Hydroxyurea and HIV: 5 years later-from antiviral to immune-modulating effects (1999)

The origins The ability of hydroxyurea to decrease intracellular levels of deoxynucleotide triphosphate (dNTP) is the property of this drug that first led us to propose using it to inhibit HIV replication [1]. At that time it was known that although HIV can infect both quiescent and activated T lymphocytes, it can replicate productively only in activated cells [2-4]. However, in a state called unintegrated latency, HIV can survive for some time in quiescent cells in an incomplete DNA form. If a quiescent cell becomes activated within 2 weeks of infection, reverse transcription is completed, the virus becomes integrated into the host cell genome, and new infectious viral particles can be produced [3,4]. Because it was not clear why HIV failed to replicate in quiescent T lymphocytes, it was postulated that the scarcity of dNTP in quiescent cells could partly be responsible. This same scarcity could also explain why our group [5] and others [6] found DNA of variable length carried by the mature virion. In fact, because viral DNA is more stable than viral RNA, a DNA form ensures longer survival of HIV in quiescent cells, while the virus awaits ‚rescue‚ by antigen-mediated T cell activation. By adding hydroxyurea to activated T lymphocytes, it was possible to reduce the levels of dNTP in these cells and to inhibit HIV reverse transcription, in a process mimicking the natural block to viral replication in quiescent T lymphocytes [1]. This experiment proved the hypothesis that HIV replication depends on adequate levels of dNTP and, at the same time, it suggested the potential for a new anti-HIV drug. In this sense, hydroxyurea belongs to a new class of anti-HIV compounds, because it inhibits the virus indirectly by interacting with a cellular protein [7]. This protein, ribonucleotide reductase, represents the rate-limiting step for dNTP synthesis, and it contains a free radical that is irreversibly quenched by hydroxyurea (for a review see [8]). The intrinsic advantage to targeting cellular proteins is that, unlike viral proteins, they are not prone to mutation. Retroviruses are particularly predisposed to mutate because of the propensity of reverse transcriptase to make mistakes during the copy-making process [9,10]. A drug that does not induce resistance would prove to be invaluable. Hydroxyurea could be such a drug: almost 40 years of clinical experience have not revealed any evidence of cellular resistance to it [11]. It is commonly believed that drugs targeting cellular factors must be toxic. Fears about toxicity of hydroxyurea, in fact, proved to be a major obstacle to the initial use of this drug by physicians treating individuals infected by HIV. However, hydroxyurea has been in the hematologists clinical arsenal since the early 1960s. The bone marrow toxicity associated with this drug is the very reason hematologists use hydroxyurea to decrease the vast excess of circulating blood cells in patients with myeloproliferative disorders. High doses of hydroxyurea (2g or more daily) are used first to induce bone marrow toxicity; after the blood cell count decreases toward normal levels, the dosage is reduced, and this dosage adjustment is sufficient to level off and maintain normal blood cell counts. Hydroxyurea can then be administered to patients for several years [11]. When administered to patients with HIV infection, hydroxyurea is given at much lower doses, usually about 1g daily, which are very well tolerated by the bone marrow. Since our original description of the antiviral activity of hydroxyurea [1], several mechanisms of action have been proposed to explain the antiviral effects of hydroxyurea. They will be discussed later in this review. Immune-modulating effects of hydroxyurea A less understood but more provocative possible explanation of the antiviral activity of hydroxyurea is that it exerts an immune-modulating effect that may help to control viremia. The main cellular consequence of inhibiting ribonucleotide reductase is a cytostatic effect. Cells treated with hydroxyurea are arrested between phases G1 and S, or they enter the early S phase and accumulate [14], because the drug blocks dNTP production and thus impairs DNA synthesis. This is why compartments characterized by high cell turnover, such as bone marrow, suffer toxic effects [15]. However, the impact of this cytostatic action on the immune system cells remains unknown. HIV infection is characterized by massive activation of T lymphocytes when they become exposed to high doses of the virus. Signs of activation include elevated levels of inflammatory/activating cytokines (for a