RESUMO
Acute myeloid leukemia (AML) is the most common acute leukemia that is becoming more prevalent particularly in the older (65 years of age or older) population. For decades, "7 + 3" remission induction therapy with cytarabine and an anthracycline, followed by consolidation therapy, has been the standard of care treatment for AML. This stagnancy in AML treatment has resulted in less than ideal treatment outcomes for AML patients, especially for elderly patients and those with unfavourable profiles. Over the past two years, six new therapeutic agents have received regulatory approval, suggesting that a number of obstacles to treating AML have been addressed and the treatment landscape for AML is finally changing. This review outlines the challenges and obstacles in treating AML and highlights the advances in AML treatment made in recent years, including Vyxeos®, midostaurin, gemtuzumab ozogamicin, and venetoclax, with particular emphasis on combination treatment strategies. We also discuss the potential utility of new combination products such as one that we call "EnFlaM", which comprises an encapsulated nanoformulation of flavopiridol and mitoxantrone. Finally, we provide a review on the immunotherapeutic landscape of AML, discussing yet another angle through which novel treatments can be designed to further improve treatment outcomes for AML patients.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/terapia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Combinada , Portadores de Fármacos , Composição de Medicamentos , Humanos , Imunoterapia , Nanopartículas/químicaRESUMO
OTS964 is an inhibitor of T-lymphokine-activated killer cell-originated protein kinase (TOPK), a protein kinase important for mitosis and highly expressed in ovarian and lung cancers. This compound demonstrated potent anti-proliferative activity in a panel of cell lines positive for TOPK; however, when administered to mouse xenograft models, adverse hematopoietic toxicities were observed. To overcome this problem, OTS964 was encapsulated into liposomes and a liposomal formulation of OTS964 is now considered a lead candidate for clinical development. To support clinical development of this formulation, it is critically important to define assays that can easily distinguish between free and liposomal OTS964. Here, we develop a new assay to determine liposomal OTS964 encapsulation (percentage of drug associated with the liposomes) and OTS964 that is dissociated from the liposomes (percentage of drug released from liposomes) by monitoring the enhanced OTS964 fluorescence after its binding to albumin. The optical properties of OTS964 were investigated and three absorbance peaks were identified (235 nm, 291 nm, and 352 nm). Fluorescence was observed at 350 nm (excitation) and 470 nm (emission). Interestingly, the fluorescence of OTS964 increased 18-fold in the presence of serum proteins and more specifically albumin. This phenomenon was used to discriminate between the amounts of drug associated with the liposomes or released from the liposomes. Controls consisting of liposomal OTS964 permeabilized with saponins or octyl glucopyranoside served to confirm that drug release could be monitored by albumin-associated increases in fluorescence. The OTS964 liposomal formulation proved to be very stable with less than 10% release after 4 days in phosphate-buffered saline at 37 °C. The quantity of drug associated with the liposomal surface but not inside the liposomes could also be estimated using this approach. These studies present a novel approach to characterize liposomal release of OTS964, in real time and in a non-invasive manner while acquiring additional information about the spatial distribution of liposomal drug.
Assuntos
Inibidores de Proteínas Quinases/química , Quinolonas/química , Albuminas/química , Fluorescência , Lipossomos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Tumours are complex systems of genetically diverse malignant cells that proliferate in the presence of a heterogeneous microenvironment consisting of host derived microvasculature, stromal, and immune cells. The components of the tumour microenvironment (TME) communicate with each other and with cancer cells, to regulate cellular processes that can inhibit, as well as enhance, tumour growth. Therapeutic strategies have been developed to modulate the TME and cancer-associated immune response. However, modulating compounds are often insoluble (aqueous solubility of less than 1 mg/mL) and have suboptimal pharmacokinetics that prevent therapeutically relevant drug concentrations from reaching the appropriate sites within the tumour. Nanomedicines and, in particular, liposomal formulations of relevant drug candidates, define clinically meaningful drug delivery systems that have the potential to ensure that the right drug candidate is delivered to the right area within tumours at the right time. Following encapsulation in liposomes, drug candidates often display extended plasma half-lives, higher plasma