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Stem Cells, Vol. 17, No. 6, 345-356, November 1999
© 1999 AlphaMed Press

In Vivo and In Vitro Comparison of the Short-Term Hematopoietic Toxicity Between Hydroxyurea and Trimidox or Didox, Novel Ribonucleotide Reductase Inhibitors with Potential Anti-HIV-1 Activity

Christopher N. Mayhewa,b, Jonathan D. Phillipsa, Richard N. Greenbergc, Nicholas J. Birche, Howard L. Elfordf, Vincent S. Gallicchiob,d

a School of Health Sciences, University of Wolverhampton, Wolverhampton, United Kingdom;
b Departments of Clinical Sciences and
c Infectious Diseases, and
d Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky, USA;
e Academic Consulting Services Ltd., Codsall, United Kingdom; eMolecules for Health Inc., Richmond, Virginia, USA

Key Words. Bone marrow • Burst-forming unit–erythroid • Colony-forming unit-granulocyte-macrophage • Didox • Hematopoiesis • HIV-1 • Hydroxyurea • Ribonucleotide reductase inhibitors • Toxicity • Trimidox

Correspondence: Dr. Vincent S. Gallicchio, University of Kentucky, CAHP, Room 220, 121 Washington Avenue, Lexington, Kentucky 40536-0003, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitors of the cellular enzyme ribonucleotide reductase (hydroxyurea, [HU]) have been proposed as a new therapeutic strategy for the treatment of HIV type-1 (HIV-1) infection. However, HU use may be limited by the frequent development of hematopoietic toxicity. We report here short-term hematopoietic toxicity in mice receiving HU when compared to either of two more potent enzyme inhibitors, didox (DX) and trimidox (TX). High dose HU, DX, and TX monotherapy (500, 460, and 220 mg/kg/day respectively) was administered by daily i.p. injection (Monday-Friday) to C57BL/6 mice for 10 weeks. Effects on hematopoiesis were established by quantitating peripheral blood indices (hematocrit, hemoglobin, mean corpuscular volume, mean cell hemoglobin, mean corpuscular hemoglobin concentration, RBC, and WBC) and numbers of colony-forming units-granulocyte-macrophage (CFU-GM) and BFU-E from bone marrow and spleen. HU produced rapid induction of a macrocytic hypochromic anemia and altered white blood cell kinetics associated with myelosuppression defined as reduced marrow organ cellularity and induction of splenic extramedullary hematopoiesis. Compared to HU, TX and DX induced fewer changes in peripheral blood indices and CFU-GM and BFU-E per hematopoietic organ. In vitro human and murine marrow CFU-GM and BFU-E colony formations were assayed in the presence of dose escalation HU, DX, or TX (0, 1, 10, 50, 100, and 200 µM). HU inhibited colony formation more than either DX or TX. These in vivo and in vitro studies suggest that novel ribonucleotide reductase inhibitors TX and DX may provide an effective alternative to HU in HIV-1 therapy because they demonstrate reduced hematopoietic toxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemotherapy for HIV type-1 (HIV-1) has been significantly improved since the introduction of highly active antiretroviral therapy (HAART), containing two reverse transcriptase inhibitors and a protease inhibitor [1, 2]. These drug regimens have been successful in producing sustained reductions in viral load and are associated with enhanced immune function and improved patient survival [3, 4]. However, in addition to the high cost and complicated administration schedules of these drug regimens, several problems associated with their use in long-term HIV-1 therapy have emerged. For example, despite effective reduction in viral load, low-level viral replication of HIV-1 displaying genotypic resistance to multiple drugs has been demonstrated in the lymph nodes of patients receiving HAART [5-7]; therefore long-term treatment may ultimately result in multidrug resistant virus leaving few options for salvage therapy. Recent reports have also shown that despite effective therapy, latent infection of CD4+ cells provides a potentially life-long reservoir for HIV-1 persistence [8, 9]. Furthermore, clinical studies have demonstrated that the use of protease inhibitors is associated with induction of peripheral insulin resistance and hyperlipidemia [10-12]. Additionally, the toxicity of long-term HAART administration has yet to be established. These observations have highlighted the importance of continued research to develop more effective drugs for HIV-1 therapy.

