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Prostratin: a Potential Adjuvant Therapy for HAART Against HIV-1 Reservoirs

by Stephen J. Brown, M.D., ARA's Director of Clinical Research

HIV reservoirs are a barrier to a cure for HIV infection. HAART often reduces HIV viral loads below detection levels; however, replication-competent HIV persists in latently infected resting CD4+ T-cells (1-3). Latently infected cells are present very early after acute HIV infection (4-6), are extremely long-lived, and may persist as a viral reservoir for up to 60 years in patients on HAART (2,3,6,7). Furthermore latently infected cells are "invisible" to the immune system, and are not susceptible to available anti-viral drugs. Thus, long-lasting viral reservoirs could represent a fundamental barrier* to the eventual elimination of HIV infection. The intentional activation of latent virus may be a critical strategic requirement for future HIV therapy.

Interest in structured treatment interruptions (STI) may partially be fueled by wishful thinking, as patients grow ever weary of the dreary prospect of never-ending pill-popping. With missed doses leading to drug resistance, non-adherence is the single most important reason for clinical failure. Growing concerns about long-term toxicities and lack of access to expensive regimens for millions of impoverished AIDS-sufferers in developing nations lend a sense of urgency to finding viable alternatives. Among the many compelling rationales for STI research, the most basic raison d'être is simply the need for a brief respite from unrelenting drug regimens and intolerable side effects.

One rationale for STIs is that the very success of HAART, ironically, may be its flaw. Based on the observations of Bruce Walker, HAART decreases HIV-specific cellular immunity by lowering the amount of HIV antigen. A treatment interruption, allowing a controlled amount of virus to be re-introduced to the immune system might, theoretically, provide sufficient antigen stimulation to trigger stronger HIV-specific and lympho-proliferative responses.

The rationale for latent virus activation strategies

The rationale (8-10) for a latent virus activation strategy is that, by stimulating latently infected cells to replicate and express infectious virus, such cells will die more rapidly and/or will present viral components (e.g., such as viral envelope gp120) on their surfaces, making such cells more detectable by the immune system. They might also be rendered more susceptible to targeted destruction by immunotoxins (10) or other potential therapeutic agents. Key to the strategy is to administer the latent virus activating agent along with antiretroviral drugs, to prevent the infection of uninfected cells by the newly expressed virus.

Most attempts or plans thus far to induce latent virus activation focus on cytokines, such as IL-2 and tumor necrosis factor, as the activating agent(s). (10-14) Other potential activating agents that have been proposed include lipopolysaccharides, bacterial superantigens and anti-CD3+ antibodies. However, most if not all of the potential activating agents considered thus far are macromolecular, highly toxic and/or have other undesirable attributes. To date, few if any potential "small-molecule" or more "drug-like" agents or compounds have been considered.

Prostratin

In 1992, National Cancer Institute (NCI) scientists reported the purification of prostratin as the active constituent of extracts of the tropical plant, Homalanthus nutans, used in traditional Samoan herbal medicine. (12) Purified prostratin demonstrates anti-viral activity against both HIV-1 and HIV-2, as well as a high in vitro therapeutic index.(16,17) Prostratin is a 12-deoxyphorbol ester (390 kDa). In contrast to most other phorbol esters, prostratin is not a tumor-promoter; indeed, it is one of the most potent known anti-tumor promoters.(18) This agent was originally targeted for drug development; however, it was subsequently determined that prostratin is a potent activator of HIV expression in "latently infected" T-cell lines (18) which, at the time, halted further development as a potential anti-HIV therapeutic. With the subsequent advent of HAART, an overall consensus view toward a general latent virus activation strategy has evolved substantially within the field of HIV therapeutics research. Indeed, many international leaders pursuing HIV and AIDS research and therapy now see latent virus activation as a critical strategic requirement for future advances in therapeutics of HIV infection and AIDS.

Based on our preliminary data shown below, prostratin appears to be an important potential candidate as an AIDS therapeutic in protocols in which virus activation is an inherent strategic element. It is already known that human exposure has occurred, although we do not know what the actual exposure in dose or duration was. (16) Preliminary experimental studies (22) in rodents have indicated that blood levels achievable by oral administration of prostratin (0.4-1.4 micromolar) are well in excess of those required for in vitro activation of HIV expression in latently infected cells.

