RESUMEN
The tumor-specific targeting of cancerostatics using polymer drug carriers represents a potential strategy to achieve an effective treatment with reduced side toxicity. Synthetic water-soluble copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) are carriers with tunable architecture and drug loading, tumor-specific accumulation of the drug, and its controlled release. We describe a combination treatment of murine EL4 T cell lymphoma with HPMA-based star conjugates (Mw 250,000gmol-1) of doxorubicin (Dox) or docetaxel (Dtx) designed for enhanced tumor accumulation and combination therapy. Although the combination of linear conjugates (Mw=28,000gmol-1) containing Dox or Dtx resulted in an additive effect in the treatment of the lymphoma, the opposite was observed in the combination of two star conjugates with Dox or Dtx, as the star Dtx conjugate decreased the treatment efficacy of the star Dox conjugate. The Dtx conjugate alone was virtually ineffective in the reduction of tumor growth or survival time extension; thus, a curative effect could be solely attributed to the Dox-containing conjugate. When Dtx was delivered to the tumor on the same polymer carrier as Dox, the efficacy of the Dox-induced treatment was reduced to a lesser extent. No reduction was found when Dtx was delivered by a linear polymer or applied as a free drug. The phenomenon was strictly related to the enhanced permeability and retention (EPR) effect, as it was not observed in BCL1 leukemia, a model without EPR. The diminished treatment outcome in the combination therapy with the two star conjugates was underlined by the significantly decreased accumulation of Dox in the tumor. The use of the drug-free polymer carrier instead of the Dtx-containing star conjugate did not reduce the treatment efficacy of the Dox conjugate. Thus, the physicochemical characteristics of the polymer carrier designed for tumor-specific drug delivery systems control the activity of the respective drug, leading to changes within the tumor microenvironment that can determine ultimate efficacy of the combination therapy.
Asunto(s)
Acrilamidas/química , Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Neoplasias/tratamiento farmacológico , Taxoides/administración & dosificación , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Docetaxel , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapéutico , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/patología , Taxoides/química , Taxoides/farmacocinética , Taxoides/uso terapéuticoRESUMEN
Multidrug resistance (MDR) is a common cause of failure in chemotherapy for malignant diseases. MDR is either acquired as a result of previous repeated exposure to cytostatic drugs (P388/MDR cells) or naturally, as some tumors are congenitally resistant to chemotherapy (CT26 cells). One of the most common mechanisms of MDR is upregulation of P-glycoprotein (P-gp) expression. Here, we used HPMA copolymer conjugates, whereby the cytostatic drug doxorubicin (Dox) or the derivative of the P-gp inhibitor reversin 121 (R121) or both were covalently bound through a degradable pH-sensitive hydrazone bond. We proved that R121, when bound to a polymeric carrier, is capable of inhibiting P-gp in P388/MDR cells and sensitizing them in relation to the cytostatic activity of Dox. Conjugate bearing both Dox and R121 was found to be far more potent in P388/MDR cells than conjugate bearing Dox alone or a mixture of conjugates bearing either Dox or R121 when cytostatic activity in vitro, cell cycle arrest, accumulation of Dox in cells and induction of apoptosis were determined. Importantly, conjugate bearing R121 is also effective in vivo as it inhibits P-gp in P388/MDR tumors after intraperitoneal administration, while both the conjugate bearing Dox and R121 induces apoptosis in P388/MDR tumors more effectively than conjugate bearing Dox alone. Only conjugate bearing Dox and R121 significantly inhibited P388/MDR tumor growth and led to the prolonged survival of treated mice. However, the most dramatic antitumor activity of this conjugate was found in the CT26 tumor model where it completely cured six out of eight experimental mice, while conjugate bearing Dox alone cured no mice.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Metacrilatos/química , Nanocápsulas/química , Nanoconjugados/química , Neoplasias Experimentales/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Citostáticos/administración & dosificación , Doxorrubicina/administración & dosificación , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Ratones , Ratones Endogámicos , Ratones Desnudos , Nanocápsulas/administración & dosificación , Nanoconjugados/administración & dosificación , Neoplasias Experimentales/patología , Resultado del TratamientoRESUMEN
Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named "iBodies", consist of an HPMA copolymer decorated with low-molecular-weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live-cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.
