Preparation and Anesthetic Properties of Propofol Microemulsions in Rats

Propofol was obtained from Albemarle Corporation (Baton Rouge, LA). Purified poloxamer 188, a nonionic coblock polymer consisting of polyethylene and polypropylene monomers, was purchased from the BASF Corporation (Florham Park, NJ). Sodium salts of fatty acid (C⁸, C¹⁰, and C¹²) were supplied by Sigma Chemical Co. (St. Louis, MO). Microemulsions were prepared by combining propofol (0–100 mg/ml) with purified poloxamer 188 (0–70 mg/ml), and a fatty acid salt (0–12.5 mg/ml) in normal saline (0.90 mg/ml NaCl) bulk media. Water was ultrapurified using a water purification system (Nanopure; Barnstead/Thermolyne, Dubuque, IA) to provide a minimal electrical resistance of 18.2 MΩ. After agitation with a magnetic stirrer, these components combined to form clear, colorless microemulsions with adjustment of pH to 7.40 using either HCl or NaOH. All experimental formulations were stored under a nitrogen headspace. To characterize the dimension of the individual droplets, the effective droplet size of the nanoparticles was measured by the dynamic light-scattering method using a submicron particle sizer analyzer (90Plus; Brookhaven Instruments Corporation, Holtsville, NY) as previously described.16 

All experimental protocols involving the use of animals were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee, Gainesville, Florida. Sprague-Dawley rats (350–500 g; either sex) were purchased from Charles Rivers Laboratories (Wilmington, MA). Before delivery of animals to the investigators by the vendor, rats underwent catheterization of the left femoral vein with subcutaneous tunneling to an exit site between the scapulae. Thus, rats were supplied by the vendor with preimplanted, heparin-bonded, femoral vein catheters and did not require additional instrumentation at the time of the experiments that might require sedation or anesthesia that could confound interpretation of results. Rats were caged singly to avoid damage to the catheter by cage mates. They were allowed unlimited access to food and water with a 12:12-h light:dark cycle. On the day of experimentation, each rat was weighed. Thereafter, the central venous line of each rat was easily accessed in conscious, unrestrained rats. All catheters were aspirated until blood was observed and then flushed with 0.5 ml normal saline. Subsequently, rats entered the animal experimental protocol.

Rats were randomized using a random number generator to receive either (1) an experimental propofol microemulsion (n = 6/microemulsion) after filtration through a 200-nm pore filter to ensure sterility or (2) a conventional macroemulsion of propofol in a soybean-based solvent (Diprivan; AstraZeneca Pharmaceuticals, Wilmington, DE). In both cases, the formulation was infused at a rate of 10 mg · kg⁻¹· min⁻¹propofol via  a microprocessor-controlled syringe pump (sp2000i; World Precision Instruments, Sarasota, FL) to avoid varying rates of infusion associated with manual injection that could potentially confound anesthetic induction time parameters. The end points of anesthetic induction were total drug dose, time to loss of exploratory behavior (i.e. , stunned), time to loss of righting reflex, time to loss of lash reflex to gentle stroking (i.e. , canthal reflex), and time to loss of reflexive withdrawal of the leg after a great toe pinch every 10 s by a metal clamp. In these experiments, the metal clamp was rubber shod to avoid tissue damage during the experiment. After loss of withdrawal to the toe pinch, the drug infusion was discontinued. End points of anesthetic recovery were return of the withdrawal response to a toe pinch, recovery of spontaneous eye blinking, recovery of a sustained head lift, and recovery of the righting reflex. After the first anesthetic, each rat recovered for at least 14 days before enrollment in the experimental limb opposite the original assignment. Thus, each rat was anesthetized with both experimental and conventional propofol formulations with random initial assignment and with a 14-day recovery interval before crossover between the formulations. Following a 14-day period of observation after participation in the crossover limb, the experiment was ended.

Measurements are reported as mean ± SD. Statistical analysis was performed with SAS 9.1 (SAS Institute, Inc., Cary, NC). Linear mixed models for incomplete randomized block design were used to analyze the data from this crossover study.17Outcome variables were evaluated for normality assumptions using the Box-Cox procedure, with transformations (i.e. , square root, inverse square root) if indicated.18Initial models included terms for order effect and crossover effect. These were not included in final models if not significant, based on the Akaike Information Criterion. Models were parameterized to include treatment versus  control differences, purified poloxamer 188 concentration category, fatty acid salt carbon chain length category, and interactions between treatment difference and propofol formulation variables. Prespecified contrasts were used to compare treatment differences at each level of the formulation variables and between levels of the formulation variables. A P  value of less than 0.05 was considered statistically significant.

