Anti-HIV-1 activity in vitro of ceftazidime degradation products.

Cephalosporins in aqueous solutions generate degradation products that inhibit in vitro HIV-1 replication in cell lines, as well as in primary cells (lymphocytes and macrophages). This effect is observed at concentrations that do not interfere with the normal functions of these cells. Upon chromatographic fractionation of an aqueous solution of hydrolysed ceftazidime, a high molecular weight fraction (MW 8000) with antiviral activity was isolated. The exact chemical nature of the active component responsible for the anti-HIV activity in vitro appears to be complex and is currently unknown. Inhibition of HIV-1 reverse transcriptase and RNase H activity was observed, however, higher concentrations than those needed to inhibit HIV replication were required. The inhibitory action of the hydrolysed ceftazidime was manifested during the early phase of the HIV-1 life-cycle. Despite a lack of a direct effect of the CD4/gp120 interaction, HIV-1 mediated cell fusion was inhibited by the hydrolysed ceftazidime, suggesting that the active principle acts in a very early stage of the viral life-cycle.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Consequences of the inhibition of Hdm2 expression in human osteosarcoma cells using antisense oligonucleotides.

The present study was performed to identify a potent and sequence-specific antisense oligonucleotide (ASO), to inhibit Hdm2 expression in human cancer cell lines and to study the downstream consequences. Ten chimeric 2'-O-methoxyethyl (MoE)-modified hemimers were synthesized that targeted various regions from the 5'- to the 3'-end of Hdm2 mRNA. The IC50 of the most potent ASO, NCH-4401, was subsequently determined and compared to the IC50 of a 2'-MoE-modified ASO, with a complete phosphorothioate backbone (NCH-4668), and to a 3 bp mismatched ASO (NCH4529). NCH4401 inhibited Hdm2 expression in SJSA-1 cells with an IC50 of 120 nm, whereas NCH-4668 was less potent with an IC50 of 180 nm. The mismatched control ASO was completely inactive, indicating a sequence-dependent mechanism of action of NCH-4401. NCH4401 was subsequently used to study the consequences of inhibiting Hdm2 expression in human osteosarcoma cells. NCH-4401 completely inhibited Hdm2 protein expression in SJSA-1 cells at a concentration of 300 nm, already 4 h after start of ASO treatment. At an ASO concentration of 300 nM, p53 protein was induced 12.5-fold and p21 was induced 8-fold over background levels, 24 h after start of ASO treatment. The dramatic induction of p53 in SJSA-1 cells prompted us to investigate whether the accumulation of p53 in these cells was followed by induction of apoptosis. However, no signs for apoptosis were detected in SJSA-1 cells, following induction of wild-type p53 using the Yopro method and the induction of caspase-3 activity. SJSA-1 cells were subsequently treated with NCH-4401 at different concentrations in combination with two well-known DNA-damaging agents, i.e. carboplatin and mitomycin C. Apoptosis induction following treatment of cells with DNA-damaging agents and NCH4401 was determined in parallel by measuring caspase-3 activation and uptake of the DNA dye Yopro. Carboplatin and mitomycin C together only slightly induced apoptosis in SJSA-1 cells to a factor of approximately 2-fold, as measured by the induction of caspase-3 activity. The downregulation of Hdm2 expression by NCH4401 did not induce apoptosis on its own and did not potentiate the mitomycin C/carboplatin-induced programmed cell death.

PCTAIRE-1: characterization, subcellular distribution, and cell cycle-dependent kinase activity.

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk) family whose function is unknown. We examined the pattern of PCTAIRE-1 protein expression in a number of normal and transformed cell lines of various origins and found that the kinase is ubiquitous. Indirect immunofluorescence indicated that PCTAIRE-1 exhibits cytoplasmic distribution throughout the cell cycle. Confocal microscopy showed that PCTAIRE-1 does not colocalize with components of the cytoskeleton or with the endoplasmic reticulum. We found that endogenous PCTAIRE-1 and ectopically expressed PCTAIRE-1 display kinase activity when myelin basic protein is used as an acceptor substrate. Similar to other members of the cyclin-dependent kinase family, PCTAIRE-1 seems to require binding to a regulatory subunit to display kinase activity. PCTAIRE-1 activity is cell cycle dependent and displays a peak in the S and G2 phases. We show that the low level of kinase activity observed until the onset of S phase correlates with elevated tyrosine phosphorylation of the molecule.

Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Profile of CGP 61755: a novel and potent HIV-1 protease inhibitor that shows enhanced anti-HIV activity when combined with other antiretroviral agents in vitro.

CGP 61755 is a novel hydroxyethylene derivative produced by a high yield 10 step chemical synthesis. It is highly specific for HIV-1 protease with an IC50 of 1 nM. The ED90 in MT-2, PBLs and macrophages is infected with laboratory strains of HIV-1 or clinical isolates is 30-100 nM. In chronically infected macrophages the ED90 is 1000 nM (1000 nM for saquinavir and 10 microM for indinavir). When the antiviral activity of CGP 61755 on HIV-1 infected lymphocytes was examined using serum free medium an ED99 of 60 nM was determined, while in the presence of 10% human serum the same activity was achieved with 120 nM. When examined in combination with RT inhibitors or protease inhibitors, either in a co-culture of CEM-SS and chronically infected H9IIIB cells or in a free virus lymphocyte infection, cooperativity of the antiviral activities was observed. Dog pharmacokinetic studies comparing p.o. and i.v. data indicate that CGP 61755 has a bioavailability between 50 and 80%. Following oral administration the area under the concentration curve (AUC) values increased in a dose proportional manner. The plasma levels of the drug at 6 hours after oral administration were above the ED90. Based on these properties we believe that CGP 61755 has an attractive profile that justifies further preclinical evaluation of the drug.

Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2.

Neu differentiation factor (NDF)-induced signaling involves the activation of members of the ErbB family of receptor tyrosine kinases. Although ectopic expression of recombinant ErbB receptors has yielded valuable insight into their signaling properties, the biological function and in vivo interplay of these receptors are still poorly understood. We addressed this issue by studying NDF signaling in various human cell lines expressing moderate levels of all known ErbB receptors. NDF-induced phosphorylation of ErbB-2 and ErbB-3 was found in the breast epithelial cell line MCF10A, the breast tumor cell lines T47D and MCF7, and the ovarian tumor cell line OVCAR3. Despite similar expression levels, NDF-induced phosphorylation of ErbB-4 was cell specific and only detected in T47D and OVCAR3 cells. Blocking cell surface expression of ErbB-2 by intracellular expression of a single-chain antibody revealed that in these two cell lines, ErbB-2 significantly enhanced phosphorylation of ErbB-4. Efficient NDF-induced phosphorylation of ErbB-3 was strictly ErbB-2 dependent in the breast tumor cell lines T47D and MCF7, while it was largely ErbB-2 independent in MCF10A and OVCAR3 cells. Consequently, NDF-stimulated intracellular signaling and induction of a biological response displayed a cell-specific requirement for ErbB-2. Thus, while ErbB-2 cooperates with NDF receptors in the breast tumor cell lines, ErbB-2 independent mechanisms seem to prevail in other cellular contexts.

The replicative restriction of lymphocytotropic isolates of HIV-1 in macrophages is overcome by TGF-beta.

In vitro exposure of human blood monocyte-derived macrophages to T-cell tropic human immunodeficiency virus (HIV) isolates fails to establish a productive viral infection. Several studies have shown that such preferential HIV-1 replication in T cells or in mononuclear phagocytes (HIV tropism) may be determined by distinct viral characteristics. In the present study it was demonstrated that transforming growth factor-beta (TGF-beta), a factor known to be produced by platelets, macrophages, and other cells present at a wound site, can act as a mediator in overcoming the lymphocytotropic restriction of several well-characterized viral isolates of HIV-1 (i.e., LAV, Z84, pLAI, NY5). Macrophages infected with these isolates show cytopathic changes comparable to those seen upon infection with the monocytotropic isolate ADA. To achieve this effect with TGF-beta, the factor must be present after the infection period. The emerging virus retains its original cellular tropism. Based on these observations the authors propose a role for TGF-beta in the establishment and progression of HIV infection and disease.

