Influence of lipid composition on the antitumor activity exerted by doxorubicin-containing liposomes in a rat solid tumor model

The effect of changes in lipid composition on the antitumor activity of doxorubicin (DXR)-containing liposomes was studied in immunoglobulin solid immunocytoma-bearing Lou/M Wsl rats. Rats bearing a tumor with a diameter between 20 and 30 mm were treated i.v. with 2 mg/kg free DXR or different DXR-containing liposome types for 5 consecutive days followed by one injection more at day 11 after start of therapy. A similar pattern of tumor regression was observed for free DXR and DXR entrapped in "fluid" liposome types. However, DXR entrapped in "solid" liposome types expressed an antitumor activity which was significantly delayed; during the first 3 days after start of therapy solid DXR-containing liposomes were less effective in inducing antitumor activity than fluid DXR-containing liposomes. In order to gain more insight into the mode of action of DXR-containing liposomes, one of the solid liposome types [composed of distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and cholesterol (chol)] was compared with one of the fluid liposome types [composed of egg phosphatidylcholine, phosphatidylserine, and chol] with respect to distribution and integrity in vivo. Results obtained after i.v. administration of [3H]inulin-labeled vesicles to tumor-bearing animals suggested that a differential liposome uptake by the tumor was not relevant for the explanation of the delayed antitumor effect. To monitor the structural integrity of liposomes after i.v. injection, the liposomes were double radiolabeled with [3H]inulin as a marker of the aqueous phase and cholesteryl [14C]oleate as a marker of the lipid phase. The bilayer structure of both liposome types remained intact during their presence in the blood compartment. Intact liposomes were taken up primarily by liver and spleen with subsequent degradation of the liposome structure. The degradation rate appeared to be dependent on the lipid composition of the liposomal membranes; phosphatidylcholine/phosphatidylserine/chol liposomes were degraded much faster than distearoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/chol liposomes. The difference in degradation rate was manifested more clearly in the spleen than in the liver. In vitro investigations on uptake and processing of liposomes by liver macrophages indicated that the difference in degradation rate between liver and spleen was caused by intrahepatic reutilization of [14C]oleate liberated from the liposome structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacology of liposomal vincristine in mice bearing L1210 ascitic and B16/BL6 solid tumours.

Vincristine pharmacokinetic, tumour uptake and therapeutic characteristics were investigated here in order to elucidate the processes underlying the enhanced efficacy observed for vincristine entrapped in small (120 nm) distearoylphosphatidylcholine/cholesterol liposomes. Plasma vincristine levels after intravenous (i.v.) injection are elevated more than 100-fold in the liposomal formulation compared with free drug in tumour-bearing as well as non-tumour-bearing mice over 24 h. Biodistribution studies demonstrate that the extent and duration of tumour exposure to vincristine is dramatically improved when the drug is administered i.v. in liposomal form. Specifically, 72 h trapezoidal area under the curve values for liposomal vincristine in the murine L1210 ascitic and B16/BL6 solid tumours are 12.9- to 4.1-fold larger, respectively, than observed for free drug. Similar to previous results with the L1210 model, increased drug delivery to the B16 tumour results in significant inhibition of tumour growth, whereas no anti-tumour activity is observed with free vincristine. Comparisons of drug and liposomal lipid accumulation in tumour and muscle tissue indicate that the enhanced efficacy of liposomal vincristine is related predominantly to drug delivered by liposomes to the tumour site rather than drug released from liposomes in the circulation. Consequently, improvements in liposomal vincristine formulations must focus on factors that increase uptake of liposomes into tumour sites as well as enhance liposomal drug retention in the circulation.     

Anti-CD19-targeted liposomal doxorubicin improves the therapeutic efficacy in murine B-cell lymphoma and ameliorates the toxicity of liposomes with varying drug release rates.

Some formulations of liposomal doxorubicin with intermediate rates of drug release have shown increased levels of toxicity in mice. Because antibody-mediated targeting of liposomal drugs influences the pharmacokinetics, mechanism of uptake, and selectivity of the associated drugs, we hypothesized that anti-CD19-mediated targeting of liposomal doxorubicin might moderate the toxicity of the problem formulations. Phosphatidylcholine/cholesterol liposomal formulations of doxorubicin having faster, intermediate, and slower drug release rates were prepared by altering the fatty acyl chain length or degree of saturation of the phosphatidylcholine component. Pharmacokinetic and biodistribution studies and in vivo drug release rates were determined in mice using liposomes dual labeled with [3H]cholesteryl hexadecylether and [14C]doxorubicin. Therapeutic studies were done in xenograft models of human B lymphoma (Namalwa cells). The rate of clearance of the liposomal lipid was similar for all formulations (average t1/2, 18 hours), but the rate of clearance of doxorubicin was dependent on the release rate of the formulation (t1/2, 2-315 hours). Liposomes with the slowest drug release rates showed no toxicity and exhibited therapeutic activity that was superior to the other formulations when targeted with anti-CD19; liposomes with the most rapid drug release rates also showed no toxicity but showed little therapeutic effect even when targeted. Liposomes with intermediate drug release rates exhibited varying degrees of toxicity. The toxicities could be reduced and even overcome by targeting with anti-CD19 antibodies. For these formulations, therapeutic effects were intermediate between those found for liposomes with the fastest and slowest drug release rates.     

Integrin-targeted paclitaxel nanoliposomes for tumor therapy

A neovessel-targeted PEGylated liposomal formulation of paclitaxel was prepared with the purpose of improving the solubility of paclitaxel and specific targeting ability of this drug to tumor vasculature. Alpha_V integrins overexpressed on the surface of new formed tumor vessels were selected to be the targets and their specific ligand, a 12-mer peptide containing a cyclic RGD sequence was used to achieve the goal. After coupled with a KGG-Palmitic acid conjugate, the RGD containing peptide was successfully integrated to the lipid bilayers. Mean particle size of the liposomes was under 100 nm and the drug entrapment efficiency was greater than 95%. Release study showed a much lower release rate of paclitaxel from liposomal formulation than from Cremophor EL-based formulation which indicated that this drug was stable in an entrapped form in vitro. Plasma distribution study showed that liposomal paclitaxel–treated groups obtained higher paclitaxel concentration than Taxol-treated group after 6 h injection. Greater cellular uptake was also found in the integrin-targeted liposomal paclitaxel–treated group compared with Taxol-treated group. Treatment of mice bearing A549 tumors with the integrin-targeted paclitaxel liposomes resulted in a lower tumor microvessel density than Taxol-treated group. Therefore, RGD-based strategy could be used to enhance tumor-specific recognition of nanocarriers. Neovessel-targeted PEGylated paclitaxel liposomes developed in present study might be a more promising drug for cancer treatment.     

Comparative pharmacokinetics of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes

The comparative pharmacokinetics of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes was evaluated in rats at a dose of 6 mg/kg i.v. Doxorubicin was entrapped in cardiolipin liposomes by using 11.2 mumol of drug, 5.6 mumol of cardiolipin, 28.5 mumol of phosphatidylcholine, 19.5 mumol of cholesterol, and 11.1 mumol of stearylamine. The peak plasma concentration with free doxorubicin at 5 min was 1.7 micrograms/ml which was reduced to 0.3 micrograms/ml by 1 h. With cardiolipin liposomes, the peak plasma concentration of doxorubicin achieved at 5 min was 20.9 micrograms/ml. The plasma levels of doxorubicin decreased gradually and by 1 h the drug concentration in plasma was 10 micrograms/ml. The plasma levels of free doxorubicin and doxorubicin entrapped in liposomes were fitted to a 3-compartment computer model. The terminal half-life with free doxorubicin in plasma was 17.3 h whereas it was 69.3 h with drug entrapped in liposomes. The area under the plasma concentration curve with liposomal doxorubicin was 81.4 micrograms X h X ml-1 compared to 1.95 micrograms X h X ml-1 observed with free doxorubicin. The steady state volume of distribution with free doxorubicin was about 23-fold higher than liposomal doxorubicin. The terminal half-life with free doxorubicin in cardiac tissue was 17.9 h compared to 12.6 h with drug encapsulated in liposomes. The terminal half-lives in liver and spleen following administration of liposomal doxorubicin were 15- and 2.3-fold higher, respectively, compared to free drug; furthermore, the concentration X time values of liposomal doxorubicin in liver were 26-fold higher and in spleen 6-fold higher than the free drug. Free doxorubicin and doxorubicin entrapped in liposomes demonstrated 17 and 20% excretion in bile of the injected dose, respectively, in rats. The present studies demonstrate that liposomal encapsulation of doxorubicin significantly alters its pharmacokinetics in plasma and tissues compared to free drug.     

