RU2259817C2 - Composition for delivering stable injectable liquids and method for delivery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the present innovation deals with delivering a stable biologically active compound for a subject including glass particles that contain the mentioned biologically active compound at their diameter ranged 0.1-100 mcm and suspended in liquid that includes, at least, one biocompatible perfluorocarbon where these mentioned particles are not dissolved. The innovation enables to create completely stable compositions being ready for injections.

EFFECT: higher efficiency.

11 cl, 2 dwg, 11 ex

Description

BACKGROUND OF THE INVENTION

Vaccines or ready-to-inject drugs in solution are, in fact, unstable and need to be cooled. The pharmaceutical industry typically solves problems regarding drug instability by drying drugs by lyophilization. This method is expensive, inconvenient and essentially dangerous, since reconstitution of dried drugs can lead to erroneous doses or to contamination of solutions. Over the past 100 years, attempts have been made to develop reliable, stable, ready-to-inject liquid compositions, but the efforts have not been successful. In fact, only low molecular weight drugs can exist in aqueous solutions for a suitable shelf life.

This problem is particularly acute in the field of vaccine production. It has been estimated that 3.6 billion vaccine doses should be administered in this world by 2005. The World Health Organization (WHO) has stated that standard vaccine forms that need to be cooled all the time cannot be put into practice ("Revolutionizing Immunizations." Jodar L., Aguado T., Lloyd J. and Lambert PH. Genetic Engineering News Feb. 15.1998). Currently, in developed countries, a network of refrigerators is used, forming a "cooling chain", which extends from vaccine-producing enterprises to provincial cities. The cost of the cooling chain for the vaccine industry and nongovernmental health organizations conducting public immunization campaigns is huge. WHO has found that the true cost of the maintenance of the cooling chain is over $ 200 million annually. In addition, immunization campaigns can only be carried out for people who are close to the last link of the cooling chain.

Vaccination campaigns for the population require trained medical personnel to ensure that the dose is administered correctly and that it is not destroyed. The need to reconstitute certain vaccines, such as measles, yellow fever and BCG, is also a serious problem in this area. The problem should be solved very clearly in order to ensure the introduction of the correct dosage and, in addition, not to introduce a possible source of contamination, which often leads to adverse clinical consequences. In addition, more than one vaccine is often required during a campaign and may require multiple injections, as individual mixtures or “multivalent” vaccines may not be available due to the chemical incompatibility of some of the components. WHO has highlighted the solution of these problems with the active support of research aimed at creating stable next-generation vaccines that do not require refrigeration ("Pre-Filled Monodose Injection Devices: A safety standard for new vaccines, or a revolution in the delivery of immunizations ? "Lloyd J. and Aguado MT WHO publication. May. 1998." General policy issues: injectable solid vaccines: a role in future immunization? "Aguado MT, Jodar L., Lloyd J., Lambert PH WHO publication No A59781).

The ideal solution to this problem is to create completely stable, ready-to-inject compositions. Such stable vaccines can be packaged in administration devices or, for mass immunization campaigns, can be transported in large volumes and administered via a needleless injector. Transdermal delivery of dry, particulate matter using a needleless gas injection system has been described in (Sarphie DF, Burkoth TL. Method for providing dense particle compositions for use in transdermal particle delivery. International publication PCT Pub No. WO 9748485 (1996)) and transdermal vaccination with dry DNA vaccines is apparently very effective ("PowderJect ^'s Hepatitis B DNA Vaccine First To Successfully Elicit Protective Immune Response In Humans" at http://www.powderject.com/pressrelases.htm (1998)).

The supersonic shock wave of helium gas, which is used to bring such powder injectors into operation, has limited power and cannot deliver a dose of fine particles intramuscularly. This is due to the fact that low molecular weight particles cannot acquire the corresponding driving force for deep penetration. While intradermal delivery of DNA vaccines applied to colloidal gold particles is sufficient to achieve immunogenicity, general vaccines supplemented with insoluble aluminum or calcium salts cause unacceptable skin irritation. They should be administered intramuscularly. The administration of such vaccines requires a flexible system capable of providing a wide range of delivery depths, from intradermal to deep intramuscular, similar to the delivery system achieved with a needle and syringe. To conduct mass vaccination campaigns, the problem was solved by developing a liquid needleless injector capable of accelerating the flow of a narrow (~ 0.15 mm diameter) stream of liquid using pressures of about 3,000 psi, in the form of a "liquid nail". This device allows you to deliver a dose painlessly through the skin deep into the subcutaneous layer or into muscle tissue by punching a tiny hole through the epidermis. The large driving force imparted by the liquid stream provides deep penetration. Injectable drugs and vaccines have so far been based on water, but due to the instability problems discussed above, the range of water-stable products available for this technique is very limited.

