Activity of bile-salt-stimulated human milk lipase in the presence of liposomes and mixed taurocholate-phosphatidylcholine micelles.

(1) The interaction of bile-salt-stimulated human milk lipase and liposomal membranes has been investigated in the presence or absence of sodium taurocholate. Freshly purified enzyme enhances the permeability of liposomal membranes but thermally inactivated enzyme does not. (2) The ability of the enzyme to catalyze the hydrolysis of a relatively hydrophilic substrate, 4-nitrophenyl acetate, and a more hydrophobic substrate, 4-nitrophenyl palmitate, has also been measured in media containing small unilamellar vesicles of egg phosphatidylcholine in both the absence and presence of taurocholate, and also in the presence of free taurocholate in the absence of liposomes. (3) The enzyme-catalyzed hydrolysis of 4-nitrophenyl acetate is enhanced in all of these systems, but 4-nitrophenyl palmitate is protected from enzymic attack in the phosphatidylcholine-bile salt systems. If free taurocholate be present in the system before 4-nitrophenyl palmitate is added, then, and only then, is enzymic activity observed. (4) These results have been interpreted in terms of the importance of the microenvironment around the substrate and the role played by the bile salt surfactant in stimulating the enzyme.

The mechanism of liposomal damage by taurocholate.

The stability of small unilamellar vesicles formed by egg-yolk phosphatidylcholine (PC) has been examined in the presence of sodium taurocholate. The permeability of the vesicular membrane changes as the total taurocholate concentration increases, until a transformation from mixed bile salt/PC vesicles to mixed micelles occurs. Based on experiments in which the bile salt-induced release of either hydrophilic (carboxyfluorescein) or hydrophobic (Bromothymol blue) probes was studied, and on fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene and turbidity measurements, a two-step process for the initial stage of liposomal damage by taurocholate is postulated.

Differential effects of liposome-entrapped desferrioxamine on proliferation and erythroid differentiation of murine erythroleukemic Friend cells.

It is known that iron chelators (such as desferrioxamine) are potent inhibitors of both cell proliferation and erythroid differentiation. We have shown with in vitro studies that in the case of tumor cells desferrioxamine is even more efficient in inhibiting cell proliferation when entrapped in liposomes consisting of egg yolk phosphatidylcholine. At the same time liposome-entrapped desferrioxamine retains only a slight effect on hexamethylenebisacetamide induction of erythroid differentiation and hemoglobin accumulation of murine erythroleukemic Friend cells. Based on these findings, we propose liposome-entrapped desferrioxamine as potential antineoplastic agent as well as a specific chemical for the study of both iron metabolism and distribution in normal and neoplastic cells. In addition, unlike free desferrioxamine, the liposome-entrapped drug could also be used in combination with inducers of differentiation. With respect to this issue, it is possible that liposome-entrapped desferrioxamine, might permit erythroid differentiation of both neoplastic cells as well as normal stem cells.

Aromatic dental monomers affect the activity of cholesterol esterase.

The dental restorative monomer, BISGMA (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane), and bisphenol A diglycidyl ether (BADGE) increase the velocity of the reaction catalyzed by pancreatic cholesterol esterase (CEase, bovine). The metabolite of these monomers, bisphenol A bis(2,3-dihydroxypropyl) ether, and a common plasticizer, di-2-ethylhexyl phthalate (DEHP), also increase the velocity of CEase-catalyzed ester hydrolysis. BISGMA at concentrations of 1.5-8.0 microM increases the velocity to 126-169% of its value in the absence of BISGMA. Increasing BISGMA above 8 microM caused no further increase in velocity. BADGE at 7-25 microM increases the velocity to 112-205% of its value without BADGE. The metabolite of BISGMA and BADGE at concentrations of 2.0-7.1 microM increases the velocity to 103-113% of its value without metabolite. DEHP at concentrations of 0.52-4.3 microM increases the velocity to 108-187% of its value without DEHP. On the other hand, bisphenol A dimethacrylate is a competitive inhibitor of CEase, with a K(i) of 3.1 microM.