review, see [16,17]). There is multiple indirect in vitro and in vivo evidence that stimulation of the immune system, in turn, boosts HIV replication. Cytokines induce HIV replication in vitro [16,17]. Infection with pathogenic organisms [18,19], administration of interleukin-2 without highly active antiretroviral therapy (HAART) [20], and immunization with several vaccines [21,22] - all conditions known to stimulate the immune system - increase viral load in vivo. In contrast, immunosuppression by cyclosporin A transiently decreases viremia during primary SIV infection [23]. There is a simple explanation of these findings. HIV enters quiescent and activated T lymphocytes with similar efficiency. However, as already mentioned, the virus replicates only in activated T lymphocytes [2-4]. The boundaries of this phenomenon have now been accurately defined: cell division must progress to the G1b phase of the cell cycle for completion of HIV reverse transcription in T lymphocytes [24]. If T lymphocytes treated with hydroxyurea remain quiescent, they become refractory to productive HIV infection. Therefore, drug-induced decreases in the number of activated T lymphocytes (cells that can support a fully productive HIV infection cycle) might decrease overall production of the virus. More simply: no cell division/activation=no virus replication (Fig. 1). Several mathematical models support this hypothesis. It has been argued that the initial decrease of CD4 T lymphocytes after primary infection might account for the decrease of viral load to a ‚set point‚ [25]. Some models predict that expansion of HIV is target-cell-limited [26,27]; other models forecast that reducing target cell availability can stifle the outgrowth of drug-resistant HIV mutants [28,29].Fig. 1: Hydroxyurea (HU) immunologic mechanism of action: the hydroxyurea-CD4 interaction hypothesis. Hydroxyurea decreases the activation of CD4 T lymphocytes, and limits HIV integration/production. HIV integration and replication depend on the activation of T lymphocytes. Through its cytostatic activity, hydroxyurea decreases the number of activated T lymphocytes, thus limiting HIV production. The same cytostatic effects explain the lack of robust CD4 cell count increase during hydroxyurea-containing regimens.A large clinical trial recently provided the most compelling confirmation of the hypothesis [30]. After an initial response to triple-drug therapy (zidovudine, lamivudine, and indinavir), 23% of patients assigned to either a maintenance course of indinavir monotherapy or zidovudine plus lamivudine lost viral suppression. Loss of viral suppression during maintenance therapy strongly correlated with greater increase in CD4 cell counts during induction therapy. These results were anticipated by mathematical simulations based on the assumption that the greatest increase in the number of ‚prey‚ (CD4 T lymphocytes) resulted in the highest risk for the return of ‚predators‚ (HIV virions, expressed as detectable HIV RNA) in the plasma during the maintenance phase [31]. Hydroxyurea limits increases in T cell counts [32-34]. Therefore, it could be an ideal candidate for a new immunologic approach to HIV therapy. The ‚predator-prey‚ model [35], the absence of viral resistance to hydroxyurea, and the ability of hydroxyurea to compensate for resistance to other drugs may all contribute to the low rate of viral rebound during treatment with hydroxyurea-containing regimens and, perhaps, after treatment suspension. Although randomized, controlled trials are needed to confirm this hypothesis, preliminary reports support it. Pilot studies involving patients treated with hydroxyurea/didanosine [36-38] reveal low or no viral rebound during several months to years of follow-up. Three reported cases of HIV-infected adults with no viral rebound after discontinuation of therapy had been treated with hydroxyurea [39,40,41]. The less than robust increase in CD4 cell counts has raised concerns that hydroxyurea may have harmful immune-suppressive effects. I think the potential for this problem has been overestimated. First, since hydroxyurea exerts cytostatic effects on all lymphocytes, the percentage of CD4 T lymphocytes as part of the total lymphocyte population increases with hydroxyurea-containing treatments as much as with non-hydroxyurea treatments [42]. Second, high doses of hydroxyurea administered to patients with hematological diseases, who are predisposed to immune deficiency, have not been associated with immunologic problems [11]. Third, accumulating evidence indicates that hydroxyurea-containing treatments increase levels of naïve CD4 and CD8 T lymphocytes [43], decrease levels of activated CD8 T lymphocytes, and