In the past year, hydroxyurea's in vitro promise has been translated into a handful of clinical trials in the United States and Europe. The drug may not be a powerhouse in itself, but researchers say it is especially potent against the human immunodeficiency virus (HIV) in combination with the nucleoside analogue didanosine, also called ddI.
On a broad scale, some researchers hope hydroxyurea will usher in an era of greater focus on cancer drugs as anti-HIV agents. More narrowly, hydroxyurea represents a new approach in combating the virus.
"Drugs developed in the last 10 years primarily target the virus. Instead, we want to target cellular factors so that we can, in effect, starve the virus," explains Franco Lori, MD, PhD, director of a new European laboratory, the Research Institute for Genetic and Human Therapy (RIGHT) in Pavia, Italy.
Lori oversees clinical studies in one of a few sites worldwide where the trials with hydroxyurea are ongoing or planned. The pharmaceutical company Aguettant Sante in Lyon, France, in collaboration with INSERM, France's equivalent of the National Institutes of Health (NIH), is conducting trials. At least two others are scheduled to begin in the United States in the next several weeks.
The Italian trial is based on studies Lori conducted as a visiting associate in the NIH laboratory of Robert Gallo, MD. In a search to discover why HIV-1 does not replicate in quiescent cells, Lori says his hypothesis focusing on the lack of deoxynucleoside triphosphates (dNTPs) in the cells proved correct. "When HIV enters a quiescent cell, there is not enough food to survive."
From previous work with chronic myelogenous leukemia patients, Lori k by the lack of dNTPs more than the cell would be," Lori says. The findings were promising. "We published the first set of data demonstrating that hydroxyurea inhibits viral replication," Lori says (Proc Natl Acad Sci. 1993;90:8915-8918). Subsequent research showed that in vitro anti-HIV activity is enhanced without increased toxicity when hydroxyurea is used with didanosine (Science. 1994;266:801-805).
What's more, Lori believes that hydroxyurea may lessen problems associated with drug resistance. Because hydroxyurea targets a cellular enzyme instead of the virus, no viral mutations occur after repeated exposure to the drug. In combination with didanosine, Lori says, hydroxyurea decreases viral replication so dramatically that, there's less virus available to mutate, possibly delaying the onset of resistance to didanosine.
In Italy, Lori has enrolled 25 asymptomatic HIV-infected patients with T-cell counts of 300 to 500 in two clinical trials assessing the anti-HIV capabilities of hydroxyurea alone and in combination with didanosine and zidovudine, also call AZT. In the first trial, 15 patients are divided into three arms: one receiving 250 mg of zidovudine twice a day, another taking 500 mg of hydroxyurea twice a day, and a third receiving the same doses of both drugs. In the second trial, patients are receiving 299 mg of didanosine twice a day; half also receive 500 mg of hydroxyurea twice a day. Enrollment in the trials is continuing; a total of 100 patients is expected.
Lori says it's too son for efficacy data, but he's optimistic about safety. "We've had no indication of significant toxicity."
Through its AIDS Clinical Trials Group, the National Institute of Allergy and Infectious Diseases (NIAID) is planning trials with hydroxyurea and didanosine to begin this fall.
"If hydroxyurea acts according to theory, it will permit a great reduction in the toxicity associated with ddI, since we would be able to combine the two at low doses," says Lawrence Fox, MD, PhD, a medical officer in NIAID's HIV research branch. But Fox says he's skeptical about hydroxyurea use with the other nucleoside analogues, particularly zidovudine, because of overlapping bone marrow toxicities.
In early reports from the French study, Fox says there appear to be therapeutic benefits, no serious adverse effects, and a drop in viral load greater than that generally associated with didanosine alone.
The Shared Medical Research Foundation of Tarzana, Calif, plans this week to start a controlled trial of hydroxyurea with didanosine and stavudine, another nucleoside analogue called d4T.
"The data in vitro are excellent," says Jeffrey Galpin, MD, chair of the community-based clinical trials organization. "The problem with this drug is that it's available; a lot of [HIV- infected] people have started to use it. So we need to know the pharmacokinetics. There may be an incredible effect when the bone marrow is in good shape, but it could be harmful later when there are bone marrow problems."