concentrations and may accumulate directly in the tumour tissue. Liposomes can normalise the tumour blood vessel structure and enhance the immunogenicity of tumour cell death; relatively unrecognised impacts associated with using liposomal formulations. This review describes liposomal formulations that affect components of the TME. A focus is placed on formulations which are approved for use in the clinic. The concept of tumour immunogenicity, and how liposomes may enhance radiation and chemotherapy-induced immunogenic cell death (ICD), is discussed. Liposomes are currently an indispensable tool in the treatment of cancer, and their contribution to cancer therapy may gain even further importance by incorporating modulators of the TME and the cancer-associated immune response.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Lipossomos/química , Neoplasias/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptores da Calcitonina/antagonistas & inibidores , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Humanos , Receptores da Calcitonina/imunologia , Células Tumorais CultivadasRESUMO
The purpose of this work was to develop an optimized liposomal formulation of topotecan for use in the treatment of patients with neuroblastoma. Drug exposure time studies were used to determine that topotecan (Hycamtin) exhibited great cytotoxic activity against SK-N-SH, IMR-32 and LAN-1 neuroblastoma human cell lines. Sphingomyelin (SM)/cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes were prepared using extrusion methods and then loaded with topotecan by pH gradient and copper-drug complexation. In vitro studies showed that SM/Chol liposomes retained topotecan significantly better than DSPC/Chol liposomes. Decreasing the drug-to-lipid ratio engendered significant increases in drug retention. Dose-range finding studies on NRG mice indicated that an optimized SM/Chol liposomal formulation of topotecan prepared with a final drug-to-lipid ratio of 0.025 (mol: mol) was better tolerated than the previously described DSPC/Chol topotecan formulation. Pharmacokinetic studies showed that the optimized SM/Chol liposomal topotecan exhibited a 10-fold increase in plasma half-life and a 1000-fold increase in AUC0-24 h when compared with Hycamtin administered at equivalent doses (5 mg/kg). In contrast to the great extension in exposure time, SM/Chol liposomal topotecan increased the life span of mice with established LAN-1 neuroblastoma tumors only modestly in a subcutaneous and systemic model. The extension in exposure time may still not be sufficient and the formulation may require further optimization. In the future, liposomal topotecan will be assessed in combination with high-dose radiotherapy such as 131 I-metaiodobenzylguanidine, and immunotherapy treatment modalities currently used in neuroblastoma therapy.
Assuntos
Neuroblastoma/tratamento farmacológico , Inibidores da Topoisomerase I/administração & dosagem , Topotecan/administração & dosagem , Animais , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Humanos , Lipossomos , Masculino , Camundongos , Neuroblastoma/metabolismo , Distribuição Tecidual , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacocinética , Inibidores da Topoisomerase I/uso terapêutico , Topotecan/química , Topotecan/farmacocinética , Topotecan/uso terapêuticoRESUMO
Topotecan is a drug that is under investigation for the treatment of neuroblastoma and has been encapsulated into liposomes to improve its therapeutic efficacy. However, liposomal formulations still need to be optimized for drug retention and new techniques to measure drug release are required to better understand this process. Here, a novel in vitro method based on fluorescence de-quenching and an automated microscopy imaging platform were developed for monitoring, in real time, the release of topotecan from a liposomal formulation. Drug release from liposomes was monitored for up to 15 h under different conditions including topotecan concentrations, fetal bovine serum amounts (0-20%), and temperatures (25 and 37 °C). A cell-based assay was used to assess liposome association with cells in culture and to quantify amounts of topotecan internalized into cells after release from liposomes. Our results show that the liposomal topotecan concentration had an influence on drug release kinetics: there was a reduction in release rate as a function of increasing concentration. Our data also show that topotecan release from the liposomal formulation was dependent on serum concentration where faster release was observed at higher serum concentrations, and on temperature where faster release was found at 37 °C. This real-time liposomal drug release assay allows for better understanding of the factors important in governing release of topotecan. The assay will be essential towards designing liposomal formulations of topotecan (and potentially of other camptothecin derivatives such as irinotecan) with optimized retention times and better therapeutic efficacy for testing in the clinic.