normalize the immune response to antigens [44]. Unlike HAART, hydroxyurea-containing regimens reconstituted T helper response specific to HIV [38]. Restoration of immune parameters and immune functions by the use of a cytostatic and potentially immune-suppressive drug represents an interesting paradox. The paradox might be explained by comparing the immune system over-activated by HIV with an overheated automobile engine; at some point it has a high chance of failing. One component of this over-activation is the CD8 T lymphocyte compartment. Percentages of activated (CD38+) CD8 T lymphocytes increase dramatically during infection and predict poor prognosis [45]. Moreover, shortened telomeres in CD8 T lymphocytes after HIV infection represent indirect evidence of increased division in this cellular compartment [46]. Excessive activation of CD8 T lymphocytes can be detrimental in several ways. Over-activation may lead to exhaustion [47], depriving the immune system of its cellular arm. On the other hand, the CD8 T lymphocyte response may mediate the immunopathology of HIV infection [47]. There are, in fact, good reasons to consider HIV as a non-cytopathic or weakly cytopathic virus [48,49]. If a non-cytopathic virus infects macrophages, antigen-presenting cells, or helper T cells, these cells would be destroyed by the immune response mediated by CD8 T lymphocytes. In this scenario, CD8 T lymphocytes, and not HIV, kill CD4 T lymphocytes and thus destroy the immune system and enable the virus to persist. We propose that a cytostatic drug such as hydroxyurea could prevent the exhaustion of CD8 T lymphocytes and limit the pathogenic effects of these cells (Fig. 2).Fig. 2: Hydroxyurea (HU) immunologic mechanism of action: hydroxyurea-CD8 interaction hypothesis. HIV infection results in depletion of CD4 T lymphocytes. If this depletion is caused by the immunopathogenetic effects of activated CD8 T lymphocytes, hydroxyurea cytostatic effects will reduce CD8 activation and will ultimately prevent CD8 exhaustion and CD4 killing.In conclusion, irrespective of the pathogenic mechanism of CD4 killing, the combined beneficial effects of hydroxyurea on both CD4 and CD8 T lymphocytes could facilitate control of HIV as well as prevent immune system damage or restore previously compromised immune functions. Antiviral effects of hydroxyurea Hydroxyurea potentiates nucleoside reverse transcriptase inhibitor (NRTI) activity Because NRTI compete with cellular dNTP for incorporation into the growing HIV DNA, using hydroxyurea to decrease the concentration of cellular dNTP creates a competitive advantage for the NRTI (Fig. 3) [1]. Consequently, a higher proportion of the NRTI eventually becomes incorporated by reverse transcriptase into viral DNA and so blocks DNA synthesis. Extensive in vitro testing revealed that hydroxyurea and didanosine appeared to be effective in curtailing viral replication in both dividing and non-dividing lymphocytes and macrophages infected with HIV [12,13,50]. Hydroxyurea plus didanosine synergy was derived mathematically [12]. The combination of these drugs proved to be a more potent combination than hydroxyurea and zidovudine [51]. This difference can be explained by the competition of didanosine with deoxyadenosine triphosphate (dATP), which hydroxyurea depletes more than deoxythymidine triphosphate, with which zidovudine competes. Furthermore, combining hydroxyurea and zidovudine exacerbates bone marrow toxicity [51].Fig. 3: Hydroxyurea (HU) potentiates the activity of nucleoside reverse transcriptase inhibitors (NRTI), especially of didanosine (ddI). Hydroxyurea decreases the deoxyadenosine triphosphate (dATP) pool more effectively than the pool of the other deoxynucleotide triphosphates (dNTP). Therefore an adenosine analog such as ddI acts synergistically with hydroxyurea.Hydroxyurea compensates for HIV resistance to adenosine analog NRTI HIV variants resistant to didanosine are significantly more sensitive to this drug in the presence of hydroxyurea (Fig. 4) [32]. This observation has been extended to other adenosine analogs, such as 9-(2-phosphonylmethoxyetyl) adenine (PMEA) and (R)-9-(2-phosphonylmethoxypropyl) adenine (PMPA) [52]. These findings can be explained by the same ‚NRTI-dNTP competition‚ theory described above. HIV variants resistant to adenosine analogs are characterized by the increased propensity of reverse transcriptase to incorporate dATP rather than adenosine analog NRTI triphosphates into nascent viral DNA. Resistance to a competitive drug, however, is not a ‚black-or-white‚ phenomenon. Therefore, if hydroxyurea decreases the dATP concentration, the adenosine analog NRTI is again placed in a favorable