Ross C. Donehower
Bruce A. Chabner
Hydroxyurea (HU), one of the simplest of the anticancer drugs, has won a supportive role in the treatment of myeloproliferative disease because of its ability to suppress proliferation of myeloid, erythroid, and platelet precursors, but its value is limited by its failure to induce bone marrow remission, and the equally rapid reversibility of its effect. However, it has other notable clinical properties, including the ability to induce β-globin synthesis in patients with sickle cell anemia and thalassemia. It has been an invaluable probe for the laboratory study of its intracellular target, ribonucleotide reductase (RR), the rate-limiting step in the de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). Other actions, including the generation of nitroxyl radicals and radiosensitizing effects, have potential clinical applications. The key features of this drug are shown in Table 10.1.
HU (Fig. 10.1) was originally synthesized in Germany in 1860,1 and was found to have inhibitory effects on granulocyte production.2 Following evaluation in the National Cancer Institute's screening system, in which it displayed myelosuppressive and antileukemic activity,3, 4 it entered clinical trials in the 1960s, and was soon recognized as a potent myelosuppressive agent with a novel mechanism of action and few side effects, properties that have earned it a limited role in cancer chemotherapy. Other inhibitors of RR have since been evaluated in the clinic, including compounds of the thiosemicarbazone series and guanazole,5, 6 but have no special therapeutic advantage and greater toxicity. The principle use of HU at present is in controlling lineage proliferation in chronic myelogenous leukemia (CML), polycythemia vera (PV), and essential thrombocythemia (ET). It has little ability to induce bone marrow remission, but provides excellent control of and prevents complications of high peripheral blood counts.7
HU was once a primary agent with interferon-a in first-line therapy against CML,8, 9, 10 but has been largely replaced by the targeted agent, Gleevec, and it now used primarily for acute control of white cell count at presentation or during blastic transformation. In PV, it effectively prevents thrombosis resulting from elevated hematocrit and high platelet count,11 and it similarly lessens the incidence of thrombosis in ET in patients with platelet counts above 1.5 million.12 Because both PV and ET are chronic, slowly progressive diseases, there is concern that HU may increase the risk of leukemic conversion, a risk that has not been substantiated thus far.13, 14 In younger patients with PV, who have the prospect of long-term treatment, prophylactic phlebotomy may be favored, and in patients with ET, anagrolide and interferon- are alternatives to HU.
A major current use of HU is in the prevention of complications of sickle cell anemia. In patients with sickle cell anemia, HU increases the production of fetal hemoglobin, ameliorates symptoms, and reduces the incidence of painful crisis and hospitalization.15, 16 In vitro incubation of HU with erythroid progenitors induces fetal hemoglobin b-globin production.17 Whether the induction of fetal hemoglobin represents a response to inhibition of DNA synthesis in red cell progenitors or a specific alteration of g-globin gene transcription is uncertain.17 Recent evidence suggests that nitroxy radicals produced by decomposition of HU may directly stimulate g-globin gene transcription.18 Convincing evidence now exists that induction of fetal hemoglobin is not the only, and perhaps not the major, contributor to the drug's efficacy. The benefit from HU may be partly from its ability to suppress both erythropoiesis and myelopoiesis, and its effects on red cell adhesion to vessel walls.19 A marked decrease in the endothelial adhesion of a patient's red blood cells is observed after 2 weeks of HU therapy, coincident with a decrease in absolute reticulocyte levels, but before fetal hemoglobin levels start to rise. A strong inverse correlation between neutrophil count and crisis rate has also been noticed.20 In terms of clinical efficacy, a randomized, double-blind study has demonstrated that long-term treatment with HU decreases the incidence of painful crisis by 44% in adult patients with sickle cell disease.21 HU treatment also reduced the frequency of acute chest syndrome and hospitalization, and also reduced the need for blood transfusion. These results establish HU as the first clinically acceptable drug shown to decrease crises in sickle cell disease. Studies have shown that HU appears to be as effective in children with sickle cell disease22 and in patients with sickle cell–b-thalassemia and sickle cell–hemoglobin C disease,23 although only a small number of patients have been treated and the studies were uncontrolled.24
TABLE 10.1 KEY FEATURES OF HYDROXYUREA
HU may serve as an important model for agents that contribute to inhibition of the replication of HIV by a mechanism other than targeting of a viral enzyme or a structural protein. The ability of HU to decrease intracellular levels of dNTPs first led Lori25 to propose using the compound to inhibit HIV replication. Subsequent studies have confirmed this effect and have focused on the anti-HIV mechanism of action of HU and its synergistic interactions with other antiretroviral compounds, particularly the nucleoside reverse transcriptase inhibitors such as didanosine.26 Currently available clinical data, including those from several uncontrolled studies and four randomized trials, reveal that HU has little activity as a single agent, but produces a pronounced inhibition of HIV replication when combined with didanosine or with didanosine plus stavudine in patients who have not been heavily pretreated. Importantly, HU appears to maintain the activity of the nucleoside reverse transcriptase inhibitors even in the presence of genotypic mutations of HIV that is characteristically associated with resistance.26
Figure 10.1 Structure of hydroxyurea.
