JP2004196776A - Composition for stable injection liquid and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition for a stable injection liquid and a method for the delivery of the composition. <P>SOLUTION: The composition for the delivery of a stable bioactive compound to a target person contains a 1st component and a 2nd component. The 1st component is composed of fine particles of sugar glass or phosphate glass containing a bioactive agent wherein the sugar glass or the phosphate glass may contain a glassification promoting compound, and the 2nd component is composed of at least one kind of biocompatible liquid perfluorocarbon in which the 1st component is insoluble and dispersible. The liquid perfluorocarbon contains a surfactant as the case may be. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

Background of the Invention

  Vaccines or drugs in injectable solutions are inherently unstable and require cooling. The pharmaceutical industry has long addressed the instability problem by freeze-drying drugs. This is expensive, inconvenient, and inherently dangerous because incorrect reconstitution of the dry drug can result in the wrong dose or contaminating solution. Many attempts to develop robust, stable, ready-to-inject liquid formulations have been unsuccessful over the last 100 years. Only drugs that are naturally strong and small molecules can remain in aqueous solution with a useful shelf life.

  This problem is particularly acute in the vaccine industry. It is estimated that by 2005, 3.6 billion doses of vaccine will have to be administered worldwide. This is not possible using a standard vaccine format that always requires cooling ("Revolutionizing Immunizations", Jodar L., Aguado T., Lloyd). J. and Lambert PH., Genetic Engineering News, Feb 15 1998). In the developing world, a "cold chain" of chillers running from vaccine factories to rural towns is currently in use. The cost of cold chains is enormous for the vaccine industry and non-governmental health organizations that are running immunization campaigns. WHO estimates that the cost of maintaining a cold chain alone will exceed $ 200 million annually. In addition, the immunization campaign may only reach those living near the end links of the cold chain.

  Vaccination campaigns require medical staff to ensure that a given dose is injected correctly and does not show any signs of degradation. The need to reconstitute some vaccines such as measles, yellow fever and BCG is also a significant concern in the art. This must be done accurately to ensure the correct dosage and can be a source of contamination that has often led to clinical catastrophe. In addition, it is often necessary to vaccinate more than once during a given time period, and this may not be possible if a particular mixture or "multivalent" vaccine is not available due to some component chemical incompatibility. May require multiple injections. WHO emphasizes these issues by encouraging the study of next generation stable vaccines that do not require cooling and do not require reconstitution ("Pre-Filled Monodose Injection Devices: A safety standard"). Lloyd J. and Aguado MTWHO publication, May 1998; ”General policy issues.“ For new vaccines, or a revolution in the delivery of immunizations? ” : injectable solid vaccines: a role in future immunization? ”(General policy issue: solid vaccine for injection: role in future immunity) Aguado MT, Jodar L., Lloyd J., Lambert PHWHO publication No A59781.

  The ideal solution for this problem would be a completely stable and ready-to-inject formulation. Such stable vaccines are placed into the injection device itself as individual doses, or are shipped in large quantities and administered by needle-free jet injectors for large-scale immunization campaigns. Transdermal delivery of dry solids by gas jet injection is described (Sarphie DF, Burkoth TL., Method for providing dense particle compositions for use in transdermal particle delivery) Method). PCT publication WO97448485 (1996)) Percutaneous inoculation with a dry DNA vaccine seems to be very effective (“PowderJect's Hepatitis B DNA Vaccine First To Successfully Elicit Protective Immune Response In Humans”). , The first Hepatitis B DNA vaccine from PowderJect) (http://www.powderject.com/pressreleases.htm (1998)).

The hypersonic shock wave of helium gas used to operate these powder injectors has limited power and cannot deliver that amount of fine particles into muscle. This is because the low-mass particles cannot reach moderate momentum as they penetrate deeply. While intradermal delivery of DNA vaccines coated with colloidal gold particles is suitable for good immunogenicity, common vaccines adjuvanted with insoluble aluminum or calcium salts produce unacceptable skin irritation. They must be administered intramuscularly. What is needed is a flexible system that can reach a range of delivery depths from intradermal to deep into the muscle, similar to those that can be achieved with current needle and syringe technology. In large vaccination campaigns, which using a pressure of about 3000 psi (about 210 kg / cm ^2), a thin (～0.15Mm diameter) liquid jet capable of accelerating the liquid flow "Liquid Nails" (liquid nail) Solved by the development of injectors. This device painlessly delivers its volume from the skin into the deep subcutaneous or muscle tissue by making a small hole in the epidermis. It can penetrate deeply due to the high momentum transmitted to the liquid stream. To date, infused drugs and vaccines have been water-based, but because of the instability problem described above, the range of stable aqueous products that can take advantage of this technology is very limited.

