Inhibition of human tumor cell growth in vitro and in vivo by a specific inhibitor of human farnesyltransferase: BIM-46068.

Oncogenic mutations of the ras gene leading to constitutive activation of downstream effectors have been detected in a large spectrum of human cancers (pancreas, thyroid, colon and NSCLC). Membrane anchorage of Ras required for functional activity in signal transduction is facilitated by post-translational modifications resulting in covalent attachment of a farnesyl group to the cysteine in the C-terminal CAAX motif. This attachment is mediated by farnesyltransferase (FTase). Here, we report a novel series of potent FTase inhibitors, where the tetrapeptide CAAX motif has been modified by incorporation of a thiazolidine carboxylic acid moiety followed by reduction of the 1st and 2nd peptide bonds to a secondary and tertiary amine, respectively. The C-terminal carboxylate was converted to esters for improved cellular penetration. These compounds showed specific inhibition of purified human FTase enzyme, inhibition of proliferation in vitro in a large spectrum of human tumor cell lines and inhibition of growth of human tumor xenografts in athymic nude mice. In addition, in regard to a panel of cell lines, using the Compare analysis to determine the Pearson coefficient correlation, the anti-proliferative spectrum of BIM-46068 has been shown to be distinct from the profile of typical chemotherapeutic agents.

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: a promising neuroprotective strategy.

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(S)-N-[4-[4-[(3,4-dihydro-6-hydroxy-2, 5,7, 8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2- thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties in vitro, affords remarkable neuronal protection in vivo. Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3-10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.

Effect of Platelet-activating Factor on in vitro and in vivo Interleukin-6 Production.

The aim of the present study was to investigate the possible effect of platelet-activating factor (PAF), by comparison with interleukin-1beta and polyriboinositic/polyribocytidylic (poly I-C) acid, on IL-6 production by L 929 mouse fibroblasts. At concentrations above 1 muM PAF, the production of IL-6 by mouse fibroblasts was enhanced in a dose dependent fashion. At 5 muM PAF, the peak increase (60.1 +/- 19.4 U/ml) was similar to that induced by 50 mug/ml poly I-C (60.0 +/- 35.0 U/ml) and higher than the one evoked by 100 U/ml IL-1beta (3.8 +/- 1.8 U/ml). The increase of 11-6 activity induced by 5 muM PAF was maximal after a 22 h incubation period with L 929 cells. Lyso-PAF (5 muM) also increased IL-6 activity from fibroblasts to a similar extent compared with 5 muM PAF. In addition, the IL-6 activity induced by 5 muM PAF was still observed when the specific PAF antagonist, BN 52021 (10 muM), was added to the incubation medium of L 929 cells. The result suggests that the production of IL-6 by L 929 cells evoked by PAF in vitro is not receptor mediated. The in vivo effect of PAF on IL-6 production was also investigated in the rat. Two hours after intravenous injection of PAF (2 to 4 mug/kg), a dramatic increase of IL-6 activity in rat serum was observed, this effect being dose dependent. The increase of IL-6 induced by 3 mug/kg PAF was not observed when the animals were treated with the PAF antagonist, BN 52021 (1 to 60 mg/kg0. These results demonstrate that PAF modulates IL-6 production and that the in vivo effect is receptor mediated.

Immunomodulatory activity of two new aza alkyl phospholipid antineoplastic drugs.

