In vivo examination of hydroxyurea and the novel ribonucleotide reductase inhibitors trimidox and didox in combination with the reverse transcriptase inhibitor abacavir: suppression of retrovirus-induced immunodeficiency disease.

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.

Platelet CD62p expression and microparticle in murine acquired immune deficiency syndrome and chronic ethanol consumption.

AIMS: Abnormal platelet counts have been noticed in acquired immune deficiency syndrome (AIDS) patients. However, the actual state of platelets in AIDS is unclear. We hypothesize that platelets are activated and platelet-derived microparticles increase in murine AIDS. METHODS: To elucidate the ethanol effects on platelets in murine AIDS, we studied four groups: control, murine AIDS, ethanol, and ethanol plus murine AIDS. Platelet CD62p as a platelet activation marker and CD61(+) microparticles as platelet microparticles (PMPs) were measured by flow cytometry. RESULTS: Platelets were significantly activated in mice with murine AIDS and chronic ethanol consumption. Increased platelet CD62p expression and increased PMPs were most pronounced in advanced stages of murine AIDS. Chronic ethanol consumption persistently enhanced platelet activation and PMP formation. CONCLUSIONS: Elevated platelet CD62p and PMPs may represent a pro-thrombotic status that have important pathological consequences.

Inhibition of murine AIDS by a heterodinucleotide of azidothymidine and 9-(R)-2-(phosphonomethoxypropyl)adenine.

Tenofovir [9-(R)-2-(phosphonomethoxypropyl)adenine (PMPA)] and zidovudine [azidothymidine (AZT)] are potent anti-HIV agents that have shown a strong synergy in in vitro studies. In this paper we have investigated both the potentiality of this synergy in vivo and the possibility to administer AZT and PMPA simultaneously as a single drug AZTpPMPA. The pharmacokinetic studies reported here have shown that AZTpPMPA administered intraperitoneally in mice performs as a prodrug, providing a slow delivery of AZT and PMPA in circulation. C57BL/6 mice infected with the retroviral complex LP-BM5 were used to evaluate the efficacy of AZTpPMPA in inhibiting disease progression. Furthermore, the effectiveness of the heterodinucleotide was compared with that of AZT and PMPA, administered as single drugs, or as a combination (AZT plus PMPA). The results obtained showed that AZTpPMPA is able to reduce lymphoadenopathy (88%), splenomegaly (64%), lymph node BM5 proviral DNA content (49%) and hypergammaglobulinaemia (40%). However, upon AZT plus PMPA administration, similar (splenomegaly and lymphoadenopathy reduction) or better results (64% hypergammaglobulinaemia reduction and 75% lymph node BM5 proviral DNA content inhibition) were obtained. Furthermore, these results overlapped those obtained upon PMPA administration. Thus, no synergy between PMPA and AZT was observed in murine AIDS and administration of AZT does not improve the antiviral results obtained by PMPA administration.

Suppression of retrovirus-induced immunodeficiency disease (murine AIDS) by trimidox and didox: novel ribonucleotide reductase inhibitors with less bone marrow toxicity than hydroxyurea.

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.

Murine AIDS induces viremia and functional and phenotypic alterations in blood cells.

BACKGROUND: Murine acquired immunodeficiency syndrome (MAIDS) is characterized by generalized lymphoproliferation and progressive immunodeficiency. It is induced by a mixture of two replication-competent murine leukemia viruses (MuLV) and a disease-causing, replication-incompetent defective MuLV. Infection leads to specific phenotypic and functional alterations of lymphocytes in lymphoid organs. METHODS: We analyzed phenotypic, virological and functional parameters in the blood of mice infected with MAIDS virus. RESULTS: Disease progression correlated with increasing viremia, a loss of mitogen responsiveness of T lymphocytes, and the appearance of CD4+ Thy1- T lymphocytes. At >9 weeks after infection, the distribution of leukocyte cell populations became very heterogeneous, and late-stage leukemic events were observed in 5 of 23 mice. CONCLUSIONS: Virus titers, mitogen responsiveness and the presence of CD4+ Thy1- T lymphocytes can efficiently be monitored in the blood and serve as diagnostic parameters to monitor disease progression. Acute leukemic events occurring at the terminal stage could be responsible for the death of at least some of the mice with MAIDS.

Liposomes as carriers of the antiretroviral agent dideoxycytidine-5'-triphosphate.

