RESUMO
Estradiol-BODIPY linked via an 8-carbon spacer chain and 19-nortestosterone- and testosterone-BODIPY linked via an ethynyl spacer group were evaluated for cell uptake in the breast cancer cell lines MCF-7 and MDA-MB-231 and prostate cancer cell lines PC-3 and LNCaP, as well as in normal dermal fibroblasts, using fluorescence microscopy. The highest level of internalization was observed with 11ß-OMe-estradiol-BODIPY 2 and 7α-Me-19-nortestosterone-BODIPY 4 towards cells expressing their specific receptors. Blocking experiments showed changes in non-specific cell uptake in the cancer and normal cells, which likely reflect differences in the lipophilicity of the conjugates. The internalization of the conjugates was shown to be an energy-dependent process that is likely mediated by clathrin- and caveolae-endocytosis. Studies using 2D co-cultures of cancer cells and normal fibroblasts showed that the conjugates are more selective towards cancer cells. Cell viability assays showed that the conjugates are non-toxic for cancer and/or normal cells. Visible light irradiation of cells incubated with estradiol-BODIPYs 1 and 2 and 7α-Me-19-nortestosterone-BODIPY 4 induced cell death, suggesting their potential for use as PDT agents.
Assuntos
Nandrolona , Neoplasias , Fotoquimioterapia , Corantes , Medicina de Precisão , Compostos de Boro/farmacologia , Estradiol , Fármacos Fotossensibilizantes , Linhagem Celular Tumoral , Corantes Fluorescentes/metabolismoRESUMO
After encouraging preclinical and human dosimetry results for the novel estrogen receptor (ER) PET radiotracer 4-fluoro-11ß-methoxy-16α-18F-fluoroestradiol (18F-4FMFES), a phase II clinical trial was initiated to compare the PET imaging diagnostic potential of 18F-4FMFES with that of 16α-18F-fluoroestradiol (18F-FES) in ER-positive (ER+) breast cancer patients. Methods: Patients diagnosed with ER+ breast cancer (n = 31) were recruited for this study, including 6 who underwent mastectomy or axillary node dissection. For each patient, 18F-FES and 18F-4FMFES PET/CT scans were done sequentially (within a week) and in random order. One hour after injection of either radiotracer, a head-to-thigh static scan with a 2-min acquisition per bed position was obtained. Blood samples were taken at different times after injection to assess each tracer metabolism by reverse-phase thin-layer chromatography. The SUVmean of nonspecific tissues and the SUVmax of the tumor were evaluated for each detected lesion, and tumor-to-nonspecific organ ratios were calculated. Results: Blood metabolite analysis 60 min after injection of the tracer showed a 2.5-fold increase in metabolic stability of 18F-4FMFES over 18F-FES. Although for most foci 18F-4FMFES PET had an SUVmax similar to that of 18F-FES PET, tumor contrast improved substantially in all cases. Lower uptake was consistently observed in nonspecific tissues for 18F-4FMFES, notably a 4-fold decrease in blood-pool activity as compared with 18F-FES. Consequently, image quality was considerably improved using 18F-4FMFES, with lower overall background activity. As a result, 18F-4FMFES successfully identified 9 more lesions than 18F-FES. Conclusion: This phase II study with ER+ breast cancer patients showed that 18F-4FMFES PET achieves a lower nonspecific signal and better tumor contrast than 18F-FES PET, resulting in improved diagnostic confidence and lower false-negative diagnoses.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Estradiol/análogos & derivados , Receptores de Estrogênio/metabolismo , Adulto , Idoso , Transporte Biológico , Estradiol/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Pessoa de Meia-Idade , Traçadores RadioativosRESUMO
To develop receptor based fluorescence ligands for imaging breast and prostate cancer, a series of estrogen-, testosterone- and 19-nortestosterone conjugates linked to BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) or aza-BODIPY, were prepared. Their synthesis involves attachment of iodo derivatives of differently substituted BODIPY and aza-BODIPY analogs to the C17α-position of the steroid moieties using either the Sonogashira coupling or Click reaction. The UV-Vis absorption spectra of the conjugates range from 500 to 710nm with fluorescence emission properties ranging from 520 to 700nm, facilitating observations in living cells and tissues. Selection of the site of substitution, as well as the type of substituents on the steroidal moiety and the use of different linkers, provides a library of fluorescing conjugates to explore the effect of structural modifications on biological properties.
