Judith Senior

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Abstract Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tcs below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tcs above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.

In vitro characterization of prostanoid receptors on human myometrium at term pregnancy

1 Prostanoid receptors present on the pregnant human myometrium in vitro have been characterized according to the receptor classification proposed by Coleman et al. (1984) using natural prostanoids and synthetic, selective analogues and antagonists where available. 2 Prostaglandin E2 (PGE2) produced a biphasic effect consisting of an initial excitation followed by a dose‐related inhibition. The EP2/EP3‐receptor agonists, rioprostil and misoprostol, produced similar effects to PGE2, however, the excitatory event of the misoprostol response was related to dose. The EP1/EP3‐receptor agonist, sulprostone, evoked a purely excitatory response which was unaffected by AH6809. The selective EP2‐receptor agonist butaprost produced a long‐lasting dose‐dependent inhibition of activity. The results from these prostanoids indicated that inhibitory EP2‐ and excitatory EP3‐receptors are present on myometrium from pregnant donors at term. 3 PGF2α and the synthetic FP‐receptor agonist, fluprostenol, caused equipotent excitatory effects, indicating the presence of contractile FP‐receptors. 4 PGD2 produced a biphasic effect of which the inhibition appeared dose‐related and was antagonized by the selective DP‐receptor antagonist BW A868C. The selective DP‐receptor agonist, BW245C, produced a potent inhibitory effect that was competitively antagonized by BW A868C (pA2 = 8.6). 5 PGI2 produced a biphasic response qualitatively similar to PGE2. The EP1/IP‐receptor agonist, iloprost, produced an occasional unquantifiable excitation and dose‐related inhibition. The selective IP‐receptor prostanoid, cicaprost, evoked only an inhibitory response. 6 The stable thromboxane A2 (TXA2)‐mimetic, U46619, produced potent excitation which was competitively antagonized by the TP‐receptor antagonist, GR32191 (pA2 = 7.2). 7 The prostanoids tested indicate that a heterogeneous population of prostanoid receptors are present on human myometrium from pregnant donors. It may be concluded that excitation is EP3‐, FP‐ and TP‐receptor‐mediated and inhibition is EP2‐, DP‐ and IP‐receptor‐mediated. Comparison of data obtained from non‐pregnant specimens indicates that the lower segment tissue from pregnant donors demonstrated more pronounced responses to EP2 and IP‐receptor activation.

In vitro characterization of prostanoid FP‐, DP‐, IP‐ and TP‐receptors on the non‐pregnant human myometrium

1 Prostaglandin F (PGF), PGD, PGI and thromboxane A2 (TXA2) receptors have been pharmacologically characterized on the non‐pregnant human myometrium in vitro in accordance with the receptor classification proposed by Coleman et al. (1984). The tools for the classification include both natural prostanoids, synthetic, selective analogues and antagonists where available. 2 The potent excitatory actions of the natural FP‐receptor prostanoid, PGF2α, and the synthetic analogue, fluprostenol, indicate the presence of FP‐receptors mediating contraction on the human myometrium. 3 PGD2 produced a biphasic response consisting of excitation followed by relaxation of spontaneous activity of the myometrium. The selective DP‐receptor agonists, BW245C, produced purely inhibitory responses illustrating the presence of inhibitory DP‐receptors in this tissue. The inhibitory responses of both PGD2 and BW245C were antagonized by the competitive DP‐receptor antagonist, BWA 868C, providing conclusive evidence for the existence of DP‐receptors. 4 PGI2 produced a biphasic response similar to PGD2. Iloprost, the EP1/IP‐receptor agonist also produced a biphasic response, whilst the IP‐receptor selective agonist, cicaprost, caused inhibition only, suggesting that inhibitory IP‐receptors exist in the non‐pregnant human myometrium. 5 The TXA2‐mimetic, U46619, produced marked stimulation of the non‐pregnant human myometrium and was approximately equipotent to PGF2α and fluprostenol in this effect. The actions of U46619 were competitively antagonized by the TP‐receptor antagonist GR32191 showing that excitatory TP‐receptors exist in this tissue. 6 All prostanoids tested, both natural and synthetic, had activity on the non‐pregnant human myometrium in vitro, supporting the existence of a heterogeneous population of prostanoid receptors in this tissue. If the results from the present study are combined with those previously reported for EP‐receptor agonists (Senior et al., 1991), it may be concluded that excitation may occur through FP‐, TP‐, EP3‐ and few EP1‐receptors, whereas inhibition may occur through DP‐, IP‐ and EP2‐receptors.

Is half-life of circulating liposomes determined by changes in their permeability?