It is now known that a wide range of bioactive molecules can be stabilized by drying in glassy sugar (Roser B. "Protection of proteins and the like" UK patent No. 2187191. Roser B. and Colaco C. "Stabilization of biological macromolecular substances and other organic compounds "PCT International Publication Pub No. WO 91/18091. Roser B. and Sen S." New stabilizing glasses ". PCT Patent Application No. 9805699.7. 1998). Such dry, stabilized active substances are not exposed to adverse environments such as high temperatures and ionizing radiation.

The mechanism underlying the significant stabilization of molecules with sugars is glass formation. Since the sugar solution containing the active molecule is dried, it can either crystallize when the sugar solubility limit is reached, or it can form a supersaturated syrup. The ability of sugar to inhibit crystallization is important for a good stabilizer. A suitable sugar for this is trehalose (Green JL. & Angel CA. Phase relations and vitrification in sacchsride water solutions and the trehalose anomaly J.Phys.Chem. 93, 2880-2882 (1989)), but it is not the only sugar. In addition, drying gradually leads to the hardening of the syrup, which reaches a glassy state with a low content of residual water. Unobtrusively active molecules pass from a liquid aqueous solution to a solid solution in dry glassy sugar. Chemical diffusion is negligible in glassy sugar and therefore the chemical reactions essentially cease. Since denaturation is a chemical change, it cannot occur in glassy sugar, and as a result, the molecules stabilize. In this form, the molecules can remain unchanged if another condition is met. This condition is the presence of the second important property of a good stabilizer, namely it must be inert and non-reactive. Many glassy substances cannot be obtained because they react with the product during storage. Obvious problems are encountered with reducing sugars, which can form glassy sugars with favorable physical properties, but then their aldehyde groups attack the amino groups on the products, carrying out a typical Maillard reaction. This fact is the main reason that many lyophilized pharmaceuticals require refrigerated storage. Non-reactive sugars produce stable foods that do not require cooling at all.

Biomolecules immobilized in glassy sugar are also stable in non-aqueous industrial solvents in which they alone, as well as sugar, do not dissolve (Cleland JL. And Jones AJS. "Excipient stabilization of polypeptides treated with organic solvents" US Pat. 5589167 (1994)). Since glassy sugar serves as an impenetrable barrier in a non-solvent liquid, biomolecules in solid solution in such sugar are protected both from the chemical reactivity of the solvent and from the environment. In the event that the liquid itself is stable, sensitive products in suspended glassy particles create a stable biphasic, liquid composition. Industrial solvents of the type described by Cleland and Jones (1994) have limited use in technology. Substitution of a biocompatible non-aqueous liquid should lead to the creation of stable liquid compositions consisting of even the most unstable drugs, vaccines and diagnostics.

The first production of stable non-aqueous fluids intended for use in drug and vaccine delivery (BJRoser and SDSen "Stable particle in liquid formulations". PCT patent application No. GB98 / 00817) related to compositions of powders of stabilizing glassy substances containing active a substance suspended in injectable oils, such as sesame, peanut or soybean oil, or in esters, such as ethyl oleate. Suspended particles of glassy sugar are extremely hydrophilic in nature, while oils are hydrophobic. Due to the strong tendency of the hydrophilic and hydrophobic phases to separate, glassy sugar particles tend to combine. In order to stabilize this type of water-in-oil suspension, the use of oil-soluble surfactants dissolved in a continuous oil phase is often required.

These low HLB surfactants (hydrophilic / lipophilic balance) are collected at the interface between the hydrophilic particles and the oil and coated with an amphiphilic layer, which is more compatible with the continuous oil phase. Since each particle of glassy sugar is separated from its neighbors by dry oil, no chemical interaction between the particles occurs. Therefore, there may be several different populations of particles, each containing an excellent potentially interacting molecule, in the same oil preparation without their interaction with each other. Complex multivalent vaccines can be obtained in the same way.

However, it was subsequently found that this method has certain disadvantages that interfere with the adoption of a universal solution. These disadvantages include the inevitable sedimentation of suspended particles, which have a density of about 1.5 g / cm ³ , in a less dense oily solvent. Applicants acknowledge the existence of such a problem and seek to solve it by reducing the particle size to a size below 1 μm in diameter so that the particles remain suspended by means of thermodynamic forces, such as Brownian motion. The requirement for particle size, which should be below 1 μm in diameter, is a disadvantage of the proposed compositions. The preparation of such powders with small particles is in no way an easy task. Improved models of the spray dryer make it possible to obtain such powders, however, the small particle size will impede the use of cyclone-type collectors, resulting in the need for a filter system to return the product.

Theoretically, particle size reduction to submicron can also be achieved after the particles are suspended in oil using a high pressure microhomogenization apparatus such as a microfluidizer (Constant Systems Inc.). Such a procedure involves the inclusion of an additional step in the process, and the applicants have found that it is not very effective for breaking the glassy sugar microspheres, spray dried, which have very high mechanical strength due to their spherical shape. It involves performing multiple passes through the apparatus. And even after such a larger procedure, the particles remain intact, and a subsequent filtration or sedimentation step is required to remove them. In addition, the high viscosity of the suspensions in conventional oily solvents makes it difficult to select them into the syringe, and they need to be introduced slowly. This circumstance prevents the rapid passage of flows through thin holes, such as those that occur in a liquid needleless injector system.

