A METHOD OF INHIBITING HIV REPLICATION

BACKGROUND OF THE INVENTION

A method of inhibiting or treating HIV infection is described.

Drugs currently available for the treatment of HIV infection include agents that directly affect the production or function of virally-encoded peptides or proteins. Many of these drugs are not only unaffordable to many patients with HIV, but also have unpleasant side-effects.

The inventor has now identified an alternative strategy for inhibiting HIV replication, by regulating gene transcription of the host cell.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided the use of a compound that is an activator of AMP-activated protein kinase (AMPK), or a pharmaceutically acceptable salt or derivative of the compound, in a method of making a medicament for use in a method of treating or inhibiting HIV infection or replication in a subject.

The medicament may contain a therapeutically effective amount of the compound or salt or derivative thereof.

Typical compounds are metformin, metformin hydrochloride and AlCAR.

The subject may be a human.

According to a second embodiment of the invention, there is provided a compound that is an activator of AMP-activated protein kinase (AMPK), or a pharmaceutically acceptable salt or derivative of the compound, for use in treating or inhibiting HIV infection or replication.

Typical compounds are metformin, metformin hydrochloride and AICAR.

The compound may be used in treating or inhibiting HIV replication in a human subject, and in particular, by administering a therapeutically effective amount of the compound to the subject.

According to a third embodiment of the invention, there is provided a method of treating or inhibiting HIV infection or replication in a subject, the method including the step of administering to the subject a compound that is an activator of AMP-activated protein kinase (AMPK), or a pharmaceutically acceptable salt or derivative of the compound.

Typical compounds are metformin, metformin hydrochloride and AICAR.

A therapeutically effective amount of the compound or salt or derivative thereof may be administered to the subject, which is typically a human.

The compound may be administered to the subject without the subject also being administered anti-retrovirals.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of inhibiting or treating HlV infection by administering a compound that is an activator of AMP-activated protein kinase (AMPK), or a pharmaceuticaliy acceptable salt or derivative of the compound, to a subject, and in particular a human.

The known AMP-activated protein kinase (AMPK) activators are AICAR and metformin).

Metformin (fyN-dimethylimidodicarbonimidic diamide) and its salt metformin hydrochloride (tyW-dimethylimidodicarbonimidic diamide hydrochloride) is the most commonly used oral agent for the treatment of type 2 diabetes and other insulin resistant syndromes. Other pharmaceutically acceptable saits of metformin are metformin p-chiorophenoxyacetate and metformin embonate (metformin pamoate). The use of metformin to reduce viral replication in HIV- positive patients has not previously been suggested or investigated.

Metformin

The adenosine analogue 5-amino-1-β-D-ribofuranosyi-1H-imidazole-4- carboxamide (also described as 5-amino-4-imidazoiecarboxamide ribonucleoside, AICAR and acadesine) is also an AMPK activator (7). AlCAR is used to reduce cardiovascular complications after cardiac surgery (8). The use of AICAR in inhibiting viral replication, and in particular HIV replication, has not previously been reported.

AICAR

Metformin or AICAR could be administered to patients instead of the current HIV treatment regime which involves the administration of anti-retrovirals (ARVs). The side effects of metformin and AICAR are much milder than ARVs ₁ which wouid a!so be an advantage over the use of ARVs.

The present invention is further described by the following examples. Such examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention.

Examples

Example 1 : The use of AICAR in inhibiting HIV replication in an immortalized cell line

The U1 cell iine is an immortalized human monocytic cell line that is chronically infected with HiV. The viral genome is inserted within the host cell DNA and can be activated by treating the cells with cytokines or sodium chloride. Activation leads to viral gene transcription and translation to yield virile particles, which can be measured using an ELISA that quantifies the level of the viral peptide, P24.

Cells from the U1 ceil line, obtained from Professor Lyn Morris, National Institute of Communicable Diseases, Johannesburg, were cultured in 24-well

plates at a concentration of a million ceils per ml, at 0.5 ml per well in the presence of 60 mmol/l sodium chloride and varying concentrations of AICAR from Sigma-Aldrich. St Louis, MO, USA (0.1 , 0.2, 0.35, 0.4 mmol/l). After 3 days at 3O ⁰ C, 25 μl of tissue culture medium was carefully removed from each of the wells and the level of P24 present was assayed using a P24 ELISA, supplied by DuPont, Medical Products, Boston MA, USA, and used as a measure of the viral level.

Control tests, in which the U1 cells were stimulated with sodium chloride in the absence of AiCAR, were also performed.

Cell viability was tested using trypan blue exclusion (10).

Example 2: The use of metformin in inhibiting HIV replication in an immortalized cell line

The procedure described in Example 1 above was repeated, except that 0, 4, 8 or 16 mmol/l metformin HCI, supplied by Sigma-Aldrich, St Louis, MO, USA, was added to the cell culture medium and the AICAR was omitted.

Results

The level of P24 obtained from each sample in the above examples was expressed as a percentage of the value obtained from the control samples, which was arbitrarily set at 100%. The results from Examples 1 and 2 are shown below in Table 1.

Table 1: P24 level of HIV-positive cells treated with AICAR or metformin

^* p<0.05 vs. control (Wilcoxoπ matched pairs test). Data is expressed as means ± SD.

The above results demonstrate that both AICAR and metformin, both activators of AMPK, inhibit HlV replication in a dose-dependent manner, as assessed by measurement of P24 levels. The cell viability assay demonstrated that the fail in viral replication was not due to U1 cell death.

Examples 3 and 4: The use of AICAR and metformin in inhibiting HIV replication in isolated human peripheral blood mononuclear cells

The experiments described in examples 1 and 2 were also performed using isolated human peripheral blood mononuclear cells (PBMCs) cultured in the presence of the human immunodeficiency virus, instead of the immortalized cell lines. This system is a closer model for the in vivo environment than that described in examples 1 and 2. The results obtained in this model were essentially the same as observed in Table 1 , with clear inhibition of HIV replication by both AICAR and metformin.

The applicant envisages that the same or similar results should be obtained when administering activators of AMPK, and in particular AICAR and/or metformin, to humans. It is believed that activation of AMPK leads to reduction of nuclear factor kappa-B (NF-κB) activity. Although human clinical trials are still to be conducted, it is envisaged that the doses of metformin and/or AICAR required to block HIV replication in human subjects may be higher than those currently administered to human subjects for the treatment of type 2 diabetes.

While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the claims without departing from the spirit and scope of the present invention.

References

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