  It is now recognized that a wide range of bioactive molecules can be stabilized by drying in sugar glass (Roser B. "Protection of proteins and the like" UK Patent 2,187,191). Roser B and Colaco C. "Stabilization of biological macromolecular substances and other organic compounds" (stabilization of biological macromolecules and other organic compounds) PCT published WO 91/18091; Roser B and Sen S. "New stabilizing glasses" ( New stabilized glass) PCT Patent Application No. 9805699.7, 1998). These dry stabilizing activators are not affected by hostile environments such as high temperatures and ionizing radiation.

  The mechanism by which sugars cause significant stabilization of the molecule is the glass transition. When the sugar solution containing the active molecule is dried, it crystallizes when it reaches the solubility limit of the sugar or becomes a supersaturated syrup. The ability of a sugar to resist crystallization is an important property of a good stabilizer. Trehalose is superior in this respect (Green JL. & Angel CA., Phase relations and vitrification in saccharide water solutions and the trehalose anomaly). .93, 2880-2882 (1989)), but not the only one. Upon further drying, the syrup gradually solidifies, which turns into glass with low residual moisture. At some point, the active molecules change from a solution in water to a solid solution in dry sugar glass. Chemical diffusion is negligible in the glass, so the chemical reaction virtually stops. Since denaturation is a chemical change, it cannot occur in glass and the molecule is stabilized. In this form, the molecule remains unchanged if another condition is met. This is the second important property of a good stabilizer, that is, it is chemically inert and non-reactive. Many glasses have failed because they react with the product during storage. An obvious problem arises with reducing sugars, which form good physical glasses, but their aldehyde groups attack the amino groups of the product in a typical Maillard reaction. This is the main reason that many freeze-dried drugs require refrigerated storage. Non-reactive sugars give a stable product requiring no cooling at all.

  Biomolecules immobilized in sugar glass are also stable in non-aqueous industrial solvents in which both themselves and the sugar are insoluble (Cleland JL. And Jones AJS. “Excipient stabilization of polypeptides treated with organic solvents”). Excipient stabilization of polypeptides treated with US Pat. No. 5,589,167 (1994)). Because sugar glass acts as an impermeable barrier in non-solvent liquids, solid solution biomolecules in the glass are protected from both solvent chemical reactions and the environment. If the liquid itself is stable, the susceptible product in the suspended glass particles will form a stable two-phase liquid formulation. Industrial solvents of the type described by Cleland and Jones (1994) have limited utility in handling. As an alternative to biocompatible non-aqueous liquids, even the most unstable drugs, vaccines and diagnostics could be formulated as stable liquid formulations.

  First generation stable non-aqueous liquids were considered for drug or vaccine delivery (BJ Roser and S.D. Sen "Stable particle in liquid formulations"). PCT patent application GB98 / 00817 describes the formulation of a stabilized glass powder containing an active agent suspended in an injection oil such as sesame, peanut or soybean oil or a simple ester such as ethyl oleate. Was. Suspended sugar glass particles are very hydrophilic, while oils are hydrophobic. Due to the strong tendency of the hydrophilic and hydrophobic phases to separate easily, the sugar glass particles tended to aggregate together. In order to stabilize such "water-in-oil" suspensions, the use of oil-soluble surfactants dissolved in the continuous oil phase has often been required.

  These low HLB (hydrophilic / lipophilic balance) surfactants concentrate at the interface between the hydrophilic particles and the oil and coat them with an amphiphilic layer that is more compatible with the continuous oil phase. Since each sugar glass particle is separated from its neighbors by dry oil, no chemical interaction occurs between the particles. Thus, it is possible to have several different groups of particles without each interacting, even though each may contain different molecules that can potentially interact in the same oil formulation. Complex multivalent vaccines can thus be produced.

However, this approach has been found to have certain drawbacks that prevent it from being a universal solution. These include unavoidable sedimentation of suspended particles with a typical density of about 1.5 g / cm ^{3 in} a less dense oily vehicle. The patent recognizes this problem and attempts to solve it by reducing the particle size to less than 1 μm in diameter in order to keep them suspended by thermodynamic forces such as Brownian motion. The requirement that all particles be 1 μm or less in diameter is a disadvantage of the proposed formulation. Obtaining such small particle powders is not an easy task. This can be achieved with an improved spray dryer design, but small particle sizes hinder the use of cyclone-type collectors and require a system of product recovery filters.

  Reducing the particles to sub-micron size can also be achieved theoretically, after the particles have been suspended in the oil with a high pressure microhomogenizer such as a Microfluidizer (Constant Systems Inc.). This adds an extra step to the process and we have found that it is not very efficient in breaking up spray-dried sugar glass microspheres that have very high mechanical strength due to the sphere. This forces multiple passes through the device. Nevertheless, this tends to leave many large particles untouched, thus requiring a later filtration or sedimentation step to remove them. Moreover, the high viscosity of the suspensions in normal oily vehicles makes it difficult to draw them into syringes and requires them to be injected slowly. It prevents fast flow through narrow nozzles, as found in liquid jet injector systems.

