ES2337252T3 - PHARMACEUTICAL LIQUID SUSPENSIONS. - Google Patents

Abstract

A pharmaceutical composition comprising an active ingredient transported by sugar crystal particles having a diameter in the range of 0.1 to 100 micrometers, these particles being suspended in a biocompatible perfluorocarbon liquid, characterized in that the particle densities and the biocompatible liquid coincide so that the particles remain in a stable suspension.

Description

Liquid pharmaceutical suspensions.

Vaccines or drugs in solution ready for injecting are inherently unstable and need refrigeration. Traditionally, the pharmaceutical industry has addressed the problem of instability through lyophilization of drugs. This is expensive, inconvenient and inherently dangerous, given that the Incorrect reconstitution of dry drugs may have as wrong dose result or contaminated solutions. During the many attempts to develop have been made over the past 100 years robust, stable and ready to inject liquid formulations with a painful lack of success. Only molecule drugs inherently resistant small can survive in solutions aqueous with a useful shelf life.

This problem is particularly acute in the vaccine industry In 2005 it has been estimated that they will have to be administered 3.6 billion doses of vaccines worldwide. The World Health Organization (WHO) has indicated that this does not it will be possible using the standard vaccine formats that they need be refrigerated at all times ("Revolutionizing Immunizations ". Jodar L., Aguado T., Lloyd J. and Lambert P-H Genetic Engineering News Feb 15 1998). A "cold chain" of refrigerators is currently being used which extends from vaccine factories to cities Provincials of the developing world. The cost of the cold chain for the vaccine industry and for health organizations Non-government directing immunization campaigns is huge. WHO has estimated that only chain maintenance costs Cold are more than 200 million US dollars annually. In addition, immunization campaigns can only reach those who They live near the last link of the cold chain.

Vaccination campaigns require medically trained personnel to ensure that the dose is injected correctly and shows no signs of degradation. The need to reconstitute some vaccines, such as measles, yellow fever and BCG, in the field is also a serious problem. This can be done precisely to ensure correct dosing and also introduces a potential source of contamination, which has often led to clinical disasters. In addition, it is often necessary to administer more than one vaccine in one session and this may require multiple injections, since it is possible that specific mixtures or "multivalent" vaccines may not be available due to the chemical incompatibility of some of the components. WHO has addressed these problems by actively stimulating the investigation of the next generation of stable vaccines that do not need refrigeration and do not need 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. ).

An ideal solution for this problem would be formulations ready to inject and completely stable. Such stable vaccines could be concentrated in the form of individual doses in an injection device or, for mass immunization campaigns, sent in larger volumes and administered by means of a needleless jet injector. Transdermal administration of dry solids by gas jet injection has been described ( Sarphie DF , Burkoth TL. Method for providing dense particle compositions for use in transdermal particle delivery. Pub. No. PCT WO 9748485 (1996)) and transdermal vaccination With dried DNA vaccines it is apparently very effective ("PowderJect's Hepatitis B DNA Vaccine First to Successfully Elicit Protective Immune Response In-Humans" at http://www.powderject.com/pressreleases.htm (1998 )).

The hypersonic shock wave of helium gas that is used to direct these powder injectors has a power limited and cannot administer your fine particle dose intramuscularly This is because the low mass particles do not They can reach a suitable moment for deep penetration. Although intradermal administration of coated DNA vaccines in colloidal gold particles is suitable for a good immunogenicity, the usual vaccines, adjuvated with salts insoluble, calcium or aluminum, induce skin irritation unacceptable. It can be administered intramuscularly. What I know requires is a flexible system capable of a series of depths of administration, from intradermal to deep intramuscular, of a way similar to what can be achieved with technology Existing needle and syringe. For campaigns of mass vaccination, this has been resolved with the development of liquid jet injector capable of accelerating a narrow (diameter of approximately 0.15 mm) liquid stream, using pressures of about 20,384.27 kPa, in a "liquid nail". This device manages your dose painlessly through the skin in the deep subcutaneous or muscular tissue through the perforation of a tiny hole through the epidermis. He high momentum imparted to the liquid stream guarantees a deep penetration To date, drugs and vaccines injected are water based, but due to the problems of instability discussed above, the range of aqueous products stable accessible to this technology is very limited.