Asunto(s)
Anticuerpos/química , Imitación Molecular , Polímeros/química , Línea Celular Tumoral , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Polymer drug carriers that are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used in the development and synthesis of high-molecular-weight (HMW) drug delivery systems for cancer therapy. In this study, we compared linear (Mw ~27kDa, Rh ~4nm) and non-degradable star (Mw ~250kDa, Rh ~13nm) HPMA copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazone bond. We determined the in vitro and in vivo toxicity of both conjugates and their maximum tolerated dose (MTD). We also compared their anti-tumour activity in mouse B-cell leukaemia (BCL1) and a mouse T-cell lymphoma (EL4) model. We found that MTD was higher for the linear conjugate (85mgDOX/kg) and lower for the star conjugate (22.5mgDOX/kg). An evaluation of the intestinal barrier integrity using FITC-dextran as a gut permeability tracer proved that no pathology was caused by the MTD of either conjugate. However, free DOX showed some damage to the gut barrier. The therapy of BCL1 leukaemia by both of the polymeric conjugates using the MTD or its fraction (i.e., equitoxic dosage) showed better results in the case of the star conjugate. On the other hand, treatment of EL4 lymphoma seemed to be more efficient when the linear conjugate was used. We suppose that the anti-cancer treatment of solid tumours and leukaemias requires different types of drug conjugates. We hypothesise that the most suitable HPMA copolymer-DOX conjugate for the treatment of solid tumours should have an HMW structure with increased Rh that would be stable for three to four days after the conjugate administration and then rapidly disintegrate in the short polymer chains, which are excretable from the body by glomerular filtration. On the other hand, the treatment of leukaemia requires a drug conjugate with a long circulation half-life. This would provide an active drug, whilst slowly degrading to excretable fragments.
Asunto(s)
Acrilamidas/química , Antibióticos Antineoplásicos , Doxorrubicina/química , Portadores de Fármacos , Neoplasias/tratamiento farmacológico , Acrilamidas/farmacocinética , Acrilamidas/uso terapéutico , Acrilamidas/toxicidad , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/uso terapéutico , Antibióticos Antineoplásicos/toxicidad , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Línea Celular Tumoral , Dendrímeros/química , Dendrímeros/farmacocinética , Dendrímeros/uso terapéutico , Dendrímeros/toxicidad , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapéutico , Doxorrubicina/toxicidad , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/uso terapéutico , Portadores de Fármacos/toxicidad , Femenino , Mucosa Intestinal/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Peso Molecular , Neoplasias/sangre , Neoplasias/metabolismo , Bazo/efectos de los fármacos , Bazo/patología , Relación Estructura-ActividadRESUMEN
Here we describe the synthesis and biological properties of two types of star-shaped polymer-doxorubicin conjugates: non-targeted conjugate prepared as long-circulating high-molecular-weight (HMW) polymer prodrugs with a dendrimer core and a targeted conjugate with the anti-CD20 monoclonal antibody (mAb) rituximab (RTX). The copolymers were linked to the dendrimer core or to the reduced mAb via one-point attachment forming a star-shaped structure with a central antibody or dendrimer surrounded by hydrophilic polymer chains. The anticancer drug doxorubicin (DOX) was attached to the N-(2-hydroxypropyl)methacrylamide (HPMA)-based copolymer chain in star polymer systems via a pH-labile hydrazone linkage. Such polymer-DOX conjugates were fairly stable in aqueous solutions at pH 7.4, and the drug was readily released in mildly acidic environments at pH 5-5.5 by hydrolysis of the hydrazone bonds. The cytotoxicity of the polymer conjugates was tested on several CD20-positive or negative human cell lines. Similar levels of in vitro cytotoxicity were observed for all tested polymer conjugates regardless of type or structure. In vivo experiments using primary cell-based murine xenograft models of human diffuse large B-cell lymphoma confirmed the superior anti-lymphoma efficacy of the polymer-bound DOX conjugate when compared with the original drug. Targeting with RTX did not further enhance the anti-lymphoma efficacy relative to the non-targeted star polymer conjugate. Two mechanisms could play roles in these findings: changes in the binding ability to the CD-20 receptor and a significant loss of the immunological properties of RTX in the polymer conjugates.