RESUMEN
Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.
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Sistemas de Liberación de Medicamentos/métodos , Paclitaxel/síntesis química , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Humanos , Masculino , RatonesRESUMEN
One of the main problems in oncology is the development of drugs that cause the death of cancer cells without damaging normal cells. Another key problem to be solved is to suppress the drug resistance of cancer cells. The third important issue is to provide effective penetration of drug molecules to cancer cells. TRAIL (TNFα-related apoptosis inducing ligand)/Apo2L is a highly selective anticancer agent. However, the recombinant TRAIL protein having high efficiency against cancer cells in vitro was not effective in clinical trials. Recently we have discovered an acquisition of TRAIL resistance by cancer cells in confluent cultures, which is apparently a manifestation of the general phenomenon of multicellular resistance. The aim of this study was to evaluate whether the anticancer effect of the recombinant protein TRAIL in vivo can be improved by the suppression of multicellular TRAIL-resistance using sorafenib and a tumor-penetrating peptide iRGD, c(CRGDKGPDC). The results testified a great increase in the resistance of human fibrosarcoma HT-1080 cells to izTRAIL both in confluent cultures and in spheroids. Sorafenib administered at nontoxic concentration effectively suppressed confluent- or spheroid-mediated TRAIL-resistance of HT-1080 cells in vitro. Sorafenib combined with iRGD significantly improved the anticancer effect of the recombinant protein izTRAIL in HT-1080 human fibrosarcoma grafts in BALB/c nude mice. Consistent with this finding, multicellular TRAIL-resistance may be a reason of inefficacy of izTRAIL alone in vivo. The anticancer effect of the recombinant protein izTRAIL in vivo may be improved in combination with sorafenib, an inhibitor of multicellular TRAIL resistance and iRGD, the tumor-penetrating peptide.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Fibrosarcoma/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Sorafenib/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Oligopéptidos/farmacología , Proteínas Recombinantes/farmacología , Sorafenib/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Propargyl-152,173-dimethoxy-131-amide of bacteriochlorin e (BChl) and a 4-(4-N,N-dimethylaminostyryl)-N-alkyl-1,8-naphthalimide bearing azide group in the N-alkyl fragment were conjugated by the copper(i)-catalyzed 1,3-dipolar cycloaddition to produce a novel dyad compound BChl-NI for anticancer photodynamic therapy (PDT) combining the modalities of a photosensitizer (PS) and a fluorescence imaging agent. A precise photophysical investigation of the conjugate in solution using steady-state and time-resolved optical spectroscopy revealed that the presence of the naphthalimide (NI) fragment does not decrease the photosensitizing ability of the bacteriochlorin (BChl) core as compared with BChl; however, the fluorescence of naphthalimide is completely quenched due to resonance energy transfer (RET) to BChl. It has been shown that the BChl-NI conjugate penetrates into human lung adenocarcinoma A549 cells, and accumulates in the cytoplasm where it has a mixed granular-diffuse distribution. Both NI and BChl fluorescence in vitro provides registration of bright images showing perfectly intracellular distribution of BChl-NI. The ability of NI to emit light upon excitation in imaging experiments has been found to be due to hampering of RET as a result of photodestruction of the energy acceptor BChl unit. Phototoxicity studies have shown that the BChl-NI conjugate is not toxic for A549 cells at tested concentrations (<8 µM) without light-induced activation. At the same time, the concentration-dependent killing of cells is observed upon the excitation of the bacteriochlorin moiety with red light that occurs due to reactive oxygen species formation. The presented data demonstrate that the BChl-NI conjugate is a promissing dual function agent for cancer diagnostics and therapy.
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BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system. METHODS: We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models. RESULTS: We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan. CONCLUSIONS: We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier - a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.