In addition, it was found that particles suspended in oil, especially those containing a low HLB surfactant, are subsequently difficult to extract into the aqueous medium because, surprisingly, they retain a firmly bound, water-repellent coating of oil around the particles, even after washing in water buffer. In order for the particles to leave the oil phase and go into the aqueous phase, very vigorous shaking and mixing or the addition of a more water-soluble detergent (this time with a high HLB) is required. Such a procedure can create big problems because the particle size is reduced. The end result is a contaminated mixed emulsion rather than two pure separated phases. In the body, such an emulsion can contribute to a slow and unpredictable release of the active substance rather than the required fast and predictable delivery. In vitro extraction into the aqueous medium leads to the spread of oil at the top of the aqueous phase containing the dissolved active substance. This result may not be acceptable for some in vitro uses, such as diagnostic kits or automatic analysis systems. In the end, most natural, FDA-approved oils that can be clinically used are susceptible to photodegradation, oxidation or other forms of degradation and require careful storage in the dark at relatively low temperatures. In addition, they are not completely chemically inert, so that they can slowly interact with suspended particles.

The United Pharmaceutical Company has investigated the use of water-soluble powder in amazing new, non-aqueous perfluorocarbon fluids (Kirkland WD Composition and method for delivering active agents, US Patent No. 5770181 (1995)). This patent mainly relates to the function of PFC as oral, contrast enhancing agents for diagnostic imaging of the intestine. The water-soluble powders used in this case were added in order to improve appetite or enhance the effect of PFC contrast in the gastrointestinal tract. However, Kirkland suggested that these fluids could also be used for drug delivery, although no examples were given in the patent. In particular, the patent discloses only the shelf life of commercially available powders. BACKGROUND OF THE INVENTION Applicants have now discovered that, in order to obtain extremely stable biphasic PFC liquid compositions for both oral and parenteral delivery, brittle active particles stabilized in glassy sugar microspheres can be obtained. This circumstance greatly expands the possibilities of applying the Kirkland patent to the delivery of parenteral drugs and vaccines in the form of ready-to-administer compositions for which cooling of any type is not required. Of particular importance is the discovery that qualities such as low viscosity, high density, and low surface tension of PFCs help these stable suspensions to be delivered using automatic devices such as liquid needleless injectors. This fact leads to the appearance of two possible additional fields of application besides the indicated one, namely, the campaign on mass immunization and also self-administration.

Perfluorocarbons (PFCs) are new, extremely stable fluids obtained by exhaustively fluorinating certain organic compounds. They cannot be classified as hydrophilic or hydrophobic, since they are in fact not miscible with oil, nor with water or any other solvent, whether it is polar or non-polar, excluding other PFCs (Rewiewed in Krafft MP & Riess JG. " Highly fluorinated amphiphiles and colloidal systems, and their applications in the biomedical field. A contribution. "Biochimie, 80, 489-514, 1998). In addition, they do not participate either in hydrophobic interactions with oils, or in hydrophilic interactions with water or hydrophilic materials. As a result, it is seen that coarse phase separation, in which hydrophilic particles are firmly assembled together in oil, does not occur in PFC. Surfactants are not required to obtain stable suspensions, but fluorocarbon (FHC) surfactants are available (Krafft & Riess, 1998) and are active at very low concentrations in PFC fluids. At such very low concentrations of FHC, surfactants contribute to the formation of true monodisperse systems of certain particles, which tend to aggregate in their absence. PFC fluids themselves are completely chemically non-reactive, and the types of such fluids with low molecular weight do not accumulate in the body, but, being volatile, eventually evaporate when breathing.

Since they are excellent solvents for gases, PFC has already been used in large quantities in very special cases of clinical use. Their ability to exchange carbon dioxide for dissolved oxygen was better than the ability of hemoglobin. The results were first demonstrated on “bloodless rats” by the RPGeyer researcher in 1968 (Geyer RP, Monroe RG & Taylor K. "Survival of rats totally perfused with perfluorocarbon-detergent preparation." In: Organ Perfusion and Preservation, JVNorman, J. Folkman, LEHardison , LERiodolf and FJVeith eds. Appleton-Century-Crofts, New York. 85-95 (1968)). Currently, perfluorooctyl bromide, in the form of a PFC emulsion in water and under the trade name Oxygent ^TM (Alliance Pharmaceutical Corp.), is used instead of blood for transfusion in certain surgical procedures. In addition, PFCs are used as liquids for inhalation into the lungs for the treatment of respiratory distress syndrome in premature infants.

