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WO1999003452A1 - Procedes d'elimination de solvant residuel dans les compositions medicamenteuses administrees par voie nasale - Google Patents

Procedes d'elimination de solvant residuel dans les compositions medicamenteuses administrees par voie nasale Download PDF

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Publication number
WO1999003452A1
WO1999003452A1 PCT/US1998/014790 US9814790W WO9903452A1 WO 1999003452 A1 WO1999003452 A1 WO 1999003452A1 US 9814790 W US9814790 W US 9814790W WO 9903452 A1 WO9903452 A1 WO 9903452A1
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WO
WIPO (PCT)
Prior art keywords
gelatin
microspheres
drug
oil
emulsion
Prior art date
Application number
PCT/US1998/014790
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English (en)
Other versions
WO1999003452A9 (fr
Inventor
Kris P. Antonsen
Rajiv Nayar
Wei Wang
Margaret Caudle
Michael A. Shearer
Neville M. Consessio
Original Assignee
Bayer Corporation
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 Bayer Corporation filed Critical Bayer Corporation
Priority to EP98935751A priority Critical patent/EP0994700A4/fr
Priority to CA002296620A priority patent/CA2296620A1/fr
Priority to IL13388298A priority patent/IL133882A0/xx
Priority to AU84935/98A priority patent/AU747211B2/en
Priority to JP2000502753A priority patent/JP2001510150A/ja
Publication of WO1999003452A1 publication Critical patent/WO1999003452A1/fr
Publication of WO1999003452A9 publication Critical patent/WO1999003452A9/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
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compositions for delivery of drugs intended to reside in the nose.
  • the invention also pertains to. methods of making such nasal drug delivery compositions and to improved methods of removing solvents from pharmaceutical preparations.
  • intranasal preparations which are specifically designed to deliver drugs across the nasal mucosal membranes to effect systemic drug administration.
  • intranasal preparations specifically designed to retain the drugs for extended periods of time in the nose without crossing the mucosal membranes.
  • Ilium et al. PCT/GB95/01735 describe a drug delivery composition for nasal administration comprising ICAM-1 and bioadhesive compositions comprising chitosan, a liquid polymeric material, or a variety of water-swellable microspheres, including gelatin.
  • Ilium discloses the concept of emulsifying a warm aqueous solution of gelatin and ICAM-1 in a vegetable oil containing a surfactant, followed by thermal gelation, hardening with acetone, harvesting the ICAM/gelatin microspheres, and drying.
  • an intranasal formulation include: (1) a pharmaceutically acceptable formulation with respect to process consistency, scalability, and GRAS (generally regarded as safe) components;
  • Criteria for a commercially suitable process for producing such a product include minimal solvent consumption and demonstrated scalability to at least 300 g.
  • the present invention provides these and other advantages.
  • the present invention provides an improved process for production of pharmaceutical gelatin-based microsphere compositions comprising one or more drugs to be delivered to and retained in the nose, and the drug-containing microspheres produced by this process.
  • the invention also concerns improved methods of removing solvents from dry powder pharmaceutical compositions.
  • the process for making the microsphere/drug composition comprises the following steps: a) preparing a low-salt or salt-free aqueous solution of gelatin and the drug to be delivered at a temperature above the gelation temperature of the gelatin;
  • step (a) emulsifying a mixture of the solution of step (a) and the solution of step (b) to generate a water-in-oil emulsion wherein the volume ratio of (a) : (b) is between 1 :2 and 1 :10;
  • the invention also comprises an improved method for removing residual solvent from pharmaceutical matrices such as dry powders by contacting said matrices with a humidified stream of a suitable gas under conditions which permit residual solvent to be entrained in the gas, and then drawing off the gas. This can be done in discreet batches or as part of a continuous flow process.
  • the resulting product is a free-flowing powder comprising gelatin microspheres associated with drug, with a volume particle diameter of between 40 - 60 ⁇ m and wherein a preponderance of the microspheres exist as unaggregated individual particles, rather than as aggregates of smaller particles.
  • the invention further comprises an intranasal drug delivery system comprising the above gelatin microspheres in association with a drug to be delivered to and retained in the nose.
  • Fig. 1 shows impellers suitable for use in the present invention: (A) the A310 is a high-efficiency axial flow impeller; (B) the A200 is a four-blade axial flow impeller; and (C) the R100 is a radial-flow impeller. All are manufactured by Lightnin Equipment.
  • Fig. 2 shows results of the gelatin:oil ratio studies of Example 2.
  • Fig 3 shows the preferred position of the impellers in the emulsification step in (A) for volumes below 3 L and (B) for volumes of 3 L and above.
  • 3" radial impeller top left quadrant, 12 deg to vertical
  • (2) 4" radial impeller (3) baffles.
  • Fig. 4 shows a schematic representation of a batch emulsification process suitable for preparation of microspheres.
  • gelatin solution 50 °C
  • oil/Span mixture 50 °C
  • water in oil emulsion (4) acetone; (5) solid microspheres suspended in oil; (6) acetone washings; (7) oil/acetone; (8) acetone; (9) microspheres; (10) vacuum; (11) 75 ⁇ m mesh sieve; (12) dried microspheres.
  • Fig. 5 shows scanning electron micrographs showing structure of tlCAM(453)/gelatin microspheres made by the process of Example 8_(panels A and
  • the mean volume particle diameter of the preparation in panels A and B is 46.4 ⁇ m, with 0.5% of particles having a volume particle diameter >100 ⁇ m and undetectable levels of particles having a volume particle diameter ⁇ 10 ⁇ m.
  • the mean volume particle diameter for panels C and D is 123 ⁇ m, with 39% >100 ⁇ m and 33% ⁇ 10 ⁇ m.
  • Fig. 6 shows appearance of the emulsion droplets and microspheres at various stages during the batch emulsification process of Example 15 using baffles, gradual cooling, and high mixing speed: (A) emulsion droplets at 50 °C; (B) emulsion droplets at 40 °C; (C) emulsion droplets at 35 °C; and (D) emulsion droplets at 30 °C.
  • Fig. 7 shows appearance of the emulsion droplets and microspheres at various stages during the batch emulsification process of Example 15 using baffles, moderate cooling, and high mixing speed: (A) emulsion droplets at 625 rpm (1 min);
  • Fig. 8 shows appearance of the emulsion droplets and microspheres at various stages during the batch emulsification process of Example 15 using baffles, rapid cooling, and a high mixing speed: (A) emulsion droplets immediately following gelatin addition at 375 rpm; (B) emulsion droplets following mixing at 625 rpm for 20 min; (C) gelatin microspheres suspended in oil following mixing for one hour below 15 °C at 625 rpm; and (D) gelatin microspheres following washing, filtration and vacuum drying.
  • Fig. 9 shows appearance of the emulsion droplets and microspheres at various stages during the low mixing speed process of Example 15 using baffles and rapid cooling: (A) emulsion at 425 rpm (30 min); (B) cooled emulsion at 15 °C; and
  • FIG. 10 shows typical particle size distribution obtained by laser particle sizing of microspheres generated by the rapid cooling method of Example 15, using baffles, higher impeller height, and lower mixing speed.
  • gelatin should be food-grade, preferably at least NF grade.
  • the gelatin should have a bloom strength of at least 80, and preferably of > 150.
  • a bloom strength of 250 is most preferred because its properties are fairly consistent from batch to batch and it is available year round. Its color is lighter and has a fainter odor than lower-quality gelatin grades. Higher bloom strengths provide superior color, odor, and nasal residence time in the final product.
  • Suitable gelatin is, e.g. P-8 grade, 250 bloom, obtained from Hormel Foods (Austin, MN).
  • the concentration of gelatin may conveniently be in a range of from about 1% to about 30% (w/w). Within this range, higher gelatin concentrations lead to reduced solvent and oil consumption, but the total solids content in the aqueous solution (including gelatin and drug to be delivered) should not exceed 30% (w/w). Because gelatin concentration did not appear to affect the ultimate volume particle diameter, 20%, which is the highest easily-handled concentration based on viscosity, is preferred.
  • the drug to be delivered may be any suitable chemical or biological pharmaceutical agent that is capable of being dissolved or suspended in a gelatin solution and that is insoluble in the oil and insoluble in the dehydrating solvent used in the process.
