Liposomal lactoferrin induced significant increase of the interferon-alpha (IFN-alpha) producibility in healthy volunteers

Interferon-alpha (IFN-alpha) producibility has been widely accepted as one of the important markers to evaluate the immune status. In this study, preliminary clinical tests were carried out to confirm the immunomodulatory activity of liposomal lactoferrin including IFN-alpha producibility and NK activity. In a primary open trial, the liposomal lactoferrin was administered to five healthy males for one week and various immunological indices were evaluated. Furthermore, ten healthy males were administered 319 mg per day of liposomal or non-liposomal lactoferrin for four weeks, and immune status was monitored at 0, 1 and 4 weeks after the intake as well as three weeks after stopping it. In this double-blinded comparative study, the IFN-alpha producibility was significantly increased only in the liposomal lactoferrin group during administration and decreased 3 weeks after stopping it, while the IFN-alpha producibility was unchanged in the non-liposomal lactoferrin group. Although the biological mechanism of IFN-alpha producibility enforced by liposomal lactoferrin has not been wholly understood, it is suggested to be a novel active constituent having preventive and therapeutic effects on inflammatory diseases, cancer and infectious diseases such as chronic hepatitis C.     

Liposomal daunorubicin as treatment for Kaposi's sarcoma

Anthracycline compounds including daunorubicin are the foundation of many modem chemotherapeutic regimens. However, the side-effects of these compounds can be severe, leading to alopecia, nausea, immune deficiency, and cardiotoxicity. For immunocompromised patients with aggressive Kaposi's sarcoma (KS), these complications often preclude the completion of appropriate chemotherapeutic regimens. This review focuses on the development and efficacy of liposomal daunorubicin (DaunoXome; DNX) carriers for the treatment of KS. Encouragingly, DNX demonstrated increased in vivo stability and specificity. As a result, KS patients benefit from higher cumulative chemotherapeutic doses without significant cardiotoxicity. Tumor response to DNX treatment surpasses that of non-encapsulated daunorubicin and is similar to that observed with conventional multi-drug therapies such as ABV (doxorubicin, bleomycin, vincristine). Moreover, some reports indicate the patient quality of life during therapy may improve with DNX treatment. Although the development of DNX represents a significant advance in KS therapy, recent data suggest that additional modification of the liposomal carrier to include pegylation or target specific antibodies may further increase daunorubicin efficacy in the future.     

Rapid determination of PEGylated liposomal doxorubicin and its major metabolite in human plasma by ultraviolet-visible high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C(16) column (250 x 3 mm I.D., 5 microm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV-visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 microg/mL, with intra-day and inter-day coefficients of variation and percent error < or =10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.     

Rapid separation of low molecular weight solutes from liposomes without dilution

Liposomes can be separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1- or 5-ml plastic syringes. Excess fluid is first removed from the Sephadex beads by centrifugation and a mixture of liposomal entrapped and free solute is applied to the column bed. The centrifugation is repeated forcing the liposomal material through the column into a test tube while the free solute is quantitatively retained in the Sephadex. The procedure is applicable to a variety of solutes and 92 to 100% recovery is achieved for both charged and neutral liposomes. This technique has advantages over other methods for separating extraliposomal solutes from liposomes. Numerous samples can be processed simultaneously within minutes with no dilution of the liposomal preparation. Nonentrapped solute within the Sephadex can be easily recovered in a small volume of water or buffer.     

Trypanocidal activity of free and carrier bound daunorubicin

Activities of a range of macromolecular conjugates of daunorubicin against Trypanosoma brucei rhodesiense in vitro and in vivo are described and compared to those of free daunorubicin. Conjugates tested were daunorubicin attached to bovine serum albumin by (i) a labile 'glutaraldehyde' linkage (D-BSAG), and (ii) a stable succinyl linkage (D-BSAS), daunorubicin covalently linked to agarose beads (D-AG), and daunorubicin adsorbed onto polyisobutylcyanoacrylate nanoparticles (D-PICA). Trypanocidal activity in vitro was retained in all except D-BSAS, whereas in vivo only D-BSAG had any activity. The results indicate that daunorubicin must be released from the conjugate before it can exert its activity.     

