LIPID EMULSIONS OF PALMITOYLRHIZOXIN: EFFECTS OF COMPOSITION ON LIPOLYSIS AND BIODISTRIBUTION

Four types of lipid emulsion for highly lipophilic antitumour agent RS-1541 (13-O-palmitoylrhizoxin) with mean particle diameters of 200--260 nm were prepared using soybean oil (SOY) or dioctanoyldecanoylglycerol (ODO) for the oil phase and lecithin (LEC) or polyoxyethylene-(60)-hydrogenated castor oil (HCO-60) for surfactants. The lipolysis rate of HCO-60-emulsified emulsions by lipoprotein lipase was much slower than that of LEC-emulsified emulsions. Particle sizes of emulsions incubated in plasma with the lipase for six hours were 75%, 79%, 101%, and 93% of initial values for SOY/LEC, ODO/LEC, SOY/HCO-60, and ODO/HCO-60 emulsions, respectively, showing an apparent size decrease for LEC-emulsified emulsions. In rats, uptake clearance values of SOY/LEC and ODO/LEC emulsions of RS-1541 in the reticuloendothelial system (RES) were 81·2 and 135·3 mL h⁻¹, respectively, and AUC values were 4·0 and 1·3 μ g h mL⁻¹, respectively. In contrast, RES uptake clearances of HCO-60 emulsions of RS-1541 were considerably lower (4·2 mL h⁻¹ for SOY/HCO-60; 2·2 mL h⁻¹ for ODO/HCO-60), resulting in high AUC values (35·4 μ g h mL⁻¹ for SOY/HCO-60; 63·9 μ g h mL⁻¹ for ODO/HCO-60). The concentrations of RS-1541 in tumour tissues after an intravenous administration of ODO/HCO-60 emulsions of RS-1541 to mice bearing solid tumour M5076 sarcoma were about ten times higher than those after the administration of SOY/LEC emulsions. These results indicate that HCO-60 emulsions, compared with conventional LEC emulsions, are more stable to lipoprotein lipase and show low uptakes by RES organs, long circulations in the plasma, and high distributions in tumours. Thus, these sterically stabilized emulsions could show potential as effective carriers for highly lipophilic antitumour agents to enhance the drug delivery in tumours.     

Enhanced Tumor Delivery and Antitumor Activity of Palmitoyl Rhizoxin Using Stable Lipid Emulsions in Mice

Purpose. A highly lipophilic antitumor agent, 13-O-palmitoyl-rhizoxin (RS-1541), was incorporated into lipid emulsions of various sizes consisting of triglyceride ODO and surfactant HCO-60. Pharmacokinetics, toxicities, and antitumor activities were evaluated after intravenous administration to mice bearing subcutaneously inoculated M5076 sarcoma cells. Methods. The levels of RS-1541 in the plasma and tissues including tumor, were determined by HPLC. The maximum tolerated dose (MTD) was estimated by toxic death and change in body weight. The decrease in tumor diameter was measured for antitumor activity. Results. There existed large variations in pharmacokinetics of RS-1541, depending on the size of emulsion particles. Compared with a colloidal solution (reference solution), the small (110nm) and medium (230nm) size emulsions showed high concentrations of RS-1541 in the tumor, while the large emulsions (350nm–630nm) exhibited low concentrations. The MTD of RS-1541 was reduced, when incorporated in the emulsions larger than 220nm in size. At MTD, each size of emulsions (70nm–380nm) effectively retarded the tumor growth and increased survival time. The maximum effect was achieved for the 220 nm emulsions. Conclusions. When particle size is properly selected, these emulsions could be promising and effective as an injectable carrier for lipophilic antitumor agents in order to enhance the tumor delivery and efficacies while reducing toxicities.     

