RESUMEN
Gamma-glutamyl transferase 1 (GGT1) is a critical enzyme involved in the hydrolysis and/or transfer of gamma-glutamyl groups of glutathione, which helps maintain cysteine levels in plasma. In this study, we synthesized L-ABBA analogs to investigate their inhibitory effect on GGT1 hydrolysis and transpeptidase activity, with the goal of defining the pharmacophore of L-ABBA. Our structure-activity relationship (SAR) study revealed that an α-COO- and α-NH3+ group, as well as a two-CH2 unit distance between α-C and boronic acid, are essential for the activity. The addition of an R (alkyl) group at the α-C reduced the activity of GGT1 inhibition, with L-ABBA being the most potent inhibitor among the analogs. Next, we investigated the impact of L-ABBA on plasma levels of cysteine and GSH species, with the expectation of observing reduced cysteine levels and enhanced GSH levels due to its GGT1 inhibition. We administered L-ABBA intraperitoneally and determined the plasma levels of cysteine, cystine, GSH, and GSSG using LCMS. Our results showed time- and dose-dependent L-ABBA changes in total plasma cysteine and GSH levels. This study is the first to demonstrate the regulation of plasma thiol species upon GGT1 inhibition, with plasma cystine levels reduced by up to â¼ 75 % with L-ABBA (0.3 mg/dose). Cancer cells are highly dependent on the uptake of cysteine from plasma for maintaining high levels of intracellular glutathione. Thus, our findings suggest that GGT1 inhibitors, such as L-ABBA, have the potential to be used in GSH reduction thereby inducing oxidative stress in cancer cells and reducing their resistance to many chemotherapeutic agents.
RESUMEN
Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.
Asunto(s)
Dipéptidos/metabolismo , Inhibidores Enzimáticos/metabolismo , gamma-Glutamiltransferasa/antagonistas & inhibidores , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dipéptidos/química , Humanos , Modelos Moleculares , Conformación Proteica , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/metabolismoRESUMEN
Inhibitors of gamma-glutamyl transpeptidase (GGT1, aka gamma-glutamyl transferase) are needed for the treatment of cancer, cardiovascular illness and other diseases. Compounds that inhibit GGT1 have been evaluated in the clinic, but no inhibitor has successfully demonstrated specific and systemic GGT1 inhibition. All have severe side effects. L-2-amino-4boronobutanoic acid (l-ABBA), a glutamate analog, is the most potent GGT1 inhibitor in vitro. In this study, we have solved the crystal structure of human GGT1 (hGGT1) with ABBA bound in the active site. The structure was interrogated to identify interactions between the enzyme and the inhibitor. Based on these data, a series of novel ABBA analogs were designed and synthesized. Their inhibitory activity against the hydrolysis and transpeptidation activities of hGGT1 were determined. The lead compounds were crystalized with hGGT1 and the structures solved. The kinetic data and structures of the complexes provide new insights into the critical role of protein structure dynamics in developing compounds for inhibition of hGGT1.
