RESUMO
Recently, we reported a new fibroblast activation protein (FAP) inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-HYNIC-D-Alanine-BoroPro)(99mTc-HYNIC-iFAP) structure for tumor microenvironment SPECT imaging. This research aimed to synthesize 68Ga-[2,2',2â³,2â´-(2-(4-(2-(5-(((S)-1-((S)-2-boronopyrrolidin-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carbothioamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid] (68Ga-DOTA-D-Alanine-BoroPro)(68Ga-iFAP) as a novel radiotracer for PET imaging and evaluate its usefulness for FAP expression in malignant and non-malignant tissues. The coupling of p-SCN-benzene DOTA with HYNIC-iFAP was used for the chemical synthesis and further labeling with 68Ga. Radiochemical purity was verified by radio-HPLC. The specificity of 68Ga-iFAP was evaluated in HCT116 cells, in which FAP expression was verified by immunofluorescence and Western blot. Biodistribution and biokinetic studies were performed in murine models. 68Ga-iFAP uptake at the myocardial level was assessed in mice with induced infarction. First-in-human images of 68Ga-iFAP in healthy subjects and patients with myocardial infarction, glioblastoma, prostate cancer, and breast cancer were also obtained. DOTA-D-Alanine BoroPro was prepared with a chemical purity of 98% and was characterized by UPLC mass spectroscopy, FT-IR, and UV-vis. The 68Ga-iFAP was obtained with a radiochemical purity of >95%. In vitro and in vivo studies demonstrated 68Ga-iFAP-specific recognition for FAP, rapid renal elimination, and adequate visualization of the glioblastoma, breast tumor, prostate cancer, and myocardial infarction sites. The results of this research justify further dosimetry and clinical trials to establish the specificity and sensitivity of 68Ga-iFAP PET for FAP expression imaging.
RESUMO
Small interfering RNAs (siRNAs) have the ability to induce selective gene silencing, although siRNAs are vulnerable to degradation in vivo. Various active pharmaceutical ingredients (APIs) are currently used as effective therapeutics in the treatment of cancer. However, routes of administration are limited due to their physicochemical and biopharmaceutical properties. This research aimed to develop oral pharmaceutical formulations based on self-nanoemulsifying drug delivery systems (SNEDDS) for optimal transport and co-delivery of siRNAs related to cancer and APIs. Formulations were developed using optimal mixing design (Design-Expert 11 software) for SNEDDS loading with siRNA (water/oil emulsion), API (oil/water emulsion), and siRNA-API (multiphase water/oil/water emulsion). The final formulations were characterized physicochemically and biologically. The nanosystems less than 50 nm in size had a drug loading above 48 %. The highest drug release occurred at intestinal pH, allowing drug protection in physiological fluids. SNEDDS-siRNA-APIs showed a twofold toxicity effect than APIs in solution and higher transfection and internalization of siRNA in cancer cells with respect to free siRNAs. In the duodenum, higher permeability was observed with SNEDDS-API than with the API solution, as determined by ex-vivo fluorescence microscopy. The multifunctional formulation based on SNEDDS was successfully prepared, siRNA, hydrophobic chemotherapeutics (doxorubicin, valrubicin and methotrexate) and photosensitizers (rhodamine b and protoporphyrin IX) agents were loaded, using a chitosan-RNA core, and Labrafil® M 1944 CS, Cremophor® RH40, phosphatidylcholine shell, forming stable hybrid SNEDDS as multiphasic emulsion, suitable as co-delivery system with a potent anticancer activity.
Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos , Emulsões , RNA Interferente Pequeno , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Humanos , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Animais , Linhagem Celular Tumoral , Liberação Controlada de FármacosRESUMO
The development of rHDL-radionuclide theragnostic systems requires evaluation of the absorbed doses that would be produced in healthy tissues and organs at risk. Technetium-99m is the most widely used radionuclide for diagnostic imaging, therefore, the design of theragnostic reconstituted high density-lipoprotein (rHDL) nanosystems labeled with Technetium-99m offers multiple possibilities. OBJECTIVE: To determine the biokinetics, radiopharmacokinetics and estimate the absorbed doses induced in healthy organs by Technetium-99m transported in the core and on the surface of rHDL. METHODS: Biokinetic and radiopharmacokinetic models of rHDL/[99mTc]Tc-HYNIC-DA (Technetium-99m in the core) and [99mTc]Tc-HYNIC-rHDL (Technetium-99m on the surface) were calculated from their ex vivo biodistribution in healthy mice. Absorbed doses were estimated by the MIRD formalism using OLINDA/EXM and LMFIT softwares. RESULTS: rHDL/[99mTc]Tc-HYNIC-DA and [99mTc]Tc-HYNIC-rHDL show instantaneous absorption in kidney, lung, heart and pancreas, with slower absorption in spleen. rHDL/[99mTc]Tc-HYNIC-DA is absorbed more slowly in the intestine, while [99mTc]Tc-HYNIC-rHDL is absorbed more slowly in the liver. The main target organ for rHDL/[99mTc]Tc-HYNIC-DA, which is hydrophobic in nature, is the liver, whereas the kidney is for the more hydrophilic [99mTc]Tc-HYNIC-rHDL. Assuming that 925 MBq (25 mCi) of Technetium-99m, carried in the core or on the surface of rHDL, are administered, the maximum tolerated doses for the organs of greatest accumulation are not exceeded. CONCLUSION: Theragnostic systems based on 99mTc-labeled rHDL are safe from the dosimetric point of view. The dose estimates obtained can be used to adjust the 99mTc-activity to be administered in future clinical trials.
Assuntos
Nanopartículas , Tecnécio , Camundongos , Animais , Lipoproteínas HDL , Distribuição Tecidual , Radiometria/métodosRESUMO
The fibroblast activation protein (FAP) is heavily expressed in fibroblasts associated with the tumor microenvironment, while the prostate-specific membrane antigen (PSMA) is expressed in the neovasculature of malignant angiogenic processes. Previously, we reported that [177Lu]lutetium sesquioxide-iFAP/iPSMA nanoparticles ([177Lu]Lu-iFAP/iPSMA) inhibit HCT116 tumor progression in mice. Understanding the toxicity of [177Lu]Lu-iFAP/iPSMA in healthy tissues, as well as at the tissue and cellular level in pathological settings, is essential to demonstrate the nanosystem safety for treating patients. It is equally important to demonstrate that [177Lu]Lu-iFAP/iPSMA can be prepared under good manufacturing practices (GMP) with reproducible pharmaceutical-grade quality characteristics. This research aimed to prepare [177Lu]Lu-iFAP/iPSMA under GMP-compliant radiopharmaceutical processes and evaluate its toxicity in cell cultures and murine biological systems under pathological environments. [177Lu]Lu2O3 nanoparticles were formulated as radiocolloidal solutions with FAP and PSMA inhibitor ligands (iFAP and iPSMA), sodium citrate, and gelatin, followed by heating at 121 °C (103-kPa pressure) for 15 min. Three consecutive batches were manufactured. The final product was analyzed according to conventional pharmacopeial methods. The Lu content in the formulations was determined by X-ray fluorescence. [177Lu]Lu-iFAP/iPSMA performance in cancer cells was evaluated in vitro by immunofluorescence. Histopathological toxicity in healthy and tumor tissues was assessed in HCT116 tumor-bearing mice. Immunohistochemical assays were performed to corroborate FAP and PSMA tumor expression. Acute genotoxicity was evaluated using the micronuclei assay. The results showed that the batches manufactured under GMP conditions were reproducible. Radiocolloidal solutions were sterile and free of bacterial endotoxins, with radionuclidic and radiochemical purity greater than 99%. The lutetium content was 0.10 ± 0.02 mg/mL (0.9 GBq/mg). Significant inhibition of cell proliferation in vitro and in tumors was observed due to the accumulation of nanoparticles in the fibroblasts (FAP+) and neovasculature (PSMA+) of the tumor microenvironment. No histopathological damage was detected in healthy tissues. The data obtained in this research provide new evidence on the selective toxicity to malignant tumors and the absence of histological changes in healthy tissues after intravenous injection of [177Lu]Lu-iFAP/iPSMA in mammalian hosts. The easy preparation under GMP conditions and the toxicity features provide the added value needed for [177Lu]Lu-iFAP/iPSMA clinical translation.
