RESUMO
Chlorambucil is an alkylating drug that finds application towards chemotherapy of different types of cancers. In order to explore the possibility of utilization of this drug as an imaging agent for early diagnosis of solid tumors, attempt was made to synthesize a 99mTc complex of chlorambucil and evaluate its potential in tumor bearing small animal model. HYNIC-chlorambucil was synthesized by conjugation of HYNIC with chlorambucil via an ethylenediamine linker. All the intermediates and final product were purified and characterized by standard spectroscopic techniques viz. FT-IR, 1H/13C-NMR as well as by mass spectrometry. HYNIC-chlorambucil conjugate was radiolabeled with [99mTc]Tc and found to be formed with > 95 % radiochemical purity via RP-HPLC studies. The partition coefficient (Log10Po/w) of the synthesized complex was found to be -0.78 ± 0.25 which indicated the moderate hydrophilic nature for the complex. Biological behaviour of [99mTc]Tc-HYNIC-chlorambucil, studied in fibrosarcoma bearing Swiss mice, revealed a tumor uptake of about 4.16 ± 1.52 %IA/g at 30 min post-administration, which declined to 1.91 ± 0.13 % IA/g and 1.42 ± 0.14 %IA/g at 1 h and 2 h post-administration, respectively. A comparison of different [99mTc]Tc-chlorambucil derivatives (reported in the contemporary literature) formulated using different methodologies revealed that tumor uptake and pharmacokinetics exhibited by these agents strongly depend on the lipophilicity/hydrophilicity of such agents, which in turn is dependent on the bifunctional chelators used for formulating the radiolabeled chlorambucils.
Assuntos
Clorambucila , Compostos de Organotecnécio , Animais , Humanos , Camundongos , Antineoplásicos Alquilantes/síntese química , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Clorambucila/química , Clorambucila/síntese química , Clorambucila/farmacologia , Estrutura Molecular , Ácidos Nicotínicos/química , Ácidos Nicotínicos/síntese química , Compostos de Organotecnécio/química , Compostos de Organotecnécio/síntese química , Compostos de Organotecnécio/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Tecnécio/química , Distribuição TecidualRESUMO
The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with 177Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 µCi (9.25 MBq) and 500 µCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.
Assuntos
Sinais de Localização Nuclear , Porfirinas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Lutécio , Camundongos Nus , Porfirinas/química , Porfirinas/farmacologia , Radioisótopos , Distribuição TecidualRESUMO
OBJECTIVE: Good's buffer or HEPES has advantages over other buffers commonly used in radiopharmaceutical preparation as it exhibits significantly lower complexation tendency with metal ions. However, use of HEPES buffer for radiolabeling reactions, meant for clinical applications, has been underrated due to the non-availability of sufficient toxicity data. The objective of the present study is to find the evidences towards safety of intravenous administration of HEPES through systemic toxicological studies in small animal model to support its safe application for clinical exploitation. EXPERIMENTAL: A pilot study was performed to investigate the lethal dose of HEPES in female Sprague Dawley rats by administering seven different doses of HEPES solution (150 to 2000 mg/kg), through intravenous pathway. Similarly, for determining maximum tolerated dose (MTD), gradually increasing doses of HEPES (50 to 950 mg/kg) were administered in the same species via similar pathway. Various hematological and clinical pathological investigations were carried out in order to find out the safe administration dose of HEPES in rats. RESULTS: No mortality was observed up to 2000 mg/kg doses of HEPES. The doses beyond 300 mg/kg resulted few temporary adverse effects, though these were found to disappear within 4-5 days of dosing. CONCLUSION: The amount of HEPES to be administered during clinical intervention is usually much lower (typically 1-2.5 mg per kg of body weight of healthy adult) than the MTD determined in rat model during present report. Hence, the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe. However, further detailed investigations may be warranted for supporting the candidature of Good's buffer for regular clinical exploitation.
Assuntos
Administração Intravenosa , Ratos Sprague-Dawley , Animais , Ratos , Feminino , Medicina Nuclear , Soluções Tampão , Dose Máxima Tolerável , SegurançaRESUMO
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.
Assuntos
Neoplasias da Mama , Radioisótopos , Humanos , Feminino , Radioisótopos/uso terapêutico , Trastuzumab , Reprodutibilidade dos Testes , Compostos Radiofarmacêuticos/uso terapêutico , Neoplasias da Mama/radioterapia , Lutécio/uso terapêuticoRESUMO
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.
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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.
Assuntos
Dendrímeros , Fibrossarcoma , Porfirinas , Animais , Camundongos , Dendrímeros/química , Dendrímeros/farmacocinética , Porfirinas/química , Porfirinas/uso terapêutico , Distribuição Tecidual , Fibrossarcoma/tratamento farmacológicoRESUMO
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.
