Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs

Abstract

A CpG-containing DNA oligonucleotide functionalized with the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group (CpG ODN fma1555) was prepared from phosphoramidites 1a–d using solid-phase techniques. The oligonucleotide behaved as a prodrug by virtue of its conversion to the well-studied immunomodulatory CpG ODN 1555 through thermolytic cleavage of the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group. Such a conversion occurred at 37°C with a half-time of 73 h. The immunostimulatory properties of CpG ODN fma1555 were evaluated in two in vivo assays, one of which consisted of mice challenged in the ear with live Leishmania major metacyclic promastigotes. Local intradermal administration of CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions over time. In a different infectious model, CpG ODN 1555 prevented the death of Tacaribe-infected mice (43% survival) when administered between day 0 and 3 post infection. Administration of CpG ODN fma1555 three days before infection resulted in improved immunoprotection (60–70% survival). Moreover, co-administration of CpG ODN fma1555 and CpG ODN 1555 in this model increased the window for therapeutic treatment against Tacaribe virus infection, and thus supports the use of thermolytic oligonucleotides as prodrugs in the effective treatment of infectious diseases.

Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates

“…Deprotection and purification of pro-D35, pro-D144 and fma -D35 ODN was carried out identically as described earlier (16). The purity of the ODN was assessed by RP-HPLC using an analytical 5 μm Supelcosil LC-18S column under conditions described elsewhere (16).…”

“…The purity of the ODN was assessed by RP-HPLC using an analytical 5 μm Supelcosil LC-18S column under conditions described elsewhere (16). …”

“…The samples were loaded on a native gel (20% polyacrylamide and 1× TBE, pH 8.3) and were electrophoresed at 250 V until the bromophenol blue dye had traveled 20 cm at ∼25°C. The gel was then stained using stains-all and processed as described elsewhere (16). …”

“…To address this issue, we havedeveloped a pro-drug form of CpG ODN type D (Pro-D ODN) in which the backbone of the poly(G) track is functionalized with 2-( N -formyl- N -methyl)aminoethyl ( fma ) groups. The incorporation of these groups into ODN is achieved during solid-phase synthesis via phosphoramidites 1a – d [Figure 1, (16,17)]. In this study we assess the structural and immunostimulatory characteristics of pro-D ODN and demonstrate that they have reduced G-tetrad formation and multimerization in solution but retain their in vitro and in vivo immunomodulatory activity.…”

Síntese de pró-fármacos dendriméricos potencialmente antichagásicos e leishmanicidas derivados de hidroximetilnitrofural, 3-hidroxiflavona e quercetina

“…Those compounds are activated only by temperature (37 o C). The half-life of thiophosphate deprotection was estimated to be 73 h and the complete deprotection was achieved within 600 h. Studies in mice infected with Leishmania major metacyclic promastigotes [83] prove that CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions in local intradermal administration. Previous studies demonstrated that administration of ODN type D, which has a 3´ and poli(G) motif [84] -a group of gaunine ribonucleotide with phosphate residues acting as bridges to form diester linkages between the ribose moieties -improves its performance in healthy and immunocompromised non-human primates challenge with Leishmania major [85;86].…”