Title:  Emulsion stability

United States Patent:  RE38,459

Issued:  March 9, 2004

Inventors:  Raynolds; Stuart (Wilmington, DE); Dettre; Robert H. (Hockessin, DE)

Assignee:  Thomas Jefferson University (Philadelphia, PA)

Appl. No.:  234959

Filed:  January 21, 1999

Abstract

An injectable physiologically acceptable aqueous phase fluorocarbon emulsion, which has substantially no free fluorocarbon, has an excellent stability is prepared by a process of the invention. Typically, more than about 99.8 wt. % of the fluorocarbon remains in the size range of about 0.2 to 0.4 microns even after being stored at room temperature for one year or more in sealed containers under a non-oxidizing atmosphere. The emulsion is useful in medical applications, for example, coronary angioplasty, cancer therapy, among others.

SUMMARY OF THE INVENTION

The present invention relates broadly to a method for preparing an emulsion wherein the quantity of free or unemulsified fluorocarbon is minimized. Without wishing to be bound by any theory or explanation, it is believed that the quantity of free fluorocarbon within an emulsion can be substantially completely eliminated by reducing, if not preventing, any interaction between the emulsion and the interior surface of a storage container. For example, it is believed that pretreating the storage container causes formation of an interior monolayer coating which can prevent such interaction. Should a fluorocarbon emulsion be introduced or injected into a body, the presence of free fluorocarbon is undesirable because free fluorocarbon may cause formation of emboli in the bloodstream.

One aspect of the present invention relates to a sterilized emulsion which can be stored under ambient conditions in sealed infusion bottles for a year or more without significant deterioration, e.g., when stored at about 24^oC. the average emulsion droplet size increases to less than about 0.60 micron. Such an emulsion would be partially valuable for emergency use at facilities which are limited or over extended, for example, in disaster relief.

In another aspect, the present invention relates to a process for preparing perfluorocarbon (PFC) emulsions in physiologically compatible saline solutions which can be stored for lengthy periods, e.g., storage for more than about 2 years at a temperature of about 4^oC. or at least about 3 months at a temperature of about 24^oC. The quality of the emulsion can be improved by pretreating the storage containers, bottles, vials, among others. Typically, greater than about 99.8 wt. % of the PFC emulsified droplets remain in the size range of about 0.2-0.4 micron, when stored for a period longer than about one year at room temperature in a non-oxidizing atmosphere within, for example, sealed bottles.

The emulsions comprise about 10 through about 50% volume/volume (v/v) of at least one liquid perfluorocarbon (PFC), which has a molecular weight in the range of about 460-520, about 1-8% weight/volume (w/v) of at least one emulsifying agent, and the balance comprising a physiologically acceptable saline solution.

A sterilization emulsion, which is prepared by the method described herein, can be stored at ambient temperatures in sealed infusion bottles for at least about one year. The substantially complete elimination of any free fluorocarbon from the present emulsions allows such emulsions to be used safely on demand for medical applications. As a result, the present invention is particularly valuable for medical emergencies, and in situations wherein the availability of hospital equipment is limited.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates broadly to minimizing the presence of free fluorocarbon in an emulsion. By minimizing the presence of free fluorocarbon, the invention can be employed as a process for preparing aqueous perfluorocarbon (PFC) emulsions which can be used in medical applications. By "perfluorocarbon" it is meant a substantially fluorinated fluorocarbon, e.g., this term encompasses completely fluorinated fluorocarbons and hydrogen-containing fluorocarbons. Further, such emulsions are stable over a period of at least one year when stored at room temperature (24^oC.), or for at least about 2 years when stored at about 4^oC. By "stable" it is meant that the droplet size of the emulsion does not increase significantly, e.g., when stored at about 4^oC. the average droplet size of the emulsion remains less than about 0.60 micron. Such emulsion are typically physiologically acceptable to the human body so that these emulsions can be employed for medical purposes.

Physiologically acceptable PFC emulsions have the ability to dissolve large volumes of gases within the human body such as oxygen and carbon dioxide. This ability enables acceptable PFC emulsions to be used for blood substitutes, and in medical treatments which are more effective when supplementary oxygen can be delivered to critical body organs such as the heart, brain, liver, kidneys, among other organs. In view of the world-wide shortage of human blood for use in transfusions, and increasing concern about its freedom from undesirable species, there is a long felt need for an artificial blood which is stable under ambient conditions, and free from infectious agents.

In addition to being effective blood substitutes, the emulsions prepared by the invention are medically useful in coronary angioplasty, cancer radiotherapy and chemotherapy, heart reperfusion, emergency treatment for stroke, among other uses. These emulsions also may be incorporated into a synthetic cerebrospinal fluid composition. For example, the PFC emulsion can be employed in acute stroke therapy by incorporating the emulsion within an oxygenated fluorocarbon-based nutrient emulsion which is administered by ventriculocisternal perfusion. In some cases, a fluorocarbon emulsion can be employed as an artificial cerebrospinal fluid (CSF), which is delivered by direct flow into the lateral ventricle of the brain. Upon effective delivery of the oxygenated fluorocarbon emulsion, the fluorocarbon emulsion may be capable of salvaging significant quantities of brain tissue.