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Polímeros , Profármacos , Rituximab/química , Rituximab/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Doxorrubicina/química , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Humanos , Linfoma/tratamiento farmacológico , Linfoma/mortalidad , Linfoma/patología , Ratones , Polímeros/química , Profármacos/química , Profármacos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin-2 (IL-2) possesses a strong stimulatory activity for activated T and NK cells and it is an attractive molecule for immunotherapy. Nevertheless, extremely short half-life and severe toxicities associated with high-dose IL-2 treatment are serious and limiting drawbacks. In order to increase IL-2 half-life in vivo, we covalently conjugated synthetic semitelechelic polymeric carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) to IL-2. Thus, we synthesized IL-2-poly(HPMA) conjugate containing 2-3 polymer chains per IL-2 molecule in average. Such conjugate has lower biologic activity in comparison to IL-2 in vitro. However, it exerts much higher activity than IL-2 in vivo as shown by expansion of memory CD8+ T, NK, NKT, γδT and Treg cells. Moreover, IL-2-poly(HPMA) extremely effectively potentiates CD8+ T cell peptide-based vaccination. IL-2-poly(HPMA) shows also much longer half-time in circulation than IL-2 (-4 h versus -5 min). Collectively, modification of IL-2 with poly(HPMA) chains dramatically improves its potency and pharmacologic features in vivo, which have implications for immunotherapy. To our knowledge, this is the first proof-of-concept report of the use of polymer/protein modification of IL-2 to obtain more pronounced biological activity.
Asunto(s)
Inmunidad Innata/inmunología , Interleucina-2/inmunología , Interleucina-2/uso terapéutico , Metacrilatos/química , Nanocápsulas/química , Nanoconjugados/uso terapéutico , Animales , Difusión , Femenino , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/química , Factores Inmunológicos/inmunología , Factores Inmunológicos/uso terapéutico , Inmunoterapia/métodos , Interleucina-2/química , Masculino , Ensayo de Materiales , Ratones Endogámicos C57BL , Nanocápsulas/administración & dosificación , Nanocápsulas/ultraestructura , Nanoconjugados/química , Nanoconjugados/ultraestructura , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The molecular weight and molecular architecture of soluble polymer drug carriers significantly influence the biodistribution and anti-tumour activities of their doxorubicin (DOX) conjugates in tumour-bearing mice. Biodistribution of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-DOX conjugates of linear and star architectures were compared in EL4 T-cell lymphoma-bearing mice. Biodistribution, including tumour accumulation, and anti-tumour activity of the conjugates strongly depended on conjugate molecular weight (MW), polydispersity, hydrodynamic radius (R(h)) and molecular architecture. With increasing MW, renal clearance decreased, and the conjugates displayed extended blood circulation and enhanced tumour accumulation. The linear conjugates with flexible polymer chains were eliminated by kidney clearance more quickly than the highly branched star conjugates with comparable MWs. Interestingly, the data suggested different mechanisms of renal filtration for star and linear conjugates. Only star conjugates with MWs below 50,000g.mo(-1) were removed by kidney filtration, while linear polymer conjugates with MWs near 70,000g.mol(-1), exceeding the generally accepted limit for renal elimination, were detected in the urine 36-96h after injection. Additionally, survival of tumour-bearing mice was strongly dependent on molecular weight and polymer conjugate architecture. Treatment of mice with the lower MW conjugate at a dose of 10mg DOX eq./kg resulted in 12% long-term surviving animals, while treatment with the corresponding star conjugate enabled 75% survival of animals.
Asunto(s)
Acrilamidas/química , Acrilamidas/farmacocinética , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/química , Doxorrubicina/farmacocinética , Linfoma/tratamiento farmacológico , Acrilamidas/uso terapéutico , Animales , Línea Celular Tumoral , Doxorrubicina/uso terapéutico , Estabilidad de Medicamentos , Estimación de Kaplan-Meier , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Peso Molecular , Trasplante de Neoplasias , Relación Estructura-Actividad , Distribución TisularRESUMEN
Traditional tissue-sectioning techniques for histological samples utilize various embedding media to stabilize the tissue on a sectioning target and to provide a smooth cutting surface. Due to the ion suppression effect in MALDI ionization and number of background peaks in the low-mass region, these media are not suitable for mass spectrometry imaging (MSI) experiments. To overcome this, droplets of water are often used to mount the tissue on a sectioning target, but the ice block formed around the tissue does not provide a good support for sectioning of fragile samples. In this work, we propose a novel embedding media, compatible with MALDI ionization and MSI experiments, based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Using a reversible addition-fragmentation chain transfer polymerization technique, well-defined pHPMA polymer with narrow mass distribution was prepared. Benefits of the resulted pHPMA-based embedding media were tested on different tissue samples.
