Formulation of patient dose of [177Lu]Lu-Trastuzumab using in-house developed freeze-dried kit: A path forward for clinical translation.

Trastuzumab is a US-FDA-approved humanized monoclonal antibody used for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The aim of the present work is to optimize a freeze-dried formulation of DOTA-Trastuzumab conjugate for the preparation of patient doses of [177Lu]Lu-Trastuzumab for radioimmunotherapy of breast cancer. The formulation of [177Lu]Lu-Trastuzumab usually takes a long time, and thus, such a process is not suitable for the routine preparation of this agent in hospital radiopharmacies. To circumvent this, a pre-synthesized DOTA-Trastuzumab conjugate as a freeze-dried formulation is proposed. In the present work, DOTA-Trastuzumab conjugate was subjected to a freeze-drying process after the addition of optimized amounts of radioprotectant and cryoprotectant. [177Lu]Lu-DOTA-Trastuzumab was prepared by incubating the lyophilized powder of the kit vial with medium-specific activity 177LuCl3. The final radiochemical purity of [177Lu]Lu-DOTA-Trastuzumab, prepared using freeze-dried kit, was determined to be >95%. To ascertain the reproducibility of the procedure, six consecutive batches of the freeze-dried formulation were prepared, radiolabeled, and evaluated by carrying out both in vitro and ex vivo studies. The consistency of the results of all the six consecutive batches confirmed the robustness and utility of the in-house optimized freeze-dried formulation for the preparation of patient doses of [177Lu]Lu-Trastuzumab at hospital radiopharmacies.

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.

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.

Effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives: Comparative in-vitro and in-vivo evaluation for possible potential in PET and PDT.

tetracationic (TMPyP) and tricationic porphyrin (TriMPyCOOHP) derivatives were synthesized, characterized and investigated for binding with DNA by Isothermal Titration Calorimetry as well as by UV-Vis spectroscopy in order to study the effect of structural variation on tumor targeting efficacy of cationically charged porphyrin derivatives. Fluorescence cell imaging studies performed in cancer cell lines corroborated the findings of aforementioned studies. Photocytotoxicity experiments in A549 cell lines revealed relatively higher light dependent cytotoxic effects exerted by TMPyP compared to TriMPyCOOHP. In-vivo experiments in tumor bearing animal model revealed relatively longer retention of 68Ga-TMPyP in tumorous lesion compared to that of 68Ga-TriMPyCOOHP. The study reveals that removal of one of the positive charges of the tetracationic porphyrin derivatives significantly reduces their DNA binding ability and cytotoxicity as well as brings changes in the pharmacokinetic pattern and tumor retention in small animal model.

Preparation of [177Lu]Lu-DOTA-Ahx-Lys40-Exendin-4 for radiotherapy of insulinoma: a detailed insight into the radiochemical intricacies.

INTRODUCTION: [177Lu]Lu-DOTA-Ahx-Lys40-Exendin-4 ([177Lu]Lu-DOTA-Exendin-4) is a potential agent for radiotherapy of insulinomas owing to its specificity towards GLP-1 (Glucagon like peptide-1) receptors over-expressed on such cancers. The objective of the present study is to optimize the various radiochemistry parameters for the consistent formulation of the agent with high radiolabeling yield using carrier added [177Lu]LuCl3 and also to evaluate its biological behaviour in small animal model. METHODS: In order to optimize the radiolabeling parameters, DOTA-Exendin-4 was radiolabeled with [177Lu]LuCl3 in two different buffer systems (sodium acetate and HEPES) at three different temperatures (45, 65 and 95 °C) using three different ligand to metal ratios (3:1, 4:1 and 5:1). The radiolabeled peptide was characterized by both paper chromatography and HPLC. The effect of addition of three different radio-protectors on complexation yield was also studied. Bio-distribution studies were carried out in healthy Swiss mice to evaluate the pharmacokinetic behaviour of the radiolabeled peptide as well as to determine the in vivo specificity of the radiotracer towards GLP-1 receptors (blocking studies). Urine and kidney lysate of the animals were analyzed at various post-administration time-points in order to determine the in vivo stability of the radiolabeled peptide. RESULTS: The [177Lu]Lu-DOTA-Exendin-4 complex could be prepared consistently with >95% radiolabeling yield using the optimized reaction conditions. Bio-distribution studies revealed early accumulation of [177Lu]Lu-DOTA-Exendin-4 in pancreas along with fast clearance via renal pathway. Significantly high accumulation of the radiotracer was observed in kidneys. Analyses of urine and kidney lysate of the animals revealed in vivo stability of [177Lu]Lu-DOTA-Exendin-4. Blocking studies showed displacement of significant amount of radiotracer from GLP-1 receptor-positive organs such as, pancreas and lungs (p <0.05) in presence of unlabeled peptide, indicating the specificity of the radiolabeled preparation towards GLP-1 receptors. CONCLUSIONS: Present study shows that [177Lu]Lu-DOTA-Exendin-4 could be formulated for radiotherapeutic application with high radiochemical purity and adequate in vivo stability using [177Lu]LuCl3 produced via direct neutron irradiation. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Findings of the present study will be helpful in preparing the patient dose of [177Lu]Lu-labeled Exendin for radiotherapy of insulinoma using carrier added [177Lu]LuCl3, produced in a medium flux reactor, without the requirement of post-labeling purification.

