WO1997003997A1 - Anti-viral guanosine-rich oligonucleotides - Google Patents
Anti-viral guanosine-rich oligonucleotides Download PDFInfo
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- WO1997003997A1 WO1997003997A1 PCT/US1996/011786 US9611786W WO9703997A1 WO 1997003997 A1 WO1997003997 A1 WO 1997003997A1 US 9611786 W US9611786 W US 9611786W WO 9703997 A1 WO9703997 A1 WO 9703997A1
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Definitions
- the present invention relates generally to the field of oligonucleotide chemistry and anti ⁇ viral pharmacotherapy. More specifically, the present invention relates to novel guanosine-rich oligonucleotides and their use as novel anti-viral agents.
- oligodeoxycytidine inhibits HIV-l. Marshall et al., PNAS (1992)
- poly SdC have characterized the interaction of poly SdC with the V3 loop of HIV-l gpl20, and postulated that the specific interaction of poly SdC with the HIV-l V3 loop may be a mechanism by which an oligonucleotide could inhibit HIV-l in vivo.
- HIV-l human immunodeficiency virus type 1
- AIDS acquired immunodeficiency syndrome
- 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.
- 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
- oligonucleotides containing only deoxyguanosine (G) and thymidine (T), synthesized with natural phosphodiester (PD) internucleoside linkages were capable of inhibiting HIV-l in culture. Ojwang, et al., J. AIDS 7:560-570 (1994). The most efficacious member of he G22 this dG-rich class of oligonucleotides, 1100-15, 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.
- 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 proviral state. Integration is catalyzed by the retroviral enzyme integrase which is encoded at the 3 '-end of the pol gene. Varmus, et al. Mobile DNA, pp. 53-108, Am. Soc. Microbiol, Washington, D.C. (1989).
- Integrase first catalyzes the excision of the last two nucleotides from each 3'-end of the linear viral DNA, leaving the terminal conserved dinucleotide CA-3'-OH at these recessed 3' ends (Fig. C-1A). This activity is referred to as the 3'-processing or dinucleotide cleavage. After transport to the nucleus as a nucleoprotein complex, Varmus, et al. Mobile DNA, pp. 53-108, Am. Soc. Microbiol, Washington, D.C. (1989), integrase catalyzes a concerted DNA strand transfer reaction by nudeophilic attack of the two viral ends onto a host chromosome.
- AZT nucleotides can inhibit HIV-l integrase, Mazumder, et al., Proc. Natl. Acad. Sci. 91, 5771-5775 (1994), and that substitution or unsaturation at the 3'-position of the deoxyribose confers potency against HIV-l integrase.
- the enzyme's nucleotide binding site could serve as a potential drug target. It has been shown that the potential stacking interactions gained from the heterocyclic rings can further enhance potency against HIV-l integrase.
- oligonucleotides composed of deoxyguanosine and thymidine have been reported to inhibit HIV-l replication. Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995); Wyatt, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994). Oligonucleotides forming intramolecular G4s did not block virus adsorption but rather inhibited viral-specific transcripts. Rando, et al., J. Biol.
- G-rich nucleic acid sequences can fold, in the presence of Na + or K + ion, to form orderly structures stabilized by guanosine tetrads.
- dimers Smith, F. W., & Feigon, J. (1992) Nature (London) 344, 410-414, Sundquist, W. I. & Klug, A. (1989) Nature (London) 334, 364-366; Kang, et al. (1992) Nature (London) 356, 126131; Balaguumoorthy, P. & Brahmachari, S. K. (1994) J. Biol. Chem. 269, 21858-21869), tetrameres (Son, D. & Gilbert, W. (1990) Nature
- T30177 forms a stable intramolecular fold which is stabilized by a pair of G-tetrads, connected by three single-stranded loops and a 1-2 base long tail to either side of the fold.
- oligomer folding was coupled to K + ion binding (Rando et al., (1995) J. Biol. Chem. 270, 1754-1760).
- Additional studies have suggested that T30177 and related derivatives are potent inhibitors of HIV-l integrase, in vitro (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35).
- oligonucleotides have been shown to have potential as drugs for the treatment of a variety of human clinical disorders (Stein and Cheng, 1993; Marshall and Caruthers, 1993, Science 259: 1564-1570; Chubb and Hogan, 1992, Trends in Biotechnology 10: 132-136; Stull and Szoka, 1995, Pharm. Res. Yl: 465- 483.
- a number of oligonucleotides have undergone pre-clinical testing, and several are in human clinical trials. One finding that has aroused some concern (Black et al., 1994, Antisense Res. Dev.
- Oligonucleotides have advanced to the stage that they are now considered as potential therapeutics for the treatment of a variety of human diseases, and several are presently in clinical trials. Pre-clinical studies have generally shown that doses up to approximately 50 mg/kg are safe, but that higher doses can cause kidney and liver damage, and death (Srinivasan and Iversen, 1995,
- oligonucleotide in one embodiment, there are provided methods and compositions useful in treating pathophysiological states caused by viruses, comprising administering a pharmacological dose of an oligonucleotide, the dose being sufficient to inhibit production of the virus, wherein the oligonucleotide contains a high percentage of guanosine bases.
- the oligonucleotide has a three dimensional structure and this structure is stabilized by guanosine tetrads.
- the oligonucleotide compositions of the invention have two or more runs of two contiguous deoxyguanosines.
- the target virus is either he ⁇ es simplex virus, human immunodeficiency virus, human papilloma virus, human cytomegalovirus, adenovirus, and hepatitis B virus.
- a guanosine-rich oligonucleotide having a three dimensional structure, wherein the three dimensional structure is stabilized by guanosine tetrads or at least two runs of two contiguous deoxyguanosines and wherein these oligonucleotides exhibit anti-viral activity.
- the oligonucleotides of the present invention have partially or fully phosphorothioated internucleoside linkages (backbones) or other chemical modifications.
- the oligonucleotides of the present invention have chemically modified or unnatural (synthetic) bases.
- Figure A-1 A shows a 1973 base pair Hind III to Eco Rl sub fragment ofthe Friend Murine Leukemia Virus (FMLV) clone 57 genome.
- Figure A-1B shows a 172 base pair (Hz ' ndlll to StuI) fragment which is an expanded portion of the 1973 base pair fragment. Within this fragment is the purine rich target to which triple helix forming oligonucleotides are directed.
- Figure A-1C shows the entire Hind III/Eco Rl FMLV fragment cloned into the pT7-2 plasmid (United States
- FIG. 1D shows the Hind III/Eco Rl FMLV fragment was cloned into pBS (Stratagene) yielding pBSFMLV.
- the Hind III site, triple helix target site and Dde I site are respectively 50, 103 and 171 base pairs downstream from the mRNA start site.
- Figure A-2 shows that G-Rich phosphorothioated-oligonucleotides induced reduction in HSV-2 viral titer.
- the filled square (B106-62) (SEQ. ID. NO.
- B 106-96 is the fully phosphorothioated version of B106-62 (SEQ. ID. NO. 5).
- B106-97 is the fully phosphorothioated version of B 106-71 (SEQ. ID. NO. 6).
- ACV (4a and 4b) is acyclovir tested against two different stock concentrations of HSV-2 strain HG52. In two experiments, after virus infection and before reapplication of oligonucleotide (BIO-96 or BIO-97), the cells were rinsed with a pH 3 buffer in order to remove all virus not yet internalized (96p3 and 97p3).
- Figure A-3 shows MT-2 cells infected with 0.01 m.o.i. of HIV-l and then treated with various concentrations of oligonucleotide or AZT or ddC.
- the data represents the number of viable cells remaining in the culture dish, i.e., not undergoing virus induced cytopathic effects (CPE). In this graph, 100% is the level of CPE occurring in cultures infected with virus but not treated with any drug.
- Figure A-4 shows the culmre media taken from NIH3T3 cells chronically infected with FMLV was mixed with various concentrations of 1100-51 (SEQ. ID. NO. 29) or 1100-12 (SEQ. ID. NO. 27) (fully phosphorothioate version of 1100-00 (SEQ. ID. NO. 20)). The mixtures were then assayed for the presence of viral reverse transcriptase. The data is presented as a percent of measurable reverse transcriptase in culmre medium not treated with oligonucleotide.
- Figures A-5A, A-5B and A-5C show the radio-labelled ( 32 P) full-length or truncated mRNA transcripts were analyzed by polyacrylamide gel electrophoresis, and then quantitated by cutting out the specific transcript and measuring the radioactivity in a scintillation counter.
- Figure A-5A shows that the reduction in full length transcripts directed by the T7 and T3 promoter when 1100-51 (SEQ. ID. NO. 29) (anti-parallel triple helix forming oligonucleotide; FMLV2ap) was added.
- Figure A-6 shows inhibition of HIV-l induced syncytia formation four days post-infection.
- SUP Tl cells were infected with HIV-1 DV for four hours and then treated with various concentrations of oligonucleotides.
- Four days post-infection cells were scored for syncytium formation. All assays were performed in quadruplicate and the average values used to plot this graph. The legend to the right of the graph indicates the symbol used for each oligonucleotide tested.
- Figure A-7 shows continued suppression of HIV-l p24 production seven days post removal of oligonucleotide.
- Figure A-8 shows a Dixon Plot of random oligonucleotide 1232 (SEQ. ID. NO. 41) obtained from kinetic analysis of inhibition of HIV-RT with respect to dNTP.
- the inhibition constant K was determined by simultaneously varying dNTP (without dATP) concentrations at the same time as inhibitor (oligonucleotide 1232).
- the Kj determination was performed at 0.125 mM, 0.25 mM and 0.5 mM dNTP concentrations with constant Primer-Template concentration of 0.2 pM. HIV-RT was used at 1 unit in each reaction. The reported values are the result of simultaneous independent duplicates determinations.
- Figure A-9A reveals PBMCs derived from HIV-l positive patients were mixed with HIV-l negative PBMCs in culture medium containing drug 1100-15 (SEQ. ID. NO. 33). On day 7 the cocultures were washed and resuspended in fresh medium containing drug. The p24 levels in medium collected on day 7 (before medium change) and day 10 were assayed for p24.
- Figure A- 9B HIV-l negative PBMCs from two different donors were infected with HIV-1 DV and then incubated in the presence of drug for 10 days at which time the culmre medium was assayed for the presence of p24 antigen.
- Figures A-10A and A-10B show inhibition of binding of V3 loop specific Mabs to HIV-l gpl20 by phosphorothioate containing oligonucleotides.
- Matched sequence oligonucleotides with either phosphodiester (PD) or phosphorothioate (PT) backbones were assayed for their ability to inhibit the interaction of V3 loop specific Mabs with the gpl20 molecule: SEQ. ID. NOS. 31 (1173) and 32 (1174); SEQ ID. NOS. 24 (1100-07) and 39 (1100-21); or SEQ. ID. NOS. 42 (1229) and 43 (1230).
- immobilized gpl20 was preincubated with oligonucleotides before washing and the addition of Mab NEA 9284 (panel A) or Mab NEA 9301 (panel B).
- Figure A-l l shows a schematic diagram of the HIV-l genome not drawn to scale.
- Figure A-12 shows analysis of DNA (PCR) and RNA (RT-PCR) extracted from SUP Tl cells three days post-infection with HIV-l. (Left Panel).
- PCR analysis of HIV-l infected drug treated SUP Tl cell DNA used 0.1 ⁇ g of total extracted DNA for each reaction.
- AZT at 0.3 ⁇ M which is 10 fold over the IC 50 value (lane 1) or 1100-15 (SEQ. ID. NO. 33) at 5.0 (lane 2) or 0.3 ⁇ M (lane 3) were added to SUP Tl cells at the same time as HIV-l.
- Lanes 4 (AZT), 5 (5.0 ⁇ M 1100-15 (SEQ. ID. NO. 33)) and 6(0.3 ⁇ M 1100-15) are the results of DNA samples obtained from cells in which drug was added 8 hours post-infection.
- Lanes 8 to 10 contain 10, 100 or 1000 ng of DNA extracted from HIV-l infected control SUP Tl cells.
- the band corresponding to 220 bp is the predicted size of the internal /J-actin control and the 200 bp fragment is the predicted size for the amplified portion of the HIV-l genome.
- the right panel contains RT-PCR analysis of extracted RNA (1 ⁇ g/reaction) obtained from cells treated in an identical fashion as those described in lanes 1-6 of the left panel.
- Lanes 7 and 8 are control HIV-l infected cell mRNA and lanes 9 and 10 are the results obtained using uninfected untreated SUP Tl cell mRNA.
- Figure A- 13 shows the results of three oligonucleotides (10 "5 M) incubated with increasing concentrations (0,7.5,15,30,60 and 120 mM) of KCI (lanes 1-6 for 1100-15 (SEQ. ID. NO. 33), 7-12 for 1100-18 (SEQ. ID. NO. 36) and 13-18 for Z106-50).
- the nucleotide markers are poly dT.
- Figures A-14A and A-14B show a line model and space filling model for 1100-15 (SEQ. ID. NO. 33).
- Oxytricha class is depicted.
- the 5 '-end of the molecule is in the bottom left hand side.
- the bases (Gs) are stacked on top of each other with the 4 bases in each plane stabilized through their hydrogen bonding with each other and their interaction with the K + ion complex in the center of the tetrad.
- the space filling model (A-14B) also has the 5' bases in the lower left hand corner.
- the lighter colored atoms are part of the G-tetrad and the darker shaded atoms are part of the loop strucmres.
- the K + ion is buried within the tetrad.
- Figure A- 15 displays a one dimensional NMR analysis of a KCI titration and thermal melting parameters for 1100-15 (SEQ. ID. NO. 33).
- FIG. B-l Dose responsive profile for T30177, AZT and ddC.
- CEM-SS cells were infected with HIV-1 RF (0.01 MOI) and treated with various concentrations of each drug for six days at which time the degree of HIV-l -induced syncytium formation (cytopathic effect, cpe) was addressed. The results shown are the averages of three or more experiments with the standard deviations indicated.
- Figure B-2 Effect of T30177 on HIV-l replication in primary macrophages. Primary macrophages were obtained from PBMC preparations and infected with HIV-1 DV for 24 hours in the presence of the indicated amount of drug. Seven days post-infection the intracellular levels of p24 were quantitated using the Coulter p24 antigen capture ELISA kit. The results shown are the averages of three or more experiments.
- Figure B-3 Effect of time of drug addition on the inhibition profile of T30177, AZT, and DS5000.
- MT4 cells, infected with HIV-1 IIIB at a MOI of 1 were treated at various times during (time 0) or post-virus-infection with the test compounds at a concentration 100-fold higher than their respective IC 50 values.
- Viral p24 levels in the culmre medium were monitored 29 hour post- infection. The results shown are the averages of three or more experiments.
- FIG. B-4 HeLa-CD4-j3-galactosidase cell assays.
- FIG. B-6 Single cycle analysis of viral DNA.
- Time 0 indicates the treatment of cell cultures with drug during virus infection.
- the DNA was extracted from the infected cells and used as a template for PCR.
- the concentration of drug used in each assay is equivalent to 10 to 100-fold over their respective IC2 50 values.
- FIG. B-7 Analysis of replicated viral DNA.
- CEM-SS cells were infected with HIV-1 SKI at an MOI of 1 and then treated with T30177.
- the drug concentrations used were 0.0, 0.01, 0.1, 1 and 10 ⁇ M corresponding to lanes 1 to 5 respectively.
- the unlabeled lane in each panel contains molecular size marker control DNA.
- FIG. 1 Inhibition of HIV-l integrase 3'-processing and strand transfer and HIV-I RJ cytopathicity by guanosine quartets.
- A Schematic diagram showing 3'-processing (3'P, which liberates a GT dinucleotide) and strand transfer (S.T., which results in the insertion of one 3'- processed oligonucleotide into another target DNA), with 5 '-end labeled (asterisk), blunt-ended oligonucleotide.
- B Left panel, Concentration-response obtained from a typical experiment. The
- DNA substrate 21mer
- STP strand transfer products
- Lane 1 DNA along; lane 2, with integrase; lanes 3-6, with integrase in the presence of the indicated concentrations of T30177.
- Right panel graph derived from quantitation (see Materials and Methods) of the dose response in the left panel showing inhibition of integrase- catalyzed 3'-processing (open squares) and strand transfer (filled squares).
- C Strucmres o guanosine quartets oligonucleotides.
- D IC 50 values for several G4 oligonucleotides against both activities of HTV integrase and HIV-I R in cell culture.
- Insertions into the parent compound T30177 are shown by an italicized and underlined nucleotide while mutations are designated by a lower case nucleotide.
- the guanosines involved in the quartets are shaded and the loops are designated by the corresponding numbers (see panel C, left).
- FIG. C-2 Inhibition of strand transfer and 3'-processing activities of HIV-l integrase by the guanosine quartet T30177.
- A Left, schematic diagram depicting the strand transfer assay using the precleaved oligonucleotide (19mer substrate). Right Phosphorimager picture showing inhibition of strand transfer with T30177. The DNA substrate (19mer) and strand transfer products (STP) are shown. Lane 1, DNA alone; lane 2, plus integrase; lanes 3-6, plus integrase in the presence of the indicated concentrations of T30177.
- FIG. C-3 Inhibition of the DNA binding activity of HIV-l integrase by guanosine quartets. DNA binding was measured after UV crosslinking of reactions in which integrase was preincubated for 30 minutes at 30 °C with the guanosine quartet prior to addition of the DNA substrate.
