Submicron emulsions as colloidal drug carriers for intravenous administration: comprehensive physicochemical characterization.

Submicron injectable emulsions have been gaining more and more attention in the last few years, mainly as a vehicle for the intravenous administration of lipophilic drugs rather than as an improved delivery system for drug targeting. Submicron emulsions are available, clinically well accepted, and successfully marketed. Novel original emulsion formulations are being extensively investigated and already exhibit improved pharmacological activity; this fact underlines the promising therapeutic properties of these colloidal drug carriers as vehicles for potent lipophilic drugs. It is therefore essential to carry out a comprehensive physicochemical characterization of these submicron emulsions. It is the objective of the present review to emphasize the need for physicochemical studies in the design of new submicron emulsion drug delivery systems and to describe the different approaches available for carrying out such a complete examination of the emulsion colloidal drug carrier system.

Salvage transplantation for allograft failure using fludarabine and alemtuzumab as conditioning regimen.

Graft failure after allogeneic blood or marrow transplantation, although generally uncommon, can be a devastating complication. This report includes the outcome of nine patients who received a salvage transplant for failure to engraft after one (n=8) or 2 (n=1) prior transplants. Eight patients received allografts from the original donor. All received fludarabine 30 mg/m(2) i.v. and alemtuzumab 20 mg i.v. daily from days -6 to -2. Daily CYA was begun on day -2, and the allograft was infused on day 0. The therapy was well tolerated with low toxicity, and all nine patients engrafted, recovering neutrophils at a median of 12 days after transplant. Four patients died: two of relapse, one of a fungal infection in the setting of GVHD and one of multiple sclerosis. The combination of fludarabine and alemtuzumab is an effective and well-tolerated salvage conditioning regimen for patients who experience graft failure after blood or marrow transplants.

Short- and long-term stability assessment of a new injectable diazepam submicron emulsion.

An innovative diazepam injectable emulsion, recently characterized, was submitted to short- and long-term stability studies. The initial pH of the diazepam emulsion decreased progressively with time. However, this pH decrease can be controlled by adjusting the initial pH of the emulsion. At initial adjusted pH of 7.4 or 8.0, the pH decrease of the emulsions was markedly diminished as confirmed by pH follow-up over 10 months storage. All the emulsions prepared using less than 25% oily phase remained stable at the various storage temperatures over a 10 month period. However, emulsion stability was highly dependent on the non-ionic emulsifier concentration, while both low and high phospholipid concentrations reduced emulsion stability. Furthermore, the diazepam remained intact in the emulsion, as confirmed by HPLC when stored at 4 degrees and 25 degrees C over a 16-month storage period. The optimal emulsion formulation was stable to conventional steam sterilization cycles. Exposure of the emulsion to mechanical stress conditions did not affect the integrity of the emulsion, indicating that the dosage form developed remained practically stable.

Efficacy evaluation of a novel submicron miconazole emulsion in a murine cryptococcosis model.

Submicron emulsions of miconazole were stabilized by using a combination of three emulsifiers comprising phospholipids, poloxamer, and deoxycholic acid (DCA). The presence of DCA was vital for prolonged emulsion stability owing to its contribution to the elevated zeta potential of the emulsion. Further, the results by the phospholipid surface labelling colorimetric technique clearly suggested that poloxamer molecules interacted with phospholipid polar-head groups of the mixed DCA-phospholipid interfacial film, resulting in the stabilization of the emulsion by a steric enthalpic entropic mechanism. The plain emulsion vehicle was well tolerated up to a dose of 0.6 ml injected i.v. to BALB/c mice. The maximum tolerated dose of miconazole was 80 and 250 mg/kg in Daktarin i.v. (a marketed product) and emulsion, respectively, showing an improved safety ratio of 1 to 3 in favor of the emulsion. These results tended to confirm that the adverse effects associated with Daktarin i.v. injection should be associated with the vehicle rather than with the miconazole itself. In a murine cryptococcosis model, only one mouse out of ten remained alive by day 15 in the infected group treated with Daktarin i.v., while in the miconazole emulsion treated group, mice began to die from day 16 up to day 25 post inoculation. Thus, the multiple-dose treatment with the miconazole emulsions improved the protection offered to the infected mice. However, the therapeutic levels of miconazole that were reached in the target organ (brain) were lower than those required for complete eradication of Cryptococcus neoformans, which is known to multiply preferentially in the brain.

Characterization of diazepam submicron emulsion interface: role of oleic acid.

Oleic acid markedly improved the physical stability of a diazepam submicron emulsion. The zeta potential of the emulsion increased with increased oleic acid concentration. This effect suggested that adjustment of the diazepam submicron emulsion pH to 7.8-8.0 led to the ionization of oleic acid molecules at the oil/water (o/w) interface without being excluded from the surface regions of the oil droplets. TEM freeze-fracturing examination revealed that a mixed-emulsifier monolayer film was established at the o/w interface of the submicron emulsion. No liquid crystal or liposome formation was detected. This was confirmed by the results of phosphatidylethanolamine surface labelling at the o/w interface of the emulsion. The improved stability properties conferred to the emulsion by oleic acid should be attributed not only to the zeta potential increase, but also to the strengthening of the molecular interactions occurring between phospholipid and poloxamer emulsifiers in the presence of an ionized form of oleic acid at the o/w interface of the emulsified oily droplets as evidenced in independent monolayer studies.

