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DOCSPER—A Synthetic Lipid Fit for In Vivo ApplicationDOCSPER [1,3-Dioleoyloxy-2-(N5-carbamoyl-spermine)-propane] is a cationic amphiphile consisting of a hydrophobic 1,3 dioleylglycerol moiety and threefold positively charged spermine head group (1). We optimised the 5-step-synthesis of the lipospermine and after up-scaling we have obtained sufficient amounts to initiate preclinical investigations. DOCSPER was tested for its ability to transfect eukaryotic cells in vitro. It has proven to possess high transfection efficiency in comparison to commercially available liposomal transfection agents. Furthermore, DOCSPER was extensively tested in several in vivo studies (23). These studies revealed a high transfection efficiency, whereas very low toxicity levels were detected. Thus, the results clearly indicate that the cationic lipid DOCSPER is a reliable, low-risk system for broad applications in gene therapy.Groth D. et al. Int J Pharm 1998; 162:143–157.Nikol S. et al. Int J Angiol 2000; 9:87–95.Armeanu S. et al. Mol Ther 2000; 1(4):366–375.

Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic LipoplexesEarlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (1). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled.The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60 of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90 of the particles was in the blood. Currently, we compare the encapsulation efficiency, stability and targetability of the CCL with stabilized antisense lipid particles (SALP) (2), while also the biological activity of these carriers is addressed. In conclusion our results demonstrate that antisense ODN can be targeted very efficiently to EC in vivo, employing plasma-stable CCL, surface modified with negatively charged albumin.ReferencesStuart DD, Allen TM. BBA 2000; 1463:219–229.Semple S. et al. BBA 2001; 1510: 152–166.

Recently we demonstrated that dietary fish oil (FO) causes changes in intrahepatic cholesterol transport and hypersecretion of cholesterol into bile in rats (J. Clin. Invest. 88: 943-951, 1991). We have now investigated in more detail the relationship between cholesterol and bile acid secretion in rats with chronic bile diversion fed purified diets supplemented (9% wt/wt) with either FO or corn oil (CO) for 2 weeks. Effects of FO on biliary cholesterol secretion (+ 400%; as compared to CO after 14 days) were much more pronounced than previously observed in rats with intact enterohepatic circulation (+50%). Biliary bile acid (+30%) and phospholipid (+120%) secretion were increased to a much lesser extent than that of cholesterol resulting in the formation of bile supersaturated with cholesterol. The biliary cholesterol/bile acid molar ratio was 0.069 and 0.032 in FO- and CO-fed rats, respectively, at noon of day 14. This ratio increased to 0.108 in FO-fed rats at midnight, when bile acid output was maximal, but remained unchanged in CO-fed rats during the day-night cycle. Intravenous administration of taurochenodeoxycholic acid (15 mumol/kg) resulted in a 2-fold increase in bile acid output and a simultaneous 1.6-fold stimulation of cholesterol secretion in both groups, implying that administration of the bile acid induced the secretion of 2-3 times as much cholesterol in FO- than in CO-fed rats. Likewise, administration of bilirubin ditaurate (30 mumol/kg), an inhibitor of bile acid-induced biliary lipid secretion, reduced cholesterol output in both groups by about 50% while bile acid output remained unchanged. It is concluded that, in rats, dietary fish oil increases the disposition of cholesterol into bile by potentiating bile acid-dependent cholesterol secretion, presumably by facilitating the recruitment of bile-destined cholesterol.

