Radio-opaque liposomes for the improved visualisation of focal liver disease by computerized tomography.

Liposomes laden with radio-opaque agents (ROA) were prepared by the reverse phase evaporation method. The liposome solutions--containing 1.7-2.5 mg iodine per mg lipid--were injected into Wistar rats. 60 min post injection the rats were scanned in a Siemens CT scanner. Following the injection of 260 mg encapsulated iodine per kg bodyweight--less than half the iodine of the normal ROA bolus injection--there was a long lasting arise in liver enhancement of 40 Hounsfield units (HU). In animals with liver tumours induced by nitrosamine lesions of 2-3 mm in diameter could be detected.

Sterilization of contrast media (Isovist) containing liposomes by ethylene oxide.

Liposomes containing Isovist were prepared by controlled detergent dialysis as well as by reverse phase evaporation. After preparation these liposomes were lyophilized and then submitted to sterilization by ethylene oxide. Prior to lyophilization, trehalose was added as a protective agent to preserve the size of the liposomes. After each step in the preparation size distribution was determined by photon correlation spectroscopy. The computer program CONTIN was adapted for the data analysis and proved to be applicable for polydisperse solutions of liposomes. Neither freeze-drying nor sterilization had negative effects on the morphological quality of the samples when using 4 g of trehalose per 1 g of lipid. In addition, the quality of the liposomes was controlled by scanning electron microscopy. At the end of seven days, no growth of microorganisms occurred in any of the samples.

In vitro inhibition of phospholipase A2 by gabexate mesilate, camostate, and aprotinine.

Gabexate mesilate (FOY, ethyl-p-(6-guanidino-hexanoyl-oxy)-benzoate-methansulfonate), camostate (N,N-dimethyl-carb-amoylmethyl-4-(guanidinobenzoyloxy)-phenyl-acet ate) methansulfonate and aprotinine (Trasylol) were tested for possible inhibition of phospholipase A2. Gabexate mesilate at a concentration of 5 x 10(-4) mol/l and camostate at a concentration of 10(-3) mol/l caused a 50% reduction in enzyme activity. There was almost no inhibition by aprotinine at clinical doses; 40 million KIU/l were necessary to reduce phospholipase A2 activity by 20%. From the therapeutic dose (4,000 mg/day per i.v. infusion) and the half-life of gabexate mesilate in blood circulation (1 min) it can be calculated that the in vitro concentration of gabexate mesilate is only 10(-6) to 10(-7) mol/l. Under these conditions gabexate mesilate cannot diminish the in vivo enzyme activity of phospholipase A2.

Gabexate mesilate and camostate: new inhibitors of phospholipase A2 and their influence on the alpha-amylase activity in serum of patients with acute pancreatitis.

The new synthetic polyvalent protease inhibitors gabexate mesilate (ethyl-p[6-guanidinohexanoyloxy]-benzoate methansulfonate) and camostate (N,N-dimethylcarbamoylmethyl-4-[4-guanidinobenzoyloxy]-phenylacetate methansulfonate) were tested for possible inhibition of phospholipase A2 activity. In a pilot study, we treated 17 patients suffering from acute pancreatitis with continuous intravenous administration of gabexate mesilate, 450 mg/d. The results were compared with a placebo group (same standard therapy) of 21 patients suffering from acute pancreatitis. In vitro experiments showed that, at concentrations between 10(-4) and 5 X 10(-4) mol/L (depending on the enzyme assay employed) for gabexate mesilate and between 10(-3) and 5 X 10(-4) mol/L for camostate, a 50% reduction in phospholipase A2 activity was effected. Comparing the two groups of acute pancreatitis patients after 6 days of treatment with gabexate mesilate, we observed a statistically significantly lower alpha-amylase activity in the serum of treated patients compared with the placebo group.

Inhibition of phospholipase A2 by gabexate mesilate, camostate and aprotinine.

Gabexate mesilate (FOY, ethyl-p-[6-guanidino-hexanoyloxy]-benzoate-methanesulfonate), camostate (N,N-dimethyl-carbamoylmethyl-4-[4-guanidinobenzoyloxy]-phenylacetate), methanesulfonate (FOYPAN) and aprotinine (Trasylol) were tested for possible inhibition of phospholipase A2. Gabexate mesilate at a concentration of 5 x 10(-4) mol/l and camostate at a concentration of 10(-3) mol/l caused a 50% reduction in enzyme activity. There was no inhibition by aprotinine at clinical doses; 40 million KIU/l were necessary to reduce phospholipase A2 activity by 20%.

