WO1995017875A2

WO1995017875A2 - MIXTURES OF DIDEOXYNUCLEOSIDES AND D-ASPARTIC ACID β-HYDROXAMATE FOR INHIBITING RETROVIRAL SPREAD

Info

Publication number: WO1995017875A2
Application number: PCT/IB1995/000006
Authority: WO
Inventors: Jorge R. Vila; Serge D. Malley; Farid Hamedi-Sangsari; Jacques M. Grange
Original assignee: Compagnie De Developpement Aguettant
Priority date: 1993-12-20
Filing date: 1994-12-20
Publication date: 1995-07-06
Also published as: WO1995017875A3; AU1249595A

Abstract

A method and composition for inhibiting the spread of a retrovirus such as HIV in a human cell population in which a retrovirus such as HIV is present has been found. The spread of the retrovirus is inhibited by treatment of the cells with a synergistic combination mixture of a ribonucleoside analog excluding AZT, and D-aspartic acid β-hydroxamate.

Description

Mixtures of Dideoxy Nucleosides and D-aspartic Acid β- Hydroxamate for Inhibiting Λetroviral Spread

FIELD OF THE INVENTION

The present invention relates to a combination of a reverse transcriptase inhibitor and D-aspartic acid β- hydroxamate (DAH) in a synergistically effective amount wherein the combination is useful in inhibiting retroviral spread.

BACKGROUND OF THE INVENTION

The expression "Acquired Immuno-Deficiency Syndrome" (AIDS) was first used in 1981 to describe a state of cellular immune deficiency in homosexuals, characterized by the appearance of opportunistic infections and Kaposi's Sarcoma evolving very aggressively (CDC (Center for Disease Control), MM R, 30 : 305-308.DC, (1981)). In 1983 a retrovirus since called HIV (Human Immunodeficiency Virus type l)was isolated among AIDS patients (Barre-Sinoussi F. et al Science , 220 : 868-870 (1983)) .

Over the past several years, researchers and clinicians have gained considerable experience in studying and caring for individuals infected with HIV throughout the often prolonged course of HIV disease and AIDS. On the basis of this experience, it has become clear that the pathogenic mechanisms underlying HIV infection and disease are not unidimensional, but rather are extremely complex (Fauci AS., Science , 239, 617,(1988)). Any attempt to design a comprehensive therapeutic strategy for HIV disease must take this fact into account. (Fauci, 1993, Science , 262:1011-1018).

After entry of the HIV virus into cells and uncoating of the HIV particle, reverse transcription of the viral RNA genome into DNA replicas occurs. Among several forms of unintegrated viral DNA (now containing the long repeats [LTRs], at both the 5' and the 3' ends), only the two-LTR linear forms can integrate into the host genome. Such a process appears strictly dependent upon cell activation/replication of T lymphocytes, although "resting" T cells are clearly susceptible to HIV infection. (Zack J.A. et al, Cell ; 61, 213-222, (1990)). Furthermore, part of the reverse transcription process also can occur in unactivated T cells, a process that results in the accumulation of incomplete DNA molecules, which may persist for several hours and remain capable of being integrated into the host genome if the cell undergoes sufficient activation (Zack J.A. et al. Cell ; 61, 213-222, (1990)). Therefore, infected "resting" CD4⁺T lymphocytes can be considered a transient viral reservoir in infected individuals (Bokrinsky M.I. et al; Science, 254, 423-427, (1991)). These observations are of particular importance in anatomic compartments such as the peripheral blood and lymphoid organs, where the CD4⁺T cell subset represents the predominant infected cell type (Schmittman S M. et al. Science, 245,305-308, (1989)); (Fox CH. et al J. Infect Diε j 164, 1051-1057, (1991)).

