RESUMEN
Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice.
Asunto(s)
Macaca fascicularis , Morfolinos , Músculo Esquelético , Distrofia Muscular de Duchenne , Animales , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/sangre , Distrofia Muscular de Duchenne/metabolismo , Morfolinos/farmacocinética , Morfolinos/administración & dosificación , Ratones , Humanos , Distribución Tisular , Músculo Esquelético/metabolismo , Masculino , Exones/genética , Modelos Animales de Enfermedad , Ratones Endogámicos mdx , Ratones Endogámicos C57BLRESUMEN
Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.
Asunto(s)
Apolipoproteínas E , Barrera Hematoencefálica , Ratones Transgénicos , Oligonucleótidos Antisentido , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/farmacocinética , Humanos , Apolipoproteínas E/metabolismo , Ratones , Morfolinos/administración & dosificación , Morfolinos/farmacocinética , Morfolinos/farmacología , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismo , Atrofia Muscular Espinal/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Péptidos/administración & dosificación , Péptidos/farmacología , Péptidos/química , Péptidos/farmacocinética , Péptidos de Penetración Celular/químicaRESUMEN
PURPOSE: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have shown promise in treating Duchenne muscular dystrophy (DMD). We evaluated a semi-mechanistic pharmacokinetic (PK) and pharmacodynamic (PD) model to capture the relationship between plasma and muscle tissue exposure/response in mdx mice treated by mouse surrogate PPMO. METHODS: A single or repeated (every 4 weeks for 20 weeks) intravenous PPMO dose was administered to mdx mice (n = 6/timepoint). A PK/PD model was built to characterize data via sequential modeling. A 2-compartment model was used to describe plasma PK. A simultaneous tissue PK/PD model was subsequently developed: 2-compartment model to describe muscle PK; linked to an indirect response model describing stimulation of synthesis of skipped transcript, which was in turn linked to stimulation of synthesis of dystrophin protein expression. RESULTS: Model performance assessment via goodness-of-fit plots, visual predictive checks, and accurate parameter estimation indicated robust fits of plasma PK and muscle PK/PD data. The model estimated a PPMO tissue half-life of 5 days-a useful parameter in determining the longevity of PPMOs in tissue and their limited accumulation after multiple doses. Additionally, the model successfully described dystrophin expression after single dosing and associated protein accumulation after multiple dosing (increasing ~ twofold accumulation from the first to last dose). CONCLUSIONS: This first PK/PD model of a PPMO in a DMD disease model will help characterize and predict the time course of PK/PD biomarkers in mdx mice. Furthermore, the model framework can be used to develop clinical PK/PD models and can be extended to other exon-skipping therapies and species.
Asunto(s)
Péptidos de Penetración Celular/química , Morfolinos/farmacocinética , Distrofia Muscular de Duchenne/tratamiento farmacológico , Animales , Área Bajo la Curva , Simulación por Computador , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Distrofina/genética , Distrofina/metabolismo , Semivida , Humanos , Masculino , Ratones Endogámicos mdx , Modelos Biológicos , Modelos Estadísticos , Morfolinos/sangreRESUMEN
A nontoxic delivery vehicle is essential for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs). Though guanidinium-rich or arginine-rich cellular transporter conjugated Vivo-PMO or PPMO has been developed for in vivo application, however, either their toxicity or stability has become an issue. Previously, we reported nonpeptidic internal guanidinium transporter (IGT) mediated delivery of PMO for gene silencing and got encouraging results. In this paper, we report the synthesis of IGT using a Hg-free method for scale up and N-terminal modification of IGT with a suitable hydrophobic or lipophilic group to improve the cell permeability, endosomal escape, and mitochondrial localization and to reduce toxicity in the MTT assay. For the delivery of PMO, IGT-PMO conjugate was synthesized to target NANOG in cells, a transcription factor required for cancer stem cell proliferation and embryonic development and is involved in many cancers. Our data shows IGT-PMO-facilitated NANOG inhibition, and thereby the prevention of EpCAM-N-Cadherin-Vimentin axis mediated epithelial to mesenchymal transition (EMT) in MCF-7 cells. Moreover, unlike taxol, NANOG inhibition influences the expression of stemness factor c-Myc, Hh-Gli signaling proteins, other cancer related factors, and their respective phenotypes in cancer cells. To the best of our knowledge, this is the first report to illustrate that the IGT-PMO-mediated NANOG inhibition increases the therapeutic potential of taxol and induces G0-G1 arrest in cancer cells to prevent cancer progression. However, it warrants further investigation in other cancer cells and preclinical platforms.