- A Phosphorimager picmre showing differential inhibition of DNA binding with T30177 and T30659. Lane 1, DNA alone (20 nM); lanes 2, 8, and 14, with integrase (200 nM); lanes 3-7, in the presence of the indicated concentrations of T30177; lanes 9-13, in the presence of the indicated concentrations of T30659. The mitigations of proteins of known molecular weight are shown to the right of the gel.
- FIG. 4 Differential activities of T30177 on wild-type and deletion mutants of HIV integrase.
- A Schematic diagram showing the three domains of HIV-l integrase.
- B Inhibition of wild-type IN 1"288 (open squares), IN 1"212 (closed squares), and IN 50"212 (open triangles) in the disintegration assay.
- C Binding of HIV-l integrase wild-type (IN 1"288 ) and deletion mutants at a final concentration of 1 ⁇ M to 32 P-end labeled guanosine quartet T30177 at a final concentration of 250 nM. The mobility of proteins of known molecular weight (in KDa) are shown to the right of each figure.
- Lane 1 T30177 alone; lanes 8-9, binding to wild-type, full-length HIV-l integrase (IN 1"288 ) in the presence of the indicted metal; lanes 2-3, binding to IN 1"212 in the presence of the indicated metal; lanes 6-7, binding to IN 50"212 in the presence of the indicated metal are lanes 4-5, binding to IN 50"288 .
- Figure C-5 DNA binding activity of the zinc finger domain of HIV-l integrase. Binding of IN 1"55 to T30177 or the viral DNA substrate (see Fig. IA, 21mer). Lanes 1, DNA alone (50 nM); lanes 2, IN 1"55 (2 ⁇ M) with no metal; lanes 3, IN 1"55 with manganese (7.5 mM); lanes 4, IN 1" 55 with magnesium (7.5 mM); lanes 5, IN 1"55 with manganese (7.5 mM) and zinc (4.2 mM); lanes 6, IN 1"55 with magnesium (7.5 mM) and zinc (4.2 mM); lanes 7-10, IN 1"55 in the presence of the indicated concentration of zinc alone.
- FIG. 6 Increased binding to and inhibition by guanosine quartets in magnesium versus manganese.
- A Phosphorimager picmre showing DNA binding of wild-type integrase to radiolabeled T30177. Lane 1, DNA alone (27 nM); lanes 2-5; binding of integrase (200 nM) in manganese buffer to the indicated concentration of T30177; lanes 6-9, binding of integrase (200 nM) in magnesium buffer to the indicated concentration of T30177. The migrations of proteins of known molecular weight are shown to the right of the gel.
- B Structures of T30177 and two analogs in which the internucleotidic linkages have been changed.
- C graph derived from quantitation (see Materials and Methods) of the inhibition of integrase-catalyzed 3'-processing in the presence of T30177 and analogs in either magnesium or manganese. Inhibition by T30177
- Figure C-7 Competition of binding to either U5 viral oligonucleotide (see Fig. C-1A, 21mer) (A) or guanosine quartet T30177.
- (B) Lanes 1, DAN alone; lanes 2, with wild-type, full- length HIV-l integrase. Lanes 3-6 in panel (A), with integrase in the presence of the indicated concentrations of T30177 added after a 5 minute preincubation with the U5 viral DNA oligonucleotide. Lanes 3-6 in panel (B), with integrase in the presence of the indicated concentrations of viral U5 DNA oligonucleotide added after a 5 minute preincubation with the guanosine quartet T30177.
- Figure C-8 Inhibition of the related retroviral integrases.
- A Inhibition of 3 '-processing and strand transfer catalyzed by HIV-l (lanes 2-8), HIV-2 (lanes 9-15), FIV (lanes 16-22), and SIV (lanes 23-29) integrases in the presence of T30177.
- Lane 1 DNA alone; lanes 2, 8, 9, 15, 16, 22, 23, and 29, with integrase; lanes 3-7, 10-14, 17-21, and 24-28, with integrase in the presence of the indicated concentrations of T30177.
- B Graph derived from quantitation (see Materials and
- Figures C-9, C-10, C-13 and C-14 Three-dimensional drawings of certain guanosine tetrad forming oligonucleotides referred to in Tables C-1 and C-2.
- Figure C-ll Percentage inhibition of 3' processing by certain oligonucleotides in Table
- Figure C-12 Inhibition of syncytium formation by certain oligonucleotides in Table C-1.
- FIG. D Figures for Section D Figure D-l. Strucmre Models.
- A. The sequence and a strucmre model for oligonucleotides used in this smdy presented All four oligomers have been modified so as to include a single phosphorothioate linkage at the 5' and 3' terminus. Proposed sites of G-quartet formation have been identified by dotted lines. The continuity ofthe phosphodiester backbone is identified by solid lines.
- B A two step kinetic model for ion induced folding of oligomers in this study. It is proposed that binding a first K + or Rb + ion equivalent, marked as a (+), occurs within the central G-octet, which has been identified by dotted lines.
- This first step is relatively fast, and is associated with higher apparent ion binding affinity. It is also associated with formation of unstacked loop domains, and the resultant net loss of UV hypochromism, as compared to the initial random coil state.
- the second step in the process involves as many as two additional K + or Rb + ion equivalents, (+), at the junction between the core octet and flanking loop regions. This second step requires significant ordering of the flanking loop domains, and is therefor associated with an increase of base stacking interaction, and a generally high activation energy.
- FIG. D-2 Thermal Stability of Oligomer Folding. Thermal denaturation of oligomers has been measured as a function of ion type, ion concentration and strand concentration. Data have been obtained at 240 nm, in 20 mM Li3P04, pH 7, as the supporting buffer. Tm values were calculated from the first derivative of a plot of absorbance vs. temperature, but similar values were obtained by using the midpoint of the overall absorbance change.
- C The strand concentration dependence of Tm has been measured at 1 mM of added
- FIG. D-3 Oligomer Folding Monitored by Circular dichroism (CD).
- CD data have been obtained at 25 °C in 20 rnM Li3P04 as a function of added ion concentration. Data have been presented as molar ellipticity in units of dmole bases.
- A. The CD spectrum of T30695 in the presence of 0 mM (curve a), 0.05 mM (curve b), or 10 mM (curve e) of added KCI.
- the change in ellipticity at 264 nm, relative to that measured in the absence of added ion is presented as a function of added KCI concentration for T30695 (curve a), T30177 (curve b) and T30676 (curve c).
- the overall midpoint of the measured KCI induced transition has been plotted for each oligomer: 0.02 mM, 0.15 mM and 0.27 mM, respectively.
- C. T30695 has been treated with increasing concentration of several different cations.
- the change in ellipticity at 264 nm was then measured as described in part B as a function of added KCI (curve a), RbCl (curve b) or NaCl (curve e).
- FIG. D-4 The Kinetics of Ion Induced Folding. Ion was added to oligomers at time zero in the standard 20 mM Li3P04 assay buffer. Data have been presented as absorbance (A) vs. time after addition of metal ion.
- Figure E-l Mean arterial pressure of cynomolgus monkeys pAor to, during and following intravenous administration of AR177 over ten minutes. Blood pressure was continuously monitored via an indwelling femoral artery catheter. The values are the mean ⁇ s.d. of three monkeys at each dose.
- FIG. E-2 Neutrophil levels in blood of cynomolgus monkeys prior to, during and following intravenous administration of ARl 77 over ten minutes. Neutrophil levels were determined pre-dose (-10 minutes), and at 10, 20, 40, 60,120 and 1440 minutes following the initiation of the ten-minute infusion of AR177 into cynomolgus monkeys. The values are the mean ⁇ s.d. of three monkeys at each dose.
- Figure E-3 aPTT versus time profile following a ten-minute infusion of ARl 77 to cynomolgus monkeys. aPTT was determined before and at various time after intravenous infusion of ARl 77 as described in the Methods section. aPTT levels returned to baseline by 24 hours in all groups. Certain aPTT values in monkeys at the 20 and 50 mg/kg dose time points, denoted by asterisks, exceeded the upper limit of the assay.
- Figure E-4 Complement factor Bb concentration versus time profile following a ten - minute infusion of AR177 to cynomolgus monkeys. Bb was determined before and at various times after intravenous infusion of AR177 as described in the Methods section. Bb levels returned to baseline by 24 hours in all groups.
- Figure E-5 CH50 levels in blood of cynomolgus monkeys prior to, during and following intravenous admimstration of AR177 over ten minutes. CH50 levels were determined pre-dose (-10 minutes), and at 10, 20, 40, 60,120 and 1440 minutes following the initiation of the ten-minute infusion of AR177 into cynomolgus monkeys.
- the values are the mean of two monkeys in the saline and 50 mg/kg groups, and three monkeys in the 20 mg/kg group. Data for the third monkey in the saline and 50 mg/kg groups, and for all of the 5 mg/kg group was not available.
- FIG. 1 Plasma C max of AR177 in cynomolgus monkeys administered AR177 as a ten-minute intravenous infusion.
- the plasma concentration of ARl 77 was determined by anion-exchange HPLC as described in the Methods section.
- FIG. 1 AR177 plasma concentration versus time profiles following a ten-minute intravenous infusion to cynomolgus monkeys.
- the plasma concentration of AR177 was determined by anion-exchange HPLC as described in the Methods section.
- the plasma AR177 concentration at 24 hours for the 5, 20 and 50 mg/kg groups were ⁇ 0.020 g/mL for the 5 and 20 mg/kg groups, and 0.24 ⁇ 0.42 ⁇ /mL for the 50 mg/kg group.
- Figure E-8 The relationship between plasma AR177 and aPTT in cynomolgus monkeys following a ten-minute intravenous infusion of 5 mg AR177/kg.
- AR177 was determined by anion-exchange HPLC as described in the Methods section.
- the baseline aPTT level (at 10 minutes prior to dosing) was 32.1 ⁇ 4.4 seconds (mean ⁇ s.d.).
- FIG. 9 The relationship between plasma AR177 and aPTT in cynomolgus monkeys following a ten-minute intravenous infusion of 20 mg AR177/kg.
- the plasma concentration of AR177 was determined by anion-exchange HPLC as described in the Methods section.
- the baseline aPTT level (at 10 minutes prior to dosing) was 41.6 ⁇ 6.7 seconds (mean ⁇ s.d.).
- FIG. 10 The relationship between plasma ARl 77 and aPTT in cynomolgus monkeys following a ten-minute intravenous infusion of 50 mg AR177/kg.
- the plasma concentration of AR177 was determined by anion-exchange HPLC as described in the Methods section.
- the baseline aPU level (at 10 minutes prior to dosing) was 33.2 ⁇ 4.8 seconds (mean ⁇ s.d.).
- FIG. F-l AR177 plasma concentration after bolus IV dose 1 or 12 versus dose amount in Cynomolgus monkeys. Cynomolgus monkeys were given intravenous doses of 2.5, 10 or 40 mg/kg/day every other day for a total of 12 doses. Blood was obtained 5, 30 and 240 minutes following doses 1 and 12. The concentration of AR177 in the plasma of every monkey was determined by anion-exchange HPLC as described in the Methods section. There were six monkeys in the 10 and 40 mg/kg groups, and eight monkeys in the 40 mg/kg group. There was a linear relationship between each dose and the plasma concentration that was achieved at each of the sampling times. Figure F-2. AR177 plasma concentration versus time profile following a bolus IV injection
- FIG. F-3 The relationship between the plasma ARl 77 concentration and aPTT in Cynomolgus monkeys following a bolus IV injection of 2.5 mg AR177/kg. Cynomolgus monkeys were given intravenous doses of 2.5 mg/kg/day every other day for a total of 12 doses. This figure shows the plasma ARl 77 concentration versus aPTT levels 5, 30 and 240 minutes following doses 1 and 12. The concentration of AR177 in the plasma was determined in every monkey by anion-exchange HPLC as described in the Methods section. There were six monkeys in the 2.5 mg/kg group. The baseline aPTT levels just prior to (pre-dose) doses 1 and 12 were 24.1 ⁇ 3.4 seconds and 22.1 + 2.2. There was no change in the aPTT levels at any of the time points after the 1st or 12th doses of AR177 at 2.5 mg/kg.
- FIG. F-4 The relationship between the plasma AR177 concentration and aPTT in cynomolgus monkeys following a bolus IV injection of 10 mg AR177/kg. Cynomolgus monkeys were given intravenous doses of 10 mg/kg/day every other day for a total of 12 doses. This figure shows the plasma ARl 77 concentration versus aPTT levels 5, 30 and 240 minutes following doses 1 and 12. The concentration of AR177 in the plasma was determined in every monkey by anion-exchange HPLC as described in the Methods section. There were six monkeys in the 10 mg/kg group. The baseline aPTT levels just prior to (pre-dose) doses 1 and 12 were 23.3 ⁇ 1.8 seconds and 21.6 ⁇ 2.2. There was a close correlation between the aPTT] levels after the 1st or 12th doses of AR177 at 10 mg/kg and the aPTT levels.
- FIG. F-5 The relationship between the plasma ARl 77 concentration and aPTT in cynomolgus monkeys following a bolus IV injection of 40 mg AR177/kg. Cynomolgus monkeys were given intravenous doses of 10 mg/kg/day every other day for a total of 12 doses. This figure shows the plasma AR177 concentration versus aPTT levels 5, 30 and 240 minutes following doses 1 and 12. The concentration of AR177 in the plasma was determined in every monkey by anion-exchange HPLC as described in the Methods section. There were eight monkeys in the 40 mg/kg group. The baseline aPTT levels just prior to (pre-dose) doses 1 and 12 were 24.8 ⁇ 3.3 seconds and 22.5 ⁇ 2.5.
- AR177 at 40 mg/kg and the aPTT levels.
- FIG. G-l AR177 pharmacokinetics following a single IV dose of 0.75 mg/kg to humans.
- Four HIV-positive human patients were administered AR177 at 0.75 mg/kg as a two-hour intravenous (IV) infusion.
- Blood samples were collected in EDTA-coated tubes at various time points during and following the IV infusion.
- Plasma was obtained following low speed centriguation of the blood.
- the concentration of ARl 77 in the plasma was determined using a validated anion-exchange HPLC method.
- Figure G-2 AR177 pharmacokinetics following a single IV dose of 1.5 mg/kg to humans.
- FIG. G-3 AR177 pharmacokinetics following a single IV dose o 3.0 mg/kg to humans.
- Two HIV-positive human patients were administered ARl 77 at 3.0 mg/kg as a two-hour intravenous (IV) infusion.
- Blood samples were collected in EDTA-coated tubes at various time points during and following the IV infusion. Plasma was obtained following low speed centriguation of the blood.
- the concentration of AR177 in the plasma was determined using a validated anion-exchange HPLC method.
- FIG. G-4 ARl 77 pharmacokinetics following a single IV dose of 0.75, 1.5 or 3.0 mg/kg to humans.
- Ten HIV-positive human patients were administered AR177 at 0.75, 1.5 or 3.0 mg/kg as a two-hour intravenous (IV) infusion.
- Blood samples were collected in EDTa-coated tubes at various time points during and following the IV infusion. Plasma was obtained following low speed centriguation of the blood. The concentration of AR177 in the plasma was determined using a validated anion-exchange HPLC method.
- FIG. 1 AR177 TVi and C MAX following single doses to humans. HIV-positive human patients were administered AR177 at 0.75, 1.5 or 3.0 mg/kg as a two-hour intravenous infusion.
- the concentration of AR177 was determined in the plasma using a validated anion-exchange HPLC method.
- the C MAX maximal plasma concentration of AR177
- plasma TVz half-life of AR177 in plasma
- Figure G-6 AR177 clearance following single doses to humans. HIV-positive human patients were administered ARl 77 at 0.75, 1.5 or 3.0 mg/kg as a two-hour intravenous infusion.
- the concentration of AR177 was determined in the plasma using a validated anion-exchange HPLC method.
- the plasma clearance was determined using PKAnalyst software (Micro Math, Salt Lake
- OLIGONUCLEOTIDE oligonucleotide
- oligonucleotide as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than ten. Its exact size will depend on many factors including the specificity and anti-viral activity of the oligonucleotide for various viruses.
- bases can refer to unnatural (synthetic) bases used in place of an A, C, T or G.
- bases herein, the term includes both the deoxyribonucleic acids and ribonucleic acids.
- the following abbreviations are used.
- A refers to adenine as well as to its deoxyribose derivative
- T refers to thymine
- U refers to uridine
- G refers to guanine as well as its deoxyribose derivative
- C refers to cytosine as well as its deoxyribose derivative.
- bases can refer to unnatural (synthetic) bases used in place of an A, C, T, or G.
- inhibitory dose or therapeutic dose of the compounds in the present invention may be determined by assessing the effects of the oligonucleotide on viral replication in tissue culmre or viral growth in an animal. The amount of oligonucleotide administered in a therapeutic dose is dependent upon the age, weight, kind of concurrent treatment and nature of the viral condition being treated.
- pharmacological dose refers to the dose of an oligonucleotide which causes a pharmacological effect when given to an animal or human.
- the pharmacological dose introduced into the animal or human to be treated will provide a sufficient quantity of oligonucleotide to provide a specific effect, e.g., (1) inhibition of viral protein or enzymes, (2) inhibition of viral-specific replication, (3) preventing the target site from functioning or (4) damaging the duplex DNA at the specific site or (5) ablating the DNA at the site or (6) inhibiting the transcription/translation of the gene under the regulation of the site being bound or (7) internal inhibition of transcription or translation of the gene containing the sequence.