The above discussion provides a sound scientific basis for blocking the initial burst of virus replication and dissemination as well as the persistent replication throughout the course of disease by treating HIV-infected individuals with anti-retroviral agents from the earliest time that HIV infection is recognized through the entire course of infection. Unfortunately, currently available agents are only partially effective in suppressing virus replication and spread, and this effect is transient (Hirsch MS, et al , New Engl. J. Med . 328 1686, (1993)). Clear cut, but limited, benefit is seen when 3'-azido-2', 3'-dideoxythymidine or azidothymidine (AZT) is given to a patient with advanced HIV disease, and the benefits of early intervention are usually only temporary and do not result in significant long-term advantages with regard to the course of disease and death. (Fauci, 1993, Science , 262:1011-1018).

A number of 2',3'-dideoxynucleosides have been found to be useful for the treatment or prophylaxis of retroviral infections and especially HIV and AIDS. Examples of such materials include: 2',3'-dideoxy-cytosine (ddC) ; 2',3'- dideoxy-adenosine (ddA) ; 2',3'-dideoxy-guanosine (ddG) ; and 2' ,3'-dideoxy-inosine (ddl) and 2',3' didexoxy- thymidine (ddT or DT4) . See European patent application 0206497 and published PCT application number WO 87/01284. DAH is a known medicine for the treatment of retroviral infections such as for example AIDS as well as for treating tumors. It is known that DAH produces substantial inhibition of the replication of viruses, such as retroviruses.

However, there still remains a need for more effective treatments for the suppression of retroviruses and, in particular, the prevention and/or inhibition of HIV and viral spread. By viral spread, it is intended to include the inhibition of viral replication, and viral integration in the host cells, and also may include the ability of inhibiting the virus to infect further host cells.

Objectives of the present invention in the search for new antiretroviral agents include:

1) the identification of compounds with less toxicity and antiviral activity greater than AZT. 2) the development of drug combinations which provide an additive or synergistic effect and decrease the probability of drug resistant isolates.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a combination of a reverse transcriptase inhibitor, excluding AZT, and DAH in a synergistic combination wherein the synergistic combination is capable of preventing and/or inhibiting the spread of retroviruses including HIV. More specifically, the present invention relates to a method of preventing and/or inhibiting the spread of retroviruses, including HIV (HIV-I and HIV-II) , HTLV-1, HTLV-II, SIV and HSV, by exposing a cell population, including cells infected by HIV, to a synergistic combination of a reverse transcriptase inhibitor and DAH. Additionally, the present invention encompasses the treatment of HIV-infected and AIDS patients with a synergistic combination of a reverse transcriptase inhibitor and DAH in order to prevent and/or inhibit the spread of HIV in these patients.

In a preferred embodiment of the present invention, the reverse transcriptase inhibitors include dideoxynucleosides, such as, for example, ddl, ddA, ddC, ddG and ddT (DT4) .

In particular and in the preferred combination of the present invention, it has been found that a synergistic combination of DAH and 2',3'-dideoxyinosine (ddl) can be formed which is especially effective in preventing and/or inhibiting HIV spread. The preferred embodiment of the invention encompasses a composition including a pharmaceutical composition comprising a synergistic combination of ddl and DAH. The pharmaceutical composition can optionally contain a pharmaceutically acceptable carrier and/or excipient and/or vehicle. The preferred method of the instant invention comprises preventing and/or inhibiting retroviral or HIV spread by treating a cell population, including cells infected with HIV, with a synergistic combination of ddl and DAH. Additionally, the preferred method comprises treating an HIV infected or AIDS patient with a synergistic combination of ddl and DAH so as to prevent and/or inhibit HIV spread in the patient.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a study of the anti-viral activity of a mixture of ddl and DAH on non-activated CD4+ lymphocytes infected with the HIV virus.

Figure 2 is a study of the antiviral activity of a mixture of AZT and DAH.

Figure 3 illustrates the inhibition of viral spread by the mixture of DAH and ddl in pre-activated peripheral blood mononuclear cells (PBMC) culture infected with the HIV virus.

DETAILED DESCRIPTION OF THE INVENTION The following examples of a specific embodiment of the present invention are offered for illustrative purposes only and are not limiting with respect to the scope of the disclosure or claim coverage.