Assuntos
Inibidores da Topoisomerase I/administração & dosagem , Topotecan/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Humanos , Cinética , Lipossomos , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Topotecan/química , Topotecan/farmacologia , TranscitoseRESUMO
Therapeutic monoclonal antibodies hold great promise in the treatment of cancer and other diseases, but their unclear mechanism of action makes it difficult to identify features that will increase their efficacy. One such feature may be antibody valence, since enhanced therapeutic efficacies have been observed using multivalent, as opposed to bivalent, antibodies. For example, multivalent antibody-lipid nanoparticles (Ab-LNPs) containing rituximab (Rtx) or trastuzumab show significantly increased therapeutic activity compared to equivalent doses of the bivalent antibodies. To more fully understand this phenomenon, we created a methodology reliant on biotin-neutravidin interactions for preparing specific valences of Ab-LNPs that shows improvements in reproducibility, preparation time and overall yield of coupled Ab (up to 80%). We subsequently prepared a series of valences of Rtx-LNPs to examine binding characteristics to CD20+ lymphoma cells, distribution of Rtx-LNPs on the cell surface, modulation of CD20 expression, cytotoxicity of the constructs and ability of the different valences to directly induce apoptosis. As the valence of Rtx-LNP was increased, the amount of Rtx bound to cells increased up to â¼10-fold higher compared to bivalent Rtx. Although more Rtx was bound to cells, there were also surprising increases in the levels of unbound CD20. This suggested the formation of Rtx-enriched microdomains that were confirmed using confocal fluorescence laser-scanning microscopy. Multivalent Rtx-LNPs were significantly more cytotoxic than Rtx; for equivalent doses of drug, Rtx-LNPs elicited apoptosis in two lymphoma cell lines in a valence-dependent manner up to levels that were 14-fold higher than bivalent Rtx. It is suggested that CD20-enriched microdomains may play a role in the mechanism of action of Rtx. This new preparation methodology can be used in future studies evaluating the mechanism of action of multivalent Ab-LNPs prepared with Rtx or other therapeutic Abs.
Assuntos
Química Farmacêutica/métodos , Lipídeos/química , Nanopartículas/química , Rituximab/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Metabolismo dos Lipídeos , Lipídeos/administração & dosagem , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Rituximab/administração & dosagem , Rituximab/metabolismoRESUMO
A passive equilibration method which relies on addition of candidate drugs to pre-formed liposomes is described as an alternative method for preparing liposome encapsulated drugs. The method is simple, rapid and applicable to liposomes prepared with high (45mol%) or low (<20mol%) levels of cholesterol. Passive equilibration is performed in 4-steps: (i) formation of liposomes, (ii) addition of the candidate drug to the liposomes in combination with a permeability enhancing agent, (iii) incubation at a temperature that facilitates diffusion of the added compound across the lipid bilayer, and (iv) quenching the enhanced membrane permeability by reduction in temperature and/or removal of the permeabilization enhancer. The method is fully exemplified here using ethanol as the permeabilization enhancer and carboplatin (CBDCA) as the drug candidate. It is demonstrated that ethanol can be added to liposomes prepared with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and Cholesterol (Chol) (55:45mol ratio) in amounts up to 30% (v/v) with no change in liposome size, even when incubated at temperatures>60°C. Super-saturated solutions of CBDCA (40mg/mL) can be prepared at 70°C and these are stable in the presence of ethanol even when the temperature is reduced to <30°C. maximum CBDCA encapsulation is achieved within 1h after the CBDCA solution is added to pre-formed DSPC/Chol liposomes in the presence of 30% (v/v) ethanol at 60°C. When the pre-formed liposomes are mixed with ethanol (30% v/v) at or below 40°C, the encapsulation efficiency is reduced by an order of magnitude. The method was also applied to liposomes prepared from other compositions include a cholesterol free formulations (containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000)) and a low Chol (<20mol%) formulations prepared with the distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) DSPG)). The cytotoxic activity of CBDCA was unaffected when prepared in this manner and two of the resultant formulations exhibited good stability in vitro and in vivo. The cytotoxic activity of CBDCA was unaffected when prepared in this manner and the resultant formulations exhibited good stability in vitro and in vivo. Pharmacokinetics studies in CD-1 mice indicated that the resulting formulations increased the circulation half life of the associated CBDCA significantly (AUC0-24h of CBDCA=0.016µg·hr/mL; AUC0-24h of the DSPC/Chol CBDCA formulation=1014.0µg·hr/mL and AUC0-24h of the DSPC/DSPG/Chol CBDCA formulation=583.96µg·hr/mL). Preliminary efficacy studies in Rag-2M mice with established subcutaneous H1975 and U-251 tumors suggest that the therapeutic activity of CBDCA is improved when administered in liposomal formulations. The encapsulation method described here has not been disclosed previously and will have broad applications to drugs that would normally be encapsulated during liposome manufacturing.
Assuntos
Antineoplásicos/administração & dosagem , Carboplatina/administração & dosagem , Etanol/química , Lipossomos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Carboplatina/química , Carboplatina/farmacocinética , Linhagem Celular Tumoral , Química Farmacêutica , Colesterol/química , Feminino , Meia-Vida , Xenoenxertos , Humanos , Camundongos , Permeabilidade , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Propriedades de Superfície , TemperaturaRESUMO
The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.