The presence and replication of the human immunodeficiency virus (HIV) in cells of the mononuclear phagocyte system (MPS) together with the preferential uptake of liposomes in macrophages suggest that liposomes can become a valuable carrier of anti-HIV agents. Moreover, liposomes reduce toxicity of encapsulated drugs and protect encapsulated drugs against rapid degradation in the blood circulation. To overcome problems associated with the administration of free nucleosides and to improve targeting to the MPS, dideoxycytidine-5'-triphosphate (ddCTP) was encapsulated in liposomes. Liposomes were stable with regard to retention of the entrapped drug, particle size and chemical stability of ddCTP. Results obtained with liposome encapsulated ddCTP in the murine acquired immunodeficiency syndrome (MAIDS) model indicate that ddCTP encapsulated in liposomes can reduce proviral DNA in cells of the mononuclear phagocyte system (MPS) in both spleen and bone marrow.

Pentoxifylline decreases brain levels of platelet activating factor in murine AIDS.

Tumor necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF) have been implicated in the pathogenesis of human immunodeficiency virus (HIV)-associated encephalopathy. The effects of pentoxifylline on brain PAF levels were examined in mice infected with the LP-BM5 murine leukemia virus (MuLV). Seven weeks after viral inoculation, significant increases in serum TNF-alpha and brain PAF levels were observed. One week of treatment with pentoxifylline initiated 6 weeks postinfection significantly reduced both serum TNF-alpha and brain PAF levels. A significant positive correlation was observed between the levels of these substances (r = 0.62; P < 0.01). This study demonstrates that pentoxifylline treatment was effective in decreasing the levels of TNF-alpha in the serum and PAF levels in the brain of mice infected with the LP-BM5 MuLV.

Effect of blood substitute, recombinant hemoglobin, on in vivo hematopoietic recovery from AZT toxicity.

We determined the in vivo ability of infused human recombinant hemoglobin 1.1 (hr-Hb) and erythropoietin to rescue the hematopoietic activity from the suppressive effects of AZT in normal and in a murine model of AIDS (MAIDS) mice. Mice were fed with AZT for 8 weeks with or without treatment in the last 4 weeks by administering various concentrations of hr-Hb and/or erythropoietin (Epo). Blood parameters, body weight (BW) and erythroid burst-forming units (BFU-E) for all mice were determined. AZT-treated normal and MAIDS mice showed a significant decrease in hematocrit (64 and 78.1%), hemoglobin (27.2 and 45.5%), BW (17.5 and 35.5%), number of white (66.9 and 42.1%) and red blood cells (65.5 and 38%), and the number of BFU-E (73 and 59%), whereas the AZT-treated normal and MAIDS mice that received hr-Hb (5 mg/kg BW/day) and/or Epo (2 U/mouse/day) showed significant alleviation of AZT cytotoxicity. This was evident by the recovery in all blood indices examined, the number of BFU-E and the BW of mice treated. BFU-E recovery in MAIDS (97%) was greater than that in normal mice (63%) as compared to their controls. hr-Hb produced a similar response as the combination, however recovery was slightly better with the latter in some hematological parameters. Higher concentrations of hr-Hb (10-15 mg) did not result in a more significant increase in most blood indices. Our results indicate that infusion with hr-Hb can alleviate AZT toxicity in normal and in immunodeficient mice, and that hr-Hb may be clinically useful in preventing severe bone marrow depression brought about by various drugs or agents such as AZT.

Effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) on hematopoiesis administered to retrovirus-infected immunodeficient mice receiving dose-escalation zidovudine (AZT).

We have previously demonstrated that continuous administration of dose-escalation zidovudine (AZT) in either normal or LP-BM5 MuLV immunodeficient virus-infected mice (MAIDS) was associated with the development of anemia, neutropenia, and thrombocytopenia. Hematopoietic growth factors/cytokines are being evaluated to determine their efficacy in ameliorating the hematopoietic toxicity associated with AZT. In normal mice receiving AZT, an increase in only plasma erythropoietin and not GM-CSF, Meg-CSF or TNF-alpha has been reported. This article describes studies that investigated the effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) administered in normal non-viral, viral-infected, and viral-infected C57BL6 mice receiving dose-escalation AZT, i.e. 0.1 mg/ml, 1.0 mg/ml, and 2.5 mg/ml placed in the drinking water. Non-viral control mice responded to IL-3/GM-CSF by increasing erythropoiesis, myelopoiesis and platelet production measured by increased bone marrow and spleen derived erythroid, myeloid and platelet precursor stem cells cultured in semi-solid media. Virus-infected control mice not receiving IL-3/GM-CSF developed pancytopenia. Administration of IL-3/GM-CSF to virus-infected mice receiving dose-escalation AZT did not ameliorate the peripheral pancytopenia associated with immunodeficiency disease and AZT treatment, even though erythroid, myeloid and platelet precursor progenitor cells were increased at certain times when compared to either normal or viral-infected mice receiving IL-3/GM-CSF. These results indicate that the combination use of IL-3 and GM-CSF in vivo is only a partially effective growth factor/cytokine treatment to ameliorate the hematopoietic toxicity associated with the use of the anti-viral drug zidovudine.

