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1、Arch Virol (1997) 142: 15851602 Beta-cyclodextrin derivatives as carriers to enhance the antiviral activity of an antisense oligonucleotide directed toward a coronavirus intergenic consensus sequence S. Abdou, J. Collomb, F. Sallas, A. Marsura, and C. Finance GEVSM, EA 1123, Universite Henri Poincar
2、e Nancy 1, Faculte de Pharmacie, Nancy, France Accepted March 17, 1997 Summary. The ability of cyclodextrins to enhance the antiviral activity of a phosphodiester oligodeoxynucleotide has been investigated. A 18-mer oligodeoxynucleotide complementary to the initiation region of the mRNA coding for t
3、he spike protein and containing the intergenic consensus sequence of an enteric coronavirus has been tested for antiviral action against virus growth in human adenocarcinoma cells. The phosphodiester oligodeoxynucleo- tide only showed a limited effect on virus growth rate (from 12 to 34% viral inhib
4、ition in cells treated with 7.5 to 25mM oligodeoxynucleotide, respectively, at a multiplicity of infection of 0.1 infectious particle per cell). In the same conditions, the phosphorothioate analogue exhibited stronger antiviral activity, the inhibition increased from 56 to 90%. The inhibitory effect
5、 of this analogue was antisense and sequence-specifi c. Northern blot analysis showed that the sequence-dependent mechanism of action appears to be the inhibition of mRNA transcription. We conclude that the coronavirus intergenic consensus sequence is a good target for an antisense oligonucleotide a
6、ntiviral action. The properties of the phosphodiester oligonucleotide was improved after its complexation with cyclodextrins. The most important increase of the antiviral activity (90% inhibition) was obtained with only 7.5mM oligonucleotide complexed to a cyclodextrin derivative, 6-deoxy-6-S-b-D-ga
7、lactopyranosyl-6-thio-cyclomalto- heptaose in a molar ratio of 1:100. These studies suggest that the use of cyclodextrin derivatives as carrier for phosphodiester oligonucleotides delivery may be an effective method for increasing the therapeutic potential of these compounds in viral infections. Int
8、roduction The use of antisense oligonucleotides is an interesting approach to study cellular and viral gene function and to block gene expression 2, 3, 60. Oligonucleotides have been considered as ideal agents for inhibiting viral replication due in part to their high specifi city for viral DNA and
9、RNA sequences. These agents have been successfully employed to block the replication and cellular expression of several viruses, including herpes simplex virus 20, cytomegalovirus 43 and especially human immunodefi ciency virus 33. However, the vulnerability of oligonuleotides to nucleases attack, c
10、ombined with their intracellular distribution and uptake properties, have limited their therapeutic potential. To avoid the problem of nucleases digestion and to improve the cellular uptake of oligonucleotides, either chemical modifi cations of the internucleoside phosphodiester linkages 35 or of th
11、e ribose sugar 25 and various oligonucleotide conjugates have been tested. One chemical analog that is widely employed for enhanced stability is the phosphorothioate oligonucleotide. But, although this analog has increased resistance to degradation in vivo and in vitro 1, 46, 59 it has exhibited seq
12、uence-specifi c and non sequence-specifi c activities 11, 54, 55. The oligonucleotide conjugates include oligonucleotide adsorbed onto poly (isobutylcyanoacrylate) nanoparticles 40, encapsulated in liposomes 23 or conjugated to the cholesterol 18, 26. These strategies have resulted in an increase of
13、 the cellular uptake of oligonucleotide and/or stability against nucleases. However, the inherent properties of the cholesterol, liposomes or nanoparticles carriers led to increase the immunogenicity of the oligonucleotide complex and the tendency of the complex to be phagocytosed by the reticuloend
14、othelial system 44. Investigations in cyclodextrin chemistry fi eld have been increased since several decades. Cyclodextrins are molecular hosts acting as carriers. It frameworks describes a group of cyclic polysaccharides containing 6 to 8 glucose units in a-(1,4) linkage (a-CD, b-CD and g-CD have
15、respectively 6, 7 or 8 glucose units). They possess an hydrophobic internal cavity which can include various small molecules and a peripheral structure containing a large number of hydroxyl groups making the molecule water soluble. The cyclodextrins form complexes with numerous compounds and b-cyclo
16、dextrin is the most employed for numerous applications. A wide variety of cyclodextrins and derivatives has been studied to enhance the solubility and the availability of various water-soluble and water-insoluble pharmacological agents 6, 9, 21, 38, 64. This approach using cyclodextrin derivatives has been recently studied to increase the cellular uptake of phosphorothioate oligonu- cleotides 65. In this work, we have investigated the ability of native b- and g- cyclodextrins and b-cyclodex