Forty-eight microemulsions of propofol were formulated using varying concentrations of propofol, purified poloxamer 188, and C⁸fatty acid salt in a normal saline bulk media to construct a pseudoternary diagram of these systems (fig. 2). Depending on the concentrations of surfactants, propofol concentrations of 12–15 mg/ml or less produced microemulsions. In contrast, propofol concentrations exceeding 20 mg/ml with these concentrations of surfactants formed opaque, white macroemulsions similar to conventional propofol formulations. For purposes of testing in rats, the final concentration of propofol in the microemulsions was selected to be 1% (10 mg/ml), a concentration equal to that in the conventional commercially available macroemulsion formulation.

Subsequently, we more closely investigated the interaction of the concentration and types of surfactants and cosurfactants necessary to form microemulsions (or macroemulsions) while holding the concentration of propofol constant at 10 mg/ml. At the greatest concentration of purified poloxamer 188 (70 mg/ml), no fatty acid cosurfactant was required to create microemulsions of propofol as shown in the binary diagram (fig. 3). However, as the concentration of purified poloxamer 188 decreased, the concentration of the fatty acid surfactant necessary to form a microemulsion increased. In addition, the nature of the fatty acid significantly affected its concentration of fatty acid salt necessary to create a microemulsion. Thus, the concentration of fatty acid salt required to formulate propofol as the oil core of a microemulsion was markedly diminished by lengthening the carbon chain of the cosurfactant fatty acid from 8 to 10 or 12 carbon atoms at any concentration of purified poloxamer 188.

The measured particle sizes for various formulations of the microemulsions varied from 11.9 to 47.7 nm (fig. 4). In general, as the concentration of purified poloxamer 188 was increased, the diameter of the particles also increased irrespective of the carbon length of the fatty acid cosurfactant. No change was noted in the dimension of the microemulsion droplets containing C⁸fatty acid salt after 4 months of storage at room temperature.

We also investigated the relative stability of these propofol microemulsions to dilution with normal saline. That is, we determined the volume of the saline diluent necessary to break the microemulsion into a macroemulsion (fig. 5). The volume of diluent required to cause these changes increased with lengthening of the carbon chain in the fatty acid salt. This increase in the diluent volume was most noticeable for the C¹²propofol microemulsions that required approximately a sixfold dilution to break the microemulsion.

Thirty-eight rats were enrolled in this protocol, but eight rats completed only one limb of this protocol because of retraction of the catheter under the skin during the recovery interval from the first limb of the study. Data from these eight rats (n = 5 in microemulsion group, n = 3 in macroemulsion group) were removed from the study. The remaining rats (n = 30) completed the crossover study and were anesthetized with these microemulsions (n = 6/experimental group; five experimental groups) and with the macroemulsion control (n = 30). All rats experienced rapid induction without apnea and recovered for at least 14 days without apparent injury. After the 14-day observation period after crossover, the experiment was terminated. The summary data from these experiments are presented in table 1.

Propofol is an organic liquid that exists as an oil at room temperature with minimal aqueous solubility. This insolubility is both a fundamental property of propofol and a significant problem for purposes of drug delivery. To address this issue, propofol was formulated in a castor bean oil macroemulsion in the early 1980s, but this solvent was subsequently abandoned because of its propensity to cause anaphylaxis.19,20Instead, a soy bean oil macroemulsion was selected as an alternative emulsification agent to deliver propofol.21Currently, these white, opaque macroemulsions available from different pharmaceutical companies are formulated with the following components: propofol (10 mg/ml), soybean oil (100 mg/ml), glycerol (22.5 mg/ml), egg lecithin (12 mg/ml), and a preservative (0.05 mg/ml disodium edetate or 0.25 mg/ml sodium metabisulfite). The requirement that propofol be dissolved in 10% soybean oil with associated surfactant has caused a number of liabilities, including support of bacterial growth, addition of egg products that is objectionable to vegans, and the necessity of preservatives.5,6,8 