For instance, efficient interaction of drug-con- taining liposomes with target cells in the intra- vascular space or access to extravascular cells is likely to be promoted by the carrier’s prolonged presence in the circulation. To this end, there has been variable success through reduction of vesicle size [2], adjustment of surface charge [3], or phos- pholipid composition [4,5], the use of phospholip- ase-resistant phospholipids [6] and, indirectly, by blocking the reticuloendothelial system [3,7,8]. However, an important prerequisite for retarded liposome clearance is resistance to the detrimental effect of blood on bilayer stability 19,101. Recent studies have shown that excess cholesterol in cer- tain types of liposomes greatly improves bilayer stability in the presence of blood and reduces their permeability

In vitro characterization of prostanoid EP-receptors in the non-pregnant human myometrium

1 Prostaglandin receptors of the PGE type have been characterized in the non‐pregnant human myometrium in vitro according to the scheme of Coleman et al. (1984) by use of the agonists PGE2, sulprostone, rioprostil, AY23626, butaprost, misoprostol, 16,16‐dimethylprostaglandin E2, enprostil and iloprost, and, the antagonist AH6809. 2 All prostanoids tested were active in non‐pregnant human myometrium either as stimulators and/or inhibitors of spontaneous activity or both. Biphasic responses to PGE2 indicate that at least two receptor types of the EP‐receptor exist, one mediating relaxation and the other mediating contraction. 3 Further evidence for the EP‐receptor mediating excitation and relaxation was provided by the action of the EP2‐/EP3‐receptor selective prostanoids rioprostil, AY23626 and misoprostol, and the EP1‐/EP2‐receptor selective agonist 16,16‐dimethylprostaglandin E2. 4 Butaprost, an EP2‐receptor selective agonist, produced potent inhibition of spontaneous activity in the tissue which was generally longer‐lasting than that evoked by the natural prostanoid PGE2. 5 The EP1‐/EP3‐receptor selective agonist sulprostone and the EP3‐receptor agonist enprostil produced potent contractile responses supporting the presence of contractile EP3‐receptors in the non‐pregnant human myometrium in vitro. 6 The EP1‐/IP‐receptor selective agonist, iloprost, produced mixed responses in non‐pregnant human myometrium. The contractile response was inhibited by the EP1‐receptor antagonist AH6809. However, responses to the EP1‐/EP3‐receptor selective agonist sulprostone were unaffected by AH6809 which may indicate that only a small population of EP1‐receptors is present. 7 Therefore it would seem that a heterogeneous population of EP‐receptors is present in the non‐pregnant human myometrium.

Fate of cholesterol-rich liposomes after subcutaneous injection into rats

The fate of small (30-60 nm) and large (about 400 nm diameter) liposomes composed of phosphatidylcholine or sphingomyelin and equimolar cholesterol and containing quenched carboxyfluorescein and 111In-labelled bleomycin after footpad injection of rats was investigated. Judging from the values of latent carboxyfluorescein in the plasma, phosphatidylcholine and sphingomyelin small liposomes entered the blood circulation intact, presumably via the lymphatics to reach 3 h after injection a peak of 16.9 and 23.1% of the dose per total blood, respectively. Of the 111In radioactivity given in phosphatidylcholine liposomes, about 32% was recovered in the liver. Hepatic uptake of 111In for sphingomyelin liposomes was lower (about 9%) reflecting their slower rate of clearance. No latent carboxyfluorescein could be detected in the blood after injection with phosphatidylcholine or sphingomyelin large liposomes and levels of 111In in the liver (and spleen) were very low. A proportionally much greater amount of liposomal 111In was intercepted by the popliteal and to a lesser extent, the lumbar lymph nodes. Such uptake was significantly higher (e.g. 463%) for small than for large (e.g. 195% per g popliteal nodes) liposomes. After foot pad injection of large liposomes into Walker 256 tumour-bearing rats, localization of 111In in the popliteal nodes was similar to that seen with control animals. However, 111In localization in the lumbar nodes was augmented more than 5-fold. These results suggest that large liposomes as used in this study, characterized by efficient drug entrapment, inability to reach the liver and spleen and improved localization in the lymph nodes of tumour-bearing animals may be preferable to vesicles of small size for the local treatment of lymphatic metastases.

Characterization of the prostanoid receptors mediating constriction and relaxation of human isolated uterine artery

1 This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating constriction and relaxation of human isolated uterine artery. 2 U‐46619 was a potent constrictor agonist on human uterine artery (EC50 [95% CL] = 3.5 [1.8‐6.7] μM). Prostaglandin E2 (PGE2α), PGF2, PGD2 and PGI2 only weakly constricted the uterine artery, being at least 100 times less potent than U‐46619. The PGE2 and PGI2 constrictor effects may be modified by the potent dilator effects of these compounds. A number of agonists which show selectivity for FP‐, DP‐ and EP‐receptors including ICI 81008, BW 245C, sulprostone, rioprostil and butaprost, failed to cause any constriction at concentrations up to 30 μM. 3 Constrictor responses induced by all agonists tested were reduced or abolished by the TP‐receptor blocking drugs, GR 32191 and EP 092. pA2 estimates for both antagonists versus U‐46619 were 8.50, values which are consistent with their affinities at TP‐receptors. 4 In preparations pre‐constricted with phenylephrine (1 μM) both PGI2 and PGE2 were potent relaxant agonists. The selective IP‐receptor agonists, cicaprost and iloprost, also dilated human uterine artery and were approximately 10 fold more potent than PGI2. The EP2‐receptor agonists, butaprost and rioprostil and the selective DP‐agonist, BW 245C, were at least 100 fold weaker than PGI2 and PGE2 suggesting that neither DP‐ nor EP2 receptors were involved. 5 We conclude that TP‐receptors mediate constriction, whereas IP‐ and possibly EP4‐receptors mediate relaxation of human uterine artery.