  It has also been found that particles suspended in oils make it difficult to extract them later into an aqueous environment, especially when they contain low HLB surfactants, because, unexpectedly, aqueous buffers Even after washing, they maintain a tightly bound oil waterproof coat around them. Therefore, they require very vigorous shaking and mixing or addition of a more water-soluble detergent (here high HLB) for the particles to leave the oil phase and enter the aqueous phase. This becomes a major problem as the particle size decreases. The end result is often an opaquely mixed emulsion, rather than two distinct phases. Within the body, this problem can result in a slow and unpredictable release of the active agent, rather than the required quick and predictable delivery. In vitro extraction into an aqueous environment causes the oil to float above the aqueous phase containing the dissolved active. This is unacceptable for certain in vitro applications such as diagnostic kits or automated assay systems. Finally, most of the clinically available natural FDA approved oils are susceptible to photolysis, oxidation or other forms of damage and require careful storage in the dark at relatively low temperatures. In addition, since they are not completely chemically inert, they can react slowly with suspended particles.

  Alliance Pharmaceutical Company considers the use of powders of water-soluble substances in a remarkable new non-aqueous perfluorocarbon liquid (Kirkland WD, Composition and method for delivering active agents) .US Patent 5,770,181 (1995)). This patent relates primarily to the function of PFC as an oral contrast enhancer for diagnostic imaging of the intestine. The exemplified water-soluble powders were added to improve the mouthfeel of the PFC or to enhance the contrast effect in the gastrointestinal tract. However, although not shown, Kirkland recognized that these liquids could also be used for drug delivery. In particular, only storage-stable commercial powders are exemplified in that patent. We have now discovered that brittle actives stabilized with sugar glass microspheres can be engineered to yield highly stable two-phase PFC liquid formulations for both oral and parenteral delivery. This broadly extends the utility of the Kirkland patent to the delivery of parenteral drugs and vaccines as ready-to-injectable formulations without the need for any kind of cooling. The finding that the low viscosity, high density and low surface tension of PFCs means that these stable suspensions are delivered by automated equipment such as liquid jet injectors is particularly beneficial. This opens up two important additional areas to this technology: large-scale immunization campaigns and self-injection.

  Perfluorocarbons (PFCs) are new, extremely stable liquids produced by the perfluorination of certain organic compounds. Because they are essentially immiscible with both oil and water or other solvents, either polar or non-polar (except for other PFCs), they are classified as either hydrophilic or lipophilic. (Reviewed in Krafft MP & Riess JG. “Highly fluorinated amphiphiles and colloidal systems, and their applications in the biomedical field.A contribution.” Use. Contribution) Biochimie, 80, 489-514, 1998). Furthermore, they do not participate in hydrophobic interactions with oils or in hydrophilic interactions with water or hydrophilic substances. As a result, the large phase separation seen when the hydrophilic particles aggregate together strongly in the oil is less likely to occur with PFC. They do not require surfactants to form stable suspensions, but fluorohydrocarbon (FHC) surfactants are available (Krafft & Riess, 1998) and at very low concentrations in PFC liquids. Active. At these very low concentrations, FHC surfactants can ensure a complete monodisperse of certain particles that tend to agglomerate in their absence. The PFC liquid itself is completely non-reactive chemically, does not accumulate in the body in low molecular weight types, is volatile, and is eventually exhaled with breath.

Because they are good solvents for gases, PFCs have already been used in large quantities in very special clinical applications. Their ability to exchange carbon dioxide for dissolved oxygen is better than for hemoglobin. This was first demonstrated in 1968 by RPGeyer in "bloodless rats" (Geyer RP, Monroe RG & Taylor K. "Survival of rats totally perfused with perfluorocarbon-detergent preparation"). Survival of perfused rats) Organ Perfusion and Preservation, JV Norman, J Folkman, LE Hardison, LERidolf and FJVeith eds. Appleton-Century-Crofts, New York, 85-95 (1968)). Perfluorooctyl bromide, in the form of a PFC-in-water emulsion, is currently being evaluated in humans as a substitute for certain surgical transfusions under the trade name Oxygent ^™ (Alliance Pharmaceutical Corp.). PFC has also been used as a liquid by inhalation into the lungs for the treatment of respiratory distress syndrome in premature babies.

Their high density, along with chemical inertness, has also proven valuable. Perfluorophenanthrene, trade name Vitreon ^™ (Vitrophage Inc.), is used to prevent eye capsule collapse during surgery and to repair detached retinas. PFCs are also used as contrast agents for magnetic resonance imaging (MRI), for which purpose hydrophilic powders are suspended in them to improve their imageability or make them more palatable. (Kirkland WD "Composition and method for delivering active agents" US Patent 5,770,181 (1998)). This patent also suggests the use of PFC as a continuous phase for delivery of particulate water-soluble drugs. Due to the limited number of parenteral drugs that are stable as dry powders at room temperature, this patent has no applicability to most injectable drugs. However, the combination of drug stability in sugar glass microsphere powder and injectable PFC as described in Roser and Garcia de Castro (1998) applies this technology to virtually all parenteral drugs and vaccines. Can.