In vitro studies have demonstrated the ribonucleotide reductase inhibitor hydroxyurea (HU) to be an effective inhibitor of HIV-1 proviral DNA synthesis [13, 14]. HU has also been shown to potentiate the activity of dideoxynucleoside drugs in vitro, particularly 2',3'-dideoxyinosine (ddI) [15-19]. In addition, several clinical trials have shown that the combination of HU with ddI can substantially reduce viral load in patients infected with HIV-1 much more effectively than ddI monotherapy can, with efficacy approaching that of protease-inhibitor-containing regimens [20-22]. The combination of HU with ddI and d4T [23] or with ddI and indinavir [24] has also been associated with reduced viral load. However, when HU was evaluated for anti-HIV-1 efficacy as monotherapy, no change in viral load was observed and patients developed significant hematological toxicity [25, 26]. The myelosuppressive effects of HU therapy have been well documented [27-30] and therefore may limit the clinical use of HU as a drug regimen component for HIV-1 therapy. In addition, the toxicity of HU may be exacerbated in AIDS patients with advanced disease who have bone marrow suppression as a consequence of infection or in those receiving other drugs that exhibit marrow toxicity (e.g., azidothymidine, AZT [31]).

HU is a relatively weak inhibitor of ribonucleotide reductase in vitro and its in vivo enzyme inhibition probably suffers because of difficulty in maintaining therapeutic levels [32]. As a result of studies designed to enhance the inhibitory effect of the hydroxamate group of HU, hydroxy-substituted benzohydroxamic acid derivatives were synthesized and found to be more potent inhibitors of ribonucleotide reductase than HU [33, 34]. Two of these compounds, trimidox ([TX]; 3,4,5-trihydroxybenzamidoxime) and didox ([DX]; 3,4-dihydroxybenzohydroxamic acid) were found to be particularly active. In one study, TX and DX were shown to inhibit the enzyme activity of ribonucleotide reductase 100 and 17 times more effectively than HU, respectively [33]. In addition, these compounds have also been shown to have antitumor activity in various mouse tumor models [34-36]. We and others have shown that TX and DX have potent antiretroviral activity when administered as monotherapy, as well as the potential to act synergistically with ddI, in several murine-retrovirus-induced immunodeficiency models [37, 38]. In view of the limitations of HU therapy because of its deleterious effects on bone marrow, and the potential efficacy of TX and DX as anti-HIV-1 agents, this study was undertaken to compare the short-term effects of TX, DX, and HU as monotherapy on hematopoiesis when administered to normal C57BL/6 mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Female C57BL/6 mice, 10-12 weeks of age, were obtained from Harlan (Indianapolis, IN) and were housed in microisolator cages in a temperature- and humidity-controlled environment. Mice were fed Purina Lab Chow and water ad libitum. The experimental animal protocol was approved by the University of Kentucky IACUC committee.

Treatment of Mice with Ribonucleotide Reductase Inhibitors
The following doses of ribonucleotide reductase inhibitors were used for the in vivo study; HU 500 mg/kg/day, DX 460 mg/kg/day, and TX 220 mg/kg/day. DX and trimidox were synthesized as previously described [33, 39]. Drugs were injected i.p., Monday-Friday, for 10 weeks. Drug doses used approximated those demonstrated in various mouse tumor and retroviral model systems to have effective antitumor and antiretroviral activity associated with acceptable toxicity [34, 37, 40-42]. All drugs were prepared weekly, sterile filtered 0.45 µm, stored at 4°C and warmed to room temperature before injection. Untreated control mice received no injections.