- the dose will be dependent upon a variety of parameters, including the age, sex, height and weight of the human or animal to be treated, the organism or gene location which is to be attacked and the location of the target sequence within the organism. Given any set of parameters, one skilled in the art will be able to readily determine the appropriate dose.
- PATHOPHYSIOLOGICAL STATE The term "pathophysiological state" as used herein refers to any abnormal, undesirable or life-threatening condition caused directly or indirectly by a virus.
- GTO means an oligonucleotide in which there is a high percentage of deoxyguanosine, or contains two or more segments (runs) of two or more deoxyguanosine residues per segment.
- GUANOSINE TETRAD As used herein, the term “guanosine tetrads” refers to the structure that is formed of eight hydrogen bonds by coordination of the four O 6 atoms of guanine with alkali cations believed to bind to the center of a quadruplex, and by strong stacking interactions.
- the strucmre of the telomere sequence repeat T 4 G 4 is the strucmre of the telomere sequence repeat T 4 G 4 , first detected in Oxytricha.
- the oxytricha repeat has been studied in oligonucleotides by NMR and by crystallographic methods. See Smith et al., Nature, 1992, 356:164-68, and Kang et al., Nature, 1992 356:126-31. As predicted from numerous previous physical and biochemical studies, both the NMR and crystallographic studies suggest that folding is mediated by square planar Hoogsteen H-bonding among G-residues, with overall antiparallel orientation of the four strand equivalents comprising the tetrad fold. As expected, the crystallography has shown that the strucmre is selectively stabilized by tight binding of a small monovalent cation to the O 6 oxygen of guanosine.
- the present invention provides methods and compositions for treating a pathophysiological state caused by a virus, comprising the step of administering a pharmacological dose of an oligonucleotide, the dose being sufficient to inhibit the replication of the virus, wherein the oligonucleotide contains sufficient contiguous guanosines so that a guanosine tetrad (inter- or intra- molecular) can form, and the three dimensional structure of the oligonucleotide is stabilized by guanosine tetrads formed at strategic locations.
- this method of treating a virus-induced pathophysiological state may be useful against any virus.
- the methods of the present invention may be useful in treating pathophysiological states caused by viruses such as he ⁇ es simplex virus, human papilloma virus, Epstein Barr virus, human immunodeficiency virus, adenovirus, respiratory syncytial virus, hepatitis B virus, human cytomegalovirus and HTLV I and
- the oligonucleotides of the present invention contain a percentage of guanosine bases high enough to ensure anti-viral efficacy.
- the guanosine is important in forming tetrads which stabilize the three dimensional strucmre of the oligonucleotides.
- the oligonucleotides of the present invention may have any percentage of guanosine bases which will allow for tetrad formation provided that the oligonucleotide exhibits anti-viral activity.
- the oligonucleotides of the present invention contain two or more segments of two or more guanosine bases, and an overall high percentage of G in order to enable the oligonucleotide to form at least one quanosine tetrad.
- the oligonucleotides of the present invention may be capped at either the 3' or the 5' terminus with a modifier.
- the modifier is selected from the group consisting of polyamine or similar compounds that confer a net positive charge to the end of the molecule, poly- L-lysine or other similar compounds that enhance uptake of the oligonucleotide, cholesterol or similar lipophilic compounds that enhance uptake of the oligonucleotide and propanolamine or similar amine groups that enhance stability of the molecule.
- the phosphodiester linkage of the oligonucleotides of the present invention may be modified to improve the stability or increase the anti-viral activity.
- a phosphodiester linkage of the oligonucleotide may be modified to a phosphorothioate linkage.
- Other such modifications to the oligonucleotide backbone will be obvious to those having ordinary skill in this art.
- the present invention also provides specific methods of treating viral states.
- the present invention provides a method of treating a pathophysiological state caused by a virus (in preferred embodiments, as specific virus such as, he ⁇ es simplex virus, human papilloma virus, Epstein Barr virus, human immunodeficiency virus, adenovirus, respiratory syncytial virus, hepatitis B virus, human cytomegalovirus and HTLV I and II), comprising the step of administering a pharmacological dose of an oligonucleotide, the dose being sufficient to inhibit the replication of the virus, wherein the three dimensional strucmre of the oligonucleotide is stabilized by the formation of guanosine tetrads.
- a virus in preferred embodiments, as specific virus such as, he ⁇ es simplex virus, human papilloma virus, Epstein Barr virus, human immunodeficiency virus, adenovirus, respiratory syncytial virus,
- This invention discloses a novel anti-viral technology.
- the total number of antiviral mechanisms by which oligonucleotides, and especially G-rich oligonucleotides, work is not completely known, although the inventors have at least narrowed the sites of action as to certain oligonucleotide drugs as will be seen below.
- G-rich oligonucleotides were able to significantly reduce virus production in each. More importantly, actual human clinical studies have demonstrated the efficacy of the drug in reducing viral replicons in AIDS patients.
- the present invention is also drawn to oligonucleotides that have three dimensional structures stabilized by the formation of guanosine tetrads.
- the present invention demonstrates poly and/or oligonucleotides inhibit growth of HrV-1, HSV1, HSV2, FMLV and HCMV and other viruses if the molecule contains a high percentage of ribo- or deoxyriboguanosine.
- the rest of the molecule is composed of thymine, cytosine, xanthosine or adenine nucleotides (ribo- or deoxyribo-), their derivatives, or other namral or synthetic bases.
- the 5' and 3' termini of the oligonucleotide can have any attachment which may enhance stability, uptake into cells (and cell nuclei) or anti-viral activity.
- the backbone which connects the nucleotides can be the standard phosphodiester linkage or any modification of this linkage which may improve stability of the molecule or anti-viral activity of the molecule (such as a phosphorothioate linkage).
- Structural formulas for representative G-rich oligonucleotides disclosed in the instant invention are listed below in Table A-1.
- Vero cells (4 x 10 4 cells/tissue culture well) were incubated with oligonucleotide(s) for 14 hours before the oligonucleotide was removed and virus (HSV-2 strain HG52) was added to the cells at a multiplicity of infection (m.o.i.) of 0.1 to 1.0 (4 x 10 3 to 4 x IO 4 PFU). The infection was allowed to proceed for 10 minutes after which the cells are washed and fresh media, containing the same oligonucleotide was added for an additional 14 hours. Then, the cells were subjected to a freeze/thaw lysis after which the released virus was titered.
- virus HBV-2 strain HG52
- HIV-l CULTURE ASSAY The SUP Tl T lymphoma cell line was infected with HIV-l strain DV at a multiplicity of infection (m.o.i.) of 0.1 for one hour at 37-C. After the infection, free virus was washed off and the newly infected cells were plated (5 x IO 4 cells) in quadruplicate in 96 well plates that had been prepared with various dilutions of oligonucleotide. The final concentration of drug varied between 0.1 and 20 uM. After 3 days of incubation at 37'C, the plates were scored for the presence of multinucleated giant cells (syncytia).
- oligonucleotides In assays designed to inhibit syncytia formation, a number of oligonucleotides exhibited anti-HIV- 1 activity.
- the oligonucleotides and their IC50 are listed in Table A-2.
- 1100-05 is the same as 1100-01 with a cholesterol group attached to the 3' end via a triglycyl-linker.
- 1100-08 is the same as 1100-00 with a cholesterol group attached to the 3' end via a triglycyl-linker.
- 1100-07 was designed as a sequence isomer to 1100-01 and 1100-06 is the cholesterol derivative of 1100-07.
- AlOO-OO is the same sequence in the opposite orientation to HIB38p (A100-50). 1100-07, originally designed as a control for 1100-01 to be used in anti-FMLV experiments, was the most efficacious oligonucleotide tested against HIV-l.
- HIV-l strain LAV was used to infect MT-2 cells at an m.o.i of 0.01. After 7 days, these cells were scored for cytopathic effects (CPE).
- CPE cytopathic effects
- anti-HFV-1 assays in which MT-2 cells were infected at an m.o.i. of 0.01 several G-Rich oligonucleotides were able to inhibit viral-induced cytopathic effects with effective dose 50 's (IC50s) in the 0.5-1.0 uM range ( Figure A-3).
- oligonucleotides shown in Figure A-3 were effective in the 0.5 to 1.0 uM range, including AlOO-00 (fflV38p) and A100-50 (HIV38ap), AlOl-00 (HIV38ctl), HIV-26ctl.
- the oligo-nucleotide HIV-26ap exhibited less efficacy in this assay with an IC50 in the 5 to 10 uM range.
- TE represents buffer alone, i.e., no drug, while AZT and ddC are control drugs.
- Friend Murine Leukemia Virus was grown in a chronically infected murine fibroblast cell line (pLRB215) or was propagated in an acute assay system by infection of NIH3T3 cells.
- pLRB215 cells were split (1 x IO 5 ) into 24 well culmre dishes and incubated 16 to 20 hours at 37°C. The media was then removed and replaced with media containing various concentrations of oligonucleotide. After 1, 3 or 5 days, culmre media was assayed for the presence of the viral reverse transcriptase enzyme.
- NIH3T3 cells were split (1 x IO 4 ) into 96 well dishes and allowed to incubate for 16-20 hours. After incubation, culture media was removed and concentrated virus stock (10 ul) was added to each well in 100 ul of completed media containing 2 ug/ml polybrene.
- the virus infection was allowed to proceed for 18 hours at which time the virus containing media was removed and complete media containing various concentrations of oligonucleotide was added. After 4 to 7 days, the culture media was assayed for the presence of viral reverse transcriptase.
- Human cytomegalovirus was cultured in the human diploid lung fibroblast cell line MRC-5. These cells were split and placed into 24 well culmre dishes and preincubated for 24 hours with various concentrations of oligonucleotide (0.5 to 20 uM) in complete media. The oligonucleotide was then washed off and virus was added to the cells (approximately 0.1 m.o.i.) for 2 hours at 37°C. The virus was then removed and complete media containing the same concentration of oligonucleotide was added. Cells were then placed at 37°C for 10-12 days at which time virus in the culmre media was titered using a standard agar overlay procedure.
- DNA was then digested with Ddel. Oligonucleotides were then incubated with the digested DNA and the mixmre was subjected to in-vitro transcription using either the T7 or T3 bacterial enzymes.
- reverse transcriptase (either MMLV or FMLV from pLRB215 culmre media) was incubated with various concentrations of oligonucleotide and then assayed using the enzyme linked oligonucleotide sorbent assay (ELOSA), the ELOSA kit which is commercially available from New England Nuclear.
- ELOSA enzyme linked oligonucleotide sorbent assay
- a recombinant plasmid containing the ⁇ SV-1 IE 175 promoter fused to the bacterial chloramphenicol acetyltransferase gene (CAT) was linearized and used as a template for run off transcription studies.
- CAT chloramphenicol acetyltransferase gene
- the oligonucleotide B 106-62 was originally designed to form a triple helix structure with a portion of the promoter region of the major immediate early protein of HSV-2 (IE175).
- the phosphorothioate derivative of two oligonucleotides were synthesized and tested for anti-viral activity against HSV-2.
- Figure A-2 shows that the B 106-62 oligonucleotide at 20 ⁇ M was able to reduce viral titers by approximately 20% whereas the phosphorothioate version (B 106-96) reduced virus by 50% in the submicromolar concentration range.
- control oligonucleotide (B106-97), the phosphorothioate backbone derivative of B 106-71, was also able to inhibit virus at the same levels as B 106-96. Even when an extensive washing procedure at a pH of 3.0 was employed to remove excess virus not internalized during the infection, incubation with both B 106-96 and B 106-97 was able to significantly reduce virus yield. Thus, the inventors concluded that the mechanism of anti-viral activity was not merely a blocking of the adso ⁇ tion of HSV-2 virions to cells.
- Figure A-2 also shows the results of acyclovir in the same molar range as the oligonucleotides. Acyclovir was tested against two different stocks of HSV-2 strain HG52, as illustrated in Figures A-4a and A-4b.
- oligonucleotides used in these examples were synthesized on a DNA synthesizer (Applied Biosystems, Inc., model 380B or 394), using standard phosphoramidite methods. All oligonucleotides were synthesized with an amino modified 3 '-terminal, which resulted in the covalent attachment of a propanolamine group to the 3 '-hydroxyl group or resulted in a cholesterol moiety attached to the 3 '-terminal via a triglycyl-linker. Oligonucleotides used in this example were capped at their 3 '-terminal with either a propanolamine or a cholesterol moiety to reduce degradation by cellular exonucleases.
- Phosphorothioate containing oligonucleotides were prepared using the sulfiirizing agent TETD or beaucauge reagent.
- the 3 '-cholesterol modified oligonucleotides were prepared and purified as described by Vu et al. (in Second International Symposium on Nucleic Acids Chemistry, Sapporo, Japan, 1993).
- oligonucleotides with either full length phosphodiester (PD) or full length phosphorothioate (PT) backbones were stable in the culmre media for 4 days, while oligonucleotides consisting of a more random composition of nucleotides were rapidly degraded.
- TC 50 concentration of oligonucleotide needed to reduce cell proliferation by 50% (TC 50 ) of selected compounds, based on the dye metabolism assay was approximately 40 to 50 ⁇ M for oligonucleotides with PD backbones and 15 to 40 ⁇ M for those compounds containing a PT backbone.
- the TC 50 for selected oligonucleotides are presented in Table A-3. Stability and toxicity tests were replaced as described below
- the cytotoxicity of selected oligonucleotides was assayed using the CellTiter 96TM Aqueous Non-Radioactivity Cell Proliferation Assay (Promega). This is a colormetric method for determining the number of viable cells in proliferation or chemosensitive assays using a solution if MTS. Dehydrogenase enzymes found in metabolically active cells convert MTS into a formazan product. The SUP Tl cells used in the cytotoxicity assays were in log phase growth at the time of the assay. Cytotoxicity profiles for GTOs with PD backbones such as 1100-15 (SEQ. ID. NO.
- TC 50 s (50% cytotoxic concentration) in the range of 30 to 50 ⁇ M while GTOs with PT backbones such as 1100-15 had TC 50 s in the 10 to 30 ⁇ M range.
- the TC 50 for AZT in this assay format was approximately 10 ⁇ M .
- oligonucleotides Blockage of the hydroxyl terminus of oligonucleotides has been shown by many investigators to greatly reduce degradation by cellular exonucleases. Therefore, all oligonucleotides used in these studies were modified at their 3'- end with either a propanolamine group or a cholesterol group.
- 10 ⁇ M of GTOs were incubated in MEM (GIBCO) supplemented with 10% FBS. Aliquots were taken after 10 min, 1 day, 2 days, 3 days and 4 days.
- oligonucleotides were 5'- end-labeled using [ ⁇ - 32 P]ATP and polynucleotide kinase. The integrity of the oligonucleotides was then analyzed on a 20% polyacrylamide gel with 7 M urea. The results indicated that a portion of each GTO with a PD backbone was present in the culture medium for three to four days while oligonucleotides composed of a more random assortment of all four nucleotides were rapidly degraded.
- oligonucleotide 1100-07 Long Term Suppression of Acute HTV-l Infections in SUP Tl cells.
- GTOs guanosine/thymidine oligonucleotides
- Table A-2 one of the sequence motifs tested (oligonucleotide 1100-07) was 10 fold more active at inhibiting HIV-l induced syncytium formation than the other motifs tested (e.g. 1100-00 shown in Table A-1). 1100-07 and its derivatives (length and chemical modifications) were further tested for their ability to inhibit virus in a dose-dependent fashion by measurement of syncytium formation and viral p24 production.
- HIV-1 DV was used to infect the SUP Tl lymphoblastoid cell line at an m.o.i. of 0.1 TCID 50 for one hour at 37 °C prior to washing and resuspension in increasing concentrations of GTOs.
- the cells (2 x IO 4 cells/well) were inoculated in triplicate in 200 ul of RPMI 1640 containing 10% fetal calf serum.
- the number of syncytia per well or the level of p24 in the medium was determined.
- Table A-4 which results indicated that GTOs with simple PD linkages were capable of inhibiting HIV-l syncytia formation and p24 production in culture.
- the duration of the viral suppression was assayed by changing the medium in HIV-l infected cultures containing 2.5 uM of various oligonucleotides to complete media without added oligonucleotide on day 4 post-viral infection.
- the production of viral p24 antigen was then assayed on day 7 and day 11 post-infection.
- the results of this experiment indicated that the shorter variants of 1100-07 (1100-15 and 1100-16) as well as the PT version of this molecule (1100-21), were capable of totally suppressing HIV-l p24 production for at least 7 days after removal of the drug from the culture medium (Table A-6). This substantial level of prolonged inhibition was > 99 for 1100-15, 1100-16 and 1100-21 when compared to the p24 antigen levels obtained for untreated HIV- 1 infected cells (Table A-6) .
- HIV-l infected SUP Tl cells for all oligonucleotides tested is presented in Table A-6.
- 1100-12 0.0 % 0.0 % 0.0 % a Level of detectable p24 in culture medium relative to control (infected but untreated SUP Tl cells after subtraction of background values.
- b Day 4 post-infection culture medium was replaced with fresh medium without oligonucleotide.
- c SUP Tl cells infected with HIV-l but not treated with oligonucleotides or AZT were used as positive control cells in this experiment.
- d 1100-21 and 1100-12 contain phosphorothioate backbone linkages (PT).
- SUP Tl cells were counted for all treated samples 7 days after removal of the oligonucleotides from the infected cell cultures. The results indicated that for cells treated with 2.5 ⁇ M of drug there was no difference in the number of cells when compared with control cultures
- PBMCs peripheral blood mononuclear cells
- HIV infection in the presence of varying concentrations of oligonucleotide.