Immunotoxicity of multiple dosing regimens of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes

We have shown that doxorubicin entrapped in cardiolipin liposomes retain antitumour efficacy in mice but had diminished cardiac uptake and cardiotoxicity. Such liposomes are preferentially taken up by spleen. In a previous study we showed that a single dose of liposomal doxorubicin is not more toxic than free doxorubicin with regard to immunologic parameters including generation of cytotoxicity for histocompatibility alloantigens and mitogenic responsiveness. In the present study, we have explored clinically relevant multiple dosing at weekly intervals, 2, 3, or 4 times. Again, despite splenic localization of liposomal doxorubicin, the depressive effect on these immunological parameters is not greater than the effect of free drug, and, in addition, the damage is repaired earlier.     

Liposomal irinotecan: formulation development and therapeutic assessment in murine xenograft models of colorectal cancer.

PURPOSE: The purpose is to demonstrate whether an appropriately designed liposomal formulation of irinotecan is effective in treating mice with liver-localized colorectal carcinomas. EXPERIMENTAL DESIGN: Irinotecan was encapsulated in 1,2-distearoyl-sn-glycero-3-phosphocholine/cholesterol (55:45 molar ratio) liposomes using an ionophore (A23187)-generated transmembrane proton gradient. This formulation was evaluated in vivo by measuring plasma elimination of liposomal lipid and drug after i.v. administration. Therapeutic activity was determined in SCID/Rag-2M mice bearing s.c. LS180 tumors or orthotopic LS174T colorectal metastases. RESULTS: Drug elimination from the plasma was significantly reduced when irinotecan was administered in the liposomal formulation. At 1 hour after i.v. administration, circulating levels of the liposomal drug were 100-fold greater than that of irinotecan given at the same dose. High-performance liquid chromatographic analysis of plasma samples indicated that liposomal irinotecan was protected from inactivating hydrolysis to the carboxylate form. This formulation exhibited substantially improved therapeutic effects. For the LS180 solid tumor model, it was shown that after a single injection of liposomal irinotecan at 50 mg/kg, the time to progress to a 400-mg tumor was 34 days (as compared with 22 days for animals treated with free drug at an equivalent dose). In the model of colorectal liver metastases (LS174T), a median survival time of 79 days was observed after treatment with liposomal irinotecan (50 mg/kg, given every 4 days for a total of three doses). Saline and free drug treated mice survived for 34 and 53 days, respectively. CONCLUSIONS: These results illustrate that liposomal encapsulation can substantially enhance the therapeutic activity of irinotecan and emphasize the potential for using liposomal irinotecan to treat liver metastases.     

Plasma clearance, biodistribution and therapeutic properties of mitoxantrone encapsulated in conventional and sterically stabilized liposomes after intravenous administration in BDF1 mice.

Mitoxantrone can be efficiently loaded into large unilamellar vesicles using a transmembrane pH gradient. Release studies indicate that these drug-loaded carriers are highly stable and even after dissipation of the residual pH gradient retain more than 85% of encapsulated mitoxantrone following dialysis at 37 degrees C for 5 days. In murine studies we have compared the plasma clearance and biodistribution of both mitoxantrone and liposomal lipid following intravenous administration of free drug or mitoxantrone encapsulated in either conventional or sterically stabilized liposomes. In contrast to the rapid blood clearance observed for free mitoxantrone, both liposomal systems provided extended circulation lifetimes, with over 90% of the drug present 1 h after administration and 15-30% remaining at 24 h. In agreement with previous reports, longer plasma half-lives were observed for sterically stabilized liposomes than for conventional systems. In addition, a strong correlation between drug and carrier biodistribution was seen, with uptake occurring mainly in the liver and spleen and paralleling plasma clearance. This would suggest that tissue disposition reflects that of drug-loaded liposomes rather than the individual components. Liposomal encapsulation also significantly reduced mitoxantrone toxicity, allowing administration of higher, more efficacious drug doses. In a murine L1210 tumour model, for example, no long-term survivors were seen in animal groups treated with free drug, whereas at the maximum therapeutic dose of liposomal mitoxantrone survival rates of 40% were observed.     