Species Variation in Pharmacokinetics and Opsonization of Palmitoyl Rhizoxin (RS-1541) Incorporated in Lipid Emulsions

Abstract

Highly lipophilic antitumor agent, palmitoyl rhizoxin (RS-1541), was incorporated into stable lipid emulsions about 100-1000nm in mean diameter consisting of triglyceride ODO and surfactant HCO-60. The pharmacokinetics of RS-1541 were studied after i.v. injection in mice, rats, rabbits, and dogs. Dog showed characteristic pharmacokinetics of RS-1541, compared with other species. RS-1541 was much more rapidly eliminated from plasma with emulsion particles in dogs than in mice, rats, and rabbits. Most amounts of injected RS-1541 were recovered in the liver six hours after administration to dogs, while less than 20% recoveries were observed for mice and rats. To clarify this species variation, opsonization of emulsion particles were evaluated. When emulsions (about 200nm in size) were opsonized by dog plasma, and intravenously injected to rats, total clearance and liver uptake of RS-1541 were increased to 1.8 fold and 2.7 fold of control values, respectively. In contrasts, emulsions opsonized by mouse, rabbit and human plasma did not show such drastic changes in pharmacokinetics of RS-1541 in rats. Furthermore, total clearance of RS-1541 for emulsions opsonized by dog plasma was increased to 1.9 fold of controls after injection to rabbits. These results indicate that opsonizing activities of dog plasma for RS-1541 emulsions are high, compared with other species. This species variation in opsonizing process probably caused the species variation in the pharmacokinetics of RS-1541 incorporated in lipid emulsions.     

In vitro release, pharmacokinetic and tissue distribution studies of doxorubicin hydrochloride (Adriamycin HCl) encapsulated in lipiodolized w/o emulsions and w/o/w multiple emulsions.

The lipiodolized w/o emulsion or w/o/w multiple emulsion containing Doxorubicin hydrochloride (1; Adriamycin HCl) with different emulsifiers was prepared to evaluate in vitro sustained-release behavior, pharmacokinetic and tissue distribution function in Sprague Dawley (SD) rats. The results of dissolution indicate that the release of 1 was significantly sustained for both emulsions when HCO-60 (polyoxyethylene (60) hydrogenated castor oil) was used as an emulsifier. The serum concentration of 1 was reduced and prolonged for both emulsions with the increase of HCO-60. The C(max) level was lowered and T(max) value was delayed after administration of w/o emulsions with higher HCO-60 concentration. The apparent terminal half-life for 1 released from some emulsions with higher concentration of HCO-60 was 3-folds higher than that of the 1 solution. The clearance of some w/o or w/o/w ADR emulsions also decreased with the increase of HCO-60. Not only the concentration of 1 in heart and kidney decreased significantly after the administration of w/o emulsions with the higher concentration of HCO-60, but also the hepatic concentration of 1 was higher and increased with HCO-60 concentration. The hepatic 1 level became lower after administration of w/o/w multiple emulsions with the increase of HCO-60; however, the concentration of 1 in heart, lung and spleen increased somewhat. The results indicate that lipiodol and HCO-60 seemed to play an important role in the prolongation and selective retention of w/o emulsion or w/o/w multiple emulsion, in vitro and in vivo.     

Factors influencing the tissue distribution of coenzyme Q10 intravenously administered in an emulsion to rats: emulsifying agents and lipoprotein lipase activity

The tissue distribution of coenzyme Q10 (CoQ10) administered intravenously in an emulsion prepared with egg yolk phosphatidylcholine (PC), egg yolk sphingomyelin (SPM) or a combination of PC and a polyoxyethylene derivative of hydrogenated castor oil (HCO-60) (PC + HCO-60) was investigated. The disappearance from the plasma of CoQ10 administered in three different emulsions of lipid particle size less than 0.5 micron varied with the particular emulsifier. Its disappearance occurred most rapidly from the PC emulsion; with the addition of HCO-60, its disappearance was much slower. In the reticuloendothelial system, the concentration of CoQ10 was higher in the spleen, for both the SPM and PC + HCO-60 emulsions than for the PC emulsion. HCO-60 reduced the CoQ10 distribution in the liver from the PC emulsion. Differences in disappearance rates from the plasma are thus considered to be due to the extent of CoQ10 distribution in the liver. CoQ10 concentration in the heart, a target organ, was greatest with the PC emulsion. Its distribution was related to lipoprotein lipase (LPL) activity in this organ. The effects caused by HCO-60, however, could not be explained by LPL activity alone. CoQ10 distribution in the adrenal gland and kidney can be explained partly by LPL activity but in the presence of HCO-60, the distribution mechanism apparently involves other factors.     

