from body via urine was calculated. For dosimetry, patients underwent post-administration whole-body scintigraphy at five-time points: 0.5 (pre-void), 2, 24, 48 and 120 h (post-void). From the time-activity curves generated by drawing regions of interest (ROIs) on the images, number of disintegrations was determined. Absorbed doses for organs and bone lesions were calculated using OLINDA 2.2.0 software. For bone marrow dose estimates, in addition to OLINDA 2.2.0 software, MIRD schema was also adopted. Hematological profile was monitored in all patients during the treatment and post-treatment follow-up (estimating complete blood counts, every 15 d for 3 months after therapy). RESULTS: The mean ± standard deviation activity of [177Lu]Lu-EDTMP administered per patient per cycle was 2.08 ± 0.45 GBq. The results demonstrated higher uptake of [177Lu]Lu-EDTMP in bone metastases compared to normal bones. Within 2 and 24 h of administration of [177Lu]Lu-EDTMP, [177Lu]Lu activity excreted from the body was 24 ± 9% and 39 ± 14%, respectively. The mean absorbed organ doses (mean ± SD) in Gy/GBq were as follows: osteogenic cells 3.15 ± 1.85, bone marrow 0.57 ± 0.31, kidneys 0.08 ± 0.05, urinary bladder 0.32 ± 0.04, and bone lesions 2.91 ± 1.88. Strong correlation was found between (a) MIRD schema and OLINDA 2.2.0 software method for estimation of bone marrow doses (r = 0.96; P = <0.0001) and (b) Bone marrow absorbed dose and hematological toxicity (r = 0.81, P = 0.0027). CONCLUSION: Radiation absorbed doses to the bone marrow and skeletal metastatic lesions, following therapeutic [177Lu]Lu-EDTMP were estimated using a convenient and non-invasive quantitative imaging method. The estimated bone marrow absorbed dose, either by MIRD schema or the OLINDA 2.2.0 software method, demonstrated strong correlation. Strong correlation was also observed between bone marrow absorbed dose and hematological toxicity.

Therapeutic Multidose Preparation of a Ready-to-Use 177Lu-PSMA-617 Using Carrier Added Lutetium-177 in a Hospital Radiopharmacy and Its Clinical Efficacy.

Introduction: [177Lu]Lu-prostate-specific membrane antigen (PSMA)-617 has emerged as a promising radiopharmaceutical for targeting PSMA in metastatic castrate-resistant prostate carcinoma (mCRPC). We have optimized the radiolabeling protocol for a multidose formulation (27-28.8 GBq equivalent to 6-7 patient-doses) of [177Lu]Lu-PSMA-617 using [177Lu]Lu3+ produced via 176Lu(n,γ)177Lu route with moderate specific activity (0.66-0.81 GBq/µg). Methods: [177Lu]Lu-PSMA-617 was synthesized using moderate specific activity [177Lu]LuCl3 (0.74 GBq/µg) with PSMA-617 having metal-to-ligand molar ratio ∼1: 2.5 in CH3COONH4 buffer (0.1 M) containing gentisic acid at pH 4.0-4.5. Human prostate carcinoma cell line LNCaP cell (high PSMA expression) was used for in vitro cell-binding studies and generating tumor xenograft models in nude mice for tissue biodistribution studies. Several batches of the present formulation have been clinically administered in mCRPC patients (single patient dose: 4.44-5.55 GBq per cycle). Results: In this study we report a consistent and reproducible protocol for multidose formulations of [177Lu]Lu-PSMA-617 for adopting in a hospital radiopharmacy setting. Although the radiochemical yield of [177Lu]Lu-PSMA-617 was found to be 97.30% ± 1.03%, the radiochemical purity was 98.24% ± 0.50% (n = 19). In vitro and serum stability of [177Lu]Lu-PSMA-617 was retained up to 72 and 120 h after radiolabeling and upon storage at -20°C with a radioactive concentration between 0.37 and 0.74 GBq/mL upon using stabilizer concentration as low as 43-48 µg/mCi. Preclinical cell-binding studies of [177Lu]Lu-PSMA-617 revealed specific binding with LNCaP cells of 17.4% ± 2.4%. The uptake in LnCaP xenografted tumor (nude mice) was 7.5 ± 2.6% ID/g for ∼1.5-2.0 cm3 tumor volume at 24-h post-injection. Post-therapy (24 h) SPECT image of mCRPC patients with prior orchidectomy and various hormone therapy showed specific localization of [177Lu]Lu-PSMA-617 in the tumor region. Conclusions: Formulation of a ready-to-use multidose formulation of [177Lu]Lu-PSMA-617 was successfully achieved and the procedure was optimized for routine preparation at a hospital radiopharmacy set-up. High degree of localization of [177Lu]Lu-PSMA-617 in post-therapy SPECT scan and the post-therapeutic response confirms its therapeutic efficacy. Clinical Trials.gov ID: RPC/51/Minutes/Final dated 16th October, 2019.