Asunto(s)
Acrilamidas/química , Polímeros/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adhesión del Tejido , Animales , Abejas , Pulmón , RatonesRESUMEN
Interaction of nine forms of human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) with two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based doxorubicin (DOX) conjugates designed for passive tumor targeting was studied using pooled human microsomes. The compounds used in this study were two high-molecular-weight HPMA copolymers bearing doxorubicin attached to the polymeric carrier by 1) hydrazone bond enabling intracellular pH-controlled drug release or 2) amide bond through enzymatically cleavable tetrapeptide GlyPheLeuGly spacer. Both polymeric conjugates differing in mechanism of their antitumor activity and the free doxorubicin as the control were tested for potential inhibition activity. Among nine cytochrome P450 forms studied, no HPMA copolymer with bound DOX caused an inhibition of potential clinical significance. The extent of inhibition of enzymatic activities of the cytochrome P450 forms studied was negligible with the exception of CYP2B6 and was apparently caused by DOX as no inhibition was observed with polymers alone, and the extent of inhibition by the complex corresponded to this of the free DOX at the same concentration. In conclusion, the polymers and their conjugates with DOX seem to be relatively safe, at least in this respect, i.e., of inhibition of the liver microsomal drug-metabolizing enzymes.
Asunto(s)
Acrilamidas/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacología , Microsomas Hepáticos/efectos de los fármacos , Acrilamidas/química , Amidas/química , Inhibidores Enzimáticos del Citocromo P-450 , Preparaciones de Acción Retardada , Humanos , Hidrazonas/química , Concentración de Iones de Hidrógeno , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismoRESUMEN
Herein, new biodegradable star polymer-doxorubicin conjugates designed for passive tumor targeting were investigated, and their synthesis, physico-chemical characterization, drug release, biodegradation, biodistribution and in vivo anti-tumor efficacy are described. In the conjugates, the core formed by poly(amidoamine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin (Dox) attached by hydrazone bonds, which enabled intracellular pH-controlled drug release. The described synthesis facilitated the preparation of biodegradable polymer conjugates in a broad range of molecular weights (200-1000g/mol) while still maintaining low polydispersity (~1.7). The polymer grafts were attached to the dendrimers through either stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high-molecular-weight polymer carrier to excretable products. Biodegradability tests in suspensions of EL4 T-cell lymphoma cells showed that the rate of degradation was much faster for reductively degradable conjugates (close to completion within 24h of incubation) than for conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (slow degradation taking several days). This finding was likely due to the differences in steric hindrance in terms of the accessibility of the small molecule glutathione and the bulky enzyme cathepsin B to the polymer substrate. Regarding drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin under mild acidic conditions that model the tumor cell microenvironment. The star polymer-Dox conjugates exhibited significantly prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice, indicating the important role of the EPR effect in its anti-cancer activity. The star polymer conjugates showed prominently higher in vivo anti-tumor activities than the free drug or linear polymer conjugate when tested in mice bearing EL4 T-cell lymphoma, with a significant number of long-term surviving (LTS). Based on the results, we conclude that a M(w) of HPMA copolymers of 200,000 to 600,000g/mol is optimal for polymer carriers designed for the efficient passive targeting to solid tumors. In addition, an expressive therapy-dependent stimulation of the immune system was observed.