It was found that their high density in combination with chemical inertness is a very valuable quality. Perftorfenantren under the tradename Vitreon ^TM (Vitrophage Inc.) is used to prevent collapse of the capsule in eye surgery for detached and implement permutation retina. In addition, PFCs are used as contrast media for nuclear magnetic resonance imaging (MRI) imaging, and to this end, researchers have shown that hydrophilic powders can be suspended in them to improve visualization or make them more pleasant (Kirkland WD "Composition and method for delivering active agents" (U.S. Patent No. 5770181, 1998). This patent also contemplates using PFC as a continuous phase for the delivery of bulk water-soluble drugs. Since the number of parenteral drugs that are stable as dry powders at room temperature is limited, this patent does not apply to most injectable drugs. However, the use of drug stabilization in glassy sugar powders in the form of microspheres, as described by Roser and Garcia de Castro (1998), as well as injectable PFCs, makes this method applicable to virtually all parenteral drugs and vaccines.

The invention relates to a composition for delivering a stable biologically active compound to a subject, comprising glass particles containing said biologically active compound and characterized in that the particles are suspended in a liquid that includes at least one perfluorocarbon in which said particles are not soluble.

The glass preferably includes sugar glass, metal carboxylate glass and / or phosphate glass.

Thus, the invention provides, as a composition for drug delivery, a two-phase system with perfluorocarbons as a continuous phase containing a discrete glass phase in suspension, as drugs for drug delivery.

SUMMARY OF THE INVENTION

The present invention includes the use of a two-phase system with PFC in the form of a continuous phase containing discrete phases of a glassy substance in suspension, as drugs for drug delivery. Perfluorocarbon-based preparations have the main advantages that various PFCs can be mixed to form final mixtures with densities ranging from about 1.5 to 2.5 g / cm ³ . This approach makes it possible to obtain particles with densities corresponding to the suspension liquid, so that these particles do not float to the surface or fall to the bottom of the container, but remain in the form of a stable suspension. Therefore, the particles should not be submicron in size, as required for oil-based preparations, to prevent sedimentation, but they can vary significantly in size. The maximum particle size is determined only by what is used or needleless injection, may contain particles with a size in the range from 0.1 to 100 micrometers, or preferably from 1 to 10 micrometers. This approach allows to greatly simplify the method of particle production and to avoid the need to obtain particles with an extremely small size by grinding. Particles can be obtained by conventional spray drying or lyophilization drying followed by simple drying or wet grinding. In the case where a high solids content is required in the suspension, it is desirable that the particles are spherical in shape. Irregular particles exhibit a much higher tendency to “bond” together, inhibiting free flow, while spherical particles have their own “lubricating ability”, which makes it possible to achieve a solids content of over 20%. Preferably, the solids content is from 1% to 40%, or more preferably from 10% to 15%. Such particles are readily obtained by spray drying, freeze drying and hardening of the emulsion. Suspended powders, if prepared appropriately, do not require surfactants to form stable suspensions, from which sugar glass particles dissolve almost instantly when shaken with water. If minor aggregation is seen as a problem, then a small amount of FHC surfactant, as described by Krafft and Riess (1998), can be added to PFC fluids before or after the addition of stable powder. In cases where a surfactant is used, it is preferably contained in a concentration of from 0.01 mass% to 10 mass%, optimally about 1% is possible. Like PFCs, these FHCs are, in fact, extremely inert and non-reactive. Thus, solvation of particles and a chemical reaction between the suspended particles and the PFC phase does not occur. Since particles of a suitable glass and PFC liquid are environmentally stable, degradation due to lighting, high temperatures, oxygen, etc. not happening. They have negligible in vivo and in vitro toxicity, and they have been extensively tested and approved by regulatory authorities for administration to animals and humans in large volumes in order to replenish blood loss. It has been reported that while high molecular weight PFCs accumulate in the liver, the low molecular weight samples used according to the present invention are eliminated from the body by respiration.

The low surface tension and low viscosity of these PFCs allow them to flow very easily through narrow openings that may be in hypodermic needles, automatic systems, or liquid needleless injectors. PFCs are excellent dielectrics, and therefore it is easy to obtain monodisperse suspensions of particles that carry the same small electrostatic charge on the surface. They are dry and completely non-hygroscopic liquids. The dryness of the suspended powders is maintained by a low water content, preventing the dissolution or degradation of the included active particles. These compounds do not have solvent properties, which makes them ideal for suspending hydrophilic or hydrophobic particles, and this means that the final suspensions are compatible with, in fact, any materials used in containers or delivery devices. Such qualities of the compounds are opposite to those of oil-based formulations, which can cause severe damage to syringes, for example, when rubber seals swell on pistons. PFCs can be obtained with densities, vapor elasticities, and volatilities in the region indicated in the table (table 1). High densities of compounds lead to sedimentation in the most common buffers, allowing easy separation from the resulting particles, which dissolve in the aqueous phase located on top. This circumstance promotes the use of these compounds in vitro, for example, for diagnosis.