Analysis of Peripheral Blood Indices and Procurement of Hematopoietic Tissues
Following 2, 4, 6, 8, and 10 weeks of drug treatment, three mice from each treated group were analyzed for the effect of drug on the hematopoietic system. Controls consisted of three untreated animals at each time point examined. Peripheral blood was obtained 72 h after the final drug injection at each time point. Peripheral blood indices were analyzed in a Baker Model 9110 hematology analyzer (Biochem ImmunoSystems; Allentown, PA). A blood film was prepared and stained with Wright-Geimsa for determination of the white cell differential count. At 4, 6, 8, and 10 weeks, mice were anesthetized with CO2 and killed by cervical dislocation. The spleen and femurs were then removed from each mouse and immediately placed on ice. These organs were then evaluated for their content of hematopoietic progenitor cells in a colony-forming assay.

Preparation of Cells for Hematopoietic Colony-Forming Assay
Mononuclear cells were prepared for hematopoietic colony-forming assay as follows. The contents of each femur were flushed with media comprising Fisher's medium (GIBCO; Grand Island, NY) supplemented with 20% horse serum (Sigma, St. Louis, MO), 14 mM sodium bicarbonate (Sigma), 80 U/ml penicillin, and 80 µg/ml streptomycin (GIBCO). Marrow from each animal per group was pooled and a single-cell suspension was obtained by repeated gentle flushing through a 19-gauge needle. Cells were resuspended in 10 ml of media, an aliquot was removed, and red cells lysed by dilution in 3% acetic acid. Nucleated cells were then counted in a hemacytometer. The mean femoral cellularity was determined by dividing the total cell count by the number of femurs pooled. Cells were then carefully layered over an equal volume of ficoll-histopaque reagent (specific gravity 1.077 g/cm3; Sigma) and centrifuged at 300 g at room temperature for 30 min to enrich for the mononuclear fraction. Low-density mononuclear cells were removed from the interface, washed twice, resuspended in media, and enumerated as described above.

Spleens were weighed and added to 10 ml of media before mechanical disruption. Pooled splenocytes were then passed through a nylon screen, an aliquot of sample taken, the red cells lysed by 1:100 dilution in 3% acetic acid, and nucleated cells enumerated as described above.

Assay of Femoral and Splenic Content of Colony-Forming Units-Granulocyte-Macrophage (CFU-GM) and BFU-E
Analysis of the numbers of committed hematopoietic progenitor cells CFU-GM and BFU-E per femur and spleen from mice treated with HU, TX, or DX and untreated controls was performed as follows: 5 x 104 femoral cells/ml or 2.5 x 105 spleen cells/ml were added to methylcellulose growth media (Stem Cell Technologies; Vancouver, BC, Canada; Cat # M3430) containing 0.9% methylcellulose in alpha-minimal essential medium, 30% fetal bovine serum (FBS), 1% bovine serum albumin (BSA), 2% pokeweed mitogen stimulated spleen cell condition media, 3 U/ml recombinant human erythropoietin, 2 mM L-glutamine, and 10-4 M ß-mercaptoethanol. After vortexing, 1 ml media was plated in duplicate in a six-well tissue culture plate and incubated at 37°C in air containing 5% CO2 for seven days. On the basis of their morphology CFU-GM and BFU-E colonies were identified and colonies consisting of more than 50 cells were scored. The number (± standard deviation [SD]) of colony-forming cells per organ was determined using the mean colony counts and the organ cellularity.

In Vitro Murine Hematopoietic Colony-Forming Assay with Dose Escalation HU, TX, or DX
Cells from the femurs of normal mice were obtained as outlined above. To deplete the adherent cell fraction, the single-cell bone marrow suspension was incubated in a 25 cm2 tissue culture flask containing Dexter's media for 1.5 h at 37°C. After gentle rotation of the flask, nonadherent cells were removed and 5 x 104 cells/ml were added to methylcellulose media. Drugs were prepared immediately before each experiment by dilution in serum- and antibiotic-free Dulbecco's modified Eagle's media (DMEM; GIBCO). Evaluations of HU, DX, or TX were made at final concentrations of 1, 10, 50, and 100 µM, with DMEM alone serving as the control for each experiment. One hundred microliters of drug solution were added to 1.9 ml of murine bone marrow in methylcellulose. Samples were then mixed by vortexing and plated in duplicate for each dose. Murine CFU-GM and BFU-E colonies were scored after 7 days incubation as described above.