Asunto(s)
Compuestos de Boro , gamma-Glutamiltransferasa , Dominio Catalítico , Ácido Glutámico , Humanos , gamma-Glutamiltransferasa/metabolismoRESUMEN
Systemic side effects and high hydrophobicity are major disadvantages of paclitaxel (PTX), one of the most popular anticancer drugs. Here, we present singlet oxygen (SO)-activatable and mitochondria-targeted PTX prodrugs to overcome these problems and boost the cytotoxic effect of photodynamic therapy (PDT). Three PTX prodrugs were prepared by conjugating PTX with various cationic groups. Hydrophobicity was determined in LogD7.4 value. Mitochondrial localization was confirmed by fluorescence confocal microscopy and uptake of mitochondria-specific fluorescence probe. Dark- and photo-toxicity were measured in AY-27 cells with MTT assay. All three prodrugs showed better hydrophilicity than PTX and improved phototoxicity when combined with protoporphyrin IX (PpIX) PDT. In conclusion, SO-activatable and higher hydrophilic PTX prodrugs were successfully prepared. This approach could be used to improve the antitumor efficacy of PDT without the systemic side effects of PTX.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Paclitaxel/uso terapéutico , Fotoquimioterapia/métodos , Profármacos/uso terapéutico , Antineoplásicos Fitogénicos/farmacología , Humanos , Paclitaxel/farmacología , Profármacos/farmacología , Oxígeno SingleteRESUMEN
The combination of photodynamic therapy (PDT) with anti-tumor agents is a complimentary strategy to treat local cancers. We developed a unique photosensitizer (PS)-conjugated paclitaxel (PTX) prodrug in which a PS is excited by near-infrared wavelength light to site-specifically release PTX while generating singlet oxygen (SO) to effectively kill cancer cells with both PTX and SO. The aim of the present study was to identify the determinants influencing the combined efficacy of this light-activatable prodrug, especially the bystander killing effects from released PTX. Using PS-conjugated PTX as a model system, we developed a quantitative mathematical model describing the intracellular trafficking. Dynamics of the prodrug and the model predictions were verified with experimental data using human cancer cells in vitro. The sensitivity analysis suggested that parameters related to extracellular concentration of released PTX, prodrug uptake, target engagement, and target abundance are critical in determining the combined killing efficacy of the prodrug. We found that released PTX cytotoxicity was most sensitive to the retention time of the drug in extracellular space. Modulating drug internalization and conjugating the agents targeted to abundant receptors may provide a new strategy for maximizing the killing capacity of the far-red light-activatable prodrug system. These results provide guidance for the design of the PDT combination study in vivo and have implications for other stimuli-responsive drug delivery systems.
Asunto(s)
Antineoplásicos/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Líquido Intracelular/metabolismo , Paclitaxel/farmacocinética , Fármacos Fotosensibilizantes/farmacocinética , Profármacos/farmacocinética , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Líquido Intracelular/efectos de los fármacos , Modelos Biológicos , Paclitaxel/química , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/química , Profármacos/química , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
Longer wavelength light (650-800nm) is desired to treat large tumors in photodynamic therapy (PDT). However, shorter wavelength light is needed in PDT for thin tumors, not to cause undesirable local side effects. We proposed a strategy for stepwise optical imaging and PDT using a bioorthogonal click chemistry and fluorescence resonance energy transfer (FRET). We prepared azidyl rhodamine (Rh-N3, clickable FD) and cyclooctynyl phthalocyanine [Pc-(DIBAC), clickable PS], with which, here, we demonstrate that the non-catalytic click chemistry is rapid and efficient in cancer cells and FRET from a fluorescence dye (FD) to a photosensitizer (PS) is sufficient to generate enough singlet oxygen killing cancer cells by using shorter wavelength light.
Asunto(s)
Química Clic , Transferencia Resonante de Energía de Fluorescencia , Neoplasias/patología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Relación Estructura-ActividadRESUMEN
Photodynamic therapy (PDT) is a cancer treatment modality where photosensitizer (PS) is activated by visible and near IR light to produce singlet oxygen ((1)O2). However, (1)O2 has a short lifetime (<40 ns) and cannot diffuse (<20 nm) beyond the cell diameter (e.g., â¼ 1800 nm). Thus, (1)O2 damage is both spatially and temporally limited and does not produce bystander effect. In a heterogeneous tumor, cells escaping (1)O2 damage can regrow after PDT treatment. To overcome these limitations, we developed a prodrug concept (PS-L-D) composed of a photosensitizer (PS), an anti-cancer drug (D), and an (1)O2-cleavable linker (L). Upon illumination of the prodrug, (1)O2 is generated, which damages the tumor and also releases anticancer drug. The locally released drug could cause spatially broader and temporally sustained damage, killing the surviving cancer cells after the PDT damage. In our previous report, we presented the superior activity of our prodrug of CA4 (combretastatin A-4), Pc-(L-CA4)2, compared to its non-cleavable analog, Pc-(NCL-CA4)2, that produced only PDT