Efficacy evaluation of a novel submicron amphotericin B emulsion in murine candidiasis.

A submicron amphotericin B (AmB) sterile emulsion with a mean droplet size of approximately 100 nm was prepared. The emulsion was stable at 4 degrees C over a period of 4 months. The acute toxicity results showed that the maximum tolerated dose of the AmB emulsion was 2.0 mg kg-1 as compared to 0.5 mg kg-1 for a commercial deoxycholate suspension, Fungizone. Efficacy evaluations of submicron AmB emulsion compared to Fungizone were performed in a murine candidiasis model using either a single or a multiple daily dose administration of 0.4 mg kg-1. Survival (100%) was observed up to 15-18 days post-infection in mice treated with AmB emulsion or Fungizone, while all control non-treated animals had died after 9 days in both single and multi-treatment experiments. Survival (50%) was obtained after 35 days in the single treatment, and 40 days in the multi-treatment, experiment with AmB emulsion, while treatment with Fungizone gave 50% survival after 15 and 20 days, respectively. The overall results indicate that in murine candidiasis, treatment with AmB submicron emulsion was more effective than Fungizone, suggesting a potential therapeutic application.

Side-effect evaluation of a new diazepam formulation: venous sequela reduction following intravenous (i.v.) injection of a diazepam emulsion in rabbits.

Diazepam has been incorporated into a stable, submicronized injectable emulsion. Venous sequela induction in rabbits following iv administration of diazepam in a marketed hydroalcoholic solution and in the emulsion were determined and compared over a 5-day period. There was a marked difference in the local reactions induced by the iv administration of the marketed diazepam hydroalcoholic solution and the diazepam emulsion, even on the first postinjection day. This difference was confirmed by pathological analysis. The highest mean venous sequela score was reached by the rabbit group injected with the marketed diazepam solution. It should be noted that no statistical difference was observed between the saline and the diazepam emulsion rabbit groups during the 5 days of the observation period. The moderate increase in the venous sequela score values compared to that for the saline solution should be attributed to the intrinsic effect produced by diazepam itself, and not to the emulsion vehicle, which was shown not to induce any vascular reaction in the present study.

Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood.

The expression of naked plasmid DNA coding for firefly luciferase (pRSVluc) or a secreted protein, human-alpha-1-antitrypsin (pRcCMVhAAT) in mouse skeletal muscle was characterized following administration by an improved intramuscular injection technique. Injection guided by intense illumination along the longitudinal axis of the mouse quadriceps muscle and parallel to the myofibers yielded 200-fold higher levels of luciferase expression than perpendicular injection. Luciferase expression was inhibited by an excess of non-coding DNA or dextran sulfate suggesting that muscle DNA uptake mechanism(s) can be saturated. Injected plasmid DNA was rapidly eliminated from the muscle as evidenced by tissue distribution studies of radiolabeled hAAT plasmid and Southern analysis. However, PCR analysis demonstrated that hAAT cDNA persisted in the muscle for at least 1 month after injection. Immunohistochemistry techniques indicated that the hAAT gene was expressed by the muscle fibers. ELISA analysis of serum samples collected from intramuscularly injected mice demonstrated that secreted hAAT protein concentration peaked in serum by day 7, started to decline by day 14 and was barely detectable 21 days post-injection. RT-PCR analysis demonstrated that hAAT transcript persisted at the site of injection for at least 1 month indicating that the decline of serum hAAT concentration 21 days post-injection was not due to the absence of hAAT transcript. However, the decline of hAAT protein concentration in the serum was inversely correlated with accumulation of murine anti-hAAT antibodies in circulation.

Intratracheal gene delivery to the mouse airway: characterization of plasmid DNA expression and pharmacokinetics.

Intratracheal administration of plasmid DNA resulted in gene expression in mouse airways in the absence of any enhancing agent. Administration of plasmid DNA encoding the chloramphenicol acetyltransferase gene (CAT) in sterile water lead to CAT transgene expression that peaked between 1 and 3 days and was detected up to 28 days after DNA administration. Transgene expression was independent of mouse gender, age and strain. Levels of expression from DNA in various isotonic solutions did not differ from levels obtained with DNA administered in water, suggesting that transfection is not dependent on damage to airway cells caused by a hypo-osmotic delivery vehicle. Pharmacokinetic studies using radiolabeled plasmid DNA showed that DNA was rapidly degraded, while higher levels of radioactivity were retained for longer duration following administration of cationic liposome-DNA complexes in the airway. Southern blot and PCR analysis confirmed that DNA complexed with DOTMA-DOPE was retained in the airways for a longer period. However, cationic liposomes DOTMA-DOPE (1:1) or DOTAP complexed with DNA, did not enhance expression over DNA alone. These results suggest that 'naked' plasmid DNA should be included as a control in all studies on intratracheal gene delivery using nonviral systems.