Currently, it has been recognized that a broad range of bioactive molecules can be stabilized by drying in crystals, particularly sugar crystals (Roser B. "Protection of proteins and the like" 5 UK Kingdom patent United No. 2,187,191. Roser B and Colaco C. "Stabilization of biological macro-molecular substances and other organic compounds "Pub. No. PCT WO 91/18091. Roser B. and Sen S. "New stabilizing glasses". PCT patent application Nº: 9805699.7. 1998). These dry and stabilized active substances do not are affected by hostile environments such as temperatures high and ionizing radiation.

The underlying mechanism to the considerable stabilization of molecules with sugars is the transformation in glass Since the sugar solution that contains a molecule active dries, it can crystallize when the limit of Sugar solubility or may become supersaturated syrup. The ability of sugar to withstand crystallization is a Crucial property of a good stabilizer. Trehalose is good for this (Green JL. & Angel CA. Phase relations and vitrification in saccharide water solution and the trehalose anomaly J. Phys. Chem. 93 2880-2882 (1989)) but no only. Additional drying progressively solidifies the syrup which, in turn, becomes a crystal at a low content of residual water. Imperceptibly, the active molecules they change from liquid solution in water to solid solution in dried sugar crystal Chemical diffusion is insignificant in a crystal and, therefore, chemical reactions cease practically. Since denaturation is a chemical change, you cannot produce in the crystal t the molecules stabilize. Of this mode, the molecules can remain unchanged with the condition that another condition is met. This is the second property crucial of a good VIZ stabilizer that is chemically inert and not reactive. Many crystals do not pass because they react with the product during storage. With the reducing sugars produce obvious problems, which can form good crystals physicists but then their aldehyde groups attack the amino groups about products in a typical Maillard reaction. This is the main reason why many pharmaceuticals Freeze-dried require refrigerated storage. The non-reactive sugars provide stable products, which do not They require no refrigeration.

The crystal immobilized biomolecules of Sugars are also stable in non-aqueous industrial solvents in which they themselves and sugar are insoluble (Cleland JL. and Jones AJS. "Excipient stabilization of polypeptides treated with organic solvents "US Patent No. 5, 589,167. (1994)). Since the sugar crystal acts as a waterproof barrier in a liquid Non-solvent, biomolecules in solid solution in the crystal they are protected from both the chemical reactivity of the solvent and of the environment As long as the liquid itself is stable, the sensitive products in suspended glass particles They constitute a stable two-phase liquid formulation. The industrial solvents of the type described by Cleland and Jones (1994) have limited utility in processing. Replace a non-aqueous biocompatible aqueous liquid would allow formulating stable liquid formulations including drugs, vaccines and more unstable diagnostic substances.

The first generation of non-aqueous liquids stable designed for use in drug administration or vaccines (B.J. Roser and S.D. Sen "Stable particle in liquid formulations ". PCT Patent Application No. GB98 / 00817) described powder formulations of stabilizing crystals containing the active product suspended in injectable oils such as oil sesame, peanut or soy, or simple esters such as ethyl oleate Suspended sugar crystals particles they are of an intensely hydrophilic nature, while the Oils are hydrophobic. Given the strong trend of the phases hydrophilic and hydrophobic to separate, the crystal particles of Sugar tend to cluster. In order to stabilize such "water in oil" type suspensions, often required the use of oil soluble surfactants dissolved in the phase of continuous oil

These low HLB surfactants (Balance hydrophilic / lipophilic) accumulate at the interface between the particles  hydrophilic and oil and coat them with an amphiphilic layer that It is more compatible with the continuous oil phase. Since each sugar crystal particle is separated from its neighbors by dry oil, can not continue any chemical interaction between the particles. Therefore, it is possible to have several populations different from particles, each with an interactive molecule potentially different, in the same oil preparation, without They can interact. In this way you can produce complex multivalent vaccines

However, afterwards it has been found that this approach has certain drawbacks that prevent it from being a universal solution These include the inevitable settling of suspended particles, which have a typical density about 1.5 g / cm3, in the less dense oily vehicle. The Patent recognizes this problem and aims to solve it reducing particle size to less than 1 µm in diameter in order to remain suspended by forces thermodynamics such as the Brownian movement. The requirement of that all particles have a diameter of less than 1 µm is a disadvantage of the proposed formulations. Reach sayings Small particle powders is by no means an easy task. With better spray desiccator designs you can achieve this, but the small particle size would avoid the use of cyclone type collectors and would require a filter system for product recovery.

