RESUMEN
Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.
Asunto(s)
Aciltransferasas/genética , Genes Supresores de Tumor , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de la Próstata/genética , Neoplasias Testiculares/genética , Aciltransferasas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Eliminación de Gen , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de Células Germinales y Embrionarias/enzimología , Neoplasias de Células Germinales y Embrionarias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/prevención & control , Interferencia de ARN , Neoplasias Testiculares/enzimología , Neoplasias Testiculares/patología , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
Carbon nanotubes (CNTs) have been proposed as one of the most promising nanomaterials to be used in biomedicine for their applications in drug/gene delivery as well as biomedical imaging. The present study developed radio-labeled iron oxide decorated multi-walled CNTs (MWNT) as dual magnetic resonance (MR) and single photon emission computed tomography (SPECT) imaging agents. Hybrids containing different amounts of iron oxide were synthesized by in situ generation. Physicochemical characterisations revealed the presence of superparamagnetic iron oxide nanoparticles (SPION) granted the magnetic properties of the hybrids. Further comprehensive examinations including high resolution transmission electron microscopy (HRTEM), fast Fourier transform simulations (FFT), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) assured the conformation of prepared SPION as γ-Fe2O3. High r2 relaxivities were obtained in both phantom and in vivo MRI compared to the clinically approved SPION Endorem®. The hybrids were successfully radio-labeled with technetium-99m through a functionalized bisphosphonate and enabled SPECT/CT imaging and γ-scintigraphy to quantitatively analyze the biodistribution in mice. No abnormality was found by histological examination and the presence of SPION and MWNT were identified by Perls stain and Neutral Red stain, respectively. TEM images of liver and spleen tissues showed the co-localization of SPION and MWNT within the same intracellular vesicles, indicating the in vivo stability of the hybrids after intravenous injection. The results demonstrated the capability of the present SPION-MWNT hybrids as dual MRI and SPECT contrast agents for in vivo use.
RESUMEN
OBJECTIVES: The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. METHODS: Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. RESULTS: In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1ß in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. CONCLUSIONS: The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.
Asunto(s)
Artritis Reumatoide , Citocinas/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Inmunoterapia/métodos , Interleucina-4/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Artritis Reumatoide/tratamiento farmacológico , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID , Imagen Multimodal , Péptidos/administración & dosificación , Tomografía de Emisión de Positrones , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Tomografía Computarizada por Rayos X , Trasplante HeterólogoRESUMEN
Sunitinib is a potent and clinically approved tyrosine kinase inhibitor that can suppress tumour growth by inhibiting angiogenesis. However, conflicting data exist regarding the effects of this drug on the growth of metastases in preclinical models. Here we use 4T1 and RENCA tumour cells, which both form lung metastases in Balb/c mice, to re-address the effects of sunitinib on the progression of metastatic disease in mice. We show that treatment of mice with sunitinib prior to intravenous injection of tumour cells can promote the seeding and growth of 4T1 lung metastases, but not RENCA lung metastases, showing that this effect is cell line dependent. However, increased metastasis occurred only upon administration of a very high sunitinib dose, but not when lower, clinically relevant doses were used. Mechanistically, high dose sunitinib led to a pericyte depletion effect in the lung vasculature that correlated with increased seeding of metastasis. By administering sunitinib to mice after intravenous injection of tumour cells, we demonstrate that while sunitinib does not inhibit the growth of 4T1 lung tumour nodules, it does block the growth of RENCA lung tumour nodules. This contrasting response was correlated with increased myeloid cell recruitment and persistent vascularisation in 4T1 tumours, whereas RENCA tumours recruited less myeloid cells and were more profoundly devascularised upon sunitinib treatment. Finally, we show that progression of 4T1 tumours in sunitinib treated mice results in increased hypoxia and increased glucose metabolism in these tumours and that this is associated with a poor outcome. Taken together, these data suggest that the effects of sunitinib on tumour progression are dose-dependent and tumour model-dependent. These findings have relevance for understanding how anti-angiogenic agents may influence disease progression when used in the adjuvant or metastatic setting in cancer patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Indoles/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Pirroles/uso terapéutico , Animales , Ratones , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones , Sunitinib , Tomografía Computarizada por Rayos XRESUMEN
Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⺠T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28⺠T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.
