Increased polyamines may downregulate interleukin 2 production in rheumatoid arthritis.

Polyamines downregulate immune reactivity. RA is associated with decreased IL 2 production. In this study, we present evidence to suggest that excessive polyamines can contribute to the IL 2 deficiency in RA. Blocking polyamine production with inhibitors of ornithine decarboxylase results in increased IL 2 production by RA PBMC. Moreover, polyamine oxidase (PAO) inhibitors and catalase also increase IL 2 production by RA PBMC. This effect of PAO inhibition is monocyte mediated. After 3 d in culture, RA PBMC produce three times more IL 2 than do normal PBMC. This rise is prevented by exogenous spermidine but only in the presence of monocytes. The concentration of polyamines in RA PBMC and synovial fluid MNC is 2-20-fold higher than in normal cells. Thus, polyamines and their oxidation products downregulate IL 2 production by RA PBMC and may account for the decreased T cell effector function seen in this disease.

Effect of polyamine depletion in vivo by DL-alpha-difluoromethylornithine on functionally distinct populations of tumoricidal effector cells in normal and tumor-bearing mice.

The objective of the present investigation was to examine the effect of in vivo polyamine depletion by DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on cell-mediated tumoricidal activity in normal and tumor-bearing (B16 melanoma) mice. DFMO treatment in vivo for 6 days reduced splenic leukocyte polyamine levels and the induction of cytotoxic T-lymphocytes (greater than 50%) in both normal and tumor-bearing mice. However, substantially less inhibition was observed in the ability to generate cytotoxic T-lymphocytes following 18 days of DFMO treatment. In contrast, DFMO treatment for 6 or 18 days did not impair splenic natural cell-mediated cytotoxicity, assessed against natural killer sensitive YAC-1 target cells and natural cytotoxic sensitive WEHI-164 target cells, in normal or tumor-bearing mice. Natural cell-mediated cytotoxicity was not observed against fresh B16 melanoma cells. However, macrophage-mediated tumoricidal activity directed against B16 melanoma cells was augmented 79% following 6 but not 18 days of DFMO treatment. These results demonstrate that DFMO can exert very selective effects on functionally distinct populations of antitumor effector cells in vivo depending upon the schedule of DFMO administration.

Potentiation of human lymphokine-activated killer cell activity by swainsonine, an inhibitor of glycoprotein processing.

Interleukin 2 (IL-2) is a secreted glycoprotein which acts as an activation and proliferative signal for lymphocytes expressing membrane-bound glycoprotein IL-2 receptors. We have recently established that swainsonine (SW), an inhibitor of mannosidase II during N-linked glycoprotein processing, augmented mitogen-induced mononuclear leukocyte IL-2 receptor expression and IL-2-induced proliferation. The objective of the present investigation was to examine the effect of SW on lymphokine-activated killer (LAK) cell induction. Human mononuclear leukocytes were treated with various concentrations of SW (0.1-10 micrograms/ml) and IL-2 (1-100 units/ml) for up to 72 h. SW augmented IL-2-induced LAK activity directed against human lung carcinoma, melanoma, and leukemia cells 2-3-fold. LAK activity generated in the presence of SW at suboptimal doses of IL-2 (10 units/ml) was similar to that observed with higher concentrations of IL-2 (100 units/ml) alone. SW treatment alone or in combination with IL-2 increased the percentage of IL-2 receptor-positive cells. Furthermore, pretreatment with SW subsequently enhanced IL-2-induced lymphocyte proliferation. SW-treated mononuclear leukocytes exhibited an increase in high-mannose type glycoproteins based upon [3H]mannose labeling, susceptibility to alpha-mannosidase, and binding to concanavalin A-Sepharose. These results indicate that modulators of glycoprotein processing may be useful in lowering the concentrations of IL-2 required for LAK induction and maintenance.

Potentiation of natural killer cell activity and tumor immunity by diacetylputrescine.