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Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Neoplasias/terapia , Polímeros/química , Timidina Quinasa/genética , Timidina Quinasa/uso terapéutico , Animales , Cationes , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ganciclovir/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Lípidos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias/patología , Polietilenglicoles/química , Polietileneimina/química , Simplexvirus/enzimologíaRESUMEN
The human iron-binding protein lactoferrin (hLf) has been implicated in a number of important physiological pathways, including those regulating immune function and tumor growth. In an effort to develop an efficient system for production of recombinant hLf (rhLf) that is structurally and functionally equivalent to the natural protein, we generated a recombinant CELO (chicken embryo lethal orphan) avian adenovirus containing an expression cassette for hLf. Embryonated chicken eggs were infected with the generated CELO-Lf virus. rhLf expression was measured in the allantoic fluid of infected eggs by ELISA three days later. The level of recombinant protein was about 0.8mg per embryo. rhLf was efficiently purified (up to 85% yield) from the allantoic fluid of infected eggs using affinity chromatography. rhLf produced in the allantoic fluid was characterized in comparison with natural hLf (nhLf) purified from human breast milk. SDS-PAGE, Western blotting and glycosylation analyzes showed that the recombinant protein had similar physical characteristics to nhLf. In addition, we demonstrated that the antioxidative and antimicrobial activity of rhLf produced in this system is equivalent to that of nhLf. Taken together, these results illustrate the utility of the described "recombinant CELO adenovirus-chicken embryo" system for production of functionally active rhLf. Efficient production of rhLf with accurate structure and function is an important step in furthering investigation of Lf as a potential human drug.
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Aviadenovirus , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Expresión Génica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alantoides , Animales , Proteínas Portadoras/aislamiento & purificación , Embrión de Pollo , Humanos , Lactoferrina , Proteínas Recombinantes/aislamiento & purificación , CigotoRESUMEN
Upconversion nanoparticles (UCNPs) are new-generation photoluminescent nanomaterials gaining considerable recognition in the life sciences due to their unique optical properties that allow high-contrast imaging in cells and tissues. Upconversion nanoparticle applications in optical diagnosis, bioassays, therapeutics, photodynamic therapy, drug delivery, and light-controlled release of drugs are promising, demanding a comprehensive systematic study of their pharmacological properties. We report on production of biofunctional UCNP-based nanocomplexes suitable for optical microscopy and imaging of HER2-positive cells and tumors, as well as on the comprehensive evaluation of their pharmacokinetics, pharmacodynamics, and toxicological properties using cells and laboratory animals. The nanocomplexes represent a UCNP core/shell structure of the NaYF4:Yb, Er, Tm/NaYF4 composition coated with an amphiphilic alternating copolymer of maleic anhydride with 1-octadecene (PMAO) and conjugated to the Designed Ankyrin Repeat Protein (DARPin 9_29) with high affinity to the HER2 receptor. We demonstrated the specific binding of UCNP-PMAO-DARPin to HER2-positive cancer cells in cultures and xenograft animal models allowing the tumor visualization for at least 24 h. An exhaustive study of the general and specific toxicity of UCNP-PMAO-DARPin including the evaluation of their allergenic, immunotoxic, and reprotoxic properties was carried out. The obtained experimental body of evidence leads to a conclusion that UCNP-PMAO and UCNP-PMAO-DARPin are functional, noncytotoxic, biocompatible, and safe for imaging applications in cells, small animals, and prospective clinical applications of image-guided surgery.
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Neoplasias Mamarias Experimentales/diagnóstico por imagen , Nanopartículas/química , Polímeros/química , Imagen de Cuerpo Entero/métodos , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Evaluación Preclínica de Medicamentos , Erbio/química , Escherichia coli/genética , Fluoruros/química , Humanos , Mediciones Luminiscentes , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Polímeros/farmacocinética , Polímeros/toxicidad , Receptor ErbB-2/genética , Propiedades de Superficie , Tulio/química , Distribución Tisular , Itrio/químicaRESUMEN
BACKGROUND: Photodynamic therapy (PDT) is a promising technique for cancer treatment; however, low tissue permeability for irradiating light and insufficient photosensitizer (PS) accumulation in tumors limit its clinical potential. Nanoparticles are engineered to improve selective drug delivery to tumor sites, but its accumulation is highly variable between tumors and patients. Identifying PS accumulation peak in a personalized manner is crucial for therapeutic outcome. Magnetic nanoparticles (MNPs) provide opportunity for tracking drug accumulation in dynamics using non-invasive magnetic resonance imaging (MRI). The purpose of the study was to evaluate MNP loaded with PS as a theranostic tool for treating cancer in mice xenograft colon cancer models. METHODS: MNPs coated with human serum albumin (HSA) were loaded with bacteriochlorine a. MRI, atomic emission spectroscopy (AES) and fluorescent imaging were used to study MNP and drug accumulation rates and dynamics in CT26 tumors. Tumor growth curves were evaluated in animals that received PDT at different time points upon MNP systemic injection. RESULTS: Peak MNP accumulation in tumors was detected by MRI 60 min post injection (pi) and the data were verified by AES and fluorescent imaging. Up to 17% of injected dose/g of tissue was delivered to malignant tissues 24 h after injection. Consistent with MRI predicted drug accumulation peak PDT performed 60 min after intravenous injection was more efficient in inhibiting tumor growth than treatment scheduled 30 min and 240 min pi. CONCLUSIONS: PS loading on HAS-coated MNPs is a perspective approach to increase drug delivery to tumor site. Tracking for MNP accumulation by MRI can be used to predict drug concentration peak in tumors and to adjust PDT time scheduling for improved antitumor response.
