PT1452171E - Pharmaceutical liquid suspensions - Google Patents

Description

ΕΡ 1 452 171 / ΡΤ

DESCRIPTION " Liquid Pharmaceutical Suspensions "

Vaccines or drugs in solution ready for injection are inherently unstable and require refrigeration. The pharmaceutical industry has traditionally faced the problem of instability through the freeze drying of drugs. This is expensive, inconvenient and inherently dangerous, since incorrect reconstitution of dry drugs can result in wrong dosages or contaminated solutions. Many attempts have been made over the past 100 years to develop robust, stable, ready-to-inject liquid formulations with a regrettable lack of success. Only inherently resistant small molecule drugs can survive in aqueous solution with a useful shelf life.

This problem is particularly acute in the vaccine industry. It was estimated that by the year 2005, 3.6 billion doses of vaccine would have to be administered worldwide. It was stated by the World Health Organization (WHO) that this would not be possible using conventional vaccine formats that have to be permanently refrigerated (" Revolutionizing Immunizations ". Jodar L.,

Aguado Ί., Lloyd J. and Lambert P-H., Genetic Engineering News Feb 15 1998). A " cold chain " of refrigerators, which runs from vaccine plants to provincial cities in developing countries. The cost of the cold chain to the vaccine industry and to non-governmental health organizations conducting immunization campaigns is enormous. The WHO estimated that only the cost of maintaining the cold chain is annually greater than US $ 200 million. In addition, immunization campaigns can only reach those living near the last link in the cold chain.

Vaccination campaigns require medically trained personnel to ensure that the dose is correctly injected and shows no signs of degradation. The need to reconstitute some vaccines, such as measles, yellow fever and BCG, is also a serious concern. 2

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This has to be done accurately to ensure proper dosing and also introduces a potential source of contamination, which has often led to clinical disasters. In addition, it is often necessary to give more than one vaccine per session and this may require multiple injections, since particular blends or " multivalent " may not be available due to the chemical incompatibility of some of the components. WHO has underlined these problems by actively encouraging research into the next generation of stable vaccines that do not require refrigeration and 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 M.T. WHO publication May 1998. " General policy issues: injectable solid vaccines: a role in future immunization? &Quot; Aguado Μ T., Jodar L., Lloyd J., Lambert P.H. WHO publication No A59781.)

An ideal solution to this problem would be completely stable ready-to-inject formulations. These stable vaccines may be packaged in single doses in the injection device itself or, for mass immunization campaigns, packaged in larger volumes and administered by means of a needleless jet injector. Transdermal delivery 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. PCT, WO 9748485 (1996)) and transdermal vaccination with vaccines of dry DNA 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 helium gaseous hypersonic shock wave that is used to drive these powder injectors has limited potency and is unable to deliver its dose of fine particles intramuscularly. This is because the particles of small mass fail to acquire the right moment for a deep penetration. Although the intradermal delivery of DNA vaccines coated with colloidal gold particles is suitable for good immunogenicity, standard vaccines, adjuvanted with insoluble calcium or aluminum salts, induce unacceptable skin irritation. They have to be given intramuscularly. What is required is a flexible system capable of a range of delivery depths, from intradermal to deep intramuscular, similar to that achieved by the existing syringe and needle technology. For mass vaccination campaigns this has been solved by the development of the liquid jet injector capable of accelerating a narrow stream (about 0.15 mm diameter) of liquid, using pressures of about 3000 psi, on a " liquid ". This device delivers its dose painlessly through the skin into the deep muscle or subcutaneous tissue by drilling a tiny hole through the epidermis. The high momentum imparted to the liquid stream ensures deep penetration. To date, the injected drugs and vaccines have been water-based but due to the instability problems discussed above, the range of stable aqueous products accessible to this technology is very limited. It is now recognized that a wide range of bioactive molecules can be stabilized by drying in glasses, particularly sugar glasses (Roser B. " Protection of proteins and the like " 5 UK patent 2,187,191, Roser B and Colaço C. " Stabilization of biological macro-molecular substances and other organic compounds " PCT Pub. WO 91/18091. Roser B. and Sen S. " New stabilizing glasses " PCT Patent Application No. 9805699.7, 1998). These active, stabilized, dry products are not affected by hostile environments such as high temperatures and ionizing radiation. The mechanism underlying the remarkable stabilization of molecules by sugars is glassy transformation. As the sugar solution containing an active molecule is dried, it may crystallize when the sugar solubility is reached, or it may become a super-saturated 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 trehalose anomaly J. Phys. Chem. 93 2880-2882 (1989)) but is not the only one. Continuation of drying progressively solidifies the syrup, which turns into a glass with a low residual water content. Imperceptibly, the active molecules change from the liquid solution in the water to the solid solution in the dry sugar glass. The chemical diffusion is negligible in a glass and therefore the chemical reactions virtually cease. As denaturation is a chemical change, it can not occur in the glass and the molecules are stabilized. In this way, the molecules can remain unchanged as long as another condition is fulfilled. This is the second crucial property of a good stabilizer viz. which is chemically inert and non-reactive. Many glasses do not work because they react with the product in storage. Obvious problems occur with reducing sugars, which can form good physical glasses but then their aldehyde groups attack amino groups on the products in a typical Maillard reaction. This is the main reason why many freeze-dried pharmaceutical products require refrigerated storage. Non-reactive sugars give rise to stable products, which do not require any refrigeration.