The objective of the present investigation was to evaluate the immunomodulating properties of tetramethylenebisacetamide (N,N' 1-diacetylputrescine, DAP), a known inducer of cellular differentiation. We examined the effect of DAP administration in vivo on splenic and nonadherent peritoneal natural killer (NK) cell activity. A single i.p. injection of DAP (100 mg/kg) enhanced cytolytic activity directed against YAC-1 and MCA-38 tumor target cells 2- to 3-fold. Cytolytic activity peaked 3 days following DAP injection. DAP treatment increased the frequency of asialo-GM1-positive splenocytes to 15% compared with 5% for vehicle treated controls. Furthermore, cytolytic activity could be eliminated by treatment with anti-asialo-GM1 antibodies and complement. Lysis of NK-resistant P815 and EL4 tumor target cells was not observed in leukocytes from DAP-treated mice. DAP treatment of mice given injections i.p. of MCA-38 tumor cells increased survival time of the mice by 37%, curing 10% of the animals of the tumor. DAP treatment of mice given injections intrasplenically of MCA-38 tumor cells reduced both the number and the size of the hepatic metastases. The antitumor effect of DAP in vivo could be eliminated by pretreating mice with anti-asialo-GM1 antibodies or utilizing NK cell deficient beige (bg/bg) mice. These results indicate that the observed anti-tumor activity of DAP is mediated, at least in part, by NK cells.

Participation of T-lymphocytes in the curative effect of a novel synthetic polyamine analogue, N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine, against L1210 leukemia in vivo.

We have recently established that combination therapy with N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine (BEPH), a synthetic polyamine analogue, and N,N'-bis-2,3-butadieneyl-putrescine, a polyamine oxidase inhibitor, eradicated L1210 leukemia in mice and induced resistance to a subsequent L1210 challenge. We now demonstrate that BEPH treatment alone, given on a more frequent schedule (5 mg/kg, day 3, 4, 5) or at a higher dose (10 mg/kg, day 3, 4), cures 100% of L1210 leukemic mice. These treated animals were subsequently immune to a second challenge with L1210 tumor cells. However, mice cured with BEPH did not reject P388 leukemic cells, although their mean survival time was slightly prolonged. In an in vivo tumor neutralization assay, splenocytes from cured mice and L1210 cells were injected into naive mice; 80% did not develop L1210 leukemia. Coculturing lymphocytes from cured mice with L1210 cells in vitro generated a potent tumor-specific cytolytic response against L1210 target cells, whereas lymphocytes from naive mice did not generate any significant cytolytic activity. Both the in vitro and in vivo activities were completely eliminated by pretreating the splenic lymphocyte population with anti-Thy-1.2 monoclonal antibodies and complement, indicating T-cells as the effector population. In T-cell-deficient nude mice BEPH treatment was not curative, increasing survival time by approximately 2-fold. We conclude from these studies that T-cell-mediated immunity plays a pivotal role in the mechanism by which synthetic polyamine analogues, such as BEPH, prevent neoplastic growth.

Effects of three irreversible inhibitors of ornithine decarboxylase on macrophage-mediated tumoricidal activity and antitumor activity in B16F1 tumor-bearing mice.

The objective of the present investigation was to compare the effects of three ornithine decarboxylase inhibitors on tumoricidal macrophage and antitumor activities in vivo. alpha-Difluoromethylornithine (DFMO), (2R,5R)-6-heptyne-2,5-diamine, and alpha-(fluoromethyl)dehydroornithine methyl ester (delta MFMOme) were administered continuously in drinking water starting on Day 1 to B16F1 tumor-bearing mice. DFMO, (2R,5R)-6-heptyne-2,5-diamine, and delta MFMOme reduced B16F1 tumor growth, measured on Day 18, up to 87, 79, and 95%, respectively. Similarly, all three ornithine decarboxylase inhibitors reduced B16F1 putrescine and spermidine levels. delta MFMOme was substantially more effective both as an antitumor agent and in reducing polyamines. Both DFMO and delta MFMOme augmented macrophage tumoricidal activity directed against B16F1 target cells. MAP had no effect on macrophage tumoricidal activity. Lipopolysaccharide-stimulated macrophages from delta MFMOme-treated mice also exhibited an increase in interleukin and tumor necrosis factor levels. Furthermore, treatment with a known macrophage activator, gamma-interferon, enhanced the antitumor activity of delta MFMOme. delta MFMOme did not alter natural killer cell activity; however, cytolytic T-lymphocyte induction was reduced by 40 to 50%. These results demonstrate that, in addition to their established antitumor activity, ornithine decarboxylase inhibitors may also potentiate specific tumoricidal effector cell generation in vivo.