Tissue distribution and tumour localization of 99m-technetium-labelled liposomes in cancer patients.

The possible use of liposomes (phospholipid vesicles) to direct cytotoxic drugs to tumours has led us to investigate the tissue localization of i.v. injected 99m-Tc-labelled liposomes in cancer patients. Twenty mg or 300 mg doses of liposomal lipid (7:2:1 molar ratio of phosphatidylcholine : cholesterol : phosphatidic acid) were used in a study of 13 patients with advanced cancer and one with polycythaemia rubra vera (PRV). In all cases except the patient with PRV the major site of uptake of the label was the liver and spleen. In the patient with PRV the liver uptake was greatly reduced and the major site of uptake was found in regions corresponding to marrow. With the exception of one patient with a primary hepatoma, there was no significant tumour uptake of the label.     

Pharmacokinetic and imaging studies in patients receiving a formulation of liposome-associated adriamycin.

Pharmacokinetic and imaging studies in 19 patients receiving liposome-entrapped adriamycin (L-ADM) were carried out within the framework of a Phase I clinical trial (Gabizon et al., 1989a). The formulation of L-ADM tested consisted of 0.2 microM-extruded multilamellar vesicles composed of egg phosphatidylcholine, egg-derived phosphatidyl-glycerol (PG), cholesterol, and ADM intercalated in the fluid lipid bilayer. Plasma clearance of total drug extracted from the plasma after L-ADM infusion followed a biexponential curve with a pattern similar to that reported for free ADM. The plasma concentration of drug circulating in liposome-associated from was also measured in a subgroup of seven patients. Liposome-associated drug was found to be rapidly cleared from plasma. Its ratio to non-liposome-associated drug appeared to correlate with liver reserve, with highest ratios in patients with normal liver function. Liposome clearance, as measured by the plasma concentration of PG in three patients was slower than the clearance of liposome-associated ADM, suggesting that liposomes lose part of their drug payload during circulation. To learn about the liposome organ distribution, imaging studies were carried out with 111Indium-deferoxamine labelled liposomes of the same composition. Liposomes were cleared predominantly by liver and spleen and to a lesser extent by bone marrow in seven out of nine patients. In two patients with active hepatitis and severe liver dysfunction, there was minimal liver uptake and increased spleen and bone marrow uptake. Except for one hepatoma patient, intrahepatic and extrahepatic tumours were not imaged by liposomes, suggesting that liposome uptake is restricted to cells of the reticulo-endothelial system (RES).(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical pharmacology of 99mTc-labeled liposomes in patients with cancer

The pharmacokinetics, organ distribution, and 24-hr urinary excretion of negatively charged 99mTc-labeled multilamellar liposomes, composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol in a 7:3 molar ratio, were studied in seven patients with cancer. The radiolabeled liposomes were administered i.v. in three doses: 150 mg/sq m of body surface area; 300 mg/sq m; and 450 mg/sq m of lipid. The dose of 99mTc was 4.8 to 7.6 mCi per patient. The plasma disappearance curve was biphasic (half-life alpha = 5.53 min, half-life beta = 289 min), suggesting a two-compartmental model of distribution. The calculated volume of distribution indicated considerable tissue retention of liposomes. This was confirmed by body imaging. Twenty-four hr after injection, liposomes were localized in organs rich in reticuloendothelial cells, i.e., liver [44.5 +/- 9.1% (S.E.)], spleen [25.5 +/- 7.7%], lung [14.5 +/- 4.9%], and bone marrow. Although the hepatic uptake accounted for more than 40% of the total uptake, the spleen retained liposomes at a higher density. Cumulative urinary excretion of radioactivity was 13.4 +/- 1.5% over 24 hr. Liposome administration was safe and devoid of any adverse side effects. The results provide a basis for the use of liposomes as potential target-specific and safe drug carriers in the treatment of pathological conditions that involve organs rich in reticuloendothelial cells.     