Biodistribution of gadolinium incorporated in lipid emulsions intraperitoneally administered for neutron-capture therapy with tumor-bearing hamsters

Emulsions containing a distearylamide (Gd-DTPA-SA) or a distearylester (Gd-DTPA-SE) of Gd (gadolinium)-diethylenetriaminepentaacetic acid (Gd-DTPA) were intraperitoneally injected in Greene's melanoma-bearing hamsters at a dose of 2.0 ml (3.0 or 6.0 mg Gd) per hamster. In the standard-Gd and high-Gd formulations used, the weight ratios of soybean oil, water, Gd-DTPA derivative (Gd-DTPA-SA or Gd-DTPA-SE), hydrogenated L-alpha-phosphatidylcholine from egg yolk (HEPC) and co-surfactant (HCO-60, Myrj 53, Myrj 59 or Brij 700) were 7.36:92:1:2:3 and 7.36:92:2:1:3, respectively. When the effects of the co-surfactants on the biodistribution of Gd from Gd-DTPA-SA-containing emulsions in the standard-Gd formulation were compared, the HCO-60 emulsion exhibited the highest Gd accumulation in tumors, possibly resulting from its fast and complete absorption, its small particle size (78 nm) and the stable coat on the particle surfaces with polyoxyethylene. Brij 700 emulsion kept the highest blood Gd concentration for a prolonged period, possibly due to particle properties similar to those of HCO-60. However, it exhibited a slower Gd accumulation in tumors, only reaching an identical level, in comparison with the HCO-60 emulsion. This suggested the tumor to be saturated with lipid particles. When Gd-DTPA-SE was used instead of Gd-DTPA-SA, its HCO-60 emulsion exhibited only very poor Gd-accumulation due to its easy degradation. The HCO-60 emulsion particles containing Gd-DTPA-SA in the high-Gd formulation (6.0 mg Gd in 2 ml) exhibited in vivo behavior identical to those in the standard-Gd formulation; then the Gd level in tumors reached 107 micrograms Gd/g tumor (wet), and the tumor:blood (T/B) and tumor:skin (T/Sk) Gd concentration ratios were 13.2 and 5.6, respectively, at 48 h after intraperitoneal administration. These results suggest that when intraperitoneally administered, this HCO-60 emulsion, and possibly also the corresponding Brij 700 emulsion, may be an excellent delivery system for accumulating Gd in tumors in neutron-capture therapy (NCT).     

Formulation Development and Antitumor Activity of a Filter-Sterilizable Emulsion of Paclitaxel

Purpose. Paclitaxel is currently administered i.v. as a slow infusion of asolution of the drug in an ethanol:surfactant:saline admixture. However,poor solubilization and toxicity are associated with this drug therapy.Alternative drug delivery systems, including parenteral emulsions, areunder development in recent years to reduce drug toxicity, improveefficacy and eliminate premedication. Methods. Paclitaxel emulsions were prepared by high-shearhomogenization. The particle size of the emulsions was measured by dynamiclight scattering. Drug concentration was quantified by HPLC and invitro drug release was monitored by membrane dialysis. The physicalstability of emulsions was monitored by particle size changes in boththe mean droplet diameter and 99% cumulative distribution. Paclitaxelpotency and changes in the concentration of known degradants wereused as chemical stability indicators. Single dose acute toxicity studieswere conducted in healthy mice and efficacy studies in B16 melanomatumor-bearing mice. Results. QW8184, a physically and chemically stable sub-micronoil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drugloading (8-10 mg/mL) having a mean droplet diameter of <100 nmand 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both inthe presence and absence of human serum albumin. Based on singledose acute toxicity studies, QW8184 is well tolerated both in miceand rats with about a 3-fold increase in the maximum-tolerated-dose(MTD) over the current marketed drug formulation. Using the B16mouse melanoma model, a significant improvement in drug efficacywas observed with QW8184 over Taxol^®. Conclusions. QW8184, a stable sub-micron o/w emulsion of paclitaxelhas been developed that can be filter-sterilized and administered i.v.as a bolus dose. When compared to Taxol^®, this emulsion exhibitedreduced toxicity and improved efficacy most likely due to thecomposition and dependent physicochemical characteristics of the emulsion.     

