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WO1999047588A1 - Production de microparticules - Google Patents

Production de microparticules Download PDF

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Publication number
WO1999047588A1
WO1999047588A1 PCT/GB1999/000685 GB9900685W WO9947588A1 WO 1999047588 A1 WO1999047588 A1 WO 1999047588A1 GB 9900685 W GB9900685 W GB 9900685W WO 9947588 A1 WO9947588 A1 WO 9947588A1
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WO
WIPO (PCT)
Prior art keywords
microparticles
polymer
emulsifier
aqueous
solvent
Prior art date
Application number
PCT/GB1999/000685
Other languages
English (en)
Inventor
Gordon Findlay Dawson
Frank Koppenhagen
Original Assignee
Cenes Drug Delivery Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cenes Drug Delivery Limited filed Critical Cenes Drug Delivery Limited
Priority to BR9908755-3A priority Critical patent/BR9908755A/pt
Priority to CA002324235A priority patent/CA2324235A1/fr
Priority to EP99907729A priority patent/EP1068257A1/fr
Priority to AU27364/99A priority patent/AU2736499A/en
Priority to JP2000536777A priority patent/JP2002506901A/ja
Publication of WO1999047588A1 publication Critical patent/WO1999047588A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/09—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in organic liquids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C08J3/14—Powdering or granulating by precipitation from solutions

Definitions

  • the present invention relates to a method of producing microparticles for use in delivering a pharmaceutically active substance, particularly a peptide, protein or polynucleotide, and to the microparticles themselves and pharmaceutical compositions thereof.
  • the present invention relates to the so-called phase separation method of producing microparticles wherein an emulsion of water droplets is formed in a continuous phase comprising a polymer dissolved in a non-aqueous solvent such as dichloro ethane.
  • the polymer is caused to coacervate out of solution around the water droplets by the addition of a non-solvent for the polymer, such as a silicone oil.
  • the process is generally carried out under vigorous agitation using a mixer to prevent coalescence of the droplets or incipient microparticles .
  • a problem of this method is that the so-called stability window (i.e. the relative ratios of the components of the mixture e.g. silicone oil non-solvent, polymer, water and non-aqueous solvent which results in the successful formation of microparticles) is rather small. This limits the practical application of the method. For 2 example, when a ternary diagram is drawn representing silicone oil, polymer and dichloromethane solvent there is only a small area representing a very restricted range of these components (typically around 36% silicone oil, 4% polymer and 60% dichloromethane) which results in microparticles. It would be desirable to have a method of producing microparticles which was operable over a wider range of concentrations, in particular so that it could be adapted for use with a variety of active substances of varying molecular weight, solubility, polarity etc.
  • so-called stability window i.e. the relative ratios of the components of the mixture e.g. silicone oil non-solvent, polymer, water and non-aque
  • the so-called water-in-oil emulsion used to prepare the microparticles is an unstable emulsion and vigorous agitation is required in order to maintain a small droplet size and prevent coalescence during the polymer precipitation phase separation step.
  • a high viscosity silicone oil is used as the non-solvent (since low viscosity oils tend to result in an even more reduced stability window) .
  • the vigorous agitation or mixing of the system leads to the generation of large amounts of heat, which can be undesirable where the pharmaceutically active material to be loaded into the microparticles is a heat sensitive material, such as a peptide or protein.
  • heat tends to promote evaporation of volatile non-aqueous solvent (e.g. dichloromethane) leading to lack of process 3 control.
  • cooling systems such as dry ice or liquid nitrogen; but such cooling methods not preferred for large scale industrial application.
  • Patent specification US3,531,418 describes the production of a polymer solution at high temperature. As the solution cools, the polymer is precipitated out of solution in the form of microparticles around a solid active agent core. Solid active agents may need to be ground to size, which may generate heat and denature heat- sensitive active agents. Moreover, use of a non-aqueous solvent in direct contact with the active agent can also result in denaturation . Alternatively, aqueous solutions may be encapsulated.
  • US Patent 4,166,800 also relates to the preparation of microspheres by low temperature (-40° to -100°C) phase separation of a polymer and a core material. US4,389,331 employs cooling to room temperature as the phase separation step.
  • Patent specification US4,622,244 describes a standard phase separation method wherein phase separation is brought about by the addition of a phase separation agent, such as a polymeric material or a non-solvent for the polymer being used to produce the microparticles. Phase separation occurs either at low temperatures of at least -30°C or at room temperature. However, isolation of the microparticles has to be carried out at a temperature of -30°C or lower. 4 US Patent 4,673,595 describes hardening of the microparticles at a temperature of between 0° and 25°C employing particular aliphatic fluorinated or fluorohalogenated hydrocarbons as hardening agents.
  • Silicone oil non-solvent is used as a phase separation agent.
  • Patent specification W089/03678 describes the use of a second non-solvent such as heptane to harden the microspheres prior to collection thereof.