Asunto(s)
Transformación Celular Neoplásica/genética , Multimerización de Proteína/efectos de los fármacos , Receptor ErbB-2/genética , Linfocitos T/metabolismo , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Femenino , Ingeniería Genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Interleucina-4/inmunología , Interleucina-4/metabolismo , Ratones , Ratones SCID , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunología , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The fibrous shape of carbon nanotubes (CNTs) raises concern that they may pose an asbestos-like inhalation hazard, leading to the development of diseases, especially mesothelioma. Direct instillation of long and short CNTs into the pleural cavity, the site of mesothelioma development, produced asbestos-like length-dependent responses. The response to long CNTs and long asbestos was characterized by acute inflammation, leading to progressive fibrosis on the parietal pleura, where stomata of strictly defined size limit the egress of long, but not short, fibers. This was confirmed by demonstrating clearance of short, but not long, CNT and nickel nanowires and by visualizing the migration of short CNTs from the pleural space by single-photon emission computed tomographic imaging. Our data confirm the hypothesis that, although a proportion of all deposited particles passes through the pleura, the pathogenicity of long CNTs and other fibers arises as a result of length-dependent retention at the stomata on the parietal pleura.
Asunto(s)
Progresión de la Enfermedad , Inflamación/complicaciones , Inflamación/patología , Nanotubos de Carbono/química , Pleura/patología , Cavidad Pleural/patología , Animales , Proliferación Celular , Epitelio/patología , Fibrosis , Ganglios Linfáticos/patología , Mediastino/patología , Ratones , Nanotubos de Carbono/ultraestructura , Nanocables/ultraestructura , Tamaño de la Partícula , Pleura/ultraestructura , Cavidad Pleural/ultraestructura , Factores de Tiempo , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos XRESUMEN
Functionalization of nanomaterials for precise biomedical function is an emerging trend in nanotechnology. Carbon nanotubes are attractive as multifunctional carrier systems because payload can be encapsulated in internal space whilst outer surfaces can be chemically modified. Yet, despite potential as drug delivery systems and radiotracers, such filled-and-functionalized carbon nanotubes have not been previously investigated in vivo. Here we report covalent functionalization of radionuclide-filled single-walled carbon nanotubes and their use as radioprobes. Metal halides, including Na(125)I, were sealed inside single-walled carbon nanotubes to create high-density radioemitting crystals and then surfaces of these filled-sealed nanotubes were covalently modified with biantennary carbohydrates, improving dispersibility and biocompatibility. Intravenous administration of Na(125)I-filled glyco-single-walled carbon nanotubes in mice was tracked in vivo using single-photon emission computed tomography. Specific tissue accumulation (here lung) coupled with high in vivo stability prevented leakage of radionuclide to high-affinity organs (thyroid/stomach) or excretion, and resulted in ultrasensitive imaging and delivery of unprecedented radiodose density. Nanoencapsulation of iodide within single-walled carbon nanotubes enabled its biodistribution to be completely redirected from tissue with innate affinity (thyroid) to lung. Surface functionalization of (125)I-filled single-walled carbon nanotubes offers versatility towards modulation of biodistribution of these radioemitting crystals in a manner determined by the capsule that delivers them. We envisage that organ-specific therapeutics and diagnostics can be developed on the basis of the nanocapsule model described here.