The biomolecules immobilized on sugar glass are also stable in non-aqueous industrial solvents in which they themselves and sugar are both insoluble (Cleland JL and Jones AJS. &Quot; Excipient stabilization of polypeptides treated with organic solvents " US Patent 5,589,167 (1994 )). As the sugar glass acts as an impermeable barrier in a non-solvent liquid, the solid solution biomolecules in the glass are protected simultaneously from the chemical reactivity of the solvent and the environment. Since the liquid itself is stable, sensitive products in suspended glass particles constitute a stable two phase liquid formulation. Industrial solvents of the type described by Cleland and Jones (1994) have a limited processing utility. Replacement of a biocompatible non-aqueous liquid will enable stable liquid formulations to be obtained from even the most unstable drugs, vaccines and diagnostic products to be formulated. The first generation of stable non-aqueous liquids designed to be used in the delivery of drugs or vaccines (BJ Roser and SD Sen " Stable particle in liquid formulations " PCT Patent Application No. GB98 / 00817) stabilizers containing the active ingredient, suspended in injectable oils such as sesame oil, peanut or soy oil or simple esters such as ethyl oleate. The suspended sugar glass particles are of an intensely hydrophilic nature while the oils are hydrophobic. Due to the strong tendency of the hydrophilic and hydrophobic phases to separate, the sugar glass particles tend to agglomerate. In order to stabilize these " water-in-oil " the use of oil soluble surfactants dissolved in the continuous oil phase was often required.

These low HLB (Hydrophile / Lipophilic Equilibrium) surfactants accumulate at the interface between the hydrophilic particles and the oil and coat them with an amphiphilic layer which is more compatible with the continuous oil phase. As each particle of sugar glass is separated from its neighbors by dry oil, there can be no chemical interaction between the particles. It is therefore possible to have several different populations of particles, each containing a different potentially interacting molecule, in the same oil preparation, without these interacting. Multivalent complex vaccines can be produced in this manner.

It has subsequently been found, however, that this approach has certain drawbacks that prevent it from being a universal solution. These include the inevitable settling of the suspended particles, which have a typical density of about 1.5 g / cm 3, in the less dense, oily vehicle. The patent recognizes this problem and aims to solve it by reducing the particle size down to 1 pm in diameter so that these remain suspended by thermodynamic forces such as Brownian motion. The requirement that all particles be less than 1 ppm in diameter is a disadvantage of the proposed formulations. Getting powders with such small particles is by no means an easy task. Improved spray dryer designs may be able to achieve this but the small particle size would preclude the use of cyclone type collectors and would require a filter system for product recovery. The reduction of the particles to a submicron size can also theoretically be achieved after the particles are suspended in the oil with high pressure micro-homogenization equipment such as Microfluidizer (Constant Systems Inc. .). This involves an extra step in the process and we have found that it was not very efficient for breaking spray-dried sugar glass microspheres which have a very high mechanical strength due to their spherical shape. This requires multiple passes through the equipment. Even so, this tends to leave several of the larger particles unchanged and therefore would require a subsequent filtration or sedimentation step to remove them. Also, the high viscosity of the suspensions in the usual oily vehicles renders them difficult to insert into the syringe and requires that they be injected slowly, prevents rapid flow through narrow nozzles such as those found in a liquid jet injector system.

