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A Cerami

Publications and source records attributed to A Cerami.

At least 37 records · Page 2Linked to original sources
PubMed1997

Studies on the mechanism of antimalarial action of a novel arylene bis(methylketone).

2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (CNI-H0294) is a novel arylene bis(methylketone) compound that displays antimalarial activity against chloroquine- and pyrimethamine-resistant Plasmodium falciparum clones. The compound has been found to be concentrated into infected erythrocytes, with 80-179 microM accumulated when parasites were cultured in the presence of 1.0 microM CNI-H0294. Uninfected erythrocytes, in contrast, only accumulated 2.5-3.4 microM CNI-H0294 under identical conditions. Using postmitochondrial supernatants from a number of parasite clones, the compound was found to inhibit dihydrofolate reductase (EC 1.5.1.3) activity with an IC50 of 243-483 microM. Thus, while CNI-H0294 is not a powerful inhibitor of plasmodial dihydrofolate reductase, the accumulation of the compound into infected erythrocytes, when correlated to the external ED50 concentration against parasite growth in vitro, reaches concentrations sufficient to inhibit the malarial enzyme.

Animals↗
PubMed1997

Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response.

The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here, we addressed the hypothesis that spermine might counterregulate the innate immune response by downregulating the synthesis of potentially injurious cytokines. When spermine was added to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1alpha, and MIP-1beta. The inhibition of cytokine synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.

Animals↗
PubMed1997

Studies on the pharmacological properties of novel arylene bis(methylketone) compounds using solid-phase extraction and high-performance liquid chromatography.

A method utilising solid-phase extraction followed by high-performance liquid chromatography has been developed to quantify novel arylene bis(methylketone) chemotherapeutics present in biological samples. The samples are extracted over cyanopropylsilane solid-phase extraction cartridges using 10 mM heptanesulfonate-10 mM tetramethylammonium chloride-4.2 mM H3PO4-95% CH3CN as the eluent. Analytical chromatography utilises a diisopropyl-C8 reversed-phase column and a 7.5-45% CH3CN gradient in 10 mM heptanesulfonate-10 mM tetramethylammonium chloride-4.2 mM H3PO4-H2O. Detection was by ultraviolet spectrophotometry at 300 or 240 nm. The linear response of the assay was found to extend from at least 100 microg/ml down to 97.66 ng/ml for a 100 microl injection. The assay system was utilised to determine the plasma kinetics of the compounds in mice, where all the drugs were found to display rapid absorption and elimination following intraperitoneal dosing. In vitro and in vivo studies of metabolism demonstrated that each of the compounds produced several metabolites, and that this conversion could be extensive in vivo.

Aniline Compounds↗
PubMed1997

Synergism of cimetidine with anti-malarial agents.

The histamine type-2 receptor antagonist and cytochrome P450 inhibitor cimetidine was examined for its antimalarial properties in the presence or absence of chloroquine or pyrimethamine. When used alone, cimetidine displayed little activity against a number of Plasmodium falciparum clones in vitro, with an IC50 of 300-700 microM. The compound was found to be highly synergistic in combination with chloroquine and also displayed a degree of synergism when used in combination with pyrimethamine. These synergistic effects were independent of the chloroquine- or pyrimethamine-resistance status of the clones. The cytochrome P450 inhibitor proadifen displayed weak synergism or antagonism with chloroquine, depending on the clone tested, and clear antagonism with pyrimethamine. The results with proadifen indicate that cimetidine was exerting its synergistic activity via a mechanism distinct from inhibition of cytochrome P450. Additional experiments have demonstrated that cimetidine interferes with neither plasmodial sterol metabolism nor heme polymerization.

Animals↗
PubMed1996

Critical role of reverse transcriptase in the inhibitory mechanism of CNI-H0294 on HIV-1 nuclear translocation.