DETAILED DESCRIPTION OF THE INVENTION

Table 1
Properties of some PFCs Perfluoro Like Weight Density (kg / l) Viscosity (MPa · s) Surface tension mN / m Steam Elasticity (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 solvents for the delivery of pharmacological agents or bioactive agents was previously proposed by Kirkland (1995). This patent has been cited as an example in relation to, indeed, only stable commercially available flavoring or effervescent powders and the like. It does not contain examples of any stabilized bioactive drugs, such as vaccines or pharmaceuticals. In addition, it does not consider the possibility of obtaining an injectable (parenteral) preparation using PFC as a suspension solvent for active particles. In order to obtain a stable composition containing essentially unstable biomolecules with a long shelf life using PFC as a non-aqueous solvent, the particles are preferably prepared with a glass-forming agent capable of stabilizing the active substance included. The specified agent may be from a number of sugars, including trehalose, lactitol, palatinite, etc., as described in PCT international publication No. WO 91/18091, or more preferably other more effective sugar alcohols consisting of monosaccharides, or with the flow-forming agents described in UK Patent Application No. 9820689.9.

In order for the particles not to float in the dense PFC phase, it is advisable to include in the particles a density controlling agent, for example an inorganic salt. Such an agent may be a soluble salt, such as sodium or potassium chloride or sulfate, or, more preferably, insoluble material, such as barium sulfate, calcium phosphate, titanium dioxide or aluminum hydroxide. Insoluble, non-toxic materials are preferred since the release of large amounts of ionic salts in the body can cause significant local pain and irritation. In some cases, such as vaccine preparations, insoluble materials may be part of the active drug as an adjuvant. The density regulator may be in solid solution in sugar glass particles or in an insoluble, granular material in suspension in sugar glass. Correctly prepared sugar glass particles have a density approximately equal to PFC liquid, have neutral buoyancy, and do not float on the surface, do not settle down, but remain in a stable suspension without the formation of lumps.

Since PFC fluids are good dielectrics with a resistivity value typically higher than 10 ¹³ Ohm · cm, small charges on the surface of suspended particles can have a significant effect on the stability of the suspension. In order to protect suspended particles from aggregation due to weak forces with a short range, they are produced with a filler, such as lysine or aspartic acid, capable of transferring a weak, residual, electrostatic charge to dry particles. As a result, the aggregation of particles is prevented by repelling their charge, similar to what happens in stable colloids. Alternatively, small amounts of FHC surfactants, such as perfluorodecanoic acid, can be dissolved predominantly in PFC to form dispersed, preferably monodisperse, suspensions.

Such particles can be produced in a number of ways, including air drying, spray drying or freeze drying, and should not be very small, but may be a heterogeneous mixture of particles with sizes ranging between 0.1 microns and 100 microns in diameter. In some cases, even millimeter-sized particles may be suitable.

The use of such stable suspensions is not limited to either parenteral administration, as described in the example above, or oral administration, as described in Kirkland (1995). Since PFC liquid solvent is non-toxic and non-reactive, it is an ideal solvent for delivery to the mucous membrane, including intrapulmonary delivery, nasal, ophthalmic, rectal and vaginal delivery. A significant achievement of this invention is the ability to produce stable, sterile and non-irritating compositions for delivery to the mucous membrane of even very unstable drugs or vaccines. In addition, by its nature, PFC fluid is the driest and most non-hygroscopic, which greatly contributes to maintaining the sterility of these preparations for a long shelf life and periodic use, since microorganisms cannot grow in the absence of water.

Since volatile perfluorocarbons and chlorofluorocarbons have long been used as propellants in inhalers designed to deliver drugs to the lungs, the stable PFC compositions described in this text are ideal for producing small fog-like STASIS droplets of liquid for intrapulmonary delivery. For such use, the size of the particles that make up the intermittent suspended phase in PFC droplets is important and should not exceed a size of 1 to 5 μm, preferably 0.1 to 1 μm in diameter. For delivery to other mucous surfaces, such as the nose or eyes, the particle size is less important and can be up to 100 microns or even several mm in diameter.

The water content of the microparticles is preferably not more than 4%, preferably less than 2% and ideally less than 1%.

Description of drawings

Figure 1

Alkaline phosphatase (Sigma Aldrich Llt.) Was stabilized in glass based on mannitol, 33.3%, calcium lactate, 33.3%, and degraded gelatin, 33.3% (Wuso C, Croda Colloids Ltd.), spray dried in the form of microspheres and was stored at 55 ° C either as a dry powder or as a stable suspension in perfluorodecalin. Activity remained at approximately 100% level (103% for 20 days and 94% for 30 days). The loss of activity was greater in a dry powder that was not suspended in PFC (about 80% of the activity remained).