It has also been found that particles suspended in an oil, especially when containing a low HLB surfactant, are difficult to subsequently extract into an aqueous environment because surprisingly they maintain a strongly water-repellent oil coating even after washing with buffer. They therefore require very vigorous stirring and mixing or the addition of even more water soluble detergent (this time with a high HLB) for the particles to leave the oil phase and enter the water phase. This becomes an increasingly serious problem as the particle size is reduced. The end result is often a rather confused mixed emulsion rather than two clearly separated phases, and in the body this problem can cause slow and unpredictable release of the active product rather than the ready and predictable delivery required. In vitro extraction into an aqueous environment results in fluctuation of the oil over the aqueous phase containing the dissolved active product. This may not be acceptable for certain in vitro applications such as diagnostic kits or automated assay systems. Finally, most of the FDA-approved natural oils that 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 the suspended particles. 7 ΕΡ 1 452 171 / ΡΤ The Alliance Pharmaceutical Company has explored the use of powders of water-soluble substances in novel non-aqueous perfluorocarbons (Kirkland WD Composition and method for delivering active agents, U.S. Patent 5,770,181 (1995)). This patent relates primarily to the role of PFCs as oral contrast enhancers for diagnostic imaging of the intestines. The water-soluble powders exemplified therein were added to improve the palatability or enhancement of the contrast effect in the gastrointestinal tract of PFCs. However, Kirkland noted perceptually that these liquids could also be used for drug delivery although no examples are given. In particular, only commercially available post-stable storage powders are exemplified in the patent. We have now found that fragile active products stabilized on sugar glass microspheres can be manipulated to produce extremely stable two phase PFC liquid formulations for oral and parenteral delivery. This greatly extends the utility of the Kirkland patent to the delivery of parenteral drugs and vaccines in ready-to-inject formulations which do not require refrigeration of any kind. Of particular value is the finding that the low viscosity, high density and low surface tension of the PFCs mean that these stable suspensions can be delivered by automatic devices such as liquid jet injectors. This opens up two important additional fields to this technology, namely mass immunization campaigns and also self-injection.

Perfluorocarbons (PFCs) are novel, extremely stable liquids produced by the complete fluorination of certain organic compounds. They can not be classified either as hydrophilic or as lipophilic, since in fact they are essentially immiscible both in oil and in water or any other solvent, whether polar or nonpolar, except in other PFCs. (Reviewed in Krafft MP & Riess JG, " Highly fluorinated amphiphiles and colloidal systems, and the applications in the biomedical field, A Contribution, Biochimie 80 489-514 1998). In addition, they do not participate in hydrophobic interactions with oils or hydrophilic interactions with water or hydrophilic materials. As a consequence, it tends not to occur in the PFCs a complete separation of phases, as is observed when hydrophilic particles agglomerate strongly in oil. They may not require surfactants to produce stable suspensions, fluorohydrocarbon surfactants (FHC) but are available (Krafft & Riess 1998) and are active at minute concentrations in PFC liquids. At these very low concentrations FHC surfactants can ensure perfect monodisperse systems of certain particles which show a tendency to aggregate in their absence. The PFC fluids themselves are completely non-chemically reactive and the lower molecular weight types do not accumulate in the body but, being volatile, are eventually exhaled in the breath.

