3-4-5-trihydroxybenzamidoxime and 3-4-dihydroxybenzohydroxamic-acid

Ganciclovir (GCV) is a deoxyguanosine analog that is effective in inhibiting human cytomegalovirus (HCMV) replication. In infected cells GCV is converted to GCV-triphosphate which competes with dGTP for incorporation into the growing DNA strand by the viral DNA polymerase. Incorporated GCV promotes chain termination as it is an inefficient substrate for elongation. Because viral DNA synthesis also relies on cellular ribonucleotide reductase (RR) to synthesize deoxynucleotides, RR inhibitors are predicted to inhibit HCMV replication. Moreover, as dGTP competes with GCV-triphosphate for incorporation, RR inhibitors may also synergize with GCV by reducing intracellular dGTP levels and there by promoting increased GCV-triphosphate utilization by DNA polymerase. To investigate potential of RR inhibitors as anti-HCMV agents both alone and in combination with GCV, HCMV-inhibitory activities of three RR inhibitors, hydroxyurea, didox, and trimidox, were determined. In both spread inhibition and yield reduction assays RR inhibitors had modest anti-HCMV activity with 50% inhibitory concentrations ranging from 36±1.7 to 221±52μM. However, all three showed significant synergy with GCV at concentrations below their 50% inhibitory and 50% toxic concentrations. These results suggest that combining GCV with relatively low doses of RR inhibitors could significantly potentiate the anti-HCMV activity of GCV in vivo and could improve clinical response to therapy.

Topics: Antiviral Agents; Benzamidines; Cell Line; Cytomegalovirus; Cytomegalovirus Infections; Drug Synergism; Ganciclovir; Humans; Hydroxamic Acids; Hydroxyurea; Inhibitory Concentration 50; Ribonucleotide Reductases

Clinically, the vascular pathobiology of human sickle cell disease includes an abnormal state of chronic inflammation and activation of the coagulation system. Since these biologies likely underlie development of vascular disease in sickle subjects, they offer attractive targets for novel therapeutics. Similar findings characterize the sickle transgenic mouse, which therefore provides a clinically relevant inflammation model.. The authors tested two polyhydroxyphenyl hydroxamic acid derivatives, didox and trimidox, in sickle transgenic mice. Animals were examined by intravital microscopy (cremaster muscle and dorsal skin fold preparations) and by histochemistry before and after transient exposure to hypoxia, with versus without preadministration of study drug. Previous studies have validated the application of hypoxia/reoxygenation to sickle transgenic mice as a disease-relevant model.. Animals pretreated with these agents exhibited marked improvements in leukocyte/ endothelial interaction, hemodynamics and vascular stasis, and endothelial tissue factor expression. Thus, these drugs unexpectedly exert powerful inhibition on both the inflammation and coagulation systems.. Each of these changes is expected to be therapeutically beneficial in systemic inflammatory disease in general, and in sickle disease in particular. Thus, these novel compounds offer the advantage of having multiple therapeutic benefits in a single agent.

Topics: Anemia, Sickle Cell; Animals; Benzamidines; Blood Coagulation; Cell Communication; Chronic Disease; Disease Models, Animal; Drug Evaluation, Preclinical; Endothelial Cells; Enzyme Inhibitors; Humans; Hydroxamic Acids; Inflammation; Leukocytes; Mice; Mice, Transgenic; Thromboplastin

The ribonucleotide reductase inhibitor hydroxyurea (HU) has demonstrated some benefit as a component of drug cocktails for the treatment of HIV-1 infection. However, HU is notoriously myelosuppressive and often administered only as salvage therapy to patients with late-stage disease, potentially exacerbating the bone marrow toxicity of HU. In this report we have compared the antiviral effects of HU and two novel RR inhibitors trimidox (3,4,5-trihydroxybenzamidoxime) and didox (3,4-dihydroxybenzohydroxamic acid) in combination with didanosine (2,3-didoxyinosine; ddI) in the LPBM5 MuLV retrovirus model (murine AIDS). We also evaluated the effects of these drug combinations on the hematopoietic tissues of LPBM5 MuLV-infected animals. The combination of RR inhibitors and ddI was extremely effective (DX>TX>HU) in inhibiting development of retrovirus-induced disease (splenomegaly, hypergammaglobulinemia, activated B-splenocytes and loss of splenic architecture). In addition, relative levels of proviral DNA were significantly lower in combination drug-treated animals compared to infected controls. Evaluation of femur cellularity, numbers of marrow-derived myeloid progenitor cells (CFU-GM and BFU-E) and peripheral blood indices revealed that TX and DX in combination with ddI were well-tolerated. However, treatment with HU and ddI induced moderate myelosuppression. These data demonstrate that RR inhibitors in combination with ddI provide significant protection against retroviral disease in murine AIDS. Moreover, the novel RR inhibitors TX and DX appear to be more effective and less myelosuppressive than HU when administered with ddI in this model.