Amelioration of azidothymidine-induced erythroid toxicity by hemin and stem cell factor in immune-suppressed mice.

Recombinant cytokines such as stem cell factor (SCF) are currently being tested for the ability to ameliorate 3'azido-3'deoxythymidine (AZT)-induced anemia in AIDS patients. Recently, we showed that SCF greatly increased burst-forming units-erythroid (BFU-E) but failed to increase hematocrits of AZT-treated immune-deficient (MAIDS) mice. We reasoned that hemin, previously shown to both enhance BFU-E proliferation and accelerate erythroid maturation, might bring about differentiation of this large SCF-induced pool of BFU-E and further protect BFU-E from AZT's toxic effect. We therefore studied, in vitro, the effect of combinations of hemin and SCF on growth of BFU-E from MAIDS mice. Hemin, at concentrations of 10 to 100 microM, ameliorated the growth-inhibitory effect of AZT. 50 microM hemin increased the ED50 of AZT from 1 x 10(-7) M to 1.7 x 10(-6) M. SCF also ameliorated AZT-induced toxicity, but to a lesser extent. SCF and hemin increased the number of BFU-E colonies observed in the presence of AZT in an additive fashion. The resistance of BFU-E to AZT's cytotoxic effect was greater in cultures receiving hemin and SCF together than in cultures receiving SCF or hemin alone. Zinc and tin protoporphyrins (Zn and Sn PP) increased the numbers of BFU-E observed. However, neither zinc nor tin protoporphyrins increased the ED50 of AZT. Combinations of SCF and hemin may prove useful in ameliorating AZT toxicity in both immune-suppressed mice and human immunodeficiency virus (HIV)-infected patients.

The significance of the pre-challenge immune status of mice for development of retrovirus-induced immunodeficiency syndrome (MAIDS).

The effects of vaccination with RNA-free viral pseudoparticles, preinfection with non-pathogenic ecotropic virus, and induction of tolerance to viral proteins in newborns on the outcome of murine immunodeficiency syndrome (MAIDS) were studied. The parameters used to follow disease progression were: lymphopenia, circulating B and T8 cells, serum IgG and IgM levels, lymphoproliferation and skin graft rejection. Immunization with RNA-free viral pseudoparticles had no effect on any of these parameters. Preinfection of adults with ecotropic virus and the induction of tolerance in newborns to virus antigens both attenuated the early symptoms of viral infection and delayed the onset of immunodeficiency and lymphoproliferation in some mice, but did not significantly alter the number of deaths due to MAIDS. Failure of immune-based therapy to produce successful protection against MAIDS suggests that immune destruction caused by the persistent virus rather than hyperimmune activity is the main pathogenic factor in this disease.

Hemopoietic recovery from AZT toxicity with recombinant hemoglobin in a murine model of AIDS.

We studied the toxic effects of azidothymidine (AZT) on the hemopoietic colony growth (CFU-E, BFU-E and CFU-GM) of bone marrow in a murine model of AIDS (MAIDS). A sparing effect by recombinant hemoglobin (r-Hb) on AZT suppression of MAIDS bone marrow was found when 10 microM of r-Hb was included in bone marrow cultures. The AZT toxicity dose response curve showed that at a concentration of 0.1 microM, AZT inhibited CFU-E by 66%, BFU-E by 55% and CFU-GM by 67%. The addition of r-Hb (10 microM) to AZT-treated cultures stimulated CFU-E, BFU-E and CFU-GM by 89, 125 and 160%, respectively, as compared with AZT-treated (control). The addition of r-Hb to non AZT-treated cultures showed further stimulation of CFU-E, BFU-E and CFU-GM to 100, 160 and 187% of the control, respectively. These results indicate that exogenous r-Hb reverses AZT-induced hemopoietic toxicity and may prove to be useful in ameliorating AZT toxicity in immunosuppressive diseases.

Regulation of megakaryocyte colony forming cell numbers and ploidy by dideoxynucleosides in immunodeficient mice.

