Somatostatin receptor targeted liposomes with Diacerein inhibit IL-6 for breast cancer therapy.

Selective targeting to the tumor niche remains a major challenge in successful cancer therapy. Somatostatin receptor 2 (SSTR2) is overexpressed in breast cancer cells thus making this receptor an attractive target for selective guidance of ligand-conjugated drug liposomes to the tumor site. In this study, a synthetic somatostatin analogue (SST) was used as SSTR2 targeting agent and Diacerein was employed as therapeutic molecule. Diacerein loaded liposomes (DNL) were prepared and they were further decorated with the synthetic and stable analogue of somatostatin (SST-DNL). Fabricated liposomes were nano-size in range and biocompatible. SST-DNL displayed significantly better anti-tumor efficacy as compared to free Diacerein (DN) and DNL in breast cancer models. Enhanced apoptosis in breast cancer cells was detected in SST-DNL treated groups as monitored by cell cycle analysis and changes in expression level of apoptotic/anti-apoptotic proteins Bcl-2, Bax, cleaved Caspase 3 and PARP. SST-DNL more effectively inhibited the oncogenic IL-6/IL-6R/STAT3/MAPK/Akt signalling pathways as compared to DN or DNL in cancer cells. In addition, SST-DNL effectively suppressed angiogenesis and cancer cell invasion. In vivo tumor growth in a MDA-MB-231 mouse xenograft model was significantly suppressed following SST-DNL treatment. In xenograft model, immunohistochemistry of Ki-67 and CD-31 indicated that SST-DNL improved the anti-proliferative and anti-angiogenic impacts of Diacerein. In vivo pharmacokinetic studies in rats showed enhanced circulation time in the DNL or SST-DNL treated groups as compared to free DN. Considering all of these findings, we conclude that SST-DNL provides a novel strategy with better efficacy for breast cancer therapy.

Paclitaxel-loaded solid lipid nanoparticles modified with Tyr-3-octreotide for enhanced anti-angiogenic and anti-glioma therapy.

UNLABELLED: Somatostatin receptors (SSTRs) especially subtype 2 (SSTR2) are overexpressed in glioma. By taking advantage of the specific expression of SSTR2 on both glioma neovasculature endothelial cells and glioma cells, we constructed Tyr-3-octreotide (TOC)-modified solid lipid nanoparticles (SLN) loaded with paclitaxel (PTX) to enable tumor neovasculature and tumor cells dual-targeting chemotherapy. In this work, a TOC-polyethylene glycol-lipid (TOC-PEG-lipid) was successfully synthesized and used as a targeting molecule to enhance anticancer efficacy of PTX loaded sterically stabilized lipid nanoparticles. The prepared PTX-loaded SLN modified with TOC (PSM) was characterized by standard methods. In rat C6 glioma cells, PSM improved PTX induced apoptosis. Both tube formation assay and CD31 staining of treated orthotopic glioma tissues confirmed that PSM significantly improved the antiangiogenic ability of PTX in vitro and in vivo, respectively. Radiolabelled PSM achieved a much higher and specific accumulation within the glioma as suggested by the biodistribution and imaging studies. Furthermore, PSM exhibited improved anti-glioma efficacy over unmodified nanoparticles and Taxol in both subcutaneous and orthotopic tumor models. These findings collectively indicate that PSM holds great potential in improving the efficacy of anti-glioma therapy. STATEMENT OF SIGNIFICANCE: Somatostatin receptors (SSTRs) especially subtype 2 (SSTR2) are overexpressed in various mammalian cancer cells. Proliferating endothelial cells of neovasculature also express SSTR2. Tyr-3-octreotide (TOC) is a known ligand for SSTR2. We have successfully prepared paclitaxel-loaded solid lipid nanoparticles modified with TOC (PSM) having diameter less than 100nm. We found that PSM improved anti-cancer efficacy of paclitaxel in SSTR2 positive glioma of rats. This improved anti-glioma efficiency of PSM can be attributed to dual-targeting (i.e. tumor cell and neovasculature targeting) efficiency of PSM and promoted anti-cancer drug accumulation at tumor site due to TOC modification of solid lipid nanoparticles. This particular study aims at widening the scope of octreotide-derivative modified nanocarrier by exploring dual-targeting potential of PSM.

Radiolabeled new somatostatin analogs conjugated to DOMA chelator used as targeted tumor imaging agent: synthesis and radiobiological evaluation.