In Vitro Human Hematopoietic Colony-Forming Assay with Dose Escalation HU, TX, or DX
The in vitro hematopoietic colony-forming ability of human bone marrow in the presence of dose escalation HU, TX, or DX was also investigated. Human bone marrow cells were a kind gift from Dr. Craig Jordan, University of Kentucky Markey Cancer Center. Samples were obtained after surgical bone marrow harvest from normal individuals donating to the marrow/stem cell transplant program at University of Kentucky Medical Center. Procurement of human marrow and processing were performed according to institutional review board approved guidelines. Identity of donors was not made available to the authors. Red cell lysed samples were enriched for mononuclear cells by ficoll-histopaque centrifugation as outlined above. Then 5 x 104 cells were plated in duplicate in methylcellulose media (Stem Cell Technologies; Cat. #H4434) consisting of 1% methylcellulose in Iscove's modified Dulbecco's medium, 30% FBS, 1% BSA, 10-4 M ß-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant human (rh) stem cell factor, 10 ng/ml rh-GM-CSF, 10 ng/ml rh interleukin 3, and 3 U/ml rh erythropoietin. Doses of 0, 10, 50, 100, and 200 µM HU, TX, and DX were used in these studies. Plates were incubated for 12 days before scoring. Human CFU-GM and BFU-E colonies were identified on the basis of their morphology and had to consist of at least 50 cells.

Statistical Analysis
To determine whether differences between experimental groups were statistically significant, a two-tailed t-test was performed. A p-value of <0.05 was considered statistically significant. In some cases, due to the experimental samples being pooled for analysis (e.g., femoral cellularity), or small sample size, statistical analysis of results was not possible, although clear trends were evident.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The hematopoietic toxicity of high dose administration of TX and DX in normal female C57BL/6 mice was compared with HU.

Gross Toxicity
All drug-treated mice survived the 10-week administration period with no signs of gross toxicity such as failure to groom or respond normally to handling. However, body weight gain of drug treated mice was slightly affected by drug treatment and is shown in Table 1Go. Drug administration was associated with a trend toward reduced body weight compared to control mice throughout the evaluation period. There was an initial retardation of growth in mice receiving TX and DX compared to untreated controls in the first two weeks (p < 0.05). Subsequently, between weeks 2 and 10 (the final observation point) TX- and DX-treated mice gained approximately 10% in body weight, such that at week 10 there was no statistically significant difference in body weight between drug-treated groups and controls.


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  		Table 1. Body weights of C57BL/6 mice receiving ribonucleotide reductase inhibitors HU, DX, and TX*
 
Peripheral Blood Indices
Changes in peripheral blood indices were evident at week 2, particularly in mice administered HU. The WBC count in animals receiving HU was 10-fold higher than control mice (p < 0.05) and was elevated in all subsequent evaluations (Fig. 1AGo). The peak WBC was reached at week 8, being 15-fold higher than the untreated group, subsequently declining to approximately threefold higher than normal at week 10. It is evident from the WBC differential (Fig. 2AGo) that the majority of these peripheral white cells were lymphocytes. Compared to HU, the effect of TX and DX on the WBC was more modest (Fig. 1AGo). DX did elevate the WBC slightly, with the only meaningful increase occurring at week 8. However, this increase was still only one-fourth of the HU-treated group level. The other time points were similar to that of the control group. The WBC of mice treated with TX was the same as controls or slightly lower throughout the duration of the experiment, with negligible changes in the white cell differential count (Fig. 2Go). Furthermore, HU also influenced the numbers of circulating neutrophils, with elevations evident at all time points, particularly week 8 where a several-fold increase was observed (Fig. 2BGo).