Asunto(s)
Antineoplásicos/administración & dosificación , Morfolinos/administración & dosificación , Proteína Homeótica Nanog/antagonistas & inhibidores , Paclitaxel/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Portadores de Fármacos/química , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Morfolinos/farmacocinética , Morfolinos/farmacología , Proteína Homeótica Nanog/genética , Paclitaxel/farmacocinética , Paclitaxel/farmacologíaRESUMEN
Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca2+-enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.
Asunto(s)
Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Oligonucleótidos/química , Oligonucleótidos/genética , Transfección/métodos , Células A549 , Animales , Secuencia de Bases/fisiología , Técnicas de Cultivo de Célula , Línea Celular , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Morfolinos/química , Morfolinos/genética , Morfolinos/farmacocinética , Conformación de Ácido Nucleico/efectos de los fármacos , Oligonucleótidos/farmacocinética , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacocinética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.
Asunto(s)
Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Neurotensina/química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinética , Animales , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Morfolinos/administración & dosificación , Morfolinos/química , Morfolinos/farmacocinética , Oligonucleótidos Antisentido/químicaRESUMEN
The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is epigenetically silenced in some tumors by MGMT gene promoter methylation. MGMT-hypermethylated solid tumors have enhanced susceptibility to the cytotoxic effects of alkylating chemotherapy such as temozolomide, compared with non-methylated tumors. In glioblastoma, subjects with MGMT hypermethylation have significantly longer survival rates after chemoradiotherapy. We report the first successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-MGMT morpholino oligonucleotide (AMON) sequences. We demonstrate >40% reduction in the in vitro proliferation index and cell viability in radiation-primed MGMT-expressing human solid tumor cells treated with a single dose of AMONs and temozolomide. We further demonstrate the feasibility of using a non-ablative dose of radiation in vivo to guide and enhance the delivery of intravenously administered AMONs to achieve 50% MGMT knockdown only at radiation-primed tumor sites in a subcutaneous tumor model. Local upregulation of physiological endocytosis after radiation may have a role in radiation-guided uptake of AMONs. This approach holds direct translational significance in glioblastoma and brain metastases where radiation is part of the standard of care; our approach to silence MGMT could overcome the significant problem of MGMT-mediated chemoresistance.
Asunto(s)
Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Neoplasias/terapia , Oligonucleótidos Antisentido/administración & dosificación , Proteínas Supresoras de Tumor/genética , Células A549 , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Quimioradioterapia , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Femenino , Humanos , Inmunohistoquímica , Morfolinos/administración & dosificación , Morfolinos/genética , Morfolinos/farmacocinética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/radioterapia , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacocinética , Ratas , Ratas Desnudas , Transfección , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
AIM: AVI-7100 (Radavirsen) is a 20-mer phosphorodiamidate morpholino oligomer (PMOplus®) for the treatment of influenza. Results/methodology: An automated solid-phase extraction method was used to extract plasma samples (200 µl). The extracts were analyzed using liquid chromatography coupled with tandem mass spectrometry under the positive ionization mode. This method was fully validated over the calibration curve range of 5.00-1000 ng/ml. The between-run precision and accuracy ranged from 0.0 to 5.2% relative standard deviation and 91.6 to 100.0% of nominal for all quality control concentrations, including the lower limit of quantitation. DISCUSSION/CONCLUSION: This is the first liquid chromatography coupled with tandem mass spectrometry method for the measurement of AVI-7100 concentrations in human plasma. It has been used to support regulated bioanalysis.
Asunto(s)
Análisis Químico de la Sangre/métodos , Cromatografía Liquida/métodos , Morfolinos/sangre , Espectrometría de Masas en Tándem/métodos , Calibración , Ensayos Clínicos como Asunto , Humanos , Morfolinos/efectos adversos , Morfolinos/aislamiento & purificación , Morfolinos/farmacocinética , Seguridad , Extracción en Fase SólidaRESUMEN
Differing from the conventional direct-targeting strategy in which a probe or payload is directly loaded onto a targeting molecule that binds to the native target, pretargeting is an improved targeting strategy. It converts the native target to an artificial target specific for a secondary targeting molecule loaded with the probe or payload (effector). The effector is small and does not accumulate in normal tissues, which accelerates the targeting process and generates high target to nontarget ratios. DNA/cDNA analogs can serve as the recognition pair, i.e., the artificial target and the secondary targeting effector. Morpholino oligomers are so far the most investigated and the most successful DNA/cDNA analog recognition pairs for pretargeting. Herein, we describe the pretargeting principles, the pretargeting strategy using Morpholino oligomers, and the preclinical success so far achieved.