  • Patent specifications EP0377477 and US5,066,436 describe the use of other hardening agents such as fatty acid esters.
  • Patent specifications US5,000,886 and US5,500,228 describe the use of volatile silicone oils as hardening agents, so as to facilitate removal thereof so that there is no residue in the microcapsules .
  • Patent specifications GB2234896 and US5,603,960 disclose the use of surfactants during microparticle production.
  • Patent specification GB2234896 discloses the use of a hardening mixture comprising heptane and Span 80 surfactant oil-in-water emulsion to harden the microspheres and to remove non-encapsulated active peptide material so as to avoid an initial burst of active when the microparticles are administered to a patient.
  • Patent specification ⁇ s ⁇ , 603,960 describes a reversal of the standard phase separation technique, wherein the aqueous dispersion is formed in silicone oil non-solvent, and the solution of polymer in dichloromethane is added thereto to 5 initiate phase separation.
  • Span 40 surfactant may be included in the aqueous dispersion of active in the silicone oil non-solvent.
  • An object of the present invention is to provide a process having a broad stability window.
  • the present invention is based on the use of a emulsifier to stabilise the system.
  • the present invention provides a method of producing microparticles, which comprises; incorporating an emulsifier in an aqueous liquid and/or a non-aqueous solution containing a polymer; forming a dispersion of the aqueous liquid in the non- aqueous solution; and agitating the dispersion and adding thereto a non- solvent for the polymer so as to form polymer microparticles .
  • the present invention includes a emulsifier in the aqueous liquid which forms the aqueous discontinuous phase of the "water-in-oil" dispersion and/or in the continuous "oil” phase; and is present at the interface between the aqueous droplets and the non-aqueous solvent.
  • a emulsifier has been found to expand the stability window, and so to provide a process which is capable of producing microparticles over a relatively wide range of conditions (especially quantities of non-aqueous solvent, aqueous liquid, polymer, non-solvent and active 6 substance). It allows larger quantities of water and of polymer to be included in the dispersion. The process is thus robust and adaptable to a variety of active substances.
  • the process should be suitable for the sustained release of small peptide molecules. It also enables lower viscosity non-solvents to be employed, which may not be possible in the absence of the emulsifier.
  • the use of a emulsifier has been found to allow non- solvents and agitation conditions to be used, which are such that undue heat is not generated during the production of the polymer microparticles thereby protecting any pharmaceutically active agent incorporated therein from thermal degradation.
  • the aqueous liquid comprises a pharmaceutically active agent suspended or dissolved therein.
  • the pharmaceutically active agent can be any active solid or liquid substance but the method of the present invention is particularly applicable to those active agents which are susceptible to thermal degradation at temperatures above room temperature (i.e.
  • active agents are proteins and peptides, such as enzymes, hormones, antigens etc. and those which exert a therapeutic or prophylactic effect, or can be used as diagnostic agents.
  • the peptides or proteins may be recombinant, synthetic or from natural sources.
  • tne peptide or protein may be lysozyme, insulin, thyrotropin releasing hormone (TRH) , luteinising hormone releasing hormone (LHRH) or analogues thereof (e.g. Leuprolide) , or cytochrome C.
  • TRH thyrotropin releasing hormone
  • LHRH luteinising hormone releasing hormone
  • analogues thereof e.g. Leuprolide
  • cytochrome C cytochrome C.
  • the emulsifier is usually incorporated in the aqueous liquid or non-aqueous solvent by being dissolved or emulsified therein.
  • the emulsifier is usually present in an amount of up to 60% by weight, generally up to 30% by weight, preferably up to 20% by weight, typically 5-15% by weight of the liquid. There is generally at least 1% by weight of emulsifier present.
  • the emulsifier is a non-ionic surfactant, such as those having a hydroxyl- containing hydrophilic portion and a long chain fatty acid lipophilic portion. Typical non-ionic surfactants are available under the trademarks Span, Tween and Brij .
  • Span type materials are partial esters of the common fatty acids (lauric, palmitic, stearic, and oleic) and hexitol and anhydrides (hexitans and hexides) , derived from sorbitol.
  • Tween type materials are derived from the Span materials by adding polyoxyethylene chains to the non-esterified hydroxyl groups. Span products tend to be oil-soluble and dispersable or insoluble in water; while Tween products are soluble or well dispersed in water.
  • Brij surfactants include polyoxyethylene ester groups.
  • Preferred non-ionic surfactants are Span 20, 40, 60, 65 and 80.
  • Anionic surfactants or cationic surfactants (such as quaternary ammonium compounds) may also be used.
  • the HLB value of the surfactant is normally in the range 2 to 9.