Assuntos
Androgênios/química , Compostos de Boro/química , Compostos de Boro/síntese química , Estrogênios/química , Técnicas de Química Sintética , Radioisótopos de Flúor , Nandrolona/química , Tomografia por Emissão de Pósitrons , Análise EspectralRESUMO
In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.
Assuntos
Compostos de Boro/química , Meios de Contraste/síntese química , Receptor alfa de Estrogênio/metabolismo , Etinilestradiol/química , Neoplasias da Mama/diagnóstico por imagem , Química Click , Meios de Contraste/química , Meios de Contraste/metabolismo , Receptor alfa de Estrogênio/química , Feminino , Humanos , Concentração Inibidora 50 , Tomografia por Emissão de Pósitrons , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.
Assuntos
Anticorpos Monoclonais/farmacologia , Núcleo Celular/efeitos dos fármacos , Ácido Cólico/farmacocinética , Endossomos/efeitos dos fármacos , Imunoconjugados/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Sinais de Localização Nuclear/metabolismo , Peptídeos/metabolismoRESUMO
UNLABELLED: Cyclotron production of 99mTc is a promising route to supply 99mTc radiopharmaceuticals. Higher 99mTc yields can be obtained with medium-energy cyclotrons in comparison to those dedicated to PET isotope production. To take advantage of this capability, evaluation of the radioisotopic purity of 99mTc produced at medium energy (20-24 MeV) and its impact on image quality and dosimetry was required. METHODS: Thick 100Mo (99.03% and 99.815%) targets were irradiated with incident energies of 20, 22, and 24 MeV for 2 or 6 h. The targets were processed to recover an effective thickness corresponding to approximately 5-MeV energy loss, and the resulting sodium pertechnetate 99mTc was assayed for chemical, radiochemical, and radionuclidic purity. Radioisotopic content in final formulation was quantified using γ-ray spectrometry. The internal radiation dose for 99mTc-pertechnetate was calculated on the basis of experimentally measured values and biokinetic data in humans. Planar and SPECT imaging were performed using thin capillary and water-filled Jaszczak phantoms. RESULTS: Extracted sodium pertechnetate 99mTc met all provisional quality standards. The formulated solution for injection had a pH of 5.0-5.5, contained greater than 98% of radioactivity in the form of pertechnetate ion, and was stable for at least 24 h after formulation. Radioisotopic purity of 99mTc produced with 99.03% enriched 100Mo was greater than 99.0% decay corrected to the end of bombardment (EOB). The radioisotopic purity of 99mTc produced with 99.815% enriched 100Mo was 99.98% or greater (decay corrected to the EOB). The estimated dose increase relative to 99mTc without any radionuclidic impurities was below 10% for sodium pertechnetate 99mTc produced from 99.03% 100Mo if injected up to 6 h after the EOB. For 99.815% 100Mo, the increase in effective dose was less than 2% at 6 h after the EOB and less than 4% at 15 h after the EOB when the target was irradiated at an incident energy of 24 MeV. Image spatial resolution and contrast with cyclotron-produced 99mTc were equivalent to those obtained with 99mTc eluted from a conventional generator. CONCLUSION: Clinical-grade sodium pertechnetate 99mTc was produced with a cyclotron at medium energies. Quality control procedures and release specifications were drafted as part of a clinical trial application that received approval from Health Canada. The results of this work are intended to contribute to establishing a regulatory framework for using cyclotron-produced 99mTc in routine clinical practice.
