RESUMO
Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10-11-10-8 M) against a variety of HER2-expressing cancer cell lines. Several peptide-linked variants of the lead compound were prepared and conjugated to trastuzumab to afford ADCs with drug-to-antibody (DAR) ratios ranging from 3 to 5. The ADCs exhibited antigen-dependent cytotoxicity in vitro and potently suppressed tumor xenograft growth in vivo in a target-dependent manner. Moreover, the ADCs were well-tolerated in both mouse and rat. This work demonstrates for the first time that PBD polyamide hybrids can serve as effective ADC payloads.
Assuntos
Antineoplásicos , Imunoconjugados , Animais , Antineoplásicos/farmacologia , Benzodiazepinas , Linhagem Celular Tumoral , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Nylons/farmacologia , Pirróis , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing. A phosphoramidate prodrug of the payload enables low ADC aggregation even at drug-to-antibody ratios of 5:1 while still delivering a bystander-capable payload that is effective in multidrug resistant (MDR)-overexpressing cell lines. The platform was comparable in xenograft studies to the clinical benchmark DNA mono-alkylator ADC platform DGN459 but with a significantly better tolerability profile in rats. Thus, the activity and tolerability profile of this new platform make it a viable option for the development of ADCs.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Ratos , Animais , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Alquilantes , Neoplasias/tratamento farmacológico , DNA/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologiaRESUMO
Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies. In this study, we show that sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in animal and human models of sepsis. Adenovirus-mediated overexpression of soluble Flt-1 (sFlt-1) in a mouse model of endotoxemia attenuated the rise in VEGF and PlGF levels and blocked the effect of endotoxemia on cardiac function, vascular permeability, and mortality. Similarly, in a cecal ligation puncture (CLP) model, adenovirus-sFlt-1 protected against cardiac dysfunction and mortality. When administered in a therapeutic regimen beginning 1 h after the onset of endotoxemia or CLP, sFlt peptide resulted in marked improvement in cardiac physiology and survival. Systemic administration of antibodies against the transmembrane receptor Flk-1 but not Flt-1 protected against sepsis mortality. Adenovirus-mediated overexpression of VEGF but not PlGF exacerbated the lipopolysaccharide-mediated toxic effects. Together, these data support a pathophysiological role for VEGF in mediating the sepsis phenotype.
Assuntos
Sepse/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Animais , Ceco/microbiologia , Modelos Animais de Doenças , Endotoxemia/sangue , Humanos , Inflamação/sangue , Lipopolissacarídeos/toxicidade , Camundongos , Fator de Crescimento Placentário , Proteínas da Gravidez/sangue , Sepse/mortalidadeRESUMO
Mitochondrial DNA (mtDNA) deletions have been reported to accumulate to high levels in substantia nigra of older humans, and these mutations are suspected of causing age-related degeneration in this area. We have compared levels of mtDNA deletions in humans and mice and report here that levels of deletions in the mouse are very significantly lower than in humans. While human mtDNA from substantia nigra contained more than 5% of deleted molecules, mouse substantia nigra contained less than 0.5%. These results imply that mtDNA deletions are unlikely to play any significant role in of murine substantia nigra aging and further call for caution in using mouse models in studies of the role of mtDNA deletions in aging and neurodegeneration. On a more general note, these results support the view that critical targets of the various aging processes may differ significantly between distant species.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Deleção de Sequência , Substância Negra/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Artefatos , Humanos , Camundongos , Doença de Parkinson/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , Especificidade da EspécieRESUMO
Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Arteriosclerose/prevenção & controle , Monóxido de Carbono/farmacologia , Rejeição de Enxerto/prevenção & controle , Animais , GMP Cíclico/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Ativação Enzimática , Guanilato Ciclase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.
Assuntos
Imunoconjugados/uso terapêutico , Oligopeptídeos/uso terapêutico , Polímeros/uso terapêutico , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos SCID , Oligopeptídeos/farmacologia , Polímeros/farmacologiaRESUMO
Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.