Hydroxyurea may not be in the same viral load--dropping class as protease inhibitors, but Galpin says its capabilities appear to be substantial. "We need to know more. Dropping viral load significantly can allow the immune system to be reborn; it can decrease the effect of the immune system on apoptosis." In fact, he adds, potential benefits from hydroxyurea "should open up a whole new field in looking at a log of cancer drugs" as anti-HIV therapies.
Another aspect of the HIV-related hydroxyurea research is that of heightened international collaboration. Lori's research at RIGHT has a US counterpart that began operations last month in Gaithersburg, MD. RIGHT;s US director is Julianna Lisziewicz, PhD, a colleague of Lori's for several years in Gallo's laboratory.
"We have the same goal," Lisziewicz says of the US and Italian facilities. "We want to develop new therapies, mainly gene therapy, to treat diseases that are life threatening. For the first couple of years we will concentrate on AIDS."
The laboratories are backed with corporate and individual funding, in addition to $9 million form the Italian government. Now operating with 18 scientists, RIGHT hopes to expand to 54 researchers, as well as become the central element of an international network operating in the United states, Europe, Canada, and Australia.
"We want to translate basic research into clinical trials. We hope to be working very closely with hospitals to implement clinically the findings from basic research," says Lisziewicz. "This is what's missing.
AU - Torres G TI - Hydroxyurea, a potential new anti-HIV agent. AB - Published evidence from the U.S. and Europe supports the effect of the anticancer drug hydroxyurea against HIV-1, and its potential synergistic effect with ddl. Approved as an oral chemotherapeutic agent for leukemia and other cancers, hydroxyurea acts as a radical free quencher, inhibiting the cellular enzyme ribonucleotide reductase as well as cellular DNA synthesis during the S phase of the cell division, and it may be through this mechanism that hydroxyurea inhibits tumor cell growth. French scientists at the Centre Leon Bernard in Lyon tested hydroxyurea and a related compound, D-aspartic acid beta-hydroxamate alone and in combination with AZT, ddl, or ddC. Total suppression of viral production and total production against the toxic effects induced by viral replication were demonstrated using the combination of either of the two hydroxamates and ddl after 14 days. In test tube experiments conducted at the National Cancer Institute, both hydroxyurea and ddl inhibited or delayed HIV replication in a dose-dependent manner; in combination, they blocked HIV replication by more than 99.9 percent. SO - Treat Issues. 1995 Jan;9(1):7-9.
AU - Lori F ; Malykh A ; Cara A ; Sun D ; Weinstein JN ; Lisziewicz J ; Gallo RC TI - Hydroxyurea as an inhibitor of human immunodeficiency virus-type 1 replication. AB - Hydroxyurea, a drug widely used in therapy of several human diseases, inhibits deoxynucleotide synthesis--and, consequently, DNA synthesis--by blocking the cellular enzyme ribonucleotide reductase. Hydroxyurea inhibits human immunodeficiency virus-type 1 (HIV-1) DNA synthesis in activated peripheral blood lymphocytes by decreasing the amount of intracellular deoxynucleotides, thus suggesting that this drug has an antiviral effect. Hydroxyurea has now been shown to block HIV-1 replication in acutely infected primary human lymphocytes (quiescent and activated) and macrophages, as well as in blood cells infected in vivo obtained from individuals with acquired immunodeficiency syndrome (AIDS). The antiviral effect was achieved at nontoxic doses of hydroxyurea, lower than those currently used in human therapy. Combination of hydroxyurea with the nucleoside analog didanosine (2',3'- dideoxyinosine, or ddl) generated a synergistic inhibitory effect without increasing toxicity. In some instances, inhibition of HIV-1 by hydroxyurea was irreversible, even several weeks after suspension of drug treatment. The indirect inhibition of HIV-1 by hydroxyurea is not expected to generate high rates of escape mutants. Hydroxyurea therefore appears to be a possible candidate for AIDS therapy. SO - Science. 1994 Nov 4;266(5186):801-5.