competitive position. Ultimately, an HIV variant carrying a genotypic mutation associated with resistance to an adenosine analog becomes phenotypically sensitive to the drug in the presence of hydroxyurea-as sensitive, in fact, as wild-type virus [32,52].Fig. 4: Hydroxyurea (HU) compensates for HIV resistance to adenosine analog nucleoside reverse transcriptase inhibitor [e.g. didanosine (ddI)]. A ddI-resistant HIV prefers to use deoxyadenosine triphosphate (dATP) over ddI. Therefore, ddI stops DNA elongation less efficiently. If the amount of dATP is decreased by hydroxyurea, ddI becomes competitive again. dNTP, deoxynucleotide triphosphate.Hydroxyurea enhances phosphorylation of NRTI The observation that hydroxyurea enhances the in vitro anti-HIV activity of thymidine or cytidine NRTI, like stavudine or lamivudine, by increasing their intracellular phosphorylation [53,54] provides a rationale for combining hydroxyurea with these drugs. To date, however, there is no proof in vivo of this concept. It is therefore advisable to add these drugs only in the context of a hydroxyurea-didanosine regimen. Such a regimen may have particular merit as a salvage strategy after disruption of NRTI phosphorylation by certain other NRTI regimens [55]. For example, stavudine or lamivudine might be combined with hydroxyurea in zidovudine-experienced patients. Hydroxyurea is active in multiple compartments Both hydroxyurea [12] and didanosine [56] exert their most potent anti-HIV activity in resting T lymphocytes and in primary macrophages the absence of viral replication for several weeks after of hydroxyurea treatment [12]. At of of HIV DNA cells was after treatment These support the use of a combination during primary HIV infection, as macrophages represent the of initial infection during or of hydroxyurea Hydroxyurea enters cells by with a of and a that to be The drug therefore enters all the system and [11]. Hydroxyurea given to patients infected with HIV is well the with a of after After administration of of hydroxyurea a the of this drug is levels of hydroxyurea to These are similar to the and to inhibit HIV in have been between hydroxyurea and zidovudine between hydroxyurea and didanosine stavudine will be in the clinical trial Because of the effects of hydroxyurea in quiescent T lymphocytes and its high in all the effects of this drug on HIV replication in the system, and other compartments the experience with hydroxyurea has our original hypothesis and initial in vitro results [12,13,50]. studies of combined hydroxyurea and didanosine the ability of this combination to reduce viral load significantly In a involving patients treated for with didanosine daily) and hydroxyurea HIV plasma levels decreased by to in and viral load in the other in plasma viremia for of treatment In involving with HIV a combination of didanosine daily) and hydroxyurea daily) resulted in an initial decrease in plasma viremia of that increased to more than 2 after weeks of treatment A of patients a decrease in plasma viral load when of hydroxyurea were administered over as an to didanosine HIV-infected with CD4 After hydroxyurea, viremia to In a controlled trial patients with CD4 cell counts of were to didanosine daily) or didanosine plus hydroxyurea After plasma viremia in the combination group decreased significantly more than in the didanosine monotherapy didanosine and hydroxyurea had decrease patients didanosine only had a decrease rebound was in some patients in the didanosine monotherapy group and in in the combination After discontinuation of the monotherapy treatment in the combination group resulted in an in plasma viremia at 40 Although CD4 cell count between treatment were not they increased more in the didanosine monotherapy than in the hydroxyurea-didanosine Hydroxyurea has been combined with didanosine and stavudine in One trial of were to didanosine plus stavudine or this combination plus hydroxyurea daily) at weeks that the group hydroxyurea a significantly greater decrease in viral load and that significantly more of these patients a in viral load to than HIV After patients with a viral load HIV were the if they had been hydroxyurea, or hydroxyurea was to the regimen if patients had been the NRTI combination regimen. After viral load was HIV in and in of the hydroxyurea-containing regimen. who hydroxyurea had a decrease in plasma viremia to A CD4 cell count increase of only with hydroxyurea, with an increase of with were trial the of hydroxyurea in combination regimens [42]. In didanosine plus hydroxyurea viremia viremia to an similar to that with nucleoside therapy or and viremia By adding hydroxyurea to a viral load of HIV was in of patients after