Assuntos
Ciclotrons , Radioquímica/métodos , Compostos Radiofarmacêuticos/química , Pertecnetato Tc 99m de Sódio/isolamento & purificação , Contaminação de Medicamentos , Isótopos , Molibdênio , Imagens de Fantasmas , Tomografia por Emissão de Pósitrons , Controle de Qualidade , Doses de Radiação , Compostos Radiofarmacêuticos/farmacocinética , Pertecnetato Tc 99m de Sódio/química , Pertecnetato Tc 99m de Sódio/farmacocinéticaRESUMO
PURPOSE: To monitor a real-time follow-up of tumor response to photodynamic therapy (PDT) by dynamic 2-deoxy-2-[(18)F]fluoro-d-glucose ((18)FDG) and positron emission tomography (PET) using two photosensitizing drugs in vivo, and to assess their mechanisms of action. METHODS: Two types of photosensitizers with different action mechanisms were used in rats implanted with two tumors: AlPcS4 mainly affecting the tumor vascular system, and ZnPcS2 largely inducing direct cell kill. Twenty-four hours after administration of either photosensitizer, one tumor served as control while the other was treated with red light during 30min within the 2h PET imaging by infusion of (18)FDG. The usual two-tissue compartment kinetic model was modified to take into account the perturbation of the treatment during imaging. RESULTS: The illumination of the tumors during PET imaging provoked a net decrease of (18)FDG uptake in tumors treated with AlPcS4 and a near total absence of (18)FDG uptake in tumors treated with ZnPcS2. After the end of illumination, the tumors regained (18)FDG uptake with a more pronounced uptake in the tumors treated with ZnPcS2. The rate constant values of the new (18)FDG kinetic model reflected the response of the tumors to the treatment in both photosensitizers. CONCLUSIONS: Dynamic PET imaging can be used to quantitatively assess in vivo and in real-time the response of tumors to treatments. It is demonstrated that the 30min of treatment was not sufficient to reduce the activity of the tumors. The technique could be extended to directly monitor the effects of drugs in vivo.
Assuntos
Indóis/farmacologia , Neoplasias/tratamento farmacológico , Compostos Organometálicos/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Tomografia por Emissão de Pósitrons , Animais , Modelos Animais de Doenças , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/farmacocinética , RatosRESUMO
Dynamic positron emission tomography (PET), combined with constant infusion of 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG), enables real-time monitoring of transient metabolic changes in vivo, which can serve to understand the underlying physiology. Here we investigated characteristic changes in the tumour FDG-uptake profiles in relation to acute localized inflammatory responses induced by photodynamic therapy (PDT). Dynamic PET imaging with constant FDG infusion was used with EMT-6 tumour bearing mice. FDG time-activity uptake curves were measured simultaneously, in treated and reference tumours, for 3 hours, before, during and after PDT light treatment. Inflammation was studied when evoked, either by PDT using a trisulfonated porphyrazine photosensitizer, or lipopolysaccharide (LPS), and inhibited using indomethacin. The distinct transient patterns, characterized by drops and subsequent recovery of tumour FDG uptake rates, were also analysed using immunohistochemical markers for apoptosis, necrosis, and inflammation. Typical profiles for tumour FDG-uptake, consisted of a drop during PDT, followed by a gradual recovery period. Tumours treated with LPS, but not with light, showed a continuous increase in FDG-uptake during the 3 h experimental period. Treatment with indomethacin, inhibited the rise in FDG-uptake observed with either LPS or PDT. Tumour FDG-uptake profiles correlated with necrosis markers during PDT, and inflammatory response markers post-PDT, but not with an apoptosis marker at any time during or after PDT. Dynamic FDG-PET imaging combined with indomethacin reveals that, the drop in the tumour FDG-uptake rate during the PDT illumination phase reflects vascular collapse and necrosis, while the increased tumour FDG-uptake rate immediately post-illumination involves an acute localized inflammatory response. Dynamic FDG infusion and PET imaging, combined with the use of selective inhibitors, provides unique insight for deciphering the complex underlying processes leading to tumour response in PDT, and allows for rapid as well as cost effective optimization of PDT protocols.
Assuntos
Fluordesoxiglucose F18 , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Fotoquimioterapia/efeitos adversos , Tomografia por Emissão de Pósitrons , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Fluordesoxiglucose F18/metabolismo , Indometacina/farmacologia , Inflamação/diagnóstico por imagem , Inflamação/etiologia , Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fatores de TempoRESUMO
In this paper, we describe the synthesis and characterization of a series of new bimodal probes combining water-soluble sulfonated zinc phthalocyanine (ZnPc) as a fluorescence imaging unit and either (68)Ga/1,4,7,10-tetraazocyclododecane-N,N'Nâ³,N'â³-tetraacetic acid (DOTA) or (64)Cu/1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for PET imaging. The two moieties were linked through aliphatic chains of different lengths to modulate amphiphilicity. Labeling of DOTA- or NOTA-ZnPc conjugates with (68)Ga (t1/2 = 68 min) and (64)Cu (t1/2 = 12.7 h) was performed at 100 °C for 15 min with >90% efficiency for all conjugates. In vitro plasma stability assays demonstrated high stability of the (64)Cu/NOTA-ZnPc conjugate, which remained intact over a 24 h time period, and reasonably high stability of the (68)Ga/DOTA-ZnPc conjugate, which released up to 7% of free (68)Ga over a 3 h period. Based on in vitro plasma stability results, we performed biodistribution studies on two (64)Cu-labeled derivatives, which allowed us to select a single candidate for preliminary in vivo experiments. Fluorescence and PET imaging confirmed the potential of these novel conjugates to act as bimodal probes.