AU - Malley SD ; Grange JM ; Hamedi-Sangsari F ; Vila JR TI - Synergistic anti-human immunodeficiency virus type 1 effect of hydroxamate compounds with 2',3'-dideoxyinosine in infected resting human lymphocytes. AB - The cellular models generally used in the in vitro evaluation of anti-human immunodeficiency virus compounds are dividing cells. A model constituted by resting lymphocytes may more accurately reflect a drug's future efficacy in humans, since viral DNA synthesis is known to take place in quiescent cells, creating a reservoir of infected cells awaiting activation to complete their viral replication cycle and to produce infectious virions. We report here the activity of 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and two hydroxamates, D-aspartic acid beta-hydroxamate and hydroxycarbamate (hydroxyurea), alone and in various combinations, in an in vitro model based on resting lymphocytes. In our model, resting peripheral blood lymphocytes were infected with human immunodeficiency virus type 1 and treated with drugs for 7 days, at which time drugs were removed and the cells were activated by phytohemagglutinin. We show that under these conditions 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, and 2',3'-dideoxycytidine, alone or in combination, neither fully inhibit viral production nor protect lymphocytes from the cytopathic effect of viral replication, at concentrations corresponding to the peak plasma levels observed in a typical treatment schedule in humans. In contrast, we report the synergistic effect of treatment by each hydroxamate with 2',3'- dideoxyinosine of infected resting lymphocytes, resulting in the total suppression of viral production, total protection against the cytopathic effect induced by viral replication, and no effect on the ability of the cells to replicate in this cell culture system. SO - Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11017-21.
AU - Gao WY ; Johns DG ; Mitsuya H TI - Anti-human immunodeficiency virus type 1 activity of hydroxyurea in combination with 2',3'-dideoxynucleosides. AB - The effects of hydroxyurea (HU), an inhibitor of ribonucleotide reductase, on the replication of human immunodeficiency virus type 1 (HIV-1) in activated peripheral blood mononuclear cells were studied. The inhibition of HIV-1 replication by HU alone was dose dependent, with a 90% inhibitory concentration of 0.4 mM, a plasma concentration tolerated by patients with oncological diseases. HU at lower concentrations (< 0.1 mM) was found to potentiate the antiviral activity of 2',3'-dideoxyinosine (ddl), 3'-azido-2',3'- dideoxythymidine, and 2',3'-dideoxycytidine against HIV-1, with the potentiation being ddl greater than 3'-azido-2',3'- dideoxythymidine = 2',3'-dideoxycytidine. In the presence of 0.1 mM HU, the 90% inhibitory concentration of ddl was reduced by 6-fold in activated peripheral blood mononuclear cells. The potentiating effect of HU on ddl action was time dependent, with the greatest inhibition of HIV-1 growth being seen when HU was present during and after virus adsorption, i.e., apparently coinciding with the time of proviral DNA synthesis. A brief incubation of activated cells with HU and ddl at low concentrations before virus exposure reduced p24 production by > 50%. Analyses using high performance liquid chromatography and enzymatic assays suggested that the greater degree of potentiation by HU of the action of ddl, compared with the other dideoxynucleosides, is due to the more effective inhibition by HU of dATP synthesis, compared with the synthesis of the other deoxynucleoside triphosphates (dGTP, dTTP, and dCTP). The present study suggests that, for appropriate agents, pharmacological reduction of deoxynucleoside triphosphate levels represents a potential therapeutic approach for inhibition of HIV-1 replication. SO - Mol Pharmacol. 1994 Oct;46(4):767-72.