- Anti-HIV activity was assessed by analyzing supernatants, collected every three days from these mixed cultures, for the presence of HIV p24.
- the PHA activated PBMCs were grown in the presence of 10 units/ml of IL-l and medium was exchanged every three days for a period of three weeks.
- HIV p24 antigen production was assayed in drug-treated as compared to untreated control specimens. It should be noted that the results in these experiments ( Figure A-9) observed for AZT were obtained when AZT was used at 12 uM which is roughly 300 fold greater than the IC 50 for this compound.
- RNA dependent DNA polymerase activity of the RT enzyme by competitive inhibition at the active site of the enzyme.
- the K j value for all of the oligonucleotides tested is presented in Table A-7.
- the data indicate that for all oligonucleotides tested the presence of the sulfur group in the backbone greatly enhanced the interaction between the oligonucleotides and the enzymes.
- the median inhibitory dose (ID 50 ) for these oligonucleotides were also calculated (Table A-7). The ID 50 results are based on the ability of these compounds to inhibit 10 nM of HIV RT.
- Short oligonucleotides (18 mers) with PD or PT backbones were assayed to determine whether the nature of the nucleotide sequence contributed to inhibition of HIV-l RT in this assay system.
- oligonucleotides 1232 (GATC) 18 PT 0.56 0.045 a
- Each pair of oligonucleotides contain the same sequence and differ only in the natore of their backbone linkage.
- Oligonucleotides 1229 and 1230 were poly dC while the 1231 and 1232 oligonucleotides were a random sequence of all four bases (GATC).
- the backbone modifications are denoted as PD for phosphodiester and PT for phosphorothioate.
- the concentration of the gpl 20 used in these studies (125 ng/ml) was determined to be within the linear range of the detection assay.
- the ability of oligonucleotides to inhibit gpl20/CD4 interactions by binding to gpl 20 was determined by preincubation of the test compounds with soluble gpl 20 before addition to the immobilized CD4.
- the results of this experiment (Table A-8) are presented as the concentration of oligonucleotide needed to reduce by 50% CD4 bound gpl 20 (ID 50 [gpl20]).
- the reciprocal experiment was then performed to measure the ability of the oligonucleotides to inhibit these interactions by binding to immobilized CD4.
- a fixed length (18 mer) set of oligonucleotides with either PD or PT backbones were assayed to determine whether the natore of the nucleotide sequence contributed to inhibition of gpl20/CD4 interactions.
- the PD versions of these molecules had little or no measurable effects on the binding of gpl20 with CD4.
- the PT versions of these oligonucleotides did yield measurable inhibitory activity.
- the 18 mer GTO (1174) interrupted gpl20/CD4 interactions at approximately 10 fold lower concentrations than poly (SdC) 18 (1230) while the random sequence 18 mer (1232) had no measurable activity (Table
- HIV-l gpl 20 (v3 loop).
- the degree of interaction was reported to be dependent on the length of the oligonucleotide studied, with a rapid decrease in binding affinity observed for compounds shorter than 18 nucleotides.
- RNA oligonucleotide interactions with the v3 loop was conducted using a v3 loop specific murine Mab, NEA-9284 ( Figure A- 10).
- PT oligonucleotides were able to inhibit binding of NEA-9284 to gpl 20.
- the presence of bound gpl 20 specific Mab was determined using a HRP-labeled goat-o--mouse antibody.
- the results of these experiments indicated that PT oligonucleotides were able to inhibit binding of NEA -9284 to gpl 20.
- the ID 50 for the most active oligonucleotide (1100-21) was approximately 4 to 7 ⁇ M.
- RNA and DNA were extracted from SUP Tl cells 36 hours after infection with 0.1 m.o.i of HIV-1 DV . In this assay, the infected cells were treated with 1100-15 or AZT at various time points before, during or after infection. Harvesting of the infected cells at 36 hr post-infection allowed for the analysis of approximately one round of viral replication.
- a schematic diagram of the positions of the PCR primers used in the DNA and RNA analysis is shown in Figure A-l l .
- Total extracted DNA was analyzed using a PCR primer set which would amplify a 200 bp portion of the viral genome spanning the repeat element (R) into the gag gene.
- the primer set detected full-length or nearly completely synthesized viral DNA. This is the last region of the minus strand of viral DNA that is synthesized. Thus, for DNA to be detected by this primer set, two template-switching events have occurred and contiguous 5 'LTR to gag sequences must be present on either the minus or plus strand of DNA.
- RNA extracted from HIV-l infected cells was analyzed by RT-PCR.
- the antisense primer of the PCR primer pairs was used with MMLV RT and extracted mRNA to synthesize cDNA strand.
- the resultant cDNA was then used as a template in PCR reactions.
- Two RNA primer sets were used to analyze unspliced (primers rl and r2) and spliced (primers rl and r3) HIV-l transcripts. Predicted sizes of the amplified products were 101 bp and 214 bp for the unspliced and spliced species respectively.
- the same /3-action primers used for the analysis of the DNA samples were used as controls in this experiment.
- the G-tetrad formation involves the formation of eight hydrogen bonds by coordination of the four O 6 atoms of guanine with alkali cations believed to bind to the center of a quadruplex, and by strong stacking interactions.
- the oligonucleotides purified using anion exchange chromatography then have an opportunity to form inter- or intra-molecular tetrads.
- the tetrad structure can be strengthened by replacing the sodium ion with potassium.
- Nondenaturing gel analysis 1100-15 (17 mer, Table A-5) was analyzed using nondenatoring polyacrylamide gel electrophoresis. In this experiment, trace concentrations of radiolabeled oligonucleotide (10 "7 M) was incubated with increasing concentrations of cold oligonucleotide (up to 10. 5 M) before gel analysis in the presence of monovalent cation. Under the gel conditions used, 1100-15 migrated as a unique band faster than a random coiled (denatored) 17 mer oligonucleotide would and it was shown to do so in a concentration independent fashion (data not shown).
- the structore was very compact, nearly spherical, with the three loop regions and the 5' "GT tail" comprising the surface of the tetrad core. Based upon this structure, it appeared likely that interaction with cellular macromolecules would be heavily dominated by the structures of these surface loops. In that regard, the inventors believe that it may be inappropriate to think of such interactions as "tetrad binding. " The inclusion of G-tetrads in such a structure may not be important as a recognition element per se, but instead provides a latticework upon which an orderly loop array is positioned. Further, although the loop regions did not appear to be under mechanical stress, they were short enough so that they possessed very high configurational freedom.
- the structores described above possessed a single G-octet core, which was known to be the minimum structore required for nucleation of tetrad formation. Therefore, when paired with the observed short loop size, the intramolecular tetrad structore proposed for 1100-15 is best described as meta-stable, relative to other more robust tetrads which have been described in the literature.
- An increase of the core from 2 to 3 stacked tetrads, or an increase in the length of flexibility of one or more loops would be expected to increase the thermodynamic and/or kinetic stability of this structure significantly.
- the observed anti-HIV activity can be improved by sequence modification which enhances the stability of the underlying tetrad latticework.
- 1100-15 and homologues display profound resistance to cellular nucleases.
- One interesting aspect of the proposed structore was that, even in the loop domains, phosphodiester linkages are generally buried from interaction with large solutes, such as a nuclease.
- the structore analysis proposed defined local phosphodiester backbone structore at low resolution. When paired with explicit biochemical analysis of phosphodiester cleavage rate, it is possible to define sites for selective introduction of backbone modification in 1100-15 homologies, for the purpose of extending the biological half life in vivo.
- the inventors By analogy with chemical shifts of other G tetrad structores, the inventors tentatively ascribed the sha ⁇ imino signals to the 8 Hoogsteen H bonds of the core octet. The broad envelope was ascribed to the G and T imino resonances contributed by the loop and 5' terminal domains.
- thermal melting analysis at 2.7 mM in strands, 6mM KCI, 20mM LiCl, pH 6.0 over the range from 300°K to 345 °K was performed.
- oligonucleotides reduced HCMV titers in tissue culture. Each of the oligonucleotides contained a different percentage of guanosine residues and a different number of total nucleotides in the polymer. The results of this assay are depicted in Table A-9. All oligonucleotides were capable of reducing viral titer in culture including G101-50 which contained only 53% G residues (16 out of 30 total nucleotides). In Table A-9, the length and percent guanosine nucleotides is indicated for each oligonucleotide tested.
- oligonucleotides (Fig. A-1) were capable of inhibiting virus production.
- oligonucleotide controls in this experiment were capable of inhibiting virus production in culture.
- the 1100-51 (FMLV2ap), attenuated full length transcription directed by either the T7 or T3 polymerases ( Figure A-5a).
- full length transcripts directed by the T7 promoter would be 131 bases long while full length transcripts directed by the T3 promoter would be 171 bases long (position of the Dde I site relative to the mRNA start site).
- T30177 is an oligonucleotide composed of only deoxyguanosine and thymidine, it is 17 nucleotides in length is the same sequence as 110075 (SEQ. ID. NO. 33), and it contains single phosphorothioate internucleoside linkages at its 5' and 3' ends for stability. This oligonucleotide does not share significant primary sequence homology with, or possess any complementary (antisense) sequence motifs to the HIV-l genome. As shown below, T30177 inhibited replication of multiple laboratory strains of HIV-l in human T-cells lines, peripheral blood lymphocytes, and macrophages.
- T30177 was also shown to be capable of inhibiting multiple clinical isolates of HIV- 1 and preventing the cytopathic effect of HIV-l in primary CD4 + T-lymphocytes.
- IC 50 median inhibitory concentration
- T30177 needed to be added to cells during, or very soon after, viral infection.
- T30177 was a potent inhibitor of HIV-l integrase reducing enzymatic activity by 50% at concentrations in the range of 0.01 to 0.10 ⁇ M.
- T30177 was also able to inhibit viral reverse transcriptase activity, however, the 50% inhibitory value obtained was in the range of 1-10 ⁇ M depending upon the template used in the enzymatic assay.
- T30177 No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 ⁇ M). In experiments in which T30177 was removed from infected cell cultures 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. Polymerase chain reaction analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of drug from the infected cell cultures.
- T30177 to inhibit both laboratory and clinical isolates of HIV-l and the experimental data suggested to the inventors that T30177 represented a novel class of integrase inhibitors, indicating that this compound was a viable candidate against evaluation as a therapeutic agent for HIV-l in humans.
- T30177 a variant of 1100-15
- T30177 is 100-fold less effective in inhibiting gp 120-induced cell fusion events than it is at inhibiting an early event in the viral life cycle, suggesting a specific point of interdiction distinct from that of blocking virus/cell interactions.
- T30177 is a potent inhibitor of the HIV-l integrase enzyme in vitro and that by blocking these events in the viral life cycle T30177 is able to suppress virus production for prolonged periods after an initial short treatment regimen with the drug.
- Zidovudine (3'-azido-3'-deoxythymidine, AZT) and the nucleoside analogs 2',3'-dideoxyionsine (ddl) and 2',3'-dideoxycytidine (ddC) were obtained from the AIDS Research and Reference Reagents Program, National Institute of Allergy and Infectious Diseases.
- Dextran sulfate (DS5000) was purchased from Sigma, and the bicyclam derivatives JM2763 and JM3100 (De Clereq, et al., Antimicrob. Agents Chemother. 38:668-674 (1994)) were obtained from Johnson Matthey (Westchester, Pennsylvania). Chicago sky blue (CSB) was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute.
- T30177 Cytotoxicity Analysis. The cytotoxicity of T30177 was assayed as described above. The concentration of drug necessary to give one-quarter (TC 25 ), one-half (TC 50 ) or 95 % (TC 95 ) of the maximum inhibition of growth response was then determined. The degree of cell proliferation was determined according to the manufacturer's instructions. In other experiments the effect of T30177 on the viability of primary human PBMCs, PBLs and macrophages was determined using the trypan blue dye exclusion technique. Griffiths, B. , IRL Press, p. 48 (1992), or by measuring the degree of [ 3 H]thymidine or [ 3 ]leucine uptake in these cells (McGrath, M.S. , et al.Proc. N ⁇ tl. Ac ⁇ d. Sci. USA 86:2844-2848 (1989)).
- HTV-l infection assays using cell lines Laboratory strains of HIV-l , HIV-2, simian immunodeficiency virus (SIV), or the low passage isolate HIV-1 DV (Ojwang, et al., J. AIDS 7:560- 570 (1994)), were used to infect established cell lines using the indicated multiplicity of infection (MOI) of virus, for one hour at 37°C prior to washing and resuspension in medium containing increasing concentrations of drug.
- MOI multiplicity of infection
- the infected cells (2 x IO 4 cells/well) were inoculated in triplicate in 200 ⁇ l of complete medium which contains RPMI 1640 (Life Technologies) supplemented with 10% FBS, penicillin (50 U/mL), streptomycin (50 ⁇ g/mL) and L-glutamine, (2 mM).
- RPMI 1640 Life Technologies
- penicillin 50 U/mL
- streptomycin 50 ⁇ g/mL
- L-glutamine (2 mM.
- drug treated and control wells were analyzed for HIV-l induced cytopathic effects, for the presence of viral reverse transcriptase (RT) or viral p24 antigen in the culture medium.
- RT reverse transcriptase
- Cytopathic effects were monitored by either direct counting of HIV-l inducted syncytium formation or by staining cells with the tetrazolium dye XT or MTT. Buckheit, et al., AIDS Research and Human Retroviruses 7:295-302 (1991).
- the AZT resistant strain of HIV-l was kindly provided by Dr. Brendan Larder and the AIDS Directed Programme
- PBMCs Peripheral blood mononuclear cells
- HBV hepatitis B virus
- HIV-l replication was analyzed using the Coulter p24 antigen-capture assay. Assays were performed in triplicate. Data was obtained by spectrophotometric analysis at 40 nm using a Molecular Devices Vmax plate reader.
- PBLs Human peripheral blood lymphocytes
- PBLs Human peripheral blood lymphocytes
- the PBLs were suspended in culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 20% FBS and 50 ⁇ g/mL gentamicin) and the cells counted using the trypan blue exclusion technique. After adjustment of cell density to 1 x IO 7 cells per mL with culture medium, the suspension was placed in a T-75 culture flask and incubated flat at 37 °C in a humidified atmosphere of 5 % C0 2 for 2 hours.
- culture medium RPMI 1640 medium supplemented with 2 mM L-glutamine, 20% FBS and 50 ⁇ g/mL gentamicin
- the non-adherent cell population was decanted into a sterile disposable flask.
- Phytohemagglutinin (PHA-P) was added to the PBL suspension at a concentration of 2 ⁇ g/mL and the PB1 preparation was then further incubated at 37 °C for 48 hours. At this time an aliquot of the culture was used for virus infectivity studies.
- PBLs (5 x IO 5 cells/well) were infected widi HIV-l isolates at an MOI of 0.2. This level of infection yielded a satisfactory virus control RT activity value result at day 7 post-infection (Buckheit, et al. , id. (1991)).
- the cells were separated from the virus by centriguation, washed twice with culture medium, and suspended in culture medium containing IL-2 at a concentration 30 units/mL and at a cell density 2 x 10 5 PHA-P-stimulated PBL cells/0.1 mL of culture medium.
- HIV-l replication was analyzed using either the RT or p24 assay systems. Data was obtained in the p24 assays by spectrophotometric analysis at 450 nm using a Molecular Devices Vmax plate reader.
- Human macrophage cultures were established as described by Crow et al. Crowe, et al. , AIDS Research and Human Retroviruses 3(2): 135-145 (1987). Briefly, PBMCs isolated from HIV-l and HBV seronegative donors was allowed to adhere to glass at 37°C for two hours in calcium and magnesium free PBS (pH 7.4). The non-adherent cells were aspirated and the adherent cells were washed three times with cold PBS. The adherent macrophages were scraped free from the plate, counted, and inoculated into 96 well plates at a concentration of IO 5 cells/well in RPMI 1640 medium supplemented with 10% human serum.
- the macrophages were cultivated in RPMI 1640 with 10% human serum. After incubation overnight at 37°C the macrophages were infected with HIV-l D v at a multiplicity of infection of 0.1 for 24 hours at 37°C in the presence of the indicated amount of drug. Unabsorbed virus was then washed off and the cells were further incubated for 7 days at 37°C in complete medium supplemented with the indicated amount of drug. On day 7 post ⁇ infection the adherent macrophages were washed extensively with PBS and lysed with detergent. Cytoplasmic HIV p24 levels were then quantitated and percent inhibition were calculated and compared to control infected but untreated cells.
- VSV Vesicular stomatitis virus
- Sindbis virus Sindbis virus
- Coxsackie virus B4 Polio virus-1
- Semliki forest virus assays were performed as described by De Clercq.
- CEM-SS cells (2 x IO 6 cells/well) in 0.5 mL of complete medium were infected with HIV-1 SKI at a MOI of 1.0 for 45 minutes on ice at which time complete culture medium (10 mL) was added to the cells. The infected cells were then pelleted (1000 RPM for 10 min. at 4°C), washed twice and aliquoted into a 24-well flat bottom plate (2 x IO 5 cells/well). The indicated amount of drug was added to the infected cell cultures at various times during or post-infection.
- the cells were harvested 12 hours post-infection at which time cell pellets were lysed in 100 ⁇ l polymerase chain reaction (PCR) lysis buffer (50 mM KCI, 10 mM Tris-HCl (pH8.3), 2.5 mM MgCl 2 , 0.1 mg/mL gelatin, 0.45% Nonidet P40, 0.45% Tween 20 and 75 ⁇ g/mL Proteinase K) at 50 °C for one hour followed by 95 °C for 10 minutes. The lysate was stored at -20 °C until use.
