title: Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo.
abstract: OBJECTIVE
  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.
authors.affiliation: AVI BioPharma, Inc., Corvallis, OR 97333, USA.
authors.forename: Brett L
authors.initials: BL
authors.lastname: Mellbye
authors.forename: Dwight D
authors.initials: DD
authors.lastname: Weller
authors.forename: Jed N
authors.initials: JN
authors.lastname: Hassinger
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Reeves
authors.forename: Candace E
authors.initials: CE
authors.lastname: Lovejoy
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Geller
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D001769:Blood / Q000382:microbiology; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development*; D004927:Escherichia coli Infections / Q000188:drug therapy; D005260:Female; D006801:Humans; D007279:Injections, Subcutaneous; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D009025:Morpholines / Q000008:administration & dosage / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D060172:Morpholinos; D010538:Peritonitis / Q000188:drug therapy; D016019:Survival Analysis
chemical_list: D000900:Anti-Bacterial Agents; D009025:Morpholines; D060172:Morpholinos