AU - Bianchi V ; Borella S ; Calderazzo F ; Ferraro P ; Chieco Bianchi L ; Reichard P TI - Inhibition of ribonucleotide reductase by 2'-substituted deoxycytidine analogs: possible application in AIDS treatment. AB - After phosphorylation to the corresponding diphosphates, 2'-azido-2'-deoxycytidine and 2'-difluorocytidine act as powerful inhibitors of ribonucleotide reductase. Phosphorylation requires deoxycytidine kinase, an enzyme with particularly high activity in lymphoid cells. Therefore, the deoxycytidine analogs can be expected to inhibit the reductase with some specificity for the lymphoid system. Pretreatment of human CEM lymphoblasts with the analogs considerably increased the phosphorylation of 3'-deoxy-3'- azidothymidine (AzT). The increased phosphorylation of AzT is caused by a prolongation of the S phase of the cell cycle. Our results suggest the possibility of a combination of 2'-substituted deoxycytidine analogs with AzT in the treatment of AIDS. Gao et al. [Gao, W.-Y., Cara, A., Gallo, R. C. & Lori, F. (1993) Proc. Natl. Acad. Sci. USA 90, 8925-8928] have suggested the use of the ribonucleotide reductase inhibitor hydroxyurea for this purpose, since the resulting decrease in the size of deoxyribonucleotide pools decreases the processivity of the HIV reverse transcriptase. From our results it would appear that the 2'-substituted deoxycytidine analogs might be preferable to hydroxyurea. SO - Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8403-7.
AU - Chamberlain MC TI - Long survival in patients with acquired immune deficiency syndrome-related primary central nervous system lymphoma. AB - BACKGROUND. Primary central nervous system lymphoma (PCNSL) is the most common brain tumor occurring in patients with acquired immune deficiency syndrome (AIDS). After diagnosis of PCNSL, the median survival time is 2-5 months with treatment with whole brain irradiation (WBI). METHODS. Four (of approximately 40) patients with AIDS and PCNSL seen by the University of California, San Diego (UCSD) Neuro-Oncology service were treated with multimodal therapy, including WBI with hydroxyurea, followed by 3 cycles of procarbazine/lomustine/vincristine (PCV) chemotherapy. RESULTS. Survival after tumor diagnosis ranged from 11 to 16 months, with a median of 13.5 months. CONCLUSION. Selected patients with AIDS and PCNSL may have long survival when treated with multimodal therapy. SO - Cancer. 1994 Mar 15;73(6):1728-30.
AU - Gao WY ; Cara A ; Gallo RC ; Lori F TI - Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. AB - Human immunodeficiency virus type 1 (HIV-1) viral DNA synthesis in quiescent and activated peripheral blood lymphocytes (PBLs) was studied. Incomplete viral DNA (previously demonstrated to be associated with HIV-1 virions) is carried by HIV-1 virions into quiescent and activated PBLs, contributing to the formation of an early viral DNA pool in these cells. The viral DNA is subsequently completed but only extremely slowly and inefficiently in quiescent PBLs compared to that in stimulated PBLs. We find that this correlates with significantly lower levels of dNTP substrates in quiescent compared to activated PBLs. At these low dNTP concentrations, HIV-1 reverse transcriptase acts in a partially distributive manner. Increasing dNTP concentrations from the levels of quiescent PBLs to the levels of activated PBLs increases the processive action of reverse transcriptase, which in turn stimulates rapid and efficient formation of full-length DNA. Furthermore, hydroxyurea treatment of stimulated PBLs decreases the dNTP levels and the DNA synthesis rate to levels comparable to quiescent PBLs. Our data therefore indicate that low levels of dNTP may explain why HIV-1 DNA is synthesized slowly and inefficiently in quiescent PBLs and suggest that pharmacologic induction of low dNTP levels represents a therapeutic approach for inhibition of HIV-1 replication. SO - Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8925-8.
AU - Getman DP ; DeCrescenzo GA ; Heintz RM ; Reed KL ; Talley JJ ; Bryant ML ; Clare M ; Houseman KA ; Marr JJ ; Mueller RA ; et al TI - Discovery of a novel class of potent HIV-1 protease inhibitors containing the (R)-(hydroxyethyl)urea isostere. SO - J Med Chem. 1993 Jan 22;36(2):288-91.