The need for a suitable alternative formulation is evidenced by past and ongoing research into other types of propofol delivery systems. Several investigators have reported favorable results using formulations composed of medium- and long-chain triglyceride macroemulsions to deliver propofol at varying concentrations of 10–60 mg/ml.11,22,23In addition, propofol has been complexed with sulfobutylether 7-β-cyclodextrin molecules to form clear, colorless formulations that are stable at a wide range of temperatures (4°–50°C), a clear benefit compared with macroemulsion technology, and that have pharmacokinetic properties similar to the conventional drug delivery system.12,24Finally, prodrug methods have been exploited to develop phosphono-2,6-diisopropylphenol, an agent that is metabolized by native alkaline phosphatase to generate the active agent, propofol, and two by-products, formaldehyde and phosphate.25In this report, we demonstrate that microemulsion methods represent another suitable technology to deliver propofol intravenously with anesthetic parameters similar to the commercially available macroemulsions.

Microemulsions have been previously used to deliver several drugs by the oral and transcutaneous routes. Currently, an oral microemulsion of cyclosporine is available for prevention and treatment of transplant rejection of solid organs.26In addition, a number of other drugs have been formulated in microemulsions for oral (e.g. , paclitaxel, heparin) and transdermal (e.g. , apomorphine, estrogen) delivery.27–30Fewer drugs have been delivered using an intravenous route but include trans -retinoic acid and flurbiprofen.31,32Unlike other oil-in-water microemulsions wherein the active drug is dissolved in an oil excipient, propofol acted in two different, complementary roles in the current investigation. That is, the need for an excipients oil for propofol dispersal was obviated by the fact that propofol is itself an oil at room and physiologic temperatures. Therefore, propofol could serve not only as the active pharmacologic agent, but also could exist as the physical platform for the preparation of these microemulsions. By doing so, the need for additional excipients oils (e.g. , soybean or castor bean oil) is eliminated along with the potential for these excipients to nourish bacteria. Although the current authors also hypothesize that propofol microemulsions will not support bacterial growth to the same extent as macroemulsions, additional investigations are needed to test this thesis.

The onset of induction with the propofol microemulsion was similar to that caused by the commercially available propofol macroemulsion. The time to achieve induction (defined by the protocol as loss of leg withdrawal to a pinch) during macroemulsion injection was 123 s, whereas the latency periods during microemulsion treatment were 160–251 s. This delay is favorable when compared with prodrug technologies that rely on metabolism, a phenomenon that may vary within any patient population.33The differences in induction times between experimental groups reported herein may be caused by differential release of propofol from the individual droplets into the blood. That is, the different propofol nanoparticles have markedly different stabilities against dilution by blood based on the emulsifier structure and concentration selected for the formulation. In general, a microemulsion is thermodynamically stable at equilibrium. One can destabilize a microemulsion by significantly changing pressure, temperature, or chemical compositions.15The last variable can be changed simply by diluting a microemulsion with saline or blood. It is well recognized that the formation of microemulsions requires an ultralow interfacial tension (e.g. , approximately 10⁻³mN/m) at the oil–water interface.15Upon dilution with saline, the interfacial tension will increase substantially as the emulsifier molecules (i.e. , both poloxamer 188 and fatty acid salt molecules) desorb from the droplet surface. This event will markedly increase the interfacial tension at the droplet surface and ultimately destabilize the microemulsion, with release of the active pharmacologic core (i.e. , propofol). However, each emulsifier film around the microemulsion droplet has inherent molecular packing and, hence, stability. The extent of dilution required to destabilize a microemulsion represents its inherent stability. Thus, a microemulsion requiring greater dilution for destabilization indicates that its emulsifier film has a greater stability. The data in figure 5suggests that the C¹²fatty acid salt microemulsions are the most stable because they require the greatest dilution to become a turbid macroemulsion. It is likely, however, that microemulsion destabilization is affected by more than just dilution in vivo  because the C¹⁰fatty acid microemulsion required the longest time to cause anesthetic induction, whereas the C¹²fatty acid microemulsion was most stable to dilution in vitro . Further understanding and selectively modifying these destabilization rates by adapting surfactant type and concentrations alludes to the possibility of controlling release times of active pharmaceuticals from nearly immediate (e.g. , propofol) to longer times (e.g. , chemotherapeutic or antifungal agents). Moreover, although an understanding of the parameters governing release rate allows further refinement of these microemulsions, only a fraction of the formulations presented herein could be considered for additional study with an aim toward clinical use. That is, the induction times for some microemulsions (e.g. , 5% purified poloxamer 188 with C¹⁰) were more than double that caused by the macroemulsion. Whether this observation is species specific or a clinically significant result remains to be determined.