Potent antitumor activity of a novel nucleoside analogue, BCH-4556 (beta-L-dioxolane-cytidine), in human renal cell carcinoma xenograft tumor models.

Beta-L-Dioxolane-cytidine (BCH-4556) is a novel anticancer nucleoside analogue with a stereochemically unnatural beta-L configuration. This compound was previously shown to have a potent antitumor activity in human prostate and hepatocellular xenograft tumor models (K. L. Grove et al., Cancer Res., 55: 3008-3011, 1995). Herein, we extended the efficacy validation of BCH-4556 to renal cell carcinoma (RCC) cell lines both in vitro and in vivo. In vitro cytotoxicity and proliferation inhibition determinations in human RCC cell lines CAKI-1, CAKI-2, 786-0, and A498 produced IC50 concentrations ranging from 15-35 nM. In vivo antitumor activity was consistent with the in vitro sensitivity. BCH-4556 was very effective in human RCC tumor xenograft models, including CAKI-1, A498, RXF-393, and SN12C carcinomas. Very good responses were observed in animals bearing CAKI-1, A498, and RXF-393 RCC tumors given i.p. doses of 10, 25, and 50 mg/kg twice a day for 5 days, with complete regression recorded in most of the animals tested. Curative activity was also observed, with 40-60% of animals remaining tumor free in all three RCC models at the day of study termination. Significant tumor shrinkage was also evident in the SN12C model. BCH-4556 efficacy evaluation in the orthotopic subrenal capsule tumor models demonstrated a potent tumor growth inhibition against human CAKI-1 xenografts and tumor stasis against mouse Renca tumors. BCH-4556 was also effective in inhibiting the growth of rebound CAKI-1 tumors after the administration of a second treatment cycle. The observed antitumor activity of BCH-4556 in several RCC human solid tumor xenografts, including the lethal RXF-393 model, warrants further investigation of this novel nucleoside analogue in clinical trials of RCC.

6-0-butanoylcastanospermine (MDL 28,574) inhibits glycoprotein processing and the growth of HIVs.

The antiviral activity of 6-0-butanoylcastanospermine (MDL 28,574) [50% inhibitory concentration (IC50: 1.1 microM)] in JM cells infected with a recent isolate of HIV-1 (GB8), was compared with other inhibitors of glycoprotein-processing enzymes. N-butyldeoxynojirimycin (BuDNJ), deoxynojirimycin (DNJ), castanospermine (CAST) or the reverse transcriptase inhibitor 2'3'-dideoxycytidine (ddC) had activities of 56, 560, 29 and 0.1 microM, respectively. MDL 28,574 was at least 50 times more active than BuDNJ and less active but better tolerated in cell culture than ddC, two compounds currently undergoing clinical trials. The CAST derivative showed good protection in H9 cells infected with HIV-1 (RF; IIIB; U455), and HIV-2 (ROD), although the potency was less than that seen in the JM/GB8 system. HIV-1 glycoproteins, gp160 and gp120, synthesized in H9 cells chronically infected with HIV-1 (RF) and treated with MDL 28,574, were characterized by an increase in relative molecular weight of approximately 7-8000 kD. The ratio of gp120 to gp160 was markedly reduced in treated cells and provided further evidence that cleavage of the gp160 precursor molecule is a major consequence of the inhibition of glycoprotein processing. The intracellular target for MDL 28,574 was verified as alpha-glucosidase-I of the processing enzymes by the analysis of high-glucose glycopeptides recovered from treated mouse cells. This activity correlated with the antiviral effect observed against the growth of a mouse retrovirus, Moloney murine leukemia virus (MOLV), in mouse cells.

Selection and characterization of HIV-1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC).