Initial clinical evaluation of indigenous 90Y-DOTATATE in sequential duo-PRRT approach (177Lu-DOTATATE and 90Y-DOTATATE) in neuroendocrine tumors with large bulky disease: Observation on tolerability, 90Y-DOTATATE post- PRRT imaging characteristics (bremsstrahlung and PETCT) and early adverse effects.

177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) alone has lesser potential in the clinical setting of neuroendocrine tumor (NET) with large bulky disease and nonhomogeneous somatostatin receptors (SSTR) distribution, owing to lower energy (Eßmax 0.497 MeV) and a shorter particle penetration range (maximum 2-4 mm) of 177Lu. In large bulky NETs, 90Yttrium (90Y) has the theoretical advantages because of a longer beta particle penetration range (a maximum soft tissue penetration of 11 mm). Therefore, a combination of 177Lu and 90Y is a theoretically sound concept that can result in better response in metastatic NET with large-bulky lesion and non-homogeneous SSTR distribution. The aim of the study was to determine the feasibility of combining 90Y-DOTATATE with 177Lu-DOTATATE PRRT as sequential duo-PRRT in metastatic NET with (≥5 cm) including the post 90Y-DOTATATE-PRRT imaging and also to determine early toxicity of the duo-PRRT approach. A total of 9 patients received combination of 177Lu-DOTATATE with 90Y-DOTATATE (indigenously prepared and approved) through sequential duo-PRRT approach. These 9 NET patients were included and analyzed in this study. All 9 patients had undergone post-PRRT 90Y-DOTATATE imaging, including a whole-body planar bremsstrahlung imaging followed by regional single-photon emission computed tomography (SPECT)-computed tomography (CT) imaging and also a regional positron emission tomography-computed tomography imaging. Grading of 90Y-DOTATATE and 177Lu-DOTATATE uptake was done on post-PRRT imaging by both modalities. The size of the lesions ranged from 5.5 cm to 16 cm with average size of 10 cm before sequential duo-PRRT was decided. Sequential duo-PRRT was administered because of stable, unresponsive disease following 177Lu-DOTATATE in 5 patients (55.6%), progressive disease after 177Lu-DOTATATE in 2 patients (22.2%), and with neoadjuvant intent in 2 patients (22.2%). The total cumulative dose of 177Lu-DOTATATE before duo-pRRT ranged from 11.84 GBq to 37 GBq per patient and average administered dose of 27.21 GBq per patient in this study. Out of 9 patients, 8 patients received single cycle of 90Y-DOTATATE (ranging from 2.66 GBq to 3.4 GBq per patient with average administered dose of 3.12 GBq per patient). One patient received two cycles of 90Y-DOTATATE (total dose of 6.2 GBq). Out of 9 patients, 8 patients showed excellent tracer concentration in lesions on post-PRRT 90Y-DOTATATE imaging and the remaining 1 patient showed fairly adequate 90Y-DOTATATE tracer uptake in lesion on visual analysis. There was matched 90Y-DOTATATE uptake with 68Ga-DOTATATE and also with 177LuDOTATATE in all 9 patients. The sequential duo-PRRT was well tolerated by all patients. Two patients (22.2%) developed mild nausea, one patient (11.1%) developed transient mild-grade hemoglobin toxicity, and one patient (11.1%) developed mild-grade gastrointestinal symptoms (loose motion and abdominal pain). No nephrotoxicity, hepatotoxicity, and other hematological toxicity was observed. The combination of the indigenous 90Y-DOTATATE with 177Lu-DOTATATE PRRT in NET as sequential duo-PRRT was well tolerated, feasible and safe in stable, unresponsive/progressive disease following single isotope 177Lu-DOTATATE therapy and also in neoadjuvant PRRT setting with large bulky lesion (≥≥5cm). Post-PRRT 90Y-DOTATATE imaging showed excellent 90Y-DOTATATE uptake in nearly all NET patients. Mild-grade early adverse effects were easily manageable and controllable in this sequential duo-PRRT approach.