Assuntos
Endocitose , Endossomos/metabolismo , Imunotoxinas/farmacologia , Lisossomos/metabolismo , Animais , Preparações de Ação Retardada , Portadores de Fármacos , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/metabolismo , Estabilidade Proteica , ProteóliseRESUMO
Triterpenoidal saponins are synthesized in the roots of Saponaria officinalis L. The same plant is also a source for the toxin Saporin, which is a ribosome-inactivating protein. Triterpenoidal saponins are known to increase the cytotoxicity of Saporin by modulating its intracellular trafficking. Here, we investigated if the combinatorial effects elicited by purified saponins and Saporin can be applied to increase the therapeutic efficacy of the immunotoxin Saporin-Rituximab. First, saponins were purified by high-performance liquid chromatography. Thereafter, their intrinsic cytotoxicity was evaluated on Ramos cells with no observed effect up to 5 µg/mL, however, saponins increased the cytotoxicity of Saporin, while no influence was observed on its N-glycosidase activity. Saporin-Rituximab bound to CD20 in Ramos cells and, in the absence of saponins, had a GI50 (concentration inhibiting cell growth to 50â%) of 7 nM. However, in the presence of a nontoxic concentration of saponins, the GI50 of Saporin-Rituximab was 0.01 nM, a nearly 700-fold increase in efficacy. Moreover, two further immunotoxins, namely Saporin-anti-CD22 and Saporin-anti-CD25, were tested in combination with saponins yielding enhancement factors of 170-fold and 25-fold, respectively. All three receptors are present in Ramos cells and the differences in cytotoxicity enhancement may be explained by the differing expression levels of the cellular receptors. The application of purified saponins from S. officinalis L. is therefore a new strategy to potentially improve the cytotoxicity and therapeutic efficacy of Rituximab-immunotoxins for the treatment of B-cell lymphoma.
Assuntos
Imunotoxinas/farmacologia , Linfoma de Células B/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Rituximab/farmacologia , Saponaria/química , Saponinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Humanos , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponinas/química , Saponinas/isolamento & purificação , SaporinasRESUMO
The therapeutic relevance of immunotoxins is based on the conjugation of monoclonal antibodies to toxins. In cancer therapies, the conjugated antibodies not only direct the binding of immunotoxins to cancer-specific receptors and mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. In the present study, the therapeutic antibodies Cetuximab (anti-EGFR, Erbitux(®)), Panitumumab (anti-EGFR, Vectibix(®)) and Trastuzumab (anti-HER2, Herceptin(®)) were chemically conjugated to the toxin dianthin. In the first instance, recombinant dianthin was characterized by mass spectrometry and its stability was analyzed by circular dichroism. Dianthin showed increased cytotoxicity on MCF-7 cells when tested in combination with a glycosylated triterpenoid (SO1861) in a real-time impedance-based cytotoxicity assay. In data obtained by live cell imaging, SO1861 specifically mediated the endo/lysosomal escape of dianthin without disrupting the plasma membrane. The purity of immunotoxins was confirmed by SDS-PAGE and Western blot. Their cytotoxicity was evaluated in the presence of SO1861 and dianthin-Cetuximab presented a GI50 (50% growth inhibition) of 5.3pM, dianthin-Panitumumab of 1.5pM, and dianthin-Trastuzumab of 23pM. Finally, the specificity of these immunotoxins was validated in a fluorescence-based real-time assay, where their binding to target cells was prevented by preincubation with an excess of label-free unconjugated antibody. Based on these data, we propose the use of dianthin and SO1861 as a new platform technology to enhance the efficacy of therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cetuximab/farmacologia , Imunotoxinas/farmacologia , Saponinas/farmacologia , Trastuzumab/farmacologia , Sequência de Aminoácidos , Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/administração & dosagem , Reagentes de Ligações Cruzadas/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Feminino , Células HCT116 , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/genética , Imunotoxinas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Dados de Sequência Molecular , Panitumumabe , Estabilidade Proteica , Proteínas Recombinantes , Saponinas/administração & dosagem , Trastuzumab/administração & dosagemRESUMO
Quillaja saponins are used as adjuvants in animal vaccines but their application in human vaccination is still under investigation. Isolation and characterization of adjuvant saponins is very tedious. Furthermore, standardization of Quillaja saponins is critical pertaining to its application in humans. In this study, a convenient method based on agarose gel electrophoresis was developed for the separation of Quillaja saponins. Six different commercial Quillaja saponins were segregated by size/charge into numerous fractions. Each of the fractions was characterized by ESI-TOF-MS spectroscopy and thin layer chromatography. Real-time impedance-based monitoring and red blood cell lysis assay were used to evaluate cytotoxicity and hemolytic activities respectively. Two specific regions in the agarose gel (delimited by specific relative electrophoretic mobility values) were identified and characterized by exclusive migration of acylated saponins known to possess immune adjuvant properties (0.18-0.58), and cytotoxic and hemolytic saponins (0.18-0.94). In vivo experiments in mice with the isolated fractions for evaluation of adjuvant activity also correlated with the relative electrophoretic mobility. In addition to the separation of specific Quillaja saponins with adjuvant effects as a pre-purification step to HPLC, agarose gel electrophoresis stands out as a new method for rapid screening, separation and quality control of saponins.