Topics: Animals; Antiviral Agents; B-Lymphocytes; Benzamidines; Didanosine; Drug Therapy, Combination; Enzyme Inhibitors; Female; Hydroxamic Acids; Hydroxyurea; Leukemia Virus, Murine; Leukemia, Experimental; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Murine Acquired Immunodeficiency Syndrome; Retroviridae Infections; Reverse Transcriptase Inhibitors; Ribonucleotide Reductases; Treatment Outcome; Tumor Virus Infections

Inhibition of ribonucleotide reductase (RR) has gained attention as a potential strategy for HIV-1 therapy through the success of hydroxyurea (HU) to potentiate the activity of the nucleoside reverse transcriptase inhibitor (NRTI) didanosine (ddI) in clinical trials. However, the use of HU has been limited by its development of hematopoietic toxicity. In this study, the novel RR inhibitors didox (DX; 3,4-dihydroxybenzohydroxamic acid), and trimidox (TX; 3,4,5-trihydroxybenzamidoxime) were evaluated along with HU for anti-retroviral efficacy in LPBM5-induced retro-viral disease (MAIDS) both as monotherapeutic regimens and in combination with the guanine containing NRTI abacavir (ABC). Anti-retroviral drug efficacy was determined by measuring inhibition of splenomegaly, hypergammaglobulinemia, and splenic levels of proviral DNA. In this study, all RRIs tested showed the ability to improve the efficacy of ABC in the MAIDS model by reducing splenomegaly, hypergammaglobulinemia, and splenic proviral DNA levels.

Topics: Animals; Antiviral Agents; Benzamidines; Bone Marrow Cells; Dideoxynucleosides; Disease Models, Animal; Drug Therapy, Combination; Hematopoietic Stem Cells; Hydroxamic Acids; Hydroxyurea; Mice; Mice, Inbred C57BL; Murine Acquired Immunodeficiency Syndrome; Ribonucleotide Reductases; Spleen; Splenomegaly

Temozolomide (TMZ), an oral alkylating agent with good penetration of the blood-brain barrier, has shown efficacy in the treatment of malignant brain tumors. Ribonucleotide reductase (RR), the rate-limiting enzyme of DNA synthesis, seems to be a complementary target for combination chemotherapy of brain tumors. Trimidox (TX) and didox (DX) are two recently synthesized specific inhibitors of RR. The combinations of TMZ with TX or DX as a basis for synergistic chemotherapy protocols were tested in this study.. The effects of the single drugs TMZ, DX, and TX, and the combinations TMZ/DX and TMZ/TX were evaluated in the human malignant glioma cell lines U87MG, T98G, LNZ308, and wt1119. In the latter, experiments were carried out in the presence or absence of wild-type p53 protein expressed under the control of a tetracycline-responsive transgene system. Cytotoxicity was evaluated by MTT assays. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between drugs.. All drugs demonstrated cytotoxicity in brain tumor cells. Synergistic cytotoxic effects (CI<1) for TMZ and TX or DX at different dose levels were demonstrated in most of the examined cell lines. In some instances, however, drug combinations resulted in additive or even antagonistic effects. Toxicity of the single agents and synergy of the combinations did not correlate with wild-type p53 expression in the tumor cells.. The tumor toxicity of TMZ as a single agent may be modified by combinations with the novel RR inhibitors DX and TX, and is synergistically enhanced in most cases. Depending on the combination ratio, the doses for each drug for a given degree of effect in the combination may be drastically reduced.