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  		Figure 1. Peripheral blood indices of C57BL/6 mice administered ribonucleotide reductase inhibitors for 10 weeks. Mice received drugs daily (Monday-Friday) and blood indices were evaluated every two weeks, 72 h after the final drug injection at each time point. Untreated control (closed circle); HU 500 mg/kg/day (open square); DX 460 mg/kg/day (open triangle); TX 220 mg/kg/day (open circle). Mean ± standard deviation; n = 3. (A) WBC = white blood cells (p < 0.05 versus untreated control, HU wk 2); (B) RBC = red blood cells (p < 0.05, HU wks 2, 4, 6, 8, and 10; DX wks 8 and 10; TX wks 6, 8, and 10); (C) MCV = mean corpuscular volume (p < 0.05, HU wks 4, 8, and 10; DX wks 4, 6, 8, and 10); (D) HCT = hematocrit (p < 0.05, HU wks 2, 6, 8, and 10; DX wks 6 and 8; TX wks 8 and 10); (E) HGB = hemoglobin (p < 0.05, HU wks 2, 6, 8, and 10; DX wks 6 and 8; TX wks 4 and 8); (F) MCH = mean cell hemoglobin (p < 0.05, HU wks 4 and 10; DX wks 4, 6 and 8; TX wk 4); (G) MCHC = mean corpuscular hemoglobin concentration (p < 0.05, HU wks 2, 6, and 10; TX wk 4).

 


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  		Figure 2. Effect of ribonucleotide reductase inhibitor administration on the absolute lymphocyte (A) and neutrophil (B) counts of C57BL/6 mice. Mice were treated daily (Monday-Friday) and every two weeks during the experiment; 72 h after the final drug administration, a peripheral blood smear was prepared. Slides were stained with Wright-Geimsa stain and a white blood cell differential count performed. Mean absolute lymphocyte and neutrophil counts for each experimental group (three mice per group) were calculated by multiplying the WBC by the respective differential count divided by 100. Untreated control (closed circle); HU 500 mg/kg/day (open square); DX 460 mg/kg/day (open triangle); TX 220 mg/kg/day (open circle). p < 0.05, absolute lymphocytes—HU wk 2; absolute neutrophils—TX wk 8.

 
Evaluation of red-cell indices demonstrated that HU induced a hypochromic macrocytic anemia first evident at week 2 (Fig. 1B, CGo). The nadir in the RBC count and the peak mean corpuscular volume (MCV) of mice receiving HU was reached at week 8 (44% and 129% of control, respectively; p < 0.05). Changes in the RBC induced by DX were not evident until week 4. However, DX reduced the RBC to 66% at week 8 (p < 0.05). After the nadir in the red blood cell parameters (Figs. 1B, D, EGo) in mice treated with HU and DX at week 8, there was a recovery in these indices evident at week 10. DX increased the MCV, with the peak at week 8 being 115% of control level, one-half the increase caused by treatment with HU. The maximum effect of TX on the RBC was a reduction to 80% of control levels at week 10 (p < 0.05), and it had minimal effect on the MCV. A comparison of the red-cell morphology observed following HU, TX, and DX treatment is demonstrated in Figure 3Go. Numerous hypochromic and macrocytic red-cells are evident in the peripheral blood smear of HU-treated mice (Fig. 3DGo). On the other hand, the smears from TX- or DX-treated mice contained fewer morphological abnormalities than those from HU-treated mice.



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  		Figure 3. Photographic demonstration of the red cell morphology of mice treated with ribonucleotide reductase inhibitors. Representative Wright-Geimsa stained peripheral blood smears from mice after an eight week drug treatment are shown at a magnification of 40 x. (A) untreated control, (B) DX 460 mg/kg/day, (C) TX 220 mg/kg/day, (D) HU 500 mg/kg/day. Note the presence of numerous macrocytic (M), and hypochromic (H) red cells on the smear from the HU treated animal (D). Some of the macrocytic red cells also appear to be polychromatic.