HIV-1 replication requires the translocation of viral genome into the nucleus of a target cell. We recently reported the synthesis of an arylene bis(methyl ketone) compound (CNI-H0294) that inhibits nuclear targeting of the HIV-1 genome and thus HIV-1 replication in monocyte cultures. Here we demonstrate that CNI-H0294 inhibits nuclear targeting of HIV-1-derived preintegration complexes by inactivating the nuclear localization sequence of the HIV-1 matrix antigen in a reaction that absolutely requires reverse transcriptase. This drug/reverse transcriptase interaction defines the specificity of its antiviral effect and is most likely mediated by the pyrimidine side-chain of CNI-H0294. After binding to reverse transcriptase, the carbonyl groups of CNI-H0294 react with the nuclear localization sequence of matrix antigen and prevent its binding to karyopherin alpha, the cellular receptor for nuclear localization sequences that carries proteins into the nucleus. Our results provide a basis for the development of a novel class of compounds that inhibit nuclear translocation and that can, in principle, be modified to target specific infectious agents.

Antiviral Agents↗
PubMed1996

An agent cleaving glucose-derived protein crosslinks in vitro and in vivo.

Glucose and other reducing sugars react with proteins by a nonenzymatic, post-translational modification process called nonenzymatic glycosylation or glycation. The sugar-derived carbonyl group adds to a free amine, forming a reversible adduct which over time rearranges to produce a class of products termed advanced-glycation end-products (AGEs). These remain irreversibly bound to macromolecules and can covalently crosslink proximate amino groups. The formation of AGEs on long-lived connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal ageing and which occurs at an accelerated rate in diabetes. AGEs can activate cellular receptors and initiate a variety of pathophysiological responses. They modify an appreciable fraction of circulating low-density lipoproteins preventing uptake of these particles by their high-affinity tissue receptors. Advanced glycation has also been implicated in the pathology of Alzheimer's disease. Because AGEs may form by a pathway involving reactive alpha-dicarbonyl intermediates, we investigated a potential pharmacological strategy for selectively cleaving the resultant glucose-derived protein crosslinks. We now describe a prototypic AGE crosslink 'breaker', N-phenacylthiazolium bromide (PTB), which reacts with and cleaves covalent, AGE-derived protein crosslinks. The ability of PTB to break AGE crosslinks in vivo points to the importance of an alpha-dicarbonyl intermediate in the advanced glycation pathway and offers a potential therapeutic approach for the removal of established AGE crosslinks.

Amyloid beta-Peptides↗
PubMed1996

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Base Sequence↗
PubMed1996

Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.

Amino Acids↗
PubMed1996

Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone.

An overproduction of proinflammatory cytokines by activated macrophages/monocytes mediates the injurious sequelae of inflammation, septic shock, tissue injury, and cachexia. We recently synthesized a tetravalent guanylhydrazone compound (CNI-1493) that inhibits cytokine-inducible arginine transport and nitric oxide (NO) production in macrophages, and protects mice against lethal endotoxemia and carrageenan-induced inflammation. During these investigations we noticed that CNI-1493 effectively prevented lipopolysaccharide (LPS)-induced NO production, even when added in concentrations 10-fold less than required to competitively inhibit L-arginine uptake, suggesting that the suppressive effects of this guanylhydrazone compound might extend to other LPS-induced responses. Here, we report that CNI-1493 suppressed the LPS-stimulated production of proinflammatory cytokines (tumor necrosis factor [TNF], interleukins 1beta and 6, macrophage inflammatory proteins 1alpha and 1beta) from human peripheral blood mononuclear cells. Cytokine suppression was specific, in that CNI-1493 did not inhibit either the constitutive synthesis of transforming growth factor beta or the upregulation of major histocompatibility complex class II by interferon gamma (IFN-gamma). In contrast to the macrophage suppressive actions of dexamethasone, which are overridden in the presence of IFN-gamma, CNI-1493 retained its suppressive effects even in the presence of IFN-gamma. The mechanism of cytokine-suppressive action by CNI-1493 was independent of extracellular L-arginine content and NO production and is not restricted to induction by LPS. As a selective inhibitor of macrophage activation that prevents TNF production, this tetravalent guanylhydrazone could be useful in the development of cytokine-suppressive agents for the treatment of diseases mediated by overproduction of cytokines.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.

AIDS Dementia Complex↗
PubMed1996

Antimalarial activity of novel arylene bis(methylketone) compounds.