Pharmacokinetics,Tissue Distribution,and Safety of P-Ethoxy Oligonucleotides Incorporated in Liposomes

Abstract

P-ethoxy oligonucleotides (oligos) are lipophilic analogs of phospho-diesters. We have used liposomes to increase the intracellular uptake of P-ethoxy oligos, and demonstrated that liposomal P-ethoxy antisense oligos specific for Bcr-Abl, Grb2, Crkl or Bcl-2 mRNA could selectively inhibit the production of the corresponding proteins, thereby inducing growth inhibition in leukemia and lymphoma cell lines. In support of studying the effectiveness of liposomal P-ethoxy antisense oligos in animal models, we had conducted a series of studies to evaluate the pharmacokinetics, tissue distribution and safety of intravenous injection of liposomal P-ethoxy oligos in normal mice. The pharmacokinetics and tissue distribution of liposomal P-ethoxy oligos are very similar to those of other liposomal compounds. The plasma clearance rate of liposomal P-ethoxy oligos was biphasic; the t_1/2 α and t_1/2 β were approximately 6.7 min and 7 h, respectively. The highest concentrations of liposomal P-ethoxy oligos were found in spleen and liver, with a t_1/2 of approximately 48 h. When up to 180 mg of P-ethoxy oligos per kg of mice's body weight were used, the administration of liposomal P-ethoxy oligos had no adverse effects on renal and hepatic functions, or on the hematological parameters studied. No major organ pathologic changes were observed. Our studies suggested that, at the doses studied, liposomal P-ethoxy oligos could be safely used in animal studies. Since liposomal P-ethoxy oligos were found to accumulate mainly in spleen and liver, which are the major organs of leukemic and lymphoma disease manifestation, we are currently investigating the use of liposomal P-ethoxy antisense oligos in experimental leukemia and lymphoma animal models.     

Pulmonary Delivery of Liposomal Drugs

Abstract

This overview will discuss our studies of liposomes aerosols to treat diseases of the lung and will entail (i) formulation and characterization of liposome aerosols, including dry liposome powder aerosols, (ii) modulation of the pharmacokinetic profile of liposomal drugs delivered by aerosol or intratracheal instillation, (iii) liposome-alveolar macrophage interactions in vitro and in vivo, and (iv) safety of liposome aerosols in vivo in mice, sheep and healthy human volunteers. Water-soluble agents can be retained in liposomes during aerosolization with air-pressure nebulizers within certain limitations of liposome composition, size, and operating conditions. Dry powder liposome aerosols have been formulated and deliver water-soluble encapsulated substances efficiently. Pharmacokinetic profiles of liposomal drugs delivered via intratracheal instillation exhibit typical slow release plasma profiles indicating that the carrier is the rate-limiting barrier for release. Accordingly, pulmonary mean residence times are significantly prolonged and systemic concentrations remain low. Liposomes do not inhibit the phagocytic activity of alveolar macrophages in vitro and in vivo, have no apparent histopathologic effects on lung architecture even after chronic administration, and do not alter dynamic compliance, lung resistance, p_aO₂ and p_aCO₂ in awake, unanesthetized sheep and in healthy human volunteers. In conclusion, liposomes are a promising innocuous aerosol delivery system for drugs to achieve prolonged localized drug concentrations in the lung or intracellular drug targeting to alveolar macrophages.     