  • the emulsifier (which may or may not be a surfactant) may be any pharmaceutically acceptable emulsifying agent o O and may be non ionic, for example gum arabic, alginic acid, cetostearyl alcohol, cetyl alcohol, a glucose fatty acid ester, glyceryl monooleate, glyceryl monostearate, hydroxypropyl cellulose, a medium chain triglyceride, low molecular weight methylcellulose , a poloxamer, a polyoxyethylene alkyl ether, a polyoxyethylene castor oil derivative, a polyoxyethylene fatty acid ester, a polyoxyethylene stearate, polyvinyl alcohol, a sorbitan fatty acid ester, or a sucrose fatty acid ester; cationic, for example cetrimide, monoethanolamine or triethanolamine; or anionic, for example a cholic acid derivative, carbomer, docusate sodium oleic acid, propylene glycol
  • the aqueous liquid is dispersed in a non- aqueous solution containing a polymer, generally a pharmaceutically acceptable polymer for pharmaceutical applications.
  • a polymer generally a pharmaceutically acceptable polymer for pharmaceutical applications.
  • the dispersion will be unstable and requires vigorous agitation. The degree of agitation may be determinative of the droplet size of the discontinuous aqueous phase.
  • the surfactant also has an effect in controlling particle size.
  • the aqueous liquid is usually 0.3-50%, generally 5-50%, especially 10-20% by weight of the dispersion. A preferred range is 1-20% by weight.
  • the polymer used for forming the polymer microparticles is generally a pharmaceutically acceptable 9 polymer such as a polyester, polyvinylchloride, polycaprolactone or the well known polylactide family of polymers, in particular a polylactide-co-glycolide polymer.
  • a pharmaceutically acceptable 9 polymer such as a polyester, polyvinylchloride, polycaprolactone or the well known polylactide family of polymers, in particular a polylactide-co-glycolide polymer.
  • the ratio of lactide to glycolide (and endcapping) and molecular weight may be varied and determines the rate of release of the active material from the microparticles.
  • the molar ratio of lactide to glycolide can vary in the range 100:0 to 0:100. However, molar ratios of 100:0 to 50:50 are preferred since the copolymers tend to be soluble in the non-aqueous solvents preferably employed.
  • the ratio is preferably between 70:30 and 35:65 (more preferably 70:30 to 50:50).
  • Preferred copolymers have a lactide to glycolide ratio of 50:50 or 75:25.
  • the number average molecular weight Mn of the polylactide polymer may be in the range 5,000 to 50,000.
  • the inherent viscosity (i.v.) is generally from below 0.2 up to 8. In vivo, such polymers undergo biodegradation by random, non-enzymatic scission to form lactic acid and glycolic acid metabolites. Thus, the bulk degradation of the polymer is determinative of the release time for most of the active agents to be included within the microparticle, such that the microparticles have a sustained release effect.
  • the sustained release period may be up to 365 days but will generally be in the range 5 to i ⁇ O days, typically 10 to 30 days.
  • the use of an emulsifier according to the present invention has been found to produce microparticles of narrow size distribution and excellent consistency of shape. Clearly, it is 10 desirable that the particles should have a consistent spherical shape, which are easier to inject, rather than irregular shapes.
  • the non-aqueous solvent for the polymer solution is generally an organic solvent.
  • the polymer may be present in an amount of up to 25% by weight, preferably m the range 0.5 to 10% by weight, more preferably 1 to 3% by weight.
  • Dichloromethane methylene chloride
  • Dichloromethane methylene chloride
  • a non-solvent for the polymer is added to the continuous non-aqueous phase in order to coacervate the polymer from solution onto the dispersed aqueous droplets.
  • the dispersion is kept in a constant state of agitation in order to prevent coalescence of the aqueous droplets or of the forming microparticles. If the non-aqueous solution has a relatively high viscosity, then the vigorous agitation results in the production of considerable heat, which must be removed by cooling in conventional processes.
  • the non-solvent is a material of relatively low viscosity which is dissolvable in the non- aqueous solvent but does not substantially increase the viscosity tnereor
  • the temperature of the method can be kept to around room temperature and is preferably in the range 10-25°C. However, no external cooling means are required in order to achieve this, thereby substantially 11 facilitating the industrial application of the process.
  • the non-solvent is a silicone oil, such as those available from Dow Corning or Fluka Chemicals (Gillingham, UK) .
  • the Dow Corning 200 series of oils are particularly preferred.
  • the viscosities lie in the range 50-150 mPa, although viscosities of up to 500 may be employed.
  • the prior art has generally employed silicone oils having viscosities of 500 to 1000 mPa and above.
  • the polymer microparticles formed may then be hardened in conventional manner, e.g. by admixing with a non-solvent for the polymer such as a liquid hydrocarbon e.g. heptane or other conventional non-solvents. Agitation may be continued to prevent coalescence of the microparticles until hardening is completed.
  • a non-solvent for the polymer such as a liquid hydrocarbon e.g. heptane or other conventional non-solvents. Agitation may be continued to prevent coalescence of the microparticles until hardening is completed.