- PCR polymerase chain reaction
- PCR analysis of viral cDNA was performed using 10 ⁇ L of total cell lysate in a 100 ⁇ L reaction buffer as previously described (Rando, et al, /. Biol. Chem. 270: 1754-1760 (1995)).
- the primers used were 5'-ATAATCCACCTATCCCAG TAGGAGAAAT-3 ' and 5'-TTTGGTCCTTGTCTTATGTCCAGAATCG-3' which will amplify a 115 bp segment of the HIV-l genome.
- the cycle conditions used were 95°C for 10 minutes to denature the DNA, followed by 30 cycles of 95 °C for 75 seconds, 60 °C for 75 seconds, and a final extension step at 60 °C for 10 minutes.
- CEM-SS cells (2 x IO 7 ) were infected with HIV-1 SKI (MOI of I) for 45 minutes at 37 °C with gentle mixing. Following virus attachment, the cells were gently pelleted, washed twice and resuspended in complete tissue culture medium. The cells were then divided into aliquots, treated with various concentrations of drug and placed in T75 culture flasks. The cells were incubated at 37°C for 18-20 hours and then harvested by centriguation.
- HIV-1 SKI MOI of I
- low- and high-molecular weight DNA were prepared from HIV-l infected cells (untreated or treated with increasing concentrations of drug) according to the protocol originally described by Hirt (Hirt, B.J., J. Mol. Biol. 26:365-369 (1967)) and modified by Gowda et al. Gowda, et al., J. Immunol. 142:773-780 (1989).
- PCR primer sets included control primers for the amplification of mitochondrial DNA (sense, 5'-GAATGTCTGCACAGCCACTTT-3'; antisense, 5'-ATAGAAAGGCTAGGACCAAAC-3'; amplified product, 427 bp); primers for the detection of early viral transcription events (M667 and AA55 primers as described by Zack et al. (Zack, et al.
- primers for the detection of the viral gag gene (sense, 5 ' -A G TGG G G G G G A C A TC AA G C A G C CA TG C A AA T-3 ' ; anti s en s e , 5 ' - TTTGGTCCTTGTCTTATGTCCAGAATG-3', amplified product 300 bp); and primers for the detection of circular proviral DNA (sense, 5'-CCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3'; antisense, 5 '-CAG TGGGTTCCCTAGTTAGC-3 ' , amplified product, 536 bp) .
- PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
- Reverse transcriptase enzyme inhibition assays Purified recombinant RT (HIV-1 BH10 ) was obtained from the University of Alabama, Center for AIDS research. The enzyme assays utilized three different template:primer systems, primed ribosomal RNA, gapped duplex DNA, and poly(rA)p(dT) 12 . lg to evaluate the inhibition of HIV-l RT as described by White et al. (White, et al., Antiviral Res. 16:257-266 (1991), and Parker et al. (Parker, et al., J. Biol. Chem. 266: 1754- 1762 (1991)). Integrase enzyme assays.
- HIV-l integrase enzyme wild-type was a generous gift from Dr. R. Craigie, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases. The enzyme (0.25 ⁇ M) was preincubated in reaction buffer at 30°C for 30 minutes. All 3'-processing and strand-transfer reactions were performed as described previously by Fresen et al. (Fresen, et al., Proc. Natl. Acad. Sci. USA 90:2399-2403
- HIV-l protease enzyme (Bachem) was diluted to 166 ug/mL in 50 mM NaOAc, 5 mM DTT, 2 mM EDTA, and 10% glycerol (pH 5.0) and stored as 10 ul aliquots at -20°C.
- HIV protease substrate I (Molecular Probes) was diluted to a working concentration of 0.32 nmol/ ⁇ L.
- Enzyme (20 ⁇ L), substrate (20 ⁇ L) and drug (20 ⁇ L) were added to each well of a microtiter plate. Positive and negative controls were evaluated in parallel. Fluorescence was quantitated on a Labsystems Fluoroskan II using 355 nm for excitation and 460 nm emission wavelengths at 37 °C at time zero and at 30 minute intervals for 2 hours.
- HeLa-CD4-/J-galactosidase cell assays Two different assays using genetically engineered HeLa cells were performed as described previously. Buckheit, et al., AIDS Research and Human Retroviruses 10: 1497-1506 (1994). These assays utilized the HeLa-CD4-LTR-
- LTR long terminal repeat
- the HL2/3 cells express both the HIV-l envelope glycoprotein and tat gene product so that co-cultivation of these cells with the HeLa-CD4-LTR-/3-galactosidase cells would allow for
- CD4- and gpl20 ⁇ mediated cell fusion The extent of cell fusion can then be monitored by the degree of tat transactivation of LTR-driven ⁇ -galactosidase expression.
- Buckheit, et al. AIDS Research and Human Retroviruses 10: 1497-1506 (1994); Ciminale, et al., AIDS Research and Human Retroviruses 6: 1281-1287 (1990). Results of the In Vitro HTV Inhibition Studies
- the anti-HIV- 1 activity in cell culture assays of the oligonucleotide (1100-15) composed entirely of G and T was established by the inventors. See also, Ojwang, et al., J. AIDS 7:560-570 (1994); Rando, et al, J. Biol. Chem. 270: 1754-1760 (1995). 1100-15 was found to inhibit HIV-1 DV in SUP Tl cells with a median inhibitory concentration (IC 50 ) of 0.125 ⁇ M. 1100-15 was synthesized with an unmodified (natural) PD internucloeside linkage and a propanolamine group attached to the 3 '-terminus to increase the stability of the oligonucleotide.
- T30177 a modified variant of 1100-15, has the same sequence as 1100-15 but contains an hydroxyl moiety at its 3 '-terminus and a single PT internucleoside linkage at both the 5'- and 3 '-ends.
- Cytotoxicity Assays The cytotoxicity of T30177 was determined using several different cell lines and primary human cells as described above. The TC 25 , TC 50 and TC 95 values obtained are shown in Table B-l . The cytotoxicity profile obtained for log phase growing cells was variable depending upon the cell line used, while the slower growing PBMCs, PBLs, and macrophages all tolerated the compound at concentrations exceeding 100 ⁇ M as monitored using the trypan blue exclusion, [ 3 H]thymidine uptake, or [ 3 H]leucine uptake techniques.
- Table B-l Cytotoxicity of T30177 in established cell lines and primary cells.
- Macrophages > 100 > 100 > 100
- TC 25 , TC 50 , and TC 95 values are the concentrations of T30177 required to inhibit 25%, 50% and 95% of growth (cell lines) or cell survival (primary human cells).
- the cytotoxicity of T30177 in human cell lines was determined using log phase growing cells.
- the cytotoxicity of T30177 in primary human cells was determined using trypan blue exclusion technique or by measuring the uptake of [ 3 H]thymidine or [ 3 H]leucine on slow growing primary cells.
- Table B-2 Inhibitory effects of T30177, AZT, and DS500 on viral replication.
- HIV-2 ran MT4 5.98 ⁇ 1.05 - 0.084 ⁇ 0.086 s ⁇ v MAC25 , MT4 1.5 ⁇ 1.2 - 0.548 ⁇ 0.48
- the IC50 value is the concentration of drug required to inhibit virus production by 50%.
- the results presented are the averages of three or more experiments.
- the MOI used for all HIV-l, HIV-2 and SIV strains tested was 0.01.
- T30177 was also tested for its ability to inhibit laboratory strains of HIV-2 and SIV.
- the results (Table B-2) from these assays indicate that T30177 is more active against the strains of HIV- 1 and SIV tested than against the two strains of HIV-2 tested (ROD and EHO).
- T30177 was found to be inactive against a variety of enveloped and nonenveloped viruses tested (Table B-3) with IC 50 values found to be grater than the highest concentration of drug tested (200 ⁇ g/mL or 37 ⁇ M). This is in contrast to DS5000 which was found to be a potent inhibitor of all of the enveloped viruses tested except Vaccinia and Semliki forest viruses (Table B-3). Table B-3. Inhibition of viral replication in cell lines treated with T30177 or DS5000.
- HSV-l (KOS) >200 2 400 >400
- HSV-2 (G) >200 2 400 >400
- HSV-l TK (B2006) >200 2 400 >400
- HSV-l TK (VMW1837) >200 2 400 >400
- Sindbis virus >200 10 200 >400
- Vesicular Stomatitis virus >200 20 400 >400
- Non Enveloped Viruses
- Activated PBMCs were infected with laboratory strains of HTV-l and cultured in the presence of T30177, AZT or ddl. Treatment of infected PBMCs with T30177 inhibited the replication of the all four HTV-l isolates tested with IC 50 values ranging from 0.12 to 1.35 ⁇ M (Table B-4). In this assay AZT was more efficacious against all HIV-l isolates tested, on a molar scale, than T30177 while at the same time T30177 was more potent than ddl against the two HTV-l strains tested. It is also interesting to note that HIV-1 IIIB was more susceptible to T30177 in assays performed using PBMCs than in assays using T-cell lines (Tables B-2 and B-4).
- Table B-4 HIV-l replication in primary human cells treated with T30177, ddl or AZT
- Concentration of drug required to inhibit viral production by 50% was determined using the Coulter p24 antigen capture or RT assays.
- Antiviral assays were performed using laboratory strains of HTV-l in peripheral blood mononuclear cells (PBMCs) or using syncytium inducing (SI) or non-syncytium inducing (NSl) clinical isolates of HTV-l in PBLs.
- PBMCs peripheral blood mononuclear cells
- SI syncytium inducing
- NSl non-syncytium inducing
- the therapeutic potential of any anti-HIV drug is dependent upon its ability to inhibit clinical isolates of the virus obtained from different geographical locations. Therefore, the inventors evaluated the ability of T30177 to inhibit the infection of PBLs using a variety of clinical isolates of HIV-l which were both syncytium inducing (SI) and non syncytium inducing (NSl) strains of HIV-l . In addition, the isolates used in this study had their origins in different geographic regions. After infection with HTV-l the PBLs were cultured in the presence of T30177, AZT or ddl for seven days.
- T30177 inhibited the viral replication of all the HTV-l isolates tested with IC 50 values ranging from 0.23 to 3.08 ⁇ M (Table B-4).
- AZT and ddl had IC 50 values ranging from 0.01 to 1.65 ⁇ M and 0.41 to 2.61 ⁇ M, respectively. It is important to note that T30177 was active against both NSl and SI isolates and was very active against he JOGA isolate which was obtained from a pediatric patient. The JOGA isolate was also observed to be relatively resistant to AZT treatment (Table B-4).
- T30177 Another major target cell of HIV-l infection is the macrophage.
- Fully differentiated macrophages were infected with HIV-1 DV and treated with T30177 or AZT.
- T30177 significantly inhibited HTV-l replication in macrophages ( Figure B-2).
- T30177 and AZT worked best when administered at concentrations above the IC 50 values obtained for these drugs in assays performed in established cell lines.
- T30177 was much less sensitive to changes in the viral MOI. For example in these assays when the MOI of HTV-IRF was changed from 0.01 to 1.28, T30177 only exhibited a 14-fold increase in its IC 50 value while at the same time the IC 50 value for AZT increased over 1000-fold (Table B-5).
- T30177 on CD4 and CD8 T-cell Subsets.
- One of the principal immunological markers correlated with progression to AIDS is the decline in T lymphocytes which express the CD4 cell determining marker (CD4).
- the change in CD4 + T-lymphocytes is usually monitored by noting changes in the ratio of CD4 + to CD8 + lymphocytes in the blood.
- CD4 and CD8 antigen expression was analyzed on the surface of cultured PBMCs seven days post-infection with either laboratory strains or clinical isolates of HTV-l .
- T30177, DS5000 or AZT was added to MT-4 cells infected with HIV-1 1IIB (MOI of 1) at various times post-infection.
- Test compounds were added at a concentration 100-fold higher than the determined IC 50 value for each drug in the standard assay performed using MT-4 cells and the IIIB strain of HIV-l (Table B-2).
- Viral p24 antigen levels were monitored 29 hour post-infection.
- the results of this assay indicate that postponing the addition of T30177 for one hour was enough to dramatically reduce the inhibitory effects of this compound in a fashion similar to that of DS5000 and clearly different from AZT which lost its protective capacity when added to the cell culture medium 3 or 4 hours post-infection ( Figure
- HeLa-CD4-3-galactosidase cell studies To differentiate the effects of T30177 on early events in the viral life cycle, through integration and subsequent production of the tat gene product, from the inhibition of HIV- 1 gp 120-mediated cell fusion two experimental protocols were employed. The first protocol monitored the effects of the drug on the ability of HIV-1 RF to infect and/or replicate within HeLa-CD4-LTR-/3-galactosidase cells and was performed as described in Methods. In this experiment drug interdiction at any step in the viral life cycle through the production of the tat gene product would cause a decrease in expression of the ⁇ - galactosidase gene, the transcription of which is regulated by the HIV-l LTR.
- T30177 is a potent inhibitor of /3-galactosidase production in this assay with an IC 50 value of 0.009 ⁇ M, while the IC 50 value obtained for CSB in the same experiment was 0.26 ⁇ M ( Figure B-4A).
- T30177 had no observable direct effect on ⁇ -galactosidase enzyme activity at concentrations up to 10 ⁇ M
- the second protocol used was a virus-free assay designed to monitor CD4- and gpl20-mediated cell fusion events.
- T30177 was able to interfere with the fusion process ( Figure B-4B).
- the observed IC 50 value (1 ⁇ M) was approximately 100-fold higher than that needed to interfere with ⁇ - galactosidase production in the virus infection assay ( Figure B-4A).
- the IC 50 value observed for CSB increased approximately 3-fold to 0.8 ⁇ M over the concentration needed to interrupt ⁇ - galactosidase production in the virus infection assay ( Figure B-4).
- T30526 an oligonucleotide in which a dA has been substituted for a dG at a position that would interrupt the formation of one of the two tetrads involved in the G-octet.
- T30526 has the same partial PT pattems as T30177 (Table B-7).
- T30526 has the same partial PT pattem as T30177 (Table B-7).
- T30526 was found to be approximately 100-fold less potent that T30177 in inhibiting HIV-1 RF production in culture assays (Table B-7), 10- 15-fold less potent at inhibiting virus-infected cell /3-galactosidase production ( Figure B-4A) and did not inhibit cell fusion at the highest concentration of drug tested (20 ⁇ M, Figure B-4B).
- Oligonucleotides were synthesized with either total phosphodiester (PD) backbone, total phosphorothioate (PT) backbone, or partial phosphorothioate (pPT) backbone, in which the 5'- and 3 '-penultimate internucleoside linkages were phosphorothioate.
- PD total phosphodiester
- PT total phosphorothioate
- pPT partial phosphorothioate
- HIV-1 1IIB infected MT-4 cells were treated with T30177, AZT, DS5000, or the bicyclam compounds JM2763 or JM3100 for four days using drug concentrations equivalent to 1 , 10 or 100-fold over their respective IC S0 values (Table B-7).
- the IC 50 values used for JM2763 and JM3100 were from previously reported results, (De Clereq, et al., Antimicrob. Agents Chemother. 38:668-674 (1994)). After four days in culture the cells were washed and then further cultured in complete medium without drug.
- the cells were monitored daily for the appearance of viral-induced syncytium formation and every second or third day for viral p24 antigen in the culture medium.
- T30177 at 100-fold over the IC 50 value (approximately 10 ⁇ M), suppression of virus P24 production was observed for at 1st 27 days after removal of drug from the infected cell culture ( Figure B-5).
- T30177 was also tested for its ability to inhibit HIV-l protease and integrase enzymes. When concentrations of T30177 up to 10 ⁇ M were used in protease inhibition assays no effect on the viral enzyme was observed (data not shown). However, when assayed for its effect on HIV-l integrase, T30177 was able to reduce both the 3'-processing and strand transfer activities of the integrase enzyme with IC 50 values of 0.092 and 0.046 ⁇ M, respectively (Table B-7).
- T30177 To determine if the sequence, three dimensional structure, chemical composition of the backbone or a combination of these parameters contributed to the observed anti-integrase activity of T30177, the inventors synthesized and tested for enzyme inhibitory activity the oligonucleotides shown in Table B-7.
- T30038, T30175, and T30526 are variations of T30177.
- T30340, T30341 and T30659 are variations ofthe thrombin-binding aptamer sequence reported by Bock et al. Bock, et al., Nature 355:564-566 (1992).
- IC 50 values for each of these oligonucleotides tested in the integrase assay are shown in Table B-7.
- the results of this experiment indicate that any of the sequence motifs tested were potent inhibitors of the HIV-l integrase enzyme when the oligonucleotides were synthesized with a PT backbone.
- the number of PT linkages in the backbone was reduced to one linkage at each end of the molecule (pPT) the thrombin binding aptamer (T30559) and the antisense sequence
- T30662 no longer displayed anti-integrase activity while the level of inhibition observed using T30177 was relatively the same as that observed using the total PT version of this molecule (T30038).
- T30177 sequence motif was able to inhibit viral integrase with IC 50 values of 170 and 125 nM for the 3'processing and strand transfer enzyme activities, respectively.
- T30526 the tetrad-disrupted mutant version of T30177, was still able to inhibit viral integrase protein in this assay, albeit at a concentration 2- to 3-fold higher than that observed using T30177.