Because of the spread of drug-resistant Plasmodium species, there is an urgent need for novel effective antimalarial agents. A series of arylene bis(methylketone) compounds were screened in vitro against a number of Plasmodium falciparum clones and in vivo against Plasmodium berghei. 2-amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (Cytokine Network Inc. [CNI]-H0294) was the most effective of the compounds in vitro, with an IC50 of 1.5-4.0 microM against parasite clones with a wide range of sensitivities to chloroquine and pyrimethamine. Other compounds in the series had in vitro IC50 values of 20-25 microM. In a 4-day test for suppression of P. berghei parasitemia in vivo, 50 mg/kg/day CNI-H0294 significantly decreased parasitemia by >90%. The compound was found to have low toxicity in mice, with an LD50 of 590 +/- 66 mg/kg intraperitoneally, and rapid plasma kinetics. These results show that CNI-H0294 has considerable antimalarial activity and merits further study.

Animals↗
PubMed1996

Cerebroprotective effects of aminoguanidine in a rodent model of stroke.

BACKGROUND AND PURPOSE: During a cerebral infarction, a complex cascade of cytotoxic events ultimately determines the volume of brain cell loss. The studies presented here demonstrate that aminoguanidine, an experimental therapeutic currently in clinical trials to prevent diabetic complications, is cerebroprotective in focal cerebral infarction. METHODS: Adult Lewis rats (n = 6 to 12 per group) were anesthetized with ketamine and subjected to focal cerebral infarction by tandem permanent occlusion of the right middle cerebral artery and ipsilateral common carotid artery (CCA), followed by temporary occlusion of the contralateral CCA. Infarct volume (cortical) was assessed 24 hours after the onset of ischemia by planimetric analysis of coronal brain slices stained with tetrazolium. RESULTS: Aminoguanidine (320 mg/kg IP) administered 15 minutes after the onset of ischemia resulted in a significant reduction of infarct volume (7.6 +/- 2.6% of hemisphere in controls versus 1.3 +/- 0.2% of hemisphere in aminoguanidine-treated rats; P < .05). Administration of aminoguanidine conferred significant cerebroprotection even when administered 1 or 2 hours after the onset of ischemia (88% and 85% reduction from control, respectively; P < .05). Cerebroprotection by aminoguanidine was independent of systemic physiological variables known to influence stroke size (eg, temperature, mean arterial blood pressure, blood glucose, and arterial pH, PCO2, and PO2). CONCLUSIONS: These results indicate that the stroke-reducing properties of aminoguanidine are dose and time dependent, with substantial cerebroprotection persisting even with drug delivery up to 2 hours after the onset of ischemia. It is now plausible to pursue development of aminoguanidine as an experimental therapeutic in stroke, and possible mechanisms of these cerebroprotective effects are under consideration.

Animals↗
PubMed1996

Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait.

BACKGROUND: Human falciparum malaria, caused by the intracellular protozoa Plasmodium falciparum, results in 1-2 million deaths per year. P. falciparum digests host erythrocyte hemoglobin within its food vacuole, resulting in the release of potentially toxic free heme. A parasite-specific heme polymerization activity detoxifies the free heme by cross-linking the heme monomers to form hemozoin or malaria pigment. This biochemical process is the target of the widely successful antimalarial drug chloroquine, which is rapidly losing its effectiveness due to the spread of chloroquine resistance. We have shown that chloroquine resistance is not due to changes in the overall catalytic activity of heme polymerization or its chloroquine sensitivity. Therefore, the heme polymerization activity remains a potential target for novel antimalarials. In this study, we investigated the ability of heme analogs to inhibit heme polymerization and parasite growth in erythrocytes. MATERIALS AND METHODS: Incorporation of radioactive hemin substrate into an insoluble hemozoin pellet was used to determine heme polymerization. Incorporation of radioactive hypoxanthine into the nucleic acid of dividing parasites was used to determine the effects of heme analogs on parasite growth. Microscopic and biochemical measurements were made to determine the extent of heme analog entry into infected erythrocytes. RESULTS: The heme analogs tin protoporphyrin IX (SnPP), zinc protoporphyrin IX (ZnPP), and zinc deuteroporphyrin IX, 2,4 bisglycol (ZnBG) inhibited polymerization at micromolar concentrations (ZnPP << SnPP < ZnBG). However, they did not inhibit parasite growth since they failed to gain access to the site of polymerization, the parasite's food vacuole. Finally, we observed high ZnPP levels in erythrocytes from two patients with beta-thalassemia trait, which may inhibit heme polymerization. CONCLUSIONS: The heme analogs tested were able to inhibit hemozoin formation in Plasmodium falciparum trophozite extracts. The increased ZnPP levels found in thalassemic erythrocytes suggest that these may contribute, at least in part, to the observed antimalarial protection conferred by the beta-thalassemia trait. This finding may lead to the development of new forms of antimalarial therapy.