Several receptor-specific radiopeptides have been developed and effective in the diagnosis of malignant diseases. Among them, somatostatin receptor (SSTR) scintigraphy with (111)In-DTPA-octreotide has become a tumor diagnostic radiopharmaceutical in nuclear medicine. However, it suffers some drawbacks concerning the imaging properties and elevated radiation burden of (111)In. Here, we report the synthesis of radiolabeled two new octapeptides with improved uptake in SSTR2-positive tumors in comparison with (99m)Tc-HYNIC-Tyr(3)-octreotide (HYNIC-TOC). Octapeptides were synthesized in high yield by Fmoc solid-phase synthesis and coupling the macrocyclic chelator DOMA(1,4,7-Tri-Boc-10-(carboxymethyl)-1,4,7,10-tetraazocyclododecane-1-yl-monoacetic acid) to these peptides for (99m)Tc labeling. New peptides DOMA-Asn(3)-octreotate(DOMA-AATE) and DOMA-Pro(3)-octreotate(DOMA-PATE) were purified, characterized by RP-HPLC, MALDI-mass, (1)H-NMR, (13)C-NMR. Labeling was performed by SnCl2 method to get products with excellent radiochemical purity (97 %). Radiopeptides were found to be substantially stable under physiological condition for 24 h. Internalization and receptor-binding studies were determined in somatostatin receptor-expressing C6-glioma cell line and rat brain cortex membrane and the results compared with HYNIC-TOC as standard. The IC50 values of (99m)Tc-DOMA-AATE(1.10 ± 0.48 nM) and (99m)Tc-DOMA-PATE(1.76 ± 0.06 nM) showed high affinity binding for SSTR2 receptor and they internalized rapidly in C6 cells. Biodistribution and imaging studies were performed in C6 tumor-bearing rat under gamma camera showing significant uptake in kidney, urine and C6 tumor. Radiopeptides exhibited fast blood clearance and rapid elimination through the urinary systems. However, (99m)Tc-DOMA-AATE exhibited the highest tumor to muscle and tumor to blood uptake ratios among three. These favorable characteristics validate (99m)Tc-DOMA-AATE as a more promising (99m)Tc-radiotracer than (99m)Tc-DOMA-PATE, (99m)Tc-HYNIC-TOC for SSTR2-positive tumor scintigraphy.

Synthesis, characterization, conformational analysis of a cyclic conjugated octreotate peptide and biological evaluation of (99m)Tc-HYNIC-His(3)-Octreotate as novel tracer for the imaging of somatostatin receptor-positive tumors.

Peptides are attracting increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. We therefore synthesized new cyclic octapeptides conjugated with HYNIC by Fmoc solid-phase peptide synthesis. These were purified and analyzed by RP-HPLC, MALDI mass, (1)H NMR, (13)C NMR, HSQC, HMBC, COSY and IR spectroscopy. Conformational analysis of the peptides was performed by circular dichroism spectroscopy, in pure water and trifluoroethanol-water (1:1), revealed the presence of strong secondary structural features like ß-sheet and random coils. Labeling was performed with (99m)Tc using Tricine and EDDA as coligands by SnCl(2) method to get products with excellent radiochemical purity >99.5 %. Metabolic stability analysis did not show any evidence of breaking of the labeled compounds and formation of free (99m)Tc. Internalization studies were done and IC(50) values were determined in somatostatin receptor-expressing C6 glioma cell line and rat brain cortex membrane, and the results compared with HYNIC-TOC as standard. The IC(50) values of (99m)Tc-HYNIC-His(3)-Octreotate (21 ± 0.93 nM) and (99m)Tc-HYNIC-TOC (2.87 ± 0.41 nM) proved to be comparable. Biodistribution and image study on normal rat under gamma camera showed very high uptake in kidney and urine, indicating kidney as primary organ for metabolism and route of excretion. Biodistribution and image study on rats bearing C6 glioma tumor found high uptake in tumor (1.27 ± 0.15) and pancreas (1.71 ± 0.03). Using these findings, new derivatives can be prepared to develop (99m)Tc radiopharmaceuticals for imaging somatostatin receptor-positive tumors.

Synthesis, radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor-positive tumor scintigraphy.

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6-hydrazinopyridine-3-carboxylic acid-Ser3-octreotate (HYNIC-SATE) radiolabeled with 99mTc, using ethylenediamine-N,N'-diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)-positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of 99mTc-HYNIC-SATE was studied in SSTR2-expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague-Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor-bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC-SATE IC50=1.60±0.05 n m) and specific internalization into rat C6 cells. After administration of 99mTc-HYNIC-SATE in C6 glioma-bearing rats, a receptor specific uptake of radioactivity was observed in SSTR-positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. 99mTc-HYNIC-SATE is a new receptor-specific radiopeptide for targeting SSTR2-positive brain tumor and might be of great promise in the scintigraphy of SSTR2-positive tumors.