We have investigated the processing of the non-exchangeable fluorescent phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine (N-Rh-PE) by rat liver cells. In the hepatocyte couplet system, N-Rh-PE was incorporated into the plasma membrane at 2 degrees C and readily internalized upon warming to 37 degrees C. Fluorescence was initially found to be concentrated in vesicles clustered throughout the cell, but subsequently it started to accumulate in pericanalicular vesicles, tentatively identified as lysosomes, and in the bile canalicular lumen. Analysis of cells and media by t.l.c. revealed the slow formation of at least two metabolites. After intravenous injection into bile-fistula rats of [9,10-3H-oleoyl]N-Rh-PE incorporated in small unilamellar liposomes, the initial rates of elimination from plasma of 3H and rhodamine label were virtually identical. However, biliary secretion of the 3H label (5.5% of dose at 2 h) was much slower than that of the rhodamine label (49.3% at 2 h). The rhodamine label in bile was chloroform-soluble, but not identical to the native molecule, and was resistant to phospholipase A2 and alkaline hydrolysis. To gain insight in the mechanism of the rapid bile secretion of this metabolite, we compared the processing of N-Rh-PE, its deacylated form [glycerophospho(N-sulphorhodamine B sulphonyl)ethanolamine; Gly-N-Rh] and the rhodamine label itself (sulphorhodamine B sulphonyl chloride; SRho). Intravenous injection of chloroform-soluble N-Rh-PE and of methanol/water-soluble Gly-N-Rh complexed with albumin both resulted in rapid bile secretion of chloroform-soluble fluorescent compounds (60.2% and 86.3% respectively at 2 h), which showed behaviour identical to that of the metabolite of liposomal N-Rh-PE on t.l.c. Methanol/water-soluble SRho was also rapidly secreted into bile (89.5% at 2 h) without being metabolized. Bile secretion of the chloroform-soluble metabolite of N-Rh-PE and of SRho was markedly impaired (-31% and -52% respectively) in GY Wistar rats, which express a genetic defect in the hepatobiliary transport of organic anions. Our data show that the rat hepatocyte is capable of modifying the structure of N-Rh-PE, a process which proceeds considerably faster in vivo than in vitro. The chloroform-soluble metabolite is subsequently rapidly removed via the bile. The canalicular organic anion transporting system, which is deficient in GY rats, appears to be involved in the excretion of this apolar product of hepatic metabolism.

3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (FUdR-dipalmitate), a lipophilic prodrug of 5-fluoro-2'-deoxyuridine (FUdR), was incorporated in different types of liposomes. The in vivo distribution and intrahepatic deacylation of liposomal FUdR-dipalmitate was found to be strongly dependent on liposome composition and on drug to lipid ratio. The use of fluid-type liposomes (egg PC/PS/CHOL) rendered FUdR-dipalmitate more susceptible to enzymatic breakdown than solid-type liposomes (DSPC/DPPG/CHOL). A decrease of the retention of the drug in the body was also obtained when FUdR-dipalmitate was incorporated in solid-type liposomes with high drug to lipid ratio (1:10) than with low ratio (1:50). In spite of these substantial differences in the rates at which FUdR was liberated from liposomes with different fluidity, size, or drug to lipid ratio, only minor differences in therapeutic effect were observed in a number of murine tumour models (P388 leukaemia, Lewis Lung carcinoma, B16 melanoma and a C26 adenocarcinoma liver metastasis model). The lipophilic prodrug of FUdR exhibited antitumour activity at 100-600 times lower doses than the free drug. However, at these therapeutic doses FUdR-dipalmitate was also far more toxic. This prohibited the use of higher doses to increase antitumour activity.

To investigate the contribution of plasma-derived phosphatidylcholine (PC) to bile PC, the hepatic processing and biliary secretion of liposome-associated PC was studied in rats. For this purpose, small unilamellar vesicles (SUV), containing trace amounts of [2-palmitoyl-9,10-3H]dipalmitoylphosphatidylcholine ([palmitoyl-3H]DPPC), [choline-14C]-dipalmitoylphosphatidylcholine ([choline-14C]DPPC), di[14C]palmitoylphosphatidylcholine ([14C]DPPC) or di[1-14C]-oleoylphosphatidylcholine ([14C]DOPC), were administered intravenously to unanaesthetized rats, equipped with permanent catheters in heart and bile duct. Biliary secretion of the 14C-head-group label of DPPC was very slow (0.3% of injected dose in 4 h), whereas the [3H]palmitoyl label was secreted at a much higher rate (16% in 4 h), but only after substantial catabolism of the acyl chain. To study the latter process in more detail, we compared hepatic metabolism and biliary secretion of [1-14C]acyl-labelled DPPC and DOPC. In rats with an 8-day bile drainage, degradation products of the oleoyl chain were utilized for synthesis of bile acids, which were subsequently secreted into the bile (2% in 6 h). A much smaller fraction (0.6% in 6 h) was secreted as PC and lyso-PC. When bile drainage was started immediately after SUV injection, i.e. a situation with a low hepatic bile acid synthesis rate and a high phospholipid secretion, the secretion of [14C]DOPC-derived radioactivity in the form of bile acids was decreased (0.2% in 6 h), and that as (lyso-)PC increased (1.5% in 6 h). Biliary secretion of DPPC palmitoyl chains in bile-diverted rats was much less than that of the oleoyl chains, and occurred predominantly as PC and lyso-PC (0.6%, compared with 0.4% as bile acids in 6 h). Breath analyses demonstrated that a considerable fraction of both acyl chains was oxidized to CO2 and expired: 25.1% of the administered label for oleoyl chains and 13.4% for palmitoyl chains respectively in a 4 h period. The results of this study indicate that liposomal PC is only minimally secreted into bile via a direct pathway; the bulk is extensively degraded in the liver. Resulting products are partly secreted into bile, as bile acid or as resynthesized PC. There appears to be a quantitative difference in the metabolism of oleoyl and palmitoyl acyl chains.