The reduction of particles to a size of submicrons can also, in theory, be achieved after the particles are suspended in the oil, with equipment high pressure micro-homogenizer, such as the Microfluidifier (Constant Systems Inc.) This involves a stage additional to the process and the inventors have found that it is not very efficient in breaking sugar crystal microspheres spray-dried, which have a very strong mechanical resistance high for its spherical shape. This forces multiple passes through of the team. Even in this case, it tends to leave a series untouched of the largest particles and therefore would require a stage additional filtration or sedimentation to eliminate them. Likewise, high viscosity of suspensions in oily vehicles usual hinders its rise in the syringe and requires that Inject slowly. This prevents rapid flows through fine nozzles such as those experienced in an injector system of liquid jet.

It has also been found that particles suspended in oil, especially when they contain a low HLB surfactant, are difficult to extract afterwards in an aqueous environment because, surprisingly, they maintain a water-repellent oil coating closely bonded around them, even after washing in aqueous buffer Therefore, they require very strong agitation and mixing or the addition of an even more water-soluble detergent (this time with a high HLB) so that the particles leave the oil phase and enter the water phase. This is increasingly a problem because the particle size is reduced. The end result is often a rather messy mixed emulsion instead of two cleanly separated phases, in the body this problem can produce a slow and unpredictable release of the active substance instead of the necessary rapid and predictable administration. In vitro extraction in an aqueous environment results in the oil floating in the upper part of the aqueous phase containing the dissolved active substance. This may not be acceptable for certain in vitro applications, such as diagnostic kits or automatic test systems. Finally, most PDA-approved natural oils, which can be used clinically, are vulnerable to photodegradation, oxidation or other forms of damage, and require careful storage in the dark at relatively low temperatures. In addition, they are not completely chemically inert so that they can react slowly with suspended particles.

The Alliance Pharmaceutical Company has explored the use of powders of water-soluble substances in the considerably new non-aqueous perfluorocarbon liquids ( Kirkland WD Composition and method for delivering active agents . U.S. Patent No. 5,770,181. (1995)). This patent mainly concerns the role of PFCs as oral contrast enhancing agents for bowel imaging tests. The water-soluble powders exemplified herein were added to improve palatability or enhance the effect of contrast in the gastrointestinal tract of PFCs. However, Kirkland realized in a perceptual way that these liquids could also be used for drug administration, although no examples are provided. In particular, only commercially available stable SHELF powders are exemplified in the patent. At present, the inventors have found that fragile active substances stabilized in sugar crystal microspheres can be engineered to produce extremely stable two-phase liquid PFC formulations for both oral and parenteral administration. This considerably extends the utility of the Kirkland patent to the administration of parenteral drugs and vaccines in ready-to-inject formulations that do not require any refrigeration of any kind. Of particular value is the discovery that the low viscosity, high density and low surface tension of PFCs means that these stable suspensions can be administered by automatic devices such as liquid jet injectors. This opens two important additional fields to this technology, namely mass immunization campaigns and also self-injection.

Perfluorocarbons (PFCs) are new extremely stable liquids produced by the complete fluoridation of certain organic compounds. They cannot be classified as hydrophilic or lipophilic, since, in fact, they are essentially immiscible in oil or water or in any other polar or apolar solvent, except for other PFCs. ( Revised 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 in hydrophobic interactions with oils or in hydrophilic interactions with water or hydrophilic materials. As a consequence, the strong phase separation, as observed when hydrophilic particles are closely grouped in oil, tends not to occur in PFCs. They may not require surfactants to produce stable suspensions, but fluorohydrocarbon (FHC) surfactants are available (Krafft & Riess 1998) and are active at minute concentrations in PFC liquids. At these very low concentrations, FHC surfactants can guarantee perfect monodispersed systems of certain particles, which show a tendency to aggregate in their absence. The PFC liquids themselves are chemically completely non-reactive and the lower molecular weight types do not accumulate in the body, but, being volatile, they are eventually exhaled by breathing.

Since they are excellent solvents for gases, PFCs have already been used in large quantities in very special clinical applications. Its ability to exchange carbon dioxide for dissolved oxygen is better than that of hemoglobin. This was first demonstrated in "bloodless rats" by RP Geyer in 1968 ( Geyer RP, Monroe RG & Taylor K. " Survival of rats totally perfused with perfluorocarbon-detergent preparation ." In: Organ Perfusion and Preservation, J. V Norman, J Folkman, LE Hardison, LE Ridolf and FJ Veith eds. Appleton-Century-Crofts, New York. 85-95 (1968) ). Perfluorooctyl bromide is currently being evaluated in the form of a PFC emulsion in water and with the Oxygent? Brand (Alliance Pharmaceutical Corp.) in humans as an alternative to blood transfusion for certain surgical procedures. PFCs have also been used by inhalation, in the form of fluids, in the lungs as a treatment for respiratory distress syndrome in premature babies.

It has also been found that its high density combined with chemical inertity is valuable. Perfluorophenanthrene, with the Vitreon ™ brand (Vitrophage Inc.), is used to prevent collapse of the eye capsule during surgery and allow reposition of detached retinas. PFCs have also been used as contrast media for magnetic resonance imaging (MRJ) and, to this end, it has been reported that hydrophilic powders can be suspended in them in order to improve their imaging properties or make them more Pleasant to taste. ( Kirkland WD . " Composition and method for delivering active agents " US Patent 5,770,181. 1998). This patent also suggests the use of PFC as the continuous phase for administering water soluble particulate drugs. Since the number of parenteral drugs that are stable in the form of dry powders at room temperature is limited, this patent has no applicability to most injectable drugs. However, the combination of drug stabilization in sugar crystals microsphere powders as described in Roser and García de Castro (1998) and injectable PFCs makes this technology somewhat applicable to virtually all parenteral drugs and vaccines.

The invention provides a composition. pharmaceutical comprising an active ingredient transported by sugar crystal particles that have a diameter in the range of 0.1 to 100 micrometers, these particles being suspended in a biocompatible perfluorocarbon liquid, which characterized in that the densities of the particles and the liquid biocompatible match so that the particles remain in a stable suspension

Perfluorocarbon based preparations they have important advantages in that they can be mixed different PFCs to obtain final mixtures with variable densities from about 1.5 to 2.5 g / cm3. This allows the particles are formulated with densities equivalent to that of the fluid in suspension so that they do not float or sink until bottom of the container, but remain in the form of a suspension stable. Therefore, the particles do not need to have a size of submicrons, as required in preparations based on oil to prevent sedimentation and may be monodispersed or vary considerably in size. The last diameter Particle is directed solely for the purpose of preparation. Preparations intended for needle injection or injection in jet could contain particles in the range of 0.1 to 100 micrometers or, preferably, 1 to 10 micrometers. This allows a great simplification in the way of manufacturing the particles and avoid the need for particle size production extremely small by grinding. Particles can manufactured by conventional spray drying or by cryo-drying, followed by simple drying or grinding wet When high content is needed in the suspension solids, it is desirable that the particles have a spherical shape. Irregularly shaped particles have a much greater tendency to "join" and inhibit free flow, while spherical particles have an inherent "lubricity" that It allows to reach solids contents well above 20%. He Preferred range is from 1% to 40% or, more preferably, from 10% to fifteen%. These particles are easily manufactured by drying by atomization, drying by atomization-cryo-drying or solidification of the emulsion.

Suspended powders, if properly formulated, do not need surfactants and produce stable suspensions from which the crystal particles dissolve almost instantaneously when agitated with water. If minor aggregation is perceived as a problem, advantageously, small amounts of an FHC surfactant can be added as described in Krafft and Riess (1998) to the PFC fluid either before or after stable powder mixing. . If a surfactant is used, it is preferably present in a concentration of 0.01% to about 10% by weight, the optimum being probably about 1%. Like PFCs, these FHCs are inherently extremely inert and non-reactive. Therefore, there is no solvation of the particles or chemical reaction between the suspended particles and the PFC phase. Since suitable particles of crystal and PFC liquid are stable at room temperature, there is no degradation by light, high temperatures, oxygen etc. They have negligible toxicity in vivo or in vitro and have been extensively tested and approved by regulatory authorities by infusion of large volumes in both animals and humans for the purpose of blood replacement. Although it has been reported that high molecular weight PFCs accumulate in the liver, the examples of lower molecular weight used in this application are ultimately removed from the body by exhalation.