  • suitable drugs include but are not limited to, e.g., small chemical entities; proteins, peptides, and polypeptides (e.g. ICAM-1 and other antiviral agents; vaccines, antigens, antibodies; lymphokines, and fragments of any of the foregoing); nucleotides (e.g. genes, DNA, RNA, and fragments of any of the foregoing) carbohydrates, etc.
  • the concentration of the drug to be delivered is similarly determined by the total allowable concentration of the loaded solids.
  • the aqueous solution of gelatin and drug may be prepared by any convenient means. Solutions of gelatin and drug may be prepared individually and then mixed, or either the gelatin or the drug may be added to a solution of the other. Persons skilled in the art will understand that buffering or adjustment of the pH may be needed to protect the drug during processing. Also, for protein drugs it is preferable that the aqueous solution be low-salt or salt-free to avoid precipitating the protein. By “low salt” is meant ⁇ 200 mOs/kg.
  • ICAM-1 When the drug is ICAM-1 , a solution of ICAM-1 in histidine buffer, pH 7, is conveniently prepared and added to an aqueous solution of gelatin. L-histidine is obtained from, e.g., Calbiochem Corp.
  • ICAM immunohistidine
  • the term "ICAM” as used herein is intended to refer to ICAM-1 and any fragments, analog or derivative of ICAM-1 which retains the ability to bind to human rhinovirus of the major receptor group and inhibit infectivity.
  • the ICAM may be prepared as set forth in Example 1 below.
  • Gelatin-containing solutions should be held at a temperature above the gelation temperature of the gelatin. For a gelatin with a bloom strength of 250, this means at 40 °C or higher.
  • the oil is preferably a refined oil, e.g. mineral or vegetable oil, preferably food-grade or NF grade vegetable oil.
  • suitable vegetable oils are corn, soybean, and safflower. Hyperrefined vegetable oils from which color bodies, hydrophilic substances, and natural surfactants have been removed do not work as well as less refined oils in this procedure.
  • Corn oil and soybean oil are preferred; corn oil is particularly preferred because it is more economical.
  • Suitable corn oil is e.g. NF grade as obtained from, e.g., Ruger Chemical Co. (Welch, Holme and Clark).
  • Suitable surfactants are Span 80 (sorbitan monooleate, obtained from Ruger Chemical Co., ICI Americas Inc.), lecithin, and pluronic L 1011 (BASF). Span 80 is preferred. In general, a hydrophilic-lipophilic balance (HLB) of 4-6 is desired. Surfactants with HLB values of 3 or below (e.g. glyceryl monooleate, sorbitan trioleate) are less effective. A surfactant concentration of 0.1% is too low, 1% is functional, and higher concentrations provide no further improvement in the result.
  • HLB hydrophilic-lipophilic balance
  • the aqueous solution of gelatin and drug is emulsified with the solution of oil and surfactant is emulsified to generate a water-in-oil emulsion.
  • emulsification Methods of emulsification are well-known in the art. Generally, the aqueous gelatin/drug solution is gradually added to the oil/surfactant solution with stirring or mixing. Proper agitation during emulsification is required to achieve the desired mean volume particle diameter (40-60 ⁇ m). It is possible to use a variety of stirrers or impellers, but it is preferred to minimize the formation of a vortex to avoid air entrainment. This can be achieved through the use of baffles or correct positioning of the impeller. Initial specific power consumption is preferably at least 1.4 watts/L. High-shear mixing should be avoided. Mixing conditions affect the final product and so care must be taken to reproduce mixing conditions to insure reproducibility of the final product.
  • the volume ratio of gelatin/drug solution to oil/surfactant solution affects the quality of the resulting product.
  • the volume ratio of gelatin/drug solution to oil/surfactant solution is between 1 :2 and 1 :10. Larger ratios are desirable because the emulsion volume, and the oil and solvent requirements, are reduced.
  • the mean volume particle diameter rises gradually with volume ratios up to 1 :2. At higher ratios, the mean volume particle diameter rises dramatically, and at even higher ratios, emulsion inversion takes place, making particle formation impossible. Volume ratios of 1:2 to 1:5 are preferred; 1 :3 to 1 :4 are particularly preferred.
  • Emulsification is continued until the target droplet size is obtained.
  • Droplet size is monitored by methods known to those in the art, e.g. by optical microscopy. Droplets of 20-80 ⁇ m are preferred; 40-60 ⁇ m are particularly preferred. For a volume of 8 L, a mean volume particle size of approximately 50 ⁇ m can be achieved in approximately 30 min ( ⁇ 10 min).
  • the temperature of the emulsion is reduced to below the gelation temperature of the gelatin at a controlled rapid rate to achieve gelation before droplet coalescence, thus allowing the formation of gelatin microspheres with associated drug at the desired particle size.
  • the emulsion is cooled to a temperature of 23 °C or lower.
  • the cooling rate is between 1-4 °C/min.
  • a cooling rate of between 2.0-2.5 °C/min is particularly preferred.
  • separation is most efficient if the microspheres are separated from as much of the oil as possible at the outset. This can be done by physical means, such as allowing the microspheres to settle under gravity or by centrifugation followed by decantation. Separation can also be accomplished by washing with suitable solvents. It is important that the emulsification be agitated gently during the washing process. If washing is used, there are several possible alternatives to remove the oil:
  • the oil is removed by washing first with volume hydrocarbon solvent such as heptane, then by washing with a water-miscible solvent such as acetone.
  • the hydrocarbon solvent is chosen as follows: i) it should be miscible with both the oil and the water-miscible wash chosen below; ii) it should not dissolve appreciable water; and iii) its density should be less than mixtures of water and the water-miscible solvent containing up to 20% water.
  • the hydrocarbon wash reduces the oil phase viscosity and density, allowing the microspheres to settle easily with gravity. This permits a large portion of the hydrocarbon phase to be decanted immediately.
  • a single wash with 0.5 emulsion volumes is sufficient. The wash is carried out by brief stirring (5 min) at room temperature. Two such washes are sufficient to remove a majority of the oil.
  • the remaining washes are with a water-miscible solvent such as acetone (HPLC grade, J.T. Baker) or low molecular weight alcohols.
  • a water-miscible solvent such as acetone (HPLC grade, J.T. Baker) or low molecular weight alcohols.
  • the initial water-miscible wash will lead to three phases (1) the gelatin microspheres, (2) a liquid phase (mostly water- miscible wash), and (3) another liquid phase floating on the water-miscible wash and containing mostly the hydrocarbon and the remaining oil.
  • This splitting of the two liquid phases is a consequence of extracting the water from the gelatin microspheres.
  • the two organic phases separate easily from each other and from the solids.
  • An advantage of this approach is that the microspheres are transferred into the water-miscible solvent-rich phase, where they will remain throughout the rest of the washes. Typically the hydrocarbon-rich phase is eliminated after the initial water-miscible solvent wash.
  • the second method is to wash only with water-miscible solvent. This process is carried out using sequential stirring, settling, and decantation steps as above. This has the virtue of requiring only one solvent, but is somewhat more complex because the initial water-miscible wash again results in a splitting of the liquid into two phases. In this case, however, the oil-rich phase is the lower one, and the beads are not separated immediately from the oil and surfactant.
  • the acetone because it also acts as a solvent, for water, does not appreciably reduce the oil-phase viscosity, and ordinarily at least two water-miscible washes of 1 emulsion volume each are necessary to remove the oil phase. Phase separation is slower than when hydrocarbon is used as in (a) above.
  • the now-dehydrated gelatin/drug microspheres, suspended in water-miscible solvent, are collected.
  • Methods of separating the microspheres from the water- miscible solvent are well-known to those in the art. Examples of suitable means are filtration using e.g. a Buechner funnel or centrifugafion using ordinary or basket centrifuges.
  • the process of the present invention is suitable for scale-up more than 100 x over that described by Ilium et al.
  • An advantage of the present process is that the microsphere product has no particles having volume particle diameters less than 10 ⁇ m as measured by laser light scattering analysis.