Preparation and evaluation of 99mTc-labeled porphyrin complexes prepared using PNP and HYNIC cores: studying the effects of core selection on pharmacokinetics and tumor uptake in a mouse model.

Porphyrins are tetrapyrrolic macrocyclic ligands known for their affinity towards neoplastic tissues and once radiolabeled with a suitable diagnostic radioisotope could potentially be used for the imaging of tumorous lesions. In the present study, an unsymmetrically substituted porphyrin derivative namely 5-(p-amino-propyloxyphenyl)-10,15,20-tris(carboxymethyleneoxyphenyl)-porphyrin was synthesized and modified further to enable radiolabeling with 99mTc using two different 99mTc-cores viz. 99mTc-HYNIC (hydrazino nicotinic acid) and 99mTc(N)PNP2 (PNP2 = bis-[(2-dimethylphosphino)ethyl]-methoxy-ethylamine) in order to study the effect of employing different 99mTc-cores on tumor affinity and pharmacokinetic behavior of the resultant 99mTc-labeled porphyrin complexes. 99mTc-Porphyrin complexes were characterized by reversed phase HPLC studies and could be prepared with >95% radiochemical purity under optimized radiolabeling conditions. Both 99mTc-complexes were found to be adequately stable in human blood serum till 3 h post-preparation. Bio-distribution studies, carried out in Swiss mice bearing fibrosarcoma tumors, revealed relatively higher tumor uptake for the 99mTc-HYNIC-porphyrin complex (3.95 ± 1.42 and 3.28 ± 0.27% IA per g) compared to that exhibited by the 99mTc(N)PNP-DTC-porphyrin complex (1.52 ± 0.53 and 1.56 ± 0.10% IA per g) at 1.5 and 3 h post-administration, although the former complex exhibited comparatively lower lipophilicity in the octanol-water system. Higher uptake and longer retention in the blood were observed for the 99mTc-HYNIC-porphyrin complex (6.63 ± 0.75 and 4.36 ± 0.25% IA per g) compared to that exhibited by the 99mTc(N)PNP-DTC-porphyrin complex (2.41 ± 0.54 and 2.30 ± 0.16% IA per g) at both 1.5 and 3 h post-administration. However, relatively lower liver uptake was observed for the former complex (19.26 ± 3.48 and 18.45 ± 1.05% IA per g) than that exhibited by the latter one (39.37 ± 3.88 and 34.15 ± 8.25% IA per g) at both 1.5 and 3 h post-administration. This study indicates that the in vivo behavior exhibited by the 99mTc-labeled porphyrins not only depends on their lipophilicity/hydrophilicity but is also governed by the Tc-cores employed for radiolabeling.

Convenient Formulation of 68Ga-BPAMD Patient Dose Using Lyophilized BPAMD Kit and 68Ga Sourced from Different Commercial Generators for Imaging of Skeletal Metastases.

