Hydroxyurea and HIV: 5 years later-from antiviral to immune-modulating effects
From the Research Institute for Genetic and Human Therapy, Pavia, Italy and Washington, DC, USA.
Requests for reprints to: F. Lori, Research Institute for Genetic and Human Therapy (RIGHT), Medical-Dental Building, SW307, 3900 Reservoir Road, NW, Washington DC 20007, USA.
Received: 3 December 1998; revised: 11 March 1999; accepted: 8 April 1999.
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 . 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  and others  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 . 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 . 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 ).
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 .
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 . 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 , 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 , 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 . 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) , 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 .
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 . 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‚ . 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].
A large clinical trial recently provided the most compelling confirmation of the hypothesis . 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 .
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 , 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 . Second, high doses of hydroxyurea administered to patients with hematological diseases, who are predisposed to immune deficiency, have not been associated with immunologic problems . Third, accumulating evidence indicates that hydroxyurea-containing treatments increase levels of naïve CD4 and CD8 T lymphocytes , decrease levels of activated CD8 T lymphocytes, and normalize the immune response to antigens . Unlike HAART, hydroxyurea-containing regimens reconstituted T helper response specific to HIV .
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 . Moreover, shortened telomeres in CD8 T lymphocytes after HIV infection represent indirect evidence of increased division in this cellular compartment .
Excessive activation of CD8 T lymphocytes can be detrimental in several ways. Over-activation may lead to exhaustion , depriving the immune system of its cellular arm. On the other hand, the CD8 T lymphocyte response may mediate the immunopathology of HIV infection . 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).
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) . 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 . The combination of these drugs proved to be a more potent combination than hydroxyurea and zidovudine . 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 .
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) . This observation has been extended to other adenosine analogs, such as 9-(2-phosphonylmethoxyetyl) adenine (PMEA) and (R)-9-(2-phosphonylmethoxypropyl) adenine (PMPA) . 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].
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 . For example, stavudine or lamivudine might be combined with hydroxyurea in zidovudine-experienced patients.
Hydroxyurea is active in multiple compartments
Both hydroxyurea  and didanosine  exert their most potent anti-HIV activity in resting T lymphocytes and macrophages. Studies in primary macrophages documented the absence of viral replication for several weeks after suspension of hydroxyurea treatment . At concentrations of 1mmol/l, <1 copy of HIV DNA per 1000 cells was detected 31 days after treatment stopped. These data support the use of a hydroxyurea-based combination during primary HIV infection, as macrophages probably represent the route of initial infection during sexual, parental, or vertical transmission [57,58].
Pharmacodynamics of hydroxyurea
Hydroxyurea enters cells by passive diffusion, with a Km of essentially zero and a Vmax that appears to be infinite . The drug therefore enters easily all body compartments, including the central nervous system and genital tract . Hydroxyurea given orally to patients infected with HIV is well absorbed from the gastrointestinal tract, with a t max of 0.85-0.96 h after ingestion . After administration of 500mg of hydroxyurea twice a day, the half-life (t 1/2) of this drug is 2.5±0.5 h . Serum levels of hydroxyurea range from 0.01 to 0.13mmol/l. These values are similar to the concentrations (between 0.01 and 0.1mmol/l) demonstrated to inhibit HIV in vitro. No pharmacological interactions have been observed between hydroxyurea and zidovudine . Interactions between hydroxyurea and didanosine and/or stavudine will be studied in the RIGHT 702 clinical trial (see below).
Because of the effects of hydroxyurea in quiescent T lymphocytes [12,13] and its high diffusibility in all body compartments, the effects of this drug on HIV replication and/or latency in the central nervous system, genital tract, and other reservoir compartments deserve in-depth evaluation.
Clinical experience: beyond the anecdote
Clinical experience with hydroxyurea has largely confirmed our original hypothesis  and initial in vitro results [12,13,50]. Small pilot studies of combined hydroxyurea and didanosine indicated the ability of this combination to reduce viral load significantly [33,36,51,61-64]. In a study involving 12 drug-naïve, asymptomatic patients treated for 90 days with didanosine (200mg twice daily) and hydroxyurea (500mg twice daily), HIV RNA plasma levels decreased by 1.4 to 2.9 logs in six patients, and viral load became undetectable in the other six . Reductions in plasma viremia lasted for >1 year of treatment . In another study involving six patients, including three with advanced HIV disease, a combination of didanosine (200mg twice daily) and hydroxyurea (250mg four times daily) resulted in an average initial decrease in plasma viremia of 0.9 logs that increased to more than 2 logs after 65 weeks of treatment . A third study of 26 patients demonstrated a substantial decrease in plasma viral load when 1000mg of hydroxyurea were administered over 1 month as an adjunct to didanosine among HIV-infected persons with 100- 350×106 CD4 cells/l . After withdrawing hydroxyurea, viremia returned to baseline values .