Their low surface tension and low viscosity allow them to flow very easily through the narrow holes that can be found in hypodermic needles, automatic systems or liquid jet injectors. PFCs are excellent electrical insulators and, therefore, it is easy to achieve monodisperse suspensions of particles that carry the same small surface electrostatic charge. They are dry and completely non-hygroscopic liquids. Its very low water content maintains the dryness of the suspended powders, which prevents dissolution or degradation of the active substances incorporated. Their unique lack of solvent properties makes them ideal for suspending hydrophilic or hydrophobic particles and means that the final suspensions are compatible with almost any material used in containers or delivery devices. This contrasts with oil-based preparations, which can produce an intense jam in the syringes by, for example, rubber seals on the plungers. PFCs can be obtained in a range of densities, vapor pressures and volatilities (Table I). Their high densities make them sink into more conventional buffers, which allows for easy separation of product particles, which dissolve in the aqueous phase that floats on top. Therefore, this facilitates its use for in vitro applications such as diagnostic products.

TABLE I Properties of some PFCs

one

The use of PFC as vehicles for the administration of pharmacological agents or bioactive agents was previously suggested in Kirkland (1995). In this patent only examples of inherently stable and commercially available flavoring or effervescent powders and the like have been made. It contained no examples of any stabilized bioactive substance, such as vaccines or pharmaceuticals. In addition, it does not consider the possibility of manufacturing an injectable (parenteral) preparation using PFC as the suspension vehicle for active particles. In order to achieve a stable formulation of inherently fragile biomolecules with a prolonged life using PFC as a non-aqueous vehicle, the particles would preferably be formulated to contain a crystal forming agent capable of stabilizing the incorporated active substance. This can be of a variety of sugars, including trehalose, lactitol, palatinit etc., as described in PCT No. WO 91/18091 or, more preferably, other monosaccharide sugar alcohols or more effective crystal forming agents, such as described in the UK patent application No. 9820689.9.

In order to prevent particles from floating in the dense phase of PFC, it is an advantage to incorporate an agent particle density regulator, for example a salt inorganic This may be a soluble salt, such as chloride or sodium or potassium sulfate, or, more preferably, a material insoluble such as barium sulfate, calcium phosphate, dioxide titanium or aluminum hydroxide. Insoluble materials are preferred non-toxic since the release of large amounts of salts ionic in the body can produce considerable local pain and irritation. In some cases, as in vaccine preparations, insoluble materials can be part of the preparation active as an adjuvant. The density regulator can be in solid solution in glass particles or a material insoluble particulate in suspension in the crystal. When they are formulated correctly, the crystal particles have a density approximately equivalent to that of the PFC liquid, they have a neutral buoyancy, neither float nor sediment but They remain in stable suspension without forming cakes.

Since PFC liquids are good electrical insulators, with a typical resistivity greater than 10 13 ohm.cm, small surface charges on the Suspended particles can have 5 significant effects on The stability of the suspension. In order to avoid aggregation of suspended particles due to weak short forces scope, preferably manufactured so that they contain a excipient such as lysine or aspartic acid capable of donating a charge Residual electrostatic weak to dry particles. This avoids the aggregation ensuring the repulsion of charges of the particles, similar to that observed in stable colloids. As an alternative, small amounts of FHC surfactants, such as acid perfluorodecanoic, can advantageously dissolve in PFCs to give dispersed suspensions, preferably monodispersed.

These particles can be manufactured from numerous modes, including air drying, atomization or cryo-drying, and they don't have to be particularly small but they can be a heterogeneous mixture of sizes varying between 0.1 µ and 100 µ in diameter. For some applications, even particle size in millimeters may be suitable.

The use of these stable suspensions is not restricted to parenteral use as exemplified by which precedes even oral use as exemplified in Kirkland (nineteen ninety five). Since the PFC liquid vehicle is so harmless and not reagent, is an idear vehicle for mucosal administration, including intrapulmonary, intraocular, intrarectal and intravaginal The capacity provided by this patent, for produce stable, sterile and non-irritating formulations for mucosal administration of even very unstable drugs or vaccines It is a considerable advance. Also, the nature is very dry and completely non-hygroscopic liquid PFC helps considerably in maintaining the sterility of these preparations during prolonged storage and use intermittent because microorganisms cannot grow absent  of water.