Assuntos
Adjuvantes Imunológicos/isolamento & purificação , Saponinas de Quilaia/isolamento & purificação , Adjuvantes Imunológicos/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ensaio de Desvio de Mobilidade Eletroforética , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Quillaja/química , Saponinas de Quilaia/química , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Glicosídeos/química , Lipídeos/química , Peptídeos/química , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Nanopartículas/química , Saponaria/química , TransfecçãoRESUMO
AIMS: The intention of this work was to lift saponin supported tumor targeted therapies onto the next level by using targeted toxins in nude mice xenotransplant models. MATERIALS & METHODS: Combined application of dianthin coupled to EGF and saponin SO-1861 was tested in a xenograft model of colon carcinoma. In vitro cytotoxicity was tested in real-time in NIH3T3 cells (no human EGF receptor expression), HER14 and human colon carcinoma HCT116 (both EGF receptor overexpressing) cells. A xenograft model was established using HCT116 cells and tumor-bearing animals were treated with SO-1861 (30 µg/treatment) and dianthin coupled to EGF (0.35 µg/treatment). Tumor progression was monitored, using (18)F-2-fluor-2-desoxy-d-glucose, by small animal PET and by x-ray computed tomography. RESULTS: In vitro results demonstrated a high-receptor specificity and the in vivo experiment showed a progressive reduction of the tumor volume and glycolytic activity in the treated group (>95% reduction; p < 0.05). CONCLUSION: This therapy has great advantage because of high specificity, low side effects and great effectiveness for future development in the treatment of colon cancer.
Assuntos
Carcinoma/patologia , Neoplasias do Colo/patologia , Imunotoxinas/farmacologia , Saponinas/farmacologia , Animais , Carcinoma/diagnóstico , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/tratamento farmacológico , Dianthus/química , Modelos Animais de Doenças , Quimioterapia Combinada , Fator de Crescimento Epidérmico , Hemólise/efeitos dos fármacos , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/efeitos adversos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons , Proteínas Inativadoras de Ribossomos Tipo 1 , Saponinas/administração & dosagem , Saponinas/efeitos adversos , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success.
Assuntos
Imunotoxinas/química , Imunotoxinas/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos/metabolismo , Animais , Humanos , Imunotoxinas/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
Saponaria officinalis L. (Caryophyllaceae), also known as fuller's herb or soapwort is a medicinal plant, which grows from Europe to Central Asia. Medicinal properties attributed to this plant include its antitussive and galactogogue properties. Recently, bisdesmodic saponins with very specific structural features from S. officinalis have been shown to strongly enhance the efficacy of specific targeted toxins (anti-tumor antibodies connected to protein toxins) in-vitro and in-vivo in a synergistic manner. In the presently reported novel approach we used preparative all-liquid high-speed countercurrent chromatography (HSCCC) to recover a total of 22 fractions using biphasic solvent system tert-butylmethylether/n-butanol/acetonitrile/water 1:3:1:5 (v/v/v/v) from a complex precipitated crude saponin mixture. Out of these 22 fractions, 3 fractions had the enhancer effect on anti-tumor toxins out of which one fraction (F7) was further tested elaborately in different cell lines. The molecular weight distribution and compound profiles of separated saponins were monitored by off-line injections of the sequentially collected fractions to an electrospray ion-trap mass-spectrometry system (ESI-IT-MS). The functional saponin fractions were mainly bisdesmosidc and contained saponin m/z 1861 amongst other. Using the bio-assay guided monitoring, the highly active fractions containing 2 to 3 bisdesmodic saponins (5µg/mL) were screened for their effectiveness in enhancing the anti-tumor activity of targeted toxin Sap3-EGF, which was determined using the impedance based real-time cell cytotoxicity evaluation. This novel combination of HSCCC fractionation, MS-target-guided profiling procedure and bio-assay guided fractionation yielded 100mg of functional saponins from a 60g crude drug powder in a rapid and convenient manner.