The Use of Transmembrane pH Gradient-Driven Drug Encapsulation in the Pharmacodynamic Evaluation of Liposomal Doxorubicin

Abstract

The toxicity and efficacy properties of doxorubicin entrapped inside liposomes are sensitive to the physical characteristics of the vesicle carrier system. Studies addressing such relationships must use preparation procedures with the ability to independently vary vesicle size, lipid composition and drug to lipid ratio while maintaining high trapping efficiencies. The transmembrane pH gradient-driven encapsulation technique allows such liposomal doxorubicin formulations to be prepared. Pharmacokinetic, toxicology and antitumour studies with these systems have revealed several important relationships between liposome physical properties and biological activity. The acute toxicity of liposomal doxorubicin is related primarily to the ability of the liposomes to retain doxorubicin after administration. Including cholesterol and increasing the degree of acyl chain saturation of the phospholipid component in the liposomes significantly decreases drug leakage in the blood, reduces cardiac tissue accumulation of doxorubicin and results in increased LD₅₀ values. In contrast, the efficacy of liposomal doxorubicin is most influenced by liposome size. Specifically, liposomes with a diameter of approximately 100 nm or less exhibit enhanced circulation lifetimes and antitumour activity. While these relationships appear to be rather straightforward, there exist anomalies which suggest that a more thorough evaluation of liposomal doxorubicin pharmacokinetics may be required in order to fully understand its mechanism of action. A key feature in this regard is the ability to differentiate between non-encapsulated and liposome encapsulated doxorubicin pools in the circulation as well as in tumours and normal tissues. This represents a major challenge that must be addressed if significant advances in the design of more effective liposomal doxorubicin formulations are to be achieved.     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG₂₀₀₀) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG₂₀₀₀. At this proportion of DSPE-PEG₂₀₀₀, the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG₂₀₀₀ in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Actin disruption inhibits endosomal traffic of P-glycoprotein-EGFP and resistance to daunorubicin accumulation

Intracellular traffic of human P-glycoprotein (P-gp), a membrane transporter responsible for multidrug resistance in cancer chemotherapy, was investigated using a P-gp and enhanced green fluorescent fusion protein (P-gp-EGFP) in human breast cancer MCF-7 cells. The stably expressed P-gp-EGFP from a clonal cell population was functional as a drug efflux pump, as demonstrated by the inhibition of daunorubicin accumulation and the conferring of resistance of the cells to colchicine and daunorubicin. Colocalization experiments demonstrated that a small fraction of the total P-gp-EGFP expressed was localized intracellularly and was present in early endosome and lysosome compartments. P-gp-EGFP traffic was shown to occur via early endosome transport to the plasma membrane. Subsequent movement of P-gp-EGFP away from the plasma membrane occurred by endocytosis to the early endosome and lysosome. The component of the cytoskeleton responsible for P-gp-EGFP traffic was demonstrated to be actin rather than microtubules. In functional studies it was shown that in parallel with the interruption of the traffic of P-gp-EGFP, cellular accumulation of the P-gp substrate daunorubicin was increased after cells were treated with actin inhibitors, and cell proliferation was inhibited to a greater extent than in the presence of daunorubicin alone. The actin dependence of P-gp traffic and the parallel changes in cytotoxic drug accumulation demonstrated in this study delineates the pathways of P-gp traffic and may provide a new approach to overcoming multidrug resistance in cancer chemotherapy. protein traffic; drug resistance in cancer; daunorubicin     