  • the microparticles may be filtered and washed. Generally, the microparticles are then dried, which has the effect of removing residual non-aqueous solvent and residual water so as to leave the microparticle consisting substantially of polymer, active agent if present and residual emulsifier (e.g. in amounts up to 5% by weight, more generally up to 2% by weight).
  • the residual emulsifier is beneficial in that it facilitates the resuspension of the microparticles in water where the microparticles are to be used in the form of an aqueous suspension, for example as an injectable sustained release pharmaceutical formulation. 12
  • the loading efficiency is defined as the amount of active substance entrapped in the microparticle divided by the amount introduced into the process .
  • microparticles comprising a pharmaceutically active agent, a emulsifier and a pharmaceutically acceptable polymer. Because of the method of production, the microparticles are substantially free of non-solvent, such as silicone oil. The substantial freedom of the microparticles from hydrophobic non-solvents such as silicone oil, together with the presence of emulsifier ensures that the microparticles of the present invention are particularly easy to resuspend in water in order to provide pharmaceutical compositions thereof.
  • Typical active agents are as follows.
  • LHRH Luteinising Hormone Releasing Hormone
  • TRH Thyrotrophin Releasing Hormone 14
  • a further aspect of the present invention provides pharmaceutical formulations, particularly for injection, comprising the microparticles suspended in an aqueous pharmaceutically acceptable liquid.
  • microparticles of the present invention may also find use in non-pharmaceutical areas such as agriculture (e.g. for the sustained release of pesticides) or in food (e.g. to incorporate sustained release flavourings into chewing gum) and this forms a further aspect of the invention.
  • the microspheres can be used for targetting active agents to specific tissue sites.
  • Drug targetting applications arise from the ability of microspheres to immobilise a depot of a pharmacologically active material in a tissue bed. This can be achieved either by direct injection into the tissue or by administration of particles having the correct size distribution into the arterial system supplying the tissue of interest, the particles then being trapped in the relevant capillary bed.
  • the median particle size Dv50 (also known as Dv0.5) of the microparticles is in the range 2-300 microns, preferably 10-300 microns, more preferably 10-100 microns and particularly 10-50 microns.
  • the Dv50 is the particle diameter having 50% of the total sample volume above and below it. It therefore represents the median volume diameter. This is relevant to active agents delivery systems, since particle volume is 15 related to the quantity of active agent that can be loaded. It is found that the loading efficiences of the active agent in the microparticles of the present invention is good and is generally in the range 5-75%.
  • Example 1 This Example illustrate the use of different emulsifiers (i . e . surfactants) .
  • the procedure of Example 1 was repeated, with the only change that different emulsifiers were used (Span 20,40 and 65), and the volume of silicone oil was 20 mi.
  • the resulting mean particle sizes are shown in Table 2A. TABLE 2A
  • Emulsifier used Particle size ( ⁇ m)
  • 600mg emulsifier was dispensed into a mixing vessel along with 25ml 3% w/v poly (D, L-lactide-co-glycolide) (50:50 ratio, i.v. 0.7) in dichloromethane, and 3ml of demineralised water. The system was then homogenised and 20ml of silicone oil (lOO Pas) added under mixing. The resulting mixture was transferred into 200ml n-heptane and stirred for 1 hour after which the product was allowed to settle. The supernatant was decanted, 200ml fresh n- heptane added, and stirring continued. The resulting product was then collected on a membrane filter.
  • Microparticles were sized by the method of laser light diffraction and the results summarised in Table 2B. This data illustrates that a range of emulsifiers can be utilised in the process.
  • Example 3 shows the effect of different oil viscosities and mixing speeds.
  • the procedure described in Example 1 was repeated, with the only change that 20 ml of silicone oil of viscosity 110 or 378 m.Pa.s was added, whilst the mixing speed was varied (6500, 8600 and 11,500 rp ) .
  • the resulting mean particle sizes are shown in Table 3.
  • silicone oil silicone oil DC200, -100 mPa.s
  • a syringe driver equipped with 50 ml glass syringe.
  • mixing was continued for 1 minute.
  • the resulting mixture was then added to 200ml stirred n- heptane.
  • Stirring was continued for a minimum of 30 minutes, whereafter stirring was stopped, and the formed microparticles allowed to settle.
  • Supernatant was decanted, another 200ml of n-heptane added, and stirring continued for at least 30 minutes.
  • stirring was stopped, and the microparticles were collected over a 21 cellulose ester filter (1.2 ⁇ m pore size).
  • microparticles were dried under vacuum overnight. Microparticle sizes were determined using laserlight diffraction, and the entrapment of BSA in the microspheres determined using a bicinchoninic acid (BCA) protein assay. The results are shown in Table 4.
  • silicone oil silicone oil DC200, ⁇ 100 mPa.s
  • a syringe driver with a 50-ml glass syringe. 22
  • mixing was continued for 1 minute.
  • the resulting mixture was then added to 200 ml stirred n-heptane. Stirring was continued for a minimum of 30 minutes, after which the microparticles were washed and collected as described in Example 4.
