Field of the Invention

This invention relates to novel compositions and meth¬ ods for treating HIV infections. In particular, this inven¬ tion relates to novel thio-containing ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) wherein the RNA and DNA derivatives contain thiol groups in reduced or oxidized for at the five position of the pyrimidine ring of monomeric units which comprise the RNA and DNA derivatives. In another aspect of the present invention, a method for treating HIV infections is presented.

Background of the Invention

HTLV I (Human T-cell leukemia lymphotropic virus) was the first human retrovirus identified as an infectious agent. This virus is etiologically associated with adult T-cell leukemia (See, for example, Gallo, et al. Blood, 60, 545 (1982)). A related but distinct retrovirus, HTLV-II was iso¬ lated from a T-cell variant of hairy cell leukemia (See Kalyanaraman, et al.. Science. 218, 571 (1982). Both of these viruses have exhibited a tropism for human 0KT4+ cells and have the capacity to immortalize and transform normal human T- cells in culture (Miyoshi, et al.. Nature (Lond. ) . 294, 770 (1981) and Popovic, et al., Proc. Natl. Acad. Sci. USA. 80, 5402 (1983)).

Because of T-cell activity, particularly OKT4+ and other biological properties similar to HTLV-I and II, the search for a similar retrovirus associated with Autoimmune Deficiency Syndrome (AIDS) was undertaken. Gallo, et al., Science. 224, 500 (1983) is recognized as the first investiga¬ tor to isolate and characterize a retrovirus associated with AIDS which is biologically and morphologically distinct from HTLV I and II. Due to certain homologous gene sequences between the AIDS associated virus and HTLV I and II, this virus has been termed HTLV III (HIV 1) . Another retrovirus, termed lymphoadenopathy virus (HIV 2) has also been shown to be associated with AIDS (Ellrodt, et al.. Lancet, 1, 1383 (1984) and Levy, et al.. Science, 225, 840 (1984)).

In comparison with the other T-lymphotropic retroviruses HTLV I and II, HTLV III (HIV-1) and lymphoadenopathy viruses are nontransforming cytopathic viruses without immortalizing activity. The viral replication process is believed to be an important event in the progress of AIDS. It is further believed that the enzyme RNA-Directed DNA polymerase or "Reverse" Transciptase may be implicated in the progress of the disease (Chandra, et al., Cancer Res. , 45, 4677s-4684s (1985) ) and that this enzyme may be an interesting target for the development of potential drugs against AIDs. The above paper includes some interesting results with selec¬ tive inhibitors (including modified polycytidylic acid (MPC)) of a reverse transriptase purified from HTLV-III and from HTLV-III infected H9 cells.

One of the more significant biological properties of the HIV-1 virus is its ability to integrate a DNA copy of its diploid RNA genome into the chromosome of the host cell. This means that once a single cell in a person has been infected with the AIDS virus, the viral genetic information will be carried indefinitely. Relatively few of the infected cells are actually killed by HIV-1, thus the cells continue to release fresh virus and infect further cells. Some infected cells have a short half-life of days or weeks, but certain infected cells, for example, dendritic macrophage cells may live for decades. Although numerous cells in circulation may be infected, perhaps only 1 cell in 10,000 is actually activated at any one time to produce the virus to infect other cells. Perhaps the greatest need at present is to develop an agent or series of agents which suppress the transcription of the genetic information from the integrated proviral DNA to produce new viral agents. (See, for example, Oxford, et al., Jour. Antimic. Chemother. , 23, Supp. A 9-27 (1989)).

It is well known that the reverse transcriptases of RNA tumor viruses can utilize complexes of certain homopolyribonucleotides with the complementary oligodeoxyribonucleotides as template-primers (See Sarngad- haran, et al., Methods Cancer Res. , 12, 3 (1976)). Numerous successful attempts to inhibit the reverse transcriptases from

various RNA tumor viruses with the use of macromolecular antimetabolites such as 5-Mercaptopolyuridylic acid (MPU) and 5-Mercaptocytidylic acid (MPC) have also been reported (See, for example, Kung, et al.. Res. Comm. Chem. Path. Pharm. , 36, 215 (1982); Bardos, T.J. , and Ho, Y.K., "An Update on Antitemplates ," In New Approaches to the Design of Antineoplastic Agents. 1982, by Elsevier Science Publishing Company, Inc.; Chandra, P. and Bardos, T.J., Res. Comm. Chem. Path. Pharm.. 4, 615 (1973); Bardos, T.J. , "Antimetabolites: Molecular Design and Mode of Action", In Topics in Current Chemistry, Volume 52, Published by Springer-Verlag, 1974; Chandra, P. , "Molecular Approaches for Designing Antiviral and Antitumor Compounds," In Topics in Current Chemistry. Volume 52, Published by Springer-Verlag, 1974).

Reverse transcriptase is a DNA polymerase located in the core fraction of the virus complexed with viral RNA. In this ribonucleoprotein containing reverse transcriptase, the transcription of viral RNA into a complementary DNA occurs (See Chandra, et al., Cancer Res. _f 45, 4677s-4684s (1985)). RNA directed DNA synthesis is unique to the life cycle of retroviruses; no similar reaction is known to occur in normal, uninfected human tissues.

Reverse transcriptase is a multifunctional enzyme, comprising at least four domains that reportedly mediate RNA dependent DNA polymerase activity, RNase H activity, and DNA dependent DNA polymerase activity and a site for nucleic acid binding. The final product of the reactions mediated by reverse transcriptase is a linear double stranded DNA contain¬ ing long terminal repeat units written as 5'-U3-TR-U5-3' pres¬ ent at both ends of the DNA. (See Chandra, et al., supra) .

Recently, Sallafranque-Andreola, et al., Eur. J. Biochem.. 184, 367 (1989) and Bordier, et al.. Nucleic Acids Res.. 18, 429 (1990) established that HIV reverse transcrip¬ tase expressed in yeast may utilize tRNA ^L y ^s as primer. Those papers indicate that the first step in retroviral replication involves the partial annealing of tRNA ^L Y ^s to the primer bind-

ing site (PBS) for the initiation cDNA synthesis via reverse transcriptase. Also reported was the inhibition of reverse transcriptase activity by tRNA ^Lvs and especially certain oligomers corresponding to the anticodon regions of tRNA ^L Y ^s .