RESUMEN
Gamma-ray emitting 111In, which is extensively used for imaging, is also a source of short-range Auger electrons (AE). While exhibiting negligible effect outside cells, these AE become highly toxic near DNA within the cell nucleus. Therefore, these radionuclides can be used as a therapeutic anticancer agent if delivered precisely into the nuclei of tumor target cells. Modular nanotransporters (MNTs) designed to provide receptor-targeted delivery of short-range therapeutic cargoes into the nuclei of target cells are perspective candidates for specific intracellular delivery of AE emitters. The objective of this study was to evaluate the in vitro and in vivo efficacy of 111In attached MNTs to kill human bladder cancer cells overexpressing epidermal growth factor receptor (EGFR). The cytotoxicity of 111In delivered by the EGFR-targeted MNT (111In-MNT) was greatly enhanced on EJ-, HT-1376-, and 5637-expressing EGFR bladder cancer cell lines compared with 111In non-targeted control. In vivo microSPECT/CT imaging and antitumor efficacy studies revealed prolonged intratumoral retention of 111In-MNT with t½ = 4.1 ± 0.5 days as well as significant dose-dependent tumor growth delay (up to 90% growth inhibition) after local infusion of 111In-MNT in EJ xenograft-bearing mice.
RESUMEN
PURPOSE: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111In for cancer therapy. METHODS: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111In in high specific activity. Cytotoxicity of the 111In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111In-labeled MNT in B16-F1 melanoma tumor-bearing mice. RESULTS: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111In by serial imaging and significant tumor growth delay (82% growth inhibition) were found. CONCLUSION: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111In-NOTA-MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111In-NOTA-MNT suggests further applications of the MNT delivery strategy should be explored.
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Antineoplásicos/farmacología , Radioisótopos de Indio/química , Nanopartículas/química , Animales , Autorradiografía , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Receptores ErbB/metabolismo , Femenino , Receptores de Folato Anclados a GPI/metabolismo , Humanos , Hormonas Estimuladoras de los Melanocitos/farmacología , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Receptores de Melanocortina/metabolismo , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos XRESUMEN
Novel hybrid molecule containing 2-mercaptoethylamine was synthesized starting from O-propyloxime-N-propoxy bacteriopurpurinimide (dipropoxy-BPI), which was readily oxidized in oxygen atmosphere yielding the corresponding disulfide analogue (disulfide-BPI). Spectral, photophysical, photodynamic, and biological properties of compound were properly evaluated. Compounds bearing disulfide moiety can directly interact with glutathione (GSH), thereby reducing its intracellular concentration. Indeed, mice sarcoma S37 cell line was treated in vitro with disulfide-BPI, yielding a CC50 value of 0.05 ± 0.005 µM. A relatively high level of singlet oxygen was detected. It was demonstrated (by fluorescence) that the PS was rapidly accumulated in a cancer nest (S37) at a relatively high level after 2 h upon intravenous administration. After 24 h, no traces of the molecule were detected in the tumor mass. Moreover, high photodynamic efficiency was demonstrated at doses of 150-300 J/cm2 against two different in vivo tumor models, achieving 100% regression of cancer growth.