Pharmacokinetics of liposome encapsulated cisplatin in rats.

The chemotherapeutic anticancer agent Cisplatin--cis-diamine-dichloroplatinum (II)--has several disadvantages, such as its extreme nephrotoxicity, fast elimination via the kidneys, and rapid binding to plasma proteins. Encapsulation into negatively charged, multilamellar liposomes (MLV), composed of egg-lecithin: cholesterol: dicetylphosphate in 5:5:1 molar ratio, causes drastic changes in behavior after a single i.v. injection: marked increase in the circulation half life paralleled by slower urinary excretion; remarkedly higher levels of free drug in the blood; increased uptake in liver, spleen and lymph nodes, and decreased uptake in skin and bones. In the kidneys a reduced and retarded uptake over 15 hr, then a higher uptake was found. The liposomal Cisplatin has a strong transitory diuretic effect, which nevertheless is not paralleled by other signs of renal injury such as decreased output of creatinine or increased output of protein or urinary enzymes. By pre-ejection of empty liposomes the diuresis can be reduced and the additional Pt deposit in the kidneys prevented.

Influence of a portacaval shunt on the distribution of 14C-chol-PC-DCP-liposomes and liposome entrapped 3H-methotrexate in the organs of rats.

The individual roles of hepatic parenchymal cells and non-parenchymal cells in the uptake of intravenously injected liposomes by the liver have not yet been clearly determined. Experimentally, it is suggested that a portacaval shunt reduces the phagocytic capacity of the liver RES. In our experiments a portacaval shunt does not influence the hepatic uptake of 14C-Chol-PC-DCP-liposomes and the liposome entrapped 3H-Methotrexate in rats. In normal rats and in rats with a portacaval shunt, most of the 14C- and 3H-radioactivity is found in the liver and the ratio between the amounts of free and liposome entrapped radioactivity 3 to 15 hours after i.v. injection is approximately 1:10. Therefore, it is concluded that the liver RES does not play an integral role in the enrichment of liposomes in the liver.

Uptake of liposomes and sheep red blood cells by the liver and spleen of rats with normal or decreased function of the reticuloendothelial system.

The distribution of negatively charged liposomes in rats with normal or depressed function of the liver RES was examined. RES activity was determined by the uptake of the sheep red blood cells (SRBC). Whereas pretreatment with colloidal carbon or dextran sulphate drastically diminishes the SRBC uptake by the liver, the liposome uptake is decreased by 12-15% only. In the spleen, such pretreatment boosts the SRBC uptake five- to sixfold, whereas liposome uptake was decreased by about 50%. This indicates that phagocytosis by the RES is only of several liposome-cell interactions. Consequently, the suppression of the RES function is of no practical use when attempting to suppress the preferential liposome uptake by the liver and spleen.

Effect of liposome-entrapped methotrexate on Ehrlich ascites tumor cells and uptake in primary liver cell tumor.

A therapeutically useful concentration (0,5 mg/ml) of Methotrexate was prepared in negatively charged artificial liposomes (lecithine: cholesterol: dicetylphosphate=5:5:1 molar ratios). Entrapment yield after separation on Sepharose 6B is nearly 100%. In contrast to free Methotrexate the liposome entrapped folic acid antagonist is eliminated only slowly by the kidneys and after intravenous application it is unevenly distributed in the body of mice, the highest concentrations being found in liver and spleen. Daily injections (7.5 mg/kg/day) of entrapped Methotrexate for 5 days into the tail vein of Ehrlich ascites tumor bearing mice reduced both tumor cell count and the production of ascites fluid about fourfold as compared to mice receiving the same dose of Methotrexate in the free form. Six hours after intravenous application of liposome entrapped Methotrexate the tissue concentration in normal liver is ca. 20-fold higher than when the same amount is applied in the free state. On the other hand, only a little uptake of liposome entrapped Methotrexate was detected in the tissue of nitrosamine-induced primary liver tumor when compared to normal liver tissue of rats.

[The in vitro and in vivo stability of the entrapment of methotrexate in negatively charged liposomes after sterile filtration (author's transl)]. / Die in vitro und in vivo Stabilität des Einschlusses von Methotrexat in negativ geladenen Liposomen nach Sterilfiltration.