  • the laser light scattering technique the angular variation in intensity of light scattered from a plume of the particles in air is measured, using as a light source a laser of defined wavelength.
  • the scattering data are deconvoluted to provide a volumetric particle size distribution. Instruments suitable for making these measurements are known to those skilled in the art and are available from e.g. Malvern Instruments, Southboro, MA.
  • the desired particle size range is achieved by use of high-bloom strength gelatin (preferably at least 150, most preferably 250 or greater), use of a low-salt or salt-free buffer for the aqueous gelatin/drug solution, consistent low-vortex or vortex-free stirring during emulsification to achieve consistent droplets of the target size, and rapid cooling upon attainment of desired droplet size to prevent aggregation.
  • high-bloom strength gelatin preferably at least 150, most preferably 250 or greater
  • use of a low-salt or salt-free buffer for the aqueous gelatin/drug solution consistent low-vortex or vortex-free stirring during emulsification to achieve consistent droplets of the target size
  • rapid cooling upon attainment of desired droplet size to prevent aggregation The improved process eliminates the need for a centrifuge and facilitates large batch processing. It also uses less solvent, is more efficient, costs less, and results in better product than the prior art processes.
  • the invention further comprises a novel method of removing volatile solvent from a pharmaceutical matrix.
  • the process involves contacting said pharmaceutical matrix with a suitable humidified gas under conditions which permit residual solvent to be entrained in the gas, and removing the solvent/gas mixture.
  • the humidified gas is passed through a fluidized bed of the pharmaceutical matrix.
  • the pharmaceutical matrix may be any pharmaceutical formulation, including but not limited to, e.g., beads, polymer or nonpolymer processed materials, drug- related raw materials, excipients and final products including biopharmaceuticals, including dry powder microspheres such as gelatin microspheres prepared according to the above process, and oil-based microspheres such as liposomes known in the art.
  • the invention is particularly suitable for use with matrices which tend to physically trap entrained solvent within the matrix, so that procedures such as heat or vacuum drying do not effectively remove the solvent.
  • the solvent may be any residual volatile solvent remaining after the basic preparation process.
  • solvents commonly used in the preparation of pharmaceutical formulations include water, acetone, and aliphatic alcohols (e.g. methanol, ethanol, isopropyl alcohol), DMSO, chloroform, methylene chloride etc.
  • the gas may be any suitable gas which is nonreactive with the pharmaceutical formulation and which is capable of removing and carrying the solvent.
  • suitable gases are air, nitrogen, argon, and carbon dioxide.
  • the relative humidity in the gas should be from about 85 to about 96 %.
  • the purpose of the humidity is to retain sufficient moisture within the pharmaceutical matrix.
  • low moisture content inhibits escape of the solvent.
  • the solvent is water
  • lower relative humidities should be used. Humidities of 0-50% are preferred, depending on the water sorption and desorption properties of the matrix.
  • the matrix could simply be placed in a gas-tight chamber with an appropriate salt solution to obtain a specific high humidity.
  • the evaporated solvent would not be able to leave the closed chamber and therefore, would equilibrate with the solvent adsorbed in the matrix.
  • stagnant dry matrix easily aggregates under high relative humidities.
  • humidified gas could be passed over the matrix but three problems would still exist: (1) a matrix such as a powder would be difficult to contain; (2) dry matrix could be stagnant and could form aggregates; and (3) the evaporated solvent could be hazardous and difficult to control.
  • a fluidized bed is prepared according to methods known to those skilled in the art, see, e.g., Porter, H. F., McCormick, P. Y., Lucas, R. L., and Wells, D. F., "Gas-Solid Systems," in Chemical Engineers' Handbook, 5 th ed., R. H. Perry and C. H. Chilton, eds. (McGraw-Hill, New York, 1973).
  • a column such as a chromatography column may be used.
  • the matrix containing the residual solvent is loaded into the column and humidified gas is passed into the bottom of the column and through the pharmaceutical matrix in order to fluidize the matrix bed.
  • Advantages to using such a column include: (1) the humidified gas can be evenly distributed through the whole area of the bottom filter support, exposing the pharmaceutical matrix homogeneously to the gas stream, and (2) the column can be easily dismantled for sample loading, unloading, and cleaning. Because of the gas flow, the pharmaceutical matrix is completely fluidized inside the column. The continuous fluidization reduces aggregation of dry matrix.
  • Residual water is then removed by passing dry gas through the matrix and removing the humidified gas.
  • dry means having less than 50% relative humidity.
  • Apparatuses suitable for use in the present invention are available commercially from, e.g., Fluid Air Inc., Aurora, IL; Niro Inc., Columbia, MD. Other variations will be apparent to those skilled in the art, for example methods of modifying the apparatus and procedure as necessary for various matrices and batch sizes.
  • a humidity of 92% at room temperature is capable of reducing residual acetone level to 200 ppm or below within 8 hours. It is believed that acetone is adsorbed and bound strongly in the gelatin matrix by hydrogen bonding, which prevents its removal from the polymer under relatively high vacuum and/or even at temperatures higher than its boiling point.
  • the hydrogen-bound acetone molecules are replaced quickly by water molecules.
  • the decreased glass transition temperature of gelatin at higher moisture contents also increases the rate of acetone diffusion out of the gelatin matrix.
  • the result is a dry powder having a pharmaceutically acceptable level of residual solvent.
  • the resulting product is a free-flowing dry powder comprising gelatin microspheres containg the desired drug. Specifically, the following parameters are preferred:
  • D(v,0.9), D(v,0.1) and D(v,0.5) are the 90th, 10th and 50th percentile volume particle diameter of the microspheres, respectively.
  • the drug microspheres of the present invention may be administered as prepared above or may be compounded in a pharmaceutical preparation in which such drug microspheres comprise the active ingredient or one of a plurality of active ingredients, or may be mixed with microspheres containing other drugs.
  • Drug- containing gelatin microspheres may be mixed with placebo gelatin microspheres containing no drug to achieve blends with lower concentration of drug per unit volume or weight.
  • Suitable pharmaceutical preparations may, for example, take the form of ointments, gels, pastes, creams, sprays (including aerosols), lotions, powders, suspensions, solutions and emulsions of the active ingredient in suitable excipients.
  • suitable excipients include pharmaceutically acceptable fillers and extenders, binding agents, moisturizing agents, agents for retarding dissolution, disintegrating agents, resorption accelerators, surface active agents, adsorptive carriers, and lubricants. It is understood that the excipient(s) must be chosen to preserve the integrity and activity of the drug microspheres and to be compatible with the selected route of administration.
  • the pharmaceutical preparations which are powders and sprays can, for example, contain appropriate diluents, e.g. lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder or mixtures of these substances.
  • Aerosol sprays can, for example, contain the usual propellants, e.g. chlorofluorohydrocarbons.
  • compositions which are ointments, pastes, creams and gels can, for example, contain appropriate diluents, e.g. animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures thereof.
  • appropriate diluents e.g. animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures thereof.
  • compositions which are solutions and emulsions can, for example, contain appropriate diluents and emulsifiers known to those in the art, e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (e.g. ground nut oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbital or mixtures thereof.
  • appropriate diluents and emulsifiers known to those in the art, e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylform
  • the pharmaceutical preparations which are suspensions can contain appropriate diluents (e.g. ethyl alcohol, propylene glycol), surface-active agents (e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbite and sorbitane esters), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures thereof.
  • appropriate diluents e.g. ethyl alcohol, propylene glycol
  • surface-active agents e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbite and sorbitane esters