Assuntos
Radioisótopos de Cobre/farmacocinética , Corantes Fluorescentes/farmacocinética , Radioisótopos de Gálio/farmacocinética , Compostos Heterocíclicos com 1 Anel/farmacocinética , Compostos Heterocíclicos/farmacocinética , Indóis/farmacocinética , Animais , Radioisótopos de Cobre/sangue , Radioisótopos de Cobre/química , Corantes Fluorescentes/química , Radioisótopos de Gálio/sangue , Radioisótopos de Gálio/química , Compostos Heterocíclicos/sangue , Compostos Heterocíclicos/química , Compostos Heterocíclicos com 1 Anel/sangue , Compostos Heterocíclicos com 1 Anel/química , Indóis/sangue , Indóis/química , Isoindóis , Camundongos , Camundongos Nus , Neoplasias/diagnóstico , Imagem Óptica/métodos , Tomografia por Emissão de Pósitrons/métodosRESUMO
PURPOSE: The aim of this study was to compare the in vivo stability, uptake, and positron emission tomography (PET) imaging performance of a novel estrogen receptor PET tracer, 4-fluoro-11ß-methoxy-16α-[(18)F]fluoroestradiol (4FMFES), with 16α-[(18)F]fluoroestradiol (FES). PROCEDURES: MC7-L1 and MC4-L2 (ER+) cell lines and their ERα-knockdown variants (ERαKD) were implanted subcutaneously in Balb/c mice. After 21 days, mice were imaged using either FES or 4FMFES. One hour post-injection, static images were acquired for 30 min and the tumor %ID/g uptake values were derived. Biodistribution data were also obtained 1 h following the injection of either FES or 4FMFES. Blood samples were taken at different times and analyzed on thin-layer chromatography to quantify the presence of radiometabolites for each radiotracer. To assess specific targeting to the estrogen receptors, mice bearing only ER+ tumors were treated with the competitive ER inhibitor fulvestrant 48 h prior to imaging with 4FMFES. RESULTS: Metabolic stability was found to be similar for both tracers in mice. Both FES and 4FMFES differentiated ER+ tumors from ERαKD tumors in biodistribution and PET imaging studies. 4FMFES achieved a significantly higher %ID/g uptake in ER+ tumors and MC4-L2 ERαKD tumors than FES in the PET imaging studies. Also, tumor-to-background ratio was higher in ER+ tumors using 4FMFES compared to FES. Dissection data showed a significantly higher %ID/g in all tested cell lines and ER-rich tissues using 4FMFES versus FES. Fulvestrant-treated mice had either low or undetectable tumor uptake. CONCLUSION: In a tumor-bearing mouse model, 4FMFES achieves better specific tumor uptake and better contrast than FES, making it a promising candidate for ER imaging.
Assuntos
Estradiol/análogos & derivados , Neoplasias Mamárias Animais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular Tumoral , Meios de Contraste , Modelos Animais de Doenças , Estradiol/farmacologia , Feminino , Fulvestranto , Humanos , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
The synthesis of a series of new zinc phthalocyanine-peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine-bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor.
Assuntos
Complexos de Coordenação/síntese química , Indóis/síntese química , Oligopeptídeos/síntese química , Fármacos Fotossensibilizantes/síntese química , Zinco , Alcinos/síntese química , Alcinos/metabolismo , Alcinos/farmacologia , Animais , Bombesina/análogos & derivados , Bombesina/síntese química , Bombesina/metabolismo , Bombesina/farmacologia , Linhagem Celular Tumoral , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Peptídeo Liberador de Gastrina/metabolismo , Humanos , Indóis/metabolismo , Indóis/farmacologia , Integrinas/metabolismo , Isoindóis , Camundongos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologiaRESUMO
Sulfonated metallo phthalocyanines (MPcS(n)) are second generation photosensitizers advanced for photodynamic therapy of various medical applications. A series of ZnPcS(n) was demetallated and subsequently converted to the corresponding [(64)Cu]CuPcS(n) in 40-50% isolated yields and >98% radiochemical purities. Tumor-bearing mice were injected with the (64)Cu-labeled products and subjected to 3-h dynamic PET imaging studies. Biodistribution patterns showed characteristic differences between the various derivatives. Tumor uptake was detected only for the amphiphilic derivatives [(64)Cu]CuPcS(2) and [(64)Cu]CuPcS(3)C(6) (1-1.5%ID/g). The biological data suggest that PET imaging with [(64)Cu]CuPc can be used to establish structure-PDT efficacy relationships for Pc-based photosensitizers.