The anti-HIV effect of HU is not consistently accompanied by an increase in the CD4+ lymphocyte count. The lack of such an increase has been attributed to the cytostatic activity of the drug and has uncertain clinical relevance. The antiviral activity of HU is induced at low doses, typically 1,000 mg/day orally, that cause minimal toxicity. Questions about the role of the compound in the treatment of HIV infection that remain to be answered include its effectiveness in boosting immune function, the most appropriate dosage regimen, the timing of individual doses, its role in salvage therapy, and the risk associated with long-term use.
MECHANISM OF ACTION AND CELLULAR PHARMACOLOGY
The primary site of cytotoxic action for HU is inhibition of the RR enzyme system. This highly regulated enzyme system is responsible for the conversion of ribonucleotide diphosphates to the deoxyribonucleotide form, which can subsequently be used in either de novo DNA synthesis or DNA repair.27 HU can be shown to inhibit RR in vitro,28 and the extent of inhibition of DNA synthesis observed in HU-treated cells correlates closely with the size of the decreased deoxyribonucleotide pools.29 This enzyme has an important role as a rate-limiting reaction in the regulation of DNA synthesis. In human and other mammalian cells, this unique enzyme consists of two different subunits, usually referred to as M-1 and M-2.30 Protein M-1 is a dimer with a molecular weight of 170 kd and contains the binding site for the substrates as well as the allosteric effector sites.30, 31 This subunit is responsible for the complex regulation of the enzyme by cellular nucleotide pools. Although considerable variability exists among enzymes from various tissue sources, the general regulatory effects are summarized in Table 10.2.31 The reduction of all substrates is inhibited and the enzyme complex dissociates in the presence of deoxyadenosine triphosphate.32Protein M-1 is present at a relatively constant level throughout the cell cycle, except in cells in G0 or those that have undergone terminal differentiation, in which it is markedly decreased.33 The gene coding for this protein can be mapped to chromosome 11.33 Protein M-2 is the catalytic subunit of the enzyme and exists as a dimer with a molecular weight of 88 kd. This unique protein contains stoichiometric amounts of iron and a stable organic free radical localized to a tyrosine residue. The fully conserved tyrosyl radical is essential to enzyme activity, and is localized in proximity to and stabilized by the binuclear nonheme iron complex.34, 35 The cellular concentration of M-2 protein is variable throughout the cell cycle; it peaks in S phase, which suggests that functional enzyme activity depends on the concentration of M-2 protein.36 The M-2 subunit sequences have been mapped to chromosome 2 in human cells and seem to be in the same amplification unit as the gene for ornithine decarboxylase.
TABLE 10.2 REGULATORY EFFECTS OF NUCLEOTIDE TRIPHOSPHATE ON RIBONUCLEOTIDE REDUCTASE
HU enters cells by passive diffusion. The inhibition of RR occurs as a result of inactivation of the tyrosyl free radical on the M-2 subunit, with disruption of the enzyme's iron-binding center.37 The fact that this inhibition can be partially reversed in vitro by ferrous iron and that cytotoxicity can be enhanced by iron-chelating agents38 emphasizes the importance of the nonheme iron cofactor in this process. HU selectively kills cells in S phase, and within an S-phase population of cells, those that are most rapidly synthesizing DNA are most sensitive.39 The cytotoxic effects of HU correlate with dose or concentration achieved, as well as with duration of drug exposure.40 Following HU exposure, cells progress normally through the cell cycle until they reach the G1-S interface. Rather than being prevented from entering S phase, as was once thought, cells enter S phase at a normal rate but are accumulated there as a result of the inhibition of DNA synthesis.41 Cells undergo apoptosis in a process mediated by both p53 and non-p53 pathways.
