JP2001510150A - Method for removing residual solvent from nasal drug delivery compositions - Google Patents

Abstract

  (57) [Summary] The present invention is also known as pharmaceutical compositions, agents, such as antiviral agents, and especially as intercellular adhesion molecule-1 (ICAM-1) for the delivery of drugs intended to reside in the nasal cavity. For the intranasal administration of antiviral agents comprising the major human rhinovirus receptor, methods of preparing said nasal drug compositions, and improved residual solvents from pharmaceutical matrices. Removal method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] BACKGROUND OF THE INVENTION 1. Field of invention relates to compositions for the delivery of drugs to be retained in the nasal cavity intended. The invention also relates to methods of making such nasal drug delivery compositions and to improved removal of solvents from pharmaceutical preparations.

[0002] In order to bring the background systemic drug administration, in particular, a number of intranasal formulations designed to deliver a drug is known through the nasal mucosa. However, there is still a need for an intranasal preparation specifically designed to retain the drug for an extended period of time in the nasal cavity without passing through the mucosa.

[0003] Cortesi, R., Esposito, E., Menegatti, E., Gambri, R., and Nastruzzi, C
., “Gelatin Microspheres as a New Approach for the Controlled Delivery
of Synthetic Nucleotides and PCR-Generated DNA Fragments ", Int. J. Pharm. 105: 181-6 (1994)," A new method for the controlled delivery of synthetic nucleotides and PCR-generated DNA fragments. " Discloses a gelatin solution emulsified in isopropyl palmitate (without surfactant) as the oil phase.Cooling the emulsion forms gel beads, which are then washed with acetone and calcined. Collected on sintered glass discs.Average volume particle diameter is 22 μm in the range of 5-42 μm.Particles smaller than 10 μm tend to accumulate in the lungs, so particle sizes in this range may not Is not suitable for the administration of intended pharmaceuticals.

PCT / GB 95/01735 to Illum et al. Discloses a bioadhesive composition comprising ICAM-1 and various water-swellable microspheres, including chitosan, liquid polymeric substances, or gelatin. A drug delivery composition for nasal administration is described. Illum announces the idea of emulsifying a warm aqueous solution of gelatin and ICAM-1 in a vegetable oil containing surfactant, followed by gelation by heat, hardening with acetone, collection and drying of the ICA M / gelatin microspheres. ing.

However, the process taught by Illum for the production of gelatin microspheres and the resulting product is not desirable for commercial scale pharmaceutical use. The method requires large amounts of oil and acetone. The resulting product was found to contain primarily agglomerates of small primary particles. These agglomerates tend to decompose during the treatment period, resulting in fragments and particles having a volume diameter of less than 10 μm of about 3.5% by volume.
A mixture of non-agglomerated particles, comprising: Particles having a volume diameter of less than 10 μm are not suitable for administration of drugs intended for nasal retention because they tend to penetrate the lower respiratory tract and lungs, which are undesirable from either a safety or commercial standpoint. Not suitable.

[0006] Thus, the delivery of drugs to the nasal cavity that maintains biological activity and physical integrity, exhibits prolonged nasal residence time, and allows for sustained release of the active ingredient into the nasal cavity It is desirable to provide a pharmaceutical preparation for the same. Desirable characteristics for intranasal preparations include: (1) a pharmaceutically acceptable formulation with respect to process consistency, weighability, and GRAS (generally considered safe) components; (3) high quality and activity of the released drug, (4) a volume diameter of 10-100 μm, preferably 20-80 μm, to minimize the worry of pulmonary delivery. Limited particle size range, (5) no irritation of mucociliary organs, (6) convenient and reproducible route of administration into the nasal cavity, (7) based on dry powder, (8) cooling And (9) residual solvent concentrations below pharmaceutically acceptable concentrations.

[0007] The criteria for a commercially viable method for producing such products include a minimum solvent consumption and a proven weighing capacity of at least 300 g.

[0008] One problem commonly encountered in pharmaceutical compositions where the production process involves a solvent is that residual solvents in the final product, such that the product does not meet safety and regulatory requirements,
Unacceptably high concentration. Traditional solvent removal methods are often unsatisfactory. For example, vacuum evaporation at room temperature may be inadequate, and at elevated temperatures may cause decomposition of the active ingredient. The alternative method of extraction with a supercritical fluid is not always effective when applied to dry powders. Accordingly, a need exists for improved methods of removing residual solvents from pharmaceutical preparations.