We have recently demonstrated that azidothymidine (AZT) elevates the levels of circulating platelets in mice made immune deficient by infection with LP-BM5 murine leukemia virus (MAIDS mice). In an attempt to elucidate the mechanisms of the AZT platelet elevating effect, we examined the number of splenic and bone marrow megakaryocyte colony-forming cells (CFU-mk) and the ploidy of megakaryocytes derived from CFU-mk using fluorescence cytophotometric methods. Two other dideoxynucleosides (ddN) 2'3'-dideoxyinosine (ddl) and 2'3'-dideoxycytidine (ddC) were assessed to determine the specificity of the effect of AZT. MAIDS mice were given ddN in drinking water for 15 days. AZT was the only ddN that significantly increased circulating platelet levels in MAIDS mice. AZT significantly increased splenic CFU-mk in MAIDS mice, but bone marrow CFU-mk were not affected. ddl and ddC failed to change either platelet levels or the numbers of splenic or bone marrow CFU-mk. The ploidy of megakaryocytes derived from splenic and bone marrow CFU-mk were examined by first identifying CFU-mk by staining for acetylcholinesterase, followed by nuclear staining with propidium iodide. The fluorescence of individual cells was then measured using a Perceptics image analysis system. Modal ploidy of CFU-mk megakaryocytes derived from spleen cells of AZT-treated immunodeficient mice was shifted upwards from 16N to 32N. Similarly, AZT treatment changed the modal ploidy number of colony megakaryocytes derived from bone marrows of immunodeficient mice from 16N to 32N. The ploidy distribution was also significantly shifted. ddl and ddC failed to significantly alter either modal ploidy number or distribution of megakaryocytes derived from splenic or bone marrow CFU-mk. These findings suggest that AZT may affect physiological processes that lead to platelet formation.

Sustained zidovudine treatment on hematopoiesis in immunodeficient mice.

Zidovudine (AZT) has been the drug of choice in the treatment of human AIDS; however, associated with the use of zidovudine has been the development of hematopoietic toxicity, the mechanism of which is not clearly defined. We report here studies designed to evaluate dose-escalation of zidovudine, i.e. 0.1 and 1.0 mg/ml placed in the drinking water on hematopoiesis in C57BL/6 normal and LP-BM5 immunodeficiency virus-infected mice. Over a 6-week evaluation period, compared to normal, non-virus-infected controls, murine immunodeficiency (MAIDS) infection was associated with reduced hematopoietic progenitors, i.e. CFU-E, BFU-E, CFU-GM, and CFU-Meg from bone marrow and spleen. Following zidovudine treatment, further suppression of marrow-derived progenitors was observed, while increased numbers of progenitors were obtained from the spleen. Spleen-derived erythroid progenitors, i.e. CFU-E, were increased by 950% (P < 0.001) from MAIDS-infected animals receiving 1.0 mg/ml of drug following 4-weeks exposure compared to non-drug-treated MAIDS control animals. Splenic BFU-E were increased 654% following 6-weeks exposure compared to non-drug-treated MAIDS-infected mice. This study suggests that the bone marrow is particularly sensitive to zidovudine toxicity which, at least early in exposure, appears to be compensated by splenic-derived hematopoiesis, in particular, erythropoiesis. Overt toxicity develops when, at least in this immunodeficiency model, the spleen is unable to provide progenitors in response to continued zidovudine exposure in vivo.

Sustained elevation of platelet counts by long-term azidothymidine treatment of immunosuppressed mice.

Azidothymidine (AZT) has been demonstrated to increase platelet counts in patients suffering from acquired immunodeficiency syndrome (AIDS). However, the ability of long-term AZT treatment to sustain increases in platelet counts is controversial. We have recently demonstrated that AZT elevates the levels of circulating platelets in both normal C57BL/6 mice and mice made immunodeficient by infection with LP-BM5 murine leukemia virus (MAIDS mice). We therefore studied the effect of long-term AZT administration on platelet formation in both normal and MAIDS mice. Peripheral blood indices, levels of femoral and splenic megakaryocyte colony forming units (CFU-MK), and plasma levels of cytokines important in platelet formation-interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF)--were examined. Platelet counts remained elevated throughout a 120-day AZT treatment period. Splenic CFU-MK were not significantly changed in MAIDS mice, except at day 15 when they were elevated. Splenic CFU-MK were significantly decreased in normal mice at days 8 and 120, and increased at day 30. Bone marrow CFU-MK were increased by AZT treatment at all time points tested in both normal and MAIDS mice. Plasma levels of GM-CSF were unchanged by AZT treatment in both normal and MAIDS mice. Plasma levels of IL-6 were unchanged in AZT-treated normal mice but decreased in AZT-treated MAIDS mice. These results indicate that long-term AZT treatment maintains elevated levels of platelets in both normal and MAIDS mice and affects CFU-MK colony formation. Our studies add to a growing body of work suggesting that AZT can ameliorate thrombocytopenia associated with HIV disease.