Summary of the invention

The present invention uses a two-phase system comprising PFC as a continuous phase containing a discontinuous glass phase in suspension as a drug delivery formulation. Can be mixed with different PFC in order to obtain a final mixture density of about 1.5~2.5g / cm ^3, the great advantage that perfluorocarbon-based formulations exhibit. Thus, the particles may be formulated at a density consistent with the suspending fluid such that the particles remain in a stable suspension without floating or sinking to the bottom of the container. Thus, the particles need not be of submicron size as required in oily formulations to prevent sedimentation, and can vary greatly in size. The ultimate particle size is determined solely by the purpose of the formulation. Formulations for needle or jet injection contain particles in the range of 0.1 to 100 micrometers, preferably 1 to 10 micrometers. This greatly simplifies the process of producing the particles and avoids the need for very small particle sizes due to grinding. The particles may be made by conventional spray-drying or freeze-drying, followed by simple dry or wet milling. When high solids are required in the suspension, the particles are preferably spherical in shape. Irregularly shaped particles have a fairly large tendency to "bind" together to impede free flowing, whereas spherical particles have an inherent "smoothness" and thus have a solids content well over 20%. Can be reached. Such particles are easily made by spray drying, spray freeze drying or emulsion solidification.

  The suspension powder, if formulated properly, does not require a surfactant and forms a stable suspension in which the sugar glass particles dissolve almost immediately when shaken with water. If small agglomeration appears to be a problem, small amounts of FHC surfactants as described in Kraft and Riess (1998) can be advantageously added to the PFC solution before or after mixing the stable powder. Like PFCs, these FHCs are inherently very inert and non-reactive. Therefore, there is no solvation of the particles and there is no chemical reaction between the suspended particles and the PFC phase. Since both the sugar glass particles and the PFC liquid are environmentally stable, they are not decomposed by light, high temperature, oxygen or the like. They have only negligible in vivo or in vitro toxicity, and have been extensively tested and approved by formal agencies on large scale infusions into both animals and humans for blood exchange purposes. Although high molecular weight PFCs have been reported to accumulate in the liver, the low molecular weight example used in this application is exhaled and eventually excreted from the body.

  Thanks to their low surface tension and their low viscosity, they can flow very easily from the small holes found in hypodermic needles, automatic systems or liquid jet injectors. PFC is an excellent electrical insulator, so it is easy to obtain a monodispersed suspension of particles with similar small surface electrostatic charges. They are dry, completely non-hygroscopic liquids. Thanks to these very low water contents, the dryness of the suspended powder can be maintained, thus preventing dissolution or decomposition of the formulated active agent. Their unique lack of solubility makes them ideal for suspending hydrophilic or hydrophobic particles, and the final suspension is compatible with virtually any substance used in containers or delivery devices Means This is in contrast to oily formulations, for example, which can cause severe clogging of the syringe by inflating the rubber seal with a plunger. PFCs are obtained in a range of densities, vapor pressures and volatility (Table I). Their high density allows them to sink in most conventional buffers, so that they can be easily separated from the product particles dissolved in the overlying aqueous phase. Thus, this facilitates their use for in vitro applications such as diagnostics.

Detailed description of the invention

Table I Properties of some PFCs Perfluoro-MW density Viscosity Surface tension Vapor pressure
(kg / L) (mPas) (mN / m) (mbar)
Hexane 338 1.682 0.656 11.1 294
-N-octane 438 1.75 1.27 16.98 52
Decalin 462 1.917 5.10 17.6 8.8
Phenanthrene 624 2.03 28.4 19 <1

  The use of PFC as a vehicle for the delivery of drugs or bioactive agents was already suggested in Kirkland (1995). This patent merely exemplifies a commercially available flavor or effervescent powder which is inherently stable. For stabilized bioactive agents such as vaccines or drugs, it did not include any examples. Furthermore, the possibility of making injectable (parenteral) formulations by using PFC as a suspending vehicle for the active agent particles is not considered therein. To obtain a stable formulation of an inherently brittle biomolecule with long shelf life using PFC as a non-aqueous vehicle, the particles are preferably formulated to contain a glass former that can stabilize the formulated active. This may be from a variety of sugars, including trehalose, lactitol, palatinit, etc., as described in PCT WO 91/18091, or more preferably, from other sugars, such as those described in UK patent application 98206899.9. It may be from an effective monosaccharide sugar alcohol or a glass former.

  It is advantageous to incorporate a density modifier into the particles so that the particles do not float in the high-density PFC phase. This can be a soluble salt, such as sodium or potassium chloride or sulfate, or more preferably, an insoluble material such as barium sulfate, calcium phosphate, titanium dioxide or aluminum hydroxide. Insoluble non-toxic substances are preferred since the release of large amounts of ionic salts in the body can cause significant local pain and irritation. The insoluble substance may in some cases be part of the active formulation as an adjuvant, for example in a vaccine formulation. The density modifier may be in a solid solution in the sugar glass particles or in an insoluble particulate material suspended in the sugar glass. When properly formulated, the sugar glass particles are approximately density-matched to the PFC liquid, are buoyant-neutral, do not float or settle, and remain a stable suspension without caking.