Jiang et al.42 have demonstrated that HU may be transformed in vivo to nitric oxide (NO), which is a known RR inhibitor. The possibility therefore exists that the RR inhibition observed after HU exposure may be both direct and mediated through the NO metabolite. Indeed, HU-borne NO may be the intermediate effector in other actions of the drug, such as its induction of fetal hemoglobin.18
Several of the enzymes involved in DNA polymerization and DNA precursor synthesis are assembled in a replitase complex during S phase of the cell cycle to channel metabolites to enzymes sequentially during the synthetic process.43 Replitase contains DNA polymerases, thymidine kinase, dihydrofolate reductase, nucleoside-5′ phosphate kinase, thymidylate synthase, and RR. Cross-inhibition is a phenomenon observed with enzymes of the replitase complex, in which inhibition of one enzyme in the complex leads to inhibition of a second, unrelated enzyme. This occurs only in intact cells and only in S phase. Evidence suggests that this is the result of a direct allosteric, structural interaction from a remote site within the complex because disruptions of deoxyribonucleotide pools do not explain the findings.44 HU appears to be able to inhibit DNA polymerases, thymidylate synthase, and thymidine kinase by this mechanism under certain conditions.
A potentially important consequence of HU action is the acceleration of the loss of extrachromosomally amplified genes that are present in double-minute chromosomes.45 Evidence indicates that such acentric extrachromosomal elements are common in the gene amplification process. Exposure to HU at clinically achievable concentrations leads to enhanced loss of both amplified oncogenes and drug-resistance genes.46 Strategies for use of this phenomenon clinically are under consideration.
MECHANISMS OF CELLULAR RESISTANCE
The principal mechanism by which cells achieve resistance to HU is elevation in cellular RR activity. As previously noted, the cellular levels of the M-1 subunit do not change during the cell cycle, whereas levels of the M-2 catalytic subunit increase during DNA synthesis. The site of action of HU specifically involves the M-2 subunit, and the increased RR activity seen in resistant cells is principally the result of overexpression of this protein.47 Transfection of the human M-2 gene into drug-sensitive KB cells confers resistance by increasing the enzyme activity, and subsequently the dNTP intracellular pools.48 Transfection of the M-1 gene does not result in a decreased sensitivity to HU, although transfected cells resist dNTP inhibition of RR activity, probably because of an alteration of the function of effector binding sites. Several different molecular mechanisms can contribute to the increased RR activity in HU-resistant cells. A number of cell lines have amplifications of the gene coding for M-2 protein accompanied by an elevation in M-2 messenger RNA and M-2 protein levels.49 It also seems that posttranscriptional modifications, such as an increase in initiation factor 4E, can occur during drug selection, which results in an increased translational efficiency. An increase in M-2 protein biosynthetic rate can then occur with no further increase in messenger RNA levels.50
In most studies, HU resistance has been associated with parallel decreased sensitivity to other RR inhibitors and often to other antimetabolites.51Interestingly, some inhibitors of the M-2 subunit, including the new compound Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, or 3-AP), retain their antitumor effect in HU-resistant cell lines.5 In addition, some of these cell lines with increased RR activity display an increased sensitivity to nucleotide analogs such as 6-thioguanine (via increased conversion to the deoxynucleotide and enhancement of its incorporation into DNA)52 or gemcitabine (via increased drug uptake by the cells).53
HU has been studied both in the laboratory and in the clinic as a modulator of cytosine arabinoside (AraC) metabolism and cytotoxicity. It causes a significant increase in formation of arabinosylcytosine triphosphate and AraC incorporation into DNA54 in HU-treated cells. The assumption was that this was the result of the decreased pools of the competitor, deoxycytidine triphosphate, expected after HU exposure. In some cell lines, deoxycytidine levels fall as well, increasing AraC conversion to ara-CMP.54 The phosphorylation of nucleoside analogs such as gemcitabine, fludarabine, and cladribine also increases in the presence of HU. No randomized clinical trial has purely assessed the contribution of HU to combination therapy with AraC. A controlled phase 3 trial, however, has shown the superiority of interferon-a and HU plus AraC over interferon-a and HU in patients with newly diagnosed CML.55 HU also has been shown to increase the toxicity of fludarabine in a small clinical pilot study.
The major clinical interest in HU in the treatment of solid tumors has been in combination with 5-fluorouracil. Synergy has been demonstrated in experimental tumor models, presumably based on the ability of HU to lower cellular pools of deoxyuridine monophosphate, the competitive substrate for inhibition of thymidylate synthase by 5-fluorodeoxyuridylate.56 A number of clinical trials of this combination have been performed, but its role remains uncertain. These two G1-S arresting agents, 5-fluorouracil and HU, have been shown in vitro to interfere with the cytotoxic effects of antimitotic agents (vinblastine sulfate, colchicine, nocodazole) that produce mitotic arrest and apoptosis.57 The antimetabolites perturb the ability of the antimitotic drugs to induce bcl-2 phosphorylation and c-raf-1 activation, and to increase the p21WAF1/CIP1 protein levels, and prevents the majority of cells from progressing to the G2-M phase.
