Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-02-03
2001-09-11
Crane, L. Eric (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S023100, C536S024500, C435S006120
Reexamination Certificate
active
06288042
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of oligonucleotide chemistry and anti-viral pharmacotherapy. More specifically, the present invention relates to therapeutically active guanosine-rich intramolecular tetrad forming oligonucleotides, to methods of treating viral diseases using said oligonucleotides, and to pharmaceutical compositions containing the novel oligonucleotides.
2. Description of the Related Art
General In Vitro Studies
Previously, it was believed that “antisense” oligonucleotides inhibit viruses by interfering with protein translation via an RNA:DNA duplex structure. More recent research, however, indicates a variety of possible mechanisms by which oligo-nucleotides inhibit viral infections. For example, oligodeoxycytidine (poly SdC) inhibits HIV-1. Marshall et al.,
PNAS
(1992) 89:6265-6269, discussed the potential mechanism (competitive inhibition) by which oligodeoxycytidine directly inhibits viral reverse transcriptase. Poly SdC also inhibited AMV reverse transcriptase and Pol I (Klenow fragment) and polymerase &agr;, &bgr; and &ggr;. Previously, Matsukura et al.,
PNAS
(1987) 84:7706-7710, used a similar phosphorothioate derivative of oligo-deoxycytidine to demonstrate inhibition of HIV-1 in culture. Marshall and Caruthers,
Science (
1993) 259:1564-1569, reported the use of diphosphorothioate oligo-nucleotides, e.g., antisense-specific, random nucleotide combinations and oligodeoxycytidine against HIV-1. In all cases, the mechanism of action was attributed to a direct inhibition of HIV-1 reverse transcriptase. Other potential mechanisms of anti-viral action of oligonucleotides were postulated by Boiziau et al.,
PNAS
(1992) 89:768-772, e.g., promotion of RNAse H activity and inhibition of reverse transcriptase initiating cDNA synthesis. In addition, Goa et al.,
Molecular Pharmacology
(1992) 41:223-229 reported that phosphorothioate oligonucleotides inhibit human DNA polymerases and RNAse H, and the adsorption or penetration of the virus into cells. Iyer et al.,
Nucleic Acids Research
(1990) 18:2855-2859 reported that if a base was removed from an anti-sense polynucleotide forming an abasic site, the compound did not lose its activity which argues against the need for the formation of an RNA:DNA antisense mediated hybrid for anti-viral activity. Stein et al. have characterized the interaction of poly SdC with the V3 loop of HIV-1 gp120, and postulated that the specific interaction of poly SdC with the HIV-1 V3 loop may be a mechanism by which an oligonucleotide could inhibit HIV-1 in vivo.
It is known that synthetic oligonucleotides may be designed which are capable of binding to duplex DNA to form triplex DNA. See U.S. Pat. No. 5,176,996 Hogan & Kessler issued Jan. 5, 1993. That patent discloses a method for making synthetic guanosine-rich oligonucleotides which are targeted to specific sequences in duplex DNA and which form collinear triplexes by binding to the major groove of the DNA duplex.
Specific In Vitro Studies/In Vitro HIV Inhibition With T30177
Infection with the human immunodeficiency virus type 1 (HIV-1) and the subsequent development of acquired immunodeficiency syndrome (AIDS), has become a threat to public health on a global scale. Preventing further spread of this disease is a major health priority worldwide. Although HIV-1 was confirmed to be the causative agent of AIDS as early as 1984, few drugs and no vaccines are effective at preventing the ultimate onset of AIDS in HIV-1 seropositive individuals. This is due, in large part, to the complexity of the causative agent itself, the dynamics of virus production and the speed at which drug-resistant mutants can arise. Ho, et al.,
Nature
373:123-126 (1995); Wei, et al.,
Nature
373:117-122 (1995).
Infection of T-cells by HIV-1 results in the insertion of proviral (double-stranded) DNA into the host cell genome. Goff, S. P.,
Annu. Rev. Genet.
26:527-544 (1992). The integration process involves both the sequence-specific and sequence independent endonucleolytic and strand transfer activities of the virally encoded integrase enzyme. Katz, et al.,
Ann. Rev. Biochem.