  • microcrystalline cellulose aluminum metahydroxide
  • bentonite agar-agar and tragacanth or mixtures thereof.
  • the pharmaceutical preparations of the present invention can also contain coloring agents and preservatives as well as perfumes.
  • the pharmaceutical preparations of the present invention contain from 0.1 to 99.5% by weight, preferably from 0.5 to 95% by weight, of the drug-containing microspheres in the total composition.
  • compositions are carried out by suitable methods well-known to those in the art.
  • viruses e.g. rhinovirus and adenovirus
  • Antiviral agents suitable for treatment of such infections are known but it is desired to provide formulations for enhanced delivery to such mucosal surfaces, particularly to the nasal passages which are one of the primary routes of infection for such viruses.
  • ICM-1 human major rhinovirus receptor
  • fragments thereof which are capable of residing in the nasal passages for extended periods of time to effect protection against rhinoviral infection in a safe and cost-effective manner.
  • the gelatin microspheres of the present invention are particularly suitable for intranasal administration.
  • intranasally and “intranasal administration” is meant administration to the nasal cavity in a form intended to remain in the nose, including dry powders, nasal drops, nasal spray, and creams, gels, or other formulations suitable for topical application to the nasal cavity.
  • the pharmaceutical gelatin/drug microspheres of the present invention may also be formulated for topical administration, e.g. as creams, lotions, salves, ointments, etc., for topical administration to e.g. to the eye, mouth, ear, skin.
  • Oral formulations may also include troches and mucoadhesive buccal tablets, etc.
  • gelatin/drug microspheres of the present invention are also suitable for topical administration of other water-soluble pharmaceutical chemical and biological agents, e.g. small chemicals such as antibiotics; proteins, peptides, and polypeptides (e.g., vaccines, antigens, antibodies, lymphokines, and fragments of any of the foregoing); nucleotides (e.g.
  • genes DNA, RNA, and fragments of any of the foregoing); carbohydrates; and antiviral agents such as Enviroxine, Pirodavir, interferon-alpha, sialidase inhibitors, acyclovir, adenosine, arabinoside, interferon and interferon -inducing agents.
  • antiviral agents such as Enviroxine, Pirodavir, interferon-alpha, sialidase inhibitors, acyclovir, adenosine, arabinoside, interferon and interferon -inducing agents.
  • Rhinoviruses are members of the picornavirus family and are responsible for 50% of colds in humans over the course of one year. During peak season (mid- September to mid-October) however, rhinoviruses account for up to 75% of all colds. Approximately 90% of all rhinoviruses bind to a major human rhinovirus receptor (HRR).
  • HRR human rhinovirus receptor
  • An antiviral agent comprising the human major rhinovirus receptor or fragments thereof is an effective inhibitor of rhinoviral infection of susceptible cells.
  • the human major rhinovirus receptor is the same as the protein known as intercellular adhesion molecule-1 (ICAM-1), with the exception of a G>A at nucleotide 1462, resulting in the amino acid substitution Glu>Lys at amino acid position 442.
  • IAM-1 intercellular adhesion molecule-1
  • ICAM-1 is a glycoprotein with a molecular weight of 45-50 kD (excluding carbohydrate) and 8 potential sites for N-linked carbohydrate attachment; the glycosylated protein has a molecular weight of 82-88 kD.
  • ICAM-1 has 507 amino acids and consists of a cytoplasmic domain, a transmembrane domain, and five extracellular immunoglobulin-like domains (amino acids 1-88, 89-185, 186-284, 285-385, and 386-453).
  • the presently preferred embodiment for antiviral purposes is a fragment consisting of the first 453 amino acids of the full HRR sequence, which retains rhinovirus binding activity.
  • tlCAM(453) Recombinant tlCAM(453) was purified from fermentation fluid of continuous cell culture of Chinese hamster ovary (CHO) cells containing cDNA coding for tlCAM(453) maintained by continuous perfusion in a customized medium free of all plasma-derived components.
  • tlCAM(453) is meant the first 453 amino acids (domains 1-5) of ICAM-1.
  • the sequence for ICAM-1 is known to those skilled in the art, as are methods of preparing the cDNA coding for tlCAM(453). See, e.g circular Staunton, D.E., S.D. Marlin, C. Stratowa, M.L. Dustin, and T.A.
  • tlCAM(453) obtained from clarified and concentrated fermentation fluid was purified by hydrophobic chromatography, metal ion chromatography, precipitation at low pH, filtration, anion exchange chromatography, hydroxyapatite adsorption, size exclusion chromatography, and a second HAP adsorption step.
  • Viral inactivation was accomplished via low pH precipitation (demonstrated to reduce MuLV and Reovirus titers by 36 and 22 logs, respectively), and by pasteurization (shown to reduce the reference virus titers by an additional 5 logs).
  • the tlCAM(453)-containing solution was sterilized by filtration to yield the liquid drug substance, which was stored at -35° C until formulated into a gelatin-based microsphere powder. Some lots were lyophilized in phosphate-buffered saline (PBS, or 0.15 M NaCI, 0.01 M Na2HPO4, adjusted to pH 7.0) and reconstituted before use.
  • tlCAM(453) was concentrated to >50 g/L and diafiltered into a low-ionic-strength 10 mM histidine buffer, pH 7.0.
  • tlCAM(453) tlCAM(453) was made as in Example 1.
  • Albumin 25% albumin (human) USP (Bayer Corporation, Elkhart, IN)
  • Grades of Gelatin Acid-type gelatin, extracted from porcine skin, was obtained from Hormel Foods (Austin, MN). Gelatin is normally graded by its Bloom strength, a measure of gel strength under defined conditions (see, e.g., US Patent No. 1 ,540,979). These studies were conducted with gelatin with Bloom strengths of 225 (Hormel grade P-7), 250 (grade P-8) and 275 (grade P-9). Oils: A variety of oils was used. Food-grade corn oil was obtained from a grocery store (e.g. Mazola®, CPC International, Englewood Cliffs, NJ). Pharmaceutical or NF grade was supplied by Ruger Chemical (Irvington, NJ). Soybean oil was either food-grade or NF grade from Ruger Chemical.
  • Span 80® sorbitan monooleate
  • Span 85® trioleate
  • Arlacel 186TM glyceryl monooleate
  • Pluronic L-1011TM was from BASF (Mt. Olive, NJ).
  • Lecithin was supplied by Central Soya (Ft. Wayne, IN).
  • Solvents Acetone and n-heptane were reagent grade, purchased from JT Baker (Phillipsburg, NJ), EMScience (Gibbstown, NJ), and Mallinkrodt (Chesterfield, MO).
  • tlCAM(453) was included in order to assess its effect on the process and to evaluate the effect of the process on molecular integrity and biological activity. Finally, the process established in the range-finding experiments was scaled up to the 150-g level to establish commercial utility.
  • Range-finding experiments were conducted by emulsifying a total volume of 200 mL in a 400-mL beaker (7.3 cm diameter). Either a 1.5" radial-flow impeller (item R100, Lightnin Equipment, Milwaukee, Wl) or a 2" high-efficiency axial-flow impeller (item A310) was used. The gelatin solution was added to the oil phase while stirring in initial experiments at 700 rpm. After 10 min, the stirring speed was then lowered to 400 rpm. The designs of the impellers is shown in Fig. 1. In other experiments, the stirring rate was held constant.
  • the initial temperature of emulsification was at least 45 °C, and cooling took place by natural convection. Washing was carried out using two 100-mL washes with heptane, followed by two 100-mL washes with acetone. For each wash, the resulting suspension was either stirred by hand or by using a motor-driven mixer. The solids were allowed to settle after each wash by gravity, and the supernatant removed by decantation. The final powder was collected by vacuum filtration on a Buechner funnel, using #3 Whatman (Fairfield, NJ) filter paper. It was dried overnight under vacuum at 34 - 40 °C.
  • the suitability of the powder was evaluated in stages. Initial evaluation required that the powder be free-flowing, without stickiness attributable to residual oil. Any lumps were removed by passing the powder through a 60- or 70-mesh sieve. Because the goal was to have a powder falling into the size range 10 - 100 ⁇ m, and preferably 20 - 80 ⁇ m, most of the powder should pass easily through either of these sieves, which have cut-off diameters of 250 and 212 ⁇ m, respectively.
  • tlCAM(453) and albumin were calculated on the following basis: i) The moisture content of the gelatin raw material and of the final product were assumed to be similar. Thus, moisture content could be ignored. ii) Only the polypeptide portion of the tlCAM(453) molecule (and not the carbohydrate portion) was taken into account.
  • This expression is based on a mass balance for the preparation of a gelatin solution containing tlCAM(453) (or albumin).
