Lymphotropic antifilarial agents derived from closantel and chlorambucil.

New closantel and chlorambucil prodrugs expected to accumulate in the lymphatic system were evaluated on the filaria Molinema dessetae. The prodrugs of closantel had a delayed effect in vitro on the infective larvae compared to the free drug. The closantel prodrugs were less toxic in vivo than closantel itself. The most active prodrug after treatment at 200 mumol/kg by the oral route was the 1,3-dipalmitoyl-2-succinyl-glycerol-closantel. The macrofilaricidal delayed effect of closantel prodrugs was of interest to prevent anaphylactic shock. In vitro, chlorambucil was active on M. dessetae infective larvae with an IC50 of 26 microM. 1,3-Dipalmitoyl-2-chlorambucil-glycerol was slightly active while the addition of a thioether function between the drug and the lymphotropic ligand canceled the activity. However, no activity with chlorambucil and its prodrugs was observed in vivo. The lymphotropism of these prodrugs has now to be verified using comparative pharmacokinetics in serum and lymph to quantify the increase in drug concentration in lymph.

Studies on lipidomimetic derivatives of alpha-difluoromethylornithine (DFMO) to enhance the bioavailability in a Trypanosoma B. brucei murine trypanosomiasis model.

DFMO, a trypanostatic drug, presents a satisfactory intestinal absorption but its elimination from the blood is rapid so that high doses are necessary to obtain to therapeutic effect. In this study, we propose a strategy to enhance the bioavailability of DFMO by using lipidomimetic derivatives. Three lipidomimetic DFMO derivatives called O-DFMO, S-DFMO and Chol-DFMO were designed to reach easily the plasma and to be cleaved preferentially by plasma esterases progressively liberating free DFMO. Chol-DFMO only could be cleaved partially whereas the other compounds appeared to be stable in reconstituted intestinal medium and mouse plasma. Nevertheless, the use of DFMO derivatives in T. b. brucei experimental chemotherapy appeared as an interesting approach. Thus, O-DFMO was trypanocidal in vitro whereas DFMO, the active principle, was only trypanostatic. Nevertheless, this compound did not release DFMO in mouse blood as expected and acted therefore not as a prodrug. Oral treatment using low doses of compound O-DFMO was able to cure 40% mice while the active principle (eflornithine) administered at 50 fold higher molarity failed to cure any mice. This indicates that compound O-DFMO acts by a specific mechanism which remains to be investigated. S-DFMO was less active and Chol-DFMO had no in vitro activity but released small amounts of DFMO in mice, however, too slight to obtain a therapeutic effect.

Synthesis and orally macrofilaricidal evaluation of niclosamide lymphotropic prodrugs.

The development of new macrofilaricidal drugs is described following a strategy for the promotion of the lymphatic transport of anthelminthic drugs by-passing the liver. The selected compound was niclosamide (CAS 50-65-7) which is very effective in vitro against infective larvae but has no significant antifilarial activity when orally administered at 200 mumol/kg. To estimate the interest of such an approach, the synthesis of 5 prodrugs was achieved in a first stage. The intrinsic antifilarial activity and the delayed effect of these compounds were evaluated in vitro. Then, in vivo tests were performed with Molinema dessetae infective larvae to select the best ligands. The prodrug V 1,3-dihexadecanamido-2-[4-chloro(2- chloro-4-nitroanilinocarbonyl)phenyloxy-carbonylpropanoyl oxy]propane (having a diamide function) was responsible for an in vitro delayed effect and an orally in vivo activity (200 mumol/kg when administered in a single dose). The biological improvement of this easily micellizable prodrug which is stable to intestinal enzymes in respect to Niclosamide confirms such a strategy.

Influence of alkyl chain lengths in dialkylglycerophosphocholines towards phospholipase A2 inhibition in macrophages.

Rat peritoneal macrophages were cultured with a specific and potent phospholipase A2 activator A 23187, with 1-stearoyl-2-[3H]arachidonoyl-sn-GPC as source of [3H] arachidonic acid, and with a dialkyl-GPC, at 2, 10 or 20 microM. Four dialkyl-GPCs were prepared by chemical synthesis. Position 2 of rac-glycerol was alkylated with an alkane chain of 8 carbons and position 1 was alkylated with various alkane chains (8, 10, 12, or 16 carbons). [3H] arachidonic acid was split, then recovered with cell nonesterified fatty acids and nonphosphorous glycerolipids after endocellular phospholipase A2 activity. It was also recovered with fatty acids and eicosanoids isolated from culture medium. Inhibition of fatty acid release and eicosanoid synthesis depended on mixed chain dialkyl-GPC structures. The highest inhibitory effect on arachidonic acid release was reached with 1-decyl-2octyl-GPC and was practically as high in culture medium (IC50 at 5 microM) as in cells (IC50 at 4 microM). 1,2-di-octyl-GPC and 1-dodecyl-2-octyl-GPC had weaker inhibitory effects (but higher in culture medium than in cells). The asymmetrical 1-hexadecyl-2-octyl-GPC poorly affected enzyme activity.

Structure-activity of three phospholipid analogues towards inhibition of phospholipase A2 in macrophages.