Assuntos
Antígenos de Neoplasias/metabolismo , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Polímeros/uso terapêutico , Animais , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos SCID , Oligopeptídeos/farmacologia , Polímeros/farmacologiaRESUMO
BACKGROUND: Heart failure is the leading cause of death in the United States. By delineating the pathways that regulate cardiomyocyte function, we can better understand the pathogenesis of cardiac disease. Many cardiomyocyte signaling pathways activate protein tyrosine kinases. However, the role of specific protein tyrosine phosphatases (PTPs) in these pathways is unknown. METHODS AND RESULTS: Here, we show that mice with muscle-specific deletion of Ptpn11, the gene encoding the SH2 domain-containing PTP Shp2, rapidly develop a compensated dilated cardiomyopathy without an intervening hypertrophic phase, with signs of cardiac dysfunction appearing by the second postnatal month. Shp2-deficient primary cardiomyocytes are defective in extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) activation in response to a variety of soluble agonists and pressure overload but show hyperactivation of the RhoA signaling pathway. Treatment of primary cardiomyocytes with Erk1/2- and RhoA pathway-specific inhibitors suggests that both abnormal Erk/MAPK and RhoA activities contribute to the dilated phenotype of Shp2-deficient hearts. CONCLUSIONS: Our results identify Shp2 as the first PTP with a critical role in adult cardiac function, indicate that in the absence of Shp2 cardiac hypertrophy does not occur in response to pressure overload, and demonstrate that the cardioprotective role of Shp2 is mediated via control of both the Erk/MAPK and RhoA signaling pathways.
Assuntos
Cardiomiopatia Dilatada/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Miócitos Cardíacos/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Cardiomegalia/enzimologia , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Síndrome LEOPARD/enzimologia , Síndrome LEOPARD/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Modelos Animais , Síndrome de Noonan/enzimologia , Síndrome de Noonan/genética , Especificidade de Órgãos , Fenótipo , Pressão , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTPRESUMO
Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. This chapter outlines the methods for collecting individual cells appropriate for analysis of mtDNA mutations by single-molecule PCR. In addition, we describe the protocols for respiratory complexes II and IV in these cells. Together, the identification of respiratory deficiency and mtDNA mutations within single cells provides a powerful means of evaluating the importance of these events in disease and aging.
Assuntos
DNA Mitocondrial/genética , Complexo II de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Mutação/genética , Análise Mutacional de DNA , Humanos , Lasers , Microdissecção , Reação em Cadeia da PolimeraseRESUMO
Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação/genética , Reação em Cadeia da Polimerase/métodos , Análise Mutacional de DNA , HumanosRESUMO
Caspase-9 is a critical regulator of mitochondria-mediated apoptosis. We found that adult cardiac myocytes, but not nonmyocytes, have high caspase-9 expression, and exhibit relative resistance to caspase-9-induced cell death. Thus, we hypothesized that cardiac myocytes possess factors that resist apoptosis. Through a yeast two-hybrid screening of adult human heart cDNA library, we identified HS-1 associated protein-1 (HAX-1), a 35-kD BH-domain containing protein localized to the mitochondria as one of the molecules that interacts with caspase-9. Recombinant HAX-1 protein inhibited caspase-9 processing in a dose-dependent manner in a cell-free caspase activation assay. Overexpression of HAX-1 in adult cardiac myocytes conferred 30% protection from apoptosis as compared with the control. Suppression of HAX-1 expression using siRNA-HAX-1 resulted in significant cell death in adult cardiac myocytes, suggesting the importance of HAX-1 in cardiac myocyte resistance to apoptotic stimulation. On apoptotic stimulation, some caspase-9 translocated to the mitochondria and co-localized with HAX-1, confirming the spatial proximity of caspase-9 and HAX-1. In summary, HAX-1 is a newly identified anti-apoptotic factor and its mechanism of action is through caspase-9 inhibition.