Randomized trial of cyclophosphamide, methotrexate, and fluorouracil chemotherapy added to tamoxifen as adjuvant therapy in postmenopausal women with node-positive estrogen and/or progesterone receptor-positive breast cancer: a report of the National Cancer Institute of Canada Clinical Trials Group. Breast Cancer Site Group.

PURPOSE AND METHODS: By the mid 1980s, tamoxifen alone was considered standard adjuvant therapy for postmenopausal women with node-positive, estrogen receptor (ER)- or progesterone receptor (PgR)-positive breast cancer. From 1984 through 1990, 705 eligible postmenopausal women with node-positive, ER- or PgR-positive breast cancer were randomized to a National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) study that compared tamoxifen 30 mg by mouth daily for 2 years (TAM) versus TAM plus chemotherapy with all-intravenous cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2 given every 21 days for eight cycles (CMF). RESULTS: There were no significant differences in overall survival, recurrence-free survival, locoregional recurrence-free survival, or distant recurrence-free survival between the two treatment arms. However, there was significantly greater severe toxicity, which included leukopenia (P < .0001), nausea and vomiting (P < .0001), and thromboembolic events (P < .0001), as well as significantly more mild or greater toxicity, which included thrombocytopenia (P = .04), anemia (P = .02), infection (P = .0004), mucositis (P = .0001), diarrhea (P = .0001), and neurologic toxicity (P = .006), in women who received TAM plus CMF. CONCLUSION: The addition of CMF to TAM adds no benefit and considerable toxicity in this group of women.

Liposome-associated retinoic acid. Increased in vitro antiproliferative effects on neoplastic cells.

The activity of liposome-associated retinoic acid was analyzed on in vitro cultured tumor cell lines and compared to the antiproliferative effects of free retinoic acid. It was found that liposome-associated retinoic acid is about 300 times more active than free retinoic acid in inhibiting in vitro cell growth of leukemic and melanoma cell lines. An increased activity of retinoic acid (10-20 times) was also obtained after premixing of this compound with empty liposomes, demonstrating that the retinoic acid efficiently interacts with liposomes which may facilitate solubility and cell uptake of retinoids.

Enzymes inside lipid vesicles: preparation, reactivity and applications.

There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-rehydration method', followed by the 'extrusion technique' has shown to be superior over other procedures. In addition to many investigations in the field of cheese production--there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in the enzyme-replacement therapy or for immunoassays)--including a few in vivo studies. In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous. Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase. Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products. Using physical (e.g. microwave irradiation) or chemical methods (e.g. addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent. A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there. There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates. Using micrometer-sized vesicles (so-called 'giant vesicles') as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules.

A phase I study of pegylated liposomal doxorubicin hydrochloride (Caelyx) in combination with cyclophosphamide and vincristine as second-line treatment of patients with small-cell lung cancer.

The purpose of this study was to determine the recommended phase II dose of liposomal doxorubicin (Caelyx ; Doxil in the United States) in combination with cyclophosphamide and vincristine for previously treated patients with good performance status with relapsed or refractory small-cell lung cancer. Twenty-one eligible patients were enrolled between November 1999 and September 2001 and received liposomal doxorubicin 25-40 mg/m2, cyclophosphamide 750-1000 mg/m2, and vincristine 1.2 mg/m2 intravenously (I.V.) every 21 days. At doses of liposomal doxorubicin 40 mg/m2, cyclophosphamide 750 mg/m2, and vincristine 1.2 mg/m2 I.V., 1 of 6 patients had dose-limiting neutropenia and fever in cycle 2 and 2 of 6 developed grade 3 hand-foot syndrome during cycle 3. Therefore, the recommended phase II doses are liposomal doxorubicin 35 mg/m2, cyclophosphamide 750 mg/m2, and vincristine 1.2 mg/m2 I.V. every 21 days. Antitumor activity was seen at all dose levels. This combination is well tolerated and has evidence of antitumor activity. A phase II evaluation is ongoing.

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.

Tumor cell growth inhibition by liposome-encapsulated aromatic polyamidines.