 
Femoral Cellularity and Spleen Weight
HU administration was associated with myelotoxicity, defined by the observation that the number of nucleated bone marrow cells was reduced to 50% of control at week 4, and was lower than control at all time points (Fig. 4AGo). Consequently the suppression of the number of cells per femur by HU at the earliest time points induced a rebound in the absolute neutrophil count, which peaked at week 8 (Fig. 2BGo). Comparatively, at week 4 TX and DX reduced the number of femoral nucleated cells to approximately 70% and 80% of control, respectively. At subsequent time points the femoral cellularity of animals receiving both TX and DX was above or close to normal.



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  		Figure 4. Femoral cellularity (A) and spleen weight (B) of C57BL/6 mice receiving ribonucleotide reductase inhibitor treatment. Mice were treated daily (Monday-Friday) and femurs and spleen excised 72 h after the final drug injection at each time point. Femur cellularity, calculated from the pooled bone marrow of each treatment group, is expressed as percent of the mean untreated control. Untreated control (closed circle); HU 500 mg/kg/day (open square); DX 460 mg/kg/day (open triangle); TX 220 mg/kg/day (open circle). Spleen weight (drug treated versus untreated control) p < 0.05, HU wks 4, 8, and 10; TX wk 6.

 
Administration of HU to animals resulted in dramatic splenomegaly (Fig. 4BGo). The mean spleen weight of untreated mice was approximately 100 mg. After four weeks of HU administration, spleens weighed approximately 275 mg (p < 0.05). Splenomegaly peaked in HU-treated mice at week 6 (400 mg) and then declined to approximately 200 mg at week 10 (p < 0.05). The changes in spleen weight associated with TX or DX administration were less pronounced. DX did, however, induce slight splenomegaly that peaked at approximately 200 mg at week 8. The spleen weight of mice treated with TX was nearly equal to or slightly lower than control at all time points.

Bone Marrow and Spleen-Derived Progenitor Cells
Administration of ribonucleotide reductase inhibitors, particularly HU and DX, reduced the numbers of femoral CFU-GM and BFU-E (Figs. 5A and BGo). At week 8, HU and DX reduced CFU-GM per femur to 25% of control. There was a slight rebound by week 10 but levels remained at 40%-50% of control. The effect of TX on femoral CFU-GM was less pronounced. At week 8 CFU-GM were 50% of control, though by week 10 levels had rebounded to approximately 90% of control levels. At weeks 4 and 8 HU and DX reduced femoral BFU-E to approximately 20%-30% of control. However, there was a rebound to approximately 40% by week 10. At week 4 TX had a negligible effect on the number of femoral BFU-E, but at week 8 levels were reduced to 20% of control. However, there was a substantial rebound at week 10 to levels above control.



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  		Figure 5. Number of femur- (A, B) and spleen- (C, D) derived hematopoietic progenitor cells (CFU-GM and BFU-E) in C57BL/6 mice receiving ribonucleotide reductase inhibitor drug treatment. Untreated control (closed circle); HU 500 mg/kg/day (open square); DX 460 mg/kg/day (open triangle); TX 220 mg/kg/day (open circle). Bone marrow and spleen cells were assayed for the numbers of CFU-GM and BFU-E colony-forming cells per organ at the indicated times during the experiment using a seven-day in vitro hematopoietic colony-forming assay.