Figure 2

A commercial tetanus vaccine (# T022 courtesy of Evans Medeva plc) was obtained in the form of a powder of suitable density using added calcium phosphate in a 20% trehalose solution. It was dried by lyophilization by spraying in liquid nitrogen using a nozzle with two jets of fluid, followed by lyophilization of a frozen powder consisting of microspheres in a Labconco freeze dryer with an initial temperature at -40 ° C for the entire duration of the main drying. The humoral immune response of six groups of 10 guinea pigs was measured 4, 8, and 12 weeks after injection with the same dose of ASSIST of a stabilized tetanus vaccine reconstituted in buffered saline / or as anhydrous preparations in oil or PFC.

Immune responses to all dried preparations were lower than to the fresh control vaccine (data not shown), indicating a significant loss of immunogenicity when spray dried. The antigenic properties of the toxoid, evaluated by ELISA uptake, did not change during the drying process. Based on the data obtained, it was concluded that more effort will be required to protect the aluminum hydroxide adjuvant during drying.

The animal's immune response to the STASIS vaccine, whose density was administered using calcium phosphate (group 3), was basically the same as the response to the control vaccine, reconstituted in aqueous buffer (group 1), and to the powder in oil vaccine (group 2), while the immune response of control animals injected with non-aqueous solvents only (groups 4 & 5) was not shown.

Ten of the following examples are presented to describe the implementation of the invention.

EXAMPLE 1:

Spray dried particles in PFC.

Particles were obtained by spray drying from an aqueous solution using a Labplant model SD 1 spray dryer using sugars and other excipients. The usual compositions were as follows:

A. Mannit 15% w / v Calcium Lactate in Water 15% w / v B. trehalose 15% w / v Calcium Phosphate in Water 15% w / v

Particles were obtained using a nozzle with two jets of fluid with a hole of 0.5 mm in inner diameter. It was found that the optimal air flow from the nozzle was half the maximum flow, and the temperature in the drying chamber was 135 ° C at the inlet and 70-75 ° C at the exit. Particles were collected in a glass cyclone and subjected to secondary drying in a vacuum oven, with a temperature drop to 80 ° C, over 4 hours. After cooling, they were suspended in PFC using sonication. It was shown that for processing approximately 75% of the powder, it was enough to carry out a 30-second release of ultrasonic energy from the titanium probe into the ultrasonic compartment of MSE MK 2, or place the powder for up to 10 minutes in an ultrasonic bath Decon FS200 Frequency sweep.

The suspension formed was monodisperse and consisted of spherical glassy particles ranging in size from about 0.5 to 30 microns with an average of about 10 microns measured with a microscope. Particles based on calcium mannitol / calcium lactate rose upward in the PFC layer for several minutes, but were easily resuspended by gentle shaking. Particles based on trehalose / calcium phosphate were selected by density using PFC and formed a stable suspension.

Spray dried powders of sugar glass particles were suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene at a concentration of 1, 10, 20 and 40% w / v. It was found that they form monodisperse suspensions with a slight tendency to aggregation. The addition of 0.1% perfluorodecanoic acid to PFC suppressed a slight tendency to aggregate on surfaces. It was found that these suspensions easily passed through a 25 g needle by aspiration or discharge.

EXAMPLE 2:

Stability of a Glass Stabilized Enzyme Suspension in PFC

Alkaline phosphatase (Sigma Aldrich Ltd.) was spray dried in a Labplant apparatus as described above. The composition contained 33.3% w / w. mannitol, 33.3% w / w calcium phosphate and 33.3% w / w. degraded gelatin (Wuso C, Croda colloids Ltd.). The dried enzyme was stored at 55 ° C as a dry powder or as a suspension in perfluorodecalin. The enzyme included in such microspheres consisting of mannitol-based sugar glass suspended in perfluorodecalin retained enzymatic activity close to 100% for more than 30 days at 55 ° C (Figure 1).

EXAMPLE 3:

In vivo efficacy

Preclinical trials of such a composition containing a clinical tetanus vaccine (kindly provided by Medeva plc) were undertaken jointly with the National Institute for Biological Standards and Control (a metrology laboratory with the right to verify and certify measuring instruments of the World Health Organization). The results of such a test showed that the stable STASIS preparation was completely equivalent to a water-based liquid vaccine with respect to its ability to immunize guinea pigs to develop a protective immune response (Figure 2). The data obtained confirm that the suspension in PFC is a ready-to-inject composition with the same in vivo bioavailability as a conventional water-based liquid composition.

EXAMPLE 4:

Particles obtained by freeze drying with spray

Particles were obtained by spraying liquid droplets in liquid nitrogen and then vacuum drying the frozen powder. The resulting particles were less dense than the spray dried powders and formed pastes in PFC at concentrations higher than 20% w / v. At lower concentrations, they formed a monodisperse suspension after sonication.