PURPOSE: 68Ga-BPAMD has recently emerged as one of the preferred radiopharmaceuticals for imaging of bone lesions due to its ability to produce high-resolution images and uncomplicated availability of 68Ga, a positron emission tomography (PET) radionuclide, from commercial 68Ge/68Ga generators. The primary objective of this work is to develop freeze-dried BPAMD kit, for the easy and convenient formulation of 68Ga-BPAMD patient dose at the hospital radiopharmacy. In addition, the kit should be compatible with 68Ga, eluted using HCl of various molarities from the 68Ge/68Ga generators sourced from different suppliers. PROCEDURES: Freeze-dried BPAMD kit, comprising 50 µg of BPAMD and 150 mg of HEPES, was prepared and evaluated using 68Ga eluted from three different 68Ge/68Ga generators. Radiochemical purity (RCP) of 68Ga-BPAMD was determined by both thin-layer chromatography and high-performance liquid chromatography studies. The maximum volume of 68Ga, which can be added in the kit, was determined. The biological behavior of 68Ga-BPAMD, prepared using the freeze-dried kit, was evaluated by both in vitro and in vivo studies. Clinical studies were also performed in limited number of patients suffering from metastatic bone cancer. RESULTS: 68Ga-BPAMD could be prepared with >95% RCP using the freeze-dried BPAMD kit and 68Ga eluted from 68Ge/68Ga generators obtained from three different suppliers. 68Ga-BPAMD, prepared using the freeze-dried kit, exhibited adequate serum stability and ∼91% binding with the hydroxyapatite particles. Biodistribution studies in normal Wistar rats exhibited selective uptake of the agent in skeleton and fast clearance of the nonaccumulated activity through urinary route. Clinical studies in cancer patients showed excellent accumulation of the agent in bone lesions. CONCLUSION: The preliminary studies exhibited the potential of the developed BPAMD kit toward its utilization for the PET scanning of skeletal metastases.

Studies towards elucidating the potential of 5,10,15,20-tetrakis(p-carboxy-methyleneoxyphenyl)porphyrin as a theranostic agent for applications in PET and PDT.

Porphyrins, owing to their inherent tendency to accumulate in tumorous lesions, are considered suitable for developing agents for theranostic applications involving tumor diagnosis and targeted tumor therapy. The aim of the present work is to study the potential of a porphyrin derivative namely, 5,10,15,20-tetrakis(p-carboxymethyleneoxyphenyl)porphyrin (SPTA) as a theranostic agent for applications in positron emission tomography (PET) and photodynamic therapy (PDT). SPTA was synthesized in-house following a three-step reaction process and characterized by using spectroscopic techniques, viz. UV-vis, FT-IR, 1H-NMR and 13C-NMR spectroscopy, as well as by mass spectrometry. SPTA was labeled with 68Ga, a generator produced PET radioisotope, and the radiolabeled product was characterized by HPLC. The 68Ga-SPTA complex was prepared with a radiochemical purity of >95% under optimized conditions. The diagnostic potential of 68Ga-SPTA was evaluated by cell uptake studies in two different tumor cell lines (HT1080 and A549) which revealed the affinity of 68Ga-SPTA towards the cancer cells. Biodistribution studies carried out in Swiss mice bearing fibrosarcoma tumors exhibited the accumulation of the radiotracer in the tumor. The therapeutic potential of SPTA was evaluated by determining its photo-cytotoxicity employing the MTT assay in HT1080 and A549 cell lines using three different light doses, which indicated the significant cytotoxicity of SPTA in the presence of light. The present study indicates the possible potential of SPTA in radionuclide imaging as well as in photodynamic therapy (PDT) thus confirming the promising theranostic nature of this porphyrin derivative.

Comparative Evaluation of Using NOTA and DOTA Derivatives as Bifunctional Chelating Agents in the Preparation of 68Ga-Labeled Porphyrin: Impact on Pharmacokinetics and Tumor Uptake in a Mouse Model.

PURPOSE: Both NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) derivatives have been used as bifunctional chelating agents (BFCAs) for the preparation of 68Ga-labeled target-specific agents having potential for positron emission tomography (PET) imaging of cancerous lesions. In the present work, the authors have attempted a comparative pharmacokinetic evaluation between 68Ga-labeled porphyrins prepared using NOTA and DOTA derivatives as the BFCAs. PROCEDURES: A symmetrical porphyrin derivative, 5,10,15,20-tetrakis(p-carboxymethyleneoxyphenyl)porphyrin, was synthesized and coupled with two different BFCAs viz. p-NH2-benzyl-NOTA and p-NH2-benzyl-DOTA. Both the porphyrin-BFCA conjugates were radiolabeled with 68Ga. A comparative bioevaluation involving pharmacokinetics and tumor affinity was performed in a tumor-bearing small animal model. RESULTS: Gallium-68-labeled porphyrin-amido-benzyl-NOTA and porphyrin-amido-benzyl-DOTA complexes were prepared with high radiochemical purity. Both radiolabeled complexes exhibited almost similar stability in human serum and near-identical tumor affinity and pharmacokinetic behavior in animal studies. CONCLUSION: The present study demonstrates that the pharmacokinetic behavior of 68Ga-labeled porphyrin derivatives, prepared using either NOTA or DOTA derivatives as BFCAs, remains almost identical and hence both NOTA and DOTA derivatives could be considered equivalent for developing 68Ga-based PET agents for imaging of tumorous lesions.