Assessment of the effect of Bacopa monnieri (L.) Wettst. extract on the labeling of blood elements with technetium-99m and on the morphology of red blood cells / Avaliação do efeito do extrato de Bacopa monnieri (L.) Wettst. na marcação de elementos sanguíneos com tecnécio-99m e na morfologia de células vermelhas do sangue

Bacopa monnieri (L.) Wettst. (BM), a traditional Ayurvedic medicine, used for centuries as a memory enhancing, anti-inflammatory, antipyretic, sedative and antiepileptic agent. BM extract have been extensively investigated by several authors for their neuropharmacological effects. In nuclear medicine, red blood cells (RBC) labeled with technetium-99m (99mTc) have several clinical applications. However, data have demonstrated that synthetic or natural drugs could modify the labeling of RBC with 99mTc. As Bacopa monnieri is extensively used in medicine, we evaluated its influence on the labeling of RBC and plasma proteins using technetium-99m (99mTc). This labeling procedure depends on a reducing agent and usually stannous chloride is used. Blood was incubated with BM extracts. Stannous chloride solution and 99mTc were added. Blood was centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P or BC were also precipitated, centrifuged and insoluble fraction (IF) and soluble fraction (SF) were separated. The percentage of radioactivity ( percentATI) in BC, IF-BC and IF-P were calculated. The percentATI significantly decreased on BC from 95.53±0.45 to 35.41±0.44, on IF-P from 80.20±1.16 to 7.40±0.69 and on IF-BC from 73.31±1.76 to 21.26±1.40. The morphology study of RBC revealed important morphological alterations due to treatment with BM extracts. We suggest that the BM extract effect could be explained by an inhibition of the stannous and pertechnetate ions or oxidation of the stannous ion or by damages induced in the plasma membrane.

Bacopa monnieri (L.) Wettst. (BM), uma planta tradicional da medicina ayurvédica, é usada por séculos para problemas de memória, antiinflamatória, antitérmica, sedativa e como agente anti-epiléptico. O extrato BM têm sido extensivamente investigada por diversos autores por seus efeitos neurofarmacológicas. Na medicina nuclear, os glóbulos vermelhos (RBC) marcados com tecnécio-99m (99mTc) tem várias aplicações clínicas. Entretanto, os dados demonstraram que drogas sintéticas ou naturais podem modificar a marcação de hemácias com 99mTc. Como Bacopa monnieri é amplamente utilizado em medicina, foi avaliada a sua influência na marcação de hemácias e proteínas plasmáticas com tecnécio-99m (99mTc). Este procedimento de marcação depende de um agente redutor e normalmente o cloreto estanoso é usado. Sangue foi incubado com os extratos BM. Solução de cloreto estanoso e 99mTc foram adicionados. O sangue foi centrifugado e o plasma (P) e células sangüíneas (CS) foram isolados. Amostras de P ou BC também foram precipitadas, centrifugadas e fração insolúvel (FI) e fração solúvel (FS) foram separadas. A porcentagem de radioatividade ( por centoATI) em BC, IF-BC e SE-P foram calculados. A As por cento ATI diminuiu significativamente em BC de 95,53±0,45-35,41±0,44, no IF-P de 80,20±1,16 para 7,40±0,69 e no IF-BC em 73,31±1,76-21,26±1,40. O estudo da morfologia de hemácias revelou alterações morfológicas importantes devido a tratamentos com extratos BM. Sugere-se que a ação do extrato BM poderia ser explicada por uma inibição dos íons estanoso e pertecnetato ou oxidação do íon estanoso ou por danos na membrana plasmática.

Synthesis, radiolabeling and biological evaluation of a neutral tripeptide and its derivatives for potential nuclear medicine applications.

Peptides are important regulators of growth and cellular functions not only in normal tissue but also in tumors. So they are becoming radioligands of increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. So development of new peptide radiopharmaceuticals is becoming one of the most important areas in nuclear medicine research. A small tripeptide derivative NH(2)PhePheCys was synthesized by Fmoc solid phase peptide synthesis using an automated synthesizer. The oxidized form, i.e. NH(2)PhePheCysCysPhePheNH(2,) was also prepared by iodine oxidation method from NH(2)PhePheCys(ACM). The ligands were analyzed by HPLC and mass spectroscopy. They were radiolabeled with (99m)Tc using SnCl(2). In vitro analytical studies and biological characterizations were performed using the peptide radiopharmaceuticals. Images taken under gamma camera showed very high uptake in the liver, lung and spleen. Significant uptake was also observed in bone marrow and brain for (99m)Tc-NH(2)PhePheCys. Metabolites were produced in vivo when the radiopharmaceuticals were injected intravenously and were identified from rat brain and liver homogenate studies. Clearance through kidney did not show any evidence of breaking of the labeled compounds and formation of free (99m)Tc. Radiopharmaceuticals prepared using tripeptide and hexapeptide ligands were transported into the brain through blood brain barrier depending on the size and sequence characteristics. Using this property of peptide new derivatives can be prepared to develop (99m)Tc radiopharmaceuticals for imaging normal brain tissues as well as for diagnosing various brain disorders.

Comparative evaluation of Tc-99m cystine and Tc-99m MAG3 in normals and patients with renal functional impairment.

Tc-99m cystine has been proved to be a good renal agent in animals for morphologic as well as the functional status of the kidney. In this study, we compared Tc-99m cystine with Tc-99m mercaptoacetyltriglycine, which is used for evaluation of renal function in normal patients, and those with various degrees of renal functional impairment. The clearance values and static images are compared with Tc-99m mercaptoacetyltriglycine. The results show that Tc-99m cystine has good radiopharmaceutical characteristics suitable for evaluation of both renal function as well as morphology.