  • f is the mass fraction (or loading) of ICAM(453) (or albumin)
  • c is the concentration, in g/L, of tlCAM(453) (or albumin) in the original solution
  • p is the density of the tlCAM(453) (or albumin) solution, in g/L
  • m i is the mass of the tlCAM(453) (or albumin) solution used.
  • the buffer salt concentration for the tlCAM(453) (or albumin) solution is given by c b (in g/L); this can be significant if the tlCAM(453) (or albumin) concentration is low and the buffer is phosphate-buffered saline. For a low-salt buffer, the correction given by c b is small.
  • the total mass of gelatin used is G g m g for the case where a gelatin solution is mixed with an tlCAM(453) (or albumin) solution.
  • m g represents the mass of gelatin solution, and G g the mass fraction (g/g) of gelatin before adding the tlCAM(453) (or albumin).
  • the solution densities were measured and found to be 1020 g/L for tlCAM(453) at 50g/L and 1070 g/L for albumin at 250 g/L.
  • oil-soluble surfactants having a variety of chemical compositions. These were Span 80 and 85, Pluronic L-1011 , Arlacel 186, and soybean lecithin. Each of these was tested in corn oil at the 1 % level in the 200-mL model system under the conditions shown in Table I:
  • gelatin-oil ratio A310 impeller, at 700/400 rpm (start/end)
  • D[4,3] mean volume particle diameter; the volume particle diameter is the diameter of a sphere having the same volume as the given particle
  • This ratio was defined as the ratio, by volume, of a solution of 20% (w/w) porcine 25 Bloom type A gelatin: corn oil containing 1% Span 80. Ratios of 1 :4 to 1:1 were examined. Higher ratios are preferred because at a given scale, oil and solvent consumption and processing volume are reduced. At the same time, a higher proportion of gelatin solution in the emulsion increases the likelihood that droplets or beads collide with each other, resulting in increased incidence of coalescence or aggregation.
  • Microspheres of the desired size were obtained at a ratio of 1 :3 in multiple runs at varying scales.
  • the difference between ratios of 1 :3 and 1 :2 is slight: at the 200-mL scale, 5 runs at a ratio of 1 :3 led to sizes of 62.8 ⁇ 14.6 ⁇ m, while a single run at 1 :2 led to 77.0- ⁇ m microspheres (Fig.2).
  • Mixing was with a 1.5" R100 (radial flow) impeller, used at 600/700 RPM (start/end).
  • the table shows that before implementing these changes, particles were large and varied in size from batch to batch. It also shows that mixing conditions during emulsion cooling do influence the final diameter.
  • the gelatin solution was set at 20% (w/w) solids. It is difficult to remove entrained air or pour gelatin solutions at higher concentrations. Lower concentrations appeared to offer little advantage, and would require extra oil and solvent per gram of final product.
  • the volume ratio of gelatin to oil was set to 1:3. It is desirable to use as little oil as possible. As described above, reducing the oil to 1 :2 made it more difficult to obtain the desired particle-size distribution.
  • tlCAM(453) was incorporated into the gelatin microspheres made by the process of Example 2 to evaluate the effect of the protein on particle size; determine protein loading; and evaluate the effect of processing conditions on bioactivity.
  • Emulsification conditions were 1 :3 volume ratio of 20% porcine 250 Bloom type A gelatin in water (w/w):1% Span 80 in corn oil.
  • Potency results are expressed as a concentration in ⁇ g/mL necessary to inhibit viral infection of susceptible cells. Thus, higher values reflect lower potency.
  • Runs 1 ,2, and 3 in Table VI were carried out in order to show consistent tlCAM(453) loading, particle size distribution, and yield.
  • bioactivity of the tlCAM(453) released from the powder was measured and found to be comparable to the starting material.
  • tlCAM(453) yield was ⁇ 100%.
  • the nephelometric assay for tlCAM(453) detects both active and inactive tlCAM(453), so the loss of material is not assay-related. The most likely point at which tlCAM(453) is lost is in the initial acetone wash. If all of the water present is extracted into the acetone phase, it will contain 17% water. This may be enough to dissolve tlCAM(453) and perhaps gelatin as well.
  • a scale of 150 g was selected as the target. At this scale (3 L emulsion), cooling under ambient conditions was too slow, therefore the mixing vessel was immersed in a refrigerated water bath at an initial temperature of 50 - 55 °C. The gelatin solution was added to the warm oil, and the refrigeration unit was immediately turned on. It took approximately 70 min before the emulsion reached its final temperature of 10°C.
  • Factors influencing particle size were expected to be: i) impeller size and type ii) impeller position iii) tank dimensions iv) temperature profile with time v) presence of tlCAM(453) (keeping solids at 20%).
  • the stirring speed is limited by the formation of a vortex in the emulsion. Because vortex formation was undesirable, particles of the desired diameter could not be obtained while stirring at constant speed, even when the impeller was placed in the optimal position. As the emulsion cooled, the emulsion viscosity increased, and it was possible to increase the stirring speed during the cooling process without increasing vortexing. When this was done, particles of the desired size were achieved. Therefore it is critical that the stirring accomplish two things: first, to provide sufficient shear to break up the gelatin solution into sufficiently small droplets, and second, to provide sufficient fluid flow so that no stagnant zones form near the walls.
  • baffles did allow an increase in mixing speed over the unbaffled configuration.
  • the gap between the baffle and the tank wall was only 1/8" (3 mm), potentially allowing a build-up of cool, slowly moving material to accumulate. If baffling is used care must be taken to avoid this problem.
  • the table shows that a VA" (8.9 cm) marine propeller with shallow pitch (-10°) served, when driven at 850 rpm, to create microspheres with a mean diameter of 50 - 70 ⁇ m. Incorporation of tlCAM(453) at 10% did not have a significant effect on the particle size.
  • the beneficial rate of cooling adds to the complexity of scale-up. Because cooling is carried out from the vessel walls, there is a boundary layer of cooler and therefore more viscous emulsion near the wall of the tank. This layer is kept thin by mixing action. At higher cooling rates, the thermal (and thus hydrodynamic) boundary layer becomes thicker, and more intensive mixing is necessary to offset this effect.
  • the drug to be delivered is a protein having an isoelectric point such that it tends to precipitate during the process of Examples 3 and 5, adjustments can readily be made to avoid such precipitation.
  • albumin precipitates when added to warm gelatin solution, because gelatin solutions at 20 wt% have pH values of approximately 5, close to the isoelectric point of albumin. Precipitation was avoided by adjusting the pH of the gelatin solution to 6.0-6.1 or by dissolving the gelatin initially in 0.019 M NaOH.
  • the gelatin/tlCAM(453) microspheres are prepared by a process that begins by mixing a solution of tlCAM(453) and gelatin in corn oil to form a water-in-oil emulsion.
  • This emulsification process uses corn oil, gelatin, Span 80, water for injection, and tlCAM(453) in L-histidine buffer as the raw materials.
  • the wet gelatin beads are subsequently washed with acetone to remove the oil and surfactant, and to dehydrate the gelatin beads into a dry microsphere powder. Residual acetone is removed and the final moisture content is fixed by fluidizing a bed of the microspheres with an air stream of controlled humidity.
  • Table X lists the quantitative composition of tlCAM(453)/gelatin microspheres and placebo (gelatin microspheres only) bulk powder preparations prepared by this process.
  • Table X Unit formula listing components for placebo and tlCAM(453)/gelatin microsphere bulk powder preparation.
  • Span 80 and corn oil are reduced to respective levels of ⁇ 0.14 and ⁇ 0.3 mg/g of gelatin microsphere powder.
  • Acetone at the end of the process complies to a limit of ⁇ 250 ppm.
  • tlCAM(453) is concentrated to > 50 g/L and diafiltered into a low-ionic strength 10 mM histidine buffer, pH 7.0.
  • the ultrafiltration/diafiltration (UF/DF) is carried out using a model S10Y30 (30,000 MWCO) tangential-flow ultrafiltration cartridge (Amicon, Beverly, MA) with at least 7 volumes of the histidine diafiltration buffer.
  • the final UF/DF formulated tlCAM(453) bulk is sterile-filtered through a 0.2- micron filter into sterile polyethylene terephthalate copolymer (PETG) bottles and stored at not more than -30°C. Aliquots of the sterile formulated bulk are used to quantitate tlCAM(453) content by immunonephelometry, tlCAM(453) integrity by SDS-PAGE, bioactivity by the cell-based plaque assay, and microbial load.