The present invention provides various advantages.

SUMMARY OF THE INVENTION The present invention provides an improved method of preparing a pharmaceutical, gelatin-based microsphere composition comprising one or more drugs to be delivered and retained in the nasal cavity, The manufactured drug-containing microspheres are provided. The invention also relates to an improved method for removing solvents from a dry powder pharmaceutical composition.

In general, the preparation of the microsphere / drug composition comprises the following steps: (a) 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 (B) preparing a solution of a suitable oil and a suitable surfactant; (c) emulsifying a mixture of the solution of step (a) and the solution of step (b);
Producing a water-in-oil emulsion wherein the volume ratio of: :( b) is between 1: 2 and 1:10; (d) continuing emulsification until the target droplet size is achieved; (e) Thereafter, the temperature of the emulsion was lowered at a controlled and rapid rate below the gelation temperature of the gelatin to achieve gelation prior to droplet aggregation and thus to the desired particle size in association with the drug. Allowing the formation of gelatin microspheres, wherein the emulsions in steps (c, d) and (e) above are mixed at a maximum velocity consistent with a circulation pattern with low or no vortex, f) separating the drug-containing microspheres from the oil / surfactant phase; and (g) dehydrating the drug-containing microspheres to produce a dry powder.

The present invention also provides for contacting the matrix with a humidified stream of a suitable gas under conditions that entrain residual solvent into the gas, and then drying the gas by aspirating the gas. Comprising an improved method of removing residual solvent from a pharmaceutical matrix such as a powder. This can be performed in separate batches or as part of a continuous flow process.

The resulting product has a volume particle diameter of between 40 and 60 μm, where the majority of the microspheres are present not as aggregates of smaller particles but as non-agglomerated discrete particles. A free flowing powder comprising zelatin microspheres combined with

[0014] The present invention further comprises an intranasal drug delivery system comprising said gelatin microspheres associated with a drug delivered and maintained in the nasal cavity.

[0015] DETAILED DESCRIPTION OF THE INVENTION 1. Preparation of Gelatin Microspheres (See FIG. 4) Preparation of Aqueous Solution of Gelatin and Drug An aqueous solution of gelatin and drug is prepared. Neither the gelatin grade nor the gelatin concentration is critical to the final microsphere particle size. Gelatin is food grade, preferably at least N
Must be F grade. Gelatin is at least 80, and preferably ≧ 1
Must have a bloom strength of 50. A bloom strength of 250 is most preferred because its properties are fairly constant from batch to batch and are available year round. Its color is paler and has a weaker odor than a lower quality gelatin grade. Higher bloom intensity provides the finished product with excellent color, odor and nasal residence time. A suitable gelatin is, for example, P-8 grade, 250 bloom obtained from Hormel Foods (Austin, MN). The concentration of gelatin is advantageously between about 1% and about 30%.
% (W / w). Within this range, higher gelatin concentrations lead to reduced solvent and oil consumption, but the total solids content (including gelatin and drug delivered) in aqueous solutions should not exceed 30% (w / w). No. Since the gelatin concentration did not appear to affect the final volume particle diameter, a viscosity-based, easily processable maximum concentration of 20% is preferred.

The drug to be delivered can be dissolved or suspended in a gelatin solution, and is insoluble in oil, insoluble in the dehydrating solvent used during the process, any suitable chemical or biological pharmaceutical It may be a substance. Examples of suitable agents include, for example, small chemicals; proteins, peptides, and polypeptides (eg, ICAM-1 and other antiviral agents, vaccines, antigens, antibodies, lymphokines, and any of the foregoing. Fragments), nucleotides (eg, gene, DNA, RNA)
, And fragments of any of the foregoing)), carbohydrates, and the like. The concentration of drug delivered is also determined by the total acceptable concentration of solids added.

[0017] The aqueous solutions of the gelatin and the drug can be prepared by any convenient means. The gelatin and drug solutions can be prepared separately and then mixed, or either the gelatin or drug can be added to the other solution. Those skilled in the art
It will be appreciated that buffering or pH adjustment may be required to protect the drug during the process. Further, for protein drugs, it is preferred that the aqueous solution has a low salt content or contains no salt to avoid precipitating the protein. "Low content of salt" means <200 mOs / kg.