Assuntos
Indóis/química , Fármacos Fotossensibilizantes/síntese química , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Animais , Radioisótopos de Cobre/química , Indóis/farmacocinética , Isoindóis , Marcação por Isótopo , Camundongos , Neoplasias/diagnóstico por imagem , Compostos Organometálicos/química , Fármacos Fotossensibilizantes/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Phthalocyanines (Pc) were conjugated with peptide moieties to improve their target selectivity for potential use as fluorescence and/or positron emission tomography (PET) probes in medical imaging. Three synthetic methods based on palladium-catalyzed cross-coupling reactions (Sonogashira, Buchwald-Hartwig, and Suzuki-Miyaura) were investigated. Using these methods, a series of peptides monofunctionalized with Pc at the N/C-terminal position or on a phenylalanine side chain was obtained in good yields and characterized.
Assuntos
Indóis/química , Paládio/química , Peptídeos/química , Catálise , Isoindóis , Tomografia por Emissão de PósitronsRESUMO
Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Imunoconjugados/uso terapêutico , Fotoquimioterapia/métodos , Porfirinas/uso terapêutico , Adenocarcinoma/imunologia , Adenocarcinoma/radioterapia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Morte Celular , Linhagem Celular Tumoral/efeitos da radiação , Neoplasias do Colo/imunologia , Neoplasias do Colo/radioterapia , Éter de Diematoporfirina/farmacologia , Éter de Diematoporfirina/uso terapêutico , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Imunoterapia/métodos , Integrina beta4/imunologia , Isotiocianatos/química , Luz , Camundongos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/química , Porfirinas/farmacologia , Resultado do TratamentoRESUMO
The impact of lipophilicity on biological parameters critical to photodynamic efficacy was analyzed for a new generation of trisulfobenzo(mononaphtho)porphyrazines. The porphyrazines were substituted on the naphtho ring with linear alkynyl side chains of various lengths. When compared to the analogous phthalocyanine structures, the added benzo ring in the porphyrazine structures increased the lipophilicity for analogs with short alkynyl chains, while this effect disappeared for analogs with longer side chains. In aqueous media, the analogous phthalocyanine series showed aggregation tendencies. In contrast, no correlation between aggregate formation and the length of the alkynyl side chain was evident in the porphyrazine series. At low concentrations, the length of the side chain did not affect cell uptake, while phototoxicity towards EMT-6 mouse tumour cells showed a parabolic relationship, where the hexynyl derivative showed the highest activity. The trisulfonated porphyrazines localized at intracellular organelles, plasma and perinuclear membranes, but could not be found in the nucleus. Total cell uptake of dye did not correlate with phototoxicity, suggesting that localization in certain intracellular organelles, and distribution into critical intracellular sites are important determinants of their photodynamic activity. The hexynyl trisulfonated zinc porphyrazine derivative (ZnNPcS(3)C(6)) showed the strongest in vitro photodynamic activity and therefore was further studied in an EMT-6 mouse tumour model. An i.v. dose of 1 micromol of ZnNPS(3)C(6) per kg, followed 24 h later by activation with light, induced 100% tumour necrosis within 24 h post-PDT. This treatment delayed tumour volume doubling time from 5 days to >2 weeks, and gave 41% tumour cure >3 weeks post-PDT. Applying the same light dose fractionated (5 min on, 2 min off), further improved tumour response, leading to a doubling time of 26 days and a 73% tumour cure. At the administered 1 micromol kg(-1) dye dose, no skin phototoxicity was observed and >90% blood clearance was observed within 5 h post-injection. Compared to the analogous trisulfo monohexynyl zinc phthalocyanine, the new trisulfobenzo(mononaphthohexynyl)porphyrazine provided a broader range of excitation wavelengths, and improved photodynamic potency, while apparently being free of unwanted systemic side effects.