The emulsions made by the process of this invention comprise about 10 through about 50% (v/v) of at least one liquid PFC which has a molecular weight in the range of about 460-520, and 1-8% (w/v) of at least one emulsifying agent, the balance being a physiologically acceptable aqueous solution of electrolytes. Normally, substantially completely all of the PFC becomes a component of the emulsion. The droplet size of the emulsion prior to sterilizing is about 0.10 micron. After sterilizing the emulsion, the particle size of the emulsion droplets ranges from about 0.2 to about 0.4 micron. The emulsion droplet size can be measured by using a Coulter N4MD sub-micron particle analyzer.

The PFCs are substantially chemically insert, and have no known adverse effect upon human physiology. Suitable PFC characteristics are such that following delivery to the body, the PFC is substantially completely expelled from the body through the respiratory system. Any suitable PFC, which is readily excreted from the body, can be used for preparing an emulsion that has substantially no free fluorocarbon. Suitable PFCs can be produced by any process which avoids contamination with physiologically unacceptable substances, or a process wherein such substances can be adquately removed by using conventional separation methods.

Specific examples of suitable PFCs are perfluorooctyl bromide (PFOB), bisperfluorobutyl ethylene (F-44E), and mixtures thereof, among others. A suitable PFC is encapsulated or emulsified by being contacted with at least one emulsifying agent such as a phospholipid, e.g., egg yolk lecithin.

The emulsion is present within an aqueous medium such as a dilute solution of salts. For example, the aqueous medium may comprise electrolytes which are present at concentrations that are sufficient to obtain, an isotonic emulsion. Typically, the aqueous electrolyte solution contains at least about 0.90 gram of electrolyte per liter of water for injection. Examples of suitable electrolytes comprise at least one member selected from the group of sodium chloride, potassium chloride, dibasic sodium phosphate, sodium bicarbanate, hydrated sodium citrate, hydrated calcium chloride, hydrated magnesium chloride, among others. For example, an aqueous electrolyte solution is obtained by preparing a buffered saline solution, e.g., about 7.4 g NaCl and 2.3 g NaHCO₃ per liter. In some cases, the electrolyte solution comprises a modified Tyrode's solution which has the following-general composition per liter:

The ingredients for a Tyrode's solution can be dissolved into sterile water for injection, and diluted further to a final volume of about one liter.

For best results, the containers and equipment, which are used for preparing and storing the emulsion and its components, are thoroughly cleaned and sterilized prior to being used. Glassware is typically first cleansed by washing with aqueous isopropanol, e.g., about 70/30 v/v isopropanol/water; followed by rinsing with deionized water which has a neutral pH. Stainless steel parts of equipment, e.g., a homogenizer, which will contact the emulsion, can be washed at room temperature with an Alconox solution (Alcanox is a biodegradable compounded alkyl aryl sodium sulfonate available from Alconox Inc., New York, N.Y.). The glass equipment can then be heated in an oven to a temperature of at about 250^oC. to ensure that the glass equipment is substantially pyrogen free. Failure to effectively clean containers and other processing equipment may introduce contaminants into the emulsion which impair the utility of the final emulsion.

When a Tyrode's solution is used as an electrolyte for preparing an emulsion, there can be a tendency for calcium carbonate to precipitate, thereby destabilizing the emulsion. For best results, calcium carbonate precipitation is reduced, if not prevented, by purging a freshly prepared electrolyte solution with carbon dioxide for about 15 to 30 minutes, and filtering the purged solution through an approximately 0.2 micron filter in a manner which assures sterility.

An electrolyte solution, which possesses an enhanced product sterility and a lower endotoxin content, can be obtained by conducting all the processing steps within a laminar flow hood. A class 100 laminar flow work space is normally satisfactory for this purpose. The laminar flow work space ensures that most particulate material above about 0.3 micron in size is continuously removed by filters, thereby providing a working atmosphere which contains less than about 100 particles above 0.3 micron per cubic foot. When the laminar flow work space is used in conjunction with conventional sterile processing techniques, the preparation of sterile, low endotoxin emulsions is enhanced.

For best results, prior to preparing the emulsion, the emulsifying agent, e.g., egg yolk lecithin, should be stored under nitrogen with dry ice refrigeration. Such storage is useful to prevent the emulsifying agent from undergoing any significant oxidative degradation, and/or microbial contamination. Oxidation typically has a detrimental effect on the stabilizing ability of the emulsifying agent.