When the drug is ICAM-1, ICAM in histidine buffer at pH 7
A-1 solution is conveniently prepared and added to an aqueous solution of gelatin. l-Histidine is obtained, for example, from Calbiochem Corp. As used herein, "ICAM"
"Means ICAM-1 and any fragments, analogs or derivatives of ICAM-1 that bind to the major receptor group of human rhinovirus and retain the ability to suppress infectivity. ICAM can be prepared as shown in Example 1 below.

The gelatin-containing solution must be maintained at a temperature above the gelatinization temperature of the gelatin. For gelatin having a bloom strength of 250, this means above 40 ° C.

Preparation of Oil and Surfactant Solutions The oil is preferably a refined oil, such as a mineral or vegetable oil, preferably food grade or NF
It is a grade of vegetable oil. Examples of suitable vegetable oils are corn, soy, and safflower. In this method, ultra-refined vegetable oils from which pigment substances, hydrophilic substances, and natural surfactants have been removed are as ineffective as poorly refined oils. Corn oil and soybean oil are preferred, and corn oil is particularly preferred because it is more economical. Suitable corn oils are, for example, Ruger Chemical Co. (Welch, Holme
and Clark), for example NF grades.

Examples of suitable surfactants are Span 80 (Ruger Chemical Co., ICI Americas Inc.
Sorbitan monooleate), lecithin, and pluronic L 1011 (
BASF). Span 80 is preferred. Generally, a hydrophilic-lipophilic balance of 4-6 (H
LB) is desirable. Surfactants having an HLB value of 3 or less (eg, glyceryl monooleate, sorbitan trioleate) are relatively ineffective. A surfactant concentration of 0.1% is too low, 1% is functional, higher concentrations do not provide further improvement in results.

4) The aqueous solution of emulsified gelatin and the drug is emulsified with the solution of the oil, and the surfactant is emulsified to form a water-in-oil emulsion.

[0023] Emulsification methods are well known in the art. Generally, an aqueous solution of gelatin / drug is
Add slowly to the oil / surfactant solution while stirring or mixing. Appropriate agitation during the emulsification period is necessary to achieve the desired average volume particle diameter (40-60 μm). Various agitators or impellers can be used, but it is preferable to minimize the formation of swirls to avoid entrainment of air. This can be achieved through the use of baffles or the precise placement of the impeller. The initial extra power consumption is preferably at least 1.4 watts / L. High mixing should be avoided. Since mixing conditions affect the final product, care must be taken to reproduce the mixing conditions to ensure reproducibility of the final product.
The volume ratio of gelatin / drug solution to oil / surfactant solution affects the quality of the resulting product. Preferably, the volume ratio of gelatin / drug solution to oil / surfactant solution is between 1: 2 and 1:10. Larger ratios are desirable because the volume of the emulsion, as well as the oil and solvent requirements, are reduced. However, the average volume particle diameter gradually increases with the volume ratio up to 1: 2. At higher ratios, the average volume particle diameter increases dramatically, and at even higher ratios, emulsion reversal occurs, rendering particle formation impossible. A volume ratio of 1: 2 to 1: 5 is preferred, with 1: 3 to 1: 4 being particularly preferred.

Emulsification is continued until the target droplet size is obtained. The droplet size is monitored by methods known to those skilled in the art, for example by an optical microscope. Droplets of 20 to 80 μm are preferred, and 40 to 60 μm are particularly preferred. For a volume of 8 L, an average volume particle size of about 50 μm can be achieved in about 30 minutes (± 10 minutes).

5) To achieve gelation prior to aggregation of the chilled droplets, the temperature of the emulsion is reduced below the gelation temperature of the gelatin at a controlled, rapid rate, thereby allowing the drug at the desired particle size Allows the formation of bound gelatin microspheres. The emulsion is cooled to a temperature of 23 ° C. or less, for example, for gelatin having a bloom strength of 250.

Preferably, the cooling rate is between 1 and 4 ° C./min. Cooling rates between 2.0 and 2.5 ° C./min are particularly preferred.