Testing of the mixture of dideoxyinosine (ddl) and DAH on the spread of the HIV virus was conducted under two types of conditions: a) CD4+ lymphocytes purified from PBMC, and infected with HIV virus without prior activation/proliferation of these cells by phytohemagglutinin (PHA) and interleukin-2 (IL-2) . b) PBMC preactivated by PHA and IL-2, then infected with the HIV virus. Example 1

Study of the activity of the association of ddl and DAH on non-activated CD4⁺ lymphocytes, infected with HIV virus.

The non-activated CD4⁺ cells were infected, then treated for 7 days by DAH, DDI or the combination of the two, then activated by PHA and IL-2 (PHA-IL-2) .

Cellular viability between 90 and 100% was observed during the first seven days after infection, both for the infected control and for the infected cells treated with the two drugs separately or in combination. Comparable proliferative cellular response was observed in the presence of PHA-IL-2 for the first 3 days (days 7-9) both in the six virus infected groups and in the non-infected, non-treated donor CD-4⁺ cells. This proliferative response is associated with cytopathic effect in the infected control group, and in the groups treated with DAH alone at 0.5 and 1.5 mM these groups had greater than 50% loss of viability compared to the uninfected control group: this effect is due to viral replication and is accompanied by large-scale production of p24-HIV in the culture supernatant-seen at day 15 (86215 pg of p24/ml for the infected control, 77581 and 51180 for 0.5 and 1.5 mM DAH treatment groups respectively) see figure 1.

The cytopathic effect was observed later for the cells treated with 5 μM ddl and reached substantially the same level of p24 production as the infected control 10 days later (101080 pg p24/ml) , see figure 1.

The association of DAH at 0.5 mM with ddl at 5 μM does not substantially change the viral replication profile as compared to ddl alone (75570 pg p24/ml at day 25) , see figure 1.

By contrast, a surprising synergistic effect is observed with the combination 1.5 mM of DAH and 5 μM of ddl, where no residual viral production is detectable (< 1 pg p24/ml) at day 7 and day 25 despite cellular proliferation which is identical to the non-treated, non- infected control (>90% cell viability measured by MTT test) .

The CD4⁺ lymphocytes were purified from PBMC with lmmunomagnetic beads (Dynabeads M450) . These cells were infected with the HIV-1 virus strain IIIB at a multiplicity of infection of 5 000 tissue culture infectious dose (TCID) per 10⁶ cells (241pg/ml p24 antigen equivalent of virus) . After 2 hours of virus-cell contact, the cells were washed twice and placed in the culture medium RPMI 1640 (supplemented with 10% FCS (fetal calf serum) 2mM gluta ine, penicillin 100 IU/ml and Streptomycin 100 μg/ml) at a density of 1.3xl0⁶ cells/ml. ddl was immediately added at a concentration of 5 μM and DAH at a concentration of 0.5 mM and 1.5 mM. The drugs and culture medium were partially renewed (50%) on day 4, maintaining the same concentration of each. On day 7, in order to remove the drugs, the cells were washed twice and put back in culture in the presence of PHA at a concentration of 1 μg/ml and recombinant 11-2 at a concentration of 20 U/ml. This culture was maintained until day 25, with partial renewal (50%) of the medium twice a week. The number of viable cells was quantified by a tetrazolium-based colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (CDC (Center for Disease Control) , MMWR,30:305-308.DC, (1981) ),and activity is expressed as a percentage of the signal in the drug-free and virus-free control. Viral replication was quantified by measuring the HIV-l p24 antigen by ELISA using the Dupont de Nemours kit. Example 2

In order to determine whether or not this synergistic effect in association with DAH is specific to ddl, or whether a similar effect could be observed with other deoxynucleoside analogs used in the treatment of AIDS, a parallel study was conducted combining DAH with azidothymidine (AZT) with surprising results.