effects. Here, we provide clear evidence demonstrating that the released anticancer drug, CA4, indeed damages the surviving cancer cells over and beyond the spatial and temporal limits of (1)O2. In the limited light illumination experiment, cells in the entire well were killed due to the effect of released anti-cancer drug, whereas only a partial damage was observed in the pseudo-prodrug treated wells. A time-dependent cell survival study showed more cell death in the prodrug-treated cells due to the sustained damage by the released CA4. Cell cycle analysis and microscopic imaging data demonstrated the typical damage patterns by CA4 in the prodrug treated cells. A time-dependent histological study showed that prodrug-treated tumors lacked mitotic bodies, and the prodrug caused broader and sustained tumor size reduction compared to those seen in the tumors treated with the pseudo-prodrug. This data consistently support that the released CA4 overcomes the spatiotemporal limitations of (1)O2, providing far superior antitumor effect.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Rayos Infrarrojos , Profármacos/farmacología , Oxígeno Singlete/metabolismo , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Células MCF-7 , Estructura Molecular , Profármacos/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Cancer-specific survival has changed remarkably little over the past half century, mainly because metastases that are occult at diagnosis and generally resistant to chemotherapy subsequently develop months, years or even decades following definitive therapy. Targeting the dormant micrometastases responsible for these delayed or occult metastases would represent a major new tool in cancer patient management. Our hypothesis is that these metastases develop from micrometastatic cells that are suppressed by normal extracellular matrix (ECM). METHODS: A new screening method was developed that compared the effect of drugs on the proliferation of cells grown on a normal ECM gel (small intestine submucosa, SISgel) to cells grown on plastic cell culture plates. The desired endpoint was that cells on SISgel were more sensitive than the same cells grown as monolayers. Known cancer chemotherapeutic agents show the opposite pattern. RESULTS: Screening 13,000 compounds identified two leads with low toxicity in mice and EC50 values in the range of 3-30 µM, depending on the cell line, and another two leads that were too toxic to mice to be useful. In a novel flank xenograft method of suppressed/dormant cells co-injected with SISgel into the flank, the lead compounds significantly eliminated the suppressed cells, whereas conventional chemotherapeutics were ineffective. Using a 4T1 triple negative breast cancer model, modified for physiological metastatic progression, as predicted, both lead compounds reduced the number of large micrometastases/macrometastases in the lung. One of the compounds also targeted cancer stem cells (CSC) isolated from the parental line. The CSC also retained their stemness on SISgel. Mechanistic studies showed a mild, late apoptotic response and depending on the compound, a mild arrest either at S or G2/M in the cell cycle. CONCLUSIONS: In summary we describe a novel, first in class set of compounds that target micrometastatic cells and prevent their reactivation to form recurrent tumors/macrometastases.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Micrometástasis de Neoplasia/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Concentración 50 Inhibidora , Dosis Máxima Tolerada , Ratones Endogámicos BALB C , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Donor-acceptor dyads featuring near-IR sensitizers derived from thieno-pyrrole-fused BODIPY (abbreviated as SBDPiR) and fullerene, C60 have been newly synthesized and characterized. Occurrence of ultrafast photoinduced electron transfer (PET) leading to the formation of charge-separated state in these dyads, capable of harvesting light energy from the near-IR region, is established from femtosecond transient absorption studies.
Asunto(s)
Fulerenos/química , Procesos Fotoquímicos , Luz , Modelos Moleculares , Estructura MolecularRESUMEN
We examined the concept of a novel prodrug strategy in which anticancer drug can be locally released by visible/near IR light, taking advantage of the photodynamic process and photo-unclick chemistry. Our most recently formulated prodrug of combretastatin A-4, Pc-(L-CA4)2, showed multifunctionality for fluorescence imaging, light-activated drug release, and the combined effects of PDT and local chemotherapy. In this formulation, L is a singlet oxygen cleavable linker. Here, we advanced this multifunctional prodrug by adding a tumor-targeting group, folic acid (FA). We designed and prepared four FA-conjugated prodrugs 1-4 (CA4-L-Pc-PEGn-FA: n = 0, 2, 18, â¼45) and one non-FA-conjugated prodrug 5 (CA4-L-Pc-PEG18-boc). Prodrugs 3 and 4 had a longer PEG spacer and showed higher hydrophilicity, enhanced uptake to colon 26 cells via FR-mediated mechanisms, and more specific localization to SC colon 26 tumors in Balb/c mice than prodrugs 1 and 2. Prodrug 4 also showed higher and more specific uptake to tumors, resulting in selective tumor damage and more effective antitumor efficacy than non-FA-conjugated prodrug 5. FR-mediated targeting seemed to be an effective strategy to spare normal tissues surrounding tumors in the illuminated area during treatment with this prodrug.