 
Splenic CFU-GM and BFU-E in animals receiving HU were increased at all time points (Fig. 4CGo). At week 4 CFU-GM was 19 times normal. At weeks 8 and 10 there was a decline in the numbers of splenic CFU-GM but levels remained higher than normal (approximately five times normal). It is evident that the increase in peripheral blood neutrophils associated with HU administration (Fig. 2BGo) is at least in part due to the development of extramedullary splenic hematopoiesis. At week 4 DX had little effect on numbers of splenic CFU-GM. However, at weeks 8 and 10 DX induced a slight increase in the splenic CFU-GM (approximately three times normal). TX had little effect on splenic CFU-GM. HU also induced an elevation in splenic BFU-E (Fig. 4DGo). At week 4 HU increased splenic BFU-E to approximately 10 times the control. At weeks 8 and 10 splenic BFU-E were increased to approximately six and eight times the control, respectively. Conversely, at week 4 splenic BFU-E were below control in mice receiving DX and TX. By week 8, DX and TX induced an elevation to 3.5 and 3 times control, respectively. However, although splenic BFU-E continued to increase in mice treated with DX (four times control at week 10), TX did not further increase splenic BFU-E at week 10.

In Vitro Murine Progenitor Cell Assays
In order to compare further the hematopoietic toxicity of HU, TX, and DX, in vitro CFU-GM and BFU-E progenitor cell assays were performed. At all doses tested, HU inhibited both CFU-GM and BFU-E colony formation more than either DX or TX (Table 2Go). At a dose of 50 µM HU inhibited CFU-GM colony formation by more than 99%. However, 50 µM DX and TX inhibited colony formation by 53.1% and 58.4%, respectively. The concentration of HU that inhibited murine CFU-GM colony formation by 50% (IC50) was 18 µM. The IC50 for DX and TX was 45 µM and 40 µM, respectively.


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  		Table 2. Inhibition of in vitro murine (A) CFU-GM and (B) BFU-E colony formation by ribonucleotide reductase inhibitors*
 
HU also inhibited in vitro BFU-E colony growth more than TX or DX. At a dose of 50 µM, HU and TX inhibited BFU-E colony formation by over 98% and 52.1%, respectively. DX was less toxic to murine BFU-E in vitro than HU and TX. At a dose of 50 µM, DX inhibited BFU-E colony formation by only 13.4%. The IC50 of HU, DX, and TX for murine BFU-E was 20, 78, and 45 µM, respectively.

In Vitro Human Progenitor Cell Assay
We also sought to determine whether HU was more inhibitory to in vitro human CFU-GM and BFU-E colony formation than DX or TX. Doses ranging from 1 to 200 µM were evaluated. HU was more toxic than DX or TX to human CFU-GM and BFU-E at all doses tested (Table 3Go). At a dose of 100 µM HU completely inhibited human CFU-GM colony formation. However, 100 µM DX and TX inhibited CFU-GM growth by 63.7% and 52.3%, respectively. The IC50 of HU, DX, and TX for human CFU-GM was 14, 80, and 90 µM, respectively.


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  		Table 3. Inhibition of in vitro human (A) CFU-GM and (B) BFU-E colony formation by ribonucleotide reductase inhibitors*
 
A similar pattern was noted for human BFU-E. There was no colony growth at a dose of 100 µM HU. However, 100 µM DX and TX inhibited human BFU-E colony formation by 77.7% and 55.9%, respectively. The ID50 for HU, DX, and TX for human BFU-E was determined to be 19, 67, and 155 µM, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Identification of the development of multiple-drug-resistant HIV-1 [5, 6] and toxicity [11] in patients receiving HAART therapy has highlighted the necessity for the development of new drug strategies for the treatment of HIV-1 infection. One approach that has received attention has been the use of HU to inhibit the cellular enzyme ribonucleotide reductase [43-45]. HU has received the most attention since it is available for clinical studies. The rationale for this approach is that inhibition of ribonucleotide reductase depletes the cellular pool of deoxynucleotide triphosphates (dNTPs) required for proviral DNA synthesis by viral reverse transcriptase (RT) [13, 14, 46]. In addition, depletion of the endogenous dNTP pool increases the anti-HIV activity of dideoxynucleotide drugs (e.g., ddI/AZT) by decreasing competition for their incorporation into the active site of RT, thereby enhancing termination of the nascent proviral DNA chain [14-18]. An added advantage of including HU in HIV-1 combination therapy is the ability to overcome the development of nucleoside analog drug resistance [47]. However, the myelotoxicity commonly associated with the use of HU [48] has limited its clinical appeal. This report demonstrates that the second-generation ribonucleotide reductase inhibitors DX and TX are less toxic to the hematopoietic system of mice than HU. We also show that TX and DX demonstrate reduced inhibition of human and murine committed progenitor cell growth in vitro compared to HU at equimolar doses.

In all aspects of in vivo murine hematopoiesis examined, DX and TX induced less dramatic changes than HU. Particularly striking in animals receiving DX and TX was the relative lack of perturbations in WBC production kinetics compared to those induced by HU. In this study, myelosuppression associated with HU administration was observed in HU-treated animals. Myelosuppression was manifested not only by a reduction in bone marrow cellularity but an increase in peripheral blood lymphocytes and neutrophils 72 h after the last drug administration at each time point. This increase in peripheral cells is indicative of the hematopoietic overshoot phase during recovery from bone marrow damage and has been well documented in mice receiving chemotherapeutic drugs [49-51]. Indeed, HU not only diminished normal hematopoiesis in the bone marrow but induced the development of extramedullary hematopoiesis in the spleen. DX and HU reduced the number of femoral CFU-GM and BFU-E committed progenitor cells to a similar degree. However, these changes in DX-treated animals were not reflected in either the absolute WBC counts or the red cell indices to the extent they were in animals receiving HU. Therefore, this observation separates DX-induced effects from those observed with HU.

The effects of HU on ribonucleotide reductase have been extensively reviewed [52, 53]. HU has been shown to kill cells that are actively synthesizing DNA in the S-phase of the cell cycle by depleting their supply of dNTPs through ribonucleotide reductase [40]. Theoretically, through their enhanced inhibitory action on ribonucleotide reductase, DX and TX should be more cytotoxic to S-phase cells than HU. Indeed, Szekeres et al. (1994) showed that DX and TX exhibited lower IC50 concentrations than HU in L1210 leukemia cells [35]. So the reduced cytotoxicity of DX and TX to normal hematopoietic cells, compared to HU, is an interesting and novel finding. The data from the in vitro studies reported here support our in vivo observations and suggest that the relative lack of in vivo toxicity of DX and TX are not due to a poor pharmacokinetic profile resulting in low drug concentrations in the bone marrow and consequently low enzyme inhibition. In additional support of this notion, the same DX and TX doses with an identical administration schedule were effective in inhibiting progression of retroviral-induced pathophysiology, indicating effective in vivo enzyme inhibition [37]. Other animal studies demonstrating the antitumor efficacy of TX and DX also suggest that enzyme inhibitory concentrations of TX and DX are achieved in vivo [33, 40, 41].

DX has been evaluated in phase I and II clinical trials for various solid tumors [54-56]. Of note was the observation by Carmichael et al. that the highest tolerated dose of DX (6 g/m2) did not induce myelosuppression [54]. Further work is currently underway to attempt to determine the mechanism for the reduced hematopoietic toxicity of DX and TX. One important feature of these two novel compounds that distinguishes them from HU is their potent free radical scavenging capabilities [34, 57].

Taken together, the results reported here demonstrate that the novel ribonucleotide reductase inhibitors DX and TX are less toxic to normal bone marrow cells than HU and are worthy of further evaluation for the treatment of human AIDS as alternatives to HU monotherapy or as a component of a combination drug treatment regimen.


   Acknowledgments
 
The authors would like to acknowledge the technical assistance of Penny Wildman, Division of Laboratory Animal Resources, and Rachael Alcock, Department of Radiation Medicine, University of Kentucky Chandler Medical Center.

This work was supported by NIH SBIR grant (R44-AI36095) awarded to HLE.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication on October 22, 1999.




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