The present work reports the modulation of immunocompetent cell functions by two aza alkyl phospholipids (AAP), BN 52205 and BN 52211. Each compound was compared with 1-O-octadecyl-2-O-rac-glycero-3-phosphocholine (ET-18-OCH3) and/or three drugs used for cancer treatment, i.e. cisplatyl (CIS), 5-fluorouracil (5-FU) and cytosine arabinoside (ARA-C). Interleukin (IL)-1 release from P388D1 cells was increased 2-fold in the presence of 5 micrograms/ml BN 52205 or BN 52211. However, these stimulations were lower than those obtained with ARA-C, 5-FU and CIS. Compared with ET-18-OCH3, CIS and 5-FU, BN 52205 and BN 52211 were more efficient in increasing tumor necrosis factor production induced by lipopolysaccharide (LPS) from human monocytes. In vitro, all compounds exhibited similar activity in enhancing IL-6 production from human monocytes stimulated with LPS, with the exception of 5-FU and CIS that were inactive. At 20 mg/kg (i.v.), a peak of IL-6 production was reached 2 h after injection of ET-18-OCH3 [> 1280 U/ml (n = 4, p < 0.001) versus 3.5 +/- 0.2 U/ml (n = 7)], whereas BN 52211 induced a maximum of IL-6 production after 4 h (77 +/- 27 U/ml, n = 5, p < 0.001). BN 52205 induced peaks of IL-6 production after 3 and 6 h (90 +/- 62 and 68 +/- 35 U/ml, respectively, p < 0.001, n = 4). The proliferation of rat splenocytes was abolished in the presence of BN 52205 and BN 52211 at 10 micrograms/ml, corresponding to only a partial reduction of IL-2 production at the same concentration. The production of interferon-gamma was stimulated 6- to 10-fold in the presence of 1-5 micrograms/ml BN 52205, BN 52211 and ARA-C. BN 52211 and BN 52205 were also potent enhancers of IL-3 production, whereas 5-FU and ARA-C were inhibitory. These results indicate that in addition to a direct antitumoral effect, AAP may also exhibit immunomodulatory activity both in vitro and in vivo.

Platelet-activating factor modulates interleukin-6 production by mouse fibroblasts.

We compared the effects of platelet-activating factor (PAF), interleukin-1 beta (IL-1 beta) and polyriboinositic-polyribocytidylic acid (poly-I:C) on IL-6 production by confluent L929 mouse fibroblasts. At concentrations above 1 microM, PAF dose-dependently enhanced IL-6 production; at 5 microM PAF this increase (72.7 +/- 19.9 U/ml) was higher than that evoked by 100 U/ml IL-1 beta (5.7 +/- 0.4 U/ml) or 50 micrograms/ml poly-I:C (39.3 +/- 6.7 U/ml). The IL-6 production induced by 5 microM PAF was not inhibited by addition of the specific PAF antagonist BN 52021 (10 microM) to the incubation medium. These results demonstrate that, as this is the case for IL-1, PAF also modulates IL-6 production.

Effect of long-term infusion of platelet-activating factor on pulmonary responsiveness and morphology in the guinea-pig.

The effect of chronic administration of platelet-activating factor (PAF) on airway reactivity, cell recruitment and lung morphology in the guinea-pig has been investigated. Alzet osmotic minipumps delivering either PAF (7.2 mg/kg/14 days) in 0.25% (w/v) bovine serum albumin in saline (saline-BSA), acetylcholine or saline-BSA alone were implanted s.c. in the neck region of guinea-pigs and connected to the jugular vein. In some experiments, implanted and non-implanted animals were treated daily with the PAF antagonist, BN 52021 (15 mg/kg, twice a day, p.o.). On day 15 after minipump implantation, the animals were anesthetized with urethane (1.2 g/kg, i.p.) and tracheal cannula was inserted for mechanical ventilation. Pulmonary inflation pressure (PIP) was monitored and airway responsiveness was assessed by administration of increasing doses of histamine (0.2-100 micrograms/kg, i.v.). As compared to saline-BSA-treated or non-implanted guinea-pigs, chronic treatment of the animals with PAF induced a significant (p less than 0.01) increase in airway response. No significant change in airway responsiveness was observed following chronic acetylcholine administration. In contrast, regardless of the treatment of the animals, no change in the threshold dose of histamine inducing alteration in PIP was noted, suggesting that PAF induces bronchopulmonary hyperreactivity rather than hyperresponsiveness. In addition, no significant difference was observed in the in vitro responsiveness to histamine of lung parenchymal strips from animals having received PAF or saline-BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the double-stranded polynucleotide complex polyadenylate-polyuridylate (poly A-U) on interleukin-6 production by mouse fibroblasts.