Asunto(s)
Receptores de Folato Anclados a GPI/metabolismo , Profármacos/administración & dosificación , Profármacos/farmacocinética , Estilbenos/administración & dosificación , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacocinética , Línea Celular Tumoral/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Diseño de Fármacos , Femenino , Receptores de Folato Anclados a GPI/química , Ácido Fólico/farmacología , Ratones Endogámicos BALB C , Estructura Molecular , Imagen Óptica/métodos , Fotoquimioterapia/métodos , Profármacos/química , Estilbenos/farmacocinética , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Design, synthesis, characterization, and photodynamic activity of mitochondria specific asymmetric ZnPc-Rh B conjugates are described. Conjugation of asymmetric ZnPc-OH chromophores 3a and 3b with rhodamine B via the corresponding DIC-activated ester gave the desired near IR-absorbing asymmetric ZnPc-Rh B conjugates 1a and 1b. Conjugates 1a and 1b were shown to produce singlet oxygen upon illumination in DMSO, MeOH and THF. Fluorescence aggregation studies of the dyes 1a, 1b, 3a and 3b in DMSO and phosphate buffered saline (PBS) solution showed that conjugates 1a and 1b were less aggregated compared to the corresponding non-conjugates 3a and 3b suggesting that incorporation of Rh B lowered aggregation of the conjugates in the PBS solution. The four dyes studied have logD7.4 values between 2.31 and 2.48, with the sulfur-containing conjugate 1b being the most hydrophobic. All the dyes showed negligible dark toxicity when colon 26 cells were treated with 5 µM of the dyes while 10-15% cell death was observed for dye concentrations of 15 µM. Illumination (700±40 nm, 45 J/cm(2), 15 min) of the cells ([dye]=15 µM) gave 70% cell death for ZnPc-Rh B conjugates 1a and 1b while no killing for non-conjugates 3a and 3b suggesting that the incorporation of the Rh B in the photosensitizer lowered the aggregation and subsequently improved cellular uptake and phototoxicity.
Asunto(s)
Indoles/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Compuestos Organometálicos/farmacología , Fármacos Fotosensibilizantes/farmacología , Rodaminas/farmacología , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Indoles/química , Células MCF-7 , Mitocondrias/metabolismo , Estructura Molecular , Compuestos Organometálicos/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Rodaminas/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Although Xenopus laevis has been a model of choice for comparative and developmental studies of the immune system, little is known about organogenesis of the thymus, a primary lymphoid organ in vertebrates. Here we examined the expression of three transcription factors that have been functionally associated with pharyngeal gland development, gcm2, hoxa3, and foxn1, and evaluated the neural crest contribution to thymus development. RESULTS: In most species Hoxa3 is expressed in the third pharyngeal pouch endoderm where it directs thymus formation. In Xenopus, the thymus primordium is derived from the second pharyngeal pouch endoderm, which is hoxa3-negative, suggesting that a different mechanism regulates thymus formation in frogs. Unlike other species foxn1 is not detected in the epithelium of the pharyngeal pouch in Xenopus, rather, its expression is initiated as thymic epithelial cell starts to differentiate and express MHC class II molecules. Using transplantation experiments we show that while neural crest cells populate the thymus primordia, they are not required for the specification and initial development of this organ or for T-cell differentiation in frogs. CONCLUSIONS: These studies provide novel information on early thymus development in Xenopus, and highlight a number of features that distinguish Xenopus from other organisms.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Cresta Neural/embriología , Organogénesis/fisiología , Timo/embriología , Factores de Transcripción/metabolismo , Xenopus laevis/embriología , Animales , Secuencia de Bases , Cartilla de ADN/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia , Especificidad de la Especie , Timo/anatomía & histología , Factores de Transcripción/genéticaRESUMEN