Separation of Drug / Microspheres from Oil / Surfactant Phase Generally, separation is most efficient when the microspheres are first separated from as much oil as possible. This can be done by physical means, such as causing the microspheres to settle under gravity, or by centrifugation followed by a gradient method. Separation can also be performed by washing with a suitable solvent. During the washing process, it is important to stir the emulsification slowly. If a washing method is used, there are several possible alternatives for removing the oil.

A) In a first method, the oil is removed by first washing with １ ／ volume of a hydrocarbon solvent such as heptane and then with a water-miscible solvent such as acetone. .

[0029] The hydrocarbon solvent is selected as follows. i) it must be miscible with both the oil and the water-miscible cleaning agent selected below, ii) it must not dissolve a significant amount of water, and iii) its density is water. And must be lower than mixtures with water-miscible solvents containing up to 20% water.

[0030] Hydrocarbon detergents reduce the viscosity and density of the oil phase, causing microspheres to settle easily by gravity. This makes it possible to decant most of the hydrocarbon phase immediately. One wash with 0.5 emulsion volume is sufficient. Washing is performed at room temperature with short stirring (5 minutes). Two such washes are sufficient to remove most of the oil.

The remaining wash is with a water-miscible solvent such as acetone (HPLC grade, JTBaker) or low molecular weight alcohol. If hydrocarbons are selected according to the above criteria, the first water-miscible detergent is three-phase (1) gelatin microspheres, (2)
A liquid phase (mostly a water-miscible detergent), and (3) another liquid phase, which floats on the water-miscible detergent and contains mostly hydrocarbons and residual oil. .
This separation of the two liquid phases is the result of extracting water from the gelatin microspheres. The two organic phases separate easily from each other and from the solid. The advantage of this method is that the microspheres migrate into a water-miscible, solvent-rich phase where they will stay for the remainder of the wash. Specifically, the hydrocarbon-rich phase is eliminated after the first water-miscible solvent wash.

(B) The second method is to wash only with a water-miscible solvent. The method is performed using continuous stirring, settling, and the decanting steps described above. This has the advantage of requiring only one solvent, but is somewhat more complicated since the first water-miscible detergent again results in the separation of the liquid into two phases. But in this case,
The greasy phase is the lower phase and the bead does not immediately separate from the oil and surfactant. Acetone does not significantly reduce the viscosity of the oil phase as it also acts as a solvent for water, and usually requires at least two water-miscible emulsions, one emulsion volume each, to remove the oil phase. Cleaning is required. Phase separation is slower when the hydrocarbon is used as in (a) above.

Dehydration of Drug / Microspheres to Generate Dry Powder The gelatin / drug microspheres immediately after dehydration, suspended in a water-miscible solvent, are recovered. Methods for separating microspheres from water-miscible solvents are well known to those skilled in the art. Examples of suitable methods are, for example, filtration using a Buchner funnel or centrifugation using a conventional or basket centrifuge.

Important factors of the above method are the choice of oil, the type of surfactant, the concentration of surfactant, the ratio of gelatin / oil, the choice of mixing conditions, the thermal history of the emulsion and the cooling rate (temperature profile ), As well as washing methods (including the choice of solvent).

The method of the present invention is suitable for scaling up to over 100 ×, as described by Illum et al.

An advantage of the present method is that the microsphere product is measured on a 10 μm
m having a volume particle diameter of less than m. In the laser light scattering method, the angular deviation of the intensity of the light beam scattered from the plume of particles in the air is measured by using a laser having a specified wavelength as a light source. Simplification of the scatter data gives a volume particle size distribution. Instruments suitable for performing these measurements are known to those of skill in the art and are available, for example, from Malvern Instrumets, Southboro, Mass. The desired particle size range should be high bloom strength gelatin (preferably at least 150
, Most preferably 250 or more), use of low salt or salt free buffers for aqueous gelatin / drug solutions, constant emulsification during emulsification to achieve constant droplets of the target particle size. Achieved with low or no swirling agitation, as well as rapid cooling when achieving the desired droplet size to prevent agglomeration.
The improved method eliminates the need for a centrifuge and facilitates large-scale batch processing. It also uses less solvent, is more efficient, less costly, and provides better products than conventional art methods.

The methods described above can be performed according to techniques known to those skilled in the art and using equipment, either as a batch process or as a continuous or in-line process; Appropriate adjustments and modifications could be made to implement each.

II. Reduction of Residual Solvents to Pharmaceutically Acceptable Concentrations The present invention further comprