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.

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.

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.

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).

Potential immunomodulatory effect of allelochemical juglone in mice vaccinated with BCG.

Juglone, a naphthoquinone is a known phytotoxic and cytotoxic allelochemical. Various anticancer studies suggest that pharmacological ROS insult and enzymes inhibited by juglone could be of therapeutic utility. In this study, we show that juglone induces immunomodulation in BCG-vaccinated mice. We have shown that juglone treatment leads to the generation of Th1 cytokines which develops an M1 phenotype in splenic macrophages. These findings were supported by a decrease in Treg population in BCG-vaccinated mice treated with juglone. Additionally, we observed juglone significantly increases CD8+ and T-helper memory population in BCG-vaccinated mice under immunomodulation. Further, BCG-challenge test showed that juglone enhances effector immune functions. Our finding was demonstrated by estimating ex-vivo Th1 and Th2 cytokines, flow cytometric analysis of Treg, T-helper memory, and CD8+ cells and determining serum IgG2a/IgG1 titer shift in BCG-vaccinated animals treated with juglone. Collectively, our findings provide evidence that juglone may act as immunomodulator when used at a low dose, metronomically.

Juglone-ascorbic acid synergy inhibits metastasis and induces apoptotic cell death in poorly differentiated thyroid carcinoma by perturbing SOD and catalase activities.

Anaplastic thyroid carcinoma (ATC) requires more innovative approaches as the current regimes for therapy are inadequate, also most anticancer drugs cause general suppression of physiological functions. However, therapy with limited nontarget tissue damage is desirable. In the present study, we show prooxidant ability of ascorbic acid, which enhances cytotoxicity induced by juglone. We decipher that juglone-ascorbate combination induces reactive oxygen species-mediated apoptosis leading to cell death in ARO cell line originated from ATC. This combination also affects enzyme activity of catalase, glutathione reductase, and superoxide dismutase destabilizing redox balance in cell and thereby making juglone effective at a lower dose. We also show that juglone-ascorbate combination suppresses cell migration, invasion, and expression of tumor-promoting, and angiogenic genes in ARO cell line, thereby disrupting epithelial-mesenchymal transition ability of the cells. Overall, we show that ascorbic acid increases cytotoxic potency of juglone through redox cycling when used in synergy.

Allium sativum Constituents Exhibit Anti-tubercular Activity In vitro and in RAW 264.7 Mouse Macrophage Cells Infected with Mycobacterium tuberculosis H37Rv.

BACKGROUND: Long duration of treatment, side-effects of currently used anti-tubercular drugs and emergence of drug-resistant forms of Mycobacterium tuberculosis (MTB) warrants the need to develop new drugs to tackle the scourge of tuberculosis (TB). Garlic is an edible plant reported to have anti-tubercular activity. However, previous researches on anti-tubercular effect of garlic were focused mostly on preliminary in vitro screening. OBJECTIVE: To identify constituents responsible for anti-tubercular activity of thiosulfinate-derivative rich extract of garlic (GE) and to evaluate activity of the most active constituent in RAW 264.7 mouse macrophage cells infected with M. tuberculosis H37Rv (MTBH). MATERIALS AND METHODS: In the present study, we have isolated eight compounds from GE by flash chromatography. The isolated compounds were characterized by 1H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry and Fourier transform infrared spectroscopy. Individual isolates and GE were screened for activity against MTBH by Resazurin Microtitre Plate Assay (REMA). RESULTS: Anti-tubercular activity of GE was superior to that of isolates when evaluated by REMA, possibly due to synergism amongst the constituents of GE. Cytotoxicity of GE was evaluated in RAW 264.7 mouse macrophage cells and it was observed that GE had a favorable selectivity index (>10). Therefore, anti-tubercular activity of GE was further evaluated by intracellular macrophage infection model. GE demonstrated concentration-dependent activity in macrophages infected with MTBH. CONCLUSION: This is the first report on intracellular anti-tubercular activity of any extract of garlic or its components. Appreciable intracellular anti-tubercular activity of GE in macrophages combined with low cytotoxicity makes it a suitable candidate for further development as an anti-tubercular agent. SUMMARY: Thiosulfinate-derivative rich extract of Allium sativum showed better activity than its isolated constituents against Mycobacterium tuberculosis H37Rv.(MTBH) when evaluated by Resazurin Microtitre Plate AssayThe extract showed least cytotoxic potential against RAW 264.7 mouse macrophage cells as compared to rifampicin, isoniazid and ethambutol when evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The extract had an appreciable selectivity indexExtract showed appreciable activity in RAW 264.7 mouse macrophage cells infected with MTBH, indicating its potential to be developed further as an anti-tubercular agent that can be administered as an adjunct to the existing anti-tubercular drug regimen. Abbreviations used: TB: Tuberculosis, MTB: Mycobacterium tuberculosis, MTBH: Mycobacterium tuberculosis H37Rv, GE: Thiosulfinate-derivative rich extract of garlic, REMA: Resazurin Microtitre Plate Assay, VD: Vinyldithiin, CFU: Colony forming unit, 1H NMR: 1H nuclear magnetic resonance spectroscopy, FT-IR: Fourier transform-infrared spectroscopy, LC-MS: Liquid chromatography-mass spectrometry, IC50: Concentration required to inhibit the cells by 50%, ANOVA: Analysis of variance.