Clinical and Preclinical Pharmacology of Liposomal Vincristine

Abstract

Vincristine is one of the most commonly administered anticancer drugs and is active in a wide range of indications including non-Hodgkin's lymphomas, acute lymphocytic leukemias and lung cancer. Administration of vincristine in long-circulating liposomes may be expected to result in increased accumulation of drug at tumor sites due to “passive targeting” or “disease-site targeting” effects arising from the more permeable vasculature in these regions. Further, for liposomes with appropriate drug release characteristics, extended exposure of tumor cells to vincristine would result from liposomal delivery. The combination of increased drug delivery and extended duration of drug exposure may be expected to result in increased efficacy, particularly because vincristine is a cell-cycle specific drug. It is shown that vincristine can be encapsulated in large unilamellar vesicles (diameter β 100 nm) using a pH gradient (interior acidic) approach. Further, the efficacy of liposomal formulations of vincristine in animal models is highly sensitive to the drug release rate in vivo. A liposomal formulation with drug retention characteristics such that more than 50% of the vincristine is retained in the carrier 24 h following i.v. injection exhibits significantly improved antitumor efficacy in A431 xenograft and P388 murine tumor models in comparison to either free drug or leakier liposomal formulations. The clinical activity of liposomal vincristine has been investigated in relapsed or refractory non-Hodgkin's lymphoma patients at a dose level of 2 mg/m² every two weeks. Of 83 registered patients, there were 24 responses in 68 evaluable patients. The responses according to histology are: Indolent-13%; Transformed-42%; Aggressive-45%. There were no serious cases of myelosuppression or any toxic deaths. It is concluded that liposomal vincristine can be given at high doses, is active and well tolerated and is rarely neurotoxic or myelosuppressive in these heavily pretreated patients. It appears that the benefits of low toxicity and enhanced efficacy noted in the tumor models are also observed in the clinical setting. A multicenter pivotal Phase II trial of liposomal vincristine in relapsed and refractory non-Hodgkin's lymphoma has been approved by the US FDA and is ongoing.     

Stability and reactivity of liposome‐encapsulated formate dehydrogenase and cofactor system in carbon dioxide gas‐liquid flow

Formate dehydrogenase from Candida boidinii (CbFDH) is potentially applicable in reduction of CO₂ through oxidation of cofactor NADH into NAD⁺. For this, the CbFDH activity needs to be maintained under practical reaction conditions, such as CO₂ gas‐liquid flow. In this work, CbFDH and cofactor were encapsulated in liposomes and the liposomal enzymes were characterized in an external loop airlift bubble column. The airlift was operated at 45°C with N₂ or CO₂ as gas phase at the superficial gas velocity U_G of 2.0 or 3.0 cm/s. The activities of liposomal CbFDH/cofactor systems were highly stable in the airlift regardless of the type of gas phase because liposome membranes prevented interactions of the encapsulated enzyme and cofactor molecules with the gas‐liquid interface of bubbles. On the other hand, free CbFDH was deactivated in the airlift especially at high U_G with CO₂ bubbles. The liposomal CbFDH/NADH could catalyze reduction of CO₂ in the airlift giving the fractional oxidation of the liposomal NADH of 23% at the reaction time of 360 min. The cofactor was kept inside liposomes during the reaction operation with less than 10% of leakage. All of the results obtained demonstrate that the liposomal CbFDH/NADH functions as a stable catalyst for reduction of CO₂ in the airlift. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Measurement of daunorubicin uptake of Yoshida sarcoma cells in culture using flow cytofluorimetry

A comparative measurement of the transport and localisation of daunorubicin into Yoshida sarcoma cells, was undertaken by a biochemical extraction process and a flow cytometric method. An advantage of this latter procedure would be to identify subpopulation of cells which have enhanced or impaired daunorubicin incorporation as well as the ability to exclude any non-specific incorporation into cell debris, which would otherwise interfere with the overall estimation.It has been possible to use the Biophysics argon ion laser at a wavelength of 488 nm which coincides with the visible absorption bands of daunorubicin and doxorubicin (adriamycin) and the cytofluorimetric estimations of daunorubicin incorporation have now been compared with biochemically determined uptake in Yoshida cells. A high lethal dose of 10 μM was required to achieve the direct measurement by cytofluorimetry procedures on the Biophysics instrument. From cell fractionation and CHCl₃/amyl alcohol extraction, it was possible to show that during a 5-h exposure period to daunorubicin (10 μM), the uptake into the nucleus was at first rapid and that into the cytoplasm was much slower. After about 3-h incubation, the level in the cytoplasm decreased, followed by a decrease from the nucleus 1 h later. This could be equated when observed microscopically to the gain in fluorescent cell debris.If all nuclear binding is to DNA, then at the level of (10 μM) concentration in the medium, the number of base pairs to daunorubicin would be 9 : 1, respectively. Cytofluorimetry showed a broad spread of intracellular daunorubin fluorescence which increases with cell size. Increasing external concentration caused a more rapid incorporation as well as a quicker release from the cells.     