As can be seen in figure 2, AZT alone at 5μM has only slight anti-viral activity less than 1 log, 88.4% inhibition (10030 pg p24/ml compared to 86215 pg p24/ml for infected control) , less than ddl at the same dose under the same conditions: 99.1% inhibition (766 pg p24/ml compared to 86215 pg p24/ml for infected control, see Figure 1) . The drug concentrations used here are easily attainable in plasma (plasma concentration achievable under treatment conditions: at 4 μM for AZT and 10 μM for ddl (Barre-Sinoussi F. et al Science , 220 : 868-870 (1983)).

Comparable proliferative cellular response was observed after stimulation by PHA-IL-2 in all groups. This proliferative response is associated with cytopathic effect in the infected control group, and in the groups treated with DAH alone at doses of 0.5 and 1.5 mM, and combined with AZT at 5 μM (these groups had greater than 50% loss of viability compared to the uninfected control group). The combination of DAH at 0.5 mM and at 1.5 mM with AZT at 5 μM (10199 and 9341 pg p24/ml respectively) does not modify the viral replication profile compared to AZT alone (Figure 2) .

The results show that the synergistic effect which eradicates HIV replication in CD4⁺ cells non-activated by PHA-IL-2 (Example 1) is not found from the association of DAH AZT.

In figure 2, there is shown a study of the activity of the association of AZT and DAH on non-activated CD4⁺ lymphocytes infected with the HIV virus. The CD4⁺ lymphocytes were purified from PBMC with immunomagnetic beads (Dynabeads M450) . These cells were infected with the HIV-1 virus strain IIIB at a multiplicity of infection of 5 000 tissue culture infectious dose (TCID) per 10⁶ cells (241pg/ml p24 antigen equivalent of virus) . After 2 hours of virus-cell contact, the cells were washed twice and placed in the culture medium RPMI 1640

(supplemented with 10% FCS, 2mM glutamine, penicillin 100 IU/ml and streptomycin 100 μg/ml) at a density of 1.3xl0⁶ cells/ml. AZT was immediately added at a concentration of 5 μM and DAH at a concentration of 0.5 mM and 1.5 mM. The drugs and culture medium were partially renewed (50%) on day 4, maintaining the same concentration of each. On day 7, in order to remove the drugs, the cells were washed twice and put back in culture in the presence of PHA at a concentration of 1 μg/ml and recombinant IL-2 at a concentration of 20 U/ml. This culture was maintained until Day 25, with partial renewal (50%) of the medium twice a week. The number of viable cells was quantified by a tetrazoliu -based colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Pauwels, R. et al J. Virol . Methds , 20, 309-321, (1988)), and activity is expressed as a percentage of the signal in the drug-free and virus-free control. Viral replication was quantified by measuring the HIV-1 p24 antigen by ELISA using the Dupont de Nemours kit. Example 3 Inhibition of viral spread in preactivated PBMC culture infected with the HIV virus, by the association of DAH and ddl.

PBMC preactivated with PHA and IL-2, were infected and treated by DAH at 1.5 mM; this concentration corresponds to the IC₅₀ (inhibitory concentration 50%) after 3 days measured by MTT test, with cellular viability >90% (this cell viability was determined by treating the cells with 2% Trypan Blue for 2 min and monitoring for dye exclusion). The combination of 1.5 mM of DAH and 10 μM of DDI does not modify the IC₅₀ and cellular viability.

As can be seen (figure 3) , the virus replicated rapidly in the non-treated culture maintaining a stable level as from day 6 (day 6 = 71815; day 12 = 72750; day 20 = 62750 pg p24/ml) . Treatment with ddl alone at 10 μM and DAH alone at 1.5 mM induces inhibition of 97.1% (2071 pg p24/ml) and 81.9% (13010 pg p24/ml) respectively at day 6. By contrast, a major synergistic effect is observed with the combination of 10 μM ddl and 1.5 mM DAH, with an inhibition of 99.6% (257 pg p24/ml) at day 6 and no residual viral production detectable (<1 pg p24/ml) at day 12 and day 20. This major synergistic effect, having been demonstrated with non-activated lymphocytes, where the combination of ddl with DAH eradicates the HIV infection from the cells, is also observed here where lymphocytes are preactivated and treated with the combination of ddl and DAH while the PBMC are replicating.