Asunto(s)
Marcación de Gen , Morfolinos/genética , Animales , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , Inmunoconjugados , Marcaje Isotópico , Ratones , Morfolinos/administración & dosificación , Morfolinos/química , Morfolinos/farmacocinética , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Distribución TisularRESUMEN
Determining the concentration of oligonucleotide in biological samples such as tissue lysate and serum is essential for determining the biodistribution and pharmacokinetic profile, respectively. ELISA-based assays have shown far greater sensitivities compared to other methods such as HPLC and LC/MS. Here, we describe a novel ultrasensitive hybridization-based ELISA method for quantitating morpholino oligonucleotides in mouse tissue lysate and serum samples. The assay has a linear detection range of 5-250 pM (R2 > 0.99).
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Morfolinos/sangre , Morfolinos/farmacocinética , Hibridación de Ácido Nucleico/métodos , Animales , Ratones , Morfolinos/química , Morfolinos/genética , Sensibilidad y Especificidad , Estadística como Asunto , Distribución TisularRESUMEN
Attempts to lock the active conformation of compound 4, a PI3Kß/δ inhibitor (PI3Kß cell IC50 0.015µM), led to the discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-ones, which showed high levels of potency and selectivity as PI3Kß/δ inhibitors. Compound 10 proved exquisitely potent and selective: PI3Kß cell IC50 0.0011µM in PTEN null MDA-MB-468 cell and PI3Kδ cell IC50 0.014µM in Jeko-1 B-cell, and exhibited suitable physical properties for oral administration. In vivo, compound 10 showed profound pharmacodynamic modulation of AKT phosphorylation in a mouse PTEN-null PC3 prostate tumour xenograft after a single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral dosing. Based on these results, compound 10 was selected as one of our PI3Kß/δ preclinical candidates.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Benzopiranos/química , Benzopiranos/uso terapéutico , Fosfohidrolasa PTEN/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Benzopiranos/farmacocinética , Benzopiranos/farmacología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Perros , Eliminación de Gen , Humanos , Masculino , Ratones Desnudos , Simulación del Acoplamiento Molecular , Morfolinos/química , Morfolinos/farmacocinética , Morfolinos/farmacología , Morfolinos/uso terapéutico , Próstata/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood-brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141-150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.
Asunto(s)
Apolipoproteínas E/farmacocinética , Morfolinos/farmacología , Morfolinos/farmacocinética , Atrofia Muscular Espinal/tratamiento farmacológico , Péptidos/farmacocinética , Animales , Animales Recién Nacidos , Apolipoproteínas E/síntesis química , Apolipoproteínas E/química , Biomarcadores/sangre , Barrera Hematoencefálica/química , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Exones , Fibroblastos/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Riñón/química , Ratones , Morfolinos/química , Morfolinos/uso terapéutico , Nanoconjugados/análisis , Nanoconjugados/química , Nanoconjugados/uso terapéutico , Péptidos/síntesis química , Péptidos/química , Fenotipo , Músculo Cuádriceps/química , Proteína 2 para la Supervivencia de la Neurona Motora/efectos de los fármacosRESUMEN
Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).
Asunto(s)
Metabolismo Energético/fisiología , Morfolinos/farmacocinética , Pez Cebra/embriología , Animales , Bioensayo , Indicadores y Reactivos/farmacocinética , Oxazinas/farmacocinética , Xantenos/farmacocinéticaRESUMEN
BACKGROUND: AVI-7288 is a phosphorodiamidate morpholino oligomer with positive charges that targets the viral messenger RNA that encodes Marburg virus (MARV) nucleoprotein. Its safety in humans is undetermined. METHODS: We assessed the efficacy of AVI-7288 in a series of studies involving a lethal challenge with MARV in nonhuman primates. The safety of AVI-7288 was evaluated in a randomized, multiple-ascending-dose study in which 40 healthy humans (8 humans per dose group) received 14 once-daily infusions of AVI-7288 (1 mg, 4 mg, 8 mg, 12 mg, or 16 mg per kilogram of body weight) or placebo, in a 3:1 ratio. We estimated the protective dose in humans by comparing pharmacokinetic variables in infected nonhuman primates, uninfected nonhuman primates, and uninfected humans. RESULTS: Survival in infected nonhuman primates was dose-dependent, with survival rates of 0%, 30%, 59%, 87%, 100%, and 100% among monkeys treated with 0 mg, 3.75 mg, 7.5 mg, 15 mg, 20 mg, and 30 mg of AVI-7288 per kilogram, respectively (P<0.001 with the use of the log-rank test for the comparison of survival across groups). No safety concern was identified at doses up to 16 mg per kilogram per day in humans. No serious adverse events were reported. Drug exposure (the area under the curve) was dose-dependent in both nonhuman primates and humans; drug clearance was independent of dose but was higher in nonhuman primates than in humans. The protective dose in humans was initially estimated, on the basis of exposure, to be 9.6 mg per kilogram per day (95% confidence interval, 6.6 to 12.5) for 14 days. Monte Carlo simulations supported a dose of 11 mg per kilogram per day to match the geometric mean protective exposure in nonhuman primates. CONCLUSIONS: This study shows that, on the basis of efficacy in nonhuman primates and pharmacokinetic data in humans, AVI-7288 has potential as postexposure prophylaxis for MARV infection in humans. (Funded by the Department of Defense; ClinicalTrials.gov number, NCT01566877.).