Pharmacokinetics of Vancomycin after Intravenous Administration of a W/O/W Emulsion to Rats

A stable water-in-oil-in-water multiple emulsion (w/o/w emulsion) was prepared, and its potential for drug delivery was evaluated. W/o/w emulsions were prepared using a Lipiodol Ultra-Fluid(r) and isopropyl myristate oil mixture for the oily phase and vancomycin (VCM) for the entrapped drug. The surfactants, HCO-40 (5% [w/v]) and Pluronic F-88 were dissolved in the oily phase and the external aqueous phase, respectively. Resultant w/o/w emulsions were evaluated for entrapment efficiency, particle size, viscosity, drug release in vitro, and disposition kinetics of the drug and the w/o/w emulsion in vivo. The particle size of the w/o/w emulsion decreased with an increase in the concentration of F-88 in the external aqueous phase and stirring speed at the second emulsification stage (the smallest being 2.9 ± 1.5 μm). Entrapment efficiency of VCM in the w/o/w emulsion decreased with an increase in the concentration of F-88 (the maximum being 65.3 ± 5.4%). VCM release from the w/o/w emulsion was prolonged and tended to be slower with an increase in the particle size of the emulsion. After intravenous administration, significant differences in pharmacokinetic parameters, such as k₂₁, k_elβ, AUC₀-±, MRT₀-₆, and MRT₀-±, were observed between the VCM solution and the w/o/w emulsion-entrapped drug. When the w/o/w emulsion with Sudan II in the oily phase was administered intravenously, the emulsion accumulated in the lung at first (the highest value was observed just after administration) and then in the liver (the highest value was observed at 60 min). The w/o/w emulsion prepared in this study is expected to be a possible carrier for the prolonged release of water-soluble drugs after intravenous administration.     

Formulation of Polyiodinated Triglyceride Analogues in a Chylomicron Remnant-Like Liver-Selective Delivery Vehicle

Purpose. A formulation methodology for the incorporation of polyiodinated triglyceride (ITG) analogues into a protein-free chylomicron remnant-like emulsion was developed to provide a vehicle for the selective hepatic delivery of these agents for contrast-enhanced X-ray computed tomography (CECT). Methods. Triglyceride emulsions (10% w/v) were prepared at various processing pressures, temperatures and times with a Microfluidizer^® 110-S using different emulsion component proportions to establish processing and compositional parameters in order to afford stable ITG emulsions (ITG-LE) approaching 200 nm mean diameter. Results. Preliminary data indicated that with a formulation composed of 2.4% dioleoyl PC with a cholesterol:DOPC mole ratio of 0.4 emulsified at 14,700 psi, 35°C for 10 min routinely afforded ITG-LE in the desired size range. The elimination of salt and amino acid from the bulk phase enhanced the stability of the ITG-LE. Incorporation of cholesterol into the monolayer was of critical importance in generating a stable emulsion near the targeted size, with a C:DOPC mole ratio of 0.4 producing a size minimum relative to higher or lower C:DOPC values. Conclusions. The ITG analogues can be readily incorporated into stable remnant-like emulsions of relatively uniform particle size. Combination of the unique ITG contrast agent with the remnant-like delivery vehicle demonstrates a high degree of hepatic selectivity in biodistribution studies and offers significant potential for selective hepatic CECT.     