At concentrations 1-20 microns in culture medium of rat peritoneal macrophages which were stimulated with ionophore A23187, the phospholipid analogues 1-decyl-2-octyl-glycerophosphocholine and 1-dodecyl-2-octanamido-2-deoxy glycerophosphocholine were found more potent inhibitors than 1-octyl-2-deoxy glycerophosphocholine to lower the phospholipase A2 activities. The inhibitory effect was measured by [3H] eicosatetraenoic acid ([3H]20:4) release in macrophages and extracellular fluids and synthesis of [3H] eicosanoids after incubation of macrophages with traces of the molecular species of lecithin 1-octadecanoyl-2-[3H] eicosatetraenoyl glycerophosphocholine. The three phospholipid analogues developed higher inhibitory effects than mepacrine, dexamethasone or bromophenacyl bromide, at corresponding concentrations in medium.

Synthesis and evaluation of the anti-inflammatory effects of niflumic acid lipophilic prodrugs in brain edema.

Five new lipophilic prodrugs of the non-steroidal anti-inflammatory drug, niflumic acid (Nifluril, CAS 4394-00-7), were synthetized and evaluated on the experimental brain edema (injection of phospholipase A2). The effect of these drugs in comparison with dexamethasone which elicits a marked effect on clinical and experimental brain edema was evaluated. Niflumic acid was vectorised by cholesterol, hexadecanol and by three 1,3-diacylglycerols. The anti-inflammatory activity of these compounds on experimental brain edema was evaluated by determination of the prostaglandin E2 (PGE2) brain tissue concentration. Niflumic acid reduced the prostaglandin E2 production more significantly than dexamethasone. Niflumic acid prodrug forms (1,3-dihexadecanoyl-2-[2-[3-(trifluoromethyl)anilino]nicotinoyl] glycerol and 1,3-dihexadecanoyl-2-[2-[3-(trifluoromethyl)anilino]nicotinoyloxybuta noyl] glycerol also showed a marked anti-inflammatory activity at low concentrations.

Dissociate arachidonic acid release from eicosanoid synthesis in rat peritoneal macrophages is possible using concomitantly eicosapentanoic acid as a phospholipid analogue.

Activated macrophages exposed to the association of eicosapentanoic acid 20:5 n-3 and a synthetic non hydrolysable phospholipid analogue maintained a discrete synthesis of active eicosanoids. 20:4 n-6 split from the internalized 20:4-GPC was accumulated in cells and extracellular fluids. This combination thus represents a novel approach to reduce the 20:4 n-6 cascade.

High rate of intestinal absorption of the phospholipid anlaogue 1-dodecyl 2-[1-14C] octanamido-sn-2-deoxy-glycero-3-phosphocholine in the rat.

The phospholipid analogue with two short fatty chains, 1-dodecyl-2-[1-14C] octanamido-sn-2-deoxy-glycero-3-phosphocholine ([14C] phospholipid analogue), with a non-hydrolyzable bond at position 2 of the glycerol, is an inhibitor of phospholipase A2. It was obtained after chemical synthesis and 0.5 micromol was solubilized in Na+ taurocholate with an equimolar amount of 1-octadecanoyl 2-[3H]eicosatetraenoyl-sn- glycero-3-phosphocholine which is the current substrate of phospholipases A2. Both molecules were introduced into the duodenum of rats in order to follow their captations by intestinal mucosa cells for 30, 60 or 90 min. The [14C] phospholipid analogue was poorly split by phospholipases A2 (pancreatic juice and intracellular enzymes). It disappeared from the intestinal contents (87% of the dose gone in 90 min) as rapidly as the tritiated lecithin (81%) but this was later split by the phospholipases at a higher rate.

Uptake of the phospholipase A2 inhibitor 1-dodecyl 2-[1-14C] octanamido-sn-2-deoxy glycero-3-phosphocholine by peritoneal macrophages.

The [14C] phospholipid analogue 1-dodecyl-2-[1-14C] octanamido-sn-2-deoxy glycero-3-phosphocholine was synthetized. With 2 short fatty chains linked by alkyl and amido bonds to positions 1 and 2 of the glycerophosphate backbone, it was an inhibitor of phospholipase A2 in ionophore A23187-stimulated macrophages. Its uptake by rat peritoneal macrophages and its resistance towards phospholipases A2 were determined at nanomolar or micromolar concentrations in the culture medium. A control substrate for phospholipases A2 activity was established with the lecithin 1-octadecanoyl 2-[3H] eicosatetraenoyl-sn-glycero-3-phosphocholine ([3H] 20:4-GPC), a source of [3H] arachidonic acid after cleavage at position 2. Non-stimulated- or ionophore A23187-stimulated macrophages incorporated extensively the [14C] phospholipid analogue added at 30-4000 nM. At 4000 nM which induced 50% inhibition of the phospholipase, 40% of the dose was found associated with the [14C] phospholipids of 2 x 10(6) stimulated macrophages after 120 min incubation, while only low amounts of [14C] non-phosphorous lipids were detected. It is concluded that the [14C] phospholipid analogue was readily taken up by the macrophages with limited hydrolysis.