In figure 3 there is shown the elimination of viral replication by the combination of DAH and ddl in preactivated PBMC culture infected with the HIV virus. The PBMC were purified from peripheral blood by discontinuous Ficoll density gradient centrif gation. The cells were grown at a density of 1.3xl0⁶ cells/ml in RPMI 1640 medium supplemented with 10% FCS 2mM glutamine, penicillin 100 IU/ml and streptomycin 100 μg/ml, in the presence of PHA at a concentration of 1 μg/ml and reco binant IL-2 at 20 U/ml for 72 hours, then infected by HIV-1 strain IIIB at a multiplicity of 5000 TCID for 10⁶cells (241pg/ml p24 antigen equivalent of virus). In figure 3, the first time point represents viral infection of the cells. After 2 hours of virus-cell contact, the cells were washed twice and placed in the culture medium containing IL-2 but without PHA. In the presence of ddl at a concentration of 10 μM and of DAH at a concentration of 1.5 mM. These cultures were maintained for 20 days, with partial renewal of the medium and of the two drugs, twice a week maintaining the initial concentration. Day 6 and day 14 fresh uninfected donor PBMC were added (5xl0⁵/ml) to replenish aged cultures (Chow et al Nature (1993) 361:650-654). The number of viable cells was quantified by a tetrazolium-based colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Pauwels, R. et al J. Virol . Methods , 20:309-321, (1988)), and activity is expressed as a percentage of the signal in the drug-free and virus-free control. Viral replication was quantified by measuring the HIV-l p24 antigen by ELISA using the Dupont de Nemours kit.

The subject of the present invention is also a new composition for the treatment of a cell population in the presence of a retrovirus. Additionally, the invention includes a pharmaceutical composition intended, in particular, for the treatment and prevention of retroviral infections, especially those linked to HIV and AIDS wherein the composition contains a synergistic combination of D-aspartic acid β-Hydroxamate (DAH) and a reverse transcriptase inhibitor, in particular a synergistic combination of a dideoxy-nucleoside except

AZT and DAH, most preferably a synergistic combination of dideoxyinosine and DAH as active principle, in a pharmaceutically acceptable vehicle. The composition of the invention can also contain inert or pharmacodynamically active additives, carriers and/or excipients.

The pharmaceutical composition of the invention can take the form of a lyophilized powder of the active substance, to be dissolved immediately before use in a physiological solution for the purpose of injection. The medicament can then be administered parenterally, for example intravenously, intraperitoneally, in the cerebrospinal fluid, and the like. For injection, the active principle is dissolved in a physiological solution until the desired concentration for administration is obtained. The pharmaceutical composition according to the invention can also take a form which is suitable for oral administration. For example, suitable forms are tablets, hard gelatin capsules, dragees, powders and granules. The formation of such oral forms is well-known to those skilled in the art. Any of the known formulations are useful in preparing the instant oral pharmaceutical compositions.

As regards the dosage of the medicament according to the invention, it will be clear to the artisan that the doses to be administered are variable according to the treatment period, and frequency of administration, the host and the nature and severity of the disease and that the dosages can be easily determined without any undue amount of experimentation.