Topics: Antineoplastic Agents, Alkylating; Benzamidines; Brain Neoplasms; Dacarbazine; Drug Synergism; Enzyme Inhibitors; Glioma; Humans; Hydroxamic Acids; Ribonucleotide Reductases; Temozolomide; Tumor Cells, Cultured

Recently, the use of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) in combination with nucleoside analogs has gained attention as a potential strategy for anti-HIV-1 therapy. However, appeal for the long-term use of HU in HIV-1 infection may be limited by its propensity to induce hematopoietic toxicity. We report a comparison of the efficacy and bone marrow toxicity of HU (400 and 200 mg/kg/day) with the novel RR inhibitors and free radical-scavenging compounds didox (DX; 3,4-dihydroxybenzohydroxamic acid; 350 mg/kg/day) and trimidox (TX; 3,4,5-trihydroxybenzamidoxime; 175 mg/kg/day) in the murine AIDS (LPBM5 MuLV) model of retrovirus infection. Infected mice received daily drug treatment for 8 weeks. Efficacy was determined by measuring drug effects on retroviral-induced disease progression (i.e. development of splenomegaly and hypergammaglobulinemia) and by evaluating splenic levels of proviral DNA. Bone marrow toxicity was evaluated by measuring peripheral blood indices (WBC, hematocrit and reticulocyte counts), femoral cellularity and by determining the numbers of hematopoietic progenitor cells (CFU-GM, BFU-E) per femur and spleen. Compared to infected controls receiving no drug treatment, disease progression was significantly suppressed by TX, DX and HU. However, HU was associated with mortality and induced significant hematopoietic toxicity in a time- and dose-dependent manner. Conversely, TX and DX effectively inhibited retrovirus-induced disease but did not induce hematopoietic toxicity. These results suggest that due to their reduced hematopoietic toxicity and ability to inhibit disease progression in murine AIDS, TX and DX may offer effective alternatives to HU therapy in HIV-1 infection.

Topics: Animals; Benzamidines; Bone Marrow Cells; DNA, Viral; Female; Femur; Free Radical Scavengers; Hematopoietic Stem Cells; Hydroxamic Acids; Hydroxyurea; Hypergammaglobulinemia; Leukemia Virus, Murine; Leukemia, Experimental; Mice; Mice, Inbred C57BL; Murine Acquired Immunodeficiency Syndrome; Proviruses; Retroviridae Infections; Ribonucleotide Reductases; Spleen; Splenomegaly; Tumor Virus Infections

We evaluated the ability of a short course of treatment with the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) and two novel RR inhibitors Trimidox (TX) and Didox (DX) to influence late-stage murine retrovirus-induced lymphoproliferative disease. LPBM5 murine leukaemia virus retrovirus-infected mice were treated daily with HU, TX or DX for 4 weeks, beginning 9 weeks post-infection, after development of immunodeficiency and lymphoproliferative disease. Drug effects on disease progression were determined by evaluating spleen weight and histology. Effects on haematopoiesis were determined by measuring peripheral blood indices (white blood cells and haematocrit) and assay of femur cellularity and femoral and splenic content of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). HU, TX and DX partially reversed late-stage retrovirus-induced disease, resulting in spleen weights significantly below pre-treatment values. Spleen histology was also improved by RR inhibitor treatment (DX>TX>HU). However, as expected, HU was significantly myelosuppressive, inducing a reduction in peripheral indices associated with depletion of femoral CFU-GM and BFU-E. In contrast, although TX and DX were moderately myelosuppressive, both drugs were significantly better tolerated than HU. In summary, short-term treatment in late-stage murine retroviral disease with HU, TX or DX induced dramatic reversal of disease pathophysiology. However, the novel RR inhibitors TX and DX had more effective activity and significantly less bone marrow toxicity than HU.