Asunto(s)
Antivirales/administración & dosificación , Enfermedad del Virus de Marburg/tratamiento farmacológico , Marburgvirus , Morfolinos/administración & dosificación , Animales , Antivirales/efectos adversos , Antivirales/farmacocinética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Estimación de Kaplan-Meier , Macaca fascicularis , Enfermedad del Virus de Marburg/mortalidad , Marburgvirus/genética , Morfolinos/efectos adversos , Morfolinos/farmacocinética , ARN Mensajero , ARN ViralRESUMEN
Antisense oligonucleotide (AON)-induced exon skipping is one of the most promising strategies for treating Duchenne muscular dystrophy (DMD) and other rare monogenic conditions. Phosphorodiamidate morpholino oligonucleotides (PMOs) and 2'-O-methyl phosphorothioate (2'OMe) are two of the most advanced AONs in development. The next generation of peptide-conjugated PMO (P-PMO) is also showing great promise, but to advance these therapies it is essential to determine the pharmacokinetic and biodistribution (PK/BD) profile using a suitable method to detect AON levels in blood and tissue samples. An enzyme-linked immunosorbent assay (ELISA)-based method, which shows greater sensitivity than the liquid chromatography-mass spectrometry method, is the method of choice for 2'OMe detection in preclinical and clinical studies. However, no such assay has been developed for PMO/P-PMO detection, and we have, therefore, developed an ultrasensitive hybridization-based ELISA for this purpose. The assay has a linear detection range of 5-250 pM (R(2)>0.99) in mouse serum and tissue lysates. The sensitivity was sufficient for determining the 24-h PK/BD profile of PMO and P-PMO injected at standard doses (12.5 mg/kg) in mdx mice, the dystrophin-deficient mouse model for DMD. The assay demonstrated an accuracy approaching 100% with precision values under 12%. This provides a powerful cost-effective assay for the purpose of accelerating the development of these emerging therapeutic agents.
Asunto(s)
Péptidos de Penetración Celular/química , Morfolinos/química , Oligonucleótidos Antisentido/química , Animales , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/farmacocinética , Ensayo de Inmunoadsorción Enzimática , Inyecciones Subcutáneas , Límite de Detección , Ratones Endogámicos mdx , Morfolinos/administración & dosificación , Morfolinos/farmacocinética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinéticaRESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. METHODS AND RESULTS: By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. CONCLUSIONS: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.
Asunto(s)
Sistema Cardiovascular/crecimiento & desarrollo , Células Endoteliales/química , Regulación del Desarrollo de la Expresión Génica/genética , Miocitos Cardíacos/química , Células Madre Pluripotentes/química , ARN Largo no Codificante/aislamiento & purificación , Vertebrados/genética , Animales , Sistema Cardiovascular/metabolismo , Diferenciación Celular , Linaje de la Célula , Mapeo Cromosómico , Desarrollo Embrionario/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Corazón Fetal/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Datos de Secuencia Molecular , Morfolinos/farmacocinética , Miocitos Cardíacos/citología , ARN Largo no Codificante/fisiología , Análisis de Secuencia de ARN , Transcriptoma , Vertebrados/crecimiento & desarrollo , Pez Cebra/embriologíaRESUMEN
Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence.