On the Separation of Yttrium-90 from High-Level Liquid Waste: Purification to Clinical-Grade Radiochemical Precursor, Clinical Translation in Formulation of 90Y-DOTATATE Patient Dose.

Introduction: The quality control parameters of in-house-produced 90Y-Acetate from high-level liquid waste (HLLW) using supported liquid membrane (SLM) technology were validated and compared with the pharmacopeia standard. The radiolabeling of DOTATATE yielding 90Y-DOTATATE in acceptable radiochemical purity (RCP), with expected pharmacological behavior in in vivo models, establish the quality of 90Y-Acetate. Clinical translation of 90Y-Acetate in formulation of 90Y-DOTATATE adds support toward its use as clinical-grade radiochemical. Methods: Quality control parameters of 90Y-Acetate, namely radionuclide purity (RNP), were evaluated using ß- spectrometry, γ-spectroscopy, and liquid scintillation counting. RCP and metallic impurities were established using high-performance liquid chromatography and inductively coupled plasma optical emission spectrometry, respectively. The suitability of 90Y-Acetate as an active pharmaceutical ingredient radiochemical was ascertained by radiolabeling with DOTATATE. In vivo biodistribution of 90Y-DOTATATE was carried out in nude mice bearing AR42J xenografted tumor. Clinical efficacy of 90Y-DOTATATE was established after using in patients with large-volume neuroendocrine tumors (NET). Bremsstrahlung imaging was carried out in dual-head gamma camera with a wide energy window setting (100-250 keV). Results: In-house-produced 90Y-Acetate was clear, colorless, and radioactive concentration (RAC) in the range of 40-50 mCi/mL. RCP was >98%. 90Sr content was <0.85 µCi/Ci of 90Y. Gross λ content was <0.8 nCi/Ci of 90Y and no γ peak was observed. Fe3+, Cu2+, Zn2+, Cd2+, and Pb2+ contents were <1.7 µg/Ci. The radiolabeling yield (RLY) of 90Y-DOTATATE was >94%, RCP was >98%. The in vitro stability of 90Y-DOTATATE was up to 72 h postradiolabeling, upon storage at -20°C. Post-therapy (24 h) Bremsstrahlung image of patients with large NET exhibit complete localization of 90Y-DOTATATE in tumor region. Conclusions: This study demonstrates that the in-house-produced 90Y-Acetate from HLLW can be used for the formulation of various therapeutic 90Y-based radiopharmaceuticals. Since 90Y is an imported radiochemical precursor available at a high cost in India, this study which demonstrates the suitability of indigenously sourced 90Y, ideally exemplifies the recovery of "wealth from waste." The Clinical Trial Registration number: (P17/FEB/2019).

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library.

Single- domain antibodies (SdAbs) have been deployed in various biomedical applications in the recent past. However, there are no reports of their use in the immunoradiometric assays (IRMA) for thyroglobulin (Tg). Tg is the precursor molecule for the biosynthesis of thyroid hormones: thyroxine and triiodothyronine, which are essential for the regulation of normal metabolism in all vertebrates. Patients with differentiated thyroid cancer (DTC) require periodic monitoring of their serum thyroglobulin levels, as it serves as a prognostic marker for DTC. Here, we report a methodology to produce SdAbs against human-Tg, by a hybrid immunization/directed-evolution approach by displaying the SdAb gene-repertoire derived from a hyperimmune camel in the T7 phage display system. We have demonstrated the immunoreactivity of anti-Tg-SdAb (KT75) in immunoassays for thyroglobulin and measured its affinity by surface plasmon resonance (KD ~ 18 picomolar). Additionally, we have shown the quantitative-binding property of SdAb for the first time in IRMA for thyroglobulin. The serum Tg values obtained from SdAb-Tg-IRMA and in-house assay using murine anti-Tg-monoclonal antibody as tracer significantly correlated, r = 0.81, p < 0.05. Our results highlight the scope of using the T7 phage display system as an alternative for the conventional M13-phage to construct single-domain antibody display libraries.