HU has been evaluated in both clinical and laboratory studies in combination with chemotherapy agents that produce DNA damage, such as alkylating agents, cisplatin, and inhibitors of topoisomerase II.56 Although synergy has been observed in preclinical testing, the clinical role for such combinations remains speculative. Synchronization in the G1-S phase drives cells to a condition of increased sensitivity to radiation. Besides, HU exerts a radiosensitizing action through other mechanisms because it selectively kills cells in the S phase of the cell cycle and significantly affects DNA repair mechanisms after radiation damage.
HU is generally administered orally, and doses are titrated in response to changes in peripheral white blood cell counts. Although significant interpatient variability is observed, peak concentrations of 0.1 to 2.0 mmol/L are achieved 1.0 to 1.5 hours after doses of 15 to 80 mg/kg.58 Oral bioavailability is excellent (80 to 100%), and comparable plasma concentrations are seen after oral and intravenous dosing.58, 59, 60, 61 (Table 10.3) After attainment of peak plasma concentrations, HU disappears rapidly from plasma. The elimination half-life ranges from 3.5 to 4.5 hours.60 Data available from a comprehensive population pharmacokinetic study of multiple oral and intravenous dosing are best described by a one-compartment model with parallel Michaelis-Menten metabolism and first-order renal excretion.60, 61 Renal clearance at standard doses is 60 to 90 mL/minute in an average patient, and pharmacokinetics are nonlinear with dose. The volume of distribution is described by the formula 0.186 (body weight) 1 25.4 L. Other studies using a unique dose level (1,000 or 2,000 mg daily) described a correct fit by a one- or two-compartment linear model.58
Several high-dose 24- to 120-hour continuous-infusion regimens for HU administration, with or without initial loading, have been evaluated.66, 67 Continuous infusion of 1 g/hr for 24 hours is capable of sustaining plasma concentrations in excess of 1 mmol/L. Doses of 0.5 g/m2 per day were tolerated for 12 weeks, 1 g/m2 per day was tolerated for 5 weeks, 1.66 g/m2 per day was tolerated for 3 weeks, and 2.5 g/m2 per day was tolerated for 1 week.68 Based on the available data, from the standpoint of pharmacokinetics and bioavailability, administering HU parenterally has no clear advantages, except in those patients with impaired gastrointestinal function.
TABLE 10.3 SUMMARY OF PHARMACOKINETIC PARAMETERS OF HYDROXYUREA
Although precise guidelines are not available, the prudent course is to modify dosages for patients with abnormal renal function until individual tolerance can be assessed. Unfortunately, pharmacokinetic studies of patients with altered renal function have not been performed to provide guidelines. The full extent and significance of HU metabolism in humans has not been established. Data from several experimental animal systems suggest that the metabolism of HU does occur, but none of these conversions has been demonstrated conclusively in humans. HU is degraded by urease, an enzyme found in intestinal bacteria.69Hydroxylamine (NH2OH), a product of this reaction, has not been identified in humans. Acetohydroxamic acid is found in the plasma of patients receiving HU therapy,70 however, and may represent the product of a reaction between hydroxylamine and acetylcoenzyme A, a major thioester in mammalian tissues. The conversion of HU to urea in mice has also been reported.71 An enzyme system capable of this conversion is found in mouse liver, with the greatest activity localized in the mitochondrial subcellular fraction. Similar activity has not been demonstrated in human liver.
HU distributes rapidly to tissues. Studies using radiolabeled HU in rats and mice demonstrate that the drug is found in body tissues in quantities proportional to weight 30 to 60 minutes after injection.72 The drug readily enters cerebrospinal fluid and third-space collections of fluid such as ascites or pleural effusions. Ratios for simultaneous plasma and cerebrospinal fluid concentrations of 4:1 to 9:1 and for plasma and ascites concentrations of 2:1 to 7.5:1 have been observed.73 The significance of these ratios is uncertain because they were single points taken at arbitrary times after drug administration, and the time course of disappearance from these extravascular sites was not evaluated.