Assuntos
Neoplasias Mamárias Experimentais/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/química , Porfirinas/uso terapêutico , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fármacos Fotossensibilizantes/farmacocinética , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacocinética , Porfirinas/farmacologia , Oxigênio Singlete/metabolismo , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Espectrofotometria , Relação Estrutura-AtividadeRESUMO
Tetrasulfonated zinc phthalocyanine (ZnPcS(4)) is a potent sensitizer for photodynamic therapy of various medical conditions. Depending on its mode of preparation the material consists of mixtures of ortho and meta sulfonated derivatives as well as regioisomers with different photodynamic potency. To study the effect of the site of substitution on biological parameters that contribute to overall photodynamic efficacy, a series of pure ortho- and meta-tetravinylsulfonated metallo phthalocyanines MPc-o/m-(VS)(4) were prepared from the corresponding tetraiodo phthalocyanines using the palladium-catalyzed cross-coupling reaction (Heck reaction). Compared to the tetrasulfonated phthalocyanines, the tetravinylsulfonated derivatives are more hydrophobic and their Q-band absorption maxima are red-shifted. While their in vitro phototoxicity is in the same range as the mixed isomeric sulfonated derivatives, ortho-substituted MPc-o-(VS)(4) are more photocytotoxic as compared to their corresponding meta analogs. Although the central metal ion and site of substitution affect aggregation, the tendency to aggregate did not correlate with photodynamic potency nor did overall cell uptake. Instead, intracellular localization at both mitochondrial and lysosomal membranes appears to be the major factor explaining the augmented activity of the ortho substituted derivatives.
Assuntos
Indóis/síntese química , Indóis/farmacologia , Paládio/química , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Animais , Benzeno/química , Transporte Biológico , Catálise , Linhagem Celular Tumoral , Feminino , Indóis/química , Indóis/metabolismo , Espaço Intracelular/metabolismo , Isoindóis , Isomerismo , Compostos Macrocíclicos/química , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Relação Estrutura-AtividadeRESUMO
With the aim to develop improved dual-action sensitizers suitable for both photodynamic therapy (PDT) and radiotherapy, we prepared a series of metal and metal-free cationic porphyrins, brominated either on beta- or N-allyl positions. Photo- and radiosensitizing efficacy was evaluated in MDA-MB-231 breast cancer cells incubated with 1 muM porphyrin and treated with graded doses of visible light or 0-6 Gy of 60Co gamma irradiation. Metabolic activity after PDT or cell survival after gamma irradiation were estimated by a colorimetric (MTT) or clonogenicity assay, respectively. The highest photo- and radiosensitizing activities were observed with the porphyrins substituted with bromines on N-allyl positions. The non-metalated N-allyl bromoporphyrin exhibited the highest photocytotoxicity (LD50=4.1+/-0.6 J cm(-2), compared to 15.3+/-2.2 J cm(-2) for the non-brominated analog). The radiosensitizing capacity of the cationic porphyrins was also affected by these substitutions with the non-metalated N-allyl bromo analog showing the best improvement (LD50=1.2+/-0.4 Gy vs. 3.6+/-0.9 Gy for the non-brominated analog). The increased photodynamic and radiosensitizing potencies due to bromine addition hold potential for the development of new, improved drugs for cancer treatment in combination with photodynamic and radiation therapy.
Assuntos
Bromo/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Luz , Modelos Moleculares , Porfirinas/efeitos da radiação , Ondas de Rádio , Oxigênio Singlete/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The 2-methoxy derivative of estradiol is currently in Phase II clinical trial as an anticancer agent while the 4-methyl derivative has been shown to interact with cytoplasmic and nuclear estrogen receptors in rat pituitary gland and hypothalamus. We hypothesize that the 16alpha-(18)F-analogs of these estrogens could be suitable radiotracers to evaluate action mechanisms of the parent compounds. In this study we report the synthesis of the 16alpha-(18)F and 16alpha-(19)F-analogs of the A-ring substituted estradiols in high yield via stereoselective opening of the intermediate 16beta,17beta-O-cyclic sulfones with [(18)F]F(-) or F(-) followed by deprotection.