PFC liquid is 10 ¹³ ohm. A small surface charge of the suspended particles can have a significant effect on suspension stability, as it is a good electrical insulator with a typical resistivity of at least cm. In order to prevent agglomeration due to weak short-range forces in the suspended particles, they are preferably made to contain excipients such as lysine or aspartic acid which can impart a weak residual electrostatic charge to the dried particles. This prevents aggregation, as is the case with stable colloids, by ensuring charge repulsion of the particles. On the other hand, small amounts of FHC surfactants, such as perfluorodecanoic acid, may advantageously be dissolved in the PFC to obtain a dispersed, preferably monodispersed, suspension.

  These particles may be produced by several techniques, including air, spray, or lyophilization, but need not be particularly small, and may be a heterogeneous mixture with a size of 0.1-100 μm in diameter. For some applications, millimeter-sized particles are also suitable.

  The use of these stable suspensions is not limited to parenteral use as described above or to oral use as exemplified in Kirkland (1995). Because the PFC liquid vehicle is non-toxic and non-reactive, it is an ideal vehicle for mucous membranes, including pulmonary, nasal, ocular, rectal and vaginal delivery. The ability afforded by this patent to produce stable, sterile, non-irritating formulations, even for mucosal delivery of highly unstable drugs or vaccines, is a significant advance. Moreover, the very dry and completely non-hygroscopic nature of PFC liquids greatly aids in maintaining the sterility of these formulations during long-term storage and intermittent use, as microorganisms cannot grow in the absence of water.

  Because volatile perfluorohydrocarbons and chlorofluorocarbons have long been used as propellants in inhalers, which have been considered for delivering drugs to the deep lung, the stable PFC formulations described here have Ideal for generating fine mist of liquid STASIS droplets for intra-delivery. For this application, the size of the particles that make up the discontinuous suspension phase with the PFC droplets is important and should not exceed a diameter of 1-5 μm, preferably 0.1-1 μm. For delivery to other mucosal surfaces by nose or eye, the particle size is not critical and may be within 100 μm in diameter or several mm.

Description of the drawings
FIG.
Alkaline phosphatase (Sigma Aldrich Ltd.) was stabilized in a glass based on 33.3% mannitol, 33.3% calcium lactate and 33.3% degraded gelatin (Byco C, Croda Colloids Ltd.) Spray dried as spheres and stored at 55 ° C. as a dry powder or as a stable suspension in perfluorodecalin. Activity remained at approximately 100% standard (103% at 20 days and 94% at 30 days). The dry powder not suspended in the PFC had a greater loss (about 80% residual activity).
FIG.
A commercially available tetanus toxoid vaccine (# T022, kindly provided by Evans Medeva plc) was formulated as a density-matched powder using calcium phosphate in a 20% trehalose solution. It was lyophilized by spraying into liquid nitrogen using a two-part nozzle, and then the frozen microsphere powder was lyophilized on a Labconco lyophilizer at an initial storage temperature of -40 ° C throughout the main drying. After injection with the same dose of ASSIST stabilized tetanus toxoid vaccine, reconstituted in saline buffer or as an anhydrous formulation in oil or PFC, the antibody response of six groups of ten guinea pigs was determined at weeks 4, 8, and 12. It was measured.
The response to all dried formulations was lower than the fresh vaccine control (not shown) and showed a significant loss of immunogenicity upon spray drying. The antigenicity of the toxoid, measured by a capture ELISA, was unchanged by the drying process. This indicated that further work was required to complete the storage of the aluminum hydroxide adjuvant upon drying.
The response to the calcium phosphate-matched STASIS vaccine (Group 3) is essentially identical to the control vaccine (Group 1) and the powder-in-oil vaccine (Group 2) reconstituted in aqueous buffer, while Control animals (Groups 4 & 5) injected with aqueous vehicle only showed no response.

Description of the preferred embodiment
Example 1 :
Spray-dried particles in PFC
Particles were prepared by spray drying from an aqueous solution with sugar and other excipients using a Labplant model SD1 spray dryer. A typical formulation was as follows:
A. Underwater mannitol 15% w / v
Calcium lactate 15% w / v
B. 15% w / v trehalose in water
Calcium phosphate 15% w / v

  Particles were produced using a two-liquid nozzle with a liquid orifice having an inner diameter of 0.5 mm. The half-maximum nozzle airflow was found to be best and the drying chamber was operated at 135 ° C inlet temperature and 70-75 ° C outlet temperature. The particles were collected in a glass cyclone and subjected to secondary drying in a vacuum oven using a temperature ramp to 80 ° C over 4 hours. After cooling, they were suspended in PFC using ultrasound. A 30 second burst of ultrasonic energy from a titanium probe in a MSE MK2 ultrasonic cabinet operating at about 75% power, or immersion in a Decon FS200 Frequency sweep ultrasonic bath within 10 seconds, has proven satisfactory.