Human immunodeficiency virus type 1 (HIV-1) variants were selected for resistance against the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC), using the infectious molecular clone HXB2D grown in the MT-4 line of human T cells. The variants selected with (+) dOTC were approximately 6-7-fold less sensitive than wild-type virus to this drug. Cloning and sequencing of the complete reverse transcriptase (RT) coding region of these variants identified the M1841 mutation and further selection with virus containing the M1841 substitution led to the appearance of an M184V mutation. In contrast, selection experiments performed with (-) dOTC yielded variants capable of growing in drug concentrations as high as 100 microM, but phenotypic analysis of these viruses revealed near wild-type 50% inhibitory concentration (IC50) values for this compound. Site-directed mutagenesis experiments in which the M1841 and M184V mutations were introduced into HXB2D confirmed the importance of these mutations when viruses were grown in MT4 cells. However, wild-type IC50 values in regard to both (-) and (+) dOTC were obtained when these recombinant viruses were grown in cord blood mononuclear cells (CBMC). Clinical isolates of HIV-1 resistant to lamivudine and containing the M184V substitution also displayed low-level resistance to both (-) and (+) dOTC when grown in CBMC. Finally, cell-free RT assays were performed in the presence of either (-) dOTC triphosphate, (+) dOTC triphosphate, or the triphosphate of a racemic mixture of (+) and (-) dOTC with wild-type and mutated M184V-containing recombinant RT. The data demonstrate chain termination effects of these compounds with regard to both wild-type and mutated enzyme and that the latter was approximately twofold less sensitive than the former to these drugs.

4',5'-unsaturated 5'-halogenated nucleosides. Mechanism-based and competitive inhibitors of S-adenosyl-L-homocysteine hydrolase.

The design and synthesis of (E)- and (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (6 and 13, respectively), a new class of mechanism-based inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase, is described. A number of analogues of 6 and 13 were synthesized in order to determine the structure-activity relationship necessary for inhibition of the enzyme. Substitution of chlorine for fluorine in 6 (i.e. 44), addition of an extra chlorine to the 5'-vinyl position (i.e. 51 and 52), modification of the 2'-hydroxyl group to the deoxy (34 and 35) and arabino (36 and 37) nucleosides provided competitive inhibitors of SAH hydrolase. Nucleosides 6 and 13, as well as 5'-deoxy-5',5'-difluoroadenosine (14) proved to be time-dependent inhibitors of SAH hydrolase. All three compounds are postulated to inhibit through the potent electrophile derived from oxidation of the 3'-hydroxyl of 6 or 13 to the ketone (i.e. 3 and/or the E-isomer). Consistent with the proposed mechanism of inactivation of SAH hydrolase by 6, 13, and 14 was the observation that incubation of purified rat liver SAH hydrolase with 6 resulted in release of 1 equiv of fluoride ion (by 19F NMR) and incubation with 14 resulted in release of 2 equiv of fluoride ion. The general synthetic route developed for the synthesis of the title nucleosides utilized the fluoro Pummerer reaction for the introduction of fluorine into the requisite precursors. Preliminary antiretroviral data from Moloney leukemia virus (MoLV) is presented and correlates with SAH hydrolase inhibition. Antiviral activity (IC50 against MoLV) ranged from 0.05 to 10 micrograms/mL.

Carbocyclic nucleosides as inhibitors of human tumor necrosis factor-alpha production: effects of the stereoisomers of (3-hydroxycyclopentyl)adenines.

A series of four structurally related carbocyclic nucleosides (6a, 6b, 10a, and 10b) were synthesized and evaluated for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) production from human primary macrophages. These compounds had little effect on the production of IL-1 beta and IL-6. It was determined that compound 10a was the most potent inhibitor of TNF-alpha production (IC50 = 10 microM), having 2-5-fold more activity compared to its enantiomer 10b or its diastereomers 6a and 6b. In addition, these compounds were also tested for their ability to protect mice against lethal challenges of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Compound 10a showed superior protective effects (100% protection) compared to its enantiomer 10b or its diastereomers 6a and 6b when it was administered to mice which were challenged with 3 times the LD100 dose of LPS.

