Design and Development of Thermolytic DNA Oligonucleotide Prodrugs

Abstract

Deoxyribonucleoside phosphoramidites functionalized with the thermolytic 2-(N-formyl-N-methyl)aminoethyl group for phosphorus protection (1a-d) have been prepared and employed in the solid-phase synthesis of CpG ODN fma1555. Given that this modified oligonucleotide can be converted to the immunomodulatory CpG ODN 1555 under neutral conditions at 37 degrees C, its biologic activity was demonstrated in vivo by studies showing that intraperitoneal administration of CpG ODN fma1555 in mice resulted in the activation of cytokine-secreting splenocytes. Furthermore, administration of CpG ODN fma1555 to mice that were challenged intradermally in the ear with live L. major metacyclic promastigotes, reduced the severity of Leishmania skin lesions over time to an extent similar to that obtained with CpG ODN 1555. In another infectious model experiment, CpG ODN fma1555 protected newborn mice from death (65% survival) when administered 3 days before infection with the aggressive Tacaribe (TCRV) virus. A comparable immunoprotection was obtained by treatment of TCRV-infected mice with CpG ODN 1555 administered on the same day of infection (45% survival). However, when TCRV-infected mice were treated with CpG ODN fma1555 on the day of infection, they died as a consequence of the relatively slow conversion of the oligonucleotide prodrug to the bioactive CpG ODN 1555. Co-administration of both CpG ODN 1555 and CpG ODN fma1555 to mice 3 days prior to TCRV infection or on the day of infection provided protection from death (45-65% survival) and thus widened the immunoprotection window against TCRV-infection.

Solid-Phase Synthesis of Thermolytic DNA Oligonucleotides Functionalized with a Single 4-Hydroxy-1-butyl or 4-Phosphato-/Thiophosphato-1-butyl Thiophosphate Protecting Group

“…The information obtained collectively from the dinucleotide prodrug models in terms of prodrug-to-drug conversion under thermolytic conditions prompted us to initiate the solid-phase synthesis of oligonucleotide CpG ODN hbu 1555, which was performed by employing deoxyribonucleoside phosphoramidites functionalized with the 2-( N -formyl- N -methyl)aminoethyl group for phosphorus protection and phosphoramidite 8b under conditions identical to those used for the preparation of CpG ODN fma 1555 5a. The average coupling efficiency of these phosphoramidites was 98−99%, as determined by colorimetric measurements of the dimethoxytrityl cation after each chain elongation step.…”

“…Upon cellular uptake of these oligonucleotide prodrugs, hydrolysis of the phosphodiester masking groups by intracellular enzymes generates bioactive oligonucleotide drugs. We recently reported a new class of DNA oligonucleotide prodrugs, which did not require intracellular enzyme(s) for prodrug-to-drug conversion. 5a,b This class of oligonucleotide prodrugs includes oligonucleoside phosphorothioates functionalized with the thermolytic 2-( N -formyl- N -methyl)aminoethyl group for thiophosphate protection (Scheme ). These DNA oligonucleotides exhibit the characteristics of oligonucleotide prodrugs in that they are uncharged to facilitate cellular delivery and are stable to hydrolytic nucleases.…”

“…These DNA oligonucleotides exhibit the characteristics of oligonucleotide prodrugs in that they are uncharged to facilitate cellular delivery and are stable to hydrolytic nucleases. A distinctive feature of this class of modified oligonucleotides is that only an aqueous environment, maintained at a temperature of 37 °C or above, is necessary for converting oligonucleoside 2-( N -formyl- N -methyl)aminoethyl phosphorothioate triesters ( 1 ) to functionally bioactive oligonucleoside phosphorothioate diesters ( 2 ) within a reasonable period of time. 5a,b

1 Thermolytic Conversion of DNA Oligonucleotide Prodrugs to Bioactive Therapeutic Oligonucleotides a a B = thymin-1-yl, cytosin-1-yl, adenin-9-yl, or guanin-9-yl.

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“…Thus, when synthetic single-stranded DNA oligonucleoside phosphorothioates containing unmethylated CpG motifs (CpG ODNs) are functionalized with 2-( N -formyl- N -methyl)aminoethyl thiophosphate protecting groups and administered to mice, an immunostimulatory response similar to that produced with traditional CpG ODN phosphodiesters in terms of the number of cells secreting cytokines, chemokines, and immunoglobulins is generated. 5a,b However, a delay in the induction of these immunostimulatory events is observed, consistent with the thermolytic conversion half-time of 2-( N -formyl- N -methyl)aminoethyl thiophosphate triesters ( t 1/2 = 73 h at 37 °C) to the biologically active phosphorothioate diesters. A noteworthy outcome of these findings relates to the co-administration of CpG ODN produgs of type 1 and conventional CpG ODN of type 2 in mice, which widened the timetable of therapeutic treatment against specific viral infections. 5a,b These observations prompted us to design CpG ODN prodrugs exhibiting either a shorter or longer half-time of thiophosphate deprotection relative to that of CpG ODN 1 for the preparation of effective and long-acting immunotherapeutic oligonucleotide formulations against various infectious diseases in animal models. However, the design of thermolytic oligonucleotide prodrugs poses a paradox in that one must consider the criticality of oligonucleotide solubility in biological media, which precludes the use of lipophilic thiophosphate protecting groups, and of cellular uptake, which is reportedly commensurate to the relative lipophilicity of oligonucleotides 2f…”

“…Given that CpG ODN fma 1555 [ d (G PS(FMA) C PS(FMA) T PS(FMA) -A PS(FMA) G PS(FMA) A PS(FMA) C PS(FMA)- G PS(FMA) T P S(FMA) T PS(FMA) -A PS(FMA) G PS(FMA) C PS(FMA) G PS(FMA) T), where PS(FMA) stands for the thermolytic 2-( N -formyl- N -methyl)aminoethyl phos-phorothioate triester function] is functional as a prodrug in mice, 5a,b we propose to evaluate the biophysical implications of replacing the thermolytic thiophosphate protecting group of the CpG within the motif G PS(FMA) A PS(FMA) C PS(FMA) G PS(FMA) -T PS(FMA) T 5c with thermolabile groups displaying slower or faster deprotection kinetics than those of the FMA group. These new heat-sensitive groups were designed to impart similar or better solubility properties in biological media than those in the FMA groups .…”