  The resulting suspension was monodisperse and, when viewed microscopically, consisted of spherical glass particles with an average size of about 10μ and a range of about 0.5-30μ. The mannitol / calcium lactate particles rose over the PFC layer in a few minutes, but could be easily resuspended with gentle shaking. The trehalose / calcium phosphate particles were nearly density-matched to the PFC and formed a stable suspension.

  The spray dried powder of sugar glass particles was suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene at 1, 10, 20 and 40% w / v. They were found to produce monodisperse suspensions with little tendency to agglomerate. Addition of 0.1% perfluorodecanoic acid to the PFC prevented a slight tendency to aggregate on the surface. These suspensions were found to easily pass through a 25 g needle by suction or injection.

Example 2 :
Stable alkaline phosphatase (Sigma Aldrich Ltd.) of a suspension of glass stabilizing enzyme in PFC was spray-dried on a Labplant as described above. The formulation contained 33.3% w / w mannitol, 33.3% w / w calcium phosphate and 33.3% degraded gelatin (Byco C, Croda Colloids Ltd.). The dried enzyme was stored at 55 ° C. as a dry powder or suspension in perfluorodecalin. Enzymes formulated with these microspheres, consisting of mannitol-based glass suspended in perfluorodecalin, show nearly 100% residual enzyme activity over 55 days at 55 ° C. (FIG. 1).

Example 3 :
The pre-clinical studies of similar formulation containing the in vivo efficacy clinical tetanus toxoid vaccine (which is kindly granted by Medeva plc), was carried out in collaboration with the National Institute of Biological standards and Control (approval Institute of World Health Organization). The results of this study showed that the stable STASIS formulation was completely equivalent to the aqueous liquid vaccine in its ability to immunize guinea pigs and develop a protective serum antibody response (FIG. 2). Thus, with in vivo bioavailability similar to conventional aqueous liquid formulations, it was demonstrated that the suspension in PFC was immediately forming an injectable formulation.

Example 4 :
Spray freeze-dried particles Particles were also produced by spraying liquid droplets into liquid nitrogen and then vacuum drying the frozen powder. These particles were of lower density than the spray-dried powder and formed a paste in PFC at concentrations higher than 20% w / v. At lower concentrations, they formed a monodispersed suspension after sonication.
The typical formulation used was as follows:
Substance final concentration w / w
A. Trehalose 100%
B. Trehalose 50%
49.5% of calcium phosphate
Aluminum hydroxide 0.5%

Example 5 :
Milled Hydrophobic Particles The hydrophobic sugar derivatives sucrose octaacetate and trehalose octaacetate immediately form a glass when quenched from the melt or quickly dried from a solution of chloroform or dichloromethane. Their use has been described as controlled release matrices for drug delivery (Roser et al "Solid delivery systems for controlled release of molecules incorporated therein and methods of making same"). And its production method) PCT publication WO 96/03978, 1994).

  Trehalose octaacetate powder was obtained by melting in a muffle furnace and quenching the melt on a stainless steel plate. The obtained glass disk was crushed with a pestle and a mortar and then with a high-speed homogenizer to obtain a fine powder. It was suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene at 1 and 10% w / v. They have been found to produce well dispersed suspensions. These suspensions were found to pass easily through a 23 g needle.

Example 6 :
Due to the properties of reconstituted stable sugar glass particles and the properties of PFCs in aqueous environments, it was expected that the active agents in these suspensions would be released rapidly in the body. To demonstrate complete release of the active substance contained, particles containing the following were formulated:
Trehalose 20% w / v
Calcium lactate 20% w / v
Lysine 0.5% w / v
Mordant Blue 9 Dye 1% w / v

  The formulation was spray dried as described above and added to perfluorophenanthrene and perfluorodecalin to prepare a 20% w / v dark blue opaque suspension. When an equal volume of water is added to the suspension and shaken, virtually all of the blue dye is released into the aqueous phase, with a clear blue interface floating on a nearly colorless PFC at a well-defined interface. It was found to form a layer.

Example 7 :
Unreacted among the particles in the suspension Because the individual microspheres are physically separated from all other particles in the PFC suspension, potentially reactive substances may be exposed to any risk that they may interact. And may be present together as separate particles in the same suspension. When the sugar glass dissolves and the molecules come together, a reaction occurs.
To prove this, a suspension containing two types of particles was prepared, (a) one with alkaline phosphatase enzyme and (b) the other with its colorless substrate, p-nitrophenyl phosphate.

The prescription was as follows:
a) Trehalose 10% w / v in 5 mM Tris / HCl buffer pH 7.6
Sodium sulfate 10% w / v
Alkaline phosphatase 20U / ml
b) Trehalose 10% w / v in 100 mM glycine buffer, pH 10.2, each containing 1 mM Zn ⁺⁺ and Mg ^++.
Sodium sulfate 10% w / v
p-Nitrophenyl phosphate 0.44% w / v

A suspension of the powder in perfluorodecalin containing 10% w / v powder "a" and 10% w / v powder "b" did not produce any color reaction and was a white suspension at 37 ° C for 3 weeks. It turned out to be something.
After addition of water and shaking, the powder dissolved in the upper aqueous phase. The enzymatic reaction occurred in a few minutes and exhibited the dark yellow color of p-nitrophenol in both freshly prepared samples and those kept at 37 ° C. for 3 weeks.