Effect of methylcellulose on the stability of oil-in-water emulsions

The objective of this study was to investigate how polymers used as auxiliary emulsifiers improve the stability of oil-in-water emulsions. One stable emulsion and three unstable emulsions were formulated with 30% mineral oil and an emulsifier blend of Tween® 40 and Span® 20. The stable emulsion (SE) contained 2% emulsifier blend optimized for maximum stability. One unstable emulsion, UEI, was formulated to contain 0.5% of the same emulsifier blend as the SE formulation. Two unstable emulsions were formulated to contain an unbalanced emulsifier blend, one with excessive hydrophilic emulsifier (UE2) and one with excessive lipophilic emulsifier (UE3). A series of emulsions was prepared containing increasing amounts of methylcellulose for each base emulsion. Creaming and change in particle size were measured to evaluate stability. The addition of the polymer to the stable emulsion caused instability leading to creaming and eventual oil separation. This effect of the polymer was more pronounced in UEI emulsions. However, the addition of the polymer improved the stability of the UE2 and UE3 series of emulsions. The polymer also caused a reduction in the particle size of UE3 emulsions and a proportionally larger increase in the viscosity of UE2 emulsions. These results suggest that (i) methylcellulose could act as a hydrophilic emulsifier only in the absence of Tween^® 40, (ii) methylcellulose and Tween^® 40 associate to form a complex and (iii) the concentration of Tween^® 40 is the determining factor for the stability of emulsions. A model of the methylcellulose-Tween^® 40 association and its effect at the mineral oil-water interface is proposed.     

Effect of Cationic Surfactant on Transport of Model Drugs in Emulsion Systems

Excess surfactant present in emulsions can influence the rates of transport of incorporated drugs by micellar solubilization, alteration of the partitioning process and by drug-surfactant complexation. Cetyltrimethylammonium bromide (CTAB), a cationic surfactant was selected to investigate these phenomena as it forms relatively stable mineral oil–water (O–W) emulsions and has the potential for ionic interaction. Phenylazoaniline, benzocaine, benzoic acid and phenol were chosen as model drugs for this study. The emulsion critical micelle concentration (CMC) for CTAB determined using a combination of a membrane equilibrium technique and surface-tension measurement was 10% w/v in 10% v/v% O-W emulsion systems. Ionic interaction between model drugs and surfactants and drug hydrophobicity affected their transport rates in the emulsion systems. The transport rates of the lipophilic drugs (benzocaine and phenylazoaniline) and the ionized hydrophillic drug (benzoic acid, pH 70) in the emulsion systems increased with increasing CTAB concentration up to 0–5% w/v micellar concentration and then decreased at higher concentrations. The rate of transport of phenol was not affected by the presence of micellar phase. Ionic interaction between surfactant and model drugs affected transport rates of model drugs in emulsion systems. The micellar phase was considered to affect the overall transport rates of model drugs.     

Nano-emulsion formulation using spontaneous emulsification: solvent, oil and surfactant optimisation

Nano-emulsions consist of fine oil-in-water dispersions, having droplets covering the size range of 100–600 nm. In the present work, nano-emulsions were prepared using the spontaneous emulsification mechanism which occurs when an organic phase and an aqueous phase are mixed. The organic phase is an homogeneous solution of oil, lipophilic surfactant and water–miscible solvent, the aqueous phase consists on hydrophilic surfactant and water. An experimental study of nano-emulsion process optimisation based on the required size distribution was performed in relation with the type of oil, surfactant and the water–miscible solvent. The results showed that the composition of the initial organic phase was of great importance for the spontaneous emulsification process, and so, for the physico-chemical properties of the obtained emulsions. First, oil viscosity and HLB surfactants were changed, -tocopherol, the most viscous oil, gave the smallest droplets size (171 ± 2 nm), HLB required for the resulting oil-in-water emulsion was superior to 8. Second, the effect of water–solvent miscibility on the emulsification process was studied by decreasing acetone proportion in the organic phase. The solvent–acetone proportion leading to a fine nano-emulsion was fixed at 15/85% (v/v) with EtAc–acetone and 30/70% (v/v) with MEK–acetone mixture. To strength the choice of solvents, physical characteristics were compared, in particular, the auto-inflammation temperature and the flash point. This phase of emulsion optimisation represents an important step in the process of polymeric nanocapsules preparation using nanoprecipitation or interfacial polycondensation combined with spontaneous emulsification technique.     