It has been 30 years since Photofrin-PDT was approved for the treatment of bladder cancer in Canada. However, Photofrin-PDT failed to gain popularity due to bladder complications. The PDT with red light and IV-administered Photofrin could permanently damage the bladder muscle. We have been developing a new combination strategy of PpIX-PDT with singlet oxygen-cleavable prodrugs for NMIBC with minimal side effects, avoiding damage to the bladder muscle layer. PpIX can be excited by either green (532 nm) or red (635 nm) light. Red light could be more efficacious in vivo due to its deeper tissue penetration than green light. Since HAL preferentially produces PpIX in tumors, we hypothesized that illuminating PpIX with red light might spare the muscle layer. PpIX-PDT was used to compare green and red laser efficacy in vitro and in vivo. The IC50 of in vitro PpIX-PDT was 18 mW/cm2 with the red laser and 22 mW/cm2 with the green laser. The in vivo efficacy of the red laser with 50, 75, and 100 mW total dose was similar to the same dose of green laser in reducing tumor volume. Combining PpIX-PDT with prodrugs methyl-linked mitomycin C (Mt-L-MMC) and rhodamine-linked SN-38 (Rh-L-SN-38) significantly improved efficacy (tumor volume comparison). PpIX-PDT or PpIX-PDT + prodrug combination did not cause muscle damage in histological analysis. Overall, a combination of PpIX-PDT and prodrug with 635 nm laser is promising for non-muscle invasive bladder cancer treatment.
RESUMEN
Mitochondria play an essential role in cancer treatment by providing apoptotic signals. Hexyl aminolevulinate, an FDA-approved diagnosis for non-muscle invasive bladder cancer, induces the production of protoporphyrin IX (PpIX) preferentially by mitochondria in cancer cells. Photosensitizer PpIX upon illumination can release active chemotherapy drugs from singlet oxygen-activatable prodrugs. Prodrugs placed close enough to PpIX formed in mitochondria can improve the antitumor efficiency of PpIX-PDT. The preferred uptake of prodrugs by cancer cells and tumors can further enhance the selective damage of cancer cells over non-cancer cells and surrounding normal tissues. Mitochondriotropic prodrugs of anticancer drugs, such as paclitaxel and SN-38, were synthesized using rhodamine, a mitochondrial-targeting moiety. In vitro, the mitochondrial targeting helped achieve preferential cellular uptake in cancer cells. In RT112 cells (human bladder cancer cells), intracellular prodrug concentrations were 2-3 times higher than the intracellular prodrug concentrations in BdEC cells (human bladder epithelial cells), after 2 h incubation. In an orthotopic rat bladder tumor model, mitochondria-targeted prodrugs achieved as much as 34 times higher prodrug diffusion in the tumor area compared to the nontumor bladder area. Overall, mitochondria targeting made prodrugs more effective in targeting cancer cells and tumors over non-tumor areas, thereby reducing nonspecific toxicity.
RESUMEN
Protoporphyrin IX (PpIX)-based photodynamic therapy (PDT) has shown limited efficacy in nonmuscle-invasive bladder cancer (NMIBC). To improve PDT efficacy, we developed singlet oxygen-cleavable prodrugs. These prodrugs, when combined with PpIX-PDT, induce cancer cell death through both PDT and drug release mechanisms. Inhibition of PpIX efflux was reported to be an effective strategy to improve PpIX-PDT in certain cancer cells. Our main goal was to investigate whether adding an efflux inhibitor to the combination of PpIX and prodrugs can improve the PpIX levels in bladder cancer cells and the release of active drugs, thus improving the overall efficacy of the treatment. We treated bladder cancer cell lines with lapatinib and evaluated intracellular PpIX fluorescence, finding significantly increased accumulation. Combining lapatinib with prodrugs led to significantly reduced cell viability compared to prodrugs or PpIX-PDT alone. The effect of lapatinib depended on the expression level of the efflux pump in bladder cancer cells. Interestingly, lapatinib increased paclitaxel (PTX) prodrug uptake by threefold compared to prodrug alone. Adding an efflux inhibitor (e.g., lapatinib) into bladder instillation solutions could be a straightforward and effective strategy for NMIBC treatment, particularly in tumors expressing efflux pumps, with the potential for clinical translation.
RESUMEN
Mitochondria-specific photosensitizers were designed by taking advantage of the preferential localization of delocalized lipophilic cations (DLCs) in mitochondria. Three DLC-porphyrin conjugates: CMP-Rh (a core modified porphyrin-rhodamine B cation), CMP-tPP (a core modified porphyrin-mono-triphenyl phosphonium cation), CMP-(tPP)(2) (a core modified porphyrin-di-tPP cation) were prepared. The conjugates were synthesized by conjugating a monohydroxy core modified porphyrin (CMP-OH) to rhodamine B (Rh B), or either one or two tPPs, respectively, via a saturated hydrocarbon linker. Their ability for delivering photosensitizers to mitochondria was evaluated using dual staining fluorescence microscopy. In addition, to evaluate the efficiency of the conjugates as photosensitizers, their photophysical properties and in vitro biological activities were studied in comparison to those of CMP-OH. Fluorescence imaging study suggested that CMP-Rh specifically localized in mitochondria. On the other hand, CMP-tPP and CMP-(tPP)(2) showed less significant mitochondrial localization. All conjugates were capable of generating singlet oxygen at rates comparable to CMP-OH. Interestingly, all cationic conjugates showed dramatic increase in cellular uptake and phototoxicity compared to CMP-OH. This improved photodynamic activity might be primarily due to an enhanced cellular uptake. Our study suggests that Rh B cationic group is better at least for CMP than tPP as a mitochondrial targeting vector.
Asunto(s)
Mitocondrias/metabolismo , Fármacos Fotosensibilizantes/síntesis química , Porfirinas/síntesis química , Rodaminas/síntesis química , Animales , Cationes/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Transferencia de Energía , Humanos , Luz , Células MCF-7 , Ratones , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/toxicidad , Porfirinas/química , Porfirinas/toxicidad , Rodaminas/química , Rodaminas/toxicidad , Oxígeno Singlete/metabolismoRESUMEN
Photodynamic therapy (PDT) initially employed red light, which caused some patients to experience permanent bladder contractions. PDT using the FDA-approved drug hexaminolevulinate (HAL), which produces protoporphyrin IX (PpIX) in the tumor, showed some promise but has low efficacy in treating non-muscle-invasive bladder cancer (NMIBC). We developed singlet oxygen-activatable prodrugs of two anticancer drugs, paclitaxel and mitomycin C, to enhance the antitumor effect of PpIX-PDT without producing systemic side effects, by promoting only local release of the active chemotherapeutic agent. Orthotopic NMIBC model was used to compare the efficacy of prodrugs only, PpIX-PDT, and prodrugs + PpIX-PDT. 532 nm laser with a total power of 50 mW for 20 min (60 J, single treatment) was used with HAL and prodrugs. Histology and microscopic methods with image analysis were used to evaluate the tumor staging, antitumor efficacy, and local toxicity. Prodrug + PpIX-PDT produced superior antitumor efficacy than PpIX-PDT alone with statistical significance. Both PpIX-PDT alone and combination therapy resulted in mild damage to the bladder epithelium in the normal bladder area with no apparent damage to the muscle layer. Overall, SO-cleavable prodrugs improved the antitumor efficacy of PpIX-PDT without causing severe and permanent damage to the bladder muscle layer.