Asunto(s)
Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapéutico , Dendrímeros/química , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Metacrilatos/química , Animales , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Línea Celular Tumoral , Dendrímeros/metabolismo , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Humanos , Linfoma/tratamiento farmacológico , Metacrilatos/metabolismo , RatonesRESUMEN
New biodegradable star polymer-doxorubicin (Dox) conjugates designed for passive tumor targeting were investigated and the present study described their synthesis, physico-chemical characterization, drug release and biodegradation. In the conjugates the core formed by poly(amido amine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin attached by hydrazone bonds, which enabled intracellular pH-controlled drug release, or by a GFLG sequence, which was susceptible to enzymatic degradation. The controlled synthesis utilizing semitelechelic copolymer precursors facilitated preparation of biodegradable polymer conjugates in a broad range of molecular weights (110-295 kDa) while still maintaining low polydispersity (â¼1.7). The polymer grafts were attached to the dendrimers either through stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high molecular weight polymer carrier to products that were able to be excreted from the body by glomerular filtration. Biodegradability tests showed that the rate of degradation was much faster for reductively degradable conjugates (completed within 4 h) than the degradation of conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (within 72 h). This finding was likely due to the difference in steric hindrance for the small molecule glutathione and the enzyme cathepsin B. As for drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin either under mild acidic conditions or in the presence of lysosomal enzyme cathepsin B, both of which modeled the tumor cell microenvironment.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Materiales Biocompatibles/química , Dendrímeros/química , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Metacrilatos/química , Antibióticos Antineoplásicos/química , Materiales Biocompatibles/síntesis química , Doxorrubicina/química , Portadores de Fármacos/síntesis química , Estabilidad de Medicamentos , Estructura Molecular , SolubilidadRESUMEN
Interleukin (IL)-2 has been approved for treatment of metastatic renal cancer and malignant melanoma. However, its unfavorable pharmacologic properties, severe side effects and the negative role of IL-2 in maintaining T regulatory cells are severe drawbacks. It has been shown that immunocomplexes of IL-2 and certain anti-IL-2 mAbs possess selective and high stimulatory activity in vivo. Here, we show that IL-2/S4B6 mAb immunocomplexes expand not only CD122(high) subsets and newly activated CD8(+) T cells but also natural killer T cells and γδ T cells. Further, we demonstrate that natural killer (NK) cells expanded by IL-2/S4B6 mAb immunocomplexes in vivo have high cytolytic activity, which can be further increased by coadministration of IL-12. We also demonstrate that IL-2/S4B6 mAb immunocomplexes possess noticeable antitumor activity in two syngeneic mouse tumor models, namely BCL1 leukemia and B16F10 melanoma, but only if administered early in tumor progression. To effectively treat established tumors, we administered the tumor-bearing mice first with N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugate, and subsequently with IL-2/S4B6 mAb immunocomplexes alone or with IL-12 to induce an efficient antitumor immune response. Importantly, we show that the conjugate has significantly lower immunosuppressive activity than free doxorubicin when using dosage with comparable antitumor activity, thus eliminating the majority of tumor cells while leaving the immune system mostly unimpaired for stimulation with IL-2/S4B6 mAb immunocomplexes. Indeed, we demonstrate that the conjugate and IL-2/S4B6 mAb immunocomplexes together have synergistic antitumor activity.
Asunto(s)
Acrilamidas/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Doxorrubicina/administración & dosificación , Inmunoconjugados/uso terapéutico , Inmunosupresores/administración & dosificación , Interleucina-2/inmunología , Interleucina-2/uso terapéutico , Células Asesinas Naturales/inmunología , Leucemia Experimental/terapia , Melanoma Experimental/terapia , Animales , Linfocitos T CD8-positivos/inmunología , Femenino , Interleucina-12/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Núcleo Celular/metabolismo , ADN/metabolismo , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Sistemas de Liberación de Medicamentos , Células 3T3 , Animales , Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Citometría de Flujo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/metabolismo , Ratones , Polímeros/metabolismo , Espectrometría de Fluorescencia , Células Tumorales CultivadasRESUMEN
To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Doxorrubicina/análogos & derivados , Linfoma de Células T/tratamiento farmacológico , Ácidos Polimetacrílicos/farmacología , Amidas/química , Animales , Apoptosis , Western Blotting , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular , Doxorrubicina/farmacología , Portadores de Fármacos , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Galectina 1/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Leucosialina/metabolismo , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , RatonesRESUMEN
Synthesis, physicochemical and biological properties and preliminary anticancer activity of new star-shaped polymer-doxorubicin (DOX) conjugates targeted with anti-CD20 monoclonal antibody were investigated. Mild reduction of antibody (Ab) with dithiothreitol (DTT) resulted in introduction of thiol groups into Ab. Polymer precursors used for the synthesis of the conjugates were based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with a functional group at the polymer chain end. The copolymers were linked to the thiol groups of the reduced Ab via one-point attachment forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. Neither reduction nor polymer modification of Ab influenced binding activity of the Ab to its specific cancer cell membrane antigen as it was confirmed in vitro by standard flow cytometry. The anticancer drug DOX was attached to the HPMA copolymer chain in an Ab-polymer system via a pH-labile hydrazone linkage or via an oligopeptide sequence degradable by lysosomal enzymes. Such Ab-polymer-DOX conjugates were fairly stable in aqueous solution at pH 7.4 and the drug was readily released in mildly acid environment at pH 5-5.5 by hydrolysis of hydrazone bond or more slowly by enzymolysis with lysosomal enzymes. The cytostatic activity of the anti-CD20 monoclonal Ab-targeted conjugates tested on several CD20-positive or negative human and mouse cancer cell lines confirmed considerable targeting capacity of the monoclonal Ab after its binding to the polymer carrier. New method of synthesis of star antibody-targeted polymer-drug conjugates with pH-controlled drug release described in this paper opens new perspectives for development of new therapeutics intended for cancer therapy.