Subcellular biosynthesis and transport of gangliosides formed from exogenous lactosylceramide in rat liver.

In order to clarify the mechanisms of ganglioside biosynthesis and transport we intravenously administered a liposomal dispersion of radiolabelled lactosylceramide (LacCer) to rats and then followed the time course of the individual gangliosides which became radioactive in the Golgi-apparatus and plasma-membrane fractions prepared from the liver. After administration of radiolabelled LacCer the liver retained a substantial amount of radioactivity, which was distributed among an organic phase (mainly residual LacCer), a fraction containing low-Mr substances (mainly 3H2O) and a ganglioside fraction. The hepatocytes were found to provide the bulk of gangliosides biosynthesized from exogenous LacCer. After subcellular fractionation, the total radioactive gangliosides increased in the Golgi apparatus up to 8 h, to then decrease and practically disappear at 24 h; in the plasma membranes they were progressively concentrated, accounting for high absolute values. Ganglioside patterns were greatly modified with time in both the Golgi apparatus and plasma membrane, but without significant differences between them. Biosynthesis in the Golgi apparatus and accumulation in the plasma membrane of each individual ganglioside followed a precursor-product relationship. The obtained results indicated that once a ganglioside is biosynthesized in the Golgi apparatus, it is in part made available for translocation to the plasma membrane, which rapidly occurs, and is in part retained in the Golgi apparatus, where it acts as a precursor for the biosynthesis of more glycosylated gangliosides.     

High-performance liquid chromatographic determination of liposomal nystatin in plasma and tissues for pharmacokinetic and tissue distribution studies

A reliable reversed-phase high-performance liquid chromatographic method was developed for the determination of liposomal nystatin in plasma. Nystatin is extracted by 1:2 (v/v) liquid–liquid extraction with methanol. Separation is achieved by HPLC after direct injection on a μBondapak™ C₁₈ analytical column with a mobile phase composed of 10 mM sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile adjusted to pH 6. Detection is by ultraviolet absorbance at 305 nm. Quantitation is based on the sum of the peak area concentration of the two major isomers of nystatin, which elute at 7.5–8.5 and 9.5–10.5 min. The assay was linear over the concentration range of 0.05 to 50 μg/ml. The lower limit of quantitation was 0.05 μg/ml, sufficient for investigating the plasma pharmacokinetics of liposomal nystatin in preclinical studies. Accuracies and intra- and inter-day precision showed good reproducibility. With minor modifications, this method also was used for assaying nystatin in various non-plasma body fluids and tissues.     

Liposomes as a Model for the Biological Membrane: Studies on Daunorubicin Bilayer Interaction

In this study the interaction of the antitumoral drug daunorubicin with egg phosphatidylcholine (EPC) liposomes, used as a cell membrane model, was quantified by determination of the partition coefficient (K _p). The liposome/aqueous-phase K _p of daunorubicin was determined by derivative spectrophotometry and measurement of the zeta-potential. Mathematical models were used to fit the experimental data, enabling determination of K _p. In the partition of daunorubicin within the membrane both superficial electrostatic and inner hydrophobic interactions seem to be involved. The results are affected by the two types of interaction since spectrophotometry measures mainly hydrophobic interactions, while zeta-potential is affected by both interpenetration of amphiphilic charged molecules in the bilayer and superficial electrostatic interaction. Moreover, the degree of the partition of daunorubicin with the membrane changes with the drug concentration, due mainly to saturation factors. Derivative spectrophotometry and zeta-potential variation results, together with the broad range of concentrations studied, revealed the different types of interactions involved. The mathematical formalism applied also allowed quantification of the number of lipid molecules associated with one drug molecule.     