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Nanotecnología/tendencias , Nanotubos de Carbono/química , Acetilglucosamina/metabolismo , Metabolismo de los Hidratos de Carbono , Glicosilación , Humanos , Marcaje Isotópico/métodos , Microscopía Electrónica de Transmisión de Rastreo/métodos , Nanotecnología/métodos , Oxidación-Reducción , Radioisótopos/metabolismo , Radioisótopos/farmacocinética , Estómago/diagnóstico por imagen , Glándula Tiroides/diagnóstico por imagen , Distribución Tisular , Tomografía Computarizada por Rayos X/métodosRESUMEN
The integrin alphavbeta6 is expressed only on epithelia and then usually only during processes of tissue remodelling including cancer, where its high expression correlates with reduced survival. Thus, alphavbeta6 represents an important target for imaging and therapy of cancer and new molecular-specific targeting agents are required. We have developed A20FMDV2, a peptide derived from the VP1 coat protein of foot-and-mouth-disease virus that binds specifically and stably to alphavbeta6. Using a newly generated pair of isogenic human cell lines that differ only in alphavbeta6 expression, it was shown, using biodistribution and SPECT imaging, that indium-111-labelled A20FMDV2 locates specifically to alphavbeta6-expressing tissues in vivo, achieving at least seven-times higher retention in alphavbeta6-positive than in alphavbeta6-negative tumours. In further studies with MCF10.DCIS.COM and MCF10A.CA1a breast carcinoma cell lines, which express alphavbeta6 endogenously, the radiopeptide achieved similar levels of tumour retention and permitted excellent discriminatory imaging of tumours. Thus, A20FMDV2 can be used for molecular-specific targeting of alphavbeta6 for imaging in vivo the often more aggressive, alphavbeta6-positive cancers. In the future, A20FMDV2 could serve also to deliver therapy to these same cancers.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Animales , Femenino , Humanos , Radioisótopos de Indio/metabolismo , Radioisótopos de Indio/farmacocinética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Pentético/farmacocinética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacocinética , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The palliation of pain due to bone metastases using targeted compounds containing beta-emitters such as rhenium-188 ((188)Re) is an accepted and effective form of treatment. Here, we describe the efficient synthesis and preclinical evaluation of (188)Re(CO)(3)-dipicolylamine(DPA)-alendronate, a novel bifunctional bisphosphonate for the palliative treatment of bone metastases. (188)Re(CO)(3)-DPA-alendronate can be easily synthesized with high specific activities and yields (18.8 GBq/mg, radiochemical yield > or =96%) in two steps using kit-based methodology, and in contrast with the clinically approved bisphosphonate (186/188)Re-HEDP, it forms inert, single species that have been well-characterized. In vivo imaging and biodistribution studies demonstrate that (188)Re(CO)(3)-DPA-alendronate is superior to (188)Re-HEDP in targeting and accumulating in areas of high metabolic bone activity while having low soft-tissue uptake. In addition to these studies, a simple and convenient new method for purifying its precursor, fac-[(188)Re(CO)(3)(H(2)O)(3)](+), is described.
Asunto(s)
Alendronato/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias Óseas/radioterapia , Neoplasias Óseas/secundario , Difosfonatos/farmacocinética , Radiofármacos/farmacocinética , Renio/farmacocinética , Alendronato/síntesis química , Alendronato/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Difosfonatos/síntesis química , Difosfonatos/química , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Radioisótopos/química , Radioisótopos/farmacocinética , Radiofármacos/síntesis química , Radiofármacos/química , Renio/químicaRESUMEN
UNLABELLED: The aim of this study was to determine the effects of assisted coordination by amino acids such as histidine and glutamic acid on the function of (99m)Tc-labeled gastrin peptide-hydrazinonicotinamide (HYNIC) conjugates and their ability to target cholecystokinin-R in small-animal models. METHODS: Three peptide-HYNIC conjugates containing the -AYGWMDF-NH2 C-terminal sequence and combinations of histidine, glutamic acid, and glycine were synthesized, radiolabeled with (99m)Tc/(99)Tc using either tricine or ethylenediaminediacetic acid as a coligand, and analyzed by the high-performance liquid chromatography and liquid chromatography-mass spectrometric techniques. Stability, receptor binding, and internalization and in vivo targeting in AR42J-bearing mice were assessed. RESULTS: When radiolabeling was performed using tricine as a coligand, the insertion of a histidine residue near the HYNIC residue resulted in the displacement of one molecule of tricine from the coordination sphere, a reduction in the number of radiolabeled species formed, an improvement in the in vitro stability, an increase in the rate of radiopeptide internalization, and a significant improvement in tumor uptake in vivo. When radiolabeling was performed using ethylenediaminediacetic acid as a coligand, no effect on coligand binding, homogeneity, or in vitro stability was observed but a significant improvement in the internalization in vitro and tumor uptake in vivo was again found. All of the complexes formed showed similar receptor affinity in competitive radioligand binding assays. CONCLUSION: The insertion of histidine into the sequence of peptide-HYNIC conjugates can result in more stable, more homogeneous complexes that show improvements in tumor-targeting performance both in vitro and in vivo.