Cultures of mouse embryonic fibroblasts (L 929) have been shown to produce a factor which promotes the growth of B cell hybridoma (hybridoma growth factor, HGF) i.e. interleukin 6 (IL-6). The aim of the present study was to investigate the effect of Poly A-U on IL-6 production by this cell type. After incubation for 48 h at 37 degrees C of confluent (1 week old) L 929 fibroblasts in the presence or in the absence of Poly A-U, IL-6-like activity in supernatants was measured by the proliferation assay of the IL-6-dependent B cell hybridoma cell line, 7TD1. Poly A-U increased IL-6 activity in supernatants in a dose-dependent manner at doses higher than 50 micrograms/ml, the maximum activity being observed at the highest concentration of Poly A-U used, i.e. 500 micrograms/ml. beta Interleukin-1 (beta IL-1) and poly-cytidylic-polyinosinic (Poly I-C) have been shown to be inducers of IL-6 in fibroblast culture and thus their effect was compared to that of Poly A-U. The IL-6 activity in supernatants induced by 500 micrograms/ml Poly I-C (58.4 +/- 16.4 U/ml; n = 4) was higher than that evoked by 100 U/ml beta IL-1 (5.7 +/- 0.4 U/ml) or 500 micrograms/ml Poly A-U (39.6 +/- 7.8 U/ml). The increased production of IL-6 by Poly A-U may explain part of its previously reported immunomodulatory effects.

Effect of long-term treatment with platelet-activating factor on IL-1 and IL-2 production by rat spleen cells.

To study the effect of the in vivo administration of platelet-activating factor (PAF) on cytokine production, alzet minipumps loaded with the mediator or solvent alone were connected to the jugular vein and placed under the skin of Sprague-Dawley rats. Over 7 days the animals received total doses of 0.5, 1, 4.5, 9, or 28 micrograms PAF or the solvent alone. The spleen mononuclear cells isolated from Ficoll gradients and the adherent cell fraction were separated before determination of basal and mitogen-stimulated IL-1 and IL-2 production, respectively. Adherent splenocytes from rats having received 28 micrograms PAF exhibited a decreased capability to produce IL-1, as compared to those from vehicle-treated animals. In contrast, adherent splenocytes from rats having received 9 and 4.5 micrograms PAF yielded higher amounts of released and cell-associated IL-1 activity upon LPS stimulation, as compared to those from solvent-treated animals. The PAF antagonist, BN 52021, given orally (5 mg/kg, twice a day throughout the experiments) inhibited the in vivo effect of 28 micrograms PAF. Statistically significant 144 +/- 43% (p less than 0.001, n = 5) and 73 +/- 33%, (p less than 0.01, n = 3) increases in IL-2 production were observed when whole spleen mononuclear cells from rats administered with 1 and 4.5 micrograms PAF, respectively, were stimulated with Con A. BN 52021 markedly inhibited the in vivo effect of 1 microgram PAF on the IL-2 release. Our study demonstrates that PAF can modulate immune functions in vivo and suggests that the specific PAF antagonist, BN 52021, may be used as an immunomodulatory agent.

Platelet-activating factor potentiates interleukin-1/epidermal cell-derived thymocyte-activating factor release by guinea-pig keratinocytes stimulated with lipopolysaccharide.

Interleukin-1 (IL-1) is a member of a family of closely related molecules playing an important role in many inflammatory and immunologic reactions. Besides macrophages, epidermal cells such as keratinocytes release, either spontaneously or on stimulation, IL-1-like molecules also referred to as epidermal cell-derived thymocyte-activating factor (ETAF). In the present study, the potential role of the potent lipid mediator, platelet-activating factor (PAF), in the regulation of IL-1/ETAF release by keratinocytes was investigated. Keratinocytes from guinea-pig ears were incubated for 24 h in the presence or absence of lipopolysaccharide (LPS) and PAF, either alone or in combination. LPS markedly increased in a dose-dependent fashion the IL-1/ETAF release by keratinocytes, as assessed by the mouse thymocyte proliferation assay. In contrast, no effect of PAF on IL-1/ETAF release was observed. Simultaneous addition of 1 microgram/ml LPS and 1 pM PAF to keratinocyte culture increased IL-1/ETAF release compared to that observed with LPS alone. When 1 pM PAF was added 1 h before or 1 h after LPS, no effect on IL-1/ETAF release by keratinocytes was noted. In contrast, regardless of the time of addition of 10 fM PAF to keratinocytes stimulated with LPS (either simultaneously or 1 h after LPS), an identical and non-significant increase was observed. In conclusion, although PAF is not able to induce IL-1/ETAF release by keratinocytes, it potentiates that induced by a stimulus like endotoxin.

