Intravitreally injected platelet activating factor induces retinitis in experimental animals.

PURPOSE: Platelet activating factor is a lipid which has been strongly implicated in anterior uveitis. In order to investigate further the role of platelet activating factor in intraocular inflammation, we have characterized the histological changes associated with the intravitreal injection of platelet activating factor, PAF analogs, or lyso-PAF in laboratory rabbits and rats. METHODS: Initial studies utilized a PAF analog (rac 1-0-octadecyl 2-0-ethyl glycero phosphoryl choline or ethoxy PAF), because this compound is relatively resistant to degradation by hydrolase, the major degradative enzyme for PAF. Doses ranging from 1 ug to 5 mg and time points from 6 hours to 7 days after injection were studied. RESULTS: In either rats or rabbits, 100 ug of ethoxy PAF consistently induced a marked uveitis with the predominance of inflammation focused in the retina and choroid. Retinitis was also induced in rabbits by either 1 mg PAF injected intravitreally or a similar dose of the PAF precursor/metabolite, lyso PAF. Retinal inflammation was not induced by an inactive lipid, 1,1-0,0-dihexadecyl-rac-glycero-3-phosphocholine, although this compound resulted in mild vitreous inflammation. The histological changes induced by PAF could be readily distinguished from the predominantly anterior inflammation induced by intravitreal injections of substances such an interleukin-1 or endotoxin. CONCLUSIONS: Recent studies indicating that PAF antagonists inhibit a variety of retinal toxicities and our own observations suggest that PAF could be a major mediator of retinal inflammation.

Tumor cell kinetics following long-term treatment with antineoplastic ether phospholipids.

Ether phospholipids are analogs of the naturally occurring 2-lysophosphatidylcholine that have been reported to have selective in vitro/in vivo antitumor activity. Their antiproliferative effect has been found against a variety of animal and human tumor cell lines. We have characterized the cytostatic activity of four ether phospholipids, the methoxy-substituted edelfosine (ET-18-OCH 3), the thio-derivative ilmofosine (BM 41.440), and two new aza-alkylphospholipids, BN 52205 and BN 52211, on a human tumor cell line derived from a colon adenocarcinoma, the HT29. A flow cytometric approach has been used and, contrary to previous studies, longer treatment times have been performed to allow multiple cell population doublings. The results confirm that the cytostatic activity of the four ether phospholipids is characterized by multiple "terminal points", as the drugs' action results in a G1 block, a slowdown of the transition from late-S to G2, followed by an accumulation of HT29 cells in the G2 phase of the cell cycle. Tumor cells in late G1 at the time of treatment progressed through S before being blocked in G2. In a similar fashion, tumor cells in late G2 at the time of treatment went through M but were then halted in G1. The long-term treatment studies indicate that the ether phospholipid cytostatic activity is partially reversible, depending on the drug concentration and the duration of the treatment.

New antineoplastic aza-phospholipids - synthesis, in-vitro cytotoxicity and differential cellular-sensitivity against 70 human tumor-cell lines.

The in vitro cytotoxicity and differential cellular sensitivity of three new synthetic anti-neoplastic aza-phospholipids has been determined in the National Cancer Institute's (NCI) primary antitumor drug screen. Based on a disease-oriented strategy, this screen incorporates seventy human cell lines representing leukemia, ovarian, brain, melanoma, colon, renal, lung, prostate and breast cancers. The analysis of the GI50 values obtained for each aza-derivative has revealed a differential cellular sensitivity among the cell lines examined. The study of the degree of differential growth inhibition has shown a statistically significant differential cell sensitivity for BN 52205 and BN 52211 for colon and melanoma tumor cells. The leukemia cell selectivity for BN 52211 was even more remarkable due to the low molar concentration at which the maximum selective effect occurred. These findings strongly encourage further investigations on the anti-neoplastic activity of aza-phospholipids.