Because they are excellent 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 that for hemoglobin, this was first demonstrated in " exanguate 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. 1 / Norman, J Folkman, LE Hardison, LE Ridolf and FJ Veith eds Appleton-Century-Crofts, New York, 85-95 (1968)). Perfluorooctyl bromide in the form of a PFC-in-water emulsion and under the trade name Oxygent ™ (Alliance Pharmaceutical Corp.) is currently being evaluated in humans as an alternative to blood transfusion for certain surgical procedures. PFCs were also used by inhalation, in the form of liquids, to the lungs, as a treatment for respiratory distress syndrome in premature infants. Its high density combined with chemical inertia was also found to be valuable. Perfluorophenanthrene, under the trade name Vitreon ™ (Vitrophage Inc.), is used to prevent collapse of the eye capsule during surgery and to allow repositioning of displaced retinas. PFCs were also used as contrast media for Magnetic Resonance Imaging (MRI) and for this purpose it has been reported that the hydrophilic powders can be suspended therein in order to improve their imaging properties, make them more palatable. (Kirkland W.D. " 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 the delivery of water soluble particulate drugs. As the number of parenteral drugs which are stable in the form of dry powders at room temperature is limited, this patent has no applicability for most injectable drugs. However, the combination of drug stabilization in powders of sugar glass microspheres as described in Roser and Garcia de Castro (1998) and injectable PFCs makes this technology applicable to virtually all parenteral drugs and vaccines. The invention provides a pharmaceutical composition comprising an active ingredient supported on sugar glass particles having a diameter in the range of 0.1 to 100 micrometers, said particles being suspended in a biocompatible perfluorocarbon liquid, characterized in that the densities of the particles and the biocompatible liquid be equalized so that the particles remain in a stable suspension.

Perfluorocarbon based preparations have great advantages in that different PFCs can be blended to obtain final blends having densities ranging from about 1.5 to 2.5 g / cm 3. This allows the particles to be formulated at densities equivalent to that of the suspending fluid so that they do not float or sink into the bottom of the vessel but remain in the form of a stable suspension. The particles therefore need not be of submicron dimension as required in oil-based preparations to avoid sedimentation, and may be monodisperse or vary greatly in size. The final particle diameter is governed solely by the purpose of the preparation. Preparations for needle injection or jet injection may contain particles in the range of 0.1 to 100 micrometers, or preferably 1 to 10 micrometers. This allows for a great simplification in the way of manufacturing the particles and avoids the need for particle production of extremely small dimensions by grinding. The particles may be prepared by conventional spray-drying or freeze-drying followed by simple drying or milling in wet. When a high solids content in the suspension is required, it is desirable for the particles to be spherical in shape. Particles of irregular shape have a much greater tendency to " mutually inhibiting free flow, while the spherical particles have a " lubricity " inherent in solids content well above 20%. The preferred range is 1% to 40% or more preferably 10% to 15%. These particles are readily prepared by spray drying, spray drying or emulsion solidification.

Post-suspensions, if suitably formulated, do not require surfactants, producing stable suspensions from which the glass particles dissolve almost instantly when shaken with water. If a minor aggregation problem is noted, small amounts of a FHC surfactant, as described in Krafft and Riess (1998), may advantageously be added to the PFC fluid either before or after mixing the stable powder. If a surfactant is employed, it is preferably present in a concentration of 0.01% to about 10% by weight, the optimum probably being around 1%. Like PFCs, these FHC are inherently extremely inert and non-reactive. There is therefore no solvation of the particles or chemical reaction between the suspended particles and the PFC phase. As suitable glass particles and PFC liquid are environmentally stable there is no degradation due to light, high temperatures, oxygen, etc. They have negligible in vivo or in vitro toxicity and have been extensively tested and approved by regulatory authorities and have been infused into animals and man in large volumes for blood replacement. Although high molecular weight PFCs have been reported to accumulate in the liver, the lower molecular weight examples used in this application are eventually removed from the body in the exhaled breath. 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 obtain monodisperse suspensions of the same small surface electrostatic charge. They are dry liquids and completely non-hygroscopic. Its very low water content maintains the dryness of the suspended powders, preventing the dissolution or degradation of the incorporated active products. Its unique absence of solvent properties makes them ideal for the suspension of hydrophilic or hydrophobic particles and means that the final suspensions are compatible with virtually any materials used in containers or delivery devices. This is in contrast to oil-based preparations which can cause severe clogging of the syringes, for example by swelling the rubber seals on the plungers. PFCs can be obtained with a range of densities, vapor pressures and volatilities, (Table I). Their high densities cause them to sink into most conventional buffers, allowing for easy separation of the product particles, which dissolve in the aqueous phase that floats on top. This therefore facilitates their use for in vitro applications such as diagnosis.