RESUMEN
Bladder cancer is the first cancer for which PDT was clinically approved in 1993. Unfortunately, it was unsuccessful due to side effects like bladder contraction. Here, we summarized the recent progress of PDT for bladder cancers, focusing on photosensitizers and formulations. General strategies to minimize side effects are intravesical administration of photosensitizers, use of targeting strategies for photosensitizers and better control of light. Non-muscle invasive bladder cancers are more suitable for PDT than muscle invasive and metastatic bladder cancers. In 2010, the FDA approved blue light cystoscopy, using PpIX fluorescence, for photodynamic diagnosis of non-muscle invasive bladder cancer. PpIX produced from HAL was also used in PDT but was not successful due to low therapeutic efficacy. To enhance the efficacy of PpIX-PDT, we have been working on combining it with singlet oxygen-activatable prodrugs. The use of these prodrugs increases the therapeutic efficacy of the PpIX-PDT. It also improves tumor selectivity of the prodrugs due to the preferential formation of PpIX in cancer cells resulting in decreased off-target toxicity. Future challenges include improving prodrugs and light delivery across the bladder barrier to deeper tumor tissue and generating an effective therapeutic response in an In vivo setting without causing collateral damage to bladder function.
Asunto(s)
Fotoquimioterapia , Profármacos , Neoplasias de la Vejiga Urinaria , Humanos , Fármacos Fotosensibilizantes/uso terapéutico , Ácido Aminolevulínico/uso terapéutico , Fotoquimioterapia/métodos , Protoporfirinas , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
We established a light-activatable prodrug strategy that produces the combination effect of photodynamic therapy (PDT) and site-specific chemotherapy. Prodrugs are activated by singlet oxygen (SO), generated from PS and visible or near IR light, in either intra- or inter-molecular manner. The goal of this study is to evaluate cytotoxic effects of nonmitochondria-targeted prodrugs of a number of anticancer drugs with different mechanisms of action. They were tested in both 2D and 3D in vitro conditions via inter-molecular activation of prodrugs by SO generated in mitochondria by protoporphyrin IX-PDT (PpIX-PDT). Prodrugs of anticancer drugs (paclitaxel, SN-38, combretastatin A4 and mitomycin C) were synthesized using facile and high-yielding reactions. Nonmitochondria-targeted prodrugs showed limited dark toxicity while all of them showed greatly enhanced phototoxicity compared to PpIX-PDT in the 2D culture model. Prodrugs generated up to about 95% cell killing at 2.5 µM when administered with hexyl-aminolevulinate (HAL) to produce Protoporphyrin IX in cancer cells in both 2D monolayer and 3D spheroids model. The data demonstrate that mitochondria-targeting of prodrugs is not fully essential for our inter-molecular activation prodrug strategy. The prodrug strategy also worked for anticancer drugs with diverse MOAs.
Asunto(s)
Antineoplásicos , Fotoquimioterapia , Profármacos , Antineoplásicos/farmacología , Línea Celular Tumoral , Irinotecán , Mitomicina , Paclitaxel/farmacología , Fármacos Fotosensibilizantes/farmacología , Profármacos/farmacología , Oxígeno SingleteRESUMEN
We demonstrated that a large primary and a small untreated distant breast cancer could be controlled by local treatment with our light-activatable paclitaxel (PTX) prodrug. We hypothesized that the treated tumor would be damaged by the combinational effects of photodynamic therapy (PDT) and locally released PTX and that the distant tumor would be suppressed by systemic antitumor effects. Syngeneic rat breast cancer models (single- and two-tumor models) were established on Fischer 344 rats by subcutaneous injection of MAT B III cells. The rats were injected with PTX prodrug (dose: 1 umole kg-1 , i.v.), and tumors were treated with illumination using a 690-nm laser (75 or 140 mW cm-1 for 30 min, cylindrical light diffuser, drug-light interval [DLI] 9 h). Larger tumors (~16 mm) were effectively ablated (100%) without recurrence for >90 days. All cured rats rejected rechallenged tumor for up to 12 months. In the two-tumor model, the treatment of the local large tumor (~16 mm) also cured the untreated tumor (4-6 mm) through adaptive immune activation. This is our first demonstration that local treatment with our PTX prodrug produces systemic antitumor effects. Further investigations are warranted to understand mechanisms and optimal conditions to achieve clinically translatable systemic antitumor effects.