In vitro assay systems for reverse transcriptase evi¬ dence the modulation of the catalytic rate of DNA synthesis by a number of agents including substrates, template-primers, and other interacting compounds. MPC and MPU have been shown to inhibit HIV reverse transcriptase in .in vitro assays. Slight variations in assay conditions of reverse transcriptase assays, however, may lead to the wrong interpretation with respect to the specificity of a particular inhibitor in the viral DNA polymerase systems. Another problem in these in vitro systems includes the interpretation of the enzymatic data with respect to the antiviral activity of the inhibitors. See Chandra, et al., supra.

Objects of the Present Invention

It is an object of the present invention to provide a novel method for treating HIV infections and HIV infected cells.

It is an additional object of the present invention to provide novel thiol-containing compositions which may be used for treating HIV infections as well as other infections, con¬ ditions and uses such as a disinfectant for HIV infected areas.

It is yet another object of the present invention to provide pharmaceutical compositions which contain novel thiol- containing compositions of the present invention.

These and other objects of the present invention may be readily gleaned from a review of the description of the present invention which follows.

Brief Description of the Present Invention

The present invention relates to a novel method for treating HIV infected cells and HIV infections comprising administering to a patient suffering from an HIV infection an effective amount of a thiol-containing composition selected from among 5-Thiolated oligo(deoxy)ribonucleotides and 5- Thiolated polynucleotides. These compositions have shown sur¬ prising activity against HIV-I infections in vivo (HIV infected human T-lymphocytes) indicating that the 5-Thiolated poly- and oligo- nucleotide compositions are therapeutically effective agents for inhibiting HIV-I infections and for treating AIDS. In addition to showing excellent activity against HIV infections, the compositions of the present inven¬ tion have evidenced a virtual absence of toxicity in human cell systems.

The present invention also relates to novel 5- Thiolated oligo and poly nucleotides compositions which are particularly useful as therapeutic agents for treating HIV infections. Compositions of the present invention have shown surprising activity in being able to inhibit HIV infections in vivo (human T-Lymphocytes) .

The present invention also relates to pharmaceutical compositions comprising the 5-Thiolated oligo- and polynucleotides of the present invention in therapeutically effective amounts, optionally in the presence of pharamceuti- cally compatible additives and/or excipients. Pharmaceutical compositions of the present invention are preferably administered via a parenteral route, for example via intravenous, intraarterial, intramuscular and subcutaneous routes.

Compositions of the present invention may also be used as disinfectants to treat HIV infected areas such as laboratory equipment, laboratory surfaces and other areas.

Brief Description <f the Figures

Figure 1 represents the chemical structure of three isoaccepting tRNA ^L y ^s (tRNA ^L y ^s l,2; tRNA ^L y ^s 3 and tRNA ^L y ^s 4 which

are thiolated to produce partially 5-Thiolated tRNA ^L y ^s of the present invention.

Definitions Used In Describing the Present Invention

As used herein, the term "5-Thiolated oligonucleotide" is used to describe a DNA or RNA molecule containing about 5 to about 100 nucleotide units wherein at least one pyrimidine nucleoside of the DNA or RNA molecule, i.e., a cytidine or uridine nucleoside is substituted at the 5 position of the pyrimidine ring with a thiol or SH group. The term as used embraces RNA and DNA molecules each containing the four most abundant naturally occurring nucleosides, respectively, i.e. guanosine, adenosine, uridine and cytidine in the case of RNA and 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxythymidine and 2 -deoxycytidine in the case of DNA, as well as other less abundant nucleosides such as, for example pseudouridine, methyl cytidine and methyl guanosine, among others.

The term "5-Thiolated oligoribonucleotide" is used to describe an RNA molecule (ribose sugar) containing about 5 to about 100 nucleotide units wherein at least one pyrimidine nucleoside is substituted at the 5 position with a thiol or SH group.

The term "5-Thiolated oligodeoxyribonucleotide" is used to describe a DNA molecule (2 ^/ -deoxyribose sugar) con¬ taining about 5 to about 100 nucleotide units wherein at least one pyrimidine 2'-deoxynucleoside (2 -deoxycytidine or 2'- deoxyuridine) is substituted at the 5 position with a thiol or SH group.

The term "5-Thiolated polynucleotide" is used to des¬ cribe a DNA or RNA molecule containing at least about 100 nucleotide units wherein at least one pyrimidine nucleoside per 100 total nucleosides (purine and pyrimidine) of the DNA or RNA molecule is substituted at the 5 position of the pyrimidine ring with a thiol or SH group.

The term "5-Thiolated polyribonucleotide" is used to

describe an RNA molecule containing at least about 100 nucleotide units wherein at least one pyrimidine nucleoside per 100 tott nucleosides of the RNA molecule is substituted at the 5 position of the pyrimidine ring with a thiol or SH group.

The term "5-Thiolated polydeoxyribonucleotide" is used to describe a DNA molecule containing at least about 100 nucleotide units wherein at least one pyrimidine nucleoside per 100 total nucleosides of the RNA molecule is substituted at the 5 position of the pyrimidine ring with a thiol or SH group.

The term "regional oligonucleotide" is used to des¬ cribe a DNA or RNA molecule corresponding to a specific region on the primer tRNA of HIV reverse transcriptase, tRNA ^L y ^s . The term includes oligonucleotides which are anti-sense to specific tRNA regions. Regional oligonucleotides for use in the present invention may include nucleotides which occur naturally in the tRNA such as the methylated nucleotides and pseudonucleotides, among others, as well as nucleotides which may function as substitutes for the naturally occuring nucleotides within the regional oligonucleotide.

The term "5-Mercaptopolycytidylate" (MPC) is used to define a polyribonucleotide comprised of more than 100 nucleotide units of cytidylic acid in general containing a majority (>50%) of unthiolated nucleotide units and a minority (<50%) of thiolated nucleotide units. "5-

Mercaptopolydeoxycytidylate" (MPdC) is the deoxy version of MPC.

The term "5-Mercaptopolyuridylate" (MPU) is used to define a polyribonucleotide comprised of more than 100 nucleotide units of uridylic acid in general containing a majority (>50%) of unthiolated nucleotide units and a minority (<50%) of thiolated nucleotide units. "5-

Mercaptopolydeoxyuridylate (MPdU) is the 2'-deoxy version of MPU.

The term "5-Mercaptooligocytidylate" (MOC) is used to define an oligoribonucleotide comprised of less than 100 nucleotide units of cytidylic acid in general containing a majority (>50%) of unthiolated nucleotide units and a minority (<50%) of thiolated nucleotide units. "5-

Mercaptooligodeoxycytidylate" (MOdC) is the deoxy version of MOC.

