Christiane Brassinne

Pilot study of amphotericin B entrapped in sonicated liposomes in cancer patients with fungal infections

A pilot study with amphotericin B incorporated in sonicated liposomes (ampholiposomes) made of egg phosphatidylcholine, cholesterol and stearylamine in a molar ratio 4:3:1 was performed in cancer patients with fungal infections. Fifteen patients received a total of 117 intravenous infusions of ampholiposomes. The total dose of amphotericin B administered per patient ranged from 20 to 1004 mg (mean 472 mg). The number of infusions per patient varied from 1 to 20 (mean 8) and the duration of treatment from 1 to 29 days (mean 10 days). Infusion of doses up to 1.8 mg/kg was well tolerated. None of the common side-effects of Fungizone, the colloidal suspension of amphotericin B, occurred; it was noteworthy that patients had no renal function impairment. Serum amphotericin B concentrations given as ampholiposomes were much higher than those obtained with Fungizone. With a daily treatment schedule, peak and trough serum amphotericin B concentrations, as measured by HPLC, were 10 to 20 micrograms/ml and 5 to 10 micrograms/ml respectively; while they did not exceed 2 micrograms/ml and 1 microgram/ml with Fungizone. Amphotericin B given as ampholiposomes had a prolonged serum beta half-life (25.3 +/- 16.0 h). Higher serum antifungal activity was observed with ampholiposomes as compared to Fungizone. We concluded that ampholiposomes have a better therapeutic index than Fungizone.

Intravenous infusion of high doses of liposomes containing NSC 251635, a water-insoluble cytostatic agent. A pilot study with pharmacokinetic data.

In patients with resistant malignant tumors, we performed a pilot trial of intravenous infusion of a water-insoluble cytostatic agent, NSC 251635, entrapped in large volumes of liposomes made of egg yolk lecithin, cholesterol, and stearylamine (4:3:1). Forty liposome infusions were given to 14 patients in 38 courses. The volume of liposomes (20 mg of lipids/mL) varied from 205 to 1,000 mL or 124 to 617 mL/m2 of body surface, and amounts of NSC 251635 varied from 82 to 456 mg/m2. Three patients received repeated single courses. Liposomal therapy was very well tolerated. Side effects observed during some infusions were mild sedation, fever, chills, lumbar pain, urticarial rash, and bronchospasm. In all patients investigated, an important activation of the complement system was observed. No objective regression of the tumors was observed. The limiting factor in the phase I study was not toxicity but the volume of liposomes that could be prepared at once because of the long time required for its preparation. Pharmacokinetic data showed that maximal serum phospholipid and NSC 251635 concentrations were obtained at the end of the liposome infusion. The drugs peak was followed by a decreasing phase leading to a kind of plateau and a prolonged presence of the drug in the blood until 120 hours after its administration. Comparison of the pharmacokinetics of phospholipids and NSC 251635 suggests a rather rapid dissociation of the drug from the liposome.

Steroid receptors in the human prostate. 2. Some properties of the estrophilic molecule of benign prostatic hypertrophy.

Abstract Some physicochemical properties of the estrophilic ‘receptor’ of human benign prostatic hypertrophy were examined by agar gel electrophoresis. 1) Competition analyses revealed the high selectivity of the molecule for the naturally occurring estrogens but not for representatives of other classes of steroid hormones (androgens, corticosteroids, progesterone). This, coupled with the failure of an estrogen ‘receptor’-rich extract to exhibit detectable tissue specific binding of ( 3 H) 5α-dihydrotestosterone suggests that prostatic androgen and estrogen receptors may have separate identities. 2) The molecule proved highly resistant to enzyme attack, a stability conferred by estradiol-17β rather than by the thiol reagent dithiothreitol. Its proteinaceous nature was finally demonstrated when extract was exposed to enzymes at 0°C prior to steroid addition. 3) Initial complex formation between estrogen and its ‘receptor’ protein was rapid and reached a plateau after 4 hours. Binding was greater at 0°C than 37°C.