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Citostáticos/metabolismo , Portadores de Fármacos/síntesis química , Sistemas de Liberación de Medicamentos , Metacrilatos/química , Polímeros/química , Acrilamidas/química , Animales , Antibióticos Antineoplásicos/química , Anticuerpos Monoclonales/inmunología , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/metabolismo , Línea Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/química , Evaluación Preclínica de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/metabolismo , Masculino , Ratones , Modelos Químicos , Peso Molecular , Solubilidad , Agua/químicaRESUMEN
BALB/c mice bearing syngeneic BCL1 leukemia, a mouse model of human chronic lymphocytic leukemia, were treated with polymer-bound doxorubicin conjugate targeted with BCL1-specific monoclonal antibody. Such treatment can cure up to 100% of mice and the cured mice show long-lasting resistance to BCL1 leukemia. We show that both CD4+ and CD8+ T cells are required for establishment of the resistance, but only CD8+ T cells are necessary for its maintenance. BCL1 cells express MHC class I and II and also costimulatory molecules CD80 and CD86, which can aid eliciting of antitumor response. On the other hand, BCL1 cells also use several immunoescape mechanisms, such as expression of PD-L1, PD-L2, and interleukin-10. BCL1 cells thus can be recognized by BCL1-specific T cells, but instead of effective priming, such T cells are anergized or deleted by apoptosis. Moreover, BCL1 leukemia progression is accompanied by robust expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells. Although it has been shown that depletion of Treg cells in tumor-bearing mice can retard tumor growth, direct evidence that expansion of Treg cells can promote tumor growth was lacking. In this study, we provide first direct evidence that expanded Treg cells can indeed promote tumor progression by using mice with selectively expanded Treg cells before inoculation of BCL1 leukemia. Finally, we have also shown that elimination of some immunoescape mechanism (e.g., deletion of Treg) can significantly improve the therapeutic outcome of chemotherapy.
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Linfocitos T CD8-positivos/inmunología , Doxorrubicina/análogos & derivados , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/inmunología , Ácidos Polimetacrílicos/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/inmunología , Doxorrubicina/farmacología , Femenino , Inmunoconjugados/inmunología , Inmunoconjugados/farmacología , Ratones , Ratones Endogámicos BALB C , Linfocitos T Reguladores/inmunología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/inmunologíaRESUMEN
Doxorubicin is one of the most potent anti-tumor drugs with a broad spectrum of use. To reduce its toxic effect and improve its pharmacokinetics, we conjugated it to an HPMA copolymer carrier that enhances its passive accumulation within solid tumors via the EPR effect and decreases its cytotoxicity to normal, noncancer cells. In this study, we compared the antiproliferative, pro-survival, and death signals triggered in EL-4 cancer cells exposed to free doxorubicin and doxorubicin conjugated to a HPMA copolymer carrier via either enzymatically (PK1) or hydrolytically (HYD) degradable bonds. We have previously shown that the intracellular distribution of free doxorubicin, HYD, and PK1 is markedly different. Here, we demonstrated that these three agents greatly differ also in the antiproliferative effect and cell death signals they trigger. JNK phosphorylation sharply increased in cells treated with HYD, while treatment with free doxorubicin moderately decreased and treatment with PK1 even strongly decreased it. On the other hand, treatment with free doxorubicin greatly increased p38 phosphorylation, while PK1 and HYD increased it slightly. PK1 also significantly increased ERK phosphorylation, while both the free doxorubicin and HYD conjugate slightly decreased it. Long-term inhibition of JNK significantly increased both proliferation and viability of EL-4 cells treated with free doxorubicin, showing that the JNK signaling pathway could be critical for mediating cell death in EL-4 cells exposed to free doxorubicin. Both activation of caspase 3 and decreased binding activity of the p50 subunit of NFkappaB were observed in cells treated with free doxorubicin and HYD, while no such effects were seen in cells incubated with PK1. Analysis of the expression of genes involved in apoptosis and regulation of the cell cycle demonstrated that free doxorubicin and HYD have very similar mechanisms of action, while PK1 has very different characteristics.