  • Mean microparticle size was determined by laserlight diffraction and found to be 17 ⁇ m.
  • the entrapment of lysozyme in the microspheres was determined using a BCA protein assay. The entrapment efficiency was 56%.
  • silicone oil silicone oil DC200, -100 mPa.s
  • syringe driver equipped with a 50-ml glass syringe.
  • mixing was continued for 1 minute.
  • the resulting mixture was then added to 200 ml stirred n-heptane. The stirring was continued for a minimum of 30 minutes, following which the microparticles were washed and collected as described in Example 4.
  • Mean microparticle size was determined by laserlight diffraction.
  • the entrapment of TRH in the microspheres was determined by 23 dissolving the microspheres in dichloromethane, extracting TRH into an aqueous phase, and determining the extracted amount of TRH by high performance liquid chromatography. The results are shown in Table 5.
  • lml of cytochrome C solution 25.32mg was mixed with approximately 1.5g Span 80 (20% w/w) or 1.5ml of water and the total aqueous phase adjusted to 3g with water.
  • 25ml of 10% w/v RG503 (a 50:50 lactide/glycolide copolymer) solution in dichloromethane was then added, and the system mixed at lOOOOrp for 1 minute.
  • 25ml Silicone oil was added at 2ml/minute whilst mixing following which mixing was continued for a further minute.
  • the resulting system was poured into 200ml of HPLC grade n-heptane and mixed for 1 hour.
  • Microsphere yield was determined gravimetrically , and the particle size distribution measured by laser light scattering (Malvern Mastersizer) . Results are given in Table 7.
  • silicone oil viscosity has little influence on microsphere yield, although it does appear to influence the particle size distribution (albeit in an unpredictable manner) .
  • the most important effect observed was the inability to produce microspheres in the absence of emulsifier (a precipitate resulting) , even with the most viscous oil at low temperatures.
  • this 26 appears to conflict with the prior art which demonstrates particle production without surfactant.
  • the conventional surfactant-free process is only able to produce microspheres within a very narrow range of conditions, and it is likely that the systems manufactured above are outside this stability window. This further illustrates the wide stability window of the process of the invention.
  • a disadvantage of the conventional phase separation process is the relatively narrow range of conditions over which microparticles can be produced and the consequently restricted range of microparticles available.
  • microparticulate systems were manufactured with and without emulsifier and over a range of polymer concentrations, aqueous phase volumes and protein loading levels.
  • Span 80 was dispensed to a mixing vessel along with RG505 50/50 ratio lactide/glycolide copolymer (PLGA) dissolved in dichlorometnane and cytochrome C solution.
  • the composition of these systems is summarised in Table 8.
  • Each system was homogenised whilst silicone oil (lOOmPa.s) was added at 2ml/min. Samples were taken at designated 27 intervals and dispersed into l00-200ml n-heptane. The product that resulted was allowed to settle, rinsed with further heptane and recovered by filtration. Samples were dried and the nature of each product determined following the addition of water and brief sonication. The presence of a microparticulate product was indicated by formation a fine dispersion.
  • microparticles can be produced over a far wider range of conditions in the presence of an emulsifier.
  • cytochrome C when high levels were used it was impossible to produce particles at the higher level of polymer concentration in the absence of an emulsifier.
  • the process was unable to form microparticles in the absence of an emulsifier when low levels of cytochrome C were used even at low polymer concentration.
  • TRH is a very small molecule (3 amino acids) and is therefore more difficult to entrap than larger species.
  • This experiment was performed using RG503 (a similar 50/50 co-polymer to RG505 but having a lower molecular weight) and a 75/25 lactide/glycolide copolymer.
  • burst release A failing of many microparticulate systems is rapid 29 load release during the very early phase of the release profile. Known as burst release, this can be due to the presence in the formulation of free active compound, or the active's association with the particle surface rather than its entrapment. The importance of burst release arises due to the high potency of the active typically incorporated into microparticles and the risk of the development of toxic plasma levels.
  • burst release can be attenuated by rinsing the product with an aqueous buffer prior to storage and administration. This, however, will result in the loss of a considerable quantity of often expensive active compound. It would be preferable for the formulation method to result in complete load entrapment.
  • TRH loaded microparticles were prepared. 3g of an aqueous phase containing 400mg Span 80 and 54mg TRH was transferred into a mixing vessel along with 25ml of lactide/glycolide copolymer in dichloromethane. This mixture was homogenised, and 20ml silicone oil added under constant mixing. The resulting system was transferred into 200 ml n-heptane under stirring, the resulting product allowed to settle, supernatant decanted, further heptane added and stirring continued. Microparticles were recovered by filtration and dried.
  • Entrapped TRH was determined by HPLC following particle dissolution in dichloromethane and peptide extraction into an aqueous phase. Microparticles were then washed with demineralised water, in order to remove any 30 non-entrapped or loosely associated active. Rinsed particles were re-dried and their TRH loading determined as above .