Assuntos
Estradiol/análogos & derivados , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Estradiol/síntese química , Estradiol/química , Marcação por Isótopo/métodosRESUMO
UNLABELLED: 4-Fluoro-11beta-methoxy-16alpha-18F-fluoroestradiol (4FMFES) is a newly developed radiolabeled estradiol analog for PET imaging of estrogen receptors (ERs) that shows improved target-to-background ratios, compared with 16alpha-18F-fluoroestradiol (FES), in small-animal models. The aim of this study was to assess the biodistribution, dosimetry, and safety of 4FMFES in healthy women. METHODS: Ten healthy subjects (6 pre- and 4 postmenopausal women) who had fasted were injected with 66-201 MBq of 4FMFES at a high effective specific activity (median, 251 GBq/micromol). During a 2-h period, each subject underwent 4 serial rapid PET acquisitions and 2 low-dose CT acquisitions on a PET/CT camera. Volumes of interest were drawn over source organs for each PET acquisition, allowing the calculation of time-activity curves, residence times, and radiation dosimetry estimates. Serial blood samples were obtained to measure blood and plasma activity clearance. 4FMFES safety was assessed by blood and urine analyses and vital-sign monitoring. RESULTS: A 4FMFES injection was well tolerated in all subjects. The liver showed high uptake, and the hepatobiliary excretion was massive. Little urinary excretion occurred. Uterus uptake was visualized in all subjects and remained relatively constant over time (maximum and mean standardized uptake values at 60 min were 5.34+/-3.32 and 2.68+/-1.89, respectively). Background activity was low and decreased over time, resulting in an increasing uterus-to-background ratio (12.1+/-2.2 at 60 min). The critical organ was the gallbladder (0.80+/-0.51 mGy/MBq), followed by the upper large intestine (0.13+/-0.04 mGy/MBq), small intestine (0.12+/-0.04 mGy/MBq), and liver (0095+/-0.019 mGy/MBq). For a typical 4FMFES dose of 185 MBq, the effective dose was calculated at 4.82+/-0.70 mSv. CONCLUSION: 4FMFES is considered safe for use in humans, and its effective dose remains well within acceptable limits. The absorbed dose to the gallbladder was relatively high and could potentially be reduced by injecting 4FMFES in patients who had not fasted. 4FMFES showed a significant, potentially estrogen receptor-mediated uterus uptake in both pre- and postmenopausal subjects.
Assuntos
Estradiol/farmacocinética , Adulto , Estradiol/análogos & derivados , Feminino , Radioisótopos de Flúor/química , Humanos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Doses de Radiação , Radiometria , Segurança , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Imagem Corporal TotalRESUMO
BACKGROUND: Oxidised ferrocenium compounds often possess antineoplastic activity. In contrast, reduced ferrocene derivatives frequently only show activity if cell components can oxidise them inside cells to the ferrocenium species. Ferrocene compounds having the lowest formal reduction potential are normally expected to be the most cytotoxic. Here we demonstrate this is not always the case. Some of the structure-related and physical properties that enhance ferrocenyl antineoplastic activity have been investigated. MATERIALS AND METHODS: Ferrocene-containing beta-diketones of the type FcCOCH2COR with Fc=ferrocenyl and R=CF3, CCl3, CH3, Ph(=C6H5, phenyl) and Fc, were tested for cytotoxicity against HeLa (human cervix epitheloid), COR L23 (human large cell lung carcinoma) and platinum resistant CoLo320DM (human colorectal) and COR L23/CPR cancer cell lines. Cell survival was measured by means of the colorometric 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay. RESULTS: The mean drug concentration from 3 experiments causing 50% cell growth inhibition, (IC50) values, varied between 4.5 and 85.0 micromol dm(-3'), with the CF3(-) containing beta-diketone being the most active. Drug activity was inversely proportional to the formal reduction potential, Eo', of the ferrocenyl group, and dependent on the R group in the general beta-diketone structure. The CF3 complex was more cytotocic than cisplatin inter alia against platinum-resistant cell lines, and at least eight times more reactive against cancer cell lines than against PHA (phytohaemagglutinin)-stimulated lymphocyte cultures. CONCLUSION: A drug activity-structural relationship exists in that ferrocenyl drugs with halogen substituents chains are more cytotoxic. Compounds with higher ferrocenyl group formal reduction potential and stronger acid strength (i.e. smaller pKa value) are more cytotoxic.