In order to further the development of prostration as a potential therapeutic, AIDS ReSearch Alliance negotiated and eventually in-licensed prostratin from the Office of Technology Transfer. Pending completion of the licensing application, ARA proceeded to form important collaborations with laboratories around the world in order to look in more detail at the activity and potential uses of prostratin in HIV-infected individuals. This has resulted in further elucidation of both the anti-HIV activity as well as its ability to activate latently infected cells, and supports the potential utility of prostratin as an adjunctive therapy for HIV. Several of the researchers working on prostratin presented abstracts at the recent Keystone Symposia on AIDS Pathogenesis, and are reproduced here (see page 4). (17-19)

Prostratin as Anti-HIV compound

One of ARA's first collaborations was with the leading antiviral researcher Eric de Clercq in Belgium, with the goal of exploring the anti-HIV mechanism of prostratin. Myriam Witvrouw in his lab confirmed the inhibitory activity of prostratin against the replication of HIV-1(NL4.3) in MT-4 cells and PBMCs with an EC50 of 0.04 µg/ml. (17) This compound was not toxic for the host cells at concentrations up to 125 µg/ml., over 3000 times higher than needed to inhibit virus. Prostratin also proved effective against a variety of HIV-1 strains [subtype B (IIIB, NL4.3) and subtype D (NDK)], a clinical HIV-1 isolate L1, different strains of HIV-2(ROD and EHO) and SIV (MAC251). Prostratin was equally active against virus strains that were resistant towards polyanionic and polycationic types of viral entry inhibitors as well as virus strains selected for resistance against nucleoside and non-nucleoside reverse transcriptase inhibitors. Time-of-addition experiments indicated that the compound had to be present from the time of infection onwards. However, prostratin did not inhibit the binding of HIV-1 (NL4.3) to MT-4 cells at concentrations that inhibited the replication of HIV-1 (NL4.3) in these cells.

Researchers in Belgium then selected prostratin-resistant HIV-1 (NL4.3) strains by passaging the virus in the presence of increasing concentrations of the compound. Genotypic and phenotypic data of these selected strains revealed mutations commonly seen in resistance to entry inhibitors such as T-20 and AMD-3100. This together with the Time of Addition experiments supports the mechanism of prostratin's action as occurring at the fusion/entry step of the viral life cycle. However, the structure of the compound (essentially non-charged) suggests the drug should work on a cellular target. Further experiments on the effect of prostratin on expression of receptors and co-receptors are underway to further clarify the antiviral mechanism of the compound.

Prostration as activator of latently infected cells

Of greatest interest to ARA was the effect of prostratin on latently infected cells. ARA developed collaborations with several researchers who have been developing distinct experimental models of HIV latency.

Initial work was done in conjunction with Dr. Jerome Zack's lab at UCLA.(18) Dr. Zack's lab has developed a model of latency in SCID-HU-THY-THY mice. Whereas previous work by others has concentrated on the effect of prostratin on T-cell lines, Dr. Zack and his group assessed the effects of prostratin on activation of highly purified primary naïve T-cells that were DR- and CD25- (indicating "quiet" or unactivated cells). They utilized a flow cytometric assay for cellular RNA and DNA content to determine the activation state of the cell population.40 These naïve T-cells do not respond greatly to stimulation by anti-CD3 or anti-CD28 alone; rather, they require co-stimulation through both these receptors for proliferation. Surprisingly, prostratin alone had minimal effects on these cells; however, it acts as a co-stimulator in the presence of CD3 stimulation, and also acts as a primary stimulatory signal if co-stimulation via CD28 is applied.

This is a very unusual pattern of activity, suggesting that prostratin might signal T-cells via a novel mechanism. Dr. Zack & colleagues have performed extensive phenotypic characterization of the activation response to prostratin. Naïve T-lymphocytes stimulated with prostratin alone express CD25 (IL-2 receptor) but not CD69. This is also very different from CD3 stimulation, which induces CD69 but not CD25 expression. It is also different from CD28 stimulation, which does not induce the expression of either marker.

Using their SCID-hu-Thy model, they next assessed whether prostratin can activate latent HIV. From infected SCID-hu-Thy mice, they purified human CD4+/CD8- thymocytes or peripheral blood and splenocytes, and placed these cells in culture for three days in the presence of protease inhibitor to prevent virus spread. The cells were either left unstimulated, or stimulated on day 0 with CD3/CD28 or with prostratin alone, and viral Gag protein was measured by ELISA of the supernatant. Co-stimulation or activation via prostratin alone induced the expression of latent virus. These data indicate that prostratin can induce replication of the latent pool of virus without initiating T-cell replication.

Additional work on the activating effect of prostratin has been done in two other models of latency

Additional experiments were done in the laboratory of Ivan Hirsch at INSERM in Marseille, France. (20) Dr. Hirsch has developed an model of latency which successively depletes cell populations from whole blood until a very pure sample of resting infected memory CD4+ cells remains. This allows Dr. Hirsch to test compounds for their ability to activate viral production from both the short-lived pre-integration state as well as the long-lived post-integration state. At the recent Keystone conference Dr. Hirsch shared his result with AIDS ReSearch Alliance. In his model, prostratin caused activation of both pre and post integration virus.