  • this example demonstrates the ability of the phase separation process of the invention to promote high entrapment efficiency.
  • SOOmg Span 80 was dispensed to a mixing vessel along with 25ml of polymer solution. Demineralised water was added and the system homoqenised. Silicone oil was added under constant mixing and the resulting system decanted into 200ml n-heptane and stirred. Stirring was halted, allowing the product to settle. The supernatant was 31 decanted, fresh n-heptane added, and stirring continued. Microparticles were then collected on a membrane filter and allowed to dry overnight. Microparticle diameters were determined by laser light diffraction. The results of this study are reported in Table 10 and indicate that the process described is flexible with respect to the polymers that can be utilised.
  • PCL Poly (caprolactone)
  • PVC Poly (vinylchloride)
  • Span 80 800mg Span 80 was dispensed into a mixing vessel along with 3ml demineralised water and 25ml of 3% (w/v) poly(D,L- lactide-co-glycolide) (50:50 molar ratio, i.v. -0.5) in dichloromethane.
  • the system was then homogenised and 20ml of coacervation agent added at a rate of 2ml per minute under constant mixing.
  • the resulting mixture was transferred into 200ml n-heptane, and stirred. Stirring was halted, allowing microparticles to settle.
  • the 32 supernatant was decanted, 200ml fresh n-heptane added, and stirring continued.
  • the microparticulate product was then collected on a membrane filter and allowed to dry overnight.
  • Microparticle size distributions were determined by laser light scattering, and the results summarised in Table 11. These data indicate that the phase separation process here described is not limited by the use of silicone oil.
  • Span 80 was transferred into a mixing vessel along with 2ml of an aqueous phase containing 20mg leuprolide and 40ml 3% (w/v) poly (D, L-lactide-co-glycolide) (50:50 molar ratio, i.v. -0.7) in dichloromethane.
  • This mixture was homogenised and 30ml silicone oil (lOOmPas) added under constant mixing.
  • the resulting system was transferred into n-heptane and stirred.
  • the resulting product was allowed to settle, the supernatant decanted, fresh heptane added and mixing continued. Microparticles were recovered by filtration. Manufacture was carried out in triplicate and the batches combined.
  • microparticle size distribution was determined by laser light diffraction as 40 ⁇ m.
  • the leuprolide content of 33 the microparticles was determined as 0.71%w/w by HPLC following particle dissolution and extraction of the peptide into an aqueous phase.
  • Leuprolide release was determined by re-suspending a quantity of microparticulate material in 10mm MES buffer (pH 7.2) and incubating the system at 37°C. At the required time, samples were drawn, centrifuged and the leuprolide present in the aqueous supernatant determined by UV absorbance. This data is summarised in Figure 3, which demonstrates the controlled release of leuprolide from microparticles over a period of 55 days.
  • leuprolide loaded microparticles manufactured as detailed in Example 13
  • blank microparticles blank microparticles with free leuprolide or free leuprolide according to the dose schedule given in Table 12.
  • Administration was by subcutaneous injection into the right flank, and injection sites were marked with a marker pen in order to facilitate their excision at necropsy.
  • Injection site tissue was finely shredded and homogenised in a mixture consisting of 10ml hexane and 5ml phosphate buffer. Following homogenisation, the homogeniser head was rinsed with phosphate buffer, 34 rinsing's being added to the sample. Samples were centrifuged, the hexane removed, 5ml dichloromethane added and then shaken overnight. Samples were then extracted with phosphate buffer several times and the leuprolide content of the combined extracts determined by HPLC.
  • LHRH 35 analog for example leuprolide
  • leuprolide results in the suppression of plasma testosterone levels following an initial surge.
  • This activity affords a convenient method for the assessment of LHRH bio-activity, and was used to assess the activity of leuprolide formulated into microspheres as detailed in Example 13.
  • Rats were treated with leuprolide loaded microparticles, blank microparticles, free leuprolide and free leuprolide co-administered with blank microparticles as outlined in Example 14. Blood samples were drawn periodically, and plasma testosterone levels determined by radioimmunoassay .
  • Leuprolide loaded microparticles were prepared by the phase separation method. 3ml of an aqueous phase containing 400mg span 80 and 20mg leuprolide was transferred to a mixing vessel to which was added 25ml of 1.5% (w/v) poly (D, L-lactide-co-glycolide) (50:50 molar ratio, i.v. -0.7) in dichloromethane. This mixture was homogenised and 25ml silicone oil added under constant mixing. The resulting system was transferred into 200 ml n-heptane under stirring and the resulting product allowed to settle, supernatant decanted, further n-heptane added and stirring continued. Microparticles were dried and their particle size determined by laserlight diffraction.
  • Entrapped leuprolide was determined by HPLC following particle dissolution in dichloromethane and the extraction of peptide into an aqueous phase. Microparticles were found to have a diameter of 3l ⁇ m and a leuprolide content of 0.75%w/w.