Examining Absorbed Doses of Indigenously Developed 177Lu-PSMA-617 in Metastatic Castration-Resistant Prostate Cancer Patients at Baseline and During Course of Peptide Receptor Radioligand Therapy.

Aim: The objective of this study was to estimate the absorbed doses to the normal organs and tumor lesions in metastatic castration-resistant prostate cancer (mCRPC) patients treated with indigenously developed 177Lu-PSMA-617 that could establish optimal treatment protocol with minimum risk to the dose-limiting organs. Furthermore, attempt was also made to compare radiation absorbed doses for normal organs and tumor lesions in subsequent cycles of 177Lu-PSMA-617 peptide receptor radioligand therapy (PRLT) in the same group of patients during the course of treatment. Methods: A total of 30 patients of proven mCRPC were enrolled for this prospective study. These patients received up to 5 cycles of treatment with 177Lu-PSMA-617 PRLT (1 cycle for 13 patients, 2 cycles for 9 patients, 3 cycles for 3 patients, and 5 cycles for 5 patients), at 11-12-week intervals between the two successive therapies. The patients underwent postadministration whole-body scintigraphy at five time points: 0.5 (prevoid), 2, 12, 24, and 72/96 h (postvoid). From time-activity curves generated by drawing regions of interests on the images, number of disintegrations was determined. Tumor masses were estimated from pretherapeutic 68Ga-PSMA-11 positron emission tomography-computed tomography images. Absorbed doses for organs and tumors were calculated using OLINDA 2.0 software. Results: The average activity of 177Lu-PSMA-617 (mean ± SD) administered per patient per cycle was 4.94 ± 0.45 GBq. The mean absorbed organ doses (mean ± SD) from first therapy cycle in Gy/GBq were as follows: kidneys 0.52 ± 0.16, spleen 0.17 ± 0.07, liver 0.08 ± 0.05, salivary glands 0.53 ± 0.30, lacrimal glands 1.45 ± 0.85, nasal mucosa membrane 0.46 ± 0.19, urinary bladder 0.23 ± 0.02, and bone marrow 0.04 ± 0.03. The mean effective dose for whole body from first therapy cycle was 0.05 ± 0.03 Sv/GBq. Among all the normal organs, lacrimal glands received the highest absorbed dose. The median dose for all lesions, bone lesions, lymph nodes, primary site, liver lesion, lung lesion, and soft tissue deposit from first therapy cycle was determined to be 4.17, 4.23, 3.96, 4.36, 10.27, 0.78, and 4.68 Gy/GBq respectively. Absorbed doses received by the normal organs in five consecutive cycles follow three different trends, (a) for kidneys, salivary glands, and nasal mucous membrane, absorbed doses increased from first therapy cycle to second therapy cycle and then slowly decreased in subsequently therapy cycles; (b) for spleen, liver, and lacrimal glands, absorbed doses decreased with the successive therapy cycles; and (c) in case of bone marrow, bladder, and whole body, mean absorbed dose almost remained constant in each therapy cycle. Absorbed doses to the lesions gradually decreased with increase of the number of therapy cycles. Conclusions: The organ and tumor absorbed doses of 177Lu-PSMA-617 in mCRPC patients were found to be comparable to the data reported in the literature. The highest absorbed organ dose was observed in lacrimal glands and being a dose limiting organ, a cumulative activity up to 32.5 GBq (878 mCi) of 177Lu-PSMA-617 in 4-5 therapy cycles appears safe and feasible to achieve full therapeutic window.

