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.

Structure-activity relationship in PAF-acether. 2. rac-1-O-Octadecyl-2-O-acetyl-3-O-[gamma-(dimethylamino)propyl]glycerol .

Two products without phosphoryl groups, 1-O-octadecyl-2-O-acetyl-3-O-[gamma-(dimethylamino)propyl]glycerol and its quaternary salt, were synthesized from 1-O-octadecyl-2-O-benzylglycerol. In comparison with PAF-acether, they lost aggregating and bronchoconstrictive activities and did not show any antagonistic effects.

Structure-activity relationship in PAF-acether. 3. Hydrophobic contribution to agonistic activity.

The synthesis of some selected PAF-acether homologues with an alkoxy-chain length from C1 to C20 in position 1 is described. All agonist activities are closely correlated among themselves and with the calculated fatty-chain hydrophobicity. After a discussion on recent published results and comparison with our data, we conclude that the ether oxide function is absolutely essential at the glycerol 1-position for potent agonist activity and that potency correlates well with hydrophobicity parameters. We indicate the importance of steric and configurational constraints.

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.

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.

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.

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.

Decreased thermogenic response to an oral glucose load in older subjects.

The thermogenic response to a 100 g oral glucose load was studied by indirect calorimetry in 13 older persons (age range, 38-68 years) and compared with that of 16 young matched controls of similar body weight (age range, 19-30 years). The glucose-induced thermogenesis measured over 180 min and expressed as a per cent of the energy content of the glucose load was found to be reduced in the older subjects, i.e., 5.8 +/- 0.3 per cent vs 8.6 +/- 0.7 per cent, P less than 0.002). This was also accompanied by a significant decrease in the glucose oxidation rate when averaged over the same three-hour period following the glucose load, i.e., 153 mg/min vs 213 mg/min in the control subjects (P less than 0.001) despite a similar time course of glycemia. This study suggests that the thermogenic response to an oral glucose load is blunted in older people, and this may represent an additional factor that contributes to the decreased energy requirement with age and therefore to the increased propensity to obesity if energy intake is not adjusted.

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.

Effects of PAF-acether and structural analogues on platelet activation and bronchoconstriction in guinea-pigs.

PAF-acether (platelet-activating factor) (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) induces platelet-dependent bronchoconstriction in the guinea-pig which correlates with its in vivo thrombocytopenic effect. We investigated the influence of modifications of the polar head group in position 3 of the glycerol skeleton of PAF-acether on guinea-pig platelet activation and bronchoconstriction. PAF-acether itself induced concentration-dependent platelet activation (EC50 for aggregation = 0.41 nM and EC20 for secretion of ATP = 0.56 nM). The 3-phosphoryl-N-methyl-morpholino ethanol analogue was slightly more active than PAF-acether and the 3-phosphoryl-N-methyl-piperidinium ethanol, 3-phopshoryl-(N-methyl-piperidino-3') methanol and 3-phosphoryl-(N-methyl-hydroxy-4') piperidine analogues were equieffective to PAF-acether in activating platelets. The 3-phosphoryl-piperidino ethanol analogue was 8 times less active than PAF-acether; the 3-phosphoryl-morpholino ethanol analogue and the 1-O-octadecyl-2-O-acetyl-3-O-[trimethyl-ammonio)-propyl) glycerol were inactive up to 1 microM. Our data show that the choline head group is not a compulsory requirement for activity. When injected i.v. to the propranolol-treated guinea-pigs, the platelet-activating analogues also induced bronchoconstriction. Two PAF-acether antagonists, compounds 48740 RP and BN 52021, inhibited PAF-acether-induced platelet activation when added to PRP at the final concentration of 0.1 mM (aggregation inhibited by 91 +/- 4 and by 94 +/- 3% respect.; secretion inhibited by 80 +/- 12 and 79 +/- 10% respectively, mean +/- S.E.M., n = 4). Both antagonists also suppressed platelet activation and in vivo bronchoconstriction, thrombocytopenia, leukopenia and hypotension induced by PAF-acether and the various analogues. Our results indicate that PAF-acether and the analogues studied trigger platelet activation and the consequent bronchoconstriction through mechanisms which share sensitivity to same antagonists.

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.

Synthesis and characterization of a radioiodinated, photoreactive and physiologically active analogue of platelet activating factor.

The multistep synthesis of a photoreactive, radioactive and aggregating analogue of platelet-activating factor (PAF)-acether is described. The photoreactive and radioactive moiety was added at the last step; the specific radioactivity was higher than 1000 Ci/mmol. The concentration of this new analogue which causes 50% of aggregation of platelets were of the same order of magnitude as for synthetic snPAF-acether, so as for two other analogues having a bulky group at the omega end of the fatty ether chain. The photoreactivity was proved by the covalent binding of the analogue to protein (BSA) after 10-min irradiation times at 300 nm. The binding was largely prevented by prior (not by later) addition of a high concentration of lyso phosphatidyl choline.

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.

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.

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.

Phosphorylation of class I HLA antigens in U-937 monocyte-like cells. Role of protein kinase C.

Phorbol esters and 1-oleoyl-2-acetylglycerol induce an increase in phosphorylation of a series of 44-kD proteins in the U-937 human monocyte-like cell line. The phenomenon is rapid at 37 degrees C, starting 0.5 min after adding the inducer, increasing to a maximum of 5-10 min and decreasing to the basal level at 30 min. It is also observed at 4 degrees C, though much more slowly. Immunoprecipitation and two-dimensional electrophoresis demonstrated that these phosphorylated proteins belong to the class I HLA antigens.

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.

[Metabolic effects of gliclazide in diabetes type II patients. Study with indirect calorimetry]. / Effets métaboliques du gliclazide chez le diabétique de type II. Etude par calorimétrie indirecte.

An attempt is made to determine the effect of treatment with gliclazide on the manner in which obese patients with noninsulin-dependent diabetes dispose of an oral glucose load. Six patients were studied on two occasions - once after one month of an isocaloric diet containing 40% carbohydrate, and two months after gliclazide had been added. Body weight remained constant throughout the three months. However, fasting plasma glucose concentration fell and mean (+/- SEM) incremental insulin response to oral glucose increased significantly (p less than 0.05) with drug therapy (51.1 +/- 14.6 microU/ml vs 17.1 +/- 1.5 microU/ml). Glucose oxidation significantly improved after gliclazide treatment (16.9 +/- 2.4 g/3 h vs 7.5 +/- 2.1 g/3 h, p less than 0.02) in parallel with the fall in plasma glucose and the increase in insulin response. In addition, glucose storage significantly increased after drug therapy (44.1 +/- 10.1 g/3 h vs 28.4 +/- 7.2 g/3 h, p less than 0.001). In contrast, lipid oxidation fell with gliclazide treatment (7.2 +/- 0.7 g/2 h vs 12.0 +/- 1.7 g/3 h, p less than 0.02). This effect may be explained by improvement of the antilipolytic effect of insulin. In conclusion, improvement in glucose oxidation (25%) and in glucose storage (55%), together with reduced lipid oxidation, was observed after two months of gliclazide therapy.