Sensitivity of drug-resistant B-cell lines from AIDS-related non-hodgkins-lymphoma to newly synthesized podophyllotoxin derivatives and aza-alkyllysophospholipids - enhanced sensitization by pretreatment with interferon-gamma.

Non-Hodgkin's lymphoma B cells (NHL-B) often become refractory to conventional chemotherapeutic drugs. The present study investigated the sensitivity of three AIDS-derived NHL-B cell lines to newly synthesized azaalkyllysophospholipids (AALP) and podophyllotoxin derivatives. All three cell lines were sensitive to 5 AALP derivatives and toxicity was detectable at 24 h of culture and was increased at 48 and 72 h of incubation. Toxicity was concentration-dependent and die extent of cytotoxicity varied slightly from one cell line to another. These cell lines were less sensitive to the podophyllotoxin derivatives VP-16, BDPTN and BEPT than to die AALP. This was shown by the extent of cytotoxicity calculated on a molar basis and by the delayed kinetics of lysis. Pretreatment of the tumor cells with IFN-gamma stimulated cell proliferation. IFN-gamma treated tumor cells were also tested for their sensitivity to the various cytotoxic agents. In all cases, the sensitivity of the IFN-gamma pretreated cell lines to the podophyllotoxin derivatives and AALP was significantly enhanced. These findings demonstrate that drug resistant NHL-B cells are sensitive to a new family of AALP and podophyllotoxin derivatives. Further, the sensitivity of the tumor cells was enhanced by treatment with IFN-gamma. The potential clinical use of these new cytotoxic agents to overcome resistance of AIDS-related cell lymphomas is discussed.

Cytotoxicity of new aza-alkyl lysophospholipids against drug-sensitive and resistant human ovarian tumor-cell lines - role of free-radicals and potentiation of cytotoxicity by tnf-alpha and cddp.

Three aza-alkyl lysophospholipids (AALP) with related chemical structures (BN52205, BN52218, BN52227) were examined for their anti-tumor cytotoxic activity when used alone or in combination with TNF-alpha or CDDP. The three compounds were cytotoxic, though to a different degree, against a battery of human ovarian tumor cell lines. The compounds were cytotoxic to both drug sensitive and drug resistant lines and were also cytotoxic to an MDR(+) tumor cell line. BN52205 was the most potent cytotoxic AALP and differed from the least cytotoxic compound BN52227 by a substitution of a methoxy group for an ethoxy group at position 1. The AALP-mediated cytotoxicity was found to be mediated in large part by free radicals as: i) treatment of the tumor cells with an inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO), augmented cytotoxicity and often resulted in synergy and ii) the addition of the anti-oxidant glutathione inhibited cytotoxicity. Since free radicals have also been involved in both TNF-alpha and CDDP-mediated cytotoxicity, we examined the potentiating effect of combination treatment of AALP with these cytotoxic agents. Depending on the cell line, there was either an additive or a synergistic activity by the combination treatment. Furthermore, combination of BN52205 and TNF-alpha resulted in a synergistic activity against the MDR(+) ovarian line, AD10, and the cis-platinum resistant line, C30. These results demonstrate that AALP are cytotoxic to tumor cell lines and can overcome drug resistance. Further, low concentrations of AALP and TNF-alpha/drug/BSO result in additive or synergistic cytotoxic activity. These findings suggest that combination treatment can be effective in the therapy of drug resistant ovarian tumors and can achieve reduced overall toxicity.

Cytotoxic properties of a new synthetic demethylpodophyllotoxin derivative, BN 58705, against human tumor cell lines.