  • Blank microparticles were prepared by the phase separation method. 3ml of an aqueous phase containing 400mg Span 80 was transferred to a mixing vessel to which was added 25ml of 3%(w/v) poly (D, L-lactide-co-glycolide) (50:50 molar ratio, i.v. -0.7) in dichloromethane. This mixture was homogenised and 20ml silicone oil added under constant mixing. Tne resulting system was transferred into 200 ml n-heptane under stirring and the resulting product allowed to settle, supernatant decanted, further n-heptane added and stirring continued. Microparticles were dried 37 and were found to have a diameter of 45 ⁇ m and to contain no entrapped leuprolide.
  • Both blank and leuprolide loaded microparticles were dispersed in a buffer consisting of 1% sodium carboxymethyl cellulose, 0.2% Tween 80, 0.14% methyl p-hydroxybenzoate, 0.014% propyl p-hydroxybenzoate and 5% sorbitol.
  • Groups of 5 male Sprague-Dawley rats were dosed with 125mg/kg of microparticles, either blank or leuprolide-loaded, 7.5 mg/kg leuprolide or an appropriate volume of vehicle. Rats were sacrificed after 28 days, the injection sites excised, and subjected to histopathological investigation.
  • microparticles manufactured by the method here described are bio-compatible and do not result in any irritation other than that expected from the injection of any foreign body.
  • Silicone oil was determined by NMR spectroscopy . 38 Standards were prepared by dispensing 50mg of PLGA into glass vials and dissolving this material in dichloromethane laced with known quantities of silicone oil (lOOmPas). Following polymer dissolution, solvent was removed by evaporation, and the standards vacuum desiccated. 1ml deuterated chloroform was then added, the material shaken overnight, and the 1 H NMR spectrum of each standard determined. Samples were handled in a similar way except that the chloroform used to dissolve microparticles was silicone oil free. The silicone oil/solvent proton was calculated from the 1 H spectrum. This ratio was used to construct a liner calibration series for standards, from which unknown's could be determined. The results of this study are summarised on Table 13 and indicate that the process described above has the potential to produce microparticles with very low levels of residual silicone oil.
  • Residual dichloromethane and n-heptane were determined by GC. Samples were weighed into glass vials and dissolved in 1,4-dioxane. Iso-octane, containing 2-butoxyethanol as an internal standard, was then added to each vial in order to precipitate the polymer. Precipitate was removed by centrifugation and the samples analysed by GC(SGE BPX5 25mx0.32mm, l ⁇ l split injection (20:1 split) at 280°C, Helium, 2ml/min) . Calibration was carried out using heptane and DCM dissolved in 1,4-dioxane and isooctane (1:2) with 2-butoxyethanol as an internal standard. The results of this study are summarised in Table 13 and 39 indicate that the microsphere production method here described has the potential to produce product with very low levels of residual solvent.
  • Residual Span 80 was determined by UV absorbance (A 232 ) following dissolution of samples in acetonitrile, and solvent extraction of the emulsifier. Quantification was carried out by reference to the absorbencies obtained for known standards. The results of this study are summarised in Table 13 and indicate the microparticles produced by this method are associated with quantity of residual emulsifier. This may act to the benefit of the product since it may increase surface hydrophilicity and therefore aid particle wetting and redispersion.

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Abstract

L'invention concerne un procédé pour former des microparticules (et tout particulièrement celles comprenant un agent pharmaceutiquement actif) qui consiste à incorporer un émulsifiant à un liquide aqueux ou à une solution non aqueuse (p.ex., à du dichlorométhane) contenant un polymère; à former une dispersion de liquide aqueux dans une solution non aqueuse; et à l'agiter en ajoutant un non-solvant (p.ex., de l'huile de silicone) pour le polymère. La présence de l'émulsifiant permet de produire des microparticules dans des conditions les plus variées.