Effect of Number of Bifunctional Chelating Agents on the Pharmacokinetics and Immunoreactivity of 177Lu-labeled Rituximab: A Systemic Study.

OBJECTIVE: Monoclonal antibodies (mAbs) have been radiolabeled with a variety of radioisotopes utilizing various kinds of bi-functional chelating agents (BFCAs) with an aim to develop suitable agents for radioimmunotherapy. The number of BFCA moieties present per antibody molecule plays a significant role in determining the pharmacokinetics and immunoreactivity exhibited by the radiolabeled antibodies. The objective of the present study is to evaluate the effect of the number of BFCA moieties present per antibody molecule on the pharmacokinetics and immunoreactivity of the 177Lu-labeled Rituximab. METHODS: Three different mAb-BFCA conjugates were prepared using different molar ratios of Rituximab (mAb) to p-NCS-benzyl-DOTA (BFCA) viz. 1:5, 1:10 and 1:50 employing different reaction conditions. Studies were carried out to determine the average number of BFCAs attached per mAb molecule. All the three mAb-BFCA conjugates were labeled with 177Lu(III) and were subsequently evaluated in normal Swiss mice to ascertain their respective pharmacokinetic behavior. In-vitro studies were also performed in Raji cell lines (human burkitt's lymphoma) for determining the effect of increasing number of BFCAs attached per mAb molecule on the immunoreactivity of the resultant 177Lu-labeled mAb-BFCA complexes. RESULTS: 177Lu-labeled mAb-BFCA complex prepared corresponding to 1:50 mAb to BFCA ratio exhibited the least non-specific uptake and rapid clearance from majority of the organs, but also exhibited least immunoreactive fraction (IRF = 19.37%). On the other hand, 177Lu-labeled mAb-BFCA complex prepared corresponding to 1:5 mAb to BFCA ratio exhibited the highest non-specific uptake and slower clearance pattern, but highest IRF (71.17%). 177Lu-labeled mAb-BFCA complex prepared corresponding to 1:10 mAb:BFCA ratio exhibited intermediate pharmacokinetic behaviour with moderate IRF (53.05%). CONCLUSIONS: The present study indicates that antibody to BFCA ratio plays a crucial role in determining the immunoreactivity and pharmacokinetic behavior of the radiolabeled antibodies and must be prudently chosen for their successful therapeutic application.

Preparation of clinical-scale 177 Lu-Rituximab: Optimization of protocols for conjugation, radiolabeling, and freeze-dried kit formulation.

Rituximab is a monoclonal chimeric antibody, which has been approved by the US Food and Drug Administration for immunotherapy of non-Hodgkin lymphoma. Bexxar and Zevalin are the two other approved radiolabeled antibodies for radioimmunotherapy of non-Hodgkin lymphoma; however, they are of murine origin that reduces their treatment efficacy. So as to circumvent this, efforts have been made to radiolabel Rituximab with various therapeutic radioisotopes. In the present study, an effort has been made to optimize the conjugation (bifunctional chelating agent and antibody) and radiolabeling procedures for the preparation of clinical-scale 177 Lu-labeled Rituximab. An attempt was also made to prepare the freeze-dried Rituximab kit for the easy and convenient clinical translation of the agent. Clinical-scale 177 Lu-Rituximab (40 mCi, 1.48 GBq) was prepared with >95% radiochemical purity using the kit. Biological evaluation of 177 Lu-Rituximab was performed by in vitro cell binding studies in Raji cell lines, which showed satisfactory binding at 4°C and 37°C. Pharmacokinetic behavior of the agent, evaluated by biodistribution studies in normal Swiss mice, revealed high blood and liver uptake at the initial time points, although it exhibited slow and gradual clearance with time. The study indicates that clinical-scale 177 Lu-Rituximab could be conveniently formulated using the methodology described in the present article.