Since perfluorohydrocarbons and volatile chlorofluorocarbons have been used for a long time as propellants in inhalers designed to achieve administration of drugs in deep areas of the lungs, the Stable PFC formulations described herein Descriptive memory are ideal for generating a fine mist of drops Liquid STASIS for intrapulmonary administration. For this application, the size of the particles that constitute the phase discontinued discontinuously in the drops of PFC is important and not should exceed 1-5 µm, preferably of 0.1 to 1 µm in diameter. For administration to others mucous surfaces, particle size is less important and it can have a diameter of up to 100 µm or even several mm

Preferably, the microparticles have a water content not exceeding 4% and preferably less than 2%, and, ideally, less than 1%.

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Description of the figures

Figure one

Alkaline phosphatase (Sigma Aldrich Ltd.) is stabilized in a glass with 33.3% mannitol, 33.3% calcium lactate and 33.3% degraded gelatin (Byco C, Croda Colloids Ltd.), were dried by atomization in the form of microspheres and stored at 55 ° C well in the form of dry powder or in the form of a stable suspension in perfluorodecalin The activity remained around the brand 100% (103% at 20d and 94% at 30d). More loss was observed in the dry powder that was not suspended in PFC (remained around the 80% of the activity).

Figure 2

A marketed toxoid vaccine from Tetanus (No. T022, kindly supplied by Evans Medeva pic) is formulated in powder form of equivalent density using phosphate Calcium added in 20% trehalose solution. They dried up by atomization in liquid nitrogen using a two nozzle fluids, followed by cryo-drying of the microsphere powder frozen in a Labconco dryer with the temperature of initial storage at -40 ° C during primary drying. The Antibody response of six groups of 10 guinea pigs was measured at 4, 8 and 12 weeks after the injection with the same dose of ASSIST stabilized tetanus toxoid vaccine reconstituted in saline buffer or as anhydrous preparations in oil or PFC

The answers to all dry preparations were lower than that of the fresh control vaccine (not shown), indicating a significant loss of immunogenicity at spray drying. The antigenicity of the toxoid, measured by capture ELISA, it was not altered by the process of desiccation. This suggested that more work was needed to improve the preservation of aluminum hydroxide adjuvant when drying The response to the STASIS vaccine with equivalent density with calcium phosphate (group 3) is essentially the same as that of the control vaccine reconstituted in aqueous buffer (group 1) and the powder in the oil vaccine (group 2), while the animals control to which non-aqueous vehicles were injected only (groups 4  and 5) showed no response.

They will be described below, by way of example, ten examples of how the invention can be realized.

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Example one

Particles dried by atomization in PFC

Particles were produced by desiccation by atomization from an aqueous solution using a desiccator by Labplant atomization model SD 1 using sugars and others excipients Five typical formulations were:

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2

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The particles were produced using a nozzle of two fluids with a hole for liquids of an internal diameter 0.5 mm It was found that an air flow in the nozzle half of the maximum was optimal and the drying chamber worked at a 135 ° C inlet temperature and 15 outlet temperature of 70-75 ° C. The particles were collected in a cyclone of glass and underwent secondary drying in an oven of vacuum using a temperature slope of up to 80 ° C for 4 hours. Upon cooling, they were suspended in PFC using ultrasound. Be found that a 30-second burst of energy was enough Ultrasonic from a titanium probe in a hood Ultrasonic MSE MK 2 operating at a power of approximately 7% or immersion in an ultrasonic scanning bath Decon FS200 frequency for up to 10 minutes.

The resulting suspension was monodispersed and It consisted of spherical glass particles of size variable of approximately 0.5 to 30 µ, with an average of approximately 10 µ, judged microscopically. The particles of mannitol / calcium lactate rose to the top of the PFC layer for several minutes, but could be resuspended with ease with gentle agitation. Trehalose / phosphate particles calcium had a density almost equivalent to that of the PFC and formed a stable suspension

Cryo-dried particulate powders sugar crystal were suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene at 1, 10, 20 and 40% w / v. Be found that they provided monodisperse suspensions with little tendency to aggregation. The addition of 0.1% acid perfluorodecanoic to PFC inhibited any slight tendency to add on surfaces. It was found that these suspensions they passed easily through a 0.51 mm needle by aspiration or ejection.