Commonly used compositions were as follows:

Substance The final concentration wt./wt. A. Trehalose 100% B. Trehalose fifty% Calcium phosphate 49.5% Aluminum hydroxide 0.5%

EXAMPLE 5:

Ground hydrophobic particles

The hydrophobic sugar derivatives sucrose octaacetate and trehalose octaacetate easily reached a glassy state either by abrupt cooling of the melt or by rapid drying from a solution of chloroform or dichloromethane. Their use has been described as a controlled release matrix for drug delivery (Roser et al "Solid delivery systems for controlled release of molecules incorporated therein and methods of making the same", international publication PCT Pub No 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 resulting glass discs were ground in a mortar and pestle and then in a high speed homogenizer to obtain a fine powder. The powder was suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene at a concentration of 1 and 10% w / v. It has been shown that they form well dispersed suspensions. It was found that such suspensions easily pass through a 23 g needle.

EXAMPLE 6:

Reconstitution in aqueous media

Based on the nature of the soluble sugar glass particles and the properties of PFC, it was suggested that the active substances in such suspensions should be rapidly released in the body. In order to achieve complete release of the incorporated active substance, particles were obtained containing:

Trehalose 20% w / v Calcium lactate 20% w / v Lysine 0.5% w / v Dye Mordant Blue 9 1% w / v

The composition was spray dried as described above and added to perfluorophenanthrene and perfluorodecalin to give 20% w / v dark blue, opaque suspensions. It was found that when water was added to an equal volume of suspensions and shaken, all of the blue dye was indeed released into the aqueous phase, which formed a transparent blue layer located on the surface of an almost colorless PFC with a clean, clear border between them.

EXAMPLE 7:

Lack of reactivity between particles in suspension

Since individual microspheres in a PFC suspension are physically isolated from all other particles, potentially reactive substances can be together in the same suspension in separate particles without any risk of their interaction. When sugar glass dissolves and the molecules come together, an interaction occurs.

In order to demonstrate this, a suspension was prepared containing two types of particles, one (a) with an alkaline phosphatase enzyme and the other (b) with its colorless substrate, paranitrophenyl phosphate.

The compositions had the following composition:

a) Trehalose 10% w / v Sodium sulfate 10% w / v Alkaline phosphatase 20 units / ml In 5 mM Tris / HCl buffer, pH 7.6 b) Trehalose 10% w / v Sodium sulfate 10% w / v Paranitrophenyl Phosphate 0.44% w / v

In 100 mM glycine buffer, pH 10.2, containing 1 mM each of Zn ⁺⁺ and Mg ⁺⁺ chloride.

It was found that in a suspension of powders in perfluorodecalin containing 10% wt./about. powder "a" and 10% wt./about. powder "b", no color reaction develops, but it remains white for 3 weeks at 37 ° C.

When water was added and shaken, the powders dissolved in the lower aqueous phase. The enzymatic reaction proceeded for several minutes, producing intense yellow staining due to the formation of p-nitrophenol, both in the freshly prepared sample and in such a sample, which was maintained at a temperature of 37 ° C for 3 weeks.

EXAMPLE 8:

Product release in tissue space model

In order to illustrate the possible behavior of PFC suspensions upon in vivo injection, a model of translucent, hydrated tissue space was prepared by preparing 0.2% agarose gels in polystyrene bottles. 0.1 ml of the perfluorodecaline suspension of Example 5 was added to the agarose gel using a 25g needle. The result is a flattened white ball of suspension. After the next 5-10 minutes, the white paint moved up from the bottom of the ball, leaving the transparent PFC ball behind. Since the enzyme and substrate were released upon dissolution of the glass particles, they interacted with each other, resulting in the formation of a yellow reaction product p-nitrophenol, which then diffused through agarose for the next 1 hour.

EXAMPLE 9:

Bringing density to the appropriate level

Sugar glass particles (i.e., trehalose) obtained by any of the conventional drying methods had typical densities of about 1.5 g / cm ³ . The tested perfluorocarbons usually had densities ranging from 1.68 to 2.03 g / cm ³ (table 1). For this reason, upon receipt of the suspension, sugar glass particles tend to adhere to the surface of the PFC layer, resulting in a preparation in which the active substance is not uniformly distributed. However, the powders can be modified to obtain a stable suspension in PFC, in which they have neutral buoyancy and do not settle and do not float on the surface. Such results can be achieved by adding high density materials to the particle formation stage. Such materials may be water soluble or insoluble in water.

Water insoluble materials

Tricalcium phosphate has a density of 3.14 g / cm ³ , is accepted as an adjuvant for vaccines and is practically insoluble in water. Powders containing about 50% calcium phosphate have an increased density of about 2 g / cm ³ and 20% of the solid particles form stable suspensions in perfluorophenanthrene.