The term "5-Mercaptooligouridylate" (MOU) is used to define an oligoribonucleotide comprised of less than 100 nucleotide units of uridylic acid in general containing a majority (>50%) of unthiolated nucleotide units and a minority (<50%) of thiolated nucleotide units. "5-

Mercaptooligodeoxyuridylate" (MOdU) is the 2'-deoxy version of MOU.

The terms "thiol" and "thiolated" are used to describe sulphur substitution at the five position of a pyrimidine nucleoside includes thiol groups (SH) , mercaptide groups (S_) and disulfides (-S-S-) formed from the oxidation of thiols or mercaptides on thiolated nucleoside units of the RNA or DNA.

The term "n-mer" is used to describe an oligomer con¬ taining n nucleotide units.

The term "homopolymer" and "homooligomer" is used to describe RNA and DNA molecules which are made from the same nucleotide. Poly(C) , poly(U) and oligo(T) are examples of homopolymers and a homooligomer, respectively.

Detailed Description of the Invention

The present invention relates to a therapeutic method for treating HIV infections. In this method of the present invention, a patient suffering from an HIV infection, includ¬ ing a patient with AIDS, is administered an effective amount of a thiol-containing composition selected from among 5- Thiolated oligo(deoxy)ribonucleotides and 5-Thiolated polynucleotides.

Among the 5-Thiolated polynucleotides that can be used in the present invention include.the 5-Thiolated homopolyribonucleotides 5-Mercaptopolycytidylic acid (MPC) and 5-Mercaptopolyuridylic acid (MPU) as well as 5-Thiolated homopolydeoxyribonucleotides 5-Mercaptopolydeoxycytidylic acid (MPdC) and 5-Mercaptopolydeoxyuridylic acid (MPdU) with MPdC and MPdU preferred because of .their significantly enhanced activity relative to MPC and MPU as anti-HIV therapeutic agents. It is an unexpected result that MPdC and MPdU exhibit such enhanced anti-HIV activity relative to MPC and MPU. It is also a surprising result that MPdU exhibits significantly more anti-HIV activity than does the corresponding MPU.

In this homopolynucleotide aspect of the present invention, at least about 1 pyrimidine nucleoside per 100 total nucleosides is thiolated. Although virtually any thiolation content will evidence at least some anti-HIV activity in this aspect of the present invention, a thiolation content of about 5 to about 15 thiolated pyrimidine nucleosides per 100 total nucleosides (thiolation content about 5% to about 15%) with a thiolation content ranging from about 8% to about 12% being especially preferred.

In addition to the above-described anti-HIV agents, numerous 5-Thiolated homooligonucleotides may also be used in the therapeutic aspect of the present invention. Oligoribonucleotides that may be used in the present invention include 5-Mercaptooligocytidylic acid (MOC) and 5- Mercaptooligouridylic acid (MOU) . Oligodeoxyribonucleotides that may be used in the present invention include 5- Mercaptooligodeoxycytidylie acid (MOdC) and 5- Mercaptooligodeoxyuridylic acid (MOdU) , with the oligodeoxyribonucleotides being significantly more active than the corresponding oligoribonucleotides. In this aspect of the present invention in which homooligonucleotides are used to treat HIV infections, the oligonucleotides range in size from about 5 nucleotide units to about 100 nucleotide units with a preferred range of about 25 to 50 units and a most preferred range of about 35 to 45 nucleotide units. In addition to the above, oligodeoxyribonucleotides evidence unexpectedly greater

anti-HIV activity than do the corresponding oligoribonucleotides and are clearly preferred for use in the present invention because of this enhanced activity. In this aspect of the present invention, the amount of thiolation generally ranges from about 2% to about 25% of the oligonucleotide, with a range of about 5% to about 10% thiola¬ tion being preferred.

The present invention also relates to 5-Thiolated oligonucleotides which correspond to certain regions of an HIV reverse transcriptase primer tRNA, and in particular tRNALy _S . For example, 5-Thiolated oligonucleotides, preferably oligodeoxyribonucleotides, corresponding to the anti-codon region (represented in its deoxy form as 5-Mercapto 5'- d(ATC/AGA/CTT/TTA/ATC/TGA)-3 ^/ ) or the 3'terminus (represented in its deoxy form as 5-Mercapto 5'-d(GTC/CCT/GTT/CGG/GCG/CCA)- 3') of the tRNALy _S , among others, are useful for treating HIV infections. In addition, the present invention relates to "anti-sense" 5-Thiolated oligonucleotides of the regions of the tRNAj _j ys described above, for example, the anti-sense to the 3'terminus region described above (represented in its deoxy form as 5-Mercapto 5'-d(TGG/CGC/CCG/AAC/AGG/GAC)- 3') or the anti-sense to the anti-codon region set forth above (represented in its deoxy form as 5-Mercapto 5'- d(TCA/GAT/TAA/AAG/TCT/GAT)-3') . In this aspect of the present invention, 5-Thiolated oligonucleotides corresponding to a region of the primer tRNA containing from about 5 to about 20 nucleotide units, and preferably from about 12 to about 18 nucleotide units are utilized. Without being limited by way of theory, it is believed that oligo(deoxy)ribonucleoitdes containing more than about 20 nucleotide units do not add to the anti-HIV activity of the composition. In this aspect of the present invention as in other aspects, the deoxyribonucleotide is preferred over the corresponding ribonucleotide because of its enhanced anti-HIV activity.

In further aspects of the present invention additional 5-Thiolated oligonucleotides are disclosed. In this aspect of the present invention, 5-Thiolated oligonucleotides comprising a non-thiolated (binding) homooligonucleotide region to pro-

mote the binding of the remaining portion of the 5-Thiolated oligonucleotides to a homopurine (or homopyrimidine) site of the viral genome via triple-helix formations, are disclosed. In this aspect of the present invention, the non-thiolated homooligonucleotide regions which promote binding to the HIV genome via triplex formation include, for example, homooligomers such as oligo(deoxy)adenylate and oligothymidy- late bound at the 5' end of 5-Thiolated oligo(deoxy)cytidylic or uridylic acid. In this aspect of the present invention, the binding homooligonucleotide portion, which is preferably not thiolated, ranges in size from about 5 nucleotide units to about 25 nucleotide units with 10-15 nucleotide units preferred. In the case of the 5-Thiolated oligomer, this por¬ tion of the compositions generally ranges in size from about 5 nucleotide units to about 50 nucleotide units, with about 20 units to about 30 nucleotide units being preferred. The amount of thiolation of this portion of the composition generally ranges from 1 thiolation to about 15% total thiola¬ tion, with a lower amount ie. , about 1 to 3 thiols per oligomer preferred. The deoxyribonucleotides are preferred over the corresponding ribonucleotides. The structures of certain representative compositions appear below. The follow¬ ing list is, of course, by no means complete.