Asunto(s)
Aumento de la Imagen/métodos , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Radiofármacos/farmacocinética , Aminoácidos/química , Animales , Marcaje Isotópico/métodos , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Oligopéptidos/síntesis química , Oligopéptidos/farmacocinética , Especificidad de Órganos , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/farmacocinética , Compuestos de Organotecnecio/síntesis química , Compuestos de Organotecnecio/farmacocinética , Cintigrafía , Radiofármacos/síntesis química , Distribución TisularRESUMEN
Fmoc-lys(HYNIC-Boc)-OH, a precursor for solid-phase synthesis of 99mTc-labeled peptides, was synthesized efficiently without HPLC purification. HPLC-ESMS showed that deprotection and decoupling of peptide from the resin with trifluoroacetic acid gave initially HYNIC-peptide, which was trifluoroacetylated upon prolonged incubation. The trifluoroacetyl-HYNIC group was hydrolyzed during 99mTc labeling, rendering deprotection unnecessary. Trifluoroacetyl-HYNIC peptide was 99mTc-labeled as efficiently, producing the same product, as HYNIC-peptide. These modifications enhance the versatility of HYNIC for 99mTc peptide labeling.
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Fluoroacetatos , Gastrinas/síntesis química , Hidrazinas/síntesis química , Ácidos Nicotínicos/síntesis química , Oligopéptidos/síntesis química , Fragmentos de Péptidos/síntesis química , Radiofármacos/síntesis química , Tecnecio , Ácido Trifluoroacético/síntesis química , Gastrinas/química , Hidrazinas/química , Hidrólisis , Marcaje Isotópico , Ácidos Nicotínicos/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Radiofármacos/química , Ácido Trifluoroacético/químicaRESUMEN
UNLABELLED: The gastrin/cholecystokinin-2 (CCK-2) receptor has been identified as a possible target for peptide receptor radionuclide imaging and therapy. Several radiolabeled peptides binding to this receptor have been explored in animal models and clinical trials but either low tumor uptake or high renal retention has been found. The aim of this study was to identify a peptide with improved tumor-to-kidney pharmacodynamics when compared with current candidates. METHODS: A small peptide-chelator library of 34 compounds based on the C-terminal sequences of CCK-8 or minigastrin was constructed. The peptides were radiolabeled with (111)In with high labeling efficiency (>90%), as determined by high-performance liquid chromatographic analysis. The labeled peptides were screened by assessing tumor and kidney uptake in pancreatic xenograft nude mouse models, including AR42J. An extensive biodistribution analysis was performed on the lead candidate from the library. RESULTS: Minigastrin analogs containing a pentaglutamate sequence showed the highest tumor uptake but very high renal retention. CCK analogs showed the lowest tumor and renal uptake. Deletion of the pentaglutamate sequence in the gastrin analogs lowered the tumor uptake by a factor of 3 but decreased the kidney uptake by a factor of 20. Insertion of histidine residues in the sequence reduced kidney uptake by a further factor of almost 2-fold. In AR42J tumor-bearing mice, the peptide with the sequence DOTA-HHEAYGWMDF-NH(2) (DOTA is tetraazacyclododecane tetraacetic acid) showed the highest tumor-to-kidney ratio of all peptides studied, with saturable uptake in target organs and low uptake by nontarget tissues other than the kidney. CONCLUSION: This peptide is a worthwhile candidate for clinical studies to determine whether it is suitable for use in peptide receptor-targeted radionuclide therapy.