One decade of 'Bench-to-Bedside' peptide receptor radionuclide therapy with indigenous [177Lu]Lu-DOTATATE obtained through 'Direct' neutron activation route: lessons learnt including practice evolution in an Indian setting.

The present treatise chronicles one decade of experience pertaining to clinical PRRT services in a large-volume tertiary cancer care centre in India delivering over 4,000 therapies, an exemplar of successful PRRT programme employing indigenous 177Lutetium production and resources. For the purpose of systematic discussion, we have sub-divided the communication into 3 specific parts: (a) Radiopharmaceutical aspects that describes 177Lutetium production through 'Direct' Neutron Activation Route and the subsequent radiolabeling procedures, (b) The specific clinical nuances and finer learning points (apart from the routine standard procedure) based upon clinical experience and how it has undergone practice evolution in our setting and (c) Dosimetry results with this indigenous product and radiation safety/health physics aspects involved in PRRT services. Initiated in 2010 at our centre, the PRRT programme is a perfect example of affordable quality health care delivery, with indigenous production of the radionuclide (177Lu) in the reactor and subsequent radiolabeling of the radiopharmaceutical ([177Lu]Lu-DOTATATE) at the hospital radiopharmacy unit of the centre, which enabled catering to the needs of a large number of patients of progressive, metastatic and advanced Neuroendocrine Neoplasms (NENs) and related malignancies.

Surface modified silk fibroin nanoparticles for improved delivery of doxorubicin: Development, characterization, in-vitro studies.

Silk fibroin nanoparticles possess the hydrophobic nature which assists them to become a good substrate for reticulo-endothelial system (RES) and macrophageal uptake. Surface coating of these nanoparticles with hydrophilic stabilizers, like Tween-80 make them long circulating and facilitate their uptake by low density lipoprotein (LDL) receptors to cross blood brain barrier (BBB). Surface modified silk fibroin nanoparticles bearing anti-cancer agent doxorubicin (DOX) were fabricated by desolvation method and coated with Tween-80 as surface modifier. The prepared nanoparticles were characterized for various physicochemical parameters, like particle size, surface charge, surface morphology by scanning electron microscope (SEM) and transmission electron microscopy (TEM), and in vitro drug release along with in vitro cell cytotoxicity, flow cytometry and cellular uptake studies by flourocytometry on glioblastoma cell lines. Entrapment efficiency for the silk fibroin nanoparticles were found to be >85% for coated and uncoated nanoparticles. Nanoparticles with average diameter less than 150 nm having negative charge were found to show no toxicity of its own. The pro-inflammatory response of nanoparticles was observed by determining the cytokines level, such as TNF-α and IL-1ß. Sustained drug release pattern from the nanoparticles with better cytotoxicty as compared to free drug was observed, signifying their potential ability to work as a drug delivery system.

Sequential Duo-Peptide Receptor Radionuclide Therapy With Indigenous 90Y-DOTATATE and 177Lu-DOTATATE in Large-Volume Neuroendocrine Tumors: Posttherapy Bremsstrahlung and PET/CT Imaging Following 90Y-DOTATATE Treatment.

The efficacy of Lu-DOTATATE in large neuroendocrine tumors (NETs) is reduced because of the lower energy (Eßmax 0.497 MeV) and shorter range of Lu. The pure ß-emitter Y with its longer ß range is more effective in larger tumors. This should be balanced with the greater risk of Y-DOTATATE-related nephrotoxicity. Sequential duo-peptide receptor radionuclide therapy may result in a better response with minimal adverse effects in large-volume heterogeneous NETs. A 56-year-old man with large rectal NET liver metastases, treated with Y-DOTATATE and Lu-DOTATATE and sequential duo-peptide receptor radionuclide therapy, presented with post-Y-DOTATATE bremsstrahlung and PET/CT in comparison with Ga-DOTATATE PET/CT and Lu-DOTATATE scans.

Clinical utility of 177 Lu-DOTATATE PRRT in somatostatin receptor-positive metastatic medullary carcinoma of thyroid patients with assessment of efficacy, survival analysis, prognostic variables, and toxicity.