Targeting of Drugs With Synthetic Systems

Mannose Binding Proteins in the Liver and Blood.- Cell Membrane Molecules on Neoplastic Cells: Their Role in Malignant Cell Transformation and Dissemination.- Tumor-Associated Glycolipid Markers: Possible Targets for Drug and Immunotoxin Delivery.- Interaction of Macromolecular Drugs with Receptors.- Endocytosis and Lysosomes: Recent Progress in Intracellular Traffic.- Peptides as Targets and Carriers.- Hepatic Functions in Health and Disease: Implications in Drug Carrier Use.- Targeting with Synthetic Polymers: A Realistic Goal.- Drug-Poly(lysine) Conjugates: Their Potential for Chemotherapy and for the Study of Endocytosis.- Targeting of Colloidal Carriers and the Role of Surface Properties.- Targetable Nanoparticles.- Polymers as Matrices for Drug Release.- Liposomes In-Vivo: A Relationship Between Stability and Clearance?.- Liposomes as Drug Carriers to Liver Macrophages: Fundamental and Therapeutic Aspects.- Alternate Delivery of Interferons.- Liposomes in Antimicrobial Therapy.- Design, Characterization and Antitumor Activity of Adriamycin-Containing Phospholipid Vesicles.- Particle Charge and Surface Hydrophobicity of Colloidal Drug Carriers.- Particle Size Analysis of Colloidal Systems by Photon Correlation Spectroscopy.- Stability of Liposomes on Storage.- Contributors.

Stability and clearance of small unilamellar liposomes studies with normal and lipoprotein-deficient mice

The effect of high density lipoproteins (HDL) on the stability and clearance of injected liposomes was investigated under conditions of abnormal lipoprotein metabolism in vivo. Small unilamellar liposomes composed of phosphatidylcholine and containing quenched carboxyfluorescein were injected intravenously or intraperitoneally into normal mice or mice previously made lipoprotein deficient with 4-aminopyrazolo[3,4d]pyrimidine (4-APP). As evidenced from quenched carboxyfluorescein values in the blood, levels of stable liposomes in the circulation were increased and clearance rates reduced considerably in lipoprotein-deficient animals indicating increased bilayer integrity. This was confirmed by the demonstration that transfer of liposomal phosphatidylcholine to HDL, occurring in the presence of normal mouse plasma, was virtually abolished in the presence of plasma from lipoprotein deficient mice. The role of other lipoprotein species in destabilizing liposomes was also investigated. Plasma from lipoprotein-deficient mice was supplemented with increasing amounts of HDL, LDL + IDL or VLDL (to cover the physiological range of lipoprotein concentrations in mouse blood) prior to the addition of phosphatidylcholine liposomes, and incubated at 37 degrees C. It was shown that among the lipoprotein species studied only HDL was detrimental to liposomal stability under the conditions employed. Our results indicate that use of liposomal drugs in the treatment of patients must take into account HDL fluctuations in their blood as these could after liposomal membrane permeability to the drugs and thus upset therapeutic efficiency.

Studies using isolated uterine and other preparations show bimatoprost and prostanoid FP agonists have different activity profiles.

1 The pharmacology of bimatoprost, a synthetic prostaglandin‐amide, was examined in prostaglandin F2α (PGF2α)‐sensitive preparations. Bimatoprost potently contracted the rabbit isolated uterus (pEC50=7.92±0.16). In contrast, bimatoprost exhibited weak excitatory activity in human myometrium from pregnant and nonpregnant donors, mouse uterus, rat uterus, and endothelium‐intact rabbit jugular veins, and did not stimulate DNA synthesis in mouse fibroblasts. 2 The possibility that the effects of bimatoprost may reflect partial agonism at prostanoid FP receptors was examined and the contractile effects of full agonists, 17‐phenyl PGF2α (FP) and U‐46619 (TP, a control), were determined in the absence and presence of 1 μM bimatoprost on the mouse uterus. Analyses of the agonist–agonist functional studies showed no antagonism, indicating that bimatoprost is not a partial agonist. 3 Bioassay metabolism studies of bimatoprost and latanoprost (FP receptor agonist prodrug) in the rabbit uterus were conducted using recipient mouse uterus. Results indicated that the potent responses to bimatoprost in the rabbit uterus are produced by the intact molecule and not by its putative free acid metabolite, 17‐phenyl PGF2α. Some hydrolysis of latanoprost to latanoprost free acid appears to have occurred in the rabbit uterus, according to biological detection. 4 The pharmacology of bimatoprost could not be explained by its interaction with known prostanoid FP receptors and was independent of species‐, tissue‐, or preparation‐related factors. The potent contractile effects of bimatoprost in the rabbit uterus provide further pharmacological evidence for the presence of a novel receptor population that preferentially recognises bimatoprost.