RESUMO
Reconstituted high-density lipoproteins (rHDLs) can transport and specifically release drugs and imaging agents, mediated by the Scavenger Receptor Type B1 (SR-B1) present in a wide variety of tumor cells, providing convenient platforms for developing theranostic systems. Usually, phospholipids or Apo-A1 lipoproteins on the particle surfaces are the motifs used to conjugate molecules for the multifunctional purposes of the rHDL nanoparticles. Cholesterol has been less addressed as a region to bind molecules or functional groups to the rHDL surface. To maximize the efficacy and improve the radiolabeling of rHDL theranostic systems, we synthesized compounds with bifunctional agents covalently linked to cholesterol. This strategy means that the radionuclide was bound to the surface, while the therapeutic agent was encapsulated in the lipophilic core. In this research, HYNIC-S-(CH2)3-S-Cholesterol and DOTA-benzene-p-SC-NH-(CH2)2-NH-Cholesterol derivatives were synthesized to prepare nanoparticles (NPs) of HYNIC-rHDL and DOTA-rHDL, which can subsequently be linked to radionuclides for SPECT/PET imaging or targeted radiotherapy. HYNIC is used to complexing 99mTc and DOTA for labeling molecules with 111, 113mIn, 67, 68Ga, 177Lu, 161Tb, 225Ac, and 64Cu, among others. In vitro studies showed that the NPs of HYNIC-rHDL and DOTA-rHDL maintain specific recognition by SR-B1 and the ability to internalize and release, in the cytosol of cancer cells, the molecules carried in their core. The biodistribution in mice showed a similar behavior between rHDL (without surface modification) and HYNIC-rHDL, while DOTA-rHDL exhibited a different biodistribution pattern due to the significant reduction in the lipophilicity of the modified cholesterol molecule. Both systems demonstrated characteristics for the development of suitable theranostic platforms for personalized cancer treatment.
Assuntos
Nanopartículas , Medicina de Precisão , Animais , Camundongos , Distribuição Tecidual , Benzeno , Lipoproteínas HDL/metabolismo , Nanopartículas/uso terapêutico , Colesterol/metabolismo , Lipoproteínas/metabolismo , Radioisótopos , Fosfolipídeos , Receptores Depuradores/metabolismoRESUMO
Gestational diabetes mellitus (GDM) is the most common metabolic disorder of pregnancy and has considerable short- and long-term consequences for the health of both the mother and the newborn. Within its pathophysiology, genetic, nutritional, epigenetic, immunological, and hormonal components have been described. Within the last two items, it is known that different hormones and cytokines secreted by adipose tissue, known collectively as adipokines, are involved in the metabolic alterations underlying GDM. Although the maternal circulating profile of adipokines in GDM has been extensively studied, and there are excellent reviews on the subject, it is in recent years that more progress has been made in the study of their expression in visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), placenta, and their concentrations in the umbilical circulation. Thus, this review compiles and organizes the most recent findings on the maternal and umbilical circulating profile and the levels of expression of adipokines in VAT, SAT, and placenta in GDM.
Assuntos
Diabetes Gestacional , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Humanos , Recém-Nascido , Gordura Intra-Abdominal/metabolismo , Gravidez , Gordura Subcutânea/metabolismoRESUMO
Although liposomal doxorubicin (LPD) is widely used for cancer treatment, knowledge concerning the toxicity induced by this drug in healthy organs and tissues is limited. LPD-induced toxicity studies relative to free doxorubicin (DOX) have focused on cardiotoxicity in tumor-bearing animals. On the other hand, the results on DOX-induced cardiotoxicity depending on gender are controversial. One of the manifestations of toxicity is tissue inflammation. 67Ga-citrate has been used for decades to assess inflammation in various pathologies. In this work, the ex vivo biodistribution of 67Ga-citrate is used to evaluate induced multi-organ toxicity in healthy 10-week-old male and female CD1 mice treated for 5 weeks with LPD. Toxicity in males, determined by 67Ga-citrate, was evident only in the target organs of liposomes (spleen, liver, kidneys, and lungs); the average weight loss was 11% and mortality was 14%. In female mice, 67Ga-citrate revealed a cytotoxic effect in practically all organs, the average weight loss was 37%, and the mortality after the last dose of LPD was 66%. These results confirm the usefulness of 67Ga-citrate and the importance of stratifying by sex in the toxicological evaluation of drugs.