Vincristine-induced dermal toxicity is significantly reduced when the drug is given in liposomes

 A problem associated with the intravenous delivery of vincristine concerns drug extravasation at the site of injection or infusion. This can result in extensive local soft-tissue damage. A new formulation of vincristine has recently been developed based on encapsulation of the drug in liposomes. The liposomal drug is somewhat less toxic and substantially more efficacious than free drug. The studies described here assessed, using a murine model of drug extravasation, whether vincristine encapsulation in liposomes influences drug-induced dermal toxicity. It was shown that subcutaneous injection of vincristine in liposomes does not result in the gross skin necrosis and ulceration observed following injection of free drug. Histological analysis of the dermal tissue surrounding the injection site suggests that free drug induces a pronounced inflammatory reaction as judged by the presence of infiltrating leukocytes. In contrast, the liposomal formulation of vincristine engenders a mild prolonged inflammatory condition. These toxicological studies were correlated with an evaluation of drug retention at the site of administration. It was shown using radiolabelled vincristine as a drug marker, that free vincristine is rapidly eliminated from the injection site. In contrast, the level of drug at the site of injection was far greater when the drug was given in liposomal form. Received: 13 December 1994 / Accepted: 14 May 1995     

Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times

We have previously reported on liposome formulations with reduced uptake by the reticuloendothelial system, prolonged circulation time, and enhanced accumulation in transplanted tumors. One of these formulations, consisting of hydrogenated phosphatidylinositol (HPI), hydrogenated phosphatidylcholine (HPC), and cholesterol (Chol) (HPI-HPC-Chol), and a control formulation, consisting of phosphatidylglycerol (PG), phosphatidylcholine (PC), and Chol (PG-PC-Chol), were loaded with doxorubicin (DXR) and injected intravenously into BALB/c mice for pharmacokinetic studies. Although both formulations were similar in vesicle size, fraction of negatively charged lipid, and drug-to-lipid ratio, there were striking pharmacokinetic differences. DXR was cleared much faster in PG-containing liposomes than in HPI-containing liposomes. Liposome-associated drug was detectable in plasma up to 5 hours after injection in the case of PG-PC-Chol and as late as 72 hours after injection in the case of HPI-HPC-Chol. In agreement with the plasma clearance curves, peak drug concentrations in the liver were observed at 1/2, 5, and 24 hours after injection for free DXR, DXR in PG-PC-Chol, and DXR in HPI-HPC-Chol, respectively. Both types of liposomes reduced considerably the amount of drug accumulating in the heart compared with that accumulating after injection of free DXR.     

Engineered peptides for the development of actively tumor targeted liposomal carriers of doxorubicin

Chemotherapy is still the treatment of choice for many types of cancer; but its effectiveness is hampered by dose limiting toxicity. Properly designed delivery systems can overcome this shortcoming by reducing the non-specific distribution and toxicity of chemotherapeutics in healthy organs and at the same time increasing drug concentrations at tumor tissue. In this study, we developed stealth liposomal formulations of doxorubicin (DOX) having a novel stable engineered peptide ligand, namely p18-4, that binds specifically to breast cancer cell line MDA-MB-435 on its surface. The coupling of p18-4 to liposomes was carried out through conventional, post insertion and post conjugation techniques and prepared liposomes were characterized for their size and level of peptide modification. The p18-4 decorated liposomal DOX formulations were then evaluated for their cellular uptake as well as cytotoxicity against the human breast cancer MDA-MB-435 cells. In this context, the effect of coupling technique on the uptake and cytotoxicity of p18-4 liposomal DOX in MDA-MB-435 cells was evaluated. The conventional and post conjugation methods of peptide incorporation were found to be more reliable for the preparation of p18-4 decorated liposomes for active DOX targeting to MDA-MB-435 cells. p18-4 decoration of liposomes by these methods did not have a notable effect on the size of prepared liposomes and DOX release, but increased the uptake and cytotoxicity of encapsulated DOX in MDA-MB-435 cells. The results show a potential for p18-4 decorated liposomes prepared by conventional and post conjugation method for tumor targeted delivery of DOX in breast tumor models.     