Topics: Animals; Antiviral Agents; Benzamidines; Blood Cell Count; Body Weight; Bone Marrow; Hydroxamic Acids; Hydroxyurea; Leukemia Virus, Murine; Lymphoproliferative Disorders; Mice; Spleen; Time Factors

Upon appropriate drug treatment, the human erythroleukemic K562 cells have been shown to produce hemoglobin and F-cells. Fetal hemoglobin (Hb F) inhibits the polymerization events of sickle hemoglobin (Hb S), thereby ameliorating the clinical symptoms of sickle cell disease. Ribonucleotide reductase inhibitors (RRIs) have been shown to inhibit the growth of myeloid leukemia cells leading to the production of Hb F upon differentiation. Of the RRIs currently in use, hydroxyurea is the most effective agent for Hb F induction. We have examined the capacity of two novel RRIs, didox (DI) and trimidox (TRI), in combination with streptozotocin (STZ), to induce hemoglobin and F-cell production. The K562 cells were cultured with different concentrations of didox-STZ or trimidox-STZ at a fixed molar ratio of 3:1 and 1:5 for 96 hr, respectively. At pre-determined time intervals, aliquots of cells were obtained and total hemoglobin (benzidine positive) levels, number of F-cells, and Hb F were determined by the differential staining technique, fetal hemoglobin assay kit, and fluorescence cytometry respectively. The effect of combined drug treatment on the growth of K562 cells was examined by isobologram analysis. Our results indicate that a synergistic growth-inhibitory differentiation effect occurred when didox or trimidox was used in combination with STZ on K562 cells. There was an increase in the number of both benzidine-positive normoblasts and F-cells, accompanied by morphologic appearances typical of erythroid maturation. On day 4, the number of benzidine-positive cells showed a 6-9-fold increase and the number of F-cells was between 2.5- and 5.7-fold higher than the respective controls. Based upon these results, treatment with a ribonucleotide reductase inhibitor, such as didox or trimidox, in combination with STZ, might offer an additional promising option in sickle cell disease therapy.

Topics: Antineoplastic Agents; Benzamidines; Cell Differentiation; Cell Division; Drug Therapy, Combination; Enzyme Inhibitors; Fetal Hemoglobin; Hemoglobin, Sickle; Humans; Hydroxamic Acids; K562 Cells; Ribonucleotide Reductases; Streptozocin

Topics: Benzamidines; HIV Infections; HIV Protease Inhibitors; HIV-1; Humans; Hydroxamic Acids; Serotyping; Treatment Outcome

Topics: Acquired Immunodeficiency Syndrome; Animals; Anti-HIV Agents; Benzamidines; Disease Models, Animal; Drug Resistance, Microbial; Humans; Hydroxamic Acids; Immunity, Cellular; Mice; Ribonucleotide Reductases; Sensitivity and Specificity

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 microM). 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.

Topics: Acquired Immunodeficiency Syndrome; Anemia; Animals; Anti-HIV Agents; Benzamidines; Cells, Cultured; Colony-Forming Units Assay; Enzyme Inhibitors; Female; Femur; Hematopoiesis; Hematopoietic Stem Cells; Humans; Hydroxamic Acids; Hydroxyurea; In Vitro Techniques; Lymphocytes; Macrophages; Mice; Mice, Inbred C57BL; Neutrophils; Organ Size; Ribonucleotide Reductases; Spleen

Ribonucleotide reductase, a key enzyme in deoxyribonucleotide synthesis, is an important target for cancer chemotherapy. Drugs that inhibit its individual components may act synergistically to block DNA synthesis. Prior work has established that gallium inhibits the R2 subunit of ribonucleotide reductase. We show that gallium acts synergistically with the ribonucleotide reductase inhibitors gemcitabine and hydroxyurea to inhibit the proliferation of CCRF-CEM cells. In contrast, combinations of gallium with the ribonucleotide reductase inhibitors amidox, didox, or trimidox produced antagonistic effects on cell growth. Spectroscopy analysis revealed that as a result of their metal-binding properties, amidox, didox and trimidox formed complexes with gallium, thus negating potential synergistic actions. Our results have important implications in the design of clinical trials using these ribonucleotide reductase inhibitors in combination.

Topics: Antineoplastic Agents; Benzamidines; Cell Division; Deoxycytidine; Drug Synergism; Enzyme Inhibitors; Gallium; Gemcitabine; Humans; Hydroxamic Acids; Hydroxyurea; Oximes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Ribonucleotide Reductases; Tumor Cells, Cultured

Amidox (AX), didox (DX) and trimidox (TX), compounds synthetized as new ribonucleotide reductase inhibitors, have been investigated by ultraviolet (UV) spectrophotometry, polarography and high performance liquid chromatography (HPLC). The experiments have been performed at various pH values. The changes in UV absorption of the compounds studied were recorded and it was demonstrated that these changes are related to the pH and to structural features of the investigated molecules. From the compounds included in our series of experiments, only amidox and trimidox are reduced during polarographic experiments in Britton-Robinson buffer. The reduction of both compounds proceeded in two one-electron steps in acidic pH. One two-electron diffuse irreversible wave was observed at basic pH. The values of the half-wave potential became more negative in accordance with the increasing pH. HPLC assay also showed changes in the retention of compounds investigated, particularly when the pH of the mobile phase was close to the dissociation constant of the particular drug. The changes of physico-chemical properties detected by the all used methods are related to different chemical structures (the most significant changes were observed in alkaline pH).

Topics: Benzamidines; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Hydrogen-Ion Concentration; Hydroxamic Acids; Oximes; Polarography; Ribonucleotide Reductases; Spectrophotometry, Ultraviolet

Toxic and genotoxic effects of three polyhydroxy-substituted benzohydroxamates (amidox, didox and trimidox), having antineoplastic activities by the mechanism of the ribonucleotid reductase activity inhibition, were evaluated by reverse mutation assay on Salmonella typhimurium strains TA97, TA98, TA100, TA102. While amidox did not exhert any toxic effect, didox and trimidox were toxic. The toxicity of the test chemicals was dependent on the structure of their molecule and the repair capacity of the test strains. Trimidox exhibited the highest toxicity, and it was proved as a direct-acting frameshift mutagen. Its mutagenic effect was increased after a metabolic activation. Amidox and didox can be classified as frameshift promutagens.

Topics: Animals; Antineoplastic Agents; Benzamidines; Enzyme Inhibitors; Frameshift Mutation; Hydroxamic Acids; In Vitro Techniques; Mutagenicity Tests; Mutagens; Oximes; Rats; Ribonucleotide Reductases; Salmonella typhimurium; Subcellular Fractions

Ribonucleotide reductase inhibitors (RRIs) have been recently shown to inhibit retroviral replication. We examined a new series of RRIs, 3,4-dihydroxybenzohydroxamic acid (Didox) and 3,4,5-trihydroxybenzohydroxamidoxime (Trimidox) for their ability to alter disease progression in murine acquired immunodeficiency syndrome (MAIDS), both alone and in combination with 2',3'-dideoxyinosine (ddI). MAIDS disease was induced by inoculation of female C57BL/6 mice with the LP-BM5 murine leukemia virus (MuLV) and disease progression characterized by extensive peripheral lymphadenopathy and splenomegaly. Efficacy of treatment with these drugs was based upon their ability to influence survival and disease pathophysiology by monitoring the development of splenomegaly. Toxicity was determined by changes in body weight, total peripheral white blood cell count and hematocrit. Didox or trimidox monotherapy was associated with increased survival and decreased disease pathophysiology, with no apparent toxicity. Combined with ddI, their ability to reduce development of viral induced splenomegaly was enhanced compared to trimidox, didox or ddI alone. These results demonstrate RRIs have potent activity in reversing the disease manifestations characteristic of MAIDS. Further studies are warranted to determine human clinical efficacy.

Topics: Animals; Antiviral Agents; Benzamidines; Biomarkers; Didanosine; Disease Progression; Drug Therapy, Combination; Female; Hydroxamic Acids; Leukemia Virus, Murine; Mice; Mice, Inbred C57BL; Murine Acquired Immunodeficiency Syndrome; Ribonucleotide Reductases; Survival Rate

Recent information on the compounds that inhibit cellular enzymes to suppress HIV and increase the antiviral activity of nucleoside analogs, such as AZT or ddI, is examined. Specific drugs addressed include didox, trimidox, fluorouracil, floxuridine, ribavirin, and dipyridamole. Also addressed are the promising combination therapies of ribavirin with ddI, and AZT with dipyridamole.

Topics: Animals; Antiviral Agents; Benzamidines; Dipyridamole; Disease Models, Animal; Floxuridine; Fluorouracil; Humans; Hydroxamic Acids; Hydroxyurea; Mice; Murine Acquired Immunodeficiency Syndrome; Ribonucleotide Reductases