Targeting of an antitumor agent, RS-1541 (palmitoyl-rhizoxin), via low-density lipoprotein receptor

RS-1541 is a 13-O-palmitoyl derivative of rhizoxin, an inhibitor of tubulin polymerization. RS-1541 has been shown to bind preferentially to plasma lipoproteins and to exhibit selective uptake by tumors in mice. We demonstrated that: (1) 99% of RS-1541 bound to human lipoproteins after incubation with human serum, among which the low-density lipoprotein (LDL) fraction attained 10%; (2) RS-1541/LDL complex was obtained by simply incubating human native LDL with a detergent-aided RS-1541 solution; (3) uptake of the [¹⁴C]RS-1541/LDL complex in cultured human skin fibroblasts was dependent on the LDL receptor activities of the cells; (4) excess amounts of native LDL or 10 μM monensin, a proton ionophore, significantly inhibited the uptake; and (5) the detergent-aided solution of [¹⁴C]RS-1541 showed very low cellular uptake. These results suggest the possibility of LDL as an endogenous targeting carrier of RS-1541 into tumor cells, which have higher LDL receptor activities.     

A novel stable supersaturated submicron lipid emulsion of tirilazad.

This paper describes a novel supersaturated submicron lipid emulsion for parenteral drug delivery. Tirilazad was used as an amphiphilic model drug for the preparation and characterization of the supersaturated emulsions with concentrations more than two times higher than the drug solubility in the emulsion. Analysis of particle size distribution using photon correlation spectroscopy indicated that the mean particle diameters of the supersaturated 10 and 20% lipid emulsions of tirilazad were approximately 210 and 240 nm, respectively, and that the mean particle size and size distribution of the supersaturated tirilazad emulsions were not different from those of the non-supersaturated tirilazad emulsions. The apparent viscosity of the supersaturated tirilazad emulsions, as measured by continuous rheometry, was similar to that of the non-supersaturated tirilazad emulsions. However, the zeta potential of the supersaturated tirilazad emulsions, as determined by the electrophoretic light-scattering technique, was found to be dependent on drug load and was lower than that of the non-supersaturated tirilazad emulsion. Fractionation by ultracentrifugation showed that at low drug loads, the amount of drug associated with the emulsion particles and the infranatant fraction appeared to increase linearly with increasing concentration of the drug. By contrast, the amount of drug associated with the emulsion particles increased rapidly, whereas the amount of drug associated with the infranatant fraction remained constant for emulsions with a high drug load. These results suggest that the excess drug in the supersaturated emulsions is largely associated with the lipid phase. Accelerated stability studies under stress conditions (i.e., autoclaving and shaking) and long-term storage stability tests demonstrated that the supersaturated tirilazad emulsions had excellent physical stability over the study period of 16 months.     

The effects of emulsifying agents on disposition of lipid-soluble drugs included in fat emulsion

The uses for drug delivery systems of two soybean oil fat emulsions prepared with an emulsifying agent, phosphatidyl choline (PC) or Pluronic F-127 (PLU), were examined comparatively in vivo and in vitro. In the presence of lipoprotein lipase (LPL) in vitro, the mean particle size of the PLU emulsion changed less than that of the PC emulsion. The production of non-esterified fatty acid (NEFA) from the PLU emulsion in the presence of LPL was smaller than that from the PC emulsion. These in vitro results indicate that the PLU emulsion is more stable than the PC emulsion. Plasma NEFA concentration following intravenous administration of the emulsions decreased with time for the PC emulsion, but was kept lower and constant for the PLU emulsion, supporting the in vitro stability data. The order of plasma cyclosporine A (CsA) concentration following intravenous administration in the above two emulsions and the mixed solution of polyethylene glycol 400 (PEG) and dimethylamide (DMA) in rats was PLU emulsion>PC emulsion>PEG/DMA solution. The plasma concentration was maintained higher and tissue distribution lower for the PLU emulsion than for other formulations. The uptake of oil violet (OV) into the rat parenchymal cells from the PLU emulsion was approximately half that from the PC emulsion, but the uptake into the Kupffer cells was almost equal in both emulsions. In conclusion, these emulsifying agents can control plasma elimination and tissue distribution of lipophilic drugs included in the emulsion. The use of the emulsion formulation makes it possible to avoid side effects through the reduction of drug uptake into non-targeted tissues.     