The in vitro cytotoxic properties of a newly synthesized demethylpodophyllotoxin derivative, 4-o-butanoyl-4'-demethylpodophyllotoxin (BN 58705), were determined by using several human tumor cell lines of different histological origin and of different sensitivity to conventional chemotherapeutic drugs (Adriamycin and cis-diammine-dichloride platinum). BN 58705 is shown to be cytotoxic against various human tumor cell lines as assessed by the MTT assay. Furthermore, BN 58705 is shown to be cytotoxic against several drug-resistant tumor cell lines. BN 58705 is cytotoxic at concentrations 100- to 1000-fold lower than those of Adriamycin or cis-diammine-dichloride platinum required to achieve similar cytotoxicity. BN 58705 did not mediate DNA fragmentation of target cells, whereas the epipodophyllotoxin-like etoposide induced DNA cleavage by stabilizing the DNA-enzyme intermediate. Like vinca alkaloids, BN 58705 induced a block in the mitotic phase of the cell cycle. By comparison, BN 58705 exerted a stronger cytotoxic activity in vitro than did either etoposide, an epipodophyllotoxin, or vincristine, a vinca alkaloid. When BN 58705 was applied in vivo in mice, it resulted in low toxicity (50% lethal dose, 150 mg/kg). These results demonstrate than BN 58705 is cytotoxic to drug-resistant human tumor cell lines and is manyfold more potent than conventional drugs. The cytotoxic potency and low toxicity of BN 58705 are important criteria to establish its potential chemotherapeutic efficacy in vivo.

Cytotoxic activity against resistant human tumor-cell lines by 2 synthetic demethylpodophyllotoxin derivatives - a single stereoisomeric change prevents cell-death by apoptosis.

The podophyllotoxin derivative, etoposide, is currently being used in patients with variant malignant diseases. However, studies show resistance to etoposide develops, and thus new agents to overcome resistance are needed. The present study investigated the cytotoxic properties of two synthetic podophyllotoxin derivatives namely 4-o-butanoyl-4'-demethylepipodophyllotoxin (BEPT) and 4-o-butanoyl-4'-demethylpodophyllotoxin (BDPTN or BN58705). Both BEPT and BDPTN are shown to be cytotoxic against a battery of human tumor cell lines. In comparison to etoposide, the magnitude of cytotoxic activity by BEPT and BDPTN was higher. Further, both compounds were cytotoxic to drug resistant lines and also were able to overcome the etoposide and cross-resistance of an MDR+ cell line. The mechanism of cytotoxicity was also examined. Like etoposide, a topoisomerase II inhibitor and inducer of apoptosis, BEPT mediated its cytotoxic activity by an apoptotic pathway. However, BDPTN did not mediate apoptosis. These findings demonstrate that a simple modification in the stereoisomeric structure results in significant modification in both the cytotoxic patterns and the induction of apoptosis. Further, the findings show that the podophyllotoxin analogs can overcome drug resistance and suggest their possible application in the therapy of resistant tumors.

Effect of paf antagonists on the cytotoxic activity of antineoplastic ether phospholipids.

The capability of the methoxy-substituted 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3 or Edelfosine) and the two aza-alkylphospholipids BN 52205 and BN 52211 to bind to the PAF receptor was analysed in rabbit platelet membranes. Ether phospholipid concentrations were tested between 10(-5) M and 10(-11) M. The results indicate that ether phospholipids are not able to bind to the PAF receptor and do not prevent PAF binding to its receptor. Moreover, the cytotoxic effect of three potent PAF antagonists, BN 52021, BN 50730 and BN 50739, were analysed in HL60 promyelocytic cells. These cells were pre-and co-treated with PAF antagonists and ether phospholipids. The data show that the three PAF antagonists failed to counteract the activity of ET-18-OCH3, BN 52205 and BN 52211 thus demonstrating that the cytotoxic effect of these new anti-neoplastic drugs is not mediated by the PAF receptor.

Evaluation of combinations of antineoplastic ether phospholipids and chemotherapeutic drugs.