In vitro effect of transforming growth factor-beta on progression of HIV-1 infection in primary mononuclear phagocytes.

In vitro-differentiated monocytes can be infected with the monocytotropic isolate of HIV-1/ADA. The infection is characterized by formation of giant cells and production of virus that can be found in cell supernatants or cell-associated. In this study, we demonstrate that the above described parameters of infection can be enhanced by a factor present in acidified M phi supernatants, suggesting that it might be transforming growth factor beta-1 (TGF-beta 1). When recombinant or purified TGF-beta were examined, similar activities were detected. This effect apparently is not because of changes in the cellular phenotype that could favor infection. The effect of TGF-beta is exerted on cells once infection is established or on cells with active virus production. The activity can be also demonstrated using U-937 cells.

TGF-beta: upregulator of HIV replication in macrophages.

TGF-beta at physiological concentrations, when added to monocyte-derived macrophages following HIV1 infection, has an enhancing effect upon the rate of virus production. This effect is observed with the monocytotropic isolate ADA, as well as with HIV1 IIIB, which poorly replicates in macrophages.

The lipophilic muramyl peptide MTP-PE is a potent inhibitor of HIV replication in macrophages.

Cultured monocyte-derived macrophages were productively infected with human immunodeficiency virus in vitro. Treatment of these cells shortly after infection and several times thereafter with the free form of MTP-PE had an inhibitory effect on virus production. When the liposomal formulation of MTP-PE was used, higher levels of protection were achieved. The drug was not only effective when added to cells immediately after infection, but it also reduced virus production by cells with an established infection. When the liposomal formulation of MTP-PE was used only one treatment was required to achieve maximal effects. During these studies it was noted that the placebo liposomes had some effect in reducing the reverse transcriptase levels found in the supernatants of infected cells. This reduction could not be explained by direct cytotoxic effect. Both free and liposomal MTP-PE lipid significantly prevented formation of giant cells during the course of infection as well as reduced the cell associated viral antigen.

An assay for the detection of interleukin-1 synthesis inhibitors: effects of antirheumatic drugs.

The monokines interleukin-1 beta and alpha (IL-1) play a central role in the connective tissue destruction of many chronic inflammatory diseases. A high capacity screening assay for the detection of inhibitors of IL-1 biosynthesis has been established. Normal human monocytes were obtained by leukapheresis and elutriation. IL-1 beta and alpha biosynthesis was stimulated with LPS, and cell-associated and secreted IL-1 beta and IL-1 alpha were measured by specific immunoassays (ELISA). The mean total IL-1 beta (cell-associated and secreted) production in 18 different donors was 11 ng/10(6) cells (range 1.2-28.8). Secreted IL-1 beta represented 31 to 86% of the total IL-1 beta. More IL-1 alpha than IL-1 beta was produced but, unlike IL-1 beta, IL-1 alpha was poorly secreted. The steroids prednisolone and dexamethasone, gold (sodium aurothiomalate) and chloroquine were potent inhibitors of the IL-1 production. Mean IC50 values of 180 nM (range 2.5 nM-1 microns), 10 microM (range 6-20 microM) and of 75 microM were found for prednisolone, gold and chloroquine, respectively. Above 5 microM, the non-steroidal anti-inflammatory compounds indomethacin and BW755C increased IL-1 beta biosynthesis. Nordihydroguaiaretic acid inhibited the level of the secreted form of IL-1 beta, but tended to increase the cell-associated level. D-Penicillamine (up to 6 mM), cyclosporin A (up to 1 microM) and methotrexate (up to 12 microM) inhibited neither cell-associated nor secreted IL-1 beta levels. This high capacity assay, which is insensitive to classical NSAIDs, may serve in the detection and characterization of new classes of anti-inflammatory compounds.