RESUMEN
OBJECTIVES: Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogues that can inhibit bacterial growth by a gene-specific, antisense mechanism. Attaching cationic peptides to PMOs enables efficient penetration through the Gram-negative outer membrane. We hypothesized that cationic groups attached directly to the PMO would obviate the need to attach peptides. METHODS: PMOs with identical 11-base sequence (AcpP) targeted to acpP (an essential gene) of Escherichia coli were synthesized with various numbers of either piperazine (Pip) or N-(6-guanidinohexanoyl)piperazine (Gux) coupled to the phosphorodiamidate linker. Peptide-PMO conjugates were made using the membrane-penetrating peptide (RXR)(4)XB (X is 6-aminohexanoic acid; B is beta-alanine). RESULTS: MICs (microM/mg/L) were measured using E. coli: 3 + Pip-AcpP, 160/653; 6 + Pip-AcpP, 160/673; 2 + Gux-AcpP, 20/88; 5 + Gux-AcpP, 10/49; 8 + Gux-AcpP, 10/56; 3 + Pip-AcpP-(RXR)(4)XB, 0.3/2; and 5 + Gux-AcpP-(RXR)(4)XB, 0.6/4. In cell-free protein synthesis reactions, all PMOs inhibited gene expression approximately the same. These results suggested that Pip-PMOs inefficiently penetrated the outer membrane. Indeed, the MICs of 3 + Pip-AcpP and 6 + Pip-AcpP were reduced to 0.6 and 2.5 microM (1.2 and 10.5 mg/L), respectively, using as indicator a strain with a 'leaky' outer membrane. In vivo, mice were infected intraperitoneally with E. coli. Intraperitoneal treatment with 50 mg/kg 3 + Pip-AcpP, 15 mg/kg 5 + Gux-AcpP or 0.5 mg/kg 3 + Pip-AcpP-(RXR)(4)XB, or subcutaneous treatment with 15 mg/kg 5 + Gux-AcpP or (RXR)(4)XB-AcpP reduced bacteria in blood and increased survival. CONCLUSIONS: Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux-PMOs were more effective than Pip-PMOs. However, neither was as effective as the equivalent PMO-peptide conjugates. Subcutaneous treatment showed that 5 + Gux-AcpP or (RXR)(4)XB-AcpP entered the circulatory system, reduced infection and increased survival.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Morfolinas/farmacología , Animales , Antibacterianos/administración & dosificación , Antibacterianos/síntesis química , Antibacterianos/farmacocinética , Sangre/microbiología , Infecciones por Escherichia coli/tratamiento farmacológico , Femenino , Humanos , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Morfolinas/administración & dosificación , Morfolinas/síntesis química , Morfolinas/farmacocinética , Morfolinos , Peritonitis/tratamiento farmacológico , Análisis de SupervivenciaRESUMEN
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.
Asunto(s)
Antivirales , Ebolavirus/efectos de los fármacos , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Morfolinas , Oligonucleótidos Antisentido , Animales , Antivirales/química , Antivirales/farmacología , Antivirales/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ebolavirus/genética , Ebolavirus/fisiología , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Morfolinas/química , Morfolinas/farmacología , Morfolinas/uso terapéutico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Conejos , Relación Estructura-Actividad , Resultado del Tratamiento , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Antisense phosphorodiamidate Morpholino oligomers (PMO) are resistant to degradation by cellular hydrolases, DNases, RNases, and phosphodiesterases, but remain sensitive to prolonged exposure to low pH. The present studies evaluate the oral fractional bioavailability, stability, and efficacy of two distinct PMO sequences targeted to c-myc and cytochrome P-450 (CYP) 3A2. The c-myc antisense 20-mer, AVI-4126 (5'-ACGTTGAGGGGCATCGTCGC-3'), slowed the regenerative process in the rat liver after a 70% partial hepatectomy (PH). Rats were administered 3.0 mg/kg AVI-4126 in 0.1 mL saline via a bolus intravenous injection or in 0.5 mL sterile phosphate-buffered saline via gavage immediately following PH. The areas under the plasma concentration versus time curves revealed a fractional oral availability of 78.8% over a period of 10 min through 24 h. Immunoblot analysis of liver tissue from rats treated orally with AVI-4126 demonstrated a sequence-specific reduction in the target protein c-Myc, as well as secondary proliferation markers: proliferating cell nuclear antigen (PCNA), cyclin D1, and p53. The CYP3A2 antisense 22-mer AVI-4472 (5'-GAGCTGAAAGCAGGTCCATCCC-3') caused a sequence-dependent reduction of approximately five-fold in the rat liver CYP3A2 protein levels and erythromycin demethylation activity in 24 h following oral administration at a dose of 2 mg/kg. It is concluded that oral administration of PMOs can inhibit c-myc and CYP3A2 gene expression in rat liver by an antisense-based mechanism of action. These studies highlight the potential for development of PMOs as orally administered therapeutic agents.
Asunto(s)
Elementos sin Sentido (Genética)/farmacocinética , Sistema Enzimático del Citocromo P-450/biosíntesis , Compuestos de Fósforo/farmacocinética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Esteroide Hidroxilasas/biosíntesis , Administración Oral , Animales , Elementos sin Sentido (Genética)/sangre , Elementos sin Sentido (Genética)/farmacología , Disponibilidad Biológica , Biomarcadores/análisis , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Hígado/química , Hígado/efectos de los fármacos , Hígado/enzimología , Regeneración Hepática/efectos de los fármacos , Regeneración Hepática/fisiología , Masculino , Compuestos de Fósforo/sangre , Compuestos de Fósforo/farmacología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Esteroide Hidroxilasas/antagonistas & inhibidores , Esteroide Hidroxilasas/metabolismoRESUMEN
The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is beta-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to luc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 h of growth (50% inhibitory concentration [IC(50)]) were 3.6, 10.8, 9.5, and 7.5 microM, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 microM AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC(50) of either AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 microM. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.