Combinations of drugs are used clinically for the therapeutic advantages they may provide over single agents. We have studied the cytotoxic interaction between four either phospholipids ET-18-OCH3, BM 41.440, BN 52205 and BN 52211, and several chemotherapeutic drugs (ADM, CDDP, VLB, VP-16, MMC, BLM and MTX) on two human tumor cell lines, A427 (lung) and HT29 (colon). We have used the MTT colorimetric assay to evaluate growth inhibition and performed isobologram analysis on the IC50 data. For both cell lines a synergistic effect has been found between each of the four ether phospholipids in association with CDDP and ADM. In both cell lines only BM 41.440 and BN 52211 act synergistically with VLB while, in A427 cells, only BN 52205 behaves similarly with MMC. These results show that a positive interaction exists between ether phospholipids, spindle poisons and DNA-interactive drugs.

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.

Reversible or irreversible modification of [3H]PAF binding on rabbit platelet membranes differentiates various PAF receptor antagonists.

[3H]Platelet-activating factor (PAF) binding to rabbit platelet membranes was examined before and after 20 min preincubation at 25 degrees C in the presence of PAF, lysoPAF, or of five different PAF receptor antagonists (L 652731, BN 52021, WEB 2086, BN 52111 and BN 52115). When platelet membranes were not washed after preincubation with PAF or PAF antagonists, no significant specific binding of [3H]PAF was observed, which suggests full occupancy of the binding sites. When membranes were extensively washed, full recovery of specific [3H]PAF binding was attained with L 652731 and partial recoveries (60%, 55% and 30%) were reached with BN 52021, WEB 2086 and PAF, respectively; no recovery was seen with the dioxolanes BN 52111 and BN 52115. Scatchard analysis of the binding data indicated that no significant change in the dissociation constant (Kd) and maximum number of binding sites (Bmax) occurred after preincubation of platelet membrane with L 652731, whereas a reduction of Bmax was observed when PAF and BN 52021 were measured. When platelet membranes were preincubated with WEB 2086, Bmax and Kd significantly increased. The data suggest differing binding properties for PAF and the PAF antagonists. Some of the PAF antagonists may tightly bind to the PAF receptor site(s) and/or irreversibly modify or downregulate PAF recognition sites. Our results also suggest that the interaction of PAF receptor antagonists with PAF receptor can be divided into at least two components, namely a reversible component and an irreversible one.

Cytostatic activity of new synthetic anti-tumor aza-alkyllysophospholipids.

Alkyllysophospholipids are analogues of the naturally occurring 2-lysophosphatidylcholine which have been reported to have selective in vitro/in vivo anti-tumor activity. Their antiproliferative effect has been found against a variety of animal and human tumor cell lines. We have characterized the cytostatic activity of 2 newly synthetized aza-alkyllysophospholipids (AALPs), the BN52205 and the BN52211, on a human tumor cell line derived from a colon adenocarcinoma, the HT29. We used 3 different flow cytometric approaches to study which phase of the cell cycle was the most sensitive to the antiproliferative activity of the 2 AALPs. By applying the biparametric analysis of 5'-bromo-2-deoxyuridine incorporation vs. DNA content we have been able to demonstrate that the 2 AALPs do not interfere with the S phase of the cell cycle. The simultaneous measurement of total nuclear protein vs. DNA content in isolated HT29 nuclei enabled us to exclude a block in the M phase of the cell cycle. Finally, stathmokinetic analysis enabled us to show that cytostatic activity of the 2 new AALPs is characterized by multiple "terminal points" as the drugs' action results in a G1 block, in a slow-down of the transition from late S to G2 followed by an accumulation of HT29 cells in the G2 phase of the cell cycle.

Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.

Effect of BN 52256 and other mediator antagonists on ouabain-induced cardiac arrhythmia in a model of anaphylaxis in guinea-pigs.