Assuntos
Apoptose/fisiologia , Células Musculares/fisiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Animais , Caspase 9 , Caspases/deficiência , Caspases/metabolismo , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas , Células Musculares/citologia , Proteínas/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: This study characterized the role of insulin receptors and resistance on vascular endothelial growth factor (VEGF) expression and myocardial vascularization in physiological conditions and after ischemia. METHODS AND RESULTS: Cardiac microvascular density was reduced by 30% in insulin-resistant Zucker fatty rats versus lean controls. This was associated with a parallel 40% inhibition of insulin-stimulated activation of both Akt and VEGF expression in the myocardium and cardiomyocytes. In contrast, the activation of Erk1/2 by insulin remained unchanged. In cultured cardiomyocytes, insulin or insulin-like growth factor (IGF)-1 increased VEGF mRNA and protein expression by 2-fold. Inhibition of PI3K/Akt, especially Akt2-mediated cascades but not the Ras/MEK/Erk pathway, using chemical inhibitors, dominant negative adenoviral constructs, or siRNA approaches suppressed VEGF mRNA expression by insulin. Ventricular tissues from muscle insulin receptor knockout (MIRKO) mice, which lack insulin receptors in the myocardium, have significant reductions in insulin but not IGF-1 signaling, VEGF expression, and vascular density before and after ischemia versus controls. CONCLUSIONS: Insulin regulates VEGF gene expression and vascularization in the myocardium specifically via insulin receptors and the activation of PI3K/Akt pathway. Selective inhibition of this pathway may lead to the decreases in VEGF expression and capillary density in the myocardium of patients with insulin resistance.
Assuntos
Resistência à Insulina , Isquemia Miocárdica/metabolismo , Neovascularização Patológica/metabolismo , Receptor de Insulina/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Androstadienos/farmacologia , Animais , Células Cultivadas , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Flavonoides/farmacologia , Humanos , Insulina/farmacologia , Masculino , Camundongos , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Neovascularização Patológica/fisiopatologia , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Ratos Zucker , Transdução de Sinais , WortmaninaRESUMO
Heart disease remains the most frequent cause of death in the general population with increasing incidence in the elderly population. The pathologic failure of the aging heart may be related to structural and functional alterations in cardiac muscle cells. However, the molecular mechanisms underlying the aging-related decline in cardiac muscle function are largely unknown. To provide the first analysis of cardiac aging at the level of gene expression, we established and compared cDNA libraries from apparently healthy young and aged mouse ventricular cardiac muscle cells. We report the identification of genes that exhibit aging-related changes of mRNA levels. Aging expression profiles in aged hearts indicate decreased cellular adaptation and protection against stress-induced injury together with the development of contractile dysfunction. The data suggest reduced activity of the mitochondrial electron transport system and reduced levels of cardiac-specific transcription regulators. The cardiomyocyte aging profile of gene expression displays similarities with known heart disorders. Genes whose mRNA levels change with aging in cardiomyocytes might profoundly affect pathological changes in the heart.
Assuntos
Envelhecimento/fisiologia , Perfilação da Expressão Gênica , Ventrículos do Coração/metabolismo , Actinina/química , Animais , Northern Blotting , Fluoresceína-5-Isotiocianato/química , Biblioteca Gênica , Ventrículos do Coração/química , Ventrículos do Coração/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Rats were trained using olfactometry and operant conditioning to discriminate among homologous fatty acids, homologous aldehydes, and a series of unrelated odors. Their memory for the positive and negative assignment of each odor (tested under extinction) was assessed before and after they had received selective lesions of the olfactory bulbs or injection of the olfactory epithelial toxin 3-methyl indole (3-MI). Response accuracy on the memory test provided a measure of the extent to which treatments altered the remembered perceptual quality of the odors. The degree of deafferentation of the bulb by treatment with 3-MI was assessed using anterograde transport of horseradish peroxidase applied to the olfactory epithelium. Rats treated with 3-MI had a detectable reaction product only in varying numbers of glomeruli on the lateral and, in some cases, posterior medial walls of the olfactory bulb. Bulbar lesions destroyed the dorsal and dorsomedial bulbar areas that have been identified in optical and electrophysiological studies as showing responses to fatty acids. Rats with bulbar lesions had good to near perfect retention on their post-treatment memory test on all odor pairs, as did 3-MI-treated rats that still had substantial input to glomeruli on the lateral or posterior medial wall of the bulb. 3-MI-treated rats with substantially fewer afferent connections had severe retention deficits, particularly for the aldehyde and fatty acid odors, but this loss was secondary to deficits in the ability to discriminate among these odors. The results indicate that input to bulbar areas that are activated by a series of homologous odors may not be essential for odor discrimination and that deafferentation of the majority of bulbar glomeruli may be primarily without effect on odor quality perception as assessed by the memory test. These outcomes point to a much higher degree of redundancy within the olfactory bulb than that envisioned by current combinatorial or odotopic hypotheses of odor quality coding or, alternatively, to mechanisms of odor coding used in the awake behaving animal that have not yet been elucidated.