BACKGROUND: The primary aim of this study was to evaluate the therapeutic efficacy and outcome of 177 Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) in somatostatin receptor-positive metastatic medullary thyroid carcinoma (MTC), including progression-free survival (PFS) and overall survival (OS), and also to determine the various prognostic variables. The secondary aim was toxicity assessment of PRRT in this group of patients. METHODS: A total of 43 somatostatin receptor-positive metastatic MTC patients, treated with 177 Lu-DOTATATE PRRT in a large tertiary care center, were included in this analysis. After receiving the therapy, post-treatment response evaluation was undertaken for symptomatic and biochemical responses (serum calcitonin) and imaging responses with 68 Ga-DOTATATE, 18 F-FDG PET-CT, CeCT (PERCIST and RECIST 1.1 criteria). Calcitonin doubling time (CtnDT) was calculated by the American Thyroid Association calculator. The adverse events were graded according to the NCI-CTCAE v5.0 criteria. The observed Kaplan-Meier curves for both PFS and OS since first PRRT were compared with CtnDT (more than 24 months vs less than 24 months) by log-rank (Mantel-Cox) test. The prognostic variables were investigated for their association with CtnDT and response to PRRT using Cox proportional-hazards model. RESULTS: The median OS was 26 months (95% CI 16.6-35.3 months) and the median PFS 24 months (95%.CI: 15.1-32.9 months). Following 177Lu-DOTATATE PRRT, the observed median PFS and OS was longer in patients who had CtnDT more than 24 months compared to those with CtnDT less than 24 months (median PFS not yet reached vs 10 months and median OS 60 months vs 20 months). Assessing from the time-point of first 177 Lu-DOTATATE PRRT cycle, the patients with CtnDT more than 24 months had a significantly longer PFS (P < .001) and OS (P < .001) compared to those with less than 24 months. Less than 5 lesions, FDG uptake in lesions (SUVmax of <5) and patients alive at the time of analysis were the significant variables for association with CtnDT (more than 24 months). Out of 43 patients, 26 were responders (61%) and 17 nonresponders (39%) based upon PERCIST criteria, and 27 were responders (62%) while 16 patients were nonresponders (38%) based upon RECIST 1.1 criteria. The univariate analysis showed significant association between responses to PRRT with following prognostic variables: (a) size of lesions (<2 cm) and (b) FDG uptake in lesions (SUVmax of <5). PRRT was well tolerated in all patients without any major grade 3 or 4 toxicity. CONCLUSION: The results demonstrated that, 177 Lu-DOTATATE is a potentially efficacious and safe therapeutic option in SSTR avid metastatic MTC patients.

Comparison of Serum Thyroglobulin Levels in Differentiated Thyroid Cancer Patients Using In-House Developed Radioimmunoassay and Immunoradiometric Procedures.

Thyroglobulin (Tg) is a proven tumor marker in the follow-up and post-operative management of patients with differentiated thyroid cancer (DTC). All assays for serum thyroglobulin (s-Tg) are based on immunoassays, however, the assay technique has a bearing on the variations seen in the estimations. We studied this using four in-house developed radioimmunoassays (RIA) and immunoradiometric assays (IRMA). Limit of detection, working range, recovery, dilution test, precision profiles and method comparison were evaluated. All four methods were used for the estimation of s-Tg in DTC patients and also compared for their performance using commercially available Tg IRMA kits from DiaSorin and Izotop. The s-Tg values measured by six different immunoassays showed very significant inter-method correlation (0.84-0.99, p < 0.001). However, among the in-house developed assays; the coated tube IRMA showed a better sensitivity and precision at the lower concentration range and hence, is preferable for the routine measurement of s-Tg in patients negative for Tg autoantibodies (TgAb). Although the second generation IRMAs offer practical benefits of having higher sensitivity, shorter turn-around time and convenience of automation, they, unfortunately, also have higher tendency for interference from both TgAb and heterophilic antibodies, if present in the sample. On the contrary, RIA is less prone to such interference and, hence, can be used in patients with TgAb. In order to effectively use this test, it is important that nuclear medicine physicians and endocrinologists understand these intrinsic technical limitations encountered during s-Tg measurement.