- T30177 was found to have a wide range of therapeutic indices (TIs) depending upon the viral strain and cell line used in a given assay. For example, when T30177 was used to inhibit HIV-1 SKI in CEM-SS cells a TI of 3680 was obtained.
- T30177 variability in efficacy of T30177 in PBMCs and PBLs, which depended upon the clinical isolate tested, was very similar to the variation in activity observed for the nucleoside analogs AZT and ddl. It is interesting to note that an approximately 20 fold variation in the IC 50 value was observed for T30177 when used to inhibit HIV-1 IIIB in CEM-SS cells (2.8 ⁇ M) versus
- PBMSc (0.12 ⁇ M) (Tables B-2 and B-3).
- results obtained using clinical isolates to infect heterogeneous populations of primary cells may be more predictive of in vivo efficacy than data generated using laboratory strains of HIV-l in established cell lines. It is unlikely that HTV-l mB is a resistant strain of HIV-l since T30177 was more effective against this virus in PBMCs than in cell lines.
- T30177 displayed some antiviral which indicated a mechanism of action similar to the known blockers of virus adso ⁇ tion or virus mediated cell fusion such as dextran sulfate and CSB ( Figures B-3 and B-4). Like CSB and DS5000, T30177 needed to be added to cells at the time of or soon after virus infection.
- T30177 is 100-fold less effective in inhibiting gp 120-induced cell fusion events than it is at inhibiting early events in the viral life cycle, suggesting a specific point of interdiction with virus distinct at least from that of CSB.
- the antiviral profile of T30177 also displayed other characteristics which distinguished T30177 from DS5000 and CSB. For example, while DS5000 is active against a wide range of enveloped viruses, T30177 appears to be a more selective inhibitor of retroviruses with maximum efficacy displayed when used to inhibit strains of HIV-l (Tables B-2 and B-3).
- T30177 is a potent inhibitor of HIV-l integrase function in vitro (Table B-7) and by the observed accumulation of circularized proviral DNA in the low-molecular weight Hirt DNA fractions ( Figure B-7).
- T30177 While the time of drug addition studies would suggest interference with virus internalization as a key mechanism of action for T30177 it is also clear that readily detectable viral nucleic acids do enter the cells. It is quite possible that T30177 inhibits HIV-l via several different mechanisms of action. Another possibility is that T30177 is carried into the cell along with the infecting virus or is slow to accumulate within cells (Bishop et al. 1996 J. Biol. Chem. 271:56988-5703) hence the need to add drug during virus infection. Experiments designed to address these possibilities are underway.
- T30177 is stable in serum and within cells, with a half-life measured in days (Bishop, et al. J. Biol. Chem.1996271:5698-5703). This information taken together with the ability of T30177 to suppress HIV-l for over four weeks after an initial treatment regimen, in culture, makes this class of compounds an attractive candidate for development of oligonucleotide-based therapeutic agents for HIV-l.
- oligonucleotide substrates and inhibitors Preparation of oligonucleotide substrates and inhibitors.
- the following HPLC purified oligonucleotides were purchased from Midland Certified Reagent Company (Midland, TX): AE117, 5'-ACTGCTAGAGATTTTCCACAC-3'; AE118, 5'-GTGTGGAAAATCTCTAGCAGT-3'; A E 157 , 5 ' - G A A A G C G A C C G C G C C C - 3 ' ; A E 146 , 5 ' - GGACGCCATAGCCCCGGCGCGGTCGCTTTC-3'; AE156, 5 ' -
- AE117, AE118, and the first 19 nucleotides of AE156 correspond to the U5 end ofthe HIV-l long terminal repeat (LTR).
- LTR HIV-l long terminal repeat
- AE118 was 5'-end labeled using T 4 polynucleotide kinase (Gibco BRL) and y-[ 32P ]-ATP (Dupont- NEN).
- the kinase was heat-inactivated and AE117 was added to the same fmal concentration. The mixture was heated at 95°C, allowed to cool slowly to room temperature, and run on a G-25 Sephadex quick spin column (Boehringer Mannheim) to separate annealed double-stranded oligonucleotide from uninco ⁇ orated label.
- AE118S was 5'- end labeled, annealed to AE117, and column purified as above.
- AE118 was 3'-end labeled using o--[ 32P ]-cordycepin triphosphate (Dupont-NEN) and terminal transferase (Boehringer Manheim). Engleman, et al, Cell 67, 1211-1221 (1991); Vink, et al., Nucleic Acids Res. 19, 6691- 6698 (1991). The transferase was heat-inactivated and RM22M was added to the same final concentration. The mixture was heated at 95 °C, allowed to cool slowly to room temperature, and run on a G-25 spin column as before.
- AE157 was 5'-end labeled, annealed to AE156, AE146, and AE117, annealed, and column purified as above. Oligonucleotides composed of deoxyguanosine and thymidine were synthesized, purified, and incubated with potassium ion to generate the G4s. The guanosine quartet (G4) forming structures were then purified as previously described. Rando, et al., J. Biol. Chem. 270, 1754- 1760 (1995).
- Integrase proteins and assays Purified recombinant wild-type HIV-l integrase, deletion mutants
- Integrase was preincubated at a final concentration of 200 (for HIV-l and HIV-2) or 600 nM (for FIV and SIV) with inhibitor in reaction buffer (50 mM Nacl, 1 mM HEPES, pH 7.5, 50 ⁇ M dithiothreitol, 10% glycerol (wt/vol), 7.5 mM MnCl 2 or MgCL, (when specified), 0.1 mg/mL bovine serum albumin, 20 mM 2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 mM MOPS, pH 7.2) at 30°C for 30 minutes.
- reaction buffer 50 mM Nacl, 1 mM HEPES, pH 7.5, 50 ⁇ M dithiothreitol, 10% glycerol (wt/vol), 7.5 mM MnCl 2 or MgCL, (when specified), 0.1 mg/mL bovine serum albumin, 20 mM 2-mercaptoethanol, 10% dimethyl s
- IC 50 100 X[l - (D - C)/(N - Q], where C, N, and D are the fractions of 2 lmer substrate converted to 19mer (3 '-processing product) or strand transfer products for DNA alone, DNA plus integrase, and integrase plus drug, respectively.
- IC 50 was determined by plotting the drug concentration versus percent inhibition and determining the concentration which produced 50% inhibition.
- UV crosslinking experiments The method used has been described by Engleman et al. Engelman, etal., 7. Virol. 68, 5911-5917 (1994). Briefly, integrase (at the indicated concentration) was incubated with substrate in reaction buffer as above for 5 minutes at 30°C. Reactions were then irradiated with a UV transilluminator (254 nm wavelength) from 3 cm above (2.4 mW/cm 2 ) at room temperature for 10 minutes. An equal volume (16 ⁇ L) of 2X SDS-PAGE buffer (100 mM Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added to each reaction.
- 2X SDS-PAGE buffer 100 mM Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol
- integrase 200 nM was preincubated with the guanosine quartet (at the indicated concentration) for 30 minutes at 30°C prior to the subsequent addition of the radiolabeled viral DNA substrate (20 nM).
- integrase 200 nM was preincubated with either the radiolabeled viral DNA substrate (20 nM) or T30177 (20 nM) for 5 minutes at 30°C prior to the addition of competitor DNA at the indicated concentration.
- Guanosine quartet oligonucleotides inhibit HIV-l integrase.
- the inhibition of HIV-l integrase by a series of oligonucleotides which can form G4s is shown in Figure C-1.
- Oligonucleotides T30177 and T30659 fold upon themselves into structures stabilized by two G4s stacked upon each other to form a guanosine octet (Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995); Schultze, et al., J. Mol. Biol.
- T30177 is active against HIV-l in cell culture and against purified HIV-l integrase in vitro (Ojwang, et al., Antimicrob. Agents Chemother. 39, 2426-2435 (1995)) while T30659 is not.
- inhibition of both the 3 '-processing and strand transfer activities of HIV-l integrase (Fig. C-1A) by T30177 was observed in the nanomolar range (see Fig.
- T30177 was effective and T30659 was not, the inventors made a series of compounds to incrementally change one compound into the other.
- the structures of these compounds are shown in panels C and D of Figure C-1.
- the differences between T30177 and T30659 i.e., the presence of additional bases at both ends, different sequences in all three loops, and extension of loop 2 manifest themselves in dramatic increases in the IC50 values (Fig. C-1D).
- the inventors first added the same 5'- and 3 '-nucleotides to T30659 as are present on T30177, yielding T30674 (Fig. C-1C). These changes did not confer potency (Fig. C-1D).
- a strand transfer assay using "preprocessed" (3'-recessed) substrate (19mer in Fig. C-2a, left panel) was also performed to determine whether the strand transfer reaction was truly being inhibited or whether the inhibition of the 3 '-processing reaction caused the decrease in the subsequent strand transfer products. Inhibition of strand transfer using this substrate was observed in the same concentration range (Fig. C-2A, right panel) as that seen with the blunt- ended, duplex oligonucleotide substrate (Fig. C-1 A, top). Therefore, G4 oligonucleotides inhibit both steps of the integrase reactions: 3'-processing and strand transfer.
- Integrase can catalyze in vitro an apparent reversal of the DNA strand transfer reaction called disintegration. Chow, et al., Science 255, 723-726 (1992). In contrast to the 3'- processing and strand transfer reactions, disintegration requires neither the N-terminal zinc-finger region nor the C-terminal DNS-binding domain of integrase. Bushman, et al., Proc. Natl. Acad. Sci. U.S.A. 90, 3428-3432 (1993).
- HIV-l integrase catalytic core domain j n 50 - 2i2 jg C-4A
- the HIV-l integrase catalytic core domain j n 50 - 2i2 jg C-4A
- T30177 required for inhibition of disintegration was higher than that required for inhibition of either 3 '-processing or strand transfer. These results are consistent with those observed with other molecules (Fesen et al., 1994, Mazumder et al, 1994). This observation suggests that the active site of HIV-l integrase may tolerate drug-induced protein or DNA distortion during the disintegration reaction, consistent with the relative tolerance of integrase to mutagenesis of either substrate features (Chow & Brown, 1994) or protein structural domains (Bushman et al., 1993) in this reaction. This is the first example of an HIV-l integrase inhibitor requiring the enzyme zinc-finger region for inhibitory activity.
- the IN 1"212 protein which has previously been shown to bind to linear viral DNA only at high concentrations (approximately 2.56 ⁇ M) and only in the presence of divalent metal ion, (Engelman, et al., J. Virol. 68, 5911-5917 (1994)), was able to crosslink to T30177 with the same efficiency as wild-type integrase in the absence or presence of added manganese (lanes 2 and 3).
- the IN 50"288 protein which contains a nonspecific DNA-binding domain, was also able to crosslink to T30177 with the same efficiency as wild-type integrase in the absence or presence of added manganese (lanes 4 and 5), consistent with its ability to bind to viral U5 DNA (Engelman et al. , 1994).
- the extent of crosslinking was significantly diminished in the case of the core mutant IN 50"212 compared to IN 1"212 in the absence or presence of manganese (compare lanes 2 and 3 with 6 and 7, faster migrating complex).
- the higher molecular weight species in lane 6, having the expected molecular weight of a dimer has been reproducibly observed, but its density has not been confirmed.
- the In 1"55 protein was able to bind to the T30177 G4 oligonucleotide, but not the viral DNA substrate, in the presence of zinc alone (left and right panels, lanes 9 and 10). These results are in accord with the known zinc-binding ability of this domain. Bushman, et al., Proc. Natl. Acad. Sci. U.S.A. 90, 3428-3432 (1993); Burke, et al., J. Biol. Chem. 267, 9639-9644 (1992). But they also suggest that the N-terminal domain of integrase has DNA binding capabilities on its own.
- T30175 has the same base sequence as T30177 but is composed entirely of phosphorothiodiester internucleotidic linkages.
- the inhibition of 3 '-processing catalyzed by HIV-l integrase by these guanosine quartets is shown in Fig. C-6C.
- Both T30175 and T30177 showed four to five-fold increases in potency when magnesium was used as the divalent metal instead of manganese.
- T30038 showed no significant increase in potency when magnesium was used as the ion (Fig. C-6D).
- the T30177-integrase complex has a molecular weight of 37,000. Neither complex could not be competed off by either competitor DNA even at concentrations where the competitor was in 500- fold excess (Fig. C-7A, lane 6). Similar results were seen when the In 1 " 212 and IN 50"212 proteins were used in competition experiments (data not shown). Therefore, the stability of the G4 oligonucleotide DNA-integrase complex is comparable to that of the viral DNA-integrase complex is comparable to that of the viral DNA-integrase complex. Ellison, et al, Proc. Natl. Acad. Sci. U.S.A. 91, 7316-7320 (1994); Vink, et al., Nuc. Acids Res. 22, 4103-4110 (1994).
- T30177 was tested for inhibition of the related retroviral integrases from HIV-2 (van Gent et al., 1992), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV) (Vink et al., 1994b).
- SIV simian immunodeficiency virus
- FV feline immunodeficiency virus
- T30177 G4 oligonucleotide displayed some selectivity among the lentiviral integrases.
- Oligonucleotides are Novel and Potent Inhibitors of HIV-l integrase. Oligonucleotides composed of deoxyguanosine and thymidine and forming guanosine-tetrads (G4) structures have previously been shown in inhibit HIV replication. Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995); Wyatt, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994). Two mechanisms have been invoked.
- oligonucleotides have been shown to bind to the V3 loop of the envelope protein gpl20 and subsequently inhibit virus adso ⁇ tion and cell fusion.
- Wyatt, et al. Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994).
- oligonucleotides such as those described in the present study also inhibit viral-specific transcripts, Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995), presumably by inhibiting viral integration. Ojwang, et al., Antimicrob. Agents Chemother. 39, 2426-2435 (1995).
- G4 oligonucleotides differ from previously published HIV-l integrase inhibitors in several ways. (Table C-1) First, they are among the most potent inhibitors to date with IC50's in the nanomolar range. Their potency range is comparable to flavone, Fesen, et al., Biochem. Pharmacol.
- G4 oligonucleotides form stable enzyme complexes that cannot be displaced by excess viral DNA oligonucleotide.
- T30720 5' g gggTggtgggTgggt -3' pPT variant T c-5 propynl dU 6 mer
- the propynyl dU building blocks contain uracil bases in which a propynyl group has been added to the c-5 position.
- the propynl dC building blocks contain cytosine b in which a propynyl group has been added to the c-5 position.
- These reagents are commercially available from Glen Research Reagent co.
- Other unusual bases used s as dl (inosine) and iodo and bromo dU are also commercially available such a.
- pPT is shorthand for ODNs in which the linkage between the ultimate and penultimate bases has been changed from phosphodiester to phosphorothioate. PT is shorth to indicate that all linkages are phosphorothiaiote.
- RNA sugars or 2'-0-methyl RNA sugars as indicated instead of 2'-deoxy sugars as found in DNA.
- the thrombin binding aptamer sequence used was the first described by Bock et al. ⁇ Nature 1992 355:564-566] and reported to fold into an structure stabilized by an intramolecular tetrad by Want et al. Biochemistry 1993 32: 1899-1904].
- T30747 A/B contain only natural phosphodiester linkages.
- T30747B is 3'-modif ⁇ ed in that the dimethoxy trityl capping group was left attached after removal of the oligonucleotide from the solid support and subsequent purification. Usually this group is removed after synthesis of the molecule.
- Compound S935833 has the following pattern of phosphorothioate (PT) linkages where * denotes the PT linkage:
- Compound T30754 has a pPT pattern in which only the 5' and 3' linkages are PT:
- the zinc finger region can selectively bind to non-B DNA structures.
- Dyda et al.
- Science 266, 1981-1984 (1994) resembles that of the Rev C Holliday junction- resolving enzyme, Ariyoshi, et al, Cell 78, 1063-1072 (1994), and of the bacteriophage Mu transposes core.
- These structurally related proteins also bind multiple double helices, generating X structure intermediates.
- HIV-l integrase mutant with the two zinc finger histidines mutated to asparagines was able to bind to G4 oligonucleotides (data not shown).
- data not shown suggest that HIV-l integrase may have two separate binding sites, one for viral DNA and one for target or "host" DNA. This scenarios would be expected if integrase were to bind both the viral and host DNA at sites which were instinct but in close proximity in vivo.
- the ⁇ subunit of the Oxytrichia telomere binding protein has bene proposed as a molecular chaperone for the formation of G4s at the end of chromosomes by enhancing the rate of a thermodynamically favored transition. Fang, et al., Cell 74, 875-885 (1993).
- the nucleocapsid protein may also act as a molecular chaperone to enhance dimer formation. Sundquist, et al, Proc. Natl. Acad. Sci. U.S.A. 90, 3393-3397 (1993). In this manner, it may facilitate the formation of and bind to the G4.
- G4s can bind to purified nucleocapsid protein (data not shown).
- G4s may be structurally important as molecular scaffolds in both retroviral preintegration complexes and telomeres, and these structures may have associated chaperones in both cases.
- a G4 containing structure may act as a negative regulator of telomere elongation in vivo due to its ability to inhibit telomerase in vitro.
- G4 structures may act to inhibit integrase (Fig. C-1) and thereby act as a block to auto integration or digestion of the viral DNA prior to insertion into the host chromosome.
- G4s in vivo has not been demonstrated. However, they have been shown to form in vitro in telomeric sequences, Sundquist, et al., Nature 342, 825-829 (1989); Smith, et al., Nature 356, 164-168 (1992); Kang, et al., Nature 356, 126-131 (1992), HIV-l RNA sequences, Sundquist, et al, Proc. Natl. Acad. Sci. U.S.A. 90, 3393-3397 (1993); Awang, et al., Biochemistry 32, 11453-11457 (1993), fragile X syndrome nucleotide repeats, Fry, et al., Proc. Natl.