Assuntos
Odorantes , Bulbo Olfatório/fisiologia , Mucosa Olfatória/fisiologia , Olfato/fisiologia , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/fisiologia , Aldeídos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Discriminação Psicológica/efeitos dos fármacos , Discriminação Psicológica/fisiologia , Ácidos Graxos/farmacologia , Feminino , Masculino , Memória/fisiologia , Transtornos do Olfato/induzido quimicamente , Bulbo Olfatório/patologia , Mucosa Olfatória/efeitos dos fármacos , Ratos , Ratos Long-Evans , Escatol , Olfato/efeitos dos fármacosRESUMO
Cardiac hypertrophy is a complex and nonhomogenous response to various stimuli. In this study, we used high-density oligonucleotide microarray to examine gene expression profiles during physiological hypertrophy, pathological hypertrophy, and heart failure in Dahl salt-sensitive rats. There were changes in 404/3,160 and 874/3,160 genes between physiological and pathological hypertrophy and the transition from hypertrophy to heart failure, respectively. There were increases in stress response genes (e.g., heat shock proteins) and inflammation-related genes (e.g., pancreatitis-associated protein and arachidonate 12-lipoxygenase) in pathological processes but not in physiological hypertrophy. Furthermore, atrial natriuretic factor and brain natriuretic protein showed distinctive changes that are very specific to different conditions. In addition, we used a resampling-based gene score-calculating method to define significantly altered gene clusters, based on Gene Ontology classification. It revealed significant alterations in genes involved in the apoptosis pathway during pathological hypertrophy, suggesting that the apoptosis pathway may play a role during the transition to heart failure. In addition, there were significant changes in glucose/insulin signaling, protein biosynthesis, and epidermal growth factor signaling during physiological hypertrophy but not during pathological hypertrophy.
Assuntos
Cardiomegalia/patologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Insuficiência Cardíaca/patologia , Animais , Apoptose , Fator Natriurético Atrial/biossíntese , Northern Blotting , Ecocardiografia , Fator de Crescimento Epidérmico/metabolismo , Hipertrofia , Inflamação , Insulina/metabolismo , Peptídeo Natriurético Encefálico/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Associadas a Pancreatite , Condicionamento Físico Animal , RNA/química , Ratos , Ratos Endogâmicos Dahl , Transdução de SinaisRESUMO
Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development.
Assuntos
Imunoconjugados/química , Imunoconjugados/farmacologia , Polímeros/química , Alcaloides de Vinca/química , Acetais/química , Animais , Antígenos CD20/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoconjugados/farmacocinética , Células MCF-7 , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Receptor ErbB-2/imunologia , Rituximab/química , Rituximab/imunologia , Fatores de Tempo , Trastuzumab/química , Trastuzumab/imunologia , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The foundations of the Mitochondrial mutational theory of aging include two assumptions: the high abundance of mitochondrial mutations and their ability to clonally expand within individual cells. The up-to-date data pertinent to these assumptions is reviewed and semi-quantitative estimates of the frequencies of mutants and intracellular expansions are offered. The incidence of mutations in various aged tissues may be on the order of one mutant per mitochondrial genome copy, and most of the cells are likely to be affected by intracellular clonal expansions of mitochondrial genomes. Thus aged tissue may be considered a mosaic of cells with different mutant mitochondrial genotypes. Interestingly, independent studies show that a wide range of aged tissues presents with a mosaic of cells with different mitochondrial phenotypes. The necessary methodologies are available to explore whether the two mosaics are causally related. The answer apparently is positive in muscle; other tissues, brain in particular, await exploration.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Mutação , Genoma Humano , Genótipo , Humanos , Modelos Genéticos , Mosaicismo , FenótipoRESUMO