The vascular compartment hampers accurate determination of teniposide penetration into brain tumor tissue

After a pre-operative 1-h i.v infusion of 150 mg/m² of teniposide (Vumon; VM26), the drug levels were determined in resected brain tumor specimens from three patients with malignant glioma and from three patients with brain metastases. Tissue dissections were performed within 0–2.5 h after drug administration in three patients and after 24 h in the other three patients. Teniposide was quantified by high-performance liquid chromatography and the levels of albumin in the resected tissue samples were quantified by radial immunodiffusion. In addition, albumin levels were quantified in normal brain tissue, in malignant glioma and in metastatic brain tumor tissue obtained post mortem from deceased patients. The albumin levels indicated that a substantial fraction (range: 0.16–0.50) of the resected brain tumor specimens consisted of blood. As the plasma concentration of teniposide during the first hours after infusion is high, the major part of the drug measured in the tumor specimens collected within 2.5 h after drug administration originated from the blood compartment. At 24 h after drug administration, when the plasma level of teniposide had declined to approximately 0.20 μg/ml, we could discern a real tissue uptake of teniposide ranging from 0.15–0.27 μg/g wet tissue weight in the resected tumor. Although the number of patients in this study is small, this work clearly illustrates that an accurate determination of the tissue concentration of teniposide is hindered by the high concurrent plasma levels. It is therefore essential that future tissue distribution studies also include a suitable procedure that establishes the contribution of drug originating from the blood compartment. Received: 4 November 1996 / Accepted: 15 February 1997     

Evaluation of incorporation characteristics of mitoxantrone into unilamellar liposomes and analysis of their pharmacokinetic properties,acute toxicity,and antitumor efficacy

Summary Mitoxantrone (MTO) was incorporated into small unilamellar liposomes by formation of a complex between the anticancer drug and negatively charged lipids. The complex was formed at a 2:1 molar ratio between the lipids and MTO, with phosphatidic acid (PA) being the strongest complex-forming lipid. Weaker complexes and lower incorporation rates of MTO resulted when liposomes containing dicetylphosphate, phosphatidyl inositol, phosphatidyl serine, phosphatidyl glycerol, oleic acid, and tridecylphosphate were used. Thus, all further experiments were performed with PA-MTO liposomes that contained 0.1–3 mg MTO/ml and had mean vesicle sizes of 40–150 nm, depending on the drug concentration and the method of liposome preparation. In vitro incubations of free and liposomal MTO with human plasma showed that the drug is slowly transferred from the liposome membranes to the plasma proteins. For liposomal MTO a transfer rate of 48% was determined, whereas 75.8% of free MTO was bound to the plasma proteins. The organ distribution of the two preparations in mice showed that higher and longer-lasting concentrations of liposomal MTO were found in the liver and spleen. The terminal elimination halflives in the liver were 77 h for liposomal MTO and 14.4 h for free MTO. In the blood, slightly higher concentrations were detected for liposomal MTO, which also had slower biphasic elimination kinetics as compared with the free drug. Drug distribution in the heart was not significantly different from that in the kidneys. The LD₂₅ of PA-MTO liposomes in mice was 19.6 mg/kg and that of free MTO was 7.7 mg/kg. The antitumor effects of PA-MTO liposomes were evaluated in murine L 1210 leukemia, in various xenografted human tumors, and in methylnitrosourea-induced rat mammary carcinoma. Generally, the liposomal application form was more effective and less toxic than the free drug. The cytostatic effects were dependent on the tumor model, the application schedule, and the drug concentration. At doses that were toxic when free MTO was used, the liposomal preparation produced strong antitumor effects in some cases. In summary, the incorporation of MTO into liposomes changes the drug's plasmabinding properties, alters its organ distribution, reduces its acute toxicity, and increases its cytostatic efficiency in various tumor models. The liposomal PA-MTO complex represents a new application form of MTO that has advantageous properties.This research was supported in part by the Krebsliga of the Kanton Zürich and by Lederle Arzneimittel, Wolfratshausen, Federal Republic of Germany     