Uptake of Concentrated Perfluorocarbon Emulsions into Rat Lymphoid Tissues

Abstract— The effects of injecting (10–30 mL kg^?1) either perfluorodecalin (FDC) emulsions of increasing phase fraction (20–60% w/v) or the commercial formulation, Fluosol, on lymphoid tissues have been studied for up to 7 days in male rats. Tissue weights increased by up to 123% (P < 0·05) in proportion with quantity of perfluorochemical (PFC) injected, with spleen responses consistently greater than those of the liver. PFC droplets recovered from these tissues at 72 h after injection of 30% (w/v) FDC emulsion (10 mL kg^?1) had mean diameters in the 1–10 μm range, with those from the spleen being larger than those from the liver. Recovered droplet diameters were considerably greater than freshly-prepared emulsion mean particle sizes (0·21–0·25 μm). These results suggest that coalescence of emulsion droplets following accumulation in lymphoid tissues is a pre-requisite for the eventual excretion of PFC vapour through the lungs.     

Formulation Optimization of Paclitaxel Carried by PEGylated Emulsions Based on Artificial Neural Network

PURPOSE: To develop paclitaxel carried by injectable PEGylated emulsions, an artificial neural network (ANN) was used to optimize the formulation--which has a small particle size, high entrapment efficiency, and good stability--and to investigate the role of each ingredient in the emulsion. METHODS: Paclitaxel emulsions were prepared by a modified ethanol injection method. A computer optimization technique based on a spherical experimental design for three-level, three factors [soybean oil (X1), PEG-DSPE (X2) and polysorbate 80 (X3)] were used to optimize the formulation. The entrapment efficiency of paclitaxel (Y1) was quantified by HPLC; the particle size of the emulsions (Y2) was measured by dynamic laser light scattering and the stability of paclitaxel emulsions was monitored by the changes in drug concentration (Y3) and particle size (Y4) after storage at 4 degrees C. RESULTS: The entrapment efficiency, particle size and stability of paclitaxel emulsions were influenced by PEG-DSPE, polysorbate 80, and soybean oil. Paclitaxel emulsions of small size (262 nm), high entrapment efficiency (96.7%), and good stability were obtained by the optimization. CONCLUSIONS: A novel formulation for paclitaxel emulsions was optimized with ANN and prepared. The contribution indices of each component suggested that PEG-DSPE mainly contributes to the entrapment efficiency and particle size of paclitaxel emulsions, while polysorbate 80 contributes to stability.     

Preparation and Evaluation of Multiple Emulsions Water-in-oil-in-water (w/o/w) as Delivery System for Influenza Virus Antigens

In this study, investigations have been carried out to prepare adjuvant active delivery systems; multiple water-in-oil-in-water (w/o/w) emulsion formulations, containing influenza virus surface antigen Hemagglutinin (HA). A modified two-stage emulsification method has been used to prepare multiple emulsions. After improving multiple (w/o/w) emulsion formulations; F1: purified antigen solution (PAS)/soybean oil, HCO-40 and span 80/pluronic F-68, F2: PAS and HPβCD/soybean oil, HCO-40 and span 80/pluronic F-68, F3: PAS/squalane, HCO-40 and span 80/pluronic F-68, formulations were selected for the stability study that continued for a 3 month duration. To evaluate the stability of these formulations, microscopic observation, osmolarities of the internal and external aqueous phases, pH, globule size and viscosity were determined. SDS-PAGE (silver staining) was used to evaluate HA and the micro-bicinchoninic acid (mBCA) assay was used to determine the in vitro release of antigen from formulations. Immune responses of formulations were investigated in Wistar Albino rats and compared with the immune response raised against the conventional vaccine. These responses were detected with Hemagglutination Inhibition (HAI) assay.