- G4 binding protein Another G4 binding protein, KEM1
- KEM1 has been isolated and implicated in recombination-type reactions in vivo. Liu, et al., Cell 11, 1083-1092 (1994).
- the catalytic activities of this protein and of the integrase protein are DNA endonucleolytic cleavage and strand transfer.
- integrase does not catalyze endonucleolytic cleavage reactions on G4s (data not shown).
- G4s may be mechanistically relevant in a diverse set of biological processes involving enzymes with similar activities.
- oligonucleotides containing intramolecular G4s are potent inhibitors of HIV-l integrase. Inhibition is dependent on the zinc finger region of integrase and on the structure and sequence of the G4s. These findings also suggest that novel AIDS therapies could be based upon G4s as inhibitors of HIV-l integrase.
- T30177 forms an intramolecular fold which is stabilized by a pair of G-tetrads, connected by three single stranded loops, with a 1-2 base tail to ether side of the fold.
- the inventors undertook studies to determine sequence dependence of the intramolecular folding mechanism, in a set of four closely related 16-17 base oligonucleotide homologues, with sequences in the range G10-12-T4-7.
- the original T30177 compound was included, along with three derivatives which were designed so as to alter the structure of loop domains, while keeping the pair of G-tetrads intact.
- the inventors were able to show that a single base alteration within the loop or tail domains can produce a very large change in folding stability.
- the K + ion dependence of these data suggested a preliminary model wherein the loop and tail domains interact to form stable metal ion-binding sites.
- T30695 A 16mer derivative (T30695) was designed within the context of that model, with the intent of enhancing the interaction between K + and the 5' terminus of the oligomer.
- the inventors showed that T30695 folding is indeed more stable than other members of the group and is highly specific for K + , as assessed from the ion dependence of thermal denaturation, CD spectra and UV detected folding kinetics.
- the inventors compared tertiary structure stability at three K + concentrations with the capacity of the folded oligomers to inhibit the HIV-l integrase enzyme in vitro, or HIV-l infection in cell culture.
- the stability and activity data are found to be highly correlated, as a function of sequence alteration, suggesting that formation of the stable intramolecular fold may be a prerequisite for both integrase inhibition and anti-HIV- 1 activity.
- the structure of the folded state has not yet been confirmed at high resolution, the data presented here suggested that the structure of the T30695 complex with K + ion may be of pharmaceutical significance and could serve as the basis for additional improvement of the observed HIV-l activity.
- Oligonucleotide Synthesis All oligonucleotides used in this study were synthesized on an Applied Biosystems Inc. DNA synthesizer, model 380B or 394, using standard phosphoramidite chemistry, or fast deblocking Expedite chemistry on a Milligen synthesizer. All oligomers possessed 2 phosphorothioate linkages (one on each terminus) which were introduced by the H-phosphonate method. Oligonucleotides were purified by preparative anion exchange HPLC, on Q-Sepharose.
- oligonucleotides Prior to UV, CD or kinetic analysis, oligonucleotides were annealed at 20 mM Li3P04, pH 7 at 3-15 uM in strands. Samples were heated to 90°C for 5 min and then incubated for 1 hour at 37°C. Metal ion could be added as the chloride either before or after the 37° C incubation, with no measurable difference in final state, as assessed by UV, CD or gel analysis. As assessed by native gel electrophoresis (not shown), this annealing method was found to produce a single product with mobility consistent with a folded monomer over the strand concentration range from 3-15 uM, at all ion concentrations described.
- CD spectra were obtained at 25°C in 20 mM Li3P04, pH 7, at 15 uM in strands, on a Jasco J-500A spectropolarimeter. Metal ion was added to the desired concentration, followed by one hour of pre-incubation at 37 °C. Each spectrum in the text represents 5 averaged scans. To conform to traditional standards, data are presented in molar ellipticity (deg-cm2-dmolE- 1) as measured in base, rather than strand equivalents.
- the RF laboratory strain of HIV- 1 was used to infect established cell lines for one hour at 37 °C prior to washing and resuspension in medium containing increasing concentrations of drug.
- Four to six days post-infection, drug treated and control wells were analyzed for HIV- 1 induced cytopathic effects, for the presence of viral reverse transcriptase (RF) or viral p24 antigen in the culture medium as previously described by Ojwang et al. (Bishop et al., (1996) J. Biol. Chem. 271, 5698-5703).
- Purified recombinant HIV-l integrase enzyme wild-type was a generous gift from Dr. Craigie, Laboratory of Molecular Biology, NIDDK.
- Tm values for T30177 are consistently higher, by 1030°C, than has been seen for other small intramolecular folds (Smith, F. W., & Feigon, J. (1992) Nature (London) 344, 410-414; Schultze, et al., J. (1994) J. Mol. Biol. 235, 1532-1547).
- T30177 differs from these other homologues only in terms of the proposed loop domains, the inventors have synthesized homologues of T30177 where the central G-octet remains constant, but where the loop domains to either side have been modified by addition or replacement of a single base.
- the T30676 homologue is identical to T30177, but has been modified so as to add an additional G into the topmost loop of the structure.
- line c this one base addition produces a 20°C decrease in Tm over the entire range of K + ion tested.
- T30677 homologue was prepared (Figure D-1A), which is identical to T30177, but has been modified so as to convert a pair of Gs in the bottommost loop domain. As seen in Figure D-2A, line d, this two base loop substitution produces a 30 °C decrease in Tm over the range of K + ion tested.
- T30695 ( Figure D-l A). As seen in Figure D-2A, line a, even though T30695 is one base shorter than the T30177 homologue, it was found to melt at approximately 10 °C higher temperature, over the entire
- Tm values for T30695 were found to be strand concentration independent, confining the general similarity of the folding process ( Figure D-2C).
- the K + ion dependence of thermal stability was very striking.
- measured Tm values increase from 40°C to 65°C over the added KCI range from 50 EM to 1 mM. Again, this ion dependence argues that the observed stabilization is likely to result from site-specific ion binding, rather than simple ion-screening effects.
- Tm values have been measured for alkaline metal ions with differing radius: Na + (0.99A), K + (1.38A), Rb + (1.49A), and Cs+ (1.69 A).
- K + ion selectivity is detected.
- Rb + is very similar to K + in general chemical properties, and differs by only +0.1 IA in ion radius, it is seen that the Rb + complex with T30695 melts at approximately 20-30°C lower temperature over the entire concentration range studied.
- Na + ion and Cs+ ion which differ from K + in ion radius by -0.37A and +0.29A, respectively, are seen to be even more destabilizing. Similar ion binding selectivity were obtained by this method for the T30177 homologue (not shown).
- Circular Dichroism In order to explore the nature of these ion binding effects, the inventors monitored the folding of T30695 by circular dichroism (CD) methods. It is known that G-quartet based folding, both intra and intermolecular, gives rise to large induced ellipticity values (Balagurumoorthy, P. & Brahmachari, S. K. (1994) J. Biol. Chem. 269, 21858-21869; Jin, et al.
- CD circular dichroism
- Stable tetrad folds are characterized by nonconservative spectra, with maxima at 264 nm (- 1x10+5 deg-cm2/dmol) and 210 rim (-5x10+4 deg-cm2/dmol) and a minima at 240 rim (-4x1044 deg-cm2/dmol).
- Figure D-3A the inventors monitored the CD spectrum of T30695 at 0, 0.05 and lOrnM
- T30177 A Relationship Between Structure and Function.
- the inventors' interest in T30177 and its derivatives has arisen because this class of oligonucleotide is a potent inhibitor of HIV infection in culture (Rando et al., (1994) J. Biol. Chem. 270, 1754-1760; Ojwang et al. (1994) J. AIDS 1, 560-570; Bishop et al., (1996) J. Biol. Chem. 271, 5698-5703; Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35), and in vitro, has been shown herein to be the most potent inhibitor of HIV-l integrase to have been identified thus far (see also Ojwang et al.
- Antiviral activity has been obtained as described herein and elsewhere (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35), and is presented as the IC50, in nM, of added oligonucleotide.
- this first ion binding step has rather modest selectivity among the alkaline metal ions (Williamson, J. R. (1994) Annul Rev. Biophys. Biomal. Struct. 27, 703-730).
- the inventors propose that the second step in the folding process involves binding of additional ion equivalents to the loop regions of the structure. It is also proposed that this second process, which occurs at higher added ion concentration ( Figure D-3) and which is associated with the slow kinetic step of Figure D-4, is coupled to a rearrangement of the loop domains to yield two additional sites for metal ion coordination.
- T m with * were obtained by a calculation according to the linear fitting functions of T m vs. Log[KCl].
- a toxicity study of AR177 was conducted with the objective of establishing the dose-response relationship between intravenous infusion of ARl 77 and hemodynamic parameters in cynomolgus monkeys.
- Intravenous infusion is the proposed route of administration of AR177 to humans.
- the present study was conducted using the short term infusion protocol recommended by the Food and Drug Administration (Black et al., 1994), with measurement of central blood pressure, serum chemistry, hematology, coagulation factors, complement factors, and plasma AR177 concentrations.
- AR177 was synthesized at Aronex on a Milligen 8800 oligonucleotide synthesizer, and made into a stock solution at 25 mg/mL in sterile phosphate-buffered saline. AR177 has a molecular weight of 5793 daltons, and is a fully neutralized sodium salt. The structure of AR177 was characterized by phosphorus and proton NMR, sequencing, base composition, laser Reso ⁇ tion mass spectrometry, anion exchange HPLC and polyacrylamide gel electrophoresis. The AR177 was approximately 94% pure according to HPLC and electrophoretic analysis. All analyses are consistent with the proposed structure.
- Arterial blood samples were drawn at -10, + 10, +20, +40, +60 and + 120 minutes relative to the initiation of infusion into EDTA-containing tubes for hematology, complement factors, coagulation assay, serum chemistry, and plasma AR177 determination.
- blood was drawn via the femoral vein into EDTA-containing tubes for these same parameters.
- concentration of ARl 77 in dosing solutions was confirmed post experiment by absorbance at 280 nm on a spectrophotometer.
- the plasma fraction was obtained by low speed centrifugation of blood, and stored at -20 'C until used.
- Electrocardiograms ECGs
- central pressure a parameter indicative of central pressure
- heart rate a parameter indicative of central pressure
- Table E-l summarizes the study design. The animals were observed twice daily for pharmacotoxic signs and general health beginning two days before dosing and for seven days following dosing. The monkeys were not necropsied at the end of the study.
- Serum chemistry parameters The following were determined: sodium, potassium, chloride, carbon dioxide, total bilirubin, direct bilirubin, indirect bilirubin alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, calcium, phosphorus, glucose, urea nitrogen, creatinine, uric acid total protein, albumin, globulin, cholesterol and triglycerides. The samples were analyzed at Sierra Nevada Laboratories (Reno, NV).
- Hematology and coagulation parameters were determined: red blood cell count and mo ⁇ hology, total and differential white blood cells, hemoglobin, hematocrit, prothrombin time, fibrinogen, mean cell hemoglobin, mean co ⁇ uscular volume, mean co ⁇ uscular hemoglobin concentration, platelet count, and activated partial thromboplastin time. Hematology parameters were determined at Sierra Nevada Laboratories (Reno, NV).
- Complement factors The complement split product Bb and total hemolytic complement CH50 were determined. The choice of measuring the Bb split product, as opposed to other complement factors, was based on a published study showing the involvement of the alternative pathway in complement activation induced by oligonucleotides (Galbraith et al, 1994). Complement determinations were performed in the laboratory of Dr. Patricia Giclas at the Complement
- AR177 was assayed in the plasma using an anion-exchange HPLC method on a Waters HPLC system with a 626 pump, 996 photodiode array detector, 717 autosampler and Millennium system software controlled by an NEC Image 466 computer.
- Buffer A consisted of 0.1 M Tris base, 20% methanol, pH 12
- Buffer B consisted of 0.1 M Tris base, 1.0 M NaBr, pH 12.
- the anion-exchange column (Gen-Pak Fax column) was equilibrated at 80% buffer A/20% buffer B for 30 minutes before each HPLC run. Fifty microliters of 0.2 ⁇ filtered, neat plasma were analyzed per run.
- the elusion conditions were: a) five-minute isoaatic run at 80% A/20% B. during which the majority of the plasma proteins eluted, b) 25-minute linear gradient to 30% A/10% B during which AR177 elutes, c) five-minute Socratic run at 30% A/70% B. d) one-minute linear gradient to 100% B. e) two- minute run at 100% B for column clean-up, and fl two-minute linear gradient to 70% A/30% B for the step in the HPLC clean-up.
- the high pH (12) of the elusion buffers was necessary to dissociate AR177 from tissue constituents, which bind AR177 tightly around physiological pH. AR177 is completely stable at pH
- a standard curve was generated by spiking AR177 into cynomolgus monkey plasma in order to achieve concentrations of 0.04 to 128 ⁇ /mL.
- the plasma standards and unspiked plasma (control) were run on the anion-exchange HPLC column using e above conditions.
- the Waters Millennium software was used to determine the area under the peak for each ARl 77 standard at
- HPLC peak area versus AR177 concentration was plotted using Cricket Graph III 1.5.1 software. There were one to three HPLC replicate runs per AR177 standard. The limit of quantitation was 25 ng/mL (50 pL injection), whereas the limit of detection was 5 ng/mL (50 pL injection). The overall correlation coefficient of the fitted lines on the standard curve plots was greater than 0.999 on two different standard curves used in this smdy. The standard curve was linear over an approximate 3, 200-fold range. The variability of the replicates was 1-2% at all concentrations. There was one HPLC run per monkey plasma sample. This method was validated. Further details about the method will be published elsewhere (Wallace et al., submitted).
- Vd volume of distribution
- aPTT values were reestablished by two hours after termination of dosing with the 20 mg/kg dose. In the 5 mg/kg group, only a small and transient rise in aPPT was observed, and there was no change in prothrombin time (PT). Similar changes have been observed with other oligonucleotides, and are believed to be, at least in part, attributable to direct and reversible binding ofthe oligonucleotide to thrombin (Henry et al., 1994, Pharmaceutical Res. 11: S353, 1994). PT was affected to a much lesser extent than aPTT in the 20 and 50 mg AR177/kg groups (data not shown), indicating little or no effect on the extrinsic pathway. Complement activation
- Plasma levels of the complement split product Bb a marker for activation of the alternative pathway, were increased 60-85% over baseline in the 5 mg/kg group, approximately 2-fold over baseline in the 20 mg/kg group, and approximately 2- to 4-fold in the 50 mg/kg group at the end of infusion. (See Figure E-4).
- the elevation in Bb persisted through the duration of the 2-hour monitoring period, but the values had returned to normal by the following day. These increases in Bb were small in magnitude.
- AR177 had no effect on complement CH50 at doses up to 236 ⁇ /mL when it was tested in vitro in human or cynomolgus monkey plasma. (See below).
- Plasma concentrations of AR177 were maximal at the end of the infusion and declined thereafter with an approximate initial half-life of 20-30 minutes ( Figure E-6).
- Another more complete study in cynomolgus monkeys has shown the terminal half-life to be approximately 24 hours (See below). These half-lives are much longer than that reported by Lee et al. ((1995) Pharmaceut. Res. 12: 1943-1947) in cynomolgus monkeys for GS-522, a 15mer oligonucleotide that has a tetrad structure similar to AR177. No metabolites of AR177 could be observed in the plasma at any time point or dose. This contrasts with the results of Lee et al.
- the initial volume of distribution (Vd) of the three doses ranged from 200-248 mL (mean + s.d.) (Table E-2) at the conclusion of the intravenous infusion.
- the mean body weight of the monkeys in the AR177 dose groups was 3.67 kg. Assuming that plasma volume is 4% of body weight (Davies and Morris, 1993, Pharmaceutical Res. 10: 1093-1095), the plasma volume would be 147 mL. Thus, the initial Vd was slightly greater than the plasma volume.
- AR177 caused a two-fold increase in aPTT at a concentration between 30 and 59 ⁇ g/mL of human plasma, whereas the compound caused a two-fold increase in aPTT at a concentration between 118 and 236 ⁇ g/mL of cynomolgus monkey plasma in vitro.
- ARl 77 had no effect on thrombin time in human plasma, but caused approximately a 2.5-fold increase in thrombin time in cynomolgus monkey plasma at 236 ⁇ g/mL.
- AR177 had no effect on either fibrinogen or complement CH50 at doses up to 236 ug/mL in human or cynomolgus monkey plasma.
- AR177 caused a 30% increase in prothrombin time in human plasma and approximately a 15% increase in prothrombin time in cynomolgus monkey plasma at 236 ⁇ g/mL.
- AR177 was very safe. Although limited conclusions can be drawn from the present study because only one partial phosphorothioate (AR177) was examined, it is possible that the lack of the cardiovascular toxicity is due to the limited number of phosphorothioate linkages (two) in ARl 77. It is also speculated that the lack of toxicity could be due to the three-dimensional (i.e. tetrad) shape of AR177 (Rando et al., 1995, J.
- AR177 does not cause toxicity when it is administered as a bolus intravenous injection to cynomolgus monkeys every other day at 40 mg/kg for a total of 12 doses (see below).