In a controlled trial , 57 patients with CD4 cell counts of 250-500×106 cells/l were randomized to receive didanosine alone (200g twice daily) or didanosine plus hydroxyurea (500mg twice daily). After 24 weeks, plasma viremia in the combination group decreased significantly more than in the didanosine monotherapy group. Patients receiving didanosine and hydroxyurea had average 1.32 logs decrease from baseline viremia, whereas patients receiving didanosine only had a 0.78 log decrease (P=0.0005). Viral rebound was observed in some patients in the didanosine monotherapy group and in none in the combination group. After discontinuation of the monotherapy arm, continued treatment in the combination group resulted in an average 1.21 log reduction in plasma viremia from baseline at 40 weeks. Although CD4 cell count differences between treatment groups were not statistically significant, they increased more in the didanosine monotherapy arm (+83 CD4×106 cells/l) than in the hydroxyurea-didanosine arm (+54 CD4×106 cells/l).
Hydroxyurea has been combined with didanosine and stavudine in larger studies. One trial randomized 144 patients, 80% of whom were antiretroviral-naïve, to didanosine plus stavudine or this combination plus hydroxyurea (500mg twice daily) . Data at 12 weeks indicate that the group receiving hydroxyurea experienced a significantly greater decrease in viral load (2.3 logs versus 1.7 logs, P<0.001) and that significantly more of these patients exhibited a reduction in viral load to fewer than 200 HIV RNA copies/ml (54% versus 28%, P<0.001). After 12 weeks, patients with a viral load >200 HIV RNA copies/ml were withdrawn from the study if they had been receiving hydroxyurea, or hydroxyurea was added to the regimen if patients had been receiving the NRTI combination regimen. After 24 weeks, viral load was <200 HIV RNA copies/ml in 79% and <20 copies/ml in 63% of those continuing the hydroxyurea-containing regimen. Those who added hydroxyurea had a further significant decrease in plasma viremia (from 1.1 logs to 1.9 logs, P<0.0001). A CD4 cell count increase of only 28×106 cells/l with hydroxyurea, compared with an increase of 107×106 cells/l with placebo (P=0.001), were reported.
Another double-blinded, randomized trial confirmed the efficacy of hydroxyurea in combination regimens . In antiretroviral-naïve patients, didanosine plus hydroxyurea suppressed viremia (-1.4 log viremia reduction) to an extent similar to that observed with dual nucleoside therapy (zidovudine-didanosine or didanosine-stavudine: -1.6 and -1.4 log viremia reduction, respectively). By adding hydroxyurea to stavudine-didanosine a viral load of <400 HIV RNA copies/ml was achieved in 76% of patients after 24 weeks, similar to that reported in studies of triple-therapy regimens that included a protease inhibitor or a non-NRTI (NNRTI). Patients in hydroxyurea-containing arms had the smallest increases in CD4 cell counts, consistent with previous studies. However, the treatment arms did not differ when CD4 cell percentages were compared: CD4 cell percentages increased up to 5% in all arms, independently of the presence of hydroxyurea.
Thus, the efficacy of hydroxyurea was consistently proven in all randomized, controlled trials.
Low doses of hydroxyurea are well tolerated by HIV-infected patients. Bone marrow toxicity is the main side-effect. However, this toxicity is dose-dependent. At the low doses commonly used for HIV infection [1-1.2g daily), bone marrow toxicity has been described in 5-7% of patients [32,34,42]. In most cases, however, the toxicity has been grade 1-2 and has not required dosage adjustment or discontinuation of treatment. Grade 3-4 toxicity has been rare.
In a small (unpublished) study, three out of four patients experienced grade 3 toxicity after treatment with a higher dose of hydroxyurea (500mg three times daily). This pattern of toxicity is typical for this drug, and so was its management. Cell counts decreased during the first few weeks of treatment and returned to normal values a few weeks after the dose was reduced to 500mg twice daily. Special care must be dedicated to patients in advanced stages of the HIV disease. Their bone marrow is, in some cases, deficient, and absolute neutrophil counts (ANC) may be low. Patients with ANC <1700×106 cells/l were at higher risk for hydroxyurea-induced bone marrow toxicity  as were patients with very advanced HIV .
Other toxicities include alopecia, mild gastrointenstinal toxicity (nausea, vomiting), transient elevation of hepatocellular enzymes, fatigue, and, after long-term exposure to the drug, cutaneous toxicity, including mucosal ulcerations . Furthermore, hydroxyurea is regarded as teratogenic drug, and it should not be used during pregnancy .
Most physicians use a 500mg twice-daily schedule. We have been successfully using 400mg three times daily in almost 100 patients. The optimal daily dose and schedule for hydroxyurea in antiretroviral regimens, however, have not been determined. RIGHT 702, a clinical trial beginning in 1999, is designed to answer two questions about hydroxyurea: what is the best daily total dose (600, 900, or 1200mg), and what is the best daily schedule (once, twice, or three times daily).