Table I. Properties of some PFCs

(M / m) Vapor (mbar) hexane 338 1.682 0.656 11.1 294 -n-octane 438 1.75 1.27 16.98 (w / w) Density Viscosity Voltage 52 decalin 462 1.917 5.10 17.6 8.8 phenanthrene 624 2, 03 28.4 19 <1 The use of PFCs as vehicles for the delivery of pharmacological agents or bioactive agents was previously suggested in Kirkland (1995). This patent exemplified only inherently stable commercially available effervescent or flavoring powders and the like. It contained no examples of any stabilized bioactive products such as vaccines or pharmaceuticals. In addition, it does not consider the possibility of making an injectable preparation (parenteral) using PFC as a suspension vehicle for the active particles. In order to achieve a stable formulation of inherently brittle biomolecules with a long shelf life using PFC as the non-aqueous carrier, the particles will preferably be formulated so as to contain a glass-forming agent capable of stabilizing the product asset. It may be from a variety of sugars, including trehalose, lactitol, palatinit, etc., as described in PCT No. WO 91/18091 or, more preferably, more effective monosaccharide aldols or glass forming agents as described in the patent application Pat . UK No. 9820689.9.

In order to prevent the particles from floating in the dense PFC phase, it is advantageous to incorporate a density regulating agent into the particles, e.g. an inorganic salt. This may 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. Non-toxic, insoluble materials are preferred since the release of large amounts of ionic salts into the body can cause considerable local pain and irritation. The insoluble materials may in some cases, such as in vaccine preparations, be part of the active preparation as adjuvants. The density regulator may be in solid solution in the glass particles or in particulate matter suspended in the glass. When correctly formulated, the glass particles have a density approximately equivalent to that of the PFC liquid, are buoyancy neutral, neither float nor sink, but remain in stable suspension without cake formation.

Since PFC liquids are good electrical insulators, with a typical resistivity of more than 1013 ohm.cm, tiny surface charges on the suspended particles can have significant effects on the stability of the suspension. In order to prevent the suspended particles from aggregating due to weak short-range forces, they are preferably fabricated containing an excipient, such as lysine or aspartic acid, capable of donating a weak residual electrostatic charge to the dried particles, this prevents aggregation by ensuring the charge repulsion of the particles, similar to what is observed in stable colloids. Alternatively, small amounts of FHC surfactants, such as perfluorodecanoic acid, may be advantageously dissolved in the PFCs to give dispersed, preferably monodisperse, suspensions.

These particles may be manufactured in a variety of ways, including air drying, spray-drying or freeze-drying, and need not be particularly small, but may have a heterogeneous blend ranging in size from 0.1 μ to 100 μ in diameter. For some applications, even particles of the order of millimeters may be suitable. The use of such stable suspensions is not restricted either to parenteral use as exemplified above or to oral use as exemplified in Kirkland (1995). As the liquid PFC vehicle is non-toxic and non-reactive, it is an ideal vehicle for mucosal delivery, including intrapulmonary, intraocular, intra rectal and intravaginal delivery. The ability of this patent to produce stable, non-irritant, sterile formulations for even very unstable mucosal delivery of drugs or vaccines constitutes a considerable advance. Also, the very dry and completely non-hygroscopic nature of the PFC liquid greatly helps in maintaining the sterility of these preparations during prolonged storage and intermittent use since the microorganisms can not grow in the absence of water.

Because perfluorohydrocarbons and volatile chlorofluorocarbons have long been used as propellants in inhalers designed to deliver drug delivery into the deep lung, the stable PFC formulations described herein are ideal for generating fine hairs of liquid STASIS droplets for intrapulmonary delivery. For this application, the size of the particles forming the discontinuous suspended phase in the PFC droplets is important and should not exceed 1 to 5 μm, preferably 0.1 to 1 μm in diameter. For delivery to other mucosal surfaces, the particle size is less important and may be up to 100 μm or even several mm in diameter.

The microparticles preferably have a water content of not more than 4% and preferably less than 2% and ideally less than 1%. 14

ΕΡ 1 452 171 / EN

Alkaline phosphatase (Sigma Aldrich Ltd.) was stabilized on a glass based on 33.3% mannitol, 33.3% calcium lactate and 33.3% degraded gelatin (Byco C, Croda Colloids Ltd.), spray-dried in the form of microspheres and stored at 55 ° C as a dry powder or as a stable suspension in Perfluorodecalin. Activity remained near the 100% mark (103% at 20d and 94% at 30d). There was more loss in dry powder that was not suspended in PFC (remained about 80% of activity).