Failure of stem cell factor to ameliorate AZT-induced anemia in immunodeficient mice.

Recent attempts to reduce 3' azido-3' deoxythymidine (AZT)-induced anemia in AIDS patients have focused both on AZT dose reduction and on the use of recombinant cytokines. The newly cloned cytokine stem cell factor (SCF) is a potent regulator of hematopoietic progenitor cell proliferation. Therefore, we attempted to ameliorate AZT-induced anemia using stem cell factor (SCF) in the LP-BM5 murine leukemia virus-induced model of AIDS (MAIDS). SCF was administered with oral AZT for up to 1 month, and effects on erythropoiesis examined. SCF alone increased both splenic BFU-E and CFU-E. AZT alone also increased the number of splenic BFU-E and CFU-E. SCF, administered to AZT-treated MAIDS mice, did not further enhance these increases. SCF increased bone marrow cellularity in AZT-treated MAIDS mice. However, the total number of bone marrow BFU-E was unaffected. In contrast, AZT, SCF, and the combination significantly decreased bone marrow CFU-E. SCF alone increased the absolute numbers of peripheral blood reticulocytes in MAIDS mice, but did not increase reticulocyte numbers in AZT-treated mice. SCF did not significantly increase hematocrits in either control or AZT-treated mice. Further studies are needed to maximize the differentiating capacity of the enlarged erythroid progenitor cell pool induced by SCF.

Modification of thymic cell subsets induced by long-term cocaine administration during a murine retroviral infection producing AIDS.

LP-BM5 murine leukemia virus (MuLV) infection and cocaine administration are known to impair the murine immune system. We have developed a murine model to study the effect of daily cocaine administration and retrovirus infection on the lymphoid cell populations of the thymus. C57BL/6 female mice were studied following chronic cocaine administration for 11 weeks with simultaneous LP-BM5 MuLV infection. Cocaine administration reduced body and thymus weight, significantly reduced the number of CD8+ cells in the thymus, and partially prevented thymus enlargement due to lymphoid cell proliferation induced by LP-BM5 MuLV infection. Retrovirus infection was associated with a decrease in the percentage and absolute number of Thy 1.2+, CD4+, and CD8+ cells in the thymus, an effect potentiated by cocaine administration. Therefore cocaine impairs thymic function by altering the number of cells expressing T cell differentiation markers in MAIDS.

Time-dependent changes induced by azidothymidine in erythroid progenitor colonies in immunodeficient mice.

The effect of azidothymidine (AZT) on erythropoiesis in C57BL/6 mice made immunodeficient by infection with LP-BM5 murine leukemia virus (MuLV) was examined. Earlier work from our laboratory indicated that long-term treatment of LP-BM5 MuLV-infected mice with AZT induced peripheral anemia but increased the number of splenic and bone marrow erythroid burst-forming units (BFUe). In contrast, other workers have demonstrated that short-term intensive AZT treatment decreases bone marrow BFUe of normal mice. The purpose of the present study was to determine the effects of short-term oral AZT treatment in immune deficient animals. LP-BM5 MuLV-infected and normal mice were given 0, 1, and 2.5 mg/ml of AZT in their drinking water. Mice were killed after 2, 4, 8, 15, and 30 days of AZT treatment. The hematocrits of all AZT-treated mice decreased in a dose- and time-dependent fashion. AZT treatment decreased the absolute numbers of circulating reticulocytes in both normal and infected mice after 4 days of treatment. In contrast, the percentage of bone marrow early erythroblasts was increased in both normal and infected animals after 4 days of treatment. AZT at both doses decreased the number of BFUe per femur in both infected and normal mice after 2, 4, and 8 days. However, after 15 days the number of bone marrow BFUe increased. In spleen, the numbers of BFUe were increased only with high-dose AZT in both normal and infected mice at all time points, although the increases were more dramatic in infected mice. Our results indicate that the effect of AZT on bone marrow BFUe is time dependent, with inhibition being observed only at early time points. These results further demonstrate the complex effects of AZT on erythropoiesis in vivo.