When to use hydroxyurea
Physicians are rapidly expanding their use of hydroxyurea for HIV infection. Some of this increase results from the popular tactic of adding hydroxyurea to any salvage treatment, at any stage of the infection. This strategy certainly marks a change from the time when clinicians viewed hydroxyurea as a nasty cancer medication. However, randomized, controlled trials enrolled mainly asymptomatic patients. Furthermore, hard clinical trial work must still to be done to determine whether hydroxyurea can be combined as successfully with other drugs as it has been with didanosine and didanosine-stavudine and, for patients with a high viral load or during acute infection, with didanosine and a protease inhibitor.
In my opinion, the best way to exploit the properties of hydroxyurea is to use it as part of a first-line combination regimen. There are several good reasons to position hydroxyurea in the front line. In the absence of a realistic hope for HIV eradication, HIV-infected patients face long-term antiretroviral therapy-as long as the patient‚s life. As the drugs available now tend to generate resistance, maximal viral suppression is required to avoid the emergence of drug-resistant mutants and to ensure long-term efficacy. Unfortunately, complicated dosing schedules and high cost make access to, and compliance with, HAART regimens difficult for most patients. Therefore, viral escape occurs frequently, leaving individuals with multiple and often cross-resistant variants of HIV. Associated toxicity limits the long-term use of these regimens further.
An attractive alternative is to start with drugs better suited for long-term treatment; hydroxyurea has several characteristics of such a drug. Resistance to hydroxyurea has not been described after decades of clinical use ; hydroxyurea compensates for resistance to NRTI adenosine analogs  and may limit the outgrowth of escape mutants [28,29]; some patients with hematological disease have been taking hydroxyurea for years ; hydroxyurea is well tolerated [32,34,42]. Initial treatment with hydroxyurea and didanosine in patients with fewer than 50000 HIV RNA copies/ml-or with hydroxyurea, didanosine, and stavudine in patients with fewer than 100000 copies/ml-would lead to undetectable levels of viremia (<500 copies/ml) in most patients, while sparing use of more potent but also more toxic drugs (protease inhibitors and NNRTI). These other drugs could be used if the initial regimen fails, with a high chance of success, given the lack of cross-resistance between the first-line and second-line drugs.
A preliminary report encourages the use of hydroxyurea in the treatment of primary HIV infection . Plasma viremia decreased to undetectable levels in 11 individuals treated with hydroxyurea, didanosine, and indinavir before complete seroconversion. Viremia remained undetectable (<50 HIV RNA copies/ml) throughout the course of treatment, for up to 17 months. HIV RNA was also undetectable (<400 copies/ml) in the semen of six out of six patients. No HIV RNA was found in 44 million lymph node cells in two out of three individuals by in situ RNA hybridization. In the third patient, HIV RNA could be found in only three out of 44 million lymph node cells.
Of the three reported cases of HIV-infected adults with no viral rebound after discontinuation of therapy, two patients had been treated only with hydroxyurea and didanosine. No viremia rebound occurred in either patient during a 1 year suspension of treatment after the year of combination therapy ; however, proviral DNA could still be detected in small amounts in either lymph node or PBMC. The viral load before treatment, however, was low in both patients (approximately 600-1000 HIV RNA copies/ml).
In a third patient, who was treated with hydroxyurea, didanosine, and the protease inhibitor indinavir early after infection, plasma viremia decreased from 90000 HIV RNA copies/ml to an undetectable level . No rebound could be observed 18 months after treatment stopped, and replication-competent virus could be detected only at very low levels (<0.1 cell carrying replication-competent provirus per 106 cells). There is no proof that hydroxyurea had a specific role in these intriguing results.
A potential role of hydroxyurea and didanosine in prophylaxis of HIV infection after accidental percutaneous exposure has also been suggested .
Much less is known about the use of hydroxyurea in advanced stages of HIV infection. Higher bone marrow toxicity has been reported in patients with ANC <1700×106 cells/l . Severe hematological toxicity with only transient virologic benefits has been described in patients with very advanced HIV infection who were treated with hydroxyurea, didanosine, and lamivudine after a median of seven previous regimens failed .
Adding hydroxyurea to regimens containing didanosine and/or didanosine plus stavudine to reduce viral load further is an interesting option supported by the Canadian and Swiss studies [33,34]. These results are explained by the in vitro observations that hydroxyurea compensates for didanosine resistance and increases phosphorylation of thymidine analogues [53,54]. This issue deserves proper attention in randomized, controlled trials.
A need for affordable treatments
Hydroxyurea is an affordable drug. It is available and already used in most of the countries. More than 90% of people living with HIV will never be able to enjoy standard of care combinations. Health care infrastructures necessary to monitor patients and manage side-effects are well beyond the means of most countries where these people live. Drug costs represent a relevant part of the problem. There is a moral obligation to look for simple and affordable treatments.
The author thanks J. Lisziewicz, scientific Co-Director of RIGHT, and inspiring contributor to the daily work and discoveries on hydroxyurea, and M. Mascolini for editorial assistance.
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