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Example 2

Stability of the crystal stabilized enzyme suspension in PFC 5

Alkaline phosphatase (Sigma Aldrich Ltd.) is Cryoddecased in a Labplant device as indicated in foregoing. The formulation contained 33.3% w / w mannitol phosphate Calcium 33.3% w / v and degraded gelatin (Byco C, Croda colloids Ltd.) 33.3%. The dried enzyme was stored at 55 ° C. well shaped dry powder or in the form of a suspension in perfluorodecalin.

The enzyme formulated in these microspheres constituted by a mannitol-based crystal suspended in perfluorodecalin show retention of about 100% of the Enzymatic activity for more than 30 days at 55 ° C (Fig. 1).

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Example 3

Efficiency in vivo

Preclinical studies of a similar formulation containing a clinical tetanus toxoid vaccine (kindly provided by Medeva pic) were carried out in collaboration with the National Institute of Biological Standards and Control (an approved laboratory of the World Health Organization). The results of this study showed that the stable STASIS preparation was completely equivalent to a water-based liquid vaccine in terms of its ability to immunize guinea pigs to develop a protective serum antibody response (Figure 2). This confirmed that the suspension in PFC constituted a formulation ready to inject with the same bioavailability in vivo as a conventional liquid water-based formulation.

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Example 4

Particles subjected to desiccation by atomization-freezing

Particles were also manufactured by spray of liquid drops in liquid nitrogen and then drying Vacuum of frozen powder. These particles were less dense than the powders dried by atomization and formed pastes in PFC to concentrations above 20% w / v. At lower concentrations formed a monodispersed suspension after sonication. The Typical formulations used were:

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3

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Example 5

Ground hydrophobic particles

Hydrophobic derivatives of sugar sucrose octaacetate and trehalose octaacetate readily form crystals when they are inactivated from the melt or dried quickly in the chloroform or dichloromethane solution. Its use has been described as controlled release matrices for drug administration ( Roser et al. " Solid delivery systems for controlled release of molecules incorporated therein and methods of making same " Pub. PCT No. WO 96/03978 1994).

A trehalose octaacetate powder was manufactured by melting in a muffle furnace and melting inactivation in A stainless steel plate. The resulting glass discs are they ground in a mortar and then in a high homogenizer speed to produce a fine powder. This one was suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene at 1 and 10% p / v. They were found to give well dispersed suspensions. Be discovered that these suspensions easily passed through a 23 g needle

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Example 6

Reconstitution in aqueous environments

Given the nature of the particles of soluble sugar crystals and the properties of PFCs, has provided that the active substances in these suspensions are they would quickly release in the body. In order to demonstrate a complete release of an active substance included, the particles were formulated so that they contained:

Trehalose

20% p / v

Calcium lactate

20% p / v

Lysine

0.5% w / v

Mordant blue coloring 9

1% p / v

The formulation was spray dried as indicated above and added to perfluorophenanthrene and perfluorodecalin to produce 20% w / v dark blue suspensions opaque After the addition of water to an equal volume of suspensions and with agitation it was found that virtually all the blue pigment was released in the water phase that formed a layer light blue floating in the almost colorless PFC with a sharp clean interface between them.

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Example 7

Absence of reactivity between suspended particles

Since the individual microspheres in a PFC suspension are physically isolated from each other particles, potentially reactive substances may appear together in the same suspension in different particles without it produce no harm to the interaction between them. When the crystal of sugar dissolves and the molecules can be put together, it is produced the reaction. In order to prove it, a suspension was prepared containing two types of particle, one with the enzyme phosphatase alkaline and the other (b) with its colorless substrate, para phosphate nitrophenyl The formulations were:

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4

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In 5 mM Tris / HCl buffer at pH 7.6

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5

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In 100 mM glycine buffer at pH 10.2 which contains 1 mM each of Zn ++ chloride and of Mg ++.

It was found that a suspension of the powders in perfluoridecalin containing 10% w / v of powder "a" and 10 in p / v of the powder "b" that did not develop any reaction of color but remained in the form of a white suspension for three weeks at 37 ° C.

After the addition of water and stirring, the powders they dissolved in the surface aqueous phase. The reaction enzymatic took place in minutes and a yellow color occurred intensity of p-nitrophenol, both in the freshly prepared sample and in which it had been stored at 37 ° C for 3 weeks.