Effect of altered temperature storage on the in vitro cellular uptake of liposome drug products

The aim of this study was to evaluate whether temperature stress conditions affect the cellular uptake of liposomal doxorubicin, Doxil^® (DXL; Ortho Biotech, Raritan, New Jersey, USA), and liposomal daunorubicin, DaunoXome^® (DXM; Gilead Sciences, San Dimas, California, USA). Uptake of these cytotoxic compounds is essential for their pharmacological effect. Commercially available DXL and DXM were stressed for 6 days under altered temperature conditions of 22 and 50°C, as compared to storage in their buffered formulations at the labeled temperature of 4°C. The cellular uptake of the liposomal drugs was measured by fluorescence intensity in human ovarian SKOV-3 and murine macrophage J774A.1 cell lines following a 4-hour exposure to DXL or DXM. There was a 5- to 10-fold increase in the cellular uptake of DXL and DXM in both cell lines after stress exposure to 50°C. Exposure of DXL to 22°C stress decreased its uptake by SKOV-3 cells, when compared to exposure of DXL to 4°C control conditions. A cell-based uptake assay may provide a means to assess changes in the functional activity of liposomes in conjunction with evaluation of their physicochemical properties in order to evaluate the stability and integrity of liposomes.     

Effect of Daunorubicin on Subcellular Pools of Glutathione in Cultured Heart Cells from Neonatal Rats

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase.

Control heart cells were found to contain 12.2 ± 1.8 nmolesGSH/IO⁶ cells. Daunorubicin causedarapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH.

By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 89 ± 1.5nmoles, 10 cells and 3.3 ± 0.6 nmoles/10⁶ cells. respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and. after a short lag period. a release of lactate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH.

No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase. whereas dithiothreitol offered partial protection.

The results provide support for an oxidative mechanism in which the decrease in the cytosolic pool of GSH may be the causative factor of daunorubicin-induced toxicity. This decrease in GSH may affect the cytosolic NADPH and various redox groups on proteins, thereby altering the permeability of the plasma membrane and finally causing cell damage.     

Pulmonary absorption of liposomal levonorgestrel

The purpose of these studies was to achieve desired bioavailability after pulmonary administration of Levonorgestrel (LN) and to provide prolonged effective concentration of the drug in plasma and to reduce reported side effects of orally administered drug. The plain drug suspension, physical mixture (plain drug with liposomal constituents), and drug-encapsulated liposomes containing 10 μg of drug were instilled intratracheally in rats. Similarly, 10-μg drug suspension (LO) was administered orally. The blood samples were withdrawn at specific time intervals and were subjected to LN analysis by spectrofluorimetric technique. The plasma drug concentration data of both the treatments were plotted, and pharmacokinetics data were calculated and compared with that of oral administration. Percentage relative bioavailability (F^*) of 97.6% 98.6%, and 109.9% were observed after pulmonary administration of plain drug formulation (LP1), physical mixture (plain drug along with constituents of liposomes [LP2], and liposomal (LP3) formulations of the drug, respectively. Following oral administration, C_max of 14.4±0.6 ng/mL was observed at 2.1±0.2 hours followed by subtherapeutic concentration beyond 30±0.2 hours, while after pulmonary administration of LP1, LP2, and LP3 formulations, C_max of 4.4±0.4 ng/mL, 4.2±0.5 ng/mL, and 4.4±0.6 ng/ML were observed at 6.0±0.2 hours, 7.0±0.2 hours, and 6.8±0.2 hours, respectively, followed by maintenance of effective plasma drug concentration up to 60±2 hours. These studies demonstrate superiority of pulmonary drug delivery with regards to maintenance of effective therapeutic concentration of the LN in the plasma over a period of 6 to 60 hours. Hence, the pulmonary delivery is expected to reduce frequency of dosing and systemic side effects associated with oral administration of LN.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.