Assuntos
Dipeptídeos/isolamento & purificação , Dipeptídeos/uso terapêutico , Compostos Heterocíclicos com 1 Anel/isolamento & purificação , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Lutécio/isolamento & purificação , Lutécio/uso terapêutico , Antígeno Prostático Específico/isolamento & purificação , Antígeno Prostático Específico/uso terapêutico , Neoplasias da Próstata/radioterapia , Radioisótopos/isolamento & purificação , Radioisótopos/uso terapêutico , Compostos Radiofarmacêuticos/isolamento & purificação , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Dipeptídeos/farmacocinética , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Liofilização , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Técnicas In Vitro , Lutécio/farmacocinética , Masculino , Farmácia Nuclear/métodos , Serviço de Farmácia Hospitalar , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Antígeno Prostático Específico/farmacocinética , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/diagnóstico por imagem , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/radioterapia , Radioquímica/métodos , Radioquímica/normas , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
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.
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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.
Assuntos
Antineoplásicos/farmacologia , Fibrossarcoma/diagnóstico por imagem , Fibrossarcoma/tratamento farmacológico , Fotoquimioterapia , Porfirinas/farmacologia , Tomografia por Emissão de Pósitrons , Células A549 , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Estrutura Molecular , Porfirinas/síntese química , Porfirinas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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 (pâ¯<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.
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Exenatida/química , Exenatida/uso terapêutico , Compostos Heterocíclicos com 1 Anel/química , Insulinoma/radioterapia , Lutécio/uso terapêutico , Radioisótopos/uso terapêutico , Animais , Exenatida/farmacocinética , Exenatida/urina , Rim/metabolismo , Camundongos , Radioquímica , Distribuição TecidualRESUMO
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.
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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 â¼91% 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.
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Amidas/uso terapêutico , Difosfonatos/uso terapêutico , Radioisótopos de Gálio/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Radioquímica/métodos , Kit de Reagentes para Diagnóstico/tendências , Amidas/farmacologia , Animais , Difosfonatos/farmacologia , Humanos , Metástase Neoplásica , Ratos , Ratos WistarRESUMO
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.
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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.
Assuntos
Quelantes/química , Fibrossarcoma/metabolismo , Radioisótopos de Gálio/química , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos/química , Porfirinas/síntese química , Compostos Radiofarmacêuticos/síntese química , Animais , Linhagem Celular Tumoral , Quelantes/farmacocinética , Modelos Animais de Doenças , Fibrossarcoma/diagnóstico por imagem , Radioisótopos de Gálio/farmacocinética , Compostos Heterocíclicos/farmacocinética , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Camundongos , Porfirinas/química , Porfirinas/farmacocinética , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos Imunológicos/farmacocinética , Quelantes/farmacocinética , Imunoconjugados/farmacocinética , Lutécio/química , Radioisótopos/química , Compostos Radiofarmacêuticos/farmacocinética , Rituximab/farmacocinética , Animais , Anticorpos Monoclonais/química , Antineoplásicos Imunológicos/química , Quelantes/química , Imunoconjugados/química , Camundongos , Radioimunoterapia , Compostos Radiofarmacêuticos/química , Rituximab/química , Distribuição TecidualRESUMO
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.
Assuntos
Composição de Medicamentos/métodos , Liofilização , Marcação por Isótopo/métodos , Lutécio/química , Rituximab/química , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Compostos Heterocíclicos com 1 Anel/química , Humanos , Camundongos , Radioisótopos , Rituximab/farmacocinética , Distribuição TecidualRESUMO
OBJECTIVE: PSMA-617 is reported to exhibit very high binding affinity towards PSMA receptors, over-expressed on prostate cancer cells and therefore, (177)Lu-labeled PSMA-617 is expected to play a pivotal role in the clinical management of patients suffering from ca prostate. The objective of the present study is to formulate the patient dose of (177)Lu-labeled PSMA-617, pre-clinical studies in animal model and clinical investigation in limited number of prostate cancer patients as well evaluating its potential for theranostic application. EXPERIMENTAL: Patient dose of 7.4 GBq (200 mCi) of (177)Lu-labeled PSMA-617 was prepared by incubating 100 µg of PSMA-617 with (177)LuCl3 at 95 °C for 15 minutes. Radiochemical purity as well as in-vitro stability of the preparation was determined by PC and HPLC methods. The pharmacokinetic behavior and in-vivo distribution of the agent were studied by carrying out biodistribution studies in normal male Wistar rats. Preliminary clinical investigation was performed in 7 patients suffering from prostate cancer. RESULTS: The complex was prepared with >98% radiochemical purity under the optimized reaction protocols and the preparation exhibited adequate in-vitro stability. Biodistribution studies revealed no significant uptake in any of the major organ/tissue along with major clearance through renal pathway. Clinical studies showed similar distribution in lesions and physiologic areas of uptake as seen in diagnostic (68)Ga-PSMA-11 PET scans performed earlier. CONCLUSION: Preliminary clinical studies indicated the promising potential of the agent for theranostic applications. However, further investigations in large pool of patients are warranted to establish the theranostic potential of the agent.