Teniposide (VM26) disposition in children with leukemia

The clinical pharmacokinetics of teniposide (VM-26, NSC 122819) has been studied in 21 children (median age, 4.7 years) with acute lymphocytic leukemia. Teniposide was administered at a dosage of 165 mg/sq m as a 30- to 60-min i.v. infusion. Patients were studied either on the first or second dosage of the drug. Plasma samples were assayed for teniposide and metabolites by high-performance liquid chromatography with electro-chemical detection. Both compartmental and noncompartmental pharmacokinetic analyses were performed. Systemic clearance and apparent volume of distribution of steady state averaged 13.82 +/- 6.0 ml/min/sq m (S.D.) and 7.9 +/- 4.0 liter/sq m, respectively. Univariate and multivariate stepwise regression analyses were used to construct mathematical models to describe the relationships between certain patient-specific demographic and laboratory values and the pharmacokinetic parameters, systemic clearance, elimination rate constant, and area under the concentration-time curve. A significant relationship between serum alkaline phosphatase and systemic clearance, elimination rate constant, and area under the concentration-time curve was found, suggesting that liver function influences the disposition of this anticancer drug in humans.     

A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1

The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo. Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung. We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle. The coated cationic liposomes had an average diameter of 188 nm and entrapped 85-95% of the asODN. The biodistribution and pharmacokinetics of an 18-mer 125I-labeled phosphorothioate ODN formulated by this method were determined after tail vein injection in mice. The majority of the asODN was cleared from blood, with a half-life of >10 hours compared with <1 hour for free asODN. When coupled with an anti-CD19 targeted antibody, this formulation was also effective at delivering an MDR1 asODN to a multidrug-resistant human B-lymphoma cell line in vitro, decreasing the activity of P-glycoprotein. No inhibition was found for nontargeted formulations or for free asODN. A number of therapeutic opportunities exist for the use of small, stable, long-circulating, and targetable liposomal carriers such as this, with high trapping efficiencies for asODN.     

Utility of liposomes coated with polysaccharide bearing 1-amino-lactose as targeting chemotherapy for AH66 hepatoma cells

The cell recognition element is very important for drug delivery systems. We synthesized cholesteryl pullulan (CHP) bearing 1-aminolactose (1-AL) and introduced a saccharide, cholesteryl pullulan bearing 1-aminolactose (1-AL/CHP), to an outer layer of the conventional liposome as a cell recognition element. Lectin recognized the beta-galactose by aggregation of 1-AL/CHP coated liposome (1-AL/CHP liposome). The uptake of this liposome to AH66 rat hepatoma cells was greater than in liposomes without 1-aminolactose in vitro. Furthermore, 1-AL/CHP liposomal adriamycin showed a stronger antitumor effect in comparison with other types of liposomal adriamycin in vitro. When in vivo tumor-targeting efficacy was investigated in AH66 tumor transplanted mice using 3H-liposome, the tumor/serum radioactivity ratio in mice injected with 1-AL/CHP liposome was higher than that of mice injected with other liposomes. These observations suggest that 1-AL is effective as a cell recognition element. As a result, 1-AL/CHP liposome is considered to be a good carrier of anticancer drugs for the active targeting of tumor cells.     

Cationic surface charge enhances early regional deposition of liposomes after intracarotid injection

Rapid first pass uptake of drugs is necessary to increase tissue deposition after intraarterial (IA) injection. Here we tested whether brain tissue deposition of a nanoparticulate liposomal carrier could be enhanced by coordinated manipulation of liposome surface charge and physiological parameters, such as IA injection during transient cerebral hypoperfusion (TCH). Different degrees of blood-brain barrier disruption were induced by focused ultrasound in three sets of Sprague–Dawley rats. Brain tissue retention was then compared for anionic, cationic, and charge-neutral liposomes after IA injection combined with TCH. The liposomes contained a non-exchangeable carbocyanine membrane optical label that could be quantified using diffuse reflectance spectroscopy (DRS) or visualized by multispectral imaging. Real-time concentration–time curves in brain were obtained after each liposomal injection. Having observed greater tissue retention of cationic liposomes compared to other liposomes in all three groups, we tested uptake of cationic liposomes in C6 tumor bearing rats. DRS and multispectral imaging of postmortem sections revealed increased liposomal uptake by the C6 brain tumor as compared to non-tumor contralateral hemisphere. We conclude that regional deposition of liposomes can be enhanced without BBB disruption using IA injection of cationic liposomal formulations in healthy and C6 tumor bearing rats.