PCT/GB1999/000685 1998-03-14 1999-03-15 Production de microparticules WO1999047588A1 (fr)

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BR9908755-3A BR9908755A (pt) 1998-03-14 1999-03-15 Processo de produzir micropartìculas, e, micropartìcula
CA002324235A CA2324235A1 (fr) 1998-03-14 1999-03-15 Production de microparticules
EP99907729A EP1068257A1 (fr) 1998-03-14 1999-03-15 Production de microparticules
AU27364/99A AU2736499A (en) 1998-03-14 1999-03-15 Production of microparticles
JP2000536777A JP2002506901A (ja) 1998-03-14 1999-03-15 微粒子の製造

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2832312A1 (fr) * 2001-11-21 2003-05-23 Inst Nat Sante Rech Med Procede de preparation de microparticules sans solvant toxique, microparticules obtenues selon ce procede, utilisations et compositions pharmaceutiques
WO2005105278A3 (fr) * 2004-05-05 2006-08-03 Akzo Nobel Nv Procede de solidification d'emulsion antisolvante
WO2006123361A3 (fr) * 2005-03-01 2007-04-12 Sun Pharmaceutical Ind Ltd Compositions pharmaceutiques a liberation prolongee
JP2011037882A (ja) * 2001-08-31 2011-02-24 Alkermes Inc 残留溶媒抽出方法および同により生成される微粒子
WO2013023007A1 (fr) * 2011-08-08 2013-02-14 Landec Corporation Particules à libération contrôlée, prolongée, pour traiter des graines et des plantes
WO2013090828A2 (fr) 2011-12-16 2013-06-20 Biofilm Ip, Llc Compositions d'injection cryogéniques, systèmes et procédés pour la modulation cryogénique de l'écoulement dans un conduit
US20140323415A1 (en) * 2002-11-19 2014-10-30 Holger Petersen Organic compounds
GR1010327B (el) * 2021-10-06 2022-10-17 Φαρματεν Α.Β.Ε.Ε., Παρατεταμενης αποδεσμευσης ενεσιμο φαρμακευτικο σκευασμα λεβοθυροξινης και μεθοδος παρασκευης αυτου

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WO1989003678A1 (fr) * 1987-10-30 1989-05-05 Stolle Research & Development Corporation Microspheres a faible solvant residuaire et procede de microencapsulage
JPH01158042A (ja) * 1987-02-03 1989-06-21 Toray Ind Inc 球状ポリマ微粉末の製造方法
WO1993025195A1 (fr) * 1992-06-16 1993-12-23 Centre National De La Recherche Scientifique (Cnrs) Preparation et utilisation de nouveaux systemes colloidaux dispersibles a base de cyclodextrine, sous forme de nanospheres
WO1994027718A1 (fr) * 1993-05-25 1994-12-08 Hagan Derek Thomas O Preparation de microparticules et procede d'immunisation

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JPH01158042A (ja) * 1987-02-03 1989-06-21 Toray Ind Inc 球状ポリマ微粉末の製造方法
WO1989003678A1 (fr) * 1987-10-30 1989-05-05 Stolle Research & Development Corporation Microspheres a faible solvant residuaire et procede de microencapsulage
WO1993025195A1 (fr) * 1992-06-16 1993-12-23 Centre National De La Recherche Scientifique (Cnrs) Preparation et utilisation de nouveaux systemes colloidaux dispersibles a base de cyclodextrine, sous forme de nanospheres
WO1994027718A1 (fr) * 1993-05-25 1994-12-08 Hagan Derek Thomas O Preparation de microparticules et procede d'immunisation

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011037882A (ja) * 2001-08-31 2011-02-24 Alkermes Inc 残留溶媒抽出方法および同により生成される微粒子
JP2013216676A (ja) * 2001-08-31 2013-10-24 Alkermes Pharma Ireland Ltd 残留溶媒抽出方法および同により生成される微粒子
JP2015227383A (ja) * 2001-08-31 2015-12-17 アルカームズ ファーマ アイルランド リミテッド 残留溶媒抽出方法および同により生成される微粒子
FR2832312A1 (fr) * 2001-11-21 2003-05-23 Inst Nat Sante Rech Med Procede de preparation de microparticules sans solvant toxique, microparticules obtenues selon ce procede, utilisations et compositions pharmaceutiques
WO2003043605A1 (fr) * 2001-11-21 2003-05-30 Institut National De La Sante Et De La Recherche Medicale Procede de preparation de microparticules sans solvant toxique, microparticules obtenues selon ce procede, utilisations et compositions pharmaceutiques
US20140323415A1 (en) * 2002-11-19 2014-10-30 Holger Petersen Organic compounds
WO2005105278A3 (fr) * 2004-05-05 2006-08-03 Akzo Nobel Nv Procede de solidification d'emulsion antisolvante
WO2006123361A3 (fr) * 2005-03-01 2007-04-12 Sun Pharmaceutical Ind Ltd Compositions pharmaceutiques a liberation prolongee
WO2013023007A1 (fr) * 2011-08-08 2013-02-14 Landec Corporation Particules à libération contrôlée, prolongée, pour traiter des graines et des plantes
WO2013090828A2 (fr) 2011-12-16 2013-06-20 Biofilm Ip, Llc Compositions d'injection cryogéniques, systèmes et procédés pour la modulation cryogénique de l'écoulement dans un conduit
US9677714B2 (en) 2011-12-16 2017-06-13 Biofilm Ip, Llc Cryogenic injection compositions, systems and methods for cryogenically modulating flow in a conduit
GR1010327B (el) * 2021-10-06 2022-10-17 Φαρματεν Α.Β.Ε.Ε., Παρατεταμενης αποδεσμευσης ενεσιμο φαρμακευτικο σκευασμα λεβοθυροξινης και μεθοδος παρασκευης αυτου

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AU2736499A (en) 1999-10-11
CA2324235A1 (fr) 1999-09-23
GB9805417D0 (en) 1998-05-06

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