Pharmacokinetics of Amphotericin B in Patients Receiving Repeated Intravenous High Doses of Amphotericin B Entrapped into Sonicated Liposomes

AbstractRepeated high doses (2-4 mg/kg/day) of amphotericin B entrapped in sonicated liposomes made of egg yolk lecithin, cholesterol, and stearylamine in the molar ratio 4:3:1 were given intravenously to cancer patients with fungal complications. High levels of the drug were measured in the serum. Pharmacokinetic analysis showed that amphotericin B distribution followed a nonlinear bicompartmental model incorporating a liposome-free drug subsystem. This model allowed us to define the Michaelis-Menten dissociation constant of uptake of the drug entrapped in the liposomes by the storage compartment, the maximal liberation rate and fractional liberation rate of free amphotericin B, and the number of binding sites in the ampholiposome storage compartment. the levels of free amphotericin B in the serum and the kinetics of saturation of the uptake of ampholiposomes by the storage compartment were also calculated.

Intracellular penetration of liposomes containing a water insoluble antimitotic drug in L1210 cells

Abstract To demonstrate the possibility of cell delivery of water insoluble products, L1210 cells were incubated with liposomes containing Nocodazole® (NCL), an experimental drug totally insoluble in water. Liposomes of various lipid compositions readily incorporated NCL, but only those formed from dipalmitoyl phosphatidylcholine, cholesterol and stearylamine (molar ratio 4:3:1) had a clear affinity for L1210 cells, as shown by fluorescence studies. This affinity is mediated by the degree of both cell and liposome membrane fluidity. Cytoplasmic and intranuclear location of these liposomes containing NCL or drug-free and subcellular changes induced by their presence were demonstrated by ultrastructural studies.

Steroid receptors in the human prostate. Detection of tissue-specific androgen binding in prostate cancer

We have searched for tissue-specific binding of 5alpha-androstan-17beta-ol-3-one (5alpha-dihydrotestosterone; 5alpha-DHT) in cytosols prepared from 25 surgically obtained benign prostatic hypertrophy (BPH) samples and in 3 tissue specimens containing prostate cancer cells. The distinction between steroid-receptor complexes and ligand binding to serum sex hormone-binding globulin (SHBG) was facilitated by combination experiments involving both sucrose gradient ultracentrifugation and agar gel electrophoresis. Gradient analysis of a cytosol prepared from a cervical lymph node (CLN) containing metastatic prostate tissue, revealed both 8-S and 4-S forms of high affinity (charcoal stable) 5alpha-[3H]DHT binding. When electrophoresis was performed on gradient fractions from these zones, anodally migrating steroid-receptor complexes were found only in the 8-S peak, the 4-S region containing radioligand bound to cathodally directed SHBG. In similar experiments with two BPH samples heavily invaded with prostate cancer cells only a single 4-S peak of radioligand binding was detected. Its multicomponent nature was uncovered electrophoretically when, in addition to SHBG, saturable, androgen binding molecules appeared anodally. Their incomplete resolution from SHBG on a gradient might have prevented their identification had this been the only method used. In contrast to the cancer-containing tissues, no saturable 5alpha-[3H]-DHT binding, other than that to SHBG, was detected in any of the BPH samples analysed. It is considered that, of the methods currently available, agar gel electrophoresis may be particularly useful for further investigations into the possible multicomponent nature of androgen binding of tissue origin in the human prostate.

Direct fibrinogenolytic and fibrinolytic activity in the human prostate

Abstract Previous work had demonstrated a direct proteolytic activity of human prostatic extract on a substrate of casein but not of fibrinogen or fibrin. In the experiments reported here, with a substrate of fibrin or fibrinogen having a higher concentration and specific activity than in previous experiments, it became possible to demonstrate such a direct proteolytic activity on these substrates. In addition, the presence of a trypsin and plasmin inhibitor in prostatic extract could be demonstrated. By ammonium sulphate fractionation, a fraction can be separated from the extract, containing the factor responsible for the direct proteolytic activity and the inhibitor.