5'-d(T) ₁₂ -MdC ₂₈ -3' 5'-d(A) ₁₂ -MdC ₂₈ -3' 5'-d(T) ₁₂ -MdU ₂₈ -3 ^/

In yet another aspect of the present invention, 5- Thiolated homooligonucleotides are bound to the previously described regional oligonucleotides of the primer tRNAj _j ys. These 5-Thiolated homooligonucleotides, which range in length from about 5 to about 50 nucleotide units and preferably about 20 to about 40 nucleotide units, may be bound to the regional oligonucleotides at the 5' or 3' end. The amount of thiola¬ tion of these homooligomers generally ranges from 1 thiolation to about 15% thiolation, with about 3% to about 10% thiolation being preferred. As in all the compositional aspects of the present invention, the deoxyribonucleotides are preferred over

the corresponding ribonucleotides because of their greater activity. The structures of certain representative composi¬ tions of the present invention appear below. Each of these compositions comprises an 18-mer regional oligonucleotide and a 22-mer thiolated homooligomer for a total of 40 nucleotide units. The following list is, of course, by no means com¬ plete.

Anti-codon Regional 5-Mercapto oligonucleotides 5'-MdC ₂₂ -d(ATC/AGA/CTT/TTA/ATC/TGA) -3' 5'-d(ATC/AGA/CTT/TTA/ATC/TGA)-MdC ₂₂ -3' 5'-MdU ₂₂ -d(ATC/AGA/CTT/TTA/ATC/TGA) -3' 5'-d(ATC/AGA/CTT/TTA/ATC/TGA)-MdU ₂₂ -3'

3' Terminus Regional 5-Mercapto oligonucleotides 5'-MdC ₂₂ -d(GTC/CCT/GTT/CGG/GCG/CCA) -3' 5'-d(GTC/CCT/GTT/CGG/GCG/CCA)-MdC ₂₂ -3' 5'-MdU ₂₂ -d(GTC/CCT/GTT/CGG/GCG/CCA) -3' 5'-d(GTC/CCT/GTT/CGG/GCG/CCA)-MdU ₂₂ -3'

Anti-Sense Anti-codon Regional

5'-MdC ₂₂ -d(TCA/GAT/TAA/AAG/TCT/GAT) -3' 5'-d(TCA/GAT/TAA/AAG/TCT/GAT)-MdC ₂₂ -3' 5'-MdU ₂₂ -d(TCA/GAT/TAA/AAG/TCT/GAT) -3' 5'-d(TCA/GAT/TAA/AAG/TCT/GAT)-MdU ₂₂ -3'

Anti-Sense 3' Terminus Regional

5'-MdC ₂₂ -d(TGG/CGC/CCG/AAC/AGG/GAC)-3' 5'-d(TGG/CGC/CCG/AAC/AGG/GAC) -MdC ₂₂ -3' 5'-MdU ₂₂ -d(TGG/CGC/CCG/AAC/AGG/GAC) -3' 5'-d(TGG/CGC/CCG/AAC/AGG/GAC) -MdU ₂₂ -3'

In addition to the above 5-Mercapto oligodeoxyribonucleotides, the ribonucleotide analogs may also be used as well as shorter regional oligomers and shorter 5- Thiolated homooligomers. These agents are presently viewed as less preferred because of the difficulties attached to the synthesis of oligoribonucleotides in general and especially those greater than about 20 units.

In a further compositional aspect of the present invention, 5-Thiolated tRNAs including, for example, tRNA ^L y ^s are useful fc - treating HIV infections. In the present inven¬ tion, mixtures of 5-Thiolated tRNAs were found to be highly effective in vivo against HIV infections. This may be due to the presence of primer 5-Thiolated tRNAs which bind to the primer binding region of the HIV-genome as well as to the HIV reverse transcriptase. Separately, 5-Thiolated tRNA ^L y ^s (iso¬ lated from sheep liver and thiolated according to literature procedure) exhibited significantly enhanced anti-HIV activity in vivo, perhaps also due to the ability of the tRNA ^L y ^s to bind to the primer binding region of the HIV proviral DNA as well as to the HIV reverse transcriptase.

Further compositions of the present invention include thiolated enzymatically lysed segments of primer tRNA, preferably tRNA ^L y ^s which correspond to segments of the primer tRNA. After enzymatic degradation of the primer tRNA, these oligomers are isolated and then thiolated, or alternatively, thiolated and then isolated. The resulting thiolated oligonucleotides correspond to sense or anti-sense regions of the primer tRNA or proviral genome and are useful as anti-HIV agents. These thiolated oligoribonucleotides range in size from about 5 nucleotide units to about 45 nucleotide units, with a range of about 20 nucleotide units to about 40 nucleotide units being preferred.

In making the compositions of the present invention procedures well known in the art are employed. For example, in the case of 5-Thiolated homopoly(deoxy)ribonucleic acid analogs such as MPC, MPdC, MPU and MPdU, the isolated homopolymer (enzymatically synthesized by the general method¬ ology described below) is thiolated according to the general chemical method (methoxybromide procedure) of Bardos, et al., Nucleic Acid Chemistry , Vol. 2, 881 (1978); Ho, et al., Biochem. Biophys. Res. Comm.. 73, 903 (1976); and Mikulski, et al., Biochim. Biophys. Acta. , 319, 294 (1973). The amount of thiolation may be readily controlled for these as well as other compositions of the present invention by the stoichiometry of the methoxybromination and thiolation reac-

tions. The homoribopolymers PolyC and Poly U may also be synthesized by the enzymatic polymerization of the correspond¬ ing monomeric ribonucleoside-5'-diphosphates using the polynucleotide phosphorylase of . coli or M^. luteus, while poly d(C) and poly d(U) are normally obtained by polymeriza¬ tion of the deoxyribonucleoside-5'-triphosphates with the enzyme terminal deoxyribonucleotidyl transferase (tdt) . Thiolation is produced by the above-referenced methoxybromide procedure.