Apart from its antiproteinase activity, the aromatic polyamidine TAPP-Br [the bromo derivative of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H)] is able to inhibit the in vitro growth of a variety of tumor cell lines, including human melanoma, and breast and kidney carcinoma. We have now shown that TAPP-Br can efficiently be encapsulated into egg phosphatidylcholine vesicles. When incorporated into these liposomes, the inhibitory effect of TAPP-Br is significantly enhanced compared with that of the free drug. Based on these promising results, a proposal is made for the delivery of this antiproliferative agent to tumor cells by using liposomes as the vehicle.

Bilayer permeability-based substrate selectivity of an enzyme in liposomes.

Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase alpha-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide)-for which the liposome bilayer was permeable-were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor.

Human skin irritation studies of a lecithin microemulsion gel and of lecithin liposomes.

Soybean lecithin microemulsion gels offer promising features for the possible use as matrices in transdermal therapeutic systems. In order to assess the skin irritancy potential of the gel, acute and cumulative irriation tests were performed in human subjects in vivo using as comparison an unilamellar soybean lecithin liposome preparation and the solvent isopropyl palmitate (IPP). Acute irritation was tested in 151 volunteers in a 48-hour patch test, whereas cumulative irritation was assessed in a 21-day human repeated insult patch test in 20 volunteers. In the acute irritation test, discrete irritation (erythema only) developed with the gel in 2 subjects (1.3%), with the liposomes in 3 subjects (2.0%), and with IPP in 2 subjects (1.3%). For the assessment of cumulative irritation, the IT50 (irritation time of 50% of the test population) was calculated. IT50 was 13 days for the gel, 14 days for the liposomes and 17 days for IPP. This study shows a very low acute and a low cumulative irritancy potential for the soybean lecithin microemulsion gel making it a candidate matrix for transdermal therapeutic systems also under toxicological aspects.

A 1H nuclear magnetic resonance method for investigating the phospholipase D-catalyzed hydrolysis of phosphatidylcholine in liposomes.

Liposomes with mean diameters between 45 and 73 nm have been prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at pH 8.0; and a new methodology is described which allows one to quantitatively follow the phospholipase D-catalyzed transformation of POPC to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid and free choline. The method does not require a special sample preparation; it takes advantage of the fact that the chemical shift of the protons of the three methyl groups in free choline differs from the chemical shift of the choline methyl protons in POPC. Measurements have been carried out under different experimental configurations and they have been paralleled by electron and light microscopy studies, partially using a fluorescently labeled phospholipid. It has been found that for a fixed concentration of the Ca2+-independent phospholipase D from Streptomyces sp. AA 586 the initial velocity and the reaction yields depend on the size of the vesicles. The smaller the vesicles, the higher the yields and the lower the initial rates. Furthermore, the size of the liposomes does not change during hydrolysis of the external POPC layer.

Modeling of enzymatic reactions in vesicles: the case of alpha-chymotrypsin.

The kinetic behavior of the alpha-chymotrypsin-catalyzed hydrolysis of the two p-nitroanilide substrates succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) and benzoyl-L-Tyr-p-nitroanilide (Bz-Tyr-pNA) was modeled and simulated for two different systems, namely for an aqueous solution and for a vesicle system, which was composed of phospholipid vesicles containing entrapped alpha-chymotrypsin. In the case of the vesicles, the substrate was added to the bulk, exovesicular aqueous phase. The experimentally determined time-dependence of product (p-nitroaniline) formation was modeled by considering the kinetic behavior of the enzyme and-in the case of vesicles-the substrate permeability across the bilayer membrane. In aqueous solution-without vesicles-the kinetic constants kcat and KS (respectively KM) were determined from fitting the model to experimental data of batch product concentration-time curves. The results were in good agreement with the corresponding values obtained from initial velocity measurements. For the vesicle system, using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), simulation showed that the substrate permeation across the bilayer was rate limiting. Using experimental data, we could obtain the substrate permeability coefficient for Bz-Tyr-pNA by parametric fitting as 2. 45 x 10(-7) cm/s.