Examples of powders which, when they contain 20% solids in PFC, form stable suspensions include:

1 In perfluorodecalin

Substance The final concentration wt./wt. A. Trehalose fifty% Calcium phosphate fifty% B. Trehalose 47.5% Calcium lactate 10.0% Calcium phosphate 42.5%

2 In perfluorophenanthrene

Substance The final concentration wt./wt. Mannitol 18.2% Inositol 18.2% Calcium lactate 18.2% Calcium phosphate 45.4%

Other water-insoluble materials used that increased density included barium sulfate and titanium dioxide. Any non-toxic and insoluble material with an appropriate density could be used.

Water soluble materials

Soluble salts such as sodium sulfate with a density of 2.7 g / cm ³ can also be used as density increasing agents. The following stable suspensions of powder in perfluorodecaline are given:

Substance The final concentration wt./wt. Trehalose fifty% Sodium sulfate fifty%

Other non-toxic, high-density, water-soluble materials may also be used. It was found that such compositions cause discomfort after subcutaneous injection in guinea pigs, possibly due to the rapid dissolution of the ion salt at high concentrations.

EXAMPLE 10:

The effect of the selected density on the active substances in suspensions

Certain vaccines are adsorbed on insoluble gels or particles that serve as adjuvants. For these purposes, aluminum hydroxide and calcium phosphate were widely used. Such insoluble adjuvants may themselves be used to increase the density of particles to be suspended. In this case, the high-density material is not completely inert, but actually adsorbs the active macromolecule from the solution. It was necessary to show that such adsorption does not denature the active substance. In order to verify this, alkaline phosphatase was used as a model of the active substance / vaccine.

The following solution was prepared

Adjuvant type calcium phosphate 10% w / v (Superphos Kemi a / s) Trehalose 10% w / v ZnCl ₂ 1 mm Alkaline phosphatase 20 units / ml

In 5 mM Tris-HCl buffer, pH 7.6

The solution was then thoroughly mixed for 10 minutes at 37 ° C in order to adsorb alkaline phosphatase using calcium phosphate. The change in absorbance per minute was determined by the steps of centrifuging calcium phosphate, sampling the supernatant and enzymatic kinetics, using p-nitrophenyl phosphate as a substrate and measuring the absorbance at a wavelength of 405 nm. The solution was spray dried to obtain a fine powder. Any desorption of the enzyme after re-hydration of the powder was measured in the supernatant. The powder was suspended at a concentration of 20% w / v. in perfluorophenanthrene and it was found that it forms a stable suspension.

Test sample d ^absorption / min (405 nm) Stock solution (25 μl) 0.409 The supernatant from the solution above (25 μl) 0,034 Rehydrated Powder (25 μl 20% w / v in water) 0.425 The supernatant from the solution above (25 μl) 0.004 20% w / v powder in perfluorodecaline (25 μl) 0.430

As a result of the experiment, it was shown: the particle density can be adjusted to the density of the PFC solvent by incorporating an adjuvant of calcium phosphate. In the process of obtaining particles, significant desorption or loss of enzymatic activity was not observed.

Claims (12)

1. A composition for delivering a stable, biologically active compound to a subject, comprising glass particles containing said biologically active compound, characterized in that the particles have a diameter in the range of 0.1-100 μm and are suspended in a liquid that includes at least one biocompatible perfluorocarbon in which these particles do not dissolve. 2. The composition according to claim 1, characterized in that the particles are obtained by spray drying. 3. The composition according to claim 1 or 2, characterized in that the particles include a mixture containing sugar and metal phosphate. 4. The composition according to any one of the preceding paragraphs, wherein said glass comprises sugar glass, metal carboxylate glass and / or phosphate glass. 5. The composition according to any one of the preceding paragraphs, characterized in that the particles contain a substance that promotes glass formation. 6. The composition according to any one of the preceding paragraphs, characterized in that the liquid includes a surfactant. 7. The composition according to any one of the preceding paragraphs, characterized in that the particles include sugar glass formed from sugar selected from the group consisting of trehalose, sucrose, raffinose, stachyose, glucopyranosyl sorbitol, glucopyranosyl mannitol, palatinite, lactitol, monosaccharide alcohol or sugar molecules, modified with hydrophobic side chains selected from the group consisting of sucrose octaacetate or trehalose octaacetate. 8. The composition according to any one of the preceding paragraphs, characterized in that the particles include phosphates of divalent metals or metal carboxylates. 9. The composition according to claim 5 or any other item dependent on it, characterized in that said compound promoting glass formation is selected from the group consisting of peptide, protein, dextran, polyvinylpyrrolidone, borate ion, calcium lactate, calcium polyphosphate and sodium silicate or acetate (salts). 10. The composition according to any one of the preceding paragraphs, characterized in that the particle density corresponds to the density of the liquid. 11. The composition according to any one of the preceding paragraphs, characterized in that it includes a filler that transfers these particles a weak residual electrostatic charge so that these particles repel each other. 12. The composition of claim 11, wherein said excipient is an amino acid.

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