The 5-Thiolated homopolynucleotide compositions may be synthesized directly by an enzymatic method. In this enzymatic procedure, MPC and MPU are synthesized via enzymatic copolymerization of the 5'-diphosphates of 5-mercaptouridine and uridine or 5-mercaptocytidine and cytidine as described by Ho, et al., Nucleic Acid Research. 8, 3175 (1980). MPdC and MPdU are synthesized by copolymerizing the respective 5'- triphosphates with the enzyme tdt described above.

5-Thiolated homooligoribonucleotides are prepared by partial alkaline hydrolysis of Poly(C) or Poly(U) followed by chromatographic separation on DEAE cellulose according to the procedure of Ho, et al., Biochem. Biophys. Res. Commun.. 73, 903 (1976) . After the respective molecular weight fractions of the homooligomers are isolated, they are subsequently thio¬ lated by the methoxybromide method referenced above to produce MOC or MOU.

5-Thiolated homooligodeoxyribonucleotides are prepared via a step-wise chemical synthesis of OdC or OdU according to the phosphoramidite procedure or related procedures as dis¬ closed by Gait, M.J., Ed.: Oligonucleotide Synthesis. A Prac¬ tice Approach. IRL Press, Oxford, England (1984) , such as via an automated DNA synthesizer (such as that system available from Applied Biosystems, Inc., USA Model 381A) or by a manual solid-phase synthetic method. After synthesis of the respec¬ tive oligomeric deoxyribonucleotide, 5-Thiolation proceeds according to the previously cited methoxybromide thiolation method according to literature methods.

Regional 5-Thiolated oligodeoxyribonucleo ^J des such as those corresponding to the anti-codon and 3'terminus regions of tRNA ^L y ^s as well as anti-sense 5-Thiolated oligodeoxyribonucleotides are synthesized via the step-wise phosphoramidite procedure as described above and the resulting oligomer is thiolated according to the general procedure referenced above and described in the literature.

5-Thiolated oligoribonucleotides such as those cor¬ responding to the anti-codon and 3'terminus regions of tRNA ^Lvs as well as 5-Thiolated oligoribonucleotides anti-sense to those regions (up to 20 nucleotide units) are synthesized via the method as disclosed by Lehmann, et al., Nucl. Acids Res., 17, 2379 (1989) as well as other methods known in the art.

5-Thiolated tRNA mixtures, especially including, for example, 5-Thiolated tRNA-^y* ³ compositions are made by first isolating mixed aminoacid-tRNAs from animal liver, for exam¬ ple, sheep liver because of its rich tRNA ^L y ^s and bovine liver according to literature procedure (Rogg, et al., Biochim. Biophys. Acta. 195, 13, (1969)) and then thiolating the tRNA mixture by the above-referenced methoxybromide procedure. In preferred embodiments according to the present invention, 5- Thiolated tRNA ^L y ^s is prepared by first isolating tRNA ^L y ^s and thiolating the tRNA. In this tRNA ^L Y ^s embodiment, the tRNA ^L y ^s is isolated from sheep by extraction of the protein with phenol to produce a protein-free mixture of tRNAs, followed by column chromatraphy (DEAE sephadex) according to the above literature reference. Two tRNA ^L Y ^s fractions are isolated, tRNA ^L y ^s _1;2 and tRNA ^L Y ^s ₃ (see figure 1) and one tRNA ^L y ^s poor fraction between the two tRNA ^L Y ^s enriched fragments is also isolated. A tRNA ^:L y ^s ₄ fragment can be isolated from rapidly proliferating cells, also isolated from Sheep liver. Once isolated, the tRNA ^L y ^s are thiolated according to general pro¬ cedure referenced above and described in the literature.

To synthesize 5-Thiolated oligoribonucleotides cor¬ responding to regions of a primer (tRNA) , a primer tRNA, preferably tRNA ^{L s} is isolated as described above and then subject to degradation by 1 RNase according to the procedure

described in Bordier, et al., Nucleic Acid Res■ , 18, 429 (1990) followed by isolation. The isolated oligoribonucleotides are subsequently thiolated according to the above-described procedure and used as anti-HIV agents.

The previously described 5-Thiolated poly or oligonucleotide compositions may* be formulated in pharmaceuti¬ cal compositions. Such compositions include an anti-HIV effective amount of the 5-Thiolated poly or oligonucleotide compositions optionally in the presence of pharmaceutically compatible excipients and additives.

The previously described compositions of the present invention may be used to treat HIV infections. In this aspect of the present invention, an amount of a 5-Thiolated poly or oligonucleotide as previously described is administered to a patient suffering from an HIV infection in an amount effective to treat the HIV infection. The previously described 5- Thiolated poly and oligonucleotides of the present invention may be administered to a subject such as a human patient, for the treatment of HIV infections. For administration to humans in the treatment of AIDS, the prescribing physician will ultimately determine the appropriate dose for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease. In additional aspects of this therapeutic method, the compositions of the present invention may be combined with 3'-azido-3'- deoxythymidine (AZT) , 2',3'-dideoxycytidine (DDC) or other anti-HIV compositions which have a mechanism of action which differs from those of the present invention as a therepeutic method. In such cases where toxic compositions such as AZT or DDC are used in combination with the compositions of the pres¬ ent invention which exhibit a substantial absence of toxicity, the amounts of the 5-Thiolated poly or oligonucleotides used as well the AZT or DDC, among other agents, will generally be less than when each agent is used alone because of the synergistic therapeutic effect exhibited by the combination of agents.

The mode of administration may determine the sites in the organism to which the compound(s) will be delivered. For instance, delivery to a specific site of activity may be most easily accomplished by parenteral administration, for example, by injection via intraperitoneal, intravenous, intraarterial, intramuscular, subcutaneous or related routes. The preferred route of administration is via an intravenous or intraarterial route. For parenteral administration, the thiol-containing compositions of the present invention may best be used in the form of liposomes or a sterile aqueous solution which may con¬ tain other solutes, for example, sufficient salts, glucose or dextrose to make the solution isotonic.

The thiol-containing compositions according to the present invention may be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. In certain preferred embodiments of the present invention, polylysine is complexed or conjugated with the 5-Thiolated compositions of the present invention.