  • Immunonephelometry is a method for assaying the concentration of a specific protein in solution based on measurement of the intensity of light scattered from precipitation formed by mixing the protein of interest with antibodies reactive against it. Automated instruments for carrying out this assay are manufactured, for example, by Behring Diagnostics, Inc. (Somerville, NJ).
  • the tlCAM(453) is thawed by placing the bottle in a water bath set at not more than 40°C.
  • a solution composition of 104.7 g of gelatin, 14.9 g of tlCAM(453) in histidine buffer from step 1 , and sufficient water for injection for a total weight of 600 g is prepared in a 1 L glass beaker.
  • This tlCAM(453) gelatin solution has a total solids content of 20% (w/w) with tlCAM(453) comprising 12.4% of the total solids.
  • the mixture is stirred while covered for not more than 30 minutes in a 50°C water bath.
  • the speed of the mixer in the oil solution is increased to 300 rpm, and the warm tlCAM(453) gelatin solution is gently poured into the oil/surfactant solution over a period of approximately 1 minute.
  • the mixer speed is then increased to 425 rpm to form gelatin-water droplets in the oil/surfactant phase.
  • the water bath set-point is reduced to 10°C and the emulsion mixed until its temperature reaches 15°C.
  • the mixer speed is increased at 10-minute intervals to the maximum speed attainable without causing air entrainment.
  • This process cools and solidifies the gelatin-tlCAM(453) aqueous droplets into gel beads.
  • the emulsion is mixed for not less than 60 minutes at a mixing speed of not more than 640 rpm. This step hardens and stabilizes the gelatin/tlCAM(453) beads.
  • the cooled emulsion is transferred into a 7.6 L stainless steel beaker and the gel beads are subsequently separated from the oil and surfactant by washing the emulsion with acetone.
  • Six washes with acetone remove the oil/surfactant phase of the emulsion and dehydrate the tlCAM(453)/gelatin into microspheres of the target particle size.
  • microspheres are collected on a 3.0-micron Teflon®-type FS filter (Miliipore, Bedford, MA) in a Buechner funnel set on a filtration flask connected to a vacuum line.
  • the microspheres are dried into a free flowing powder by drawing a vacuum until no further filtrate emerges from the funnel.
  • All the microspheres from the Buechner funnel are transferred to a 60-mesh stainless steel sieve and sieved into a collecting pan.
  • This powder corresponds to one of two sub-batches of the tlCAM(453)/gelatin microsphere bulk powder batches that are combined prior to the next manufacturing step.
  • the powder is transferred into a type-Ill flint glass bottle capped with a
  • Teflon®-lined lid and stored at 2-8°C until removal of the acetone as set forth in Example 15 below.
  • Placebo gelatin microspheres are prepared by a procedure which follows steps 4 to 7 of the procedure of Example 8 above, omitting the addition of tlCAM(453).
  • the process steps used for manufacturing both tlCAM(453)- loaded microspheres and placebo microspheres are identical except that slightly different gelatin:oil ratios are employed in the emulsification processes for tlCAM(453)-loaded microspheres compared with the placebo gelatin microspheres in order to obtain similar particle sizes. This is achieved by changing the gelatin:oil ratio from 1 :4 (for tlCAM(453)-loaded microspheres) to 1 :3 (for placebo gelatin microspheres) while keeping the emulsion volume constant for both.
  • Albumin/gelatin microspheres are prepared according to the process of
  • Example 8 with the following changes to the preparation of the gelatin solution: A solution of gelatin and albumin is prepared by dissolving 104.8 g of 250-Bloom gelatin in 436.3 g of 0.019 M NaOH at 50 °C. 58.7 g of 25% albumin (human) USP (Bayer Corp, Elkhardt, IN) is added. This corresponds to a solids concentration of 20% and an albumin loading of 12.3% and leads to an albumin loading of 10% in the final product. The presence of the NaOH in the albumin/gelatin solution is needed to prevent isoelectric precipitation of albumin. The gelatin solufion at this concentration would otherwise have a pH of 4-5. This albumin/gelatin solufion is emulsified in corn oil and processed as in steps 4-7 of Example 8.
  • tlCAM(453) also increased the mean volume particle diameter.
  • experiments were carried out at an intermediate scale, i.e., with an emulsion volume of 800 mL in a 12.5-cm vessel and a 2" R100 impeller (40 g dry weight).
  • an intermediate scale i.e., with an emulsion volume of 800 mL in a 12.5-cm vessel and a 2" R100 impeller (40 g dry weight).
  • albumin loaded microspheres were only slightly larger than placebo microspheres (73 versus 62 ⁇ m).
  • albumin-loaded microspheres had a mean volume particle diameter of 57 ⁇ m compared to 47 ⁇ m for placebo.
  • both albumin and tlCAM(453)-loaded microspheres were larger than the placebo microspheres, but more importantly, both the albumin-loaded and tlCAM(453)-loaded microspheres exhibited a pharmaceutically acceptable size distribution.
  • Gelatin-tlCAM(453) solution weight 600 g Beaker diameter 17.3 cm
  • Impeller 76 mm R100 in off-center position, shaft 10-15° from vertical Speed: 400, increased to 640 rpm during cooling Temperatures
  • Example 8 The product of Example 8 (240 g theoretical weight) is charged into a 13-cm Amicon Vantage® S2 column (Amicon, Beverly, MA) connected to a sterile air supply and two gas washing bottles. One bottle is immersed in a water bath set at 24°C, and the second bottle is set as a moisture trap. The air is humidified during its passage through the first bottle at a flow rate of 15 L/min. The relative humidity is set to 90-95% by adjusting the water bath temperature. At 15 L/min, the powder evenly fluidizes the powder bed. Treatment is carried out for approximately 11 hours, by which time the residual acetone level in the powder is reduced to below 250 ppm.
  • Example 12 A similar setup as in Example 12 is employed to reduce the moisture content of the microspheres.
  • the relative humidity is 35-40%, controlled by setting the water-bath temperature to 2-4 °C, and the target range for the final moisture content in the microspheres is 12-18%.
  • the bulk tlCAM(453) microspheres are removed from the column and stored in type-Ill flint glass jars with Teflon®-lined lids at 2-8°C.
  • Porcine gelatin (type A) was purchased from Hormel Foods (Austin, MN).
  • Corn oil and Span 80 (sorbitan monooleate, NF grade) were supplied by Ruger Chemical Co. Inc. (Irvington, NJ). Acetone (reagent grade) was purchased from EM Science (Gibbstown, NJ). Purified ICAM was prepared according to Example 1 in a 10mM histidine buffer pH 7.0 ( ⁇ 50mg/ml). Human serum albumin (HAS Lot # 684PO67A , commercial stock) was obtained from Bayer Corporation, Biological Products, Clayton NC. The vessels, impellers, shafts and baffles used in these experiments were made of Stainless steel (SS 316).
  • R is the radius of the bucket and H is the emulsion height.
  • V k ⁇ (D
  • V L /V S (D L 3 /D S 3 )
  • the vessel diameter to be used for that larger scale can be determined.
  • Equations 1 and 2 were used to calculate the geometric scaled up parameters for various larger scales ranging from 300 to 1000g. These values are listed in Table XIV and account for a proportionate increase in emulsion volume and impeller diameter to generate a similar mixing power per unit volume within the emulsion as that generated at the 150g scale: Table XIV
  • an emulsification system is more complex than a simple mixing process, and increased shear forces are important in addition to mixing forces. Mixing times were not kept constant since they were are dependent on the batch size and rate of cooling.
  • Gelatin Oil Volume of Oil Amount of Span 80 Ratio (ml) (g)
  • the emulsification process at the 400g scale required a 9.3" diameter vessel with a 4.0" radial impeller at an initial mixing speed of 343rpm, as seen in Table XIV.
  • a gelatin solution was prepared at a mixing speed of 200 rpm using a 3" radial impeller in a 3 L stainless steel vessel placed in a water bath maintained at 50°c.
  • a mixture of corn oil and Span 80 was mixed at 250 rpm using a 4" radial impeller in a 12 L stainless steel vessel placed in a water bath maintained at 50°C.
  • the dissolved gelatin solution was added to the oil/Span mixture and the emulsification was performed under various experimental conditions (with and without baffles) and cooling rates (gradual, moderate and rapid) to generate microspheres in the desired 50 ⁇ m size range.
  • Photographs of the emulsion at various points during the emulsification process were captured by an optical microscope (Zeiss, Germany) fitted with a camera attachment (Polaroid Microcam, UK) to evaluate the effect of various process parameters on the size of the emulsion droplets and microspheres generated.
  • the microspheres were acetone washed to remove traces of oil and moisture from the microsphere preparation.