Figure 2

A commercial tetanus toxoid vaccine (# T022 kindly provided by Evans Medeva pic) was formulated as a powder of equivalent density using calcium phosphate added in 20% trehalose solution. It was freeze-dried to liquid nitrogen using a two-fluid atomizer followed by freeze-drying of the frozen microsphere powder in a Labconco freeze drier with the initial shelf temperature at -40øC throughout the entire main drying. The antibody response of six groups of 10 guinea pigs was measured 4, 8 and 12 weeks after being injected with the same dose of reconstituted ASSIST stabilized tetanus toxoid vaccine in saline buffer or as anhydrous preparations in oil or PFC.

Responses to all dried preparations were lower than that of the fresh vaccine control (not shown) indicating a significant loss of immunogenicity in spray drying. The antigenicity of the toxoid, as measured by capture ELISA, was not altered by the drying process. This suggests that further work is needed to improve the conservation of the adjuvant aluminum hydroxide on drying. The response to the STASIS vaccine with calcium phosphate-adjusted density (group 3) is essentially the same as that of the control vaccine reconstituted in aqueous buffer (group 1) and the powdered oil vaccine (group 2) while control animals 15 ΕΡ 1 452 171 / ΡΤ injected with non-aqueous vehicles alone (groups 4 & 5) showed no response.

Ten examples of how the invention may be carried out will now be described by way of illustration. EXAMPLE 1: PFC spray dried particles,

The particles were produced by spray drying from aqueous solution using a Labplant SD 1 spray drier using sugars and other excipients. Typical formulations were: 15% w / v mannitol calcium lactate in water 15% w / v B trehalose 15% w / v calcium phosphate in water 15% w / v

The particles were produced using a two-fluid atomizer with a liquid orifice of 0.5 mm internal diameter. It was found that a semi-peak air flow in the atomizer was optimal and the drying chamber was operated at an inlet temperature of 135øC and an outlet temperature of 70-75øC. The particles were collected in a glass cyclone and subjected to secondary drying in a vacuum oven using a ramp of temperatures up to 80øC over 4 hours. After cooling they were suspended in PFC using ultrasound. Whether a 30 second pulse of ultrasonic energy from a titanium probe in an MSE MK 2 ultrasonic booth operating at about 75% of power or immersion in an ultrasonic bath with Decon FS200 frequency scanning for up to 10 minutes, were sufficient. The resulting suspension was monodisperse and consisted of spherical polymer particles ranging in size from about 0.5 to 30μ with an average of about 10μ as assessed microscopically. The mannitol / calcium lactate particles rose to the top of the PFC layer over several minutes but could readily be resuspended with gentle stirring. The trehalose / calcium phosphate particles had a density practically equivalent to that of the PFC and formed a stable suspension.

Powder-dried powders of sugar glass particles were suspended in perfluorhexane, perfluorodecalin and perfluorophenanthrene at 1, 10, 20 and 40% w / v. They were found to give rise to monodisperse suspensions with little tendency to aggregate. Addition of 0.1% perfluorodecanoic acid to the PFC inhibited any slight tendency for aggregation to the surfaces. These suspensions were found to pass easily through a 25 g needle by aspiration or ejection. EXAMPLE 2: Stability of Glass Stabilized Enzyme Suspension in PFC 5 Alkaline phosphatase (Sigma Aldrich Ltd.) was spray-dried on a Labplant machine as 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 in either the dry powder form or as a suspension in perfluorodecalin. The enzyme formulated in these microspheres consisting of a mannitol-based glass suspended in perfluorodecalin exhibits retention of almost 100% of the enzymatic activity for more than 30 days at 55øC (Fig 1). EXAMPLE 3: In vivo efficacy

Preclinical trials 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 (a laboratory approved by the World Health Organization). The results of this assay showed that the stable STASIS preparation was completely equivalent to a liquid water-based 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 ready-to-inject formulation

With the same bioavailability in vivo as a conventional liquid water-based formulation. EXAMPLE 4: Dry particles by spray-drying with spray

Particles were also prepared by spraying liquid droplets to liquid nitrogen and then drying under vacuum the frozen powder. These particles were less dense than the spray-dried powders and formed slurries in PFCs in concentrations greater than 20% w / v. At lower concentrations they formed a monodisperse suspension after sonication. Typical formulations used were