Animals↗
PubMed1995

High-performance liquid chromatographic analysis of biological and chemical heme polymerization.

Free hematin can be converted to a stable polymer both chemically, by heating hematin in acid suspensions, or biologically, in the food vacuoles of malaria. A high-performance liquid chromatographic assay has been developed which can separate and quantitate both free hematin and the polymer (beta-hematin), based on the differential solubility of the two compounds. Ion-pair reverse-phase chromatography, utilizing tetramethylammonium chloride and heptane sulfonate as the ion-pair agents in the presence of 40% acetonitrile, was performed on a polymeric-resin-based column with a phenyl bonded phase. Initiating the runs at pH 2.5 led to elution only of the free hematin, and a subsequent shift to pH 12.0 converted the beta-hematin back to hematin which then eluted separately. The method was found to have a linear range of detection from 78 pmol to 20 nmol injected hematin and intra- and interday variations of 9.71 and 12.46%, respectively. The assay was used to study several basic aspects of heme polymerization in vitro, including effects of hematin and beta-hematin concentration on the rate of polymerization.

Animals↗
PubMed1995

Verapamil reversal of chloroquine resistance in the malaria parasite Plasmodium falciparum is specific for resistant parasites and independent of the weak base effect.

Verapamil increases the net uptake and cytotoxicity of structurally diverse hydrophobic molecules in many multidrug-resistant mammalian cell lines. This compound has also been reported to reverse chloroquine resistance in the human malaria parasite Plasmodium falciparum (Martin, S.K., Oduola, A.M.J., and Milhous, W.K. (1987) Science 235, 899-901). Although the mechanism of this reversal is unknown, it apparently involves an increase in the amount of chloroquine present in erythrocytes infected with the resistant parasites. Chloroquine is a diprotic weak base that accumulates in acidic organelles as a function of the pH gradient present between the organelle and the external medium. By changing the external medium pH, this property of chloroquine was used to alter the cytotoxicity phenotype of genetically chloroquine-sensitive and -resistant trophozoites. Verapamil was also found to be toxic for malaria trophozoites, although this toxicity was independent of external pH and consistently about 3-4-fold higher against resistant strains. When verapamil was tested for its effects on chloroquine cytotoxicity under conditions of phenotypic reversal, it was still found to exert only a measurable effect on the genetically resistant trophozoites. In short time incubations, verapamil was found to increase net chloroquine accumulation in erythrocytes infected with both chloroquine-sensitive and -resistant organisms, but only to affect the chloroquine susceptibility of the latter. Analysis of our data demonstrates that verapamil works independently of the overall pH gradient concentrating chloroquine into a trophozoite's lysosome. Instead, we propose that it inhibits the activity of a membrane ion channel indirectly responsible for determining chloroquine transit within the parasite's cytoplasm.

Animals↗
PubMed1995

MIF as a glucocorticoid-induced modulator of cytokine production.

Glucocorticoid hormones are important for vital functions and act to modulate inflammatory and immune responses. Yet, in contrast to other hormonal systems, no endogenous mediators have been identified that can directly counter-regulate their potent anti-inflammatory and immunosuppressive properties. Recent investigations of the protein macrophage migration inhibitory factor (MIF), which was discovered originally to be a T-lymphocyte-derived factor, have established it to be a pro-inflammatory pituitary and macrophage cytokine and a critical mediator of septic shock. Here we report the unexpected finding that low concentrations of glucocorticoids induce rather than inhibit MIF production from macrophages. MIF then acts to override glucocorticoid-mediated inhibition of cytokine secretion by lipopolysaccharide (LPS)-stimulated monocytes and to overcome glucocorticoid protection against lethal endotoxaemia. These observations identify a unique counter-regulatory system that functions to control inflammatory and immune responses.

Animals↗