  • the acetone washed microspheres were then vacuum filtered using a Buechner funnel and sieved using a 75 ⁇ m mesh sieve to eliminate large particles and aggregates from the microsphere preparation. This was followed by an acetone removal step performed by fluidizing the powder bed with humidified air (RH ⁇ 90%) for ⁇ 16 hours in a chromatography column. Following acetone removal the moisture level of the microspheres was adjusted to 13 -15 % by circulating clean air (RH ⁇ 35%) through the column. The dry microspheres were stored in glass containers.
  • the manufacturing process used was similar to that used at the smaller 150g scale.
  • a 4.0" radial impeller was placed in the top left quadrant and positioned off center at a 12 degree angle to the vertical in a 9.25" diameter vessel.
  • the cooling process used was similar to that used at the smaller 150g scale.
  • the gelatin solution 50°C was added to the vessel containing the oil/Span mixture (placed in a 50°C water bath) and mixed at the preset initial mixing speed.
  • the water bath set-point temperature was changed from 50 to 10°C and the mixing speed was increased as the emulsion gradually cooled to 10°C.
  • a rapid cooling rate ( ⁇ 2.3 deg./min) was achieved as follows. Immediately following the addition of the gelatin solution to the oil/Span mixture at 50°C, the initial mixing speed was increased to a predetermined rpm. The emulsion was mixed at this speed for a fixed time (20-40 min) at 50° C to generate droplets in the 50 ⁇ m size range. The emulsion was then rapidly cooled to 15°C by draining the warm water from the water bath and replacing it with a slurry of ice and water. The bath temperature was set to 0.1 °C. The mixing speed was increased as the emulsion rapidly cooled from 50 to 15°C. When the emulsion reached a temperature of 15°C, the bath temperature was increased to 10°C and the microspheres were held at this temperature and stirred for 1 hour at 525 rpm.
  • the impeller was positioned at a height of 1.5 " from the bottom of the vessel, and three initial mixing speeds of 600, 625 and 650 rpm were evaluated, with a final mixing speed of 780 rpm. In addition, initial mixing times of 20, 30 and 40 minutes at 650 rpm were tested.
  • the impeller was raised to 2.5" from the bottom, and an initial mixing speed of 425 rpm with a final rpm of 605 during cooling was evaluated.
  • ICAM/gelatin microspheres 20mg of ICAM/gelatin microspheres were weighed into a 12X75mm polypropylene test tube and 2.0 ml of phosphate buffered saline (pH 7.2) was added to the tube. The tube was vortexed briefly to suspend the microspheres and was placed in a 40°C water bath. The tube was vortexed at intervals of 5 minutes for a total of 1.0 hr. The resulting solution was filtered through a syringe filter (0.2 ⁇ m, 25mm Gelman Acrodisk (Fisher Scientific, Norcross, GA), low protein binding membrane) and submitted in duplicate for ICAM determination by immunonephelometry
  • Gelatin microspheres and ICAM/gelatin microspheres were prepared by an emulsification process carried out in a 9.25" diameter vessel with a 4" radial impeller at a predetermined initial mixing speed, under various experimental conditions (with and without baffles) utilizing several cooling methods (gradual, moderate and rapid cooling). The results obtained under these varying conditions are summarized below.
  • the average particle size of the microspheres generated by all three runs was large (> 70 ⁇ m), which were much higher than the acceptable product specification of ⁇ 50 ⁇ m.
  • the larger emulsion volume (8L) at the 400g scale required ⁇ 2.5 hr to gradually cool from 50 to 15°C. This corresponded to a cooling rate of 0.25°C/min which was much slower than 0.47°C/min observed at the 150g scale (3L emulsion volume).
  • the three mixing speeds evaluated were insufficient to provide the shear necessary to produce and maintain small emulsion droplets. These droplets coalesced to large droplets, which on cooling resulted in large particles.
  • a high percentage (10-20%) of the microspheres was above 100 ⁇ m, which was outside the acceptable limit of ⁇ 10%. No microspheres below 10 ⁇ m were generated by this process.
  • a high shear rate associated with a high mixing speed could not be achieved without a secondary source to enhance turbulence and shear in the system.
  • Baffles were used to increase the mixing speed and shear in the system during the emulsification step to produce smaller droplets and subsequently smaller microspheres.
  • a schematic representation of the batch emulsification process with baffles can be seen in Figure 4. Higher initial mixing speeds up to 650 rpms with minimal splashing and air entrapment were achieved using baffles.
  • microspheres prepared possessed average diameters > 65 ⁇ m (Table
  • Figure 7 shows the appearance of the emulsion droplets and solid microspheres at various stages during the moderate cooling process.
  • Emulsion droplets in the desired size range ( ⁇ 50 ⁇ m) were achieved during the emulsification step, with a higher mixing speed of 625 rpm.
  • the small emulsion droplets coalesced to yield large microspheres.
  • a higher initial mixing speed could have resulted in smaller droplets which were thermodynamically unstable resulting in a greater degree of coalescence. This could explain the higher particle sizes and greater percentages of particles above 100 ⁇ m seen in the moderate cooling process (Table XVII) over that observed using the gradual cooling process (Table XVI).
  • a rapid cooling rate of -2.3 deg./min was achieved under two different experimental conditions (high mixing and low mixing), as described earlier.
  • the emulsion cooled from 50 to 15°C in -15 min.
  • the process to be transferred to the pilot plant required a range of initial emulsification times to be specified, rather than a rigid 20 min mixing time.
  • This process was repeated for an emulsification time of 40 min, followed by rapid cooling which generated particles with a size of 63 ⁇ m (Table XVIIIa). Since the process worked at 20 and 40min., an initial emulsification time of 30 ⁇ 10 min. at 650rpm could be specified.
  • Three replicates performed with a 30 min. emulsification time followed by rapid cooling resulted in average particles sizes of - 55 ⁇ m.
  • microspheres were then acetone washed, filtered and dried.
  • the high mixing speed process resulted in splashing during the emulsification process, and air entrapment during the holding step (780 rpm for 1 hr at 15°C).
  • the impeller was raised to 2.5" from the bottom of the vessel and was used at a lower mixing speed.
  • the initial mixing speed evaluated was 425 rpm with a final rpm of 605 during the cooling process.
  • microspheres were then acetone washed, filtered and dried.
  • Gelatin microspheres, albumin/gelatin microspheres, and ICAM/gelatin microspheres (varying loads) prepared by the low mixing speed process were in 50m size range, with spans of around 0.8, no fines below 10 ⁇ m, and less than 3.5% above 100 ⁇ m.
  • ICAM/gelatin microspheres prepared with varying theoretical loads of 12.5, 10.5, 10.0, 5.0, 2.5 and 1.5 % were dissolved as described above in this example, and assayed for ICAM by immunonephelometry. All batches exhibited -100% actual loading, within assay variability.
  • a ICAM/gelatin microsphere preparation was dissolved, and the sample bioassayed.
  • the ICAM/gelatin microsphere formulation was comparable to ICAM standard (103% of standard).
  • the ED 50 was 0.298 compared to 0.303 for the standard.
  • the strategy for scaling up the microsphere manufacturing process to the 400g scale was a geometric scale-up with constant mixing power per unit volume, based on a water-in-oil emulsification process developed at the smaller 150g scale.
  • Several experimental conditions with and without baffles
  • various cooling methods grade, moderate, and rapid were evaluated.
  • the gradual cooling process without baffles required -2.5 hr to cool from 50 to 15°C.
  • the mixing speeds evaluated (343, 375 and 415 rpm) did not provide the shear necessary to produce and maintain small emulsion droplets, which on cooling resulted in large microspheres.
  • the mixing speed could not be increased above 415 rpm without causing significant splashing and air entrapment.
  • Baffles were introduced into the system to increase the mixing speed and shear forces in the system.
  • the emulsification speeds were increased to 650 rpm resulting in emulsion droplets in the 50m size range.
  • moderate cooling 35 min cooling time
  • a rapid cooling process was developed to gel the 50 ⁇ m emulsion droplets rapidly before they coalesced. This process was evaluated under two conditions; a high mixing speed with a low impeller height and a low mixing speed at a higher impeller height. Both methods consistently produced microspheres in the 50 ⁇ m size range.
  • Three factors were identified as critical for achieving microspheres in the desired 50 ⁇ m size range. The include; initial emulsification time ( ⁇ 30min), appropriate vessel geometry and mixing speeds, and a rapid cooling rate (-2.3 deg./min).

