Inhibition of murine AIDS by pro-glutathione (GSH) molecules.

Antioxidant molecules can be used both to replenish the depletion of reduced glutathione (GSH) occurring during HIV infection, and to inhibit HIV replication. The purpose of this work was to assess the efficacy of two pro-GSH molecules able to cross the cell membrane more easily than GSH. We used an experimental animal model consisting of C57BL/6 mice infected with the LP-BM5 viral complex; the treatments were based on the intramuscular administration of I-152, a pro-drug of N-acetylcysteine and S-acetyl-beta-mercaptoethylamine, and S-acetylglutathione, an acetylated GSH derivative. The results show that I-152, at a concentration of 10.7 times lower than GSH, caused a reduction in lymph node and spleen weights of about 55% when compared to infected animals and an inhibition of about 66% in spleen and lymph node virus content. S-acetylglutathione, at half the concentration of GSH, caused a reduction in lymph node weight of about 17% and in spleen and lymph node virus content of about 70% and 30%, respectively. These results show that the administration of pro-GSH molecules may favorably substitute for the use of GSH as such.

The interaction of phosphorylated sugars with human hexokinase I.

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.

Inhibition of murine retrovirus-induced immunodeficiency disease by dideoxycytidine and dideoxycytidine 5'-triphosphate.

The antiretroviral activity of many nucleoside analogues depends not only on their ability to inhibit the virus reverse transcriptase but also on the specific cellular pools of natural deoxynucleosides and on the level of the enzymes responsible for their phosphorylation. In an attempt to overcome these limitations, we have tested the efficacy of the oral administration of 2',3'-dideoxycytidine (DDC) and the administration of its phosphorylated derivative, 2',3'-dideoxycytidine 5'-triphosphate (DDCTP) encapsulated into autologous red blood cells in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). The results obtained showed that both single treatments are quite effective in preventing the typical signs of MAIDS. Combined treatment with both oral DDC and encapsulated DDCTP yields an additive response in some, but not all the parameters investigated. Furthermore, animals receiving the simultaneous administration of DDC and DDCTP show a reduction of animal body weight, a persistent high concentration of IgM, and a high titer of anti-LP-BM5 gag immunoglobulins. Thus, the administration of the same drug in different molecular forms and/or with different delivery systems should be carefully evaluated in preclinical animal models because of the unpredictability of the effects of these treatments from the conclusion drawn by studies on single treatment.

Human erythrocyte phosphoglucomutase: comparison of the kinetic properties of PGM1 and PGM2 isoenzymes.

Kinetic properties of PGM1 and PGM2 phosphoglucomutase "primary" isoenzymes from human erythrocytes were studied. The two enzyme forms share a "ping-pong" kinetic mechanism and show similar Km for substrate (glucose 1-P) and cofactor (glucose 1,6-P2). Micromolar concentrations of fructose 1,6-P2 and glycerate 2,3-P2 inhibit both PGM1 and PGM2 isoenzymes to a similar extent. The sole PGM2 form is affected by ribose monophosphates (ribose 1-P and ribose 5-P) that act as mutase inhibitors vs. glucose 1,6-P2 and as apparent activators vs. glucose 1-P. The interaction between PGM2 isoenzyme and ribose monophosphates is discussed in the light of the ability of this form to also display phosphoribomutase activity.

Antiretroviral effect of combined zidovudine and reduced glutathione therapy in murine AIDS.

A combination of antiretroviral drugs acting at different points in the virus replication cycle was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). Intramuscular administration of high doses of reduced glutathione (GSH, 100 mg/mouse/day) and AZT (0.25 mg/ml in drinking water) was found to reduce lymphoadenopathy (92%), splenomegaly (80%), and hypergammaglobulinemia (90%) significantly more than AZT alone. Combined treatment resulted in a reduction in proviral DNA content of 69, 66, and 60%, respectively, in lymph nodes, spleen, and bone marrow. Furthermore, the stimulation index of B cells was also significantly higher in animals receiving GSH and AZT whereas additional responses were not observed in the T cell stimulation index and blood lymphocyte phenotype analyses. In conclusion, the administration of high doses of GSH and AZT, a new combination of antiviral drugs, seems to provide additional advantages compared to single-agent therapy.

Inhibition of murine AIDS by reduced glutathione.

The imbalance of the redox state in cells and body fluids in HIV-1-infected patients may result in progression of the disease as well as in immunologic disfuctions. In this report, we have evaluated whether the direct administration of high doses of reduced glutathione (GSH) exerts any antiviral activity and/or improves immune functions in a murine immunodeficiency animal model. Intramuscular administration of 50 or 100 mg GSH/mouse for five consecutive days weekly to LP-BM5-infected mice did not show local or systemic signs of acute toxicity. During the first 3 weeks from infection, a period in which clinical signs of disease were not yet detectable, GSH significantly reduced the viral load in lymph nodes and spleen as evaluated by a PCR semiquantitative assay of the proviral DNA content. At 10 weeks a GSH concentration-dependent reduction of splenomegaly, lymphadenopathy and hypergammaglobulinemia was evident in all treated mice. Evaluation of proviral DNA content showed that GSH was effective in inhibiting LP-BM5 infectivity in lymph nodes, spleen, and bone marrow at 100 mg/day, while it was less effective when administered at 50 mg/day. At 10 weeks some animals receiving the highest GSH dose died, thus only the mice receiving 50 mg GSH were followed up to 15 weeks without signs of toxicity. In this case, almost not significant differences among infected untreated or treated animals were observed. Thus, GSH is effective in reducing the proviral DNA load in the first period of infection. These data and the failure of sulfhydril supplementation to further counteract the progression of disease after 10 weeks of infection suggest that combinations of GSH and other antiviral agents may be useful for improving current antiviral therapies.