Disruption of Plasmodium falciparum-infected erythrocyte cytoadherence to human melanoma cells with inhibitors of glycoprotein processing.

Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule-1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600-700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM-1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis.

A high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors.

A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 has been developed for human cytomegalovirus (HCMV) antiviral susceptibility testing and adapted to a microtiter plate format. Optimal conditions were determined and the standard routine assay was calibrated with a viral input of 0.05-0.10 plaque forming unit (p.f.u.)/cell with a density of 2000 cells/well in a 96-well microtiter plate for an incubation period of 7 days. Ganciclovir (9-(2-hydroxy-1(hydroxymethyl) ethyoxymethyl) guanine; DHPG), and cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; HPMPC) were used as positive control test compounds to validate the assay. The effective EC50 concentration values obtained with the two antiviral compounds in the present assay were in good agreement with plaque reduction assay results performed in parallel experiments. This method presents the advantage of being easy and rapid to perform, reliable, reproducible, and convenient for use in a high throughput screening capacity.

Inhibition of glycoprotein processing and HIV replication by castanospermine analogues.

Inhibitors of glycoprotein processing enzymes have been shown to have activity against HIV. Several analogues of the known glucosidase I inhibitor, castanospermine (CAST), were synthesized and evaluated for their inhibitory effect on glucosidases and for antiviral activity against Moloney murine leukemia virus (MOLV) and HIV-1. The most effective analogue was 6-O-butanoyl CAST (B-CAST, MDL 28,574) with an IC50 of 0.05 micrograms/mL against MOLV. A correlation between inhibition of glucosidase I and MOLV replication was observed. This analogue was further evaluated against HIV-induced syncytial formation in HeLa T4+ cells and against productive infection in JM cells infected with HIV 1 (GB8 strain). B-CAST showed an IC50 of 0.3 micrograms/mL in the HeLa T4+ assay, compared to CAST at 11 micrograms/mL. The compound also was more potent (IC50:0.15 micrograms/mL) than CAST (4-6 micrograms/mL) in JM cells. The antiretroviral activity of B-CAST was further confirmed in Friend leukemia virus (FLV) infection in mice. B-CAST showed equivalent activity to AZT and was more potent than CAST in inhibiting FLV-induced splenomegaly in mice. The data presented herein suggest the potential of these novel glucosidase inhibitors as anti-HIV agents.

Identification of a specific receptor for interleukin-1 in vascular smooth muscle cells: regulation by interleukin-1 and interleukin-6.

A fluorescently labeled ligand was utilized to establish the existence of an interleukin-1 (IL-1) receptor in vascular smooth muscle. The binding of the phycoerythrin-labeled IL-1 beta to the murine T cell line, EL-4, was examined as a positive control. The phycoerythrin-labeled IL-1 beta identified a specific IL-1 receptor in the EL-4 cells. Vascular smooth muscle cells were also positively stained by the fluorescent ligand. The binding of phycoerythrin-labeled IL-1 beta to these cells was saturable and reversed by 100-fold excess unlabeled IL-1 beta. Incubation of the vascular smooth muscle cells with IL-1 beta (25 ng/ml) or IL-6 (250 ng/ml) for 18 h increased and decreased, respectively, the percentage of cells positively stained by phycoerythrin-labeled IL-1 beta which suggests these cytokines regulate IL-1 receptor expression in these cells. These data indicate a specific receptor for IL-1 exists in vascular smooth muscle cells.

Activation of adenosine A3 receptors on macrophages inhibits tumor necrosis factor-alpha.