Example 8 :
Product Release in the "Tissue Gap" Model To demonstrate the kinetics that can occur with PFC suspensions when injected in vivo, place 0.2% agarose gel in a polystyrene decorative bottle by adding clear water. A model of Japanese tissue gap was prepared. 0.1 ml of the perfluorodecalin suspension of Example 5 was injected into an agarose gel through a 25 g needle. This resulted in flattened white spheres of the suspension. Over the next 5 to 10 minutes, the white color disappeared upward from the bottom of the sphere, leaving a clear sphere of PFC. As the enzyme and substrate were released by dissolution of the glass particles, they reacted together to produce the yellow color of p-nitrophenol, which then diffused throughout the agarose over an hour.

Example 9 :
Density match:
Sugar glass particles (ie, trehalose) obtained by any of the conventional drying methods exhibit a typical density of about 1.5 g / cm ³ . Perfluorocarbon we tested typically have densities of 1.68~2.03g / cm ³ (Table I). For this reason, when formulated in suspension, the sugar glass particles tend to float in the PFC layer, resulting in a formulation in which the active agent is not evenly distributed. However, they may be modified such that the powders have a neutral buoyancy and do not settle or float, resulting in a stable suspension in the PFC. This can be achieved by the addition of a high density material prior to particle formation. These may be water-soluble or insoluble.

Water-insoluble substance orthophosphate tribasic calcium has a density of 3.14 g / cm ^3, is approved as an adjuvant for vaccines, insoluble in actual tap water. Powders prepared to contain about 50% calcium phosphate show an increased density of about 2 g / cm ³ and form a stable suspension in perfluorophenanthrene at 20% solids.

Examples of powders that form stable suspensions at 20% solids in PFC include:
In 1 perfluorodecalin
Substance final concentration w / w
A. Trehalose 50%
50% calcium phosphate
B. Trehalose 47.5%
Calcium lactate 10.0%
Calcium phosphate 42.5%

2 In perfluorophenanthrene
Substance final concentration w / w
Mannitol 18.2%
Inositol 18.2%
Calcium lactate 18.2%
Calcium phosphate 45.4%

  Other density increasing water insoluble materials that have been used include barium sulfate and titanium dioxide. Any non-toxic, insoluble material can be used, provided that it is of the appropriate density.

Soluble salts such as sodium sulphate with a water-soluble density of 2.7 g / cm ³ may also be used as density- enhancing agents . The following powder formed a stable suspension in perfluorodecalin:
Substance final concentration w / w
Trehalose 50%
Sodium sulfate 50%

Other non-toxic high-density water-soluble substances may also be used. These formulations were found to cause discomfort after subcutaneous injection in guinea pigs, presumably due to the rapid dissolution of high concentrations of ionic salts.

Example 10 :
Vaccines with active agent in suspension and density matching are formulated adsorbed on insoluble gels or particles that act as adjuvants. Aluminum hydroxide and calcium phosphate are widely used for this purpose. These insoluble adjuvants can themselves be used to increase the density of the suspended particles. In this case, the high density material is not completely inert and effectively adsorbs the active polymer from solution. It is necessary to prove that this adsorption does not denature the activator. To test this, alkaline phosphatase was used as an activator / vaccine model.

The following solutions were prepared:
Adjuvant grade calcium phosphate 10% w / v (Superphos Kemi a / s) in 5 mM Tris HCl buffer pH 7.6
Trehalose 10% w / v
ZnCl ₂ 1 mM
MgCl ₂ 1 mM
Alkaline phosphatase 20U / ml

  The solution was mixed well at 37 ° C. for 10 minutes, and the alkaline phosphatase was adsorbed by calcium phosphate. This change in absorption per minute was determined by centrifuging the calcium phosphate, sampling the supernatant, and measuring its enzymatic reaction rate at a wavelength of 405 nm using p-nitrophenyl phosphate as a substrate. The solution was spray dried to produce a fine powder. Enzyme desorption was measured in the supernatant as described above after rehydration of the powder. The powder was found to form a stable suspension when suspended at 20% w / v in perfluorophenanthrene.

Test sample d ^absorbance / min (405nm)
Stock solution (25 μL) 0.409
The above supernatant (25 μL) 0.034
Rehydrated powder (25 μL of 20% w / v in water) 0.425
The above supernatant (25 μL) 0.004
20% w / v powder in perfluorodecalin (25 μL) 0.430

Experiments have proven that:
The density of the particles can be matched with that of a PFC vehicle by the inclusion of a calcium phosphate adjuvant.
No significant desorption or loss of enzyme activity occurred during the formulation process.