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Example 8

Product release in the "tissue space" model

In order to illustrate the possible behavior of PFC suspensions when injected in vivo , a transparent and hydrated tissue space model was prepared by pouring 0.2% agarose gels into bijoux polystyrene bottles. With a 25 g needle, 0.1 ml of the perfluorodecalin suspension of example 5 was injected into the agarose gel. This produced a white flattened sphere of the suspension. For the next 5-10 minutes, the white color 25 cleared from the bottom of the sphere upwards, leaving a transparent PFC sphere behind. As the enzyme and the substrate were released by dissolution of the crystal particles, they reacted together to produce a yellow color of p-nitrophenol, which was then diffused through the agarose gel during the next hour.

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Example 9

Density equivalence

The particles of sugar crystals (i.e. trehalose) obtained by any of the methods of Conventional drying show typical densities of around 1.5 g / cm3. Perfluorocarbons analyzed by inventors normally have varying densities of 1.68 to 2.03 g / cm3 (Table I). For this reason, when formulated in a suspension, sugar crystal particles tend to float over the PFC layer, which leads to a preparation in which the active substance is not distributed homogeneously. However, the powders can be modified in order to produce a suspension stable in PFC where they have a neutral buoyancy and not They settle or float. This can be achieved by adding High density materials before particle formation. These can be soluble or insoluble in water.

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Water insoluble materials

Tricalcium orthophosphate has a density of 3.14 g / cm3, is approved as an adjuvant for vaccines and is practically insoluble in water. The powders prepared for contain about 50% calcium phosphate show a higher density around 2 g / cm3 and at 20% solids form stable suspensions in perfluorophenanthrene. Examples of powders which at 20% solids content in PFC form suspensions Stable include:

1. In perfluorodecalin

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2. In perfluorophenanthrene

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Among other water insoluble materials that increase the density that have been used include sulfate Barium and titanium dioxide. You can use any material not Toxic and insoluble with the right density.

Water soluble materials

Soluble salts such as sodium sulfate with a density of 2.7 g / cm3 as the density increase. The following powder formed suspensions stable in perfluorodecalin:

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Other soluble materials can also be used in non-toxic and high density water. It has been discovered that you are formulations cause discomfort after subcutaneous injection in guinea pigs, possibly because of the rapid dissolution of high concentrations of ionic salt.

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Example 10

Effect of density equivalence on active substances in suspensions

Certain vaccines are formulated absorbed in gels or insoluble particles that act as adjuvants. Hydroxide Aluminum and calcium phosphate are widely used for this purpose. These insoluble adjuvants can be used to increase the density of the particles to be suspended. In this case, the material of high density is not completely inert, but, in fact, absorbs the active macromolecule of the solution. It is necessary to show that This absorption does not denature the active substance. To analyze this alkaline phosphatase was used as a vaccine / active substance model.

The following solution was prepared

Grade Calcium Phosphate adjuvant

10% w / v (Superphos Kemi ace)

Trehalose

10% p / v

ZnCl2

1 mM

MgCl2

1 mM

Phosphatase alkaline

20 U / ml

In 5 mM Tris / HCl buffer at pH 7.6

Then, the solution was mixed well for 10 minutes at 37 ° C to allow alkaline phosphatase to be absorbed by calcium phosphate. This change in absorption per minute is measured by centrifugation of calcium phosphate, taking sample of the supernatant and measuring its enzymatic kinetics using phosphate of p-nitrophenyl as a substrate and a wavelength 405 nm The solution was subjected to spray drying to produce a fine powder. Any defection of the enzyme after powder rehydration was measured in the supernatant as it has been cited above. The powder was suspended at 20% w / v in perfluorophenanthrene and was found to produce a suspension stable.

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The experiment demonstrates:

?

Particle density can be made equivalent to that of the PFC vehicle by inclusion of adjuvant calcium phosphate.

?

During the process of formulation does not take place any desorption or loss of activity significant enzymatic.

Claims (4)

1. A pharmaceutical composition comprising an active ingredient transported by sugar crystal particles having a diameter in the range of 0.1 to 100 micrometers, these particles being suspended in a biocompatible perfluorocarbon liquid, characterized in that the densities of the particles and the biocompatible liquid coincide so that the particles remain in a stable suspension. 2. A composition according to claim 1, characterized in that the particles contain an inorganic salt as a density regulating agent. 3. A composition according to claim 2, characterized in that the inorganic salt is calcium phosphate. 4. A composition according to any of the preceding claims, characterized in that the particles contain calcium lactate.