In yet a third model of latency developed by Dr. Jose Alcami of Spain (21), prostratin also was an effective activator of latent virus. In Dr. Alcami's model, he takes PBMC's and transfectes them with a full-length infectious HIV-1 provirus in which the Luciferase gene has been cloned in the place of Nef. This virus is fully competent for replication. Results show that the transfected cells were activated immediately after treatment with Prostatin at doses ranging from 0.1 to 10 micromolar. Luciferase activity in cell lysates and p24 production in viral supernatants were measured 24 hours after transfection and showed robust activation.

Further outside confirmation of the activity of prostratin has come from the laboratory of Dr. Pomerantz from Jefferson University in Philadelphia. (19) In data also presented at the recent Keystone Conference on AIDS Pathogenesis, Dr. Pomerantz took PBMC's from HIV infected patients and using prostratin, activated virus from the patient samples.

Future Development Plans

AIDS ReSearch Alliance has formulated a scientific development plan that encompasses future pre-clinical work through the early phases of clinical (human) trials. It includes all steps to ensure that patients involved in the human clinical trials will receive a compound thoroughly tested for safety. We have developed a very aggressive and rapid timeline for our first year of pre-clinical work.

Important additional studies of prostratin need to be done. In the next phase of proposed studies, ARA and UCLA will further characterize the effects of prostratin on various human T-cell populations in vivo. We will determine the effects of prostratin on normal human T-cell differentiation, and on HIV pathogenesis and latency in vivo, using UCLA's SCID-hu-Thy mouse model. This will be done in the presence and absence of HAART, to allow a more complete characterization of the antiviral properties of this agent.

Recent evidence has established several additional potential reservoirs and facilitators of latent infection. Replication competent HIV has been isolated from CD8+ cell, and bone marrow epithelial cells. AIDS ReSearch Alliance is in the process of establishing collaborations with prominent laboratories working in these two areas. Lastly, recent work has shown the importance of a receptor on dendritic cells called DC-SIGN, which captures virus and delivers it to T-cells that are subsequently infected. Since prostratin has the effect of down-regulating several receptors such as CD4 and CKCR-4 the effect of prostratin on DC-SIGN in of interest. One laboratory that is currently looking at the importance of DC-SIGN has agreed to look at the effect of prostratin on this receptor.

We anticipate that these studies will provide important pre-clinical data to determine whether prostratin should proceed to clinical trials as a means of eliminating the reservoir of latent HIV in T-lymphocytes of infected individuals. In addition, we hope to generate important information relevant to any similar agent being contemplated for this type of adjunctive treatment approach. Thus, this work may help to pave the way for strategies to totally eliminate HIV from the human body.

In summer of 2001, AIDS ReSearch Alliance will meet with FDA officials to determine the required studies necessary to file an IND for a Phase I study.

REFERENCES

1. Chun TW, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA 94(24): 13193-13197, 1997.

2. Finzi D, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278(5341): 1295-1300, 1997.

3. Wong JK, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278(5341):1291-1295, 1997.

4. Chun TW, et al. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc Natl Acad Sci USA 95(15): 8869-8873, 1998.

5. Chun TW, et al. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med 1(12): 1284-1290, 1995.

6. Chun TW, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387(6629): 183-188, 1997.

7. Finzi D, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5(5):512-517, 1999.

8. Ho DD. Toward HIV eradication or remission: the tasks ahead. Science 280(5371):1866-1867, 1998.

9. Cohen OJ, Fauci AS. HIV/AIDS in 1998--gaining the upper hand? JAMA 280(1): 87-88, 1998.

10. Berger EA, et al. Reconsidering targeted toxins to eliminate HIV infction: you gotta have HAART. Proc Natl Acad Sci USA 95(20):11511-11513, 1998.

11. Chun TW, et al. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J Exp Med 188(1): 83-91, 1998.

12. Gulakowski RJ, et al. Antireplicative and anticytopathic activities of prostratin, a non-tumor-promoting phorbol ester, against human immunodeficiency virus (HIV). Antiviral Res 33(2): 87-97, 1997.

13. Chun TW, Fauci AS. Latent reservoirs of HIV: obstacles to the eradication of virus. Proc Natl Acad Sci USA 96(20):10958-10961, 1999.

14. Prins JM, et al. Immuno-activation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy. AIDS 13(17):2405-2410, 1999.

15. Chun TW, et al. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy. Nat Med 5(6):651-655, 1999. Gustafson KR, et al. A nonpromoting phorbol from the samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1. J Med Chem 35(11):1978-1986, 1992.

16. Brown S, et al. Broad-spectrum Antiretroviral Activity and Mechanism of Anti-HIV Action of Prostratin, a non-tumor-promoting phorbol ester. In Keystone Symposia: HIV Pathogenesis, 2001; Keystone, CO.

17. Zack abstract Pomerantz abstract

18. Boyd M. NCI Initial Toxicity and Pharmakokinetic Data of Prostratin. 2000.

19. Personal communication Hirsch, 2001

10. Personal communication Alcami, 2001

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