Assuntos
Antibióticos Antineoplásicos , Cardiotoxicidade , Animais , Antibióticos Antineoplásicos/toxicidade , Cardiotoxicidade/tratamento farmacológico , Ácido Cítrico/toxicidade , Doxorrubicina/análogos & derivados , Doxorrubicina/toxicidade , Feminino , Inflamação , Lipossomos/farmacologia , Masculino , Camundongos , Polietilenoglicóis , Distribuição Tecidual , Redução de PesoRESUMO
Actinium-225 and other alpha-particle-emitting radionuclides have shown high potential for cancer treatment. Reconstituted high-density lipoproteins (rHDL) specifically recognize the scavenger receptor B type I (SR-BI) overexpressed in several types of cancer cells. Furthermore, after rHDL-SR-BI recognition, the rHDL content is injected into the cell cytoplasm. This research aimed to prepare a targeted 225Ac-delivering nanosystem by encapsulating the radionuclide into rHDL nanoparticles. The synthesis of rHDL was performed in two steps using the microfluidic synthesis method for the subsequent encapsulation of 225Ac, previously complexed to a lipophilic molecule (225Ac-DOTA-benzene-p-SCN, CLog P = 3.42). The nanosystem (13 nm particle size) showed a radiochemical purity higher than 99% and stability in human serum. In vitro studies in HEP-G2 and PC-3 cancer cells (SR-BI positive) demonstrated that 225Ac was successfully internalized into the cytoplasm of cells, delivering high radiation doses to cell nuclei (107 Gy to PC-3 and 161 Gy to HEP-G2 nuclei at 24 h), resulting in a significant decrease in cell viability down to 3.22 ± 0.72% for the PC-3 and to 1.79 ± 0.23% for HEP-G2 at 192 h after 225Ac-rHDL treatment. After intratumoral 225Ac-rHDL administration in mice bearing HEP-G2 tumors, the biokinetic profile showed significant retention of radioactivity in the tumor masses (90.16 ± 2.52% of the injected activity), which generated ablative radiation doses (649 Gy/MBq). The results demonstrated adequate properties of rHDL as a stable carrier for selective deposition of 225Ac within cancer cells overexpressing SR-BI. The results obtained in this research justify further preclinical studies, designed to evaluate the therapeutic efficacy of the 225Ac-rHDL system for targeted alpha-particle therapy of tumors that overexpress the SR-BI receptor.
Assuntos
Nanopartículas , Neoplasias , Partículas alfa/uso terapêutico , Animais , Lipoproteínas HDL/química , Camundongos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Receptores DepuradoresRESUMO
Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.
Assuntos
Endopeptidases/metabolismo , Neoplasias Hepáticas Experimentais , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Tecnécio , Animais , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Compostos de Organotecnécio/farmacologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Tecnécio/química , Tecnécio/farmacocinética , Tecnécio/farmacologiaRESUMO
Reconstituted high-density lipoprotein (rHDL) nanoparticles are excellent transporters of molecules and very useful for targeted therapy as they specifically recognize the scavenger receptor, class B1 (SR-B1) that is present on the surface of a wide range of tumor cells. However, they have rarely been employed to transport photosensitizers (PS) for photodynamic therapy (PDT). Rhodamine (R) compounds have been dismissed as useful PSs for PDT due to their low 1O2 production, excitation wavelengths with little tissue penetration, and poor selectivity for tumor cells. It was recently demonstrated that when irradiating at 532 nm or with Cerenkov radiation (CR) from a ß-emitting radionuclide, R123, R6G, and RB undergo electron transfer reactions (type I reaction) with folic acid. R6G also produces type I reactions with O2. In this work, the photodynamic effects of the rHDL-R system were evaluated in vitro. rHDL nanoparticles loaded with R123, R6G, and RB were synthesized, and the PS was internalized into T47D tumor cells. When cells were irradiated with a 532-nm laser in the presence of an rHDL-R systems, a cytotoxic photodynamic effect was obtained in the order R6G > R123 > RB. In the presence of CR from a 177Lu source, cytotoxicity showed the order R6G > RB > R123. The higher cytotoxicity induced by R6G in both cases corresponds to higher cellular internalization and larger production of type I and II reactions. Thus, in this work, it is proposed that rHDL-R/177Lu system can be applied in theragnostics as a multimodal radiotherapy-PDT-imaging system (imaging by SPECT or Cerenkov) and in hypoxic solid tumors in which external radiation is not effective and 177Lu-CR acts as light source.