In certain cases, an oral mode of administration may be contemplated, in which case the thiol-containing composi¬ tions of the present invention can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspension, and the like. In the case of tablets, carriers which can be used include lac¬ tose, sodium citrate, and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents, for example, stearic acid may be used. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, certain sweetening and/or flavoring agents can be added. It is to be noted that in certain cases, compositions of the present invention which are intended to be administered via an oral route may be unstable to the harsh conditions of the gastrointestinal tract, especially due to the existence of nucleases which tend to degrade the composi¬ tions of the present invention. In such cases, one of

ordinary skill in the art will recognize to formulate the oral formulations to avoid the release of the compositions under conditions which render them unstable, for example, the extremely acidic conditions of the stomach. In certain cases, in order to resist enzymatic degradation, the compositions according to the present invention may be administered as the phosphotriester prodrug form (methyl or ethyl ester) . Alter¬ natively, the use of calcium or quarternary ammonium salts of the phosphates including the cetyltrimethylammonium salt, among others may advantageously be used orally.

The thiol-containing compositions described hereinabove, including their pharmaceutically acceptable salts, are contemplated for use in the present invention. Determination of the amounts and compatibilities of the above listed biologically active agents which are used to formulate the pharmaceutical compositions of the present invention are well within the ordinary skill in the formulation art. The stability and applicability of individual pharmaceutical agents are well within the ordinary skill of the practitioner in this art.

In general, the amount of the thiol-containing com¬ positions which are administered to the patient will be an effective amount and will range from about O.lmg. to about 100 mg. per kg. weight of the patient to be treated. Preferably, the dosage administered to the patient to be treated will range from about 0.1 mg. to about 50 mg. per kg. Most preferably, the amount of thiol-containing composition administered to the patient will range from about 1 mg. to about 20 mg. per kg. Dosages within these ranges should attain blood levels of active agent of at least about 1-10 urn. In certain cases, in the treatment of HIV infections in humans, the concentration of active agent may attain blood levels of 50-100 um. or more.

It is noted that the compositions according to the present invention have exhibited low toxicity to normal cells. Certain Thiol-containing compositions according to the present invention have been screened for their potential toxic effects

on uninfected PHA-stimulated human PBM cells using a radioac¬ tive thymidine uptake method. In that method, cells in a 96- well plate were grown in the presence of drug for 24 hours, and then 1 uCi of [ ³ H]-thymidine was added to each well. After 24 hours, the cells were harvested on glass fibers, washed, dried, and the amount of radioactivity associated with the cells was determined. Cycloheximide was included as a control for toxicity in every assay. Inhibitory concentra- . tions (ID5 ₀ ) values for normal human PBM cells were derived from a computer-generated median effect plot of dose-effect data. Compositions according to the present invention exhibited very low toxicity (ID5 ₀ ranged from 38.2 to > 100 mg./ml. and in most instances was > 100 mg./ml.).

It will be appreciated that the actual preferred amounts of the thiol-containing compositions of the present invention utilized in a specific case may vary according to the severity of a disease condition and the expected pharmacokinetics of the compositions in the individual patient. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject bioactive agent by means of an appropriate, conventional pharmacological protocol. In the therapeutic aspect of the present invention, a patient with an HIV infection, will be treated by administering an effective amount of a thiol-containing com¬ position of the present invention by an oral, topical or parenteral, preferably intravenous route. The duration of treatment may vary considerably, but will generally range as a function of the response of the individual patient to the treatment schedule.

EXAMPLES

The following examples are provided for purposes of illustration only and are not to be viewed as a limitation of the scope of the invention.

Example 1

Preparation of Partially Thiolated Polynucleotides (MPC. MPU. MPdC and MPdU)

The partially thiolated polynucleotides (MPC, MPU, MPdC and MPdU) were chemically synthesized according to the general literature method described in Bardos, et al., Nucleic Acid Chemistry. Vol. 2, 881 (1978); Ho, et al., Biochem. Biophys. Res. Comm.. 73, 903 (1976); and Mikulski, et al., Biochim. Biophys. Acta.. 319, 294 (1973) . Briefly, poly(C) , Poly(U) and Poly (dC) obtained from Pharmacia, New Jersey, USA or alternatively via enzymatic synthesis according to lit¬ erature procedures, was converted to the cetyltrimethylam- monium salt using cetyltrimethylammonium bromide (dropwise addition) . The precipitated cetyltrimethylammonium poly(cytidylate) was collected by centrifugation, washed with distilled water, dissolved in methanol and evaporated to dry- ness. The residue was dried in vacuo overnight. To a solu¬ tion of the dry salt in anhydrous methanol is added methyl hypobromite and the mixture was stirred for 30 minutes at 0°C. Dry N,N-dimethylacetamide was added and then nitrogen was passed through the solution for 20 minutes. Finely ground NaSH-2H ₂ 0 was added at 0°C and the solution was stirred under nitrogen for 1.5 hours. The polynucleotide was converted to the sodium salt with 3M NaCl solution (added dropwise at 2°C with continuous stirring) , dissolved in water and purified on a column of Sephadex G-100 (Available from Pharmacia, Inc. , New Jersey, USA) . These 5-Thiolated polynucleotide were sub¬ sequently tested in vivo to determine the anti-HIV activity. MPdU is obtained by the same procedure and tested for anti-HIV activity.

Example 2

Enzymatic Preparation of Partially Thiolated Polynucleotides

(MPC and MPU)

MPC and MPU were synthesized according to the methods described by Ho, et al., in Nucleic Acids Res.. 8, 3175

(1980). Briefly, Uridine 5'-diphosphate (UDP) or Cytidine 5'- diphosphate (CDP) is mixed with its respective 5-Thiolated diphosphate, i.e. either 5-Mercaptouridine-5'-diphosphate

(hs ⁵ UDP) or 5-Mercaptocytidine-5'-diphosphate (hs ⁵ CDP) by the literature method. Mixtures of the thiolated and unthiolated nucleoside diphosphate were polymerized with polynucleotide phosphorylase according to the general procedure of Grunberg-

Manago, Prog. Nucleic Acid Res.. 1, 93 (1963) .

The reaction mixture was incubated at 48°C for about 8 hours The percentage of polymerization was followed by assaying for inorganic phosphate by the method of Chen, et al., Anal. Chem. , 28, 1756 (1956); blank was subtracted from the readings. After incubation, the solution was deprotenized by repeated extraction with chloroform-isoamyl alcohol (5:3). The polynucleotide was purified by chro atography through Sephadex G-100 in the presence of mercaptoethanol.

The relative amounts of thiolated base incorporated into the polymeric products (expressed as a percent of the total pyrimidine bases) were determined based on the UV absor- bance of the 5-mercaptopyrimidine derivative at 334 nm in the presence of dithiothreitol at pH 8.0. The resulting thiolated polynucleotides were used in the assay of example 7, below.