Example 11 :
A STASIS formulation of mannitol-based glass as in Example 1 is suspended in perfluorodecalin and used in a clinically clean, surgically clean, routinely used to deliver oxymetazoline nasal decongestants (Sudafed, Warner Lambert). , Pumped into a polypropylene atomizer. The suspension was sprayed twice into each nostril of human volunteers and asked about the degree of discomfort they felt. The volunteers did not complain at all. No side effects of administration were observed.

Alkaline phosphatase (Sigma Aldrich Ltd.) was stabilized in a glass based on 33.3% mannitol, 33.3% calcium lactate and 33.3% degraded gelatin (Byco C, Croda Colloids Ltd.) Spray dried as spheres and stored at 55 ° C. as a dry powder or as a stable suspension in perfluorodecalin. Activity remained at approximately 100% standard (103% at 20 days and 94% at 30 days). The dry powder not suspended in the PFC had a greater loss (about 80% residual activity). A commercially available tetanus toxoid vaccine (# T022, kindly provided by Evans Medeva plc) was formulated as a density-matched powder using calcium phosphate in a 20% trehalose solution. It was lyophilized by spraying into liquid nitrogen using a two-part nozzle, and then the frozen microsphere powder was lyophilized on a Labconco lyophilizer at an initial storage temperature of -40 ° C throughout the main drying. After injection with the same dose of ASSIST stabilized tetanus toxoid vaccine, reconstituted in saline buffer or as an anhydrous formulation in oil or PFC, the antibody response of six groups of ten guinea pigs was determined at weeks 4, 8, and 12. It was measured. The response to all dried formulations was lower than the fresh vaccine control (not shown) and showed a significant loss of immunogenicity upon spray drying. The antigenicity of the toxoid, measured by a capture ELISA, was unchanged by the drying process. This indicated that further work was required to complete the storage of the aluminum hydroxide adjuvant upon drying. The response to the calcium phosphate-matched STASIS vaccine (Group 3) is essentially identical to the control vaccine (Group 1) and the powder-in-oil vaccine (Group 2) reconstituted in aqueous buffer, while Control animals (Groups 4 & 5) injected with aqueous vehicle only showed no response.

Claims (18)

A composition for delivering a stable bioactive compound to a subject, comprising a first component and a second component,
The first component comprises the above-mentioned bioactive agent-containing fine particles of sugar glass, metal carboxylate glass or phosphate glass, and the sugar glass, metal carboxylate glass or phosphate glass optionally promotes glass formation. Agent compound,
The second component comprises at least one biocompatible liquid perfluorocarbon in which the first component is insoluble and dispersed, the liquid perfluorocarbon optionally containing a surfactant. And the above composition.   The sugar glass comprises trehalose, sucrose, raffinose, stachyose, glucopyranosyl sorbitol, glucopyranosyl mannitol, palatinit, lactitol, a sugar selected from the group consisting of monosaccharide alcohols, or sucrose octaacetate or trehalose octaacetate The composition of claim 1, wherein the composition is formed from a sugar molecule modified by the addition of a hydrophobic side chain selected from the group.   The composition of claim 1, wherein the phosphate glass or carboxylate glass is formed from a mixture of divalent metal phosphate or metal carboxylate.   The composition of claim 1, wherein the optional glass formation promoter is selected from the group consisting of peptides, proteins, dextran, polyvinylpyrrolidone, borate, calcium lactate, sodium polyphosphate, and silicic acid or acetate.   The composition of claim 1, wherein the sugar glass microparticles have a diameter of about 0.1 to about 100 micrometers.   The composition according to claim 5, wherein the diameter ranges from 1 to 10 micrometers.   Composition according to claim 1, wherein the microparticles have a water content of less than 4%, preferably less than 2%, ideally less than 1%.   The composition of claim 1, wherein the first component is monodispersed in the second component.   The composition of claim 1, wherein the concentration of the first component in the second component is from about 1 to about 40% by weight.   10. The composition according to claim 9, wherein the concentration range is from 10 to 15%.   The composition of claim 1, wherein the concentration of the surfactant in the second component is from about 0.01 to about 10%.   The composition of claim 11, wherein the concentration is about 1%.   The composition of claim 1, further comprising microparticles of the first component, wherein the inorganic salt is added in an amount effective to provide the microparticles with a density consistent with that of the liquid phase of the second component. .   2. The composition of claim 1, further comprising an excipient that imparts a weak residual electrostatic charge to the microparticles such that the microparticles repel each other.   15. The composition according to claim 14, wherein the excipient is an amino acid.   The composition of claim 1, wherein the perfluorocarbon is selected from the group consisting of perfluorohexane, perfluorodecalin, and perfluorophenanthrene.   The composition according to claim 1, wherein the bioactive compound is a vaccine, drug, enzyme or diagnostic reagent. (A) producing fine particles of the first component according to claim 1;
(B) suspending the microparticles in the second component perfluorocarbon of claim 1; and (c) administering the mixture to a non-human subject. For delivering a biologically active substance to an individual.

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