Chemotherapeutic efficacy of Nocodazole encapsulated in liposomes on L1210 murine leukemia.

The use of sonicated phospholipid vesicles (liposomes) as carriers of methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (Nocodazole), a water insoluble antimitotic compound active on mouse L1210 leukemia was investigated. Nocodazole was incorporated in dipalmitoyl-phosphatidylcholine: cholesterol: stearylamine (4:3:1) liposomes that were stable at room temperature for at least 48 hr. No drug leakage nor lipid exchange occurred after a 4 hr incubation at 37 degrees C with RPMI 1640 medium supplemented with 10% fetal calf serum. L1210 cells preincubated (2 x 10(6) cells/ml) at 37 degrees C for 3 hr with various concentrations of micronized Nocodazole or liposome-entrapped Nocodazole were injected i.p. into normal CDF1 mice (10(5) cells/mouse). Longest mean survival times and long-time survivors were observed in the group inoculated with L1210 cells preincubated with liposomes containing Nocodazole. CDF1 mice bearing i.p. or i.v. L1210 leukemia were treated i.p. on days 1, 5 and 9 with micronized or liposome-entrapped Nocodazole. Administration of this latter preparation induced a 50% increase in animal life span at the dosage (25 mg/kg/day) half the one required with the free compound (50 mg/kg/day). The present data indicate that enclosing Nocodazole, a water insoluble antimitotic compound, in liposomes results in an enhanced therapeutic activity against L1210 murine leukemia.

Characterization of two direct fibrinogenolytic activities and of one proteolytic inhibitor activity in the human prostate

Abstract The gel filtration of a fraction prepared by precipitation of a crude extract of human benign hyperplastic prostate tissue resulted in two separated peaks of direct proteolytic activity (DPA1 and DPA2) devoid of any kinase or plasminogen activator activity. A third peak showing proteolytic inhibitory activity (PIf) was also obtained. DPA1 was active on fibrinogen as well as on casein, although the proteolysis of fibrinogen was limited as shown by the electrophoretic pattern of breakdown products. This peak had an esterase activity on p-tosyl-Larginine methyl ester (TAME) but not on L-lysine methyl ester (LME). DPA2 was devoid of esterase activity and was much more active on fibrinogen than on casein, releasing large fragments of fibrinogen. DPA1 and DPA2 appeared to be different from plasmin, to have an active serine center and not to be dependent on thiol groups for their activity. Their possible role in the pathogenesis of the hemorrhagic diathesis associated with some prostatic disorders should be envisaged. Although the human prostate crude extract contained an antiplasmin and an antitrypsin activity, PIf was ineffective against plasmin and did not inhibit either one of the two DPAs. PIf had identical antigenic determinants as plasma α 1 -antitrypsin

Metabolic Activation of 6-Nitrochrysene and 6-Aminochrysene in Vitro and in Vivo

The well-documented mutagenicities and tumorigenicities of many nitro polycyclic aromatic hydrocarbons (nitro PAHs) in experimental models have raised the issue of the potential hazards of exposure to these compounds to humans. While several nitro PAHs have become objects of study due to their quantitative importance in the environment or their extremely potent mutagenic activities in Salmonella tester strains, 6-nitrochrysene (6-NC) came to be of interest primarily because of its highly potent carcinogenic activity in the preweanling mouse bioassay (1–5). However, when applied to mouse skin prior to repeated doses of the tumor promoter 12-0-tetradecanoylphorbol 13-acetate, 6-NC was found to be a weak initiator and, in contrast to the situation in the preweanling mouse model, was less active than the parent compound, chrysene (6,7). It is thus of considerable interest to determine the reason for the high carcinogenic potency of 6-NC in the preweanling mouse and the meaning of the results of the mouse bioassays in terms of assessing the potential risk of 6-NC and related compounds to humans.