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Penetratins 47
2.5.3.2 Oligonucleotides
As in every antisense strategy, there is no general rule for obtention of a biological
effect. For example, the abundance of the target mRNA, the stability of the cognate
protein, and the percentage of inhibition necessary to obtain an effect are usual
limiting factors. However, several parameters must be considered. The design of the
oligonucleotide must minimize the possibility of secondary structure formation and
of cross-hybridization with other RNAs. The site of translation start is often chosen
as a target but, when possible, it is preferable to test several oligonucleotides that
hybridize with different portions of the target mRNA. Fifteen to twenty-five base
oligonucleotides are usually adequate. Longer oligonucleotides are not always more
efficient and can reduce the efficacy of internalization. Phosphorothioate analogues
of oligonucleotides with a longer half-life can be used, but precipitation with the
vector often occurs during the coupling reaction. The concentration of coupled
oligonucleotide hybrids is variable; as a general rule, the coupled oligonucleotide
is 100- to 1000-fold more active than the uncoupled oligonucleotide. Generally,
maximal effects are obtained with 10 to 100 nM and targeted proteins are downregulated
after 1 to 3 h of treatment.
2.5.3.3 Peptide Nucleic Acids
According to the technical data available for the PNA–penetratin hybrids, no toxicity
was observed for concentrations up to 150 µM with 21 mer PNAs. Concentrations
used range ex vivo from 35 to 350 nM; the only in vivo study reports the use of
150 µM of PNA–penetratin conjugate delivered by intrathecal injections to target
the galanin receptor in rat CNS. Biological effects were obtained ex vivo from 30
min to 5 h following treatment, and in vivo after 48 h of treatment. 67
2.5.3.4 Drugs
Two publications reported targeting penetratin-coupled anticancer drug doxorubicin
ex vivo in transformed K562 human cells and in vivo through BBB of rats. Maximal
biological effect (killing of transformed cells) was obtained ex vivo after 48 h of
treatment (4 µM of hybrid peptide vs. 120 µM of free doxorubicin). 73 Doxorubicin
transfer through BBB was 20-fold more efficient when coupled to penetratin than
uncoupled. Delivery into the brain was observed rapidly after the intravenous injection
of 2.5 mg peptide/kg body weight, within 30 min after the injection. 56,57,78,80-83
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