Example 3

Preparation of 5-Thiolated Oligoribonucleotides

(MOC and MOU)

These compounds are prepared by the partial alkaline hydrolysis of poly(C) or poly(U) which are subsequently sepa¬ rated by column chromatography (DEAE Sephadex) and subse¬ quently thiolated according to the procedure described in Example 1. Briefly, Poly(C) was hydrolyzed by a chemical method in accordance with the procedure published by Ho, Kung & Bardos, Biochem. Biophys. Res. Comm.. 73, 903 (1976). The hydrolyzed poly(C) was loaded onto a column of DEAE cellulose (bicarbonate form, 2.5 cm X 20 cm) and eluted with 250 ml each of 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7 & 1.0 M TEAB buffer (pH 7.6-8.0). These fractions were monitored by TLC (solvent system (n-propanol/NH ₃ /H ₂ 0 55:33:10 v/v) against the standard 5'-CMP and poly(C) . Each fraction contains several oligomers of chain-lengths within 10 monomer units (+ 10-mers) . These different chain-length oligomers were separated using DEAE cellulose (DE-52 ^tm , available from Whatman Biosystems, Ltd., Kent, England) . Elution of these different oligomers in each fraction was carried out in a linear gradient of 0.15M to 0.85M TEAB buffer pH 7.6-8.0 (1000 ml) followed by 500 ml of 1.0 M TEAB buffer. The chain lengths of these purified oligomers were determined by Pt/Pi ratio as described by Chen,

et al. Anal. Chem.. 28, 1756 (1955).

Partial thiolation of the oligomers (oligo C5, oligo dCs and oligo C*j_ ₈ ) was carried out in accordance with the standard procedure as described by Bardos, .J. , et al. , Biochim. Biophys. Acta. 319, 294 (1973) . This procedure resulted in the formation of 5-Thiolated oligoribonucleotides which are subsequently tested. in vivo for anti-HIV activity.

Example 4

Preparation of 5-Thiolated Oligonucleotides (MOdC and MOdU)

These compounds were prepared by step-wise chemical synthesis using the phosphoramidite procedure (phosphotriester method, cyanoethyl protecting group, using a an Automated DNA Synthesizer (Model 381A available from Applied Biosystems, Inc.). Briefly, the phosphoramidate procedure was followed using the synthesizer which uses a solid phase synthesis chemistry in which the growing DNA chain remains covalently attached to an insoluble matrix, the support. Each step of the synthesis adds one nucleotide unit to the oligonucleotide. All reagents and solvents flow through the matrix which is contained within a column (the reaction chamber) . After com¬ pletion of the synthesis, the oligoDNA was removed from the synthesizer. The following sequences of oligonucleotides were prepared using the DNA Synthesizer and the separated oligonucleotides were, in certain cases, subsequently thio¬ lated using the method described in Example 1. This resulted in the synthesis of the following three oligonucleotides:

M(dC)4o- a 40 mer oligodeoxycytidylic acid with about 10% thiolation (4 nucleoside units per 40 unit strand)

Non-thiolated 5'-d(GTC/CCT/GTT/CGG/GCG/CCA)-3'

5-Mercapto 5'-d(GTC/CCT/GTT/CGG/GCG/CCA)-3'

Example 5

Preparation of Additional 5-Thiolated Oligonucleotides

The following oligonucleotides are synthesized by the same method as described in Example 4, above and tested for

anti-HIV activity.

5'-d(T) ₁₂ -MdC ₂₈ -3' 5'-d(A) ₁₂ -MdC ₂₈ 5'-d(T) ₁₂ -MdU ₂₈ 5'-d(A) ₁₂ -MdU ₂₈

The following oligonucleotides were synthesized by the method described in Example 4, above and were tested for anti- HIV activity as described in Example 8.

5'-d(T) ₁₂ -MdC ₂₂ -3' 5'-d(T) ₁₂ -MdC ₂₂ -3'

The following oligodeoxyribonucleotides corresponding to the anti-codon or 3' terminus region of tRNAj _j ys or anti- sense oligodeoxyribonucleotides to the same regions are synthesized by the method described above and tested for anti- HIV activity jLn vivo as described in the following examples 7 and 8:

Anti-codon Regional 5-Mercapto oligonucleotides

5'-MdC ₂₂ -d(ATC/AGA/CTT/TTA/ATC/TGA) -3'

5'-d(ATC/AGA/CTT/TTA/ATC/TGA) -MdC ₂₂ -3'

5'-MdU ₂₂ -d(ATC/AGA/CTT/TTA/ATC/TGA)-3'

5'-d(ATC/AGA/CTT/TTA/ATC/TGA)-MdU ₂₂ -3'

3' Terminus Regional 5-Mercapto oligonucleotides

5'-MdC ₂₂ -d(GTC/CCT/GTT/CGG/GCG/CCA) -3' Synthesized by the above general method. Exhibited strong anti-HIV activity as indicated in Example 8, below.

5'-d(GTC/CCT/GTT/CGG/GCG/CCA) -MdC ₂₂ -3'

5'-MdU ₂₂ -d(GTC/CCT/GTT/CGG/GCG/CCA)-3'

5'-d(GTC/CCT/GTT/CGG/GCG/CCA) -MdU ₂₂ -3'

Anti-Sense Anti-codon Regional

5'-MdC ₂₂ -d(TCA/GAT/TAA/AAG/TCT/GAT) -3'

5'-d(TCA/GAT/TAA/AAG/TCT/GAT)-MdC ₂₂ -3'

5'-MdU ₂₂ -d(TCA/GAT/TAA/AAG/TCT/GAT)-3' 5'-d(TCA/GAT/TAA/AAG/TCT/GAT)-MdU ₂₂ -3' Anti-Sense 3' Terminus Regional

5'-MdC ₂₂ -d(TGG/CGC/CCG/AAC/AGG/GAC)-3' 5'-d(TGG/CGC/CCG/AAC/AGG/GAC)-MdC ₂₂ -3' 5'-MdU ₂₂ -d(TGG/CGC/CCG/AAC/AGG/GAC)-3' 5'-d(TGG/CGC/CCG/AAC/AGG/GAC)-MdU ₂₂ -3'

Example 6

Preparation of 5-Thiolated tRNA

A mixture of tRNAs is separated from sheep liver according to the procedure of Rogg, et al., Biochim. Biophys. Acta, 195, 13 (1969) . A portion of this mixture is subse¬ quently thiolated by the procedure set forth in example 1. The 5-Thiolated tRNAs were subsequently tested for anti-HIV activity in vivo.