The results of this study demonstrated that HA was well entrapped in the multiple (w/o/w) emulsion formulations. Molecular weight and antigenicity of the entrapped HA were not affected by the emulsification procedure. These results suggest that multiple emulsion formulations entrapping influenza antigen may have potential for immunization studies as one of the vaccine delivery system with adjuvant properties.     

Contribution of serum lipoproteins as carriers of antitumour agent RS-1541 (palmitoyl rhizoxin) in mice

The tumour uptake as well as the anti-tumour activity of RS-1541 (palmitoyl rhizoxin), a potent antineoplastic agent, were investigated in mice bearing M5076 sarcoma. After intravenous administration, ¹⁴C-RS-1541 preferentially bound to the lipoproteins, to which ¹⁴C-rhizoxin did not bind. ¹⁴C-RS-1541 showed persisting high concentrations of radioactivity in the plasma (T_1/2α, 4·9 h). The uptake of radioactivity by the tumour was second to those by the liver and spleen, and several times greater than those by the other tissues. Selective and sustained uptake by the tumour was also demonstrated by wholebody autoradiography. A considerable amount of rhizoxin was detected only in the tumour after administration of ¹⁴C-RS-1541, and the area under the tissue-concentration-time curve (AUC_t) and the mean residence time (MRT) of rhizoxin in the tumour were much higher than those after administration of ¹⁴C-rhizoxin itself. The rhizoxin formation in the tumour was significantly reduced by chloroquine, a lysosomal enzyme inhibitor. RS-1541 showed a higher therapeutic activity than rhizoxin. At a 4 mg kg^?1 dose, the maximum growth inhibition was 92% for RS-1541 and 41% for rhizoxin. These results indicate that RS-1541, but not rhizoxin, is taken up by the tumour via endocytosis, most likely via the low-density-lipoprotein receptor, after binding to lipoproteins. Thus, RS-1541 was considered to exhibit sustained high concentration in tumours and potent anti-tumour activity.     

Intestinal absorption of penclomedine from lipid vehicles in the conscious rat: contribution of emulsification versus digestibility

While the inclusion of highly lipophilic compounds in self-emulsifying drug delivery systems (SEDDS) is often reported to result in strongly enhanced oral absorption, it is still controversial whether further lipolysis of the dispersed lipidic material is required for final transfer to the enterocyte membranes.

In order to assess the relative roles of lipid vehicle dispersion and vehicle digestibility in the oral absorption of penclomedine (Pcm), a series of formulations of Pcm in medium chain triglyceride (MCT)/tocophersolan (TPGS) was developed having three sizes (160 nm, 720 nm, and mm-sized (‘crude’ oil)); with or without the inclusion of tetrahydrolipstatin (THL), a known lipase-inhibitor.

Oral absorption of Pcm was studied after administration of small volumes of these formulations in the conscious rat. Kinetic evaluation was performed using population analysis. Formulations with particle size 160 nm had the highest relative bioavailability (set at F=1), whereas administration in particle size 720 nm had slightly lower bioavailability (F=0.79). Co-inclusion of THL yielded similar bioavailability for these two SEDDS. ‘Crude’ oil formulations had F=0.62 (without THL) and 0.25 (with THL).

The data in the current investigation emphasize the prominent role of increased vehicle dispersion relative to digestibility in the absorption of Pcm from MCT-TPGS in submicron emulsions. Only with Pcm administered as undispersed MCT, absorption was more dependent on the action of lipase as bioavailability was inhibited two-fold by the co-incorporation of THL.