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Gastrinas/química , Neoplasias/tratamiento farmacológico , Receptores de Péptidos/química , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Humanos , Riñón/metabolismo , Masculino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Ratas , Receptor de Colecistoquinina B/químicaRESUMEN
Pancreatic cancer has a very poor prognosis with a less than 5% survival rate at 5 years. Neither external beam radiation nor chemotherapy, alone or in combination, have given encouraging results so far. A possible solution might come from the use of targeted therapy such as radioimmunotherapy. We present here the results obtained from the preclinical development of a new monoclonal antiferritin antibody (Ab), AMB8LK. Ferritin is overexpressed in pancreatic cancer and could thus be used as a target for the delivery of radioactivity at the tumour sites. The AMB8LK Ab was conjugated to three chelating agents: the 2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (PSCN-Bz-DTPA), the (R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (p5CN-Bz-CHX-A"-DTPA) and the 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (pSCN-Bz-DOTA). Radiolabelling of the three immunoconjugates with indium 111 and yttrium 90 as well as in vitro stability and immunoreactivity against pure ferritin and cells expressing ferritin were analysed. In vivo biodistribution studies were conducted on normal and on human pancreatic adenocarcinoma CAPAN-1 tumour bearing mice. These experiments demonstrated good radiolabelling (>95%), stability and immunoreactivity of the three compounds. In the biodistribution studies, differences between the three immunoconjugates were apparent in the rate of blood clearance and in tumour, liver and bone uptake. A very good pancreatic adenocarcinoma tumour targeting was observed especially with the Bz-DTPA-AMB8LK: 20% of the injected dose of the indium-labelled compound 3 days after injection; 15% of the injected dose 5 days after that of the yttrium-labelled Ab. Altogether, these results in animal models suggest that (90)Y-Bz-DTPA-AMB8LK is a good candidate for further therapeutic efficacy studies.
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Ferritinas/inmunología , Compuestos Heterocíclicos con 1 Anillo/farmacocinética , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Ácido Pentético/farmacocinética , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral , Femenino , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/uso terapéutico , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Especificidad de Órganos , Neoplasias Pancreáticas/radioterapia , Ácido Pentético/química , Ácido Pentético/uso terapéutico , Tomografía de Emisión de Positrones/métodos , Radiofármacos/química , Radiofármacos/farmacocinética , Radiofármacos/uso terapéutico , Distribución TisularRESUMEN
The specialised medical field of Nuclear Medicine is concerned with the use of unsealed sources of radioactivity either to diagnose or treat a range of diseases. In this regard it can be distinguished from the field of Radiotherapy which uses sealed radioactive sources for treatment. The range of diseases in which Nuclear Medicine plays a role is wide and includes, among others, the fields of microbiology, endocrinology, neurology, oncology and cardiovascular medicine. However, cancer probably represents the most important and growing area of application for this modality. Nuclear Medicine employs radiopharmaceuticals. These are radiolabelled ligands that have the ability to interact with molecular targets that are relevant in the aetiology or treatment of cancer and in many respects Nuclear Medicine can be considered the archetype for the application of 'Molecular Medicine'. An example of a Nuclear Medicine Positron Emission Tomography (PET) scan is shown in Fig. 2. There is great interest in developing new radioligands that allow us to image the expression of the ever increasing range of biological pathways being discovered in the post-genomic area. Designing effective radiopharmaceuticals, however, requires an understanding of a number of radiopharmaceutical sciences including aspects of chemistry, physics, cell and molecular biology, and physiology.
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Diseño de Fármacos , Marcaje Isotópico/métodos , Neoplasias/diagnóstico , Neoplasias/terapia , Radiofármacos , Animales , Humanos , Ligandos , Redes y Vías Metabólicas , Ratones , Estructura Molecular , Neoplasias/diagnóstico por imagen , Medicina Nuclear , Radiofármacos/uso terapéutico , Tomografía Computarizada de EmisiónRESUMEN
A DOTA-gastrin analogue (APH070) which, when labelled with (111)In, has high affinity for the gastrin/CCK-2 receptor (3nM) and low tumour to kidney ratio in animal models, has been formulated and manufactured for a clinical study. Oxidation of the peptide methionine residue greatly reduces receptor affinity, therefore development work focused on producing a stable intermediate drug product (iDP) whilst ensuring that the formulation, container, closure and manufacturing process did not inhibit the extremely sensitive radiolabelling reaction (itself a source of oxidation). Stress testing revealed that APH070 was stable at 2-8 degrees C at pH 6-9. Addition of an antioxidant (monothioglycerol) to the peptide formulation reduced stability when compared to buffer alone. Use of FluroTec (4023/50) stoppers (rather than FluroTec Plus (4110/40)) increased both the stability and radiolabelling efficiency of APH070. Long term stability (6 months) of the final formulation (1mg/ml APH070 in 0.01 M pH 7.2 phosphate buffer) stored at 5 degrees C in type I glass vials with FluroTec (4023/50) stoppers was 98.6+/-0.2% and 98.4+/-0.1% for upright and inverted samples, respectively. Clinical scale radiolabelling of the final formulation routinely achieves the specification of >85% (111)In-APH070 (unoxidised) stable for up to 2h after dilution with 0.9% w/v saline solution. Specific uptake of the radiopharmaceutical in CCK-2R-expressing AR42J tumours in nude mice has been demonstrated.