Assuntos
Distribuição Contracorrente/métodos , Extratos Vegetais/química , Saponaria/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Antineoplásicos , Sinergismo Farmacológico , Imunotoxinas , Saponinas/análise , Saponinas/química , Saponinas/isolamento & purificaçãoRESUMO
Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Humanos , Imunotoxinas/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Saponinas/química , Saporinas , Trastuzumab , Triterpenos/químicaRESUMO
Macromolecular interaction of protein toxins with certain plant triterpenoids holds potential for application in tumor therapy. The ability of only certain saponins to enhance the endosomal escape of toxins specifically in tumor cells was evaluated and set into correlation with the electrophoretic mobility. Saponins from Saponaria officinalis Linn, were selected as a lead to understand this evolutionarily conserved principle in detail. Agarose gel electrophoresis was utilized to procure pure saponin fractions with different electrophoretic mobility, which were tested for their ability to enhance the toxicity by live cell monitoring. Five fractions (SOG1-SOG5) were isolated with a relative electrophoretic mobility of (-0.05, 0.41, 0.59, 0.75 and 1.00) and evaluated using thin layer chromatography, HPLC, and mass spectroscopic analysis. Cytotoxicity experiments revealed highest effectiveness with SOG3. Live cell imaging experiments with SOG3 revealed that this saponin with a specific REM of 0.59 could assist in the lyso/endosomal release of the toxic payload without affecting the integrity of plasma membrane and could lead to the induction of apoptosis. This charge dependent enhancement was also found to be highly specific to type I ribosome inactivating proteins compared to bacterial toxins. Charge interaction of plant toxins and saponins with tumor cells, plays a major role in toxin specific modulation of response. The finding opens up newer ways of finding protein saponin interaction conserved evolutionarily and to test their role in endosomal escape of therapeutic molecules.
Assuntos
Saponinas/química , Toxinas Biológicas/química , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Camundongos , Extratos Vegetais/química , Saponaria/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion proteins is well reported, the achievement of higher protein yields in heterologous expression systems through innovative strategies is of major interest. In the present study, the targeted toxins (his)saporin-EGF (SE) and (his)dianthin-EGF (DE) were expressed as fusion proteins under identical expression conditions. However, the total amount of DE was nearly two-times higher than SE. The identity of the heterologously expressed targeted toxins was confirmed by mass spectrometric studies. Their biological specific activity, monitored in real time, was almost equal. Sequence alignment shows 84% identity and a structural comparison revealed five major differences, two of which affect the secondary structure resulting in a loop (SE) to ß-strand (DE) conversion and one introduces a gap in SE (after position 57). In conclusion, these structural variations resulted in different protein expression levels while codon usage and toxicity to bacteria were excluded as a cause. Minor structural differences identified in this study may be considered responsible for the protection of DE from bacterial proteases and therefore may serve as a lead to modify certain domains in type I RIP-based targeted toxins.
Assuntos
Antineoplásicos/metabolismo , Imunotoxinas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/química , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Imunotoxinas/genética , Imunotoxinas/metabolismo , Imunotoxinas/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Reprodutibilidade dos Testes , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Saponins are a group of plant and marine derived glycosides with numerous biological functions. Two important characteristics of certain plant saponins are their ability to enhance cytotoxicity of type I ribosome inactivating proteins and stimulation of the immune system. The main objective of the present study was to investigate in real-time the permeabilizing effects of saponins on cell membrane. A set of oleanane saponins (glycyrrhizinic acid, Gypsophila, Saponaria and Quillaja saponins) and a steroid saponin (digitonin) were tested. The effects of these saponins on lysosomal membranes and hemolysis, along with their charge were also studied. Real-time monitoring of cell membrane permeabilization facilitated a highly sensitive analysis of the cellular kinetics. Saponins showed variable permeabilizing effects on cellular and lysosomal membranes at concentrations from 6 µM and hemolysis from 3 µM. Further, the results suggest that charge of the saponin may be relevant for permeabilizing effects of oleanane saponins.