Macrophage protection by addition of glutathione (GSH)-loaded erythrocytes to AZT and DDI in a murine AIDS model.

Monocyte-macrophages play a central role in HIV-1 infection because they are among the first cells to be infected and because later they are important reservoirs for the virus. Thus, newly designed therapies should take into account the protection of this cell compartment. Herein, we report the results obtained in a murine AIDS model, by the addition to AZT+DDI of a system (GSH-loaded erythrocytes) able to protect macrophages against HIV-1 infection. Five groups of LP-BM5-infected mice were treated as follows: one group was treated by AZT, one group was treated by DDI, one group was treated by the combination of both, another by GSH-loaded erythrocytes, and finally, one by the combination of all three. After 10 weeks of infection the parameters of the disease were studied and the proviral DNA content in different organs and in macrophages of bone marrow and of the peritoneal cavity was quantified. The results obtained show that mice treated with AZT+DDI+GSH-loaded erythrocytes showed proviral DNA content in the brain and in macrophages of bone marrow that was significantly lower than in mice treated with AZT+DDI. This study may help developing strategies aimed at blocking HIV-1 replication in its reservoirs in the body.

2',3'-Dideoxycytidine induced drug resistance in human cells.

2',3'-Dideoxycytidine (ddC) is a nucleoside analogue that inhibits HIV-1 replication in vitro and is currently used in AIDS therapy. This compound exerts a delayed cytotoxicity due to inhibition of mitochondrial DNA (mDNA) synthesis. We have found that long term exposure of U937 human monoblastoid cells to ddC allowed the selection of a drug-resistant cell line (U937-R) with 66% mDNA, normal ddC transport and altered deoxycytidine kinase kinetic properties. In this paper we show that U937-R cells contain an increased number of mitochondria per cell and a reduced copy number of mDNA/mitochondria. Furthermore, the intracellular concentrations of deoxycytidine 5'-triphosphate (dCTP) and 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) are also reduced although with a higher dCTP/ddCTP ratio in U937-R compared to the parental cells. This mechanism of drug resistance, with drug-resistance based on viral mutations, can provide an explanation for drug failure in antiviral therapy.

In vitro and in vivo toxicity of 2',3'-dideoxycytidine in mice.

3T3 mouse embryo fibroblast cell growth was inhibited in a concentration dependent manner by 2',3'-dideoxycytidine (ddCyd), a strong inhibitor of human immunodeficiency virus. Cell growth inhibition was associated with an increased incorporation of ddCyd into cell DNA. In contrast SP2/0-Ag14 (a mouse myeloma) cell growth is not inhibited by 100 microM ddCyd both in the presence or absence of hypoxanthine and thymidine. Furthermore, in vitro spleen cell proliferation, upon phytohemagglutinin (PHA) addition, was much more affected by ddCyd in C57BL/6 mice than in Swiss albino mice. That indeed ddCyd affects spleen cell proliferation was confirmed by studies on splenocytes obtained from C57BL/6 mice that received ddCyd for 2 weeks in drinking water. These results suggest that ddCyd toxicity in mice is cell and strain dependent and that the toxicity mechanism is related to the incorporation of the drug in cell DNA.

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.

Quantitation of electrophoretic eluted proteins.

Quantitation of stained, electroeluted proteins by the classical Lowry and Bradford protein assay is not possible because of some different interferences. In particular we have found that the substance interfering in the Lowry method cannot be removed by trichloroacetic acid precipitation nor can be compensated for by the appropriate blank. Interferences in the Bradford protein assay are due to detergents and pH of the protein buffer as well as to Coomassie brilliant blue R250 electroeluted with the protein sample. However, while these interferences can be compensated for by appropriate blank and standard curves, others (probably due to acrylamide fines) cannot be corrected. All these problems can be overcome by concentration and dialysis of electroeluted samples which permit the removal of interfering substances and the use of Bradford and Lowry protein assay in the 1-20 micrograms range, respectively. Successful applications are described for electroeluted bovine serum albumin, human hexokinase and phosphoglucomutase.

Role of hexokinase in the regulation of glucose metabolism in human erythrocytes.

Red blood cell glucose metabolism was studied in erythrocytes from a patient with trisomy 10 p which resulted in + 50% hexokinase specific activity, in normal controls and in cases of heterozygous hexokinase deficiency. The results obtained show that the hexokinase activity level is an important factor in the control of the erythrocyte's glycolytic rate while having no appreciable effect on the hexose monophosphate pathway under resting conditions. No clear conclusion could be drawn when an oxidative stress was present.