Asunto(s)
Embalaje de Medicamentos , Gastrinas/síntesis química , Radioisótopos de Indio , Marcaje Isotópico , Oligopéptidos/síntesis química , Radiofármacos/síntesis química , Receptor de Colecistoquinina B/metabolismo , Tecnología Farmacéutica/métodos , Animales , Línea Celular Tumoral , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Gastrinas/metabolismo , Concentración de Iones de Hidrógeno , Masculino , Metionina/química , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Oligopéptidos/metabolismo , Oxidación-Reducción , Radiofármacos/metabolismo , Reproducibilidad de los Resultados , Solubilidad , Soluciones , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: Preclinical single-photon emission computed tomography (SPECT)/CT imaging studies are hampered by low throughput, hence are found typically within small volume feasibility studies. Here, imaging and image analysis procedures are presented that allow profiling of a large volume of radiolabelled compounds within a reasonably short total study time. Particular emphasis was put on quality control (QC) and on fast and unbiased image analysis. METHODS: 2-3 His-tagged proteins were simultaneously radiolabelled by 99mTc-tricarbonyl methodology and injected intravenously (20 nmol/kg; 100 MBq; n = 3) into patient-derived xenograft (PDX) mouse models. Whole-body SPECT/CT images of 3 mice simultaneously were acquired 1, 4, and 24 h post-injection, extended to 48 h and/or by 0-2 h dynamic SPECT for pre-selected compounds. Organ uptake was quantified by automated multi-atlas and manual segmentations. Data were plotted automatically, quality controlled and stored on a collaborative image management platform. Ex vivo uptake data were collected semi-automatically and analysis performed as for imaging data. RESULTS: >500 single animal SPECT images were acquired for 25 proteins over 5 weeks, eventually generating >3500 ROI and >1000 items of tissue data. SPECT/CT images clearly visualized uptake in tumour and other tissues even at 48 h post-injection. Intersubject uptake variability was typically 13% (coefficient of variation, COV). Imaging results correlated well with ex vivo data. CONCLUSIONS: The large data set of tumour, background and systemic uptake/clearance data from 75 mice for 25 compounds allows identification of compounds of interest. The number of animals required was reduced considerably by longitudinal imaging compared to dissection experiments. All experimental work and analyses were accomplished within 3 months expected to be compatible with drug development programmes. QC along all workflow steps, blinding of the imaging contract research organization to compound properties and automation provide confidence in the data set. Additional ex vivo data were useful as a control but could be omitted from future studies in the same centre. For even larger compound libraries, radiolabelling could be expedited and the number of imaging time points adapted to increase weekly throughput. Multi-atlas segmentation could be expanded via SPECT/MRI; however, this would require an MRI-compatible mouse hotel. Finally, analysis of nuclear images of radiopharmaceuticals in clinical trials may benefit from the automated analysis procedures developed.