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Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Administración Intravenosa , Animales , Línea Celular Tumoral , Técnicas de Química Sintética , Disulfuros/química , Femenino , Glutatión/metabolismo , Ratones , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/química , Ratas , Sarcoma Experimental/tratamiento farmacológico , Oxígeno Singlete/química , Distribución TisularRESUMEN
Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.
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Leucemia P388 , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/farmacocinética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Femenino , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Dosificación Letal Mediana , Leucemia P388/tratamiento farmacológico , Leucemia P388/metabolismo , Leucemia P388/patología , Lípidos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Oxígeno Singlete/metabolismo , Distribución TisularRESUMEN
Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.
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Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/farmacocinética , Distribución Tisular/efectos de los fármacos , Distribución Tisular/efectos de la radiación , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Femenino , Ratones , Microscopía Confocal , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Porfirinas/química , Relación Estructura-Actividad , Factores de Tiempo , Distribución Tisular/fisiologíaRESUMEN
The 13,15-N-(3'-hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell-killing activity. It is 43-, 61- and 110-fold more active than chlorin p6, 3-formyl-3-devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002-0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 +/- 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3- to 4-fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 +/- 5 min. The time for 50% release of CIC from the cells is 60 +/- 10 min. A 10-fold decrease in CIC intracellular penetration at 22 degrees C proves that temperature-sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2-1.6 microM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic-type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near-IR photosensitizer.
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Fármacos Fotosensibilizantes/química , Porfirinas/química , Modelos Moleculares , Estructura Molecular , Espectrofotometría InfrarrojaRESUMEN
Comparative study of 13,15-[N-(2-hydroxyethyl)]cycloimide chlorin p6 (2), 13,15-(N-acetoxy)cycloimide chlorin p6 (3), 13,15-(N-hydroxy)cycloimide chlorin p6 methyl ester (4) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (5) together with the previously investigated 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (1) was performed. The dependence of the key photodynamic properties of 1-5 on the introduced substituents was analyzed. The photoinduced cell-killing activity of 4 is 100- and 280-fold higher than that of chlorin p6 and Photogem, respectively, as estimated on A549 human lung adenocarcinoma cells. The activity is reduced eight times in the order 4 > 5 > 1 > 2 > 3. The intracellular accumulation of 1-5 occurs in cytoplasm in a monomeric form bound to the lipids of cellular membranes. This form of 1, 2, 3, 4 and 5 is characterized by the high quantum yield of singlet oxygen generation, which depends on the introduced substituents, 0.66, 0.59, 0.35, 0.51 and 0.73, respectively. The photostability is two-fold less for 1 and four-fold less for 2, 3 and 5 than for 4. The rates of cellular uptake and efflux of 1-5 vary widely, thus providing the way to optimize the pharmacological properties of the photosensitizer (PS) using the respective substituents. Modifying the substituents, 1-5 were targeted to different cellular organelles. The enhanced accumulation in the Golgi apparatus and mitochondria complemented with diffuse staining of intracellular membranous structures is a property of 1-4. Compound 5 accumulates selectively in the lipid droplets and stains weakly perinuclear structures. Temperature-sensitive mechanisms of transport are responsible for the 1-4 uptake. Diffusion can play a role in the internalization of 5 but not of 1-4. Endocytosis via caveolae, clathrin-dependent and adenosine triphosphate-dependent pathways are not noticeably involved in the 1-5 internalization. Independently from their intracellular localization 1, 4 and 5 are highly efficient near-IR PS, which induce predominantly an apoptotic type of cell death under conditions providing ca 50% level of phototoxicity and necrosis at the 100% level of phototoxicity.