Substance Final Concentration w / w Trehalose 100% B Trehalose 50% Calcium phosphate 49.5% Aluminum hydroxide 0.5% EXAMPLE 5: Hydrophobic particles ground

The hydrophobic sugar derivatives octa-acetate sucrose and octa-acetate trehalose readily form glass when extinguished from the melt or dried rapidly from solution in chloroform or dichloromethane. Their use has been described as controlled delivery matrices for drug delivery (Roser et al. &Quot; Solid delivery systems for controlled release of molecules incorporated therein & methods of making same &quot; PCT Pub No. WO 96/03918 1994).

A trehalose octaacetate powder was prepared by melting in a muffle and quenching the melt on a stainless steel plate. The resulting glass disks were milled with mortar and pestle and then in a high speed homogenizer to produce a fine powder. This was suspended in perfluorohexane, perfluorodecalin and perfluorophenanthrene 10% w / v ale. They were found to give rise to well dispersed suspensions. 18 ΕΡ 1 452 171 / ΡΤ

These suspensions were found to pass easily through a 23g needle. EXAMPLE 6: Reconstitution in aqueous environments.

Due to the nature of the soluble sugar glass particles and the properties of the PFCs, it was anticipated that the active compounds in these suspensions would release rapidly in the body. In order to demonstrate the complete release of an included active ingredient, particles were formulated which contained: 20% w / v 20% w / v 0.5% w / v 1% w / v

Trehalose

Calcium Lactate Mordant Blue Dye Lysine 9 The formulation was spray-dried as above and added to perfluorophenanthrene and perfluorodecalin to produce opaque, dark blue, 20% w / v suspensions. Upon addition of water to an equal volume of the suspensions and stirring, it was found that virtually all of the blue dye had been released into the water phase which formed a clear blue layer which floated on the practically colorless PFC with a very clear interface between them . EXAMPLE 7: Non-reactivity between suspended particles

As the individual microspheres in a PFC suspension are physically isolated from all other particles, the potentially reactive substances may be present together in the same suspension in separate particles without any danger of interacting. When the sugar glass is dissolved and the molecules can join, the reaction takes place. In order to demonstrate this, a suspension was made to contain two types of particles, one (a) with the alkaline phosphatase enzyme and the other (b) with its colorless substrate, paranitrophenyl phosphate. The formulations were: Î »E 1 452 171 / ΡΤ a Trehalose 10% w / v Sodium sulfate 10% w / v Alkaline phosphatase 20 U / 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 a suspension of the perfluorodecalin powders containing 10% w / v powder &quot; a &quot; and 10% w / v powder &quot; b &quot; does not develop any colored reaction but remains in the white suspension form for three weeks at 37øC.

Upon addition of water and stirring, the powders were dissolved in the upper aqueous phase. The enzymatic reaction occurred in a matter of minutes, producing an intense yellow color of p-nitrophenol in both the freshly prepared sample and the one that had been maintained at 37 ° C for 3 weeks. EXAMPLE 8: Product release in &quot; tissue space &quot;

In order to illustrate the possible behavior of the PFC suspensions when injected in vivo, a clear, hydrated tissue space template was prepared by molding 0.2% agarose gels into polystyrene bottles. 0.1 ml of the perfluorodecalin suspension of Example 5 was injected through a 25 g needle into the agarose gel. This produced a flat white ball of suspension. Over the next 5-10 minutes the white color became clear from the bottom of the ball up leaving a clear sphere of PFC. As the enzyme and the substrate were released by dissolving the glass particles, they reacted mutually to produce a yellow color of p-nitrophenol, which then diffused throughout the agarose over the next hour. 20 ΕΡ 1 452 171 / ΡΤ EXAMPLE 9: Equivalence of densities,

Sugar glass particles (i.e trehalose) obtained by the conventional drying methods exhibit typical densities of about 1.5 g / cm 3. The perfluorocarbons we have tested typically have densities ranging from 1.68 to 2.03 g / cm 3 (Table I). For this reason, when formulated in a suspension, the sugar glass particles tend to float over the PFC layer, leading to a preparation in which the active ingredient is not homogeneously distributed. The powders may, however, be modified so as to produce a stable suspension in PFCs in which they have a neutral buoyancy and neither set nor float. This can be achieved by the addition of high density materials prior to particle formation. These may be soluble or insoluble in water.