It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible for some aspects of the aging process. However, most cells contain hundreds to thousands of mtDNA molecules. Any nascent somatic mutant therefore appears as a single copy among a majority of wild-type species. A single mutant molecule is unlikely to influence the physiology of the cell and thus cannot play a role in the aging process. To affect cellular physiology, the nascent somatic mutants must somehow accumulate clonally in the cell to significant levels. The evidence supporting the view that, indeed, clonal expansion of mtDNA mutations is a widespread process in various human tissues, and the mechanisms by which clonal expansions may affect the aging process, are reviewed. Originally, clonal expansion was demonstrated for mtDNA with large deletions in muscle. Cell-by-cell analysis of human cardiomyocytes and buccal epithelial cells revealed that clonal expansion affects point mtDNA mutations as well as deletions. Expansions are not limited to muscle, but likely are present in most tissues, and almost every cell of an aged tissue is likely to be affected by an expansion. While the very existence of clonal expansion is beyond doubt, the mechanisms driving this process are largely controversial. The hypotheses explaining expansion includes random or various selective mechanisms, or both. We show that the spectra of expanded point mutations are drastically different in cardiomyocytes and epithelial cells. This suggests that the mechanisms of expansion in these tissues are different. In particular, we propose random segregation and positive selection models for epithelial and muscle cells, respectively.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Mucosa Bucal/citologia , Mutação Puntual , Envelhecimento/fisiologia , Separação Celular , Células Clonais , DNA Mitocondrial/metabolismo , Células Epiteliais/fisiologia , Coração/fisiologia , Humanos , Mucosa Bucal/fisiologia , Miocárdio/citologiaRESUMO
Combretastatin A-4 phosphate (CA4P) is a novel and promising anti-neoplastic agent. However, it is associated with transient hypertension in both animal and human models. In this study, we examined the potential cardiac toxicity and hypertensive effects of CA4P, and defined the most effective pharmacological inhibition of CA4P-induced hypertension in rats. There was a significant, concentration dependent increase in mean arterial blood pressure with a maximum increase of about 60% of the baseline MAP at 30 mg/kg of CA4P compared to the saline control. However, there was no significant increase in the cardiac troponin I level after CA4P injection. Nitroglycerin and the calcium channel blocker diltiazem effectively blocked the hypertensive effects of CA4P while the beta blocker metoprolol was ineffective. Furthermore, sublingual nitroglycerin administration demonstrated an additional anti-hypertensive effect in a setting of a low dose diltiazem infusion (10 microg/kg/min). We conclude that CA4P treatment resulted in a concentration dependent increase in blood pressure without significant myocardial damage in healthy rats. The hypertensive effect of CA4P was effectively blocked by both nitroglycerin and diltiazem, but not metoprolol.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Diltiazem/farmacologia , Relação Dose-Resposta a Droga , Masculino , Metoprolol/farmacologia , Nitroglicerina/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Estilbenos/antagonistas & inibidores , Troponina I/sangueRESUMO
The phosphoinositide 3-kinase (PI3-kinase)-protein kinase B (Akt) signaling pathway is essential in the induction of physiological cardiac hypertrophy. In contrast, protein kinase C beta2 (PKCbeta2) is implicated in the development of pathological cardiac hypertrophy and heart failure. Thus far, no clear association has been demonstrated between these two pathways. In this study, we examined the potential interaction between the PI3-kinase and PKCbeta2 pathways by crossing transgenic mice with cardiac specific expression of PKCbeta2, constitutively active (ca) PI3-kinase, and dominant-negative (dn) PI3-kinase. In caPI3-kinase/PKCbeta2 and dnPI3-kinase/PKCbeta2 double-transgenic mice, the heart weight-to-body weight ratios and cardiomyocyte sizes were similar to those observed in caPI3-kinase and dnPI3-kinase transgenic mice, respectively, suggesting that the regulation of physiological developmental hypertrophy via modulation of cardiomyocyte size proceeds through the PI3-kinase pathway. In addition, we observed that caPI3-kinase/PKCbeta2 mice showed improved cardiac function while the function of dnPI3-kinase/PKCbeta2 mice was similar to that of the PKCbeta2 group. PKCbeta2 protein levels in both dnPI3-kinase/PKCbeta2 and PKCbeta2 mice were significantly upregulated. Interestingly, however, PKCbeta2 protein expression was significantly attenuated in caPI3-kinase/PKCbeta2 mice. PI3-kinase activity measured by Akt phosphorylation was not affected by PKCbeta2 overexpression. These data suggest a potential interaction between these two pathways in the heart, where PI3-kinase is predominantly responsible for the regulation of physiological developmental hypertrophy and may act as an upstream modulator of PKCbeta2 with the potential for rescuing the pathological cardiac dysfunction induced by overexpression of PKCbeta.