RESUMEN
INTRODUCTION: This study describes a novel and convenient route for the preparation of a trastuzumab-streptavidin conjugate such as might be used in a pre-targeting system and its in-vitro and in-vivo evaluation. METHODS: Trastuzumab was irradiated with UV light in the presence of stannous ions to reduce a number of the disulfide bridges to free thiol groups. A range of irradiation times were studied in order to quantify the number of thiols produced and to optimize the reduction process. The conjugate was then prepared by reaction with succinimidyl 4-(N-maleimidomethy cyclohexane)-1-carboxylate (SMCC)-linked streptavidin. RESULTS: Initial conjugation reactions in phosphate buffer were inefficient, producing low conjugate yields, but conjugation reactions in triethanolamine-based buffer showed greatly increased conjugation yields. A high purity product (approximately 100%) was obtained following purification by gel-filtration HPLC as determined by subsequent size exclusion HPLC analysis. The conjugate was shown to possess an essentially identical immunoreactivity to that of the native, unconjugated antibody and an unaltered biotin binding stoichiometry. Shedding and internalization by Her-2-expressing cells were low and the uptake in vivo by Her-2-expressing xenografts in nude mice was similar to that of labelled antibody. CONCLUSION: Our results demonstrate a new, simple and effective method for the successful synthesis of antibody-streptavidin conjugates which could also be applied to many other heterodimeric protein conjugation reactions.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Mama/metabolismo , Radioisótopos de Yodo/farmacocinética , Radioisótopos de Yodo/uso terapéutico , Estreptavidina/farmacocinética , Estreptavidina/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/radioterapia , Línea Celular Tumoral , Femenino , Humanos , Marcaje Isotópico , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Especificidad de Órganos , Radioinmunoterapia/métodos , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Radiofármacos/uso terapéutico , Distribución Tisular , TrastuzumabRESUMEN
We evaluated and compared a new bombesin analog [Tyr-Gly5, Nle(14)]-BBN(6-14) conjugated to DOTA or DTPA and radiolabeled with In-111 in low and high GRPR expressing tumor models. Both peptides were radiolabeled with high radiochemical purity and specific activity. In vitro assays on T-47D, LNCaP and PC-3 cells showed that the affinity of peptides is similar and a higher binding and internalization of DOTA-peptide to PC-3 cells was observed. Both peptides could target PC-3 and LNCaP tumors in vivo and both tumor types could be visualized by microSPECT/CT.
Asunto(s)
Bombesina/análogos & derivados , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Compuestos Heterocíclicos con 1 Anillo , Ácido Pentético/análogos & derivados , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/metabolismo , Radiofármacos , Receptores de Bombesina/metabolismo , Animales , Bombesina/química , Línea Celular Tumoral , Estabilidad de Medicamentos , Femenino , Xenoinjertos , Humanos , Técnicas In Vitro , Radioisótopos de Indio , Masculino , Ratones SCID , Trasplante de Neoplasias , Cintigrafía , Radiofármacos/química , Distribución TisularRESUMEN
UNLABELLED: In this study, a human carcinoembryonic antigen (CEA) transgenic (CEA.Tg) mouse model was evaluated for the preclinical assessment of agents directed against CEA. METHODS: Cell-type and organ-specific expression of CEA was studied in CEA.Tg mice derived from a colony that carries the complete human CEA gene together with flanking regulatory sequences and also in wild-type controls. Biodistribution studies were performed on wild-type and CEA.Tg mice by intravenous injection of 125I-labeled anti-CEA (PR1A3) or isotype control (IC) murine monoclonal antibodies (mAbs). Studies were also performed on tumor-bearing CEA.Tg and nude mice. RESULTS: As with humans, the CEA.Tg mice had low serum levels of CEA (mean, 8.8 +/- 5.52 ng/mL), and cell-surface CEA expression was primarily localized in the gastrointestinal tract. Both mAbs showed similar biodistribution patterns in the wild-type mice, whereas in the CEA.Tg mice, PR1A3 specifically localized to the CEA-expressing tissues. In the gastrointestinal tract, the percentage injected dose for PR1A3 was significantly higher than that for IC mAb at all the time points sampled. In CEA.Tg mice bearing a murine tumor transfected with human CEA, PR1A3 targeted tissues with constitutive CEA expression and was retained at the tumor site at high levels, whereas in nude mice, PR1A3 localized only to the site of the transplanted tumor. CONCLUSION: These results demonstrate the targeting potential of the anti-CEA antibody, PR1A3, and emphasize the value of using a transgenic model in preclinical studies.