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Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Transporte Biológico , Muerte Celular , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Fotoquimioterapia , Fármacos Fotosensibilizantes/toxicidad , Porfirinas/análisis , Porfirinas/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Solubilidad , Espectrometría de Fluorescencia , Coloración y Etiquetado , Relación Estructura-Actividad , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/patología , TemperaturaRESUMEN
It is generally assumed that a central metal is essential for the efficiency of phthalocyanines in photodynamic therapy (PDT) of cancer. Contrary to the set opinion, the results of the present study indicate that the metal-free sulfonated phthalocyanines (H2PcSn, where n is the number of sulfonate groups per molecule) possess a considerable photoactivity. The relative phototoxicities of H2PcS1.5, H2PcS2.4, H2PcS3.1 and H2PcS3.8 on HEp2 human epidermoid carcinoma cells were 3.3, 20, 3.3 and 1, respectively, thus demonstrating dependence of the activity on the sulfonation degree, known for metallo-PcSn. A significant delay in tumor growth and a decrease in tumor regrowth rate were observed in mice after PDT with H2PcS2.4. The antitumor effect declined in the order H2PcS2.4 > H2PcS3.1 > H2PcS1.5 and vanished for H2PcS3.8. We demonstrate here that the high photodynamic activity of H2PcS2.4 can be explained by its physicochemical properties in living cells and tissues. Thus, H2PcSn (n is about 2) can be considered as a new alternative in PDT of light-accessible neoplasms and further clinic-oriented studies are warranted.
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Antineoplásicos/toxicidad , Indoles/toxicidad , Metales/toxicidad , Fotoquimioterapia , Animales , División Celular/efectos de los fármacos , Humanos , Isoindoles , Linfoma , Fármacos Sensibilizantes a Radiaciones/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales CultivadasRESUMEN
The confluence-dependent resistance of human larynx carcinoma HEp-2 cells to hydrogen peroxide and a new antitumor drug based on the combination of vitamins C and B12b was studied. It was found that this resistance in growing cells is suppressed by the disruption of intercellular contacts by EGTA and is related neither to the activity of P-glycoprotein nor to the content of intracellular glutathione and the activities of glutathione S-transferases, glutathione peroxidase and glutathionine reductase. Here we showed that the level of expression of the small heat shock protein hsp27, which is known to protect cells from a variety of stresses associated with apoptosis, in growing confluent cells both in the presence and absence of the vitamins B12b and C is much higher (about 20-25 times) than in non-confluent cells. Taken together, the results suggest that the confluence-dependent resistance of cells to the combination of vitamins C and B12b and to hydrogen peroxide is mediated by hsp27 overexpression, which is activated via cell-cell adhesion.
Asunto(s)
Carcinoma/metabolismo , Resistencia a Antineoplásicos/fisiología , Proteínas de Choque Térmico/fisiología , Neoplasias Laríngeas/metabolismo , Proteínas de Neoplasias/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácido Ascórbico/farmacología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Ácido Egtácico/farmacología , Glutatión/metabolismo , Proteínas de Choque Térmico HSP27 , Humanos , Peróxido de Hidrógeno/farmacología , Hidroxocobalamina/farmacología , Neoplasias Laríngeas/tratamiento farmacológico , Neoplasias Laríngeas/patología , Chaperonas Moleculares , Células Tumorales CultivadasRESUMEN
Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H(2)PcS(2.4)) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H(2)PcS(2.4) with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H(2)PcS(2.4) intravenous injection revealed that H(2)PcS(2.4) was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H(2)PcS(2.4) was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H(2)PcS(2.4) less than adjacent skin structures, whereas EC and LLC accumulated H(2)PcS(2.4) alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H(2)PcS(2.4) accumulation. The H(2)PcS(2.4) concentration in remote healthy tissues (skin, muscles and connective tissue) was 2-3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H(2)PcS(2.4) predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Indoles/farmacocinética , Leucemia Linfoide/metabolismo , Melanoma Experimental/metabolismo , Fármacos Fotosensibilizantes/farmacocinética , Animales , Femenino , Indoles/síntesis química , Inyecciones Intravenosas , Isoindoles , Metales , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Fotoquimioterapia , Fármacos Fotosensibilizantes/síntesis química , Distribución TisularRESUMEN
Physico-chemical properties, biodistribution in animal tissues, and PDT efficacy of bacteriochlorin photosensitizers, namely cationic salts of synthetic meso-tetrakis(N-alkyl-3-pyridyl)bacteriochlorins were studied in ÐÐÑ2 cell line and in the LLC mouse model. The tested compounds showed high stability in the dark and high in vitro phototoxicity against ÐÐÑ2 cells (the half maximal inhibitory concentration LD50 in the range from 0.34±0.03 to 1.5±0.03µm). Synthetic bacteriochlorins rapidly accumulate in mouse tumor tissue with tumor-to-normal tissue fluorescence contrast ratios of 2.3-3.3, possess high PDT activity against LLC cells: inhibition of tumor growth, TGI 85.8-100%, increase in life span, ILS 105.7-129.2%, response rate, RR 50-100%. The highest PDT efficacy was found for meso-tetrakis[1-(4'-bromobutyl)-3-pyridyl]bacteriochlorin tetrabromide (IC50 0.34±0.03µm in vitro, TGI and RR 100% in vivo).