Modulation of the priming effects of platelet-activating factor on the release of interleukin-1 from lipopolysaccharide-stimulated rat spleen macrophages.

The pharmacologic modulation of the effect of platelet-activating factor (PAF) on the interleukin-1 (IL-1) activity present in the supernatants from lipopolysaccharide (LPS)-stimulated macrophages was investigated. Rat spleen macrophages were isolated by centrifugation on a Ficoll-Hypaque gradient followed by adherence on plastic petri dishes for 60 min at 37 degrees C under a 5% CO2/95% air atmosphere. The IL-1 content in the cell-free supernatants was assessed using the mouse thymocyte proliferation assay. Preincubation of macrophages for 10 min with 10 fM PAF prior to stimulation with 20 micrograms/ml LPS for 24 h markedly increased the IL-1 activity present in the supernatants from macrophages whereas no direct effect of PAF was noted. Although they had no direct effect, addition of L-651,392 (10 microM), a lipoxygenase inhibitor, or the oxygen-derived free radical scavenger mannitol (10 microM) during the 10-min preincubation period with PAF reversed by 105.0% and 79.9%, respectively, the action of the autacoid on IL-1 activity. Pertussis toxin (PT, 1 microgram/ml) decreased by 30% the LPS-induced IL-1 activity. Association of PT with PAF suppressed the enhancing effect of 10 fM PAF on the IL-1 activity present in the supernatants from LPS-stimulated macrophages. Thus, the enhancing effect of PAF on IL-1 release appears to be due to the production of lipoxygenase metabolites, leading to superoxide production and alterations of cAMP levels.

Effect of long-term treatment with platelet-activating factor on cytokine production by rat spleen cells.

The long-term in vivo effect of platelet-activating factor (PAF) production of interleukin-1 and -2 (IL-1, IL-2) was investigated. Alzet infusion minipumps loaded with PAF or solvent were placed under the back skin of Sprague-Dawley rats and connected to the jugular vein. Lymphocytes from animals having received 1, 4.5 or 9 micrograms PAF/7 days showed an increased capacity to produce IL-1 and IL-2. In contrast, splenocytes from rats receiving 28 micrograms PAF/7 days exhibited decreased capacity to produce IL-1, whereas IL-2 was unaffected. The decrease in IL-1 synthesis induced by 28 micrograms PAF and the increase in IL-2 production evoked by 1 microgram PAF were not observed in rats treated daily with the PAF antagonist, BN 52021. Thus, PAF appears to play a role in the regulation of the immune response. The reversal of the effect of PAF by BN 52021 indicates that the mediator is acting via specific binding sites similar to those reported on other cell types. These data also suggest that PAF antagonists may be used as immunomodulatory drugs.

Immune regulation by platelet-activating factor. I. Induction of suppressor cell activity in human monocytes and CD8+ T cells and of helper cell activity in CD4+ T cells.

Platelet-activating factor (PAF) is a powerful mediator of inflammation. We have recently described a potential role for PAF in immune reactions, as it inhibits T cell proliferation and IL-2 production in response to mitogens. To further define the mechanism through which this inhibition is exerted, we used a coculture system in which PBML are preincubated with increasing concentrations of PAF for 24 h, followed by washing, treatment with mitomycin C and addition to fresh autologous PBML stimulated with PHA. In this context, a significant (40 to 60%) inhibition of proliferation was observed. In parallel, PAF-pre-treated cells induced a reduction (30 to 50%) of IL-2 production by PHA-stimulated lymphocytes. The PAF receptor antagonist BN52021 could partially block the PAF-induced suppressor cell activity, but also showed some suppressor cell-inducing properties of its own (20 to 30%). The expression of suppressor cell function during the co-culture could be partially abrogated by the inclusion of indomethacin, suggesting that cycloxygenase metabolites of arachidonic acid were involved in this phase of suppression. When PBML were fractionated into monocytes, lymphocytes, or T cell subsets before pre-incubation with PAF, indomethacin-sensitive suppressor cell function was generated in the monocyte population. Monocyte-depleted lymphocytes showed slight helper effect, whereas CD8+ T cells were induced to become indomethacin-resistant suppressor cells. CD4+ T cells, in contrast, were activated to exert very marked helper effect. When incubated with PAF for 24 h, monocyte-depleted lymphocytes showed a 30% decrease in CD4+ T cell numbers and a 50% increase in CD8+ T cell numbers. Our data suggest a novel immunoregulatory role for PAF and potentially important interactions of this lipid mediator of inflammation with lymphocyte and monocyte functions.