The compositions of the invention are administered in substantially non-toxic dosage concentrations sufficient to insure the release of a sufficient dosage unit of the present synergistic combination into the patient to provide the desired inhibition of the spread of the retrovirus. The actual dosage administered will be determined by physical and physiological factors such as age, body weight, severity of condition, and/or clinical history of the patient. With these considerations in mind, the dosage of the instant synergistic combination for a particular subject can be readily determined by the physician. It might be noted that in extreme cases a dosage approaching the toxic level may be the acceptable treatment protocol. The invention also covers the use of DAH and dideoxyinosine in combination with other medicinal compositions intended for the treatment of retroviral infections and tumors. Immunostimulants and immunomodulations such as for example cytokines, including IL-2, IL-12 and interferon molecules can be used in combination with the present invention. A preferred range for in vitro administration of the compositions of the present invention includes DAH in a concentration greater than 0.5 mM and less than or equal to 3.OmM in combination with a dideoxynucleoside such as ddl at concentrations which are generally known and used in the art. A preferred embodiment of the present invention utilizes DAH at 1.5 mM and the dideoxynucleoside such as ddl in a range of between about 5μM to about 10 μM.

All of the references cited hereinabove are expressly incorporated herein, in toto, by reference thereto.

The invention has been described with reference to specific and preferred embodiments. It will be recognized by those skilled in the art that numerous changes and substitutions may be made without departing from the spirit and scope of the invention.

Claims

We Claim:

1. A composition useful for inhibiting the spread of a retrovirus in a cell population comprising a mixture of at least one dideoxynucleoside excluding AZT and D- aspartic acid β-hydroxamate wherein the D-aspartic acid β-hydroxamate (DAH) is present in a synergistically effective amount with respect to the amount of the dideoxynucleoside.

2. A composition according to claim 1, wherein the dideoxynucleoside is at least one of ddl, ddA, ddC, ddG or ddT.

3. A composition according to claim 2, wherein the dideoxynucleoside is ddl.

4. A composition according to any one of claims 1, 2 or 3, wherein the retrovirus is HIV.

5. A composition according to claim 4, wherein the retrovirus is HIV-1.

6. A composition according to claims 1, 2 or 3, wherein the amount of dideoxynucleoside is such as to provide a concentration of about .01 μM to 100 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about the amount 2.0 mM.

7. A composition according to claim 6, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 2.5 μM to

25 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

8. A composition according to claim 6, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM to 10 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

9. A composition according to claim 1, 2 or 3, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM and the amount of the DAH such as to provide a concentration of about 1.5 mM.

10. A composition according to claim 4, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.

11. A composition according to claims 1, 2 or 3, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 10 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.

12. A composition according to claims 4, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 10 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.

13. A composition according to claims 4, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 0.01 μM to 100 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

14. A composition according to claim 13, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 2.5 μM to 25 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

15. A composition according to claim 13, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM to 10 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

16. A method of inhibiting spread of a retrovirus in a human cell population in the presence of the retrovirus comprising administering to the human cell population an effective spreading inhibiting amount of a composition according to claim 1.

17. A method according to claim 16, wherein the dideoxynucleoside comprises at least one of ddl, ddA, ddC, ddG or ddT.

18. A method according to claim 17, wherein the dideoxynucleoside is ddl.

19. A method according to any one of the claims 16, 17 or 18, wherein the retrovirus is HIV.

20. A method according to claim 19, wherein the retrovirus is HIV-1.

21. A method according to any one of the claims 16, 17 or 18, wherein the amount of dideoxynucleoside is such as to provide a concentration of about

0.01 μm to 100 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about

2.0 mM.

22. A method according to claim 19, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 0.01 μM to 100 μM and the amount of the DAH is such as to provide a concentration greater than about .5 mM and equal to or less than about 2.0 mM.

23. A method according to claims 21 or 22, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 2.5 μM to 25 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

24. A method according to claims 21 or 22, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM to 10 μM and the amount of the DAH is such as to provide a concentration greater than about 0.5 mM and equal to or less than about 2.0 mM.

25. A method according to any one of the claims 16, 17 or 18, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.

26. A method according to claim 19, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 5 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.

27. A method according to any one of the claims 16, 17, or 18, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 10 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.

28. A method according to claim 19, wherein the amount of dideoxynucleoside is such as to provide a concentration of about 10 μM and the amount of the DAH is such as to provide a concentration of about 1.5 mM.