- AR177 does not cause mortality, cardiovascular toxicity, or alterations in clinical chemistry in cynomolgus monkeys receiving doses up to 50 mg/kg as a ten-minute intravenous infusion. However, there was a reversible prolongation of coagulation time at doses of 20 and 50 mg/kg. Taken together, the data suggest that AR177 does not have the hemodynamic toxicities that are associated with total phosphorothioate oligonucleotides, and can be administered safely as an intravenous infusion over ten minutes.
- Table E-2 Plasma AR177 Cm,x, aPTT and complement Bb levels.
- the AR177 plasma C MAX , aPTT, and Bb values are the means ⁇ standard deviations of data at the + 10 minute nine point (end of the infusion).
- the baseline (10 minutes prior to dosing) aPTT levels were 32.1 + 4.4, 41.6, 6.7 and 33.2 + 4.8 seconds for the 5, 20, and 50 mg/kg doses, respectively (mean ⁇ s.d.).
- the baseline (10 minutes prior to dosing) Bb levels were 0.44 ⁇ 0.14, 0.78 + 0.46 and 0.49 + 0.21 ⁇ /mL for Me 5, 20, and 50 mg/kg doses.
- Volume of distribution (Vd) dose/plasma C MAX , where the dose is the total mg of AR177. Serum Chemistry Values
- Serum chemistry was evaluated at pre-dose, and 1 and 24 hours following initiation of intravenous AR177 infusion. Values represent the mean ⁇ s.d. of 3 monkeys.
- Hematology was evaluated at pre-dose, at 10, 20, 40, 60 and 120 minutes and 24 hours following initiation of intravenous AR177 infusion. Values represent the mean ⁇ s.d. of 3 mo
- AR177 a partial phosphorothioate anti-HTV oligonucleotide (AR177) following bolus intravenous administration to cynomolgus monkeys AR177 is a 17-mer partial phosphorothioate oligonucleotide with the sequence
- ARl 77 does not cause the characteristic hypotension or neutropenia of other oligonucleotides (Cornish et al., 1993; Galbraith et al., 1994) following a ten-minute intravenous infusion, at doses up to 50 mg/kg (Wallace et al., submitted, 1996).
- an intravenous toxicity study of AR177 was conducted in cynomolgus monkeys with the objective of establishing the clinical and histopathological changes that occur following repeated doses.
- AR177 was synthesized at Biosearch, a division of PerSeptive Biosystems, on a Milligen 8800 oligonucleotide synthesizer, and vialed at 25 mg/mL in phosphatebuffered saline.
- AR177 has a molecular weight of 5793, and is a fully neutralized sodium salt.
- the structure of ARl 77 was characterized by phosphorus and proton NMR, sequencing, base composition, laser Resorption mass spectrometry, anion exchange HPLC and polyacrylamide gel electrophoresis. All analyses were consistent with the proposed structure.
- the AR177 was approximately 94% pure according to HPLC and electrophoresis analysis.
- the monkeys used in this study were laboratory bred (CV. Primates, Indonesia or Yunnan National Laboratory, China) and were experimentally naive prior to the study.
- the age of the monkeys was 3 to 61/2.
- AR177 was administered intravenously over 1-2 minutes into unsedated monkeys every other day for 23 days (12 doses) by injection into the femoral vein. (See Table E-l). The monkeys were not sedated, but were restrained during dosing. The highest dose level (40 mg/kg/injection) was selected based on observations in a previous single-dose study of pronounced anticoagulant activity of AR177 at a dose of 50 mg/kg infused over 10 minutes (Wallace et al., submitted). A comparable or greater degree of anticoagulation was expected to occur with fast (1-2 minute) infusion of 40 mg/kg, and was confirmed by the results of this study.
- the dosing schedule (every other day) was chosen in order to avoid excessive accumulation of the test material, which, based on pharmacokinetic data obtained in rats (Wallace et al., submitted), would be expected to occur with daily administration.
- the monkeys were observed twice daily for general health, changes in appetite and clinical signs of adverse events. Body weights were measured within a few days prior to the first dose (Day 1) and approximately weekly thereafter. Electrocardiographic (ECG) recordings were obtained from all animals prior to the study and on Day 22, and from recovery animals on Day 35.
- Blood samples were collected for evaluation of serum chemistry, hematology and coagulation parameters from all animals prior to the initiation of the study, on the first day of dosing (Dose 1; Day 1), and on the last day of dosing (Dose 12; Day 23).
- the sample collection on Days 1 and 23 was timed relative to dose administration in order to characterize possible acute effects on hematology parameters.
- An additional clinical pathology evaluation was conducted for all animals on Day 24, as well as for recovery animals on Day 37. Blood was collected from all animals at 5 minutes, 30 minutes and 4 hours post-dosing on Days 1 and 23 for analysis of the plasma AR177 concentration.
- Serum chemistry was determined pre-study, on day 24 (one day after the 12th dose), and on day 37 in the recovery monkeys. The following were determined: sodium, potassium, chloride, carbon dioxide, total bilirubin, direct bilirubin, indirect bilirubin alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, gammaglutamyltransferase, calcium, phosphorus, glucose, urea nitrogen, creatinine, uric acid total protein, albumin, globulin, cholesterol and triglycerides. Serum chemistry was determined at Sierra Nevada Laboratories (Reno, NV).
- Hematology and coagulation parameters were determined 9-11 days prior to the start of the study, just prior to administering doses 1 (day 1) and
- red blood cell count and morphology total and differential white blood cells, hemoglobin, hematocrit, prothrombin time, fibrinogen, mean cell hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet count, activate partial thromboplastin time, and D-dimer. Hematology was determined at Sierra Nevada Laboratories (Reno, NV).
- AR177 plasma HPLC analysis Blood was taken for plasma analysis of AR177 just prior to, and at 5, 30 and 240 minutes following administration of doses 1 and 12. The plasma fraction was obtained by low speed centrifugation of blood, and stored at -20 °C until analyzed for the ARl 77 concentration. Plasma AR177 concentrations were assayed using an anion-exchange HPLC method on a Waters HPLC system with a 626 pump, 996 photodiode array detector, 717 autosampler and
- Buffer A was 0.1 M Tris base, 20% methanol, pH 12, and Buffer B was 0.1 M Tris base, 1.0 M NaBr, pH 12.
- anion-exchange column tGen-Pak Fax column was equilibrated at 80% buffer A/20% buffer B for 30 minutes before each HPLC run. Fifty microliters of plasma were analyzed per run. The elusion conditions were: a) five-minute isocratic run at 80% A/20% B. during which the majority of the plasma proteins eluted, b) 30-minute linear gradient to 30% A/70% B during which the AR177 eluted, c) five-minute isocratic run at 30% A/70% B.
- AR177 tightly around physiological pH. AR177 is completely stable at pH 12. This method can clearly distinguish between the full length AR177 and n-l, n-2, etc. species, which are potential metabolic products.
- the W detection wavelength was 260 nm.
- the flow rate was 0.5 mL/minute in all steps. All runs were performed at room temperature. Column clean up between runs was performed by a 500 ⁇ L bolus injection of 0.1 M Tris base, 2 M NaCl, pH 10.5, followed by: a) ten-minute linear gradient to 60% A/40% B. b) one-minute linear gradient to 100% B. c) a three-minute isocratic run at 100% B and d) one-minute linear gradient to 80% A/20% B.
- ARl 77 was spiked into cynomolgus monkey plasma in order to achieve concentrations of 0.0635 to 125 ⁇ g/mL for the standard curve.
- the plasma standards and unspiked plasma (control) were run on the anion-exchange HPLC column using the above conditions.
- the Waters Millennium software was used to determine the area under the peak for each AR177 standard at 260 nm.
- the HPLC area versus AR177 concentration was plotted using Cricket Graph m 1.5.1 software. There were two HPLC replicate runs per ARl 77 standard. The areas which represented the lowest concentration were at least two times the background area at 260 nm.
- the overall correlation coefficient of the fitted lines on the standard curve plots was greater than 0.999. There was a linear concentration versus A260 relationship over a minimum 6,500fold range. The variability of the replicates was 1-2%. This method was validated.
- Necropsy and histopathology A complete necropsy was conducted on all monkeys, and included examination of the external surface of body (body orifices; dosing site; cranial, nasal, paranasal, thoracic, abdominal and pelvic cavities), and the external surface of the brain and spinal cord.
- the organ weights of the adrenals, epididymies, liver, pituitary, spleen, thyroids, parathyroids, brain, heart, lungs, prostate, testes, uterus, cervix, kidney, ovaries, seminal vesicles, and thymus were recorded.
- a histopathological assessment was made of 46 hematoxylin and eosin-stained tissues by a veterinary pathologist. These included tissues from the cardiovascular, digestive, respiratory, urogenital, lymphoid/hematopoietic, skin/musculoskeletal and nervous systems, and all major organs.
- ECG ECG, clinical chemistry, urinalysis and hematology.
- FIG. 1 Plasma AR177 concentration.
- Figure F-l shows that there was no difference between the ARl 77 plasma concentrations that were achieved after the first and twelfth (last) doses at either 2.5, 10 or
- aPTT in the 10 mg/kg group was increased to approximately twice the pre-dose value by 5 minutes post-dosing, but had returned to baseline levels by four hours.
- mean aPTT in the 40 mg/kg group exceeded the upper limit of the assay five minutes after dosing.
- aPTT values in the 40 mg/kg group had declined to approximately 2 to 4-fold above the pre-dose level.
- the aPTT had returned to the pre-dose levels in all but one monkey.
- the relationship between the ARl 77 plasma concentration and aPTT is also shown in Figures F-3, F-4, and F-5 for doses 2.5, 10, and 40 mg/kg, respectively.
- Doubling of aPTT was observed at plasma AR177 concentrations of approximately 100-220 ⁇ g AR177/mL.
- Tripling of aPTT was observed at plasma AR177 concentrations of approximately 220-300 ⁇ g AR177/mL, after which no correlation was possible because the aPTT values were off-scale.
- AR177 administered as bolus intravenous injections up to 40 mg/kg every other day for 12 doses, did not cause mortality, histopathological or cardiovascular events that have been described for other oligonucleotides (Galbraith et al., 1994; Srinivasan and Iversen, 1995). The only significant change that was observed was a prolongation of aPTT, which was reversible. To our knowledge, this is the first oligonucleotide that has not been observed to cause liver and kidney toxicity following intravenous administration. The structure of AR177 may contribute to its lack of general toxicity. AR177 contains only two phosphorothioate bonds at the 3' and 5' termini.
- AR177 phosphorothioate bonds were designed to help prevent endonuclease-induced cleavage of AR177.
- the small number of sulfurs may have reduced the propensity to bind to proteins, a phenomenon that has been observed for full phosphorothioates, which has been speculated to cause toxicity (Srinivasan and Iversen, 1995).
- AR177's three-dimensional shape may also contribute to its lack of toxicity.
- AR177 has been shown to form a structure in which hydrogen bonds form between deoxyguanosine residues to create a "G-tetrad" (Rando et al., 1995).
- This tetrad structure imparts a compact shape which makes it resistant to degradation (Bishop et al., 1996) and may make it relatively non-toxic by minimizing reactive sites.
- the resistance to degradation has been noted in single and repeat dose pharmacokinetics studies in rodents (Wallace et al., submitted), and in a more complete pharmacokinetic study in cynomolgus monkeys which showed a terminal plasma half-life of greater than 24 hours (data not shown).
- results of the ARl 77 plasma analysis demonstrated that there was no difference between the AR177 plasma concentrations that were achieved after the first or twelfth (last) doses of 2.5, 10 or 40 mg/kg. These results can be inte ⁇ reted to mean that AR177 does not induce metabolic enzymes that would, if they were induced, reduce the concentration of ARl 77 by increasing its metabolism. This has the important implication that repeat doses of ARl 77, at least when given every other day for 23 days, will not result in pharmacokinetic tolerance.
- Table 1 Monkey dosing information. Cynomolgus monkeys were given bolus intravenous injections of AR177 at 2.5,10 or 40 mg/kg/day at a constant volume every other day for a total of 12 doses. Control monkeys received sterile saline. There were 8 monkeys per group, evenly split between males 15 and females, except for the placebo group, which inadvertently had an extra female. The main group was sacrificed on day 25 following initiation of dosing, which was two days following the twelfth dose on day 23. Two monkeys in the placebo and 40 mg/kg groups were in a recovery group. The recovery group monkeys were sacrificed two weeks (on day 38 after initiation of dosing) after the other monkeys.
- Serum chemistry was evaluated at pre-study, and at days 24 and 37 (recovery monkeys only) following initiation of intravenous ARl 77 administration. Values represent the ⁇ -._ ⁇ s.d. of 2-8 monkeys. There were two monkeys per group in the saline and 40 mg/kg recovery groups, and six monkeys per group in the non-recovery groups.
- Plasma was obtained by low speed centrifiigation of the blood, and was stored frozen until analyzed by HPLC for AR177 concentration.
- concentration of AR177 was determined in patient plasma using a validated anion-exchange HPLC method at the Division of Clinical Pharmacy of the University of California, San Francisco. This method has a limit of quantitation of 15 ng/mL in human plasma.
- the C max value in the 0.75 mg/kg group was 5.1 ⁇ 1.4 ⁇ g/mL and the C max value in the 3.0 mg/kg group was 37.5 ⁇ 0.1 ⁇ g/mL, approximately a seven-fold increase (Figure G-5).
- the AUC value in the 0.75 mg/kg group was 703.6 ⁇ 154.7 ⁇ g-min/mL and the AUC value in the 3.0 mg/kg group was 8,277.8 ⁇ 2.937.4 ⁇ g-min/mL, approximately a 12-fold increase (Table 1).
- the plasma clearance and Vd values reflected the C max and AUC data.
- the plasma clearance in the 0.75 mg/kg group was 1.1 ⁇ 0.2 mL/min kg and the clearance in the 3.0 mg/kg group was 0.4 ⁇ 0.2 mL/min/kg, approximately a 65% decrease (Figure 6).
- the initial and steady- state volumes of distribution in the 0.75 mg/kg group were 0.16 ⁇ 0.05 L/kg and 0.14 ⁇ 0.05 L/kg, respectively, whereas the initial and steady-state volumes of distribution (Vd) in the 3.0 mg/kg group were 0.08 ⁇ 0.00 L/kg and 0.05 ⁇ 0.03 L/kg, respectively (Table 1).
- Vd ss (L/kg) 0.09 0.10 0.12 0.09 0.10 ⁇ 0.01
Abstract
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US60/001,505 | 1995-07-19 | ||
US08/535,168 | 1995-10-23 | ||
US1368896P | 1996-03-19 | 1996-03-19 | |
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Cited By (5)
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US5912332A (en) * | 1996-07-26 | 1999-06-15 | Hybridon, Inc. | Affinity-based purification of oligonucleotides using soluble multimeric oligonucleotides |
EP0968226A1 (en) * | 1996-12-27 | 2000-01-05 | ICN Pharmaceuticals, Inc. | G-rich oligo aptamers and methods of modulating an immune response |
EP0971944A1 (en) * | 1997-02-04 | 2000-01-19 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich tetrad forming oligonucleotides |
US6288042B1 (en) | 1993-04-23 | 2001-09-11 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich tetrad forming oligonucleotides |
WO2007044851A3 (en) * | 2005-10-06 | 2007-11-22 | Univ Delaware | G-rich polynucleotides for the treatment of huntington's disease |
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US5523389A (en) * | 1992-09-29 | 1996-06-04 | Isis Pharmaceuticals, Inc. | Inhibitors of human immunodeficiency virus |
US5567604A (en) * | 1993-04-23 | 1996-10-22 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich oligonucleotides |
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1996
- 1996-07-17 AU AU69506/96A patent/AU725936B2/en not_active Ceased
- 1996-07-17 EP EP96930490A patent/EP0871643A4/en not_active Withdrawn
- 1996-07-17 CA CA002227867A patent/CA2227867A1/en not_active Abandoned
- 1996-07-17 WO PCT/US1996/011786 patent/WO1997003997A1/en not_active Application Discontinuation
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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US6288042B1 (en) | 1993-04-23 | 2001-09-11 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich tetrad forming oligonucleotides |
US5912332A (en) * | 1996-07-26 | 1999-06-15 | Hybridon, Inc. | Affinity-based purification of oligonucleotides using soluble multimeric oligonucleotides |
EP0968226A1 (en) * | 1996-12-27 | 2000-01-05 | ICN Pharmaceuticals, Inc. | G-rich oligo aptamers and methods of modulating an immune response |
EP0968226A4 (en) * | 1996-12-27 | 2001-02-14 | Icn Pharmaceuticals | G-rich oligo aptamers and methods of modulating an immune response |
EP0971944A1 (en) * | 1997-02-04 | 2000-01-19 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich tetrad forming oligonucleotides |
EP0971944A4 (en) * | 1997-02-04 | 2005-01-26 | Aronex Pharmaceuticals Inc | Anti-viral guanosine-rich tetrad forming oligonucleotides |
WO2007044851A3 (en) * | 2005-10-06 | 2007-11-22 | Univ Delaware | G-rich polynucleotides for the treatment of huntington's disease |
US7807648B2 (en) * | 2005-10-06 | 2010-10-05 | University Of Delaware | G-rich polynucleotides for the treatment of Huntington's Disease |
Also Published As
Publication number | Publication date |
---|---|
AU725936B2 (en) | 2000-10-26 |
CA2227867A1 (en) | 1997-02-06 |
EP0871643A4 (en) | 2000-12-06 |
AU6950696A (en) | 1997-02-18 |
EP0871643A1 (en) | 1998-10-21 |
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