Animal Experiments—An Essential Component for the Development of Liposomal Anticancer AgentsDuring several years, in our institute more than a dozen of established or novel anticancer compounds have been encapsulated in liposomes and their pharmacological behavior has been tested in in vitro and in vivo experimental models.It was revealed, that for each substance a tailored liposomal system had to be developed. Animal experiments designed to determine both the antitumor activity and side effects of liposomal in comparison to the free drugs have shown that in the majority of cases a benefit for the vesicular formulation could be obtained. In 712 liposomal compounds tested (Bleomycin, Daunorubicin, Cisplatin, Carboplatin, Cyclophosphamide, CCNU, Alkylphospholipids) a substantial decrease of toxicity, mainly due to changed pharmacokinetic data could be observed. The therapeutic efficacy could be increased by use of liposomes for Bleomycin, Taxol, and Mitoxantrone while in other examples no change (Daunorubicin, Methotrexate, TNF) or even a decrease of activity (Cisplatin, Cyclophosphamide, CCNU) was registered.Carboplatinis one example in which by liposomal encapsulation the pharmcological properties were decisively changed. While the free drug leads to leuko- and thrombopenia, the Carboplatin-liposomes (CPL) revealed after only one i.p. or i.v. injection into mice a substantial and long-standing leukocytosis. That effect was paralleled by a release of cytokines from macrophages into the serum, an increased number of peripheral blood stem cells and colony forming activity. The anticancer activity of carboplatin was remarkably improved especially in breast cancer xenografts by using the liposomal formulation. We hypothesise that CPL of specific size and constitution are efficiently taken up by macrophagesmonocytes. That leads to the induction of growth factors inducing secondarily a stimulation of haematopoiesis.Another example is the encapsulation of Tamoxifen(Tam), an antiestrogen used mainly as first line therapy in estrogen receptor positive breast cancer. Tamoxifen-containing LUVETs prepared from egg phosphocholine, dicetylphosphate and an alkylphospholipid (OPP) had a higher in vitro cytotoxicity both in Tam-sensitive and –resistant breast cancer lines. In vivo testing in xenografts with inherited or acquired Tam-resistance showed that in 24 models resistance could be overcome by an oral treatment with appropriate liposomes.These both examples impressively document that only by inclusion of a consequent in vivo testing procedure the surprising pharmacological effects of liposomal anticancer agents can be revealed.

To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor [G22(infinity)] of the 111In label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower.

Cholesterol (C) needed for bile acid synthesis is recruited from de novo synthesized C as well as lipoprotein C. It is not known whether C used for the primary bile acids and biliary C originates from one or more hepatic C-pools. By using different C vehicles, i.e. lipoproteins and liposomes, we tried to discriminate between metabolic pathways for the formation of bile acids in the liver. Studies were undertaken in rats with a permanent biliary drainage. Bile acids were analyzed by HPLC and capillary GC. (3H-)C was administered intravenously in three vehicles: lipoproteins (LP), multilamellar vesicles (MLV) and small unilamellar vesicles (SUV). For all vehicles over 75% of C was excreted in bile as bile acids after 120 hrs. Initial biliary excretion was highest for LP (5.7% at 1 h after injection), followed by MLV and SUV (1.3 and 0.9% resp.). No differences were detectable between the specific activities of the excreted bile acids 12 h after injection, but at lh the specific activity of murocholic acids was markedly increased in experiments with SUV-C, but not with LMV- or LP-2. Also the gly/tau conjugation ratio was increased for SUV-C at 1 h.We conclude that more than one C pool in the hepatocytes exists from which C is recruited for bile acid synthesis, with SUV-C preferentially used for muricholic acids synthesis. Zonal heterogeneity might be responsible for the observed differences.