The dose-limiting toxicity of HU is myelosuppression.61, 66 This is the direct result of inhibition of DNA synthesis in bone marrow, and megaloblastic changes can be detected in granulocyte and erythroid precursors within 48 hours of the first dose. In patients with nonhematologic malignancies, the peripheral white blood cell count begins to fall in 2 to 5 days. Patients with leukemia or myeloproliferative syndromes have a more rapid fall in white blood cell counts. The rapidity of the effect on the circulating leukemia cell population and the brief duration of its action have been the basis for the use of HU in patients with acute myelogenous leukemia who present with markedly elevated peripheral blood blast counts, or in patients with dangerously elevated platelet counts, as in cases of essential thrombocythemia. Whether this provides an advantage over the prompt institution of standard AraC–containing leukemia therapy has not been demonstrated conclusively.
All patients on dosages of 80 mg/kg per day become leukopenic within 14 days, whereas the incidence is 70% for patients receiving half that dose. Intermittent dosing with the higher doses decreases the hematologic toxicity, but the impact on therapeutic effect has not been fully evaluated. Reversal of the HU effect on peripheral white blood cell counts occurs rapidly, but the nadir in platelet count may occur 7 to 10 days later. Treatment of the myeloproliferative syndromes usually begins with much lower dosages of 0.5 to 2.0 g/day, which are titrated to the clinical response.
The gastrointestinal side effects of nausea, vomiting, anorexia, and either diarrhea or constipation rarely require discontinuation of therapy at the dosages that are commonly used clinically. Oral mucositis and ulceration of the gastrointestinal tract are less common, but may be higher in patients receiving concomitant radiation and HU than in those receiving either therapy alone.
Patients who have taken HU for an extended period may develop one of several dermatologic changes, including hyperpigmentation, erythema of the face and hands, a more diffuse maculopapular rash, or dry skin with atrophy.74 Changes in the nails may include atrophy or the formation of multiple pigmented nail bands. More severe skin reactions include an ulcerative dermatitis resembling lichen planus.75 Skin ulcerations, usually on the legs, may occur in patients undergoing long-term treatment for myeloproliferative diseases.76 Healing or improvement of these ulcers requires cessation of treatment. Topical GM-CSF reportedly accelerates healing.77 When concomitant radiation therapy is given, patients receiving HU seem to have an increased tissue reaction and may have a recurrence or “recall” of erythema or hyperpigmentation in previously irradiated areas.78 Alopecia has been seen rarely.
A number of other, less frequent, drug-related effects have been either reported anecdotally or mentioned briefly in the clinical studies already discussed. Transient abnormalities of renal function have been noted in a number of studies, and include elevations of serum urea nitrogen and creatinine, proteinuria, and an active urine sediment. Renal failure or severe, prolonged periods of kidney dysfunction have not been reported. Liver function test abnormalities, on the other hand, have been more significant, and occasionally the patient has progressed to clinical jaundice. A more typical pattern has been transient elevation of hepatocellular enzymes.79 Headache, drowsiness, confusion, and dizziness also have been reported but are of uncertain significance. The frequency of sensorial neuropathy, rarely seen with single-agent HU therapy, is significant in regimens combining HU with didanosine, stavudine, or both.80Several cases of acute interstitial lung disease and alveolitis have been reported.81 Drug-induced fever has also been noted.82
Because of the mechanism of action of HU, of particular concern are the effects on growth and development and its mutagenic potential (teratogenic and carcinogenic), especially in patients with nonmalignant diseases, who frequently need long-term drug administration. Although the number of studies is limited and the median follow-up is short, no growth failures or chronic organ damage has been observed in children with sickle cell disease who are treated with HU. A single study has demonstrated an increased incidence of chromosomal abnormalities in patients treated with HU.83 HU treatment, especially when HU is combined with other agents, is associated with an incidence of leukemia of 3 to 12% in patients with myeloproliferative syndromes. This risk is possibly higher than in patients not treated with cytotoxic drugs and appears to be proportional to the intrinsic leukemogenic risk of the underlying condition (higher in polycythemia vera [PV] than in essential thrombocythemia [ET]). To date, no secondary leukemia has occurred in patients with sickle cell disease who have been treated with HU.23
Because of the limited available data, HU should be considered to have uncertain carcinogen potential and should be used with caution to treat nonmalignant diseases. HU is a potent teratogen in all animal species tested so far, and qualifies as a universal teratogen; it should not be used in women of childbearing age unless the possibility of pregnancy can be excluded. However, a number of patients who conceived while receiving HU for a variety of hematologic conditions completed normal pregnancies after discontinuation of the drug.84 The reader is referred to Chapter 4 (“Infertility after Cancer Chemotherapy”) and Chapter 5 (“Carcinogenesis of Anticancer Drugs”) for further discussion of these side effects.
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