similar to that reported in studies of regimens that a inhibitor or a in hydroxyurea-containing had the increases in CD4 cell with However, the treatment not when CD4 cell were CD4 cell increased to in all of the presence of hydroxyurea. the of hydroxyurea was in all randomized, controlled Hydroxyurea toxicity doses of hydroxyurea are well tolerated by HIV-infected patients. marrow toxicity is the main However, this toxicity is At the low doses commonly used for HIV infection bone marrow toxicity has been described in of patients In most however, the toxicity has been and has not dosage adjustment or discontinuation of toxicity has been In a of patients toxicity after treatment with a higher of hydroxyurea This of toxicity is for this drug, and so was its counts decreased during the first weeks of treatment and to normal a weeks after the was to must be to patients in of the HIV bone marrow in some and counts may be with were at higher risk for bone marrow toxicity as were patients with very HIV include toxicity of and, after to the drug, [11]. Furthermore, hydroxyurea is as drug, and it not be used during [11]. dosage physicians use a We have been using in almost patients. The and for hydroxyurea in antiretroviral however, have not been a clinical trial in is to about is the total or and is the or When to use hydroxyurea are their use of hydroxyurea for HIV infection. Some of this increase results the of adding hydroxyurea to any salvage at any of the infection. This strategy a the time when hydroxyurea as a However, randomized, controlled trials patients. Furthermore, clinical trial must to be to hydroxyurea can be combined as with other drugs as it has been with didanosine and and, for patients with a high viral load or during infection, with didanosine and a In the to the of hydroxyurea is to use it as part of a combination regimen. There are several good reasons to hydroxyurea in the In the absence of a for HIV HIV-infected patients antiretroviral as the the drugs now to viral suppression is to the of drug-resistant mutants and to and high make and regimens for most patients. Therefore, viral individuals with multiple and variants of HIV. toxicity limits the use of these regimens is to with drugs for hydroxyurea has several of such a drug. Resistance to hydroxyurea has not been described after of clinical use hydroxyurea compensates for resistance to NRTI adenosine analogs and may limit the outgrowth of mutants some patients with hematological have been hydroxyurea for years hydroxyurea is well tolerated treatment with hydroxyurea and didanosine in patients with than HIV with hydroxyurea, and stavudine in patients with than lead to levels of viremia in most while use of more potent but also more toxic drugs inhibitors and These other drugs could be used if the initial regimen with a high chance of given the lack of between the and drugs. A preliminary the use of hydroxyurea in the treatment of primary HIV infection viremia decreased to levels in individuals treated with hydroxyurea, and indinavir HIV the course of for to HIV was also in the of of patients. HIV was found in cells in of individuals by in In the HIV could be found in only of the reported cases of HIV-infected adults with no viral rebound after discontinuation of patients had been treated only with hydroxyurea and viremia rebound in either during a of treatment after the of combination therapy however, DNA could be in in either or The viral load however, was low in both patients HIV In a who was treated with hydroxyurea, and the inhibitor indinavir early after infection, plasma viremia decreased HIV to an level rebound could be months after treatment and virus could be only at very low levels cell carrying There is no proof that hydroxyurea had a specific in these A potential of hydroxyurea and didanosine in of HIV infection after has also been suggested less is known about the use of hydroxyurea in of HIV infection. bone marrow toxicity has been reported in patients with hematological toxicity with only has been described in patients with very HIV infection who were treated with hydroxyurea, and lamivudine after a of regimens failed hydroxyurea to regimens didanosine didanosine plus stavudine to reduce viral load is an interesting by the and studies These results are explained by the in vitro that hydroxyurea compensates for didanosine resistance and increases phosphorylation of thymidine This in randomized, controlled A for treatments Hydroxyurea is an drug. It is and already used in most of the More than of with HIV will be to of to patients and are well the of most these represent a part of the There is a to for simple and The of and to the and on hydroxyurea, and for