Asunto(s)
Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Porfirinas/uso terapéutico , Animales , Carcinoma Pulmonar de Lewis/patología , Femenino , Ratones , Fármacos Fotosensibilizantes/química , Porfirinas/química , Sales (Química) , Carga Tumoral/efectos de los fármacosRESUMEN
Four monocationic cycloimide derivatives of chlorin p(6) (CICD) were studied as photosensitizers and compared to a structurally similar neutral derivative. Cationic CICD are highly photostable (quantum yield of photobleaching is about 1 x 10(-5), generate singlet oxygen under irradiation (quantum yields are 0.3-0.45), can be involved in a photo-induced substrate-dependent generation of superoxide radicals, but do not produce OH . 17,18-delta-lacton 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) () and 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) methyl ester () possess high cancer cell killing photodynamic activity, but they provide no photoinduced bactericidal effect. Substitution of an ethyl group with a hydroxyethyl or acetyl group at position 3 of the macrocycle results in a decrease in extinction and intracellular accumulation that finally leads to the reduced photocytotoxicity. Cationic CICD are targeted to lysosomes, and their intracellular penetration occurs most probably via caveolae-dependent endocytosis. Photodynamic treatment with cationic CICD results in the cell death via necrosis at both sub-phototoxic (40-70% of dead cells) and phototoxic (90-100% of dead cells) regimes of cell treatment. Irradiation induces lysosome damage, leakage of CICD from lysosomes and development of protease activity in cytoplasm, whereas mitochondria are not affected with irradiation. A positive charge of cationic CICD modified drastically an internalization pathway, sites of intracellular localization and mechanisms of photoinduced cytotoxicity as compared to previously studied neutral and anionic CICD. Our experiments with different CICD show that varying charge and structure of substituents it is possible to modulate many cellular properties of CICD in order to find the best molecular template of the advanced near-IR photosensitizer for photodynamic therapy.
Asunto(s)
Lisosomas/efectos de los fármacos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Porfirinas/química , Porfirinas/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Transporte Biológico Activo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Estabilidad de Medicamentos , Escherichia coli/efectos de los fármacos , Humanos , Lisosomas/metabolismo , Micrococcus luteus/efectos de los fármacos , Fotobiología , Fotoquímica , Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/farmacocinética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The N. Blokhin National Cancer Research Center is one of the few Russian scientific institutions in which hybridoma technology of monoclonal antibody (mAb) production has been successfully established. Using this technology, several dozens of mAbs to various antigens of human leukocytes have been elaborated. These mAbs are widely used for immune status evaluation and for differential diagnostics of leukemias. Two mAbs were used to develop therapeutic drugs. Imuteran is a pharmaceutical form of mAb ICO-25 against a mucin-like antigen of human milk fat globules and proposed for treatment of epithelial cell-originating cancers (breast, intestinal, ovarian, lung cancer, etc.). ThePhase II clinical study of this agent is now nearly completed, and preliminary results suggest Imuteran to be a promising anticancer agent with tumor-stabilizing activity, but patients should be carefully monitored for signs of allergic reactions. mAb ICO-90 against the CD3 antigen of human T lymphocytes was used to develop the therapeutic agent Atemonate proposed for treatment of acute transplant rejection. At present, the Phase II clinical study of this agent is over, and the results confirm the drug safety and efficacy for this indication. The drug is being registered at the Ministry of Healthcare and Social Development, and transfer to serial production is expected shortly.