AU - Lori F ; Malykh A ; Cara A ; Sun D ; Lisziewicz J ; Gallo RC TI - Hydroxyurea as a novel potent inhibitor of HIV-1 replication. AB - Hydroxyurea, a drug widely used in therapy of several human diseases, inhibits deoxynucleotides (dNTP) synthesis by blocking the cellular enzyme ribonucleotide reductase, and acts as a cytostatic drug. We have recently demonstrated that hydroxyurea inhibits human immunodeficiency virus type 1 (HIV-1) DNA synthesis in activated peripheral blood lymphocytes by decreasing the amount of intracellular dNTP (Gao et al. PNAS 90, 8925-8928), thus suggesting a potential antiviral effect of this drug. Here we demonstrate that hydroxyurea blocked HIV-1 replication in a variety of acutely infected primary human cells (quiescent and activated lymphocytes/macrophages) as well as in in vivo infected cells obtained from acquired immunodeficiency syndrome (AIDS) patients. The antiviral effect was achieved by using non-toxic hydroxyurea doses, lower than those currently used in human therapy. Furthermore, we demonstrated that combination of hydroxyurea with either Zidovudine or Didanosine generated a strong synergistic inhibitory effect without increasing toxicity. Hydroxyurea therefore appears as a novel, potent and safe candidate for AIDS therapy. SO - Int Conf AIDS. 1994 Aug 7-12;10(1):8 (abstract no. 007B).
AU - Meyerhans A ; Vartanian JP ; Hultgren C ; Eriksson S ; Wain-Hobson S TI - The intracellular nucleotide pool affects HIV replication. AB - OBJECTIVE: To ascertain the impact of intracellular nucleotide pool changes on HIV-1 replication ex vivo. METHODS: PHA-stimulated peripheral blood mononuclear cells (PBMC) from seronegative blood donors were incubated with HIV-1 Lai primary isolate at 4 degrees C for 30 min. After shifting to 37 degrees C for 15 min 0 to 10mM hydroxyurea was added. Deoxyadenosine, known to reverse the effect of hydroxyurea, was added in concentrations of 0 to 1 mM. After 5 hours cells were extensively washed and cultured for up to 3 weeks under standard culture conditions. Reverse transcriptase was tested every 3 days. Cell viability was not altered by hydroxyurea or deoxyadenosine treatment. RESULTS: The effect of hydroxyurea was dose dependent. Greater than 90% inhibition of virus replication could be achieved by 10mM hydroxyurea. Inhibition was partially reversed by deoxyadenosine, which on its own also inhibited HIV replication. DISCUSSION AND CONCLUSIONS: Hydroxyurea may substantially reduce HIV-1 replication in a dose dependent manner. The concentrations used make it an impractical drug for anything other than ex vivo studies. The reversal by deoxyadenosine suggest that HIV-1 replication is sensitive to alterations in the nucleotide pools of the cell however other targets cannot be ruled out. The nature of this effect is under investigation. SO - Int Conf AIDS. 1992 Jul 19-24;8(2):A22 (abstract no. PoA 2118).
AU - Nusbaum NJ ; Joseph P TI - Effect of simultaneous AZT and hydroxyurea (HU): study in an HL-60 model system (Meeting abstract). AB - Patients (pts) with AIDS-related neoplasms may be considered for treatment with AZT, other antiretroviral agents, therapy for opportunistic infections, and/or cancer chemotherapy. These multiple medications are often poorly tolerated in this pt population, and so there is often interest in trying gentler chemotherapy than one might use in a similar cancer pt without HIV infection. It is suggested that, as protocols are designed that may expose AIDS pts simultaneously to multiple drugs that can affect DNA synthesis, the possibility of drug-drug interactions be carefully considered. In this regard, the current study explored the effect on HL-60 cells of exposure to AZT and/or HU, followed by resuspension of the cells in drug-free media. On the basis of wt, HU was considerably more toxic than AZT for the HL-60 line. As an example, 72-hr exposure to HU 0.1 mg/ml caused a marked drop in viable cell count, but with brisk growth resuming within a week in drug-free media. A combination of 0.1 mg/ml AZT and 0.1 mg/ml HU was only slightly more toxic than the single-agent HU. A single experiment with 48-hr drug exposure had suggested that there might be an element of negative synergy between the two drugs, but this isolated observation was refuted by multiple other experiments. SO Proc Annu Meet Am Soc Clin Oncol; 12:A15 1993.
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