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  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Otolaryngology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Pulmonology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Manufacturing Of Micro-Capsules (AREA)

Abstract

L'invention concerne des compositions pharmaceutiques pour l'administration de médicaments destinés à résider dans le nez, et des compositions pour l'administration de médicaments par voie nasale, p. ex. d'agents antiviraux, et en particulier d'agents antiviraux comprenant le récepteur principal de rhinovirus humain, également connu sous le nom de molécule d'adhésion intercellulaire-1 (ICAM-1). L'invention concerne en outre des procédés de préparation pour ces compositions médicamenteuses administrées par voie nasale et un procédé amélioré pour éliminer le solvant résiduel des matrices pharmaceutiques.
PCT/US1998/014790 1997-07-18 1998-07-17 Procedes d'elimination de solvant residuel dans les compositions medicamenteuses administrees par voie nasale WO1999003452A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP98935751A EP0994700A4 (fr) 1997-07-18 1998-07-17 Procedes d'elimination de solvant residuel dans les compositions medicamenteuses administrees par voie nasale
CA002296620A CA2296620A1 (fr) 1997-07-18 1998-07-17 Procedes d'elimination de solvant residuel dans les compositions medicamenteuses administrees par voie nasale
IL13388298A IL133882A0 (en) 1997-07-18 1998-07-17 Process for making an intranasal drug delivery system
AU84935/98A AU747211B2 (en) 1997-07-18 1998-07-17 Methods of removing residual solvent from nasal drug delivery compositions
JP2000502753A JP2001510150A (ja) 1997-07-18 1998-07-17 鼻腔用薬剤送達組成物からの残留溶媒の除去法

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US5296497P 1997-07-18 1997-07-18
US60/052,964 1997-07-18
US5662597P 1997-08-20 1997-08-20
US60/056,625 1997-08-20
US94240397A 1997-10-01 1997-10-01
US08/942,403 1997-10-01

Publications (2)

Publication Number Publication Date
WO1999003452A1 true WO1999003452A1 (fr) 1999-01-28
WO1999003452A9 WO1999003452A9 (fr) 1999-04-29

Family

ID=27368315

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/014790 WO1999003452A1 (fr) 1997-07-18 1998-07-17 Procedes d'elimination de solvant residuel dans les compositions medicamenteuses administrees par voie nasale

Country Status (6)

Country Link
EP (1) EP0994700A4 (fr)
JP (1) JP2001510150A (fr)
AU (1) AU747211B2 (fr)
CA (1) CA2296620A1 (fr)
IL (1) IL133882A0 (fr)
WO (1) WO1999003452A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1243247A1 (fr) * 2001-03-22 2002-09-25 Cognis Iberia, S.L. Emulsions tu type W/O ou O/W/O comprenant des microcapsules constituées d'agents actifs et d'hétéropolysaccharides ou de protéines thermogélifiables
WO2002080881A2 (fr) * 2001-04-05 2002-10-17 UNIVERSITé LAVAL Procede de fabrication d'une matrice d'administration et utilisations
EP1430947A1 (fr) * 2002-12-21 2004-06-23 Cognis Iberia, S.L. Microparticules a compartiments multiples contenant des cristaux liquides
WO2006125620A2 (fr) * 2005-05-27 2006-11-30 Stratosphere Pharma Ab Noyaux et microcapsules pouvant etre administres par voie parenterale et leur procede de fabrication
US8017152B2 (en) 2005-05-27 2011-09-13 Stratosphere Pharma Ab Cores and microcapsules suitable for parenteral administration as well as process for their manufacture
CN102716469A (zh) * 2012-07-07 2012-10-10 北京三元基因工程有限公司 干扰素α的干粉吸入剂
WO2016021835A1 (fr) * 2014-08-08 2016-02-11 (주)비씨월드제약 Procédé de préparation de microparticules à libération prolongée contenant un médicament

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3578498A (en) * 1967-06-12 1971-05-11 Cpc International Inc Desolventizing of starch
US5629011A (en) * 1992-02-05 1997-05-13 Danbiosyst Uk Limited Composition for nasal administration
US5725852A (en) * 1992-04-17 1998-03-10 Takeda Chemical Industries, Ld. Transmucosal therapeutic composition

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9414966D0 (en) * 1994-07-26 1994-09-14 Danbiosyst Uk Pharmaceutical compositions for the nasal administration of antiviral agents

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3578498A (en) * 1967-06-12 1971-05-11 Cpc International Inc Desolventizing of starch
US5629011A (en) * 1992-02-05 1997-05-13 Danbiosyst Uk Limited Composition for nasal administration
US5725852A (en) * 1992-04-17 1998-03-10 Takeda Chemical Industries, Ld. Transmucosal therapeutic composition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0994700A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1243247A1 (fr) * 2001-03-22 2002-09-25 Cognis Iberia, S.L. Emulsions tu type W/O ou O/W/O comprenant des microcapsules constituées d'agents actifs et d'hétéropolysaccharides ou de protéines thermogélifiables
WO2002076407A1 (fr) * 2001-03-22 2002-10-03 Cognis Iberia S.L. Emulsions de type eau dans huile (w/o) ou huile dans eau dans huile (o/w/o) renfermant des microcapsules de substances actives et d'heteropolysaccharides ou de proteines thermogelifiants
WO2002080881A2 (fr) * 2001-04-05 2002-10-17 UNIVERSITé LAVAL Procede de fabrication d'une matrice d'administration et utilisations
WO2002080881A3 (fr) * 2001-04-05 2003-07-17 Univ Laval Procede de fabrication d'une matrice d'administration et utilisations
EP1430947A1 (fr) * 2002-12-21 2004-06-23 Cognis Iberia, S.L. Microparticules a compartiments multiples contenant des cristaux liquides
WO2006125620A2 (fr) * 2005-05-27 2006-11-30 Stratosphere Pharma Ab Noyaux et microcapsules pouvant etre administres par voie parenterale et leur procede de fabrication
EP1726299A3 (fr) * 2005-05-27 2007-04-18 StratoSphere Pharma AB Noyaux et microcapsules pour l' administration parenterale et procédé pour leur fabrication
WO2006125620A3 (fr) * 2005-05-27 2007-08-02 Stratosphere Pharma Ab Noyaux et microcapsules pouvant etre administres par voie parenterale et leur procede de fabrication
US8017152B2 (en) 2005-05-27 2011-09-13 Stratosphere Pharma Ab Cores and microcapsules suitable for parenteral administration as well as process for their manufacture
CN102716469A (zh) * 2012-07-07 2012-10-10 北京三元基因工程有限公司 干扰素α的干粉吸入剂
CN102716469B (zh) * 2012-07-07 2013-09-11 北京三元基因工程有限公司 干扰素α的干粉吸入剂
WO2016021835A1 (fr) * 2014-08-08 2016-02-11 (주)비씨월드제약 Procédé de préparation de microparticules à libération prolongée contenant un médicament
KR101738127B1 (ko) 2014-08-08 2017-05-22 (주)비씨월드제약 약물 함유 서방성 미립자의 제조 방법

Also Published As

Publication number Publication date
EP0994700A1 (fr) 2000-04-26
WO1999003452A9 (fr) 1999-04-29
AU8493598A (en) 1999-02-10
CA2296620A1 (fr) 1999-01-28
JP2001510150A (ja) 2001-07-31
AU747211B2 (en) 2002-05-09
EP0994700A4 (fr) 2000-10-18
IL133882A0 (en) 2001-04-30

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