Different mediators released by anaphylaxis seem to be involved in different pathophysiological conditions, including cardiac arrhythmia. Histamine, 5-HT and platelet-activating factor (PAF) could participate in the enhanced arrhythmogenicity during anaphylaxis in guinea-pigs. The threshold dose of ouabain-induced arrhythmia is decreased in actively sensitized guinea-pigs by i.p. administration of ovalbumin. The purpose of the present paper was to investigate the effect of different mediator antagonists. Antagonists of PAF (WEB 2170), histamine (clemastine) and 5-HT (cyproheptadine) in doses of 5.0 mg/kg, 5.0 mg/kg and 0.5 mg/kg, respectively, can increase the threshold dose of ouabain-induced arrhythmias signalling an antiarrhythmic effect. A combination of WEB 2170 and clemastine, each of them in inactive doses (2.0 mg/kg and 1.0 mg/kg, respectively) showed a statistically significant antiarrhythmic effect. A combination of the same dose of WEB 2170 and cyproheptadine (0.1 mg/kg) under the same conditions induced an antiarrhythmic effect, too. BN 52256 is a new antiallergic drug synthesized on the basis of a novel concept of combining inhibitory activity against various inflammatory mediators in one molecule. BN 52256 in doses of 20-80 micrograms/kg exhibited a statistically significant antiarrhythmic effect. BN 52256 needed a 12.5-125 fold lower dose to induce the same antiarrhythmic effect compared to the antagonists of PAF, histamine or 5-HT investigated in this study. Depending on the pathophysiological conditions, different mediators seem to be involved in the occurrence of cardiac arrhythmia. A complex inhibition of these mediators could induce a more specific influence on such kinds of cardiac arrhythmias.

Membrane cholesterol content and sensitivity of human carcinoma-cells to antineoplastic ether phospholipids.

Ether phospholipids represent a new class of anti-cancer drugs which appear to exert their tumoricidal activity through a direct and indirect cytotoxic effect against tumor cells of different origins. The chemotherapeutic interest in these new drugs is based on the finding that, contrary to the majority of anti-cancer drugs, ether phospholipids do not interfere with DNA synthesis, are anti-invasive and induce tumor cell differentiation. There is increasing experimental evidence that the direct cytotoxic effect of these new drugs is mediated by the cell membrane. We have measured the lipid membrane composition of three human carcinoma cell lines that have been found to possess different sensitivity to the tumoricidal activity of four antitumor ether phospholipids. A statistically significant difference has been found in the membrane cholesterol content of the three cell lines and a positive correlation has been established between the membrane cholesterol level and the carcinoma cell sensitivity to ether phospholipids. These findings emphasize previous data obtained with leukemic cells and reinforce the interest in ether phospholipids whose cytotoxic properties may represent a new step towards a more promising anti-cancer chemotherapy.

Cytotoxic activity of synthetic aza alkyl lysophospholipids against drug sensitive and drug resistant human tumor cell lines.

The anti-tumor cytotoxic activity of four newly synthesized aza alkyl lysophospholipids (AALP), namely BN 52205, BN 52207, BN 52208 and BN 52211, was investigated. Using the 51Cr release assay, the four compounds were endowed with cytotoxic activity, in a concentration-dependent fashion, against various human tumor cell lines of different histological origin. Two different mechanisms appear to be involved in the AALP-mediated cytotoxicity. A rapid membrane damaging effect was observed in less than one hour's incubation of tumor cells with AALP and cytotoxicity was temperature-independent when AALP were used at greater than or equal to 200 micrograms/ml. A slower cytotoxic mechanism was observed after 18 hours incubation at 37 degrees C when AALP were used at concentrations of 30-100 micrograms/ml. The pattern and magnitube of the cytotoxic activity achieved with all the 4 AALP compounds tested were similar and the cytotoxicity mediated by combination of two compounds was additive. In addition to the cytotoxic effect, the AALP compounds also exerted a cytostatic anti-tumor effect, as assessed by inhibition of 3H TdR incorporation. Using a variety of human tumor cell lines as targets, the cytotoxic effect observed with the AALP was noted with tumor cells that were either sensitive or resistant to TNF-alpha and/or chemotherapeutic drugs such as mitomycin C, adriamycin and cis-platinum. The LD50 toxicity in mice was 100-125 mg/kg. The present findings demonstrate that AALP are cytotoxic to a variety of human tumor cell lines and do not appear to discriminate between drug/cytokine sensitive or resistant cells. Thus the present study suggests that some aza alkyl lysophospholipids may be considered as potential anticancer agents.