Example 7

Anti-HIV Activity of 5-Thiolated Oligo and Polynucleotides

Several 5-Thiolated polynucleotides were synthesized and tested against HIV 1 virus in vivo. In brief, these modified polynucleotides were synthesized by the thiolation (methoxybromide method) of preformed homopolynucleotides or by co-polymerization of 5-Mercapto-Uridine-5'-diphosphate and uridine-5'-diphosphate or adenosine-5'-diphosphate by polynucleotide phosphorylase as described in Example 2, above. The following 5-Thiolated poly(deoxy)nucleotides were made and tested: poly(U ₉₁ ,hs5u ₉ )

To test the above-described 5-Thiolated polynucleotides the following assay was utilized. H ₉ human lymphocytes were infected with diluted (1 to 10 and 1 to 32) standard HIV 1 (HTLV-III ₃ ) suspension (2000 virus particles per cell). After a 90 minute incubation period at 37°C the unadsorbed viruses were rinsed off. The washed cells were then resuspended in culture media (10 ⁵ cells/ml) and dispensed

in 2 ml por ^t ions onto Costar 24-well plates. Cc.εrol cells were treated similarly but were not exposed to the virus. To determine the inhibitory activities of various chemically modified polynucleotides and control materials (AZT and Phosphonoformic acid (PFA or Foscarnet) ) the indicated con¬ centrations of drugs were added (See Table 1, below) .

The inhibitory activites of the drugs on replication of HIV 1 were evaluated by the following methods.

a) Morphological evaluation- All infected and uninfected cell cultures were checked every second day to detect any degenerations due to the toxic effects of the com¬ pounds and to detect the cytopathic effect of the virus. (See, for example Mitsuya & Broder, Proc. Natl. Acad. Sci. USA 83, 1911 (1986)).

b) Indirect Immunofluorescence Analysis- After 6 and 12 days of incubation aliquots were removed from the cultures washed in phosphate-buffered saline spotted on glass slides and fized in acetone at -20"C for 15 min. The fixed cells were reacted with serum from an assymptomatic HIV-1 seroposi- tive patient. After 30 minutes at 37°C, cells were reacted with fluorescein isothiocyanate-conjugated goat anti-human IgG and virus antigen positive cells were counted.

c) Assay of reverse transcriptase activity- After 12 days of incubation, the cells were removed by centrifugation and the viruses were sedimented from the supernatant by high speed centrifugation. The virus pellets were treated with detergent and their RT activity determined using poly(A) -oligo(dT) ι ₂ as template'primer and [ ³ H]dTTP as sub¬ strate (See St. Clair, et al., Anti icrob. Agents Chemother.. 31, 1972 (1977)).

On the bases of the results of these assays (mor¬ phological observation, IIf and RT assay) the drug concentra¬ tions necessary for 50% inhibition were calculated and pre¬ sented below.

Table 1

Activities of Chemically Modified Polynucleotides

Against HIV

Cone.

Polynucleotide fuM Mon. Unit) Unit) poly(U ₉₁ ,hs ⁵ U ₉ ) 1

10

100 poly(U ₉₁ ,hs ⁵ U ₉ ) 'Poly(A) ^b 1

10

100 poly(A ₉₅ ,hs ⁵ U ₅ ) 1

10

100 poly(C ₇₉ ,hs ⁵ C ₂₁ ) ^c 1

10

100

PFA 1

10

100

AZT 0.01 0.1

1

^'s Percentage of fluorescent cells

^• °- Double stranded polymer ^c - Chemically thiolated polynucleotide (methoxybromide method)

Note- in the assays performed, no toxicity of the modified polynucleotides has been observed even at the highest con¬ centrations (lOOuM) used in the assays.

Example 8

Anti-HIV Activity of 5-Thiolated Oligo and Poly nucleotides

A number of Oligo- and Polynucleotides prepared above were tested for anti-HIV activity .in vivo (HIV infected human peripheral blood mononuclear cells (PBM cells) by the method of Schinazi, et al., Antimicob. Agents Chemother.. 32, 1784 (1989) . Briefly, the assay was performed according to the literature method in primary human lymphocytes infected with human immunodeficiency virus type 1. The infected control had 2.14 million dpm/ml 6 days after infection and the blanks were

less than .,800 dpm. All compounds were evaluated at con¬ centrations ranging from 0.001 to.10 ug/ml.

The results of the assay appear in Table 2, below. Although all compositions showed anti-HIV activity, the most active compositions included MPdC, MOdC (40 Mer) and 5- Thiolated tRNA. Of particular.note is the fact that MPdC exhibited an almost 40 fold enhancement relative to MPC and in general, the deoxyribonucleotides exhibited enhanced results relative to the corresponding ribonucleotides, with the excep¬ tion of tRNAs. This may be explained by the significantly enhanced stability evidenced by the deoxyribonucleotides rela¬ tive to ther ribonucleotides in vivo. Based upon these results, we expect MPdU to be significantly more active than the corresponding MPU. In Table 2, below, the slope represents a median effect plot which is a plot of log frac¬ tion affected cells/fraction unaffected cells (Fa/Fu) versus log concentration.

0 . 31 0 . 89

>10*

8 . 1*

6. 9*

17 . 1*

Note- In this same assay, AZT used as a positive control had an EC5 ₀ of 0.0021 uM (slope = 1.2). Compounds with the lower EC5 ₀ values were more potent.

*- Using the same general methodology, but Virus concentration was about 63,000 disintegrations per minute of RT activity per 10 ⁷ cells per mL of medium. The drugs were added about 45 minutes after infection. The cultures were maintained in a humidified 5% C0 ₂ - 95% air incubator at 37°C for six days after infection at which point all cultures were sampled for supernatant RT activity. The supernatant was clarified, and the virus particles were pelleted at 40,000 rpm for 30 minutes using a rotor (70.1 Ti; Beckman Instr. , Fullerton, CA.) and suspended in virus disrupting buffer. The RT assay was per-

formed in 96-well microdiluation plates using poly(rA) _n -(dT)- 12-1 _2; ^as template- ^p rimer. 3'-Azido-3'-deoxythymidine (AZT) was included in ee h as- y as a positive control. d(/CAG/ACT/TTT/AAT/CTG/AGC) is the anticodon analog of t- RNA ^L Y ^S 3

It will be understood by those skilled in the art that the foregoing description and examples are illustrative of prac¬ ticing the present invention, but are in no way limiting. Variations of the detail presented herein may be made without departing from the spirit and scope of the present invention.