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Fiebre Hemorrágica Ebola/tratamiento farmacológico , Enfermedad del Virus de Marburg/tratamiento farmacológico , Morfolinos/farmacocinética , Adulto , Animales , Área Bajo la Curva , Método Doble Ciego , Ebolavirus/efectos de los fármacos , Ebolavirus/genética , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Infusiones Intravenosas , Masculino , Enfermedad del Virus de Marburg/virología , Marburgvirus/efectos de los fármacos , Marburgvirus/genética , Persona de Mediana Edad , Morfolinos/efectos adversos , Morfolinos/sangre , Placebos , Adulto JovenRESUMEN
INTRODUCTION: The differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose. We believe some individual differences in biodistribution may be circumvented through this approach by calculating organ level ratios. METHODS: A model system of MORF/cMORF pretargeting (MORF/cMORF is a complementary pair of DNA analogues) was employed in connection with an on-going tumor therapeutic project. Human LS174T cells were implanted into the flank of severely immuno-compromised NOD-scid IL2rg(null) mice. The tumor was confirmed to express TAG-72 antigens. At 16 days post tumor inoculation, mice received IV 60 µg of MORF-conjugated CC49 (an antiTAG-72 antibody), followed 2 days later by a low-mass-dose IV coinjection containing 2.5 µg of (90)Y-cMORF and 2.5 µg of (99m)Tc-cMORF. At 3 h post radioactivity injection, the distribution of (99m)Tc was imaged on a SPECT/CT camera and then organs were excised and counted for (90)Y and (99m)Tc. Because the two labeled cMORFs do not react or interact with each other in vivo, the two groups of (90)Y and (99m)Tc data enabled a conventional group comparison. In a new effort, (90)Y/(99m)Tc ratios were calculated. Student's t-test and retrospective power analysis were performed for both approaches. In the new approach, the ratios were set at 1 as the null hypothesis. RESULTS: The Student's t-test in the conventional group approach indicated that the two labeled cMORFs distributed similarly, but significant differences were observed in salivary gland and large intestines. The coinjection-ratio approach certainly did not subvert the results of the conventional approach but revealed subtler differences. The P values were reduced, the powers were increased in most organs, and more significant differences were observed. The increased sensitivity was due to the reduced CV%s (SD/average*100%) of the (90)Y/(99m)Tc ratios. Therefore, some individual differences were circumvented and notably the ratio approach differentiated individual differences into ratio-correctable and ratio-uncorrectable. CONCLUSIONS: Although the conventional approach is reliable, the coinjection-ratio approach using organ level ratios is more sensitive and therefore is recommended whenever possible. In addition, it differentiates individual differences into "coinjection correctable" and "coinjection uncorrectable".
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Morfolinos , Radioisótopos de Itrio , Animales , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Interacciones Farmacológicas , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Inyecciones , Ratones , Morfolinos/genética , Morfolinos/farmacocinética , TecnecioRESUMEN
PURPOSE: Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents. METHODS AND RESULTS: Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of (99m)Tc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8pmol, respectively, per 10(10) cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90min for both (99m)Tc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of (99m)Tc-study MORF was significantly higher (p=0.009) than that of the control in the thigh with sterile inflammation. CONCLUSION: A (99m)Tc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.
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Morfolinos/química , Compuestos de Organotecnecio/química , ARN Bacteriano/metabolismo , Radiofármacos/química , Animales , Infecciones Bacterianas/diagnóstico , Escherichia coli/genética , Semivida , Hibridación Fluorescente in Situ , Klebsiella pneumoniae/genética , Ratones , Microscopía Fluorescente , Morfolinos/farmacocinética , ARN Ribosómico 16S/metabolismo , Radiofármacos/farmacocinética , Staphylococcus aureus/genética , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos XRESUMEN
A simple and fast homogeneous fluorescent polarization immunoassay (FPIA) was developed for the determination of furaltadone and its metabolite 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ). Monoclonal antibody with high cross-reactivity to furaltadone and the nitrophenyl derivative of AMOZ (NPAMOZ) were produced against a novel immunogen and the effects of several synthesized tracers on FPIA sensitivity studied. The proposed FPIA, using an optimum antibody and tracer pair, had an IC50 of 4.3 µg L⻹ and limit of detection at 0.6 µg L⻹ for furaltadone, and 2.7 µgL⻹ and 0.3 µg L⻹ for NPAMOZ. Recoveries of furaltadone from animal feeds by FPIA ranged from 79.6 to 87.7%, while recoveries of AMOZ from animal tissues ranged from 72.9 to 83.1%. Good correlation (R>0.99) between the results of this FPIA and a standard analytical method was obtained. The FPIA does not require separation or washing steps and the total time required for equilibrium of the antibody-tracer interaction is only 10 min. These results indicated that the proposed FPIA offers great potential and utility for the high throughput screening of furaltadone residues in animal feed and its metabolite AMOZ residues in animal tissues.