Structure-activity relationships in platelet-activating factor (PAF). 5. Synthesis and in vitro antagonistic activities of ketophosphonates.

The synthesis of new ketophosphonate isosteres of biosynthetic precursors of ether glycerophospholipids resistant to phospholipase C is described following two routes depending on whether the alkoxy chain is introduced before or after the phosphonic moiety. The common intermediates are ketophosphonic acids: better yields were obtained by attaching the n-octadecyl chain to epichlorohydrin, opening and oxidation, blockage of the resulting ketone as the chlorohydrazone, followed by an Arbuzov reaction or by azoene formation and Michael addition. These ketophosphonates differing in chain length in position 3 exhibit potent agonistic activities on rabbit platelets which increase with the number of methylene groups between the phosphonate and the ammonium moieties.

Action of platelet-activating factor (PAF) antagonists on the bronchopulmonary effects of PAF in the guinea-pig.

Intravenous injection of BN 52021 in anesthetized guinea-pigs, 5 min before challenge, inhibited in a dose-dependent fashion with an IC50 of 0.90 mg/kg the bronchoconstriction induced by PAF (60 ng/kg i.v.). However, BN 52021 did not prevent the leukopenia following PAF injection but significantly inhibited the thrombocytopenia induced by PAF. The dioxolan compound, BN 52111, dose-dependently reduced the bronchoconstriction (IC50 = 0.27 mg/kg) and at doses higher than 1 mg/kg partially antagonized the decrease in the number of circulating platelets and leukocytes induced by PAF. BN 52115 also markedly inhibited the bronchoconstriction (IC50 = 0.36 mg/kg) as well as the thrombocytopenia induced by PAF, but was without significant effect on the leukopenia. These results demonstrate that the two dioxolan compounds, BN 52111 and BN 52115, are more potent than BN 52021 in inhibiting the in vivo bronchopulmonary alterations induced by PAF. Since these alterations are related to the activation of platelets by the autacoid, these blood elements are probably the targets of BN 52111 and BN 52115. Injection of PAF (10 and 100 ng) via the pulmonary artery of ventilated and perfused guinea-pig lungs induced dose-dependent increases in pulmonary inflation pressure (PIP) and pulmonary perfusion pressure (PPP), associated with a dose-dependent release of thromboxane B2 (TxB2). Addition of BN 52021, BN 52111 or BN 52115 (0.1, 1 or 10 microM) to the perfusion medium, 15 min before challenge, dose-dependently inhibited the bronchopulmonary effects of PAF. Although BN 52111 was the more potent in inhibiting the PAF-induced increase in PIP, BN 52021 was the more active with respect to the PAF-evoked generation of TxB2, suggesting that the two phenomena are not directly related.

Structure-activity relationship in PAF-acether. 4. Synthesis and biological activities of carboxylate isosteres.

The synthesis and biological characterization of some 3-carboxylate isosteres of PAF-acether structurally modified in positions 1 (ether, carbamate), 2 (acetoyl, ethoxy), and 3 (chain length and polar head group) are reported. All derivatives present antagonist activities against PAF-acether-induced effects in vitro (platelet aggregation) and in vivo (bronchoconstriction and thrombocytopenia in guinea pig and, to a lesser extent, hypotension in rat). The functional modifications presented here do not modify dramatically the potency of antagonist activities, and there is no enantioselectivity. All of the isosteres are specific PAF-acether antagonists, except the 1-carbamoyl analogue, which is also potent against acetylcholine-induced hypotension and bronchoconstriction.