The nonionic and ionic surfactants used to formulate propofol microemulsions were carefully selected not only for their ability to combine with the drug to form stable microemulsions, but also because both surfactants have been previously injected intravenously into humans without reported adverse incidents. Justification for choosing these surfactants is presented subsequently.

The selection of purified poloxamer 188, an ethylene oxide–propylene oxide block copolymer, was justified based on a number of characteristics related to chemical and medical requirements. First, this surfactant allowed rapid and reproducible preparation of propofol microemulsions that possessed good stability over time. In general, microemulsions are thermodynamically stable and hence exhibit infinite shelf life. These formulations are destabilized upon dilution with blood or plasma or saline beyond its tolerance limit. Second, purified poloxamer 188 has been previously administered intravenously in large doses to humans, without any apparent adverse effect. For example, this surfactant was infused in patients experiencing chest crisis due to sickle cell disease in an attempt to reduce the duration and severity of the crisis.34,35Although not efficacious to treat chest crisis at large doses of purified poloxamer 188 (e.g. , 88 g), no apparent untoward effects were noted in the subjects. Third, this surfactant is primarily (> 95%) excreted by kidneys and does not stimulate or inhibit human cytochrome P-450 systems.36In fact, others have hailed this coblock surfactant as a model surfactant for pharmacologic use, although additional toxicologic evaluation may be needed before more widespread use in the clinical arena.36For example, the current investigators have also demonstrated that poloxamer surfactants may reduce platelet function in vitro  as assessed by thromboelastography.37Additional work is needed to determine whether this effect on platelets translates into functional defects in thrombosis.

For the experiments examining microemulsions formulated with C⁸, C¹⁰, or C¹², the authors held the purified poloxamer 188 concentration constant at 5% (vis-à-vis  3% or 7%). The 5% concentration of purified poloxamer 188 was selected for further study because microemulsions formulated with 7% purified poloxamer 188 required minimal to no fatty acid surfactant (fig. 2), whereas microemulsions with 3% purified poloxamer 188 required concentrations of fatty acid necessary to form stable propofol microemulsion likely to be unacceptable for in vivo  use in humans.

Sodium caprylate was chosen as the best ionic surfactant for the following reasons. First, the surfactant allows reliable synthesis of propofol microemulsions with particle diameters of less than 50 nm range. Second, sodium caprylate is already present in many blood products (e.g. , human albumin) currently in clinical use to stabilize proteins.38,39Third, the smaller length of the carbon backbone reduces the risks of cellular injury associated with fatty acids with longer carbon chains such as sodium oleate that is used to cause lung injury as a model of acute respiratory distress syndrome.40For these reasons, sodium caprylate is the best choice currently available for additional investigations using microemulsions of propofol, although the information from use of C¹⁰and C¹²allowed us to understand how surfactant selection can affect the dose–response of propofol microemulsions. In addition to the selection of surfactant type, however, the concentration of this ionic surfactant must be carefully considered. In the future, the current investigators hope to determine whether the concentrations of sodium caprylate used in this study interact with erythrocyte membranes or cause hemolysis as can occur in vitro  with higher concentrations (EC⁵⁰of 213 mm) of sodium caprylate.37 

In conclusion, we report the successful preparation, characterization, and use of propofol microemulsions in rats to induce anesthesia similar to that caused by propofol macroemulsions. Formulation of propofol microemulsions was achieved although no excipient oil (e.g. , soybean or castor bean oil) was used because propofol exists as an oil and can serve as its own core of an oil-in-water microemulsion. Thus, propofol was used both to solubilize itself and as an active pharmaceutical. The differential diluent volumes in vitro  and destabilization times in vivo  based on changing the concentration and type of surfactant allude to the possibility of selectively modifying the dose–response properties of propofol and potentially other lipophilic drugs. Future work will be aimed toward exploring these differences for propofol and other lipophilic agents and toward determining whether these propofol microemulsions reduce the liabilities associated with soybean oil–based macroemulsions.