63:133-173 (1994); Vink, et a.,
Trends in Genetics
9:433-438 (1993). Once the proviral state is established, the infection may manifest itself in several ways including a latent infection in which viral replication is not measurable until the cell becomes activated or through a chronic infection in which dividing or non-dividing cells persistently release virus in the absence of any cytopathic effect. In addition, recent reports on the kinetics of virus production (and clearance) indicate a dynamic process in which virtually a complete replacement of wild-type virus by drug-resistant virus in plasma can occur after only two to four weeks of drug therapy. Ho, et al.,
Nature
373:123-126 (1995); Wei, et al.,
Nature
373:117-122 (1995). For this reason it is of utmost importance to develop new anti-HIV-1 agents which can complement, by additive or synergistic activity, current therapies.
One relatively new approach used in the development of antiviral therapeutics for HIV-1 is the use of oligonucleotides designed as antisense agents. Letsinger, et al.,
Proc. Natl. Acad. Sci.
USA 86:6553-6556 (1989); Lisziewicz, et al.,
Proc. Natl. Acad. Sci.
USA 90:3860-3864 (1993); Milligan, et al.,
J. Med. Chem.
36:1923-1937 (1993). While much effort is being spent on rationally designed oligonucleotides such as antisense agents there have also been recent findings of multiple alternative mechanisms by which oligonucleotides can inhibit viral infections. Gao, et al.,
J. B. C.
264:11521-11526 (1989); Marshall, et al.,
Proc. Natl. Acad. Sci.
USA 89:6265-6269 (1992); Ojwang, et al.,
J. AIDS
7:560-570 (1994); Rando, et al,
J. Biol. Chem.
270:1754-1760 (1995). For example, Stein et al. (Stein, et al.,
Antisense Research and Development
3:19-31 (1993)) have characterized the interaction of oligodeoxycytidine, containing a phosphorothioate (PT) backbone (poly (SdC)) with the V3 loop of HIV-1 gp 120. It was determined that poly (SdC)
28
specifically interacted with the positively charged V3 loop with a Kd of approximately 5×10
−7
M. Stein et al. (
Antisense Research and Development
3:19-31 (1993)) then postulated that the interaction of poly (SdC) with the HIV-1 V3 loop may be a mechanism by which poly (SdC) could inhibit HIV-1 in vivo. More recently, Wyatt et. al. (Wyatt, et al.,
Proc. Natl. Acad. Sci.
USA 91:1356-1360 (1994)) have described the interaction of a short G-rich oligonucleotide, synthesized with a total PT backbone, which also interacts with the v3 loop of HIV-1 gp 120. In addition, we have previously reported that oligonucleotides containing only deoxyguanosine (G) and thymidine (T), synthesized with natural phosphodiester (PD) internucleoside linkages, were capable of inhibiting HIV-1 in culture. Ojwang, et al.,
J. AIDS
7:560-570 (1994). The most efficacious member of this dG-rich class of oligonucleotides, I100-15, was found capable of folding upon itself to form a structure stabilized by the formation of two stacked guanosine-tetrads which yielded a guanosine-octet. Rando, et al,
J. Biol. Chem.
270:1754-1760 (1995). Furthermore, it was observed that the positions of the guanosine bases in the I100-15 sequence, found in both the tetrads and connecting loops in that structure, were extremely important to the overall anti-HIV-1 activity of the oligonucleotide. Rando, et al,
J. Biol. Chem.
270:1754-1760 (1995).
Site of Activity Studies-Viral Integrase Inhibition
Two events which are characteristic of the life cycle of retroviruses can be utilized for therapeutic intervention. One is reverse transcription, whereby the single-stranded RNA genome of the retrovirus is reverse transcribed into singled-stranded cDNA and then copied into double-stranded DNA. The next event is integration, whereby the double-stranded viral DNA generated by reverse transcriptase is inserted into a chromosome of the host cell, establishing the provira
Cossum Paul A.
Hogan Michael E.
Ojwaug Joshua O.
Rando Robert F.
Wallace Thomas L.
Aronex Pharmaceuticals, Inc.
Conley & Rose & Tayon P.C.
Crane L. Eric
McDaniel C. Steven
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