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Acrilamidas/farmacología , Doxorrubicina/farmacología , Linfoma de Células T/metabolismo , Acrilamidas/análisis , Acrilamidas/química , Animales , Apoptosis/genética , Caspasa 3/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Doxorrubicina/análisis , Doxorrubicina/química , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Concentración 50 Inhibidora , Linfoma de Células T/química , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , FosforilaciónRESUMEN
Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox-PHPMA-HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10-20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naïve recipients in in vivo neutralization assay by spleen cells or CD8(+) lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.
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Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/análogos & derivados , Tolerancia Inmunológica , Inmunoglobulinas/uso terapéutico , Linfoma de Células T/tratamiento farmacológico , Ácidos Polimetacrílicos/uso terapéutico , Animales , Doxorrubicina/uso terapéutico , Portadores de Fármacos , Femenino , Humanos , Linfoma de Células T/inmunología , Linfoma de Células T/prevención & control , Masculino , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Tasa de Supervivencia , Células Tumorales Cultivadas/trasplanteRESUMEN
Polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) have been tested as potential carrier for anticancer drug - doxorubicin (Dox). Two types of conjugates were synthesized: (a) conjugates containing Dox bound through an amidic bond to an oligopeptidic side-chain (usually GFLG) and (b) hydrolytically cleavable conjugates wherein Dox is bound to the polymeric carrier through a pH sensitive bond. The mechanism of action of both conjugates is different and reflects the diverse way and intensity of their intracellular accumulation. All conjugates containing doxorubicin bound via an amidic bond directly penetrate the plasma membrane and are detectable in all associated cellular membranes, i.e. membranes of the endocytic compartment, a nuclear membrane as well as membranes of Golgi and endoplasmic reticulum. We have never been able to detect released doxorubicin inside the nuclei of the treated cells. The cytotoxicity of these conjugates seems to be primarily caused by the damage of cellular membranes. Necrosis is the main mechanism of the cell death. Conjugates containing hydrolytically bound doxorubicin are internalized by endocytosis and fluid phase pinocytosis and doxorubicin is cleaved from the polymeric carrier at low pH in late endosomes and lysosomes. An apoptosis is the main mechanism of the cell death. The spacer influences the rate of the intracellular release of the drug rather than the rate of internalization.
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Acrilamidas/química , Proliferación Celular/efectos de los fármacos , Doxorrubicina/farmacología , Células 3T3 , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Dextranos/química , Dextranos/metabolismo , Relación Dosis-Respuesta a Droga , Doxorrubicina/química , Doxorrubicina/farmacocinética , Endocitosis/fisiología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Líquido Intracelular/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Metanol/farmacología , Ratones , Microscopía Fluorescente , Pinocitosis/fisiologíaRESUMEN
A novel class of anti-cancer therapeutics - polymeric conjugates of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers and doxorubicin with pH-controlled release of the drug - is highly efficient in killing tumor cells in vitro and is potent in eradicating growing tumors in vivo. Moreover, in comparison with low-molecular-weight drugs, the macromolecular therapeutics show decreased acute as well as delayed adverse side-toxicity. More importantly, the polymeric conjugates trigger the onset of specific anti-tumor immune response and this anti-tumor immunity can be transferred with splenocytes to naïve recipients. In other terms, chemotherapy based on conjugates of HPMA copolymer with doxorubicin possesses immunomodulating properties. This finding might also have wider implications for the management of relapsing tumors in human patients.