Materials not soluble in water The tricalcium orthophosphate has a density of 3.14 g / cm 3, is approved as an adjuvant for vaccines and is practically insoluble in water. Powders prepared to contain about 50% calcium phosphate exhibit an increased density of about 2 g / cm 3 and at 20% solids form stable suspensions in perfluorophenanthrene. Examples of powders that 20% solids in PFCs form stable suspensions include: 1. In perfluorodecalin

Substance Final Concentration w / w Trehalose 50% Calcium Phosphate 50% B. Trehalose 47.5% Calcium Lactate 10.0% Calcium Phosphate 42.5% 21 ΕΡ 1 452 171 / ΡΤ 2.In perfluorophenanthrene:

Substance Final Concentration w / w Mannitol 18.2% Inositol 18.2% Calcium Lactate 18.2% Calcium Phosphate 45.4%

Other non-water soluble density enhancing materials which have been used include barium sulfate and titanium dioxide. Any insoluble and non-toxic material of appropriate density may be used.

Water soluble materials

Soluble salts such as sodium sulfate having a density of 2.7 g / cm 3 may also be used as density increasing agents. The following powder formed stable suspensions in perfluorodecalin:

Substance Final concentration w / w Trehalose 50% Sodium sulphate 50%

Other high-density non-toxic water-soluble materials may also be used. These formulations have been found to cause discomfort after subcutaneous injection into guinea pigs, possibly due to the rapid dissolution of high ionic salt concentrations. EXAMPLE 10: Effect of density matching on active substances in suspensions

Certain vaccines are formulated adsorbed onto gels or insoluble particles which act as adjuvants. Aluminum hydroxide and calcium phosphate are widely used for this purpose. These insoluble adjuvants may themselves be used to increase the density of the particles which are to be suspended. In this case the high density material is not completely inert but in fact adsorbs the active macromolecule from the solution. It is necessary to demonstrate that this adsorption does not denature the active substance. To test this, alkaline phosphatase was used as the active substance / vaccine model.

The following solution was prepared

10% w / v Adjuvant Quality Calcium Phosphate (Superphos Kemi a / s) Trehalose 10% w / v 1mM 1mM 20 U / ml

ZnCl2

MgCl2

Alkaline phosphatase

In Tris 5mM-HCl buffer, pH 7.6 The solution was then well mixed for 10 minutes at 37 ° C to allow alkaline phosphatase to be adsorbed by calcium phosphate. This change in the absorption per minute was measured by centrifugation of calcium phosphate, sampling the supernatant and measuring the enzymatic kinetics using p-nitrophenyl phosphate as the substrate and a wavelength of 405 nm. The solution was spray-dried to give a fine powder. Any desorption of the enzyme after rehydration of the powder was measured in the supernatant as above. The powder was suspended at 20% w / v in perfluorophenanthrene and found to produce a stable suspension.

(25 μΐ) 0.409 Supernatant from above (25 μΐ) 0.034 Rehydrated powder (25 μΐ from 20% w / v in water) 0.425 Supernatant from above (25 μΐ) 0.004 powder at 20 ° C % w / v in perfluorodecalin (25 μΐ) 0.430 23 ΕΡ 1 452 171 / ΡΤ Experience shows: • The particle density can be equated to that of the PFC vehicle by the inclusion of the calcium phosphate adjuvant. • There is no significant desorption or loss of enzymatic activity during the formulation process.

Lisbon, 2010-03-01

Claims (4)

A pharmaceutical composition comprising an active ingredient carried by sugar glass particles having a diameter in the range of 0.1 to 100 micrometers, said particles being suspended in a biocompatible perfluorocarbon liquid, characterized in that the densities of the particles and the biocompatible liquid are equalized so that the particles remain in a stable suspension. Composition according to Claim 1, characterized in that the particles contain an inorganic salt as a density regulating agent. Composition according to Claim 2, characterized in that the inorganic salt is calcium phosphate. Composition according to any one of the preceding claims, characterized in that the particles contain calcium lactate. Lisbon, 2010-03-01