The emulsion preparation process is begun by dispersing of intermixing the electrolyte solution and the emulsifying agent. The emulsifying agent can be dispersed within an electrolyte solution at room temperature by using a homomixer, e.g., supplied by Eppenbach, Greerco, Baldor/Boehm. The homomixer functions to apply a shear force or agitate the emulsion, thereby admixing the electrolyte and emulsifying agent to create an electrolyte/emulsfying agent dispersion which has a relatively small droplet size. Dispersing the emulsifying agent into the electrolyte solution typically produces a milky electrolyte/emulsifying agent dispersion. The dispersion can be heated to about 55^o-60^oC. while under a nitrogen atmosphere, and then homogenized by using a Microfluidizer (supplied by Microfluidics, Inc.), or a Manton-Gaulin homogenizer, thereby producing a substantially translucent dispersion. The translucent dispersion is typically cooled to about 15^o-20^oC. The average size of the dispersion particles or vesicles, which can be determined by using a Coulter N4MD sub-micron particle size analyzer, typically ranges between about 0.08-0.1 micron.

Prior to introducing the PFC into the translucent dispersion described previously, the PRC should be purged with carbon dioxide for about 30 minutes to ensure that substantially no calcium carbonate is precipitated in the emulsion, e.g., all of the calcium carbonate, if any, is converted to a soluble calcium bicarbonate. After purging the PFC with carbon dioxide, the PFC can be added slowly to the translucent dispersion while rapidly agitating the dispersion and maintaining the temperature at about 15^o-20^oC. (an Eppenbach Homomixer is effective for agitating the dispersion.) An emulsion is usually obtained in about 15 minutes. The emulsion can be homogenized by being passed five to ten times through a Microfluidizer, or a Manton-Gaulin homogenizer. The emulsion can also be filtered by using a 10-12 micron filter to remove coarse particles.

The filtered emulsion is ready for storage, e.g., within glass infusion bottles. The presence of free fluorocarbon within the emulsion is substantially completely avoided, if not prevented, by pretreating the storage bottles or containers. For example, when storing the emulsion within a glass infusion bottle, the presence of free fluorocarbon within the emulsion can be prevented by pretreating the interior surface of the bottle. The interior of the bottles can be pretreated by being coated or sprayed at room temperature with, for example, a saline lecithin dispersion. The pretreated bottles can be inverted to drain the pretreating dispersion or solution, and then filled with the PFC emulsion. After being filled with the PFC emulsion, the bottles are typically back-filled or purged with nitrogen, and sealed.

In one aspect of the invention, the storage containers may be pretreated with a medical grade of silicone oil, e.g., Dow-Corning medical grade silicone oil no. 360. For example, the interior surface of a storage bottle is coated with silicone by filling the bottles .[.With.]. .Iadd.with .Iaddend.silicone oil. After draining the silicone oil, the bottles can be depyrogenated by baking in an oven at a temperature of about 250^oC. Without wishing to be bound by any theory or explanation, it is believed that pretreating the storage containers causes formation of a firmly bound polymeric monolayer on the glass surface which reduces the interaction between the bottle and the emulsion, thereby avoiding, if not completely preventing, the presence of free PFC. For example, it is believed that a surface coating of silicone oil may react with the interior glass surface of an infusion bottle, thereby forming a non-extractable silicone-containing monolayer which minimizes the interaction between the emulsion and the bottle. While particular emphasis has been placed upon using a lecithin dispersion and silicone oil for pretreating the emulsion containers, any pretreating fluid may be employed which does not adversely effect the utility of the emulsion. However, when the emulsion is employed for medical purposes, the pretreating fluid must be physiologically acceptable.

Another advantageous result which is obtained by pretreating the bottles is that should the presence of free fluorocarbon be detected, the free fluorocarbon can be substantially completely re-emulsified by agitating or shaking the container or bottle.

The sealed emulsion-containing bottles can be sterilized by any suitable method which does not adversely affect the emulsion. For example, a rotary or stationary autoclave, e.g., which is operated at a temperature of about 121^oC., can be used for achieving an acceptable Lethality Factor of about F_O 21.5. Lethality Factor is discussed in "Disinfection, Sterilization, and Preservation", edited by Seymour S. Block, second edition, 1977; the content of which is incorporated by reference. In other words, the sealed bottles are heated in a manner which is capable of providing a quantity of heat that is equivalent to being exposed to a temperature of about 121^oC. for about 21 minutes.

When the emulsions of the invention are stored under ambient conditions, the emulsions are normally stable for at least about one year. However, the useful shelf life of the emulsions can be extended further by refrigerating the emulsion at temperatures no lower than about 4^oC.

Claim 1 of 19 Claims

The following is claimed:

1. A method for making a stable emulsion having substantially completely no unemulsified fluorocarbon comprising the steps of:

preparing an aqueous electrolyte solution,

preparing a dispersion by introducing at least one emulsifying agent into the solution,

admixing at least one perfluorocarbon into the dispersion to form an emulsion,

applying an interior coating comprising silicone oil or lecithin to a storage container,

storing the emulsion in the container which has a non-oxidizing atmosphere, wherein said emulsion contains substantially completely no unemulsified perfluorocarbon, and;

optionally agitating the emulsion.

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