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes.

PAF-acether, at doses ranging from 1pM to 0.1 microM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 +/- 39% and synthesis by 87 +/- 27% whereas at 0.1 microM a decrease of IL1 release of 52 +/- 9% and synthesis of 46 +/- 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase of inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1 microM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocytes functions, possibly via specific binding sites.

Lung maturation in the hyperinsulinemic rat fetus.

Hyperinsulinemic rat fetuses were obtained either by repeated in utero injections of long-acting insulin (resulting in fetal hypoglycemia) or by chronically infusing intravenous glucose to the mother (resulting in fetal hyperglycemia). Fetuses were examined at term. In insulin-injected fetuses (n = 15), surfactant (S) fraction phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) were significantly decreased (3.6 +/- 0.1 nmol Pi/mg tissue; p less than 0.001 and 2.8 +/- 0.1 nmol/mg; p less than 0.025, respectively) as compared with their saline-injected controls (4.8 +/- 0.2 and 3.3 +/- 0.1 nmol/mg, respectively, n = 19). However, residual (R) fraction was unchanged, and there was no difference in whole-lung phospholipids (combined S and R fractions). These results are consistent with morphological data showing a lower lamellar body area per type II cell profile in insulin-injected fetuses as compared with their controls [1.41 +/- 0.13 micron 2 (n = 72) versus 1.99 +/- 0.14 micron 2 (n = 129) p less than 0.01]. Glycogen content was slightly higher in insulin-injected fetuses (18.5 +/- 1.0 micrograms/mg, n = 17) than in their controls (15.1 +/- 0.8 micrograms/mg, n = 18; p less than 0.05). In the second model, changes in S fraction PC and DSPC were similar to those observed after insulin-injections: 4.3 +/- 0.25 and 3.4 +/- 0.2 nmol Pi/mg in fetuses of glucose-infused rats (n = 10) versus 5.7 +/- 0.45 and 4.3 +/- 0.3 nmol Pi/mg, respectively, in controls (n = 10, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021.

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.

Changes in surfactant phospholipids in fetal rat lungs from normal and diabetic pregnancies.

The purposes of this study were to adapt and evaluate further a pulmonary surfactant isolation method applicable to unperfused fetal rat lung, to quantitate key phospholipids phosphatidylcholine (disaturated phosphatidylcholine, and phosphatidylglycerol) of the isolated material during the last 3 days of gestation, and to determine if abnormalities in surfactant phospholipids were present in fetuses of diabetic pregnancies. A simplified scheme of sucrose gradient centrifugation proved useful for small scale preparations of material enriched in the phospholipids most characteristic of pulmonary surfactant. It was shown that fetal blood phospholipids did not contaminate the surfactant fraction and therefore would not produce artifacts in assessment of lung maturational changes. Analyses of subcellular fractions isolated at 19.5, 20.5, and 21.5 days revealed that the percentages of disaturated phosphatidylcholine relative to total phospholipids were 23-44% in the surfactant preparations and 14-21% in the residual (nonsurfactant) fractions, while the disaturated phosphatidylcholine/phosphatidylcholine ratios were 0.62 +/- 0.06 and 0.41 +/- 0.03, respectively. Summation of the amounts of individual phospholipids in the two fractions yielded data that were nearly identical to the concentrations of these compounds in whole fetal lung samples analyzed independently, implying that losses during the surfactant isolation technique were negligible. The concentrations of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, and total phospholipids increase markedly (more than 10-fold) and progressively in surfactant fractions prepared from normal fetal rat lung at 19.5, 20.5, and 21.5 days of gestation. In contrast, the residual fractions showed no changes from 19.5 to 20.5 days and then relatively modest increases from 20.5 to 21.5 days, except for phosphatidylglycerol, which increased markedly.(ABSTRACT TRUNCATED AT 250 WORDS)