The preparation of sterile artificial liposomes (phosphatidylcholine:cholesterol:dicethylphosphate=5:5:1) by Millipore filtration is described. Conditions for storing liposome entrapped Methotrexate have been found which allow storage of the drug in the entrapped form over a period of at least 4 months. It is shown that intravenously injected liposome entrapped Methotrexate is retained for at least 15 hours in the particulate form. During this time no free Methotrexate is detectable in the blood.

[The carrier potential of liposomes for methotrexate. Changing of the tissue levels of methotrexate in the organs of mice (author's transl)]. / Liposomen als Träger für Methotrexat. Anderung der Gewebsspiegel von Methotrexat in Mäuseorganen.

Drugs entrapped in liposomes (artificial lipid vesicles) exhibit different pharmacokinetics after intravenous application than drugs injected in a free form. The folidacidantagonist methotrexate can be entrapped in liposomes in a therapeutically useful concentration (0.5 mg MTX/ml) and can be stored with high stability of entrappment. After intravenous injection into the tail vein of mice liposomes entrapped methotrexate is found more enriched in cell systems with high rate of endocytosis and not eliminated by the kidneys within 3 h like free methotrexate. It can be shown, that for the organs liver, spleen, kidney, gut, lung, and blood over a 6 h period liposomes entrapped methotrexate is enriched in the tissues and that for example after 6 h the methotrexate level in the liver is 20 fold higher in comparison to free injected methotrexate.

[Enzyme diagnosis in lipaemic sera before and after polyanion precipitation with heparin and magnesium chloride (author's transl)]. / Enzymdiagnostik in lipämischen Seren vor und nach Polyanionenpräzipitation mit Heparin und Magnesiumchlorid.

The method for the determination of enzymic activity in turbid, lipaemic sera, which involves clearing by polyanion precipitation with heparin and magnesium chloride, was critically reviewed. In the diagnosis of diseases of the liver and pancreas, which are frequently associated with hyperlipoproteinaemia, only residual enzyme activities are measured in the cleared serum after polyanion treatment. In the measurement of glutamate dehydrogenase and in the Phadebas test for alpha-amylase, the enzymes are inactivated by treatment with heparin and magnesium chloride. On the other hand, as a result of polyanion precipitation gamma-glutamyl transferase is transferred, together with lipoproteins and chylomicrons, to the lipid-rich supernatant. Acid phosphatase also exhibits only residual activity in cleared serum. The activity of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, leucine arylamidase, cholinesterase, creatine kinase, lactate dehydrogenase, and alpha-hydroxybutyrate dehydrogenase, and the activity of alpha-amylase in the Merckotest are not affected by polyanion treatment of the serum.

Studies on the multiple forms of gamma-glutamyltransferase.

In sera of 53 patients with highly raised gamma-glutamyltransferase (gamma-GT) (EC 2.3.2.2) activity, the gamma-GT was separated by column chromatography on Sephadex DEAE A-50 into two fractions. In 35 sera only one gamma-GT fraction was found at a conductivity of 2.0--2.5 mS and in 18 sera two gamma-GT fractions were found at a conductivity of 0.2 mS and again at 2.0--2.5 mS. There was no opvious correlation to the underlying disease. In 20 sera the additional parameters, protein, protein electrophoresis, free and esterified cholesterol, bilirubin, triglycerides, lipoprotein pattern and lipoprotein X were examined. We recognized, that the two forms of the gamma-GT are not true isoenzymes but that the separation of the gamma-GT into two fractions is caused by a different distribution of the cholesterol containing lipoprotei,s.

[The electrophoretic pattern of gamma-glutamyl transferase in serum and its alteration by chylomicrons (author's transl)]. / Das elektrophoretische Muster der gamma-Glutamyltransferase im Serum und seine Anderung durch Chylomikronen

Using the sera from 20 patients with elevated gamma-glutamyl transferase activity (EC 2.3.2.2) due to intra- or posthepatic cholestasis, the enzyme was separated into four bands by cellulose acetate foil electrophoresis. The gamma-glutamyl transferase pattern permitted no differentiation between intra- or posthepatic cholestasis. Protein and lipid electrophoresis was performed simultaneously for each serum. It was found that a gamma-glutamyl transferase band, which appeared at the origin in 11 sera, was only observed in lipaemic sera containing chylomicrons. This band of gamma-glutamyl transferase does not, however, represent a true isoenzyme, because it results from a complex between chylomicrons and gamma-glutamyl transferase, which is separated with the chylomicrons, or is produced by the mixing of sera.