Murine macrophage-derived tumor necrosis factor alpha (TNF-alpha) gene expression has been shown to be dramatically induced by bacterial lipopolysaccharide, and to be dependent upon nuclear factor-kappa B (NF-kappa B) binding sites in its promoter for the lipopolysaccharide induction. Murine J774.1 macrophage cells were found to predominantly express the adenosine A3 receptor RNA relative to adenosine A1 receptor or adenosine A2 receptor RNA. Adenosine receptor agonists, in a dose-dependent manner characteristic of the adenosine A3 receptor, blocked the endotoxin induction of the TNF-alpha gene and TNF-alpha protein expression in the J774.1 macrophage cell line. The adenosine A3 receptor antagonist BW-1433 dose-dependently reversed this adenosine inhibitory effect on TNF-alpha gene expression. Thus, the binding of adenosine receptor agonists to the adenosine A3 receptor interrupts the endotoxin CD14 receptor signal transduction pathway and blocks induction of cytokine TNF-alpha, revealing a novel cross-talk between the murine adenosine A3 receptor and the endotoxin CD14 receptor in J774.1 macrophages.

Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC].

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.

Antiretroviral activity of mechanism-based irreversible inhibitors of S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA), (E)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (EDDFA), (Z)-4',5'-didehydro-5'-deoxy-5'-chloroadenosine (ZDDCA) and 5'-deoxy-5'-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, greater than 10.0 and 3.30 microM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC50 values of 0.05, 0.25 and 3.30 micrograms/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT1/2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 microgram/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 microgram/ml) treated C3H1OT1/2 cells. Incorporation of a [3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT1/2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.

Antitumor activity of norspermidine, a structural homologue of the natural polyamine spermidine.

The structural specificities of the natural polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) for cell growth are rather stringent, suggesting that appropriate structural analogues of these polycations could serve as potential antineoplastic agents via polyamine antagonism. Norspermidine (Nspd), a homologue of spermidine, had significant antitumor activity against L1210 leukemia, 3LL carcinoma and EL4 lymphoma in mice. The observed antitumor activity of the compound was potentiated by administration of a - difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO treatment alone, or in combination with Nspd reduced tumoral Put and Spd levels by greater than 50% in all three tumor models. In animals receiving both Nspd and DFMO, Nspd accumulation in the tumor cells was increased by 50% or more compared to cells from animals receiving Nspd only. Co-administration of Spd, but not Put, abolished the antitumor activity of L1210 observed with DFMO and Nspd treatment, and also reduced the tumoral accumulation of Nspd. These results indicate that appropriate structural analogues of the natural polyamines may be useful as antineoplastic agents.

Antitumor activity of a novel synthetic polyamine analogue, N,N'-bis-[3-(ethylamino)-propyl]-1-7-heptane diamine: potentiation by polyamine oxidase inhibitors.

The requirement of the natural polyamines, putrescine, spermidine and spermine, for cell growth suggests that appropriate structural analogues of these compounds could serve as potential antiproliferative agents acting via polyamine antagonism. In this investigation, the antiproliferative activity of N, N'-Bis[3-(ethylamino)-propyl]-1-7-heptane diamine (BEPH), a synthetic polyamine analogue, was investigated employing HeLa cells in culture and L1210 leukemia in mice. BEPH inhibited the growth of HeLa cells with an IC50 of 0.25 microM during a four day culture period. This concentration of the compound was cytotoxic to the cells as evidenced by an 80% reduction in cloning efficiency. Only marginal changes in intracellular polyamine concentrations were observed during incubation with 0.25 microM BEPH. In both HeLa cells and L1210 cells in culture, incorporation of radioactive precursors into DNA, RNA and protein were reduced by BEPH. Inhibition of protein synthesis was discernible prior to inhibition of RNA and DNA in these cells. In mice inoculated i.p. with 10(5) L1210 cells on day 0, i.p. administration of 10.0 mg/kg of BEPH qd(X5) beginning on day 1 prolonged the survival time by 84% compared to controls. The same dose of the compound, in combination with 10.0 mg/kg of N,N'-bis-2-3-butadienylputrescine, an inhibitor of the polyamine catabolizing enzyme polyamine oxidase (PAO), produced a 100% cure rate. Similar results were obtained when BEPH was combined with N-methyl-N'-2-3-butadienylputrescine, another PAO inhibitor. Furthermore, animals cured of the leukemia by the combination chemotherapy were resistant to a subsequent challenge with L1210 cells, indicating the development of tumor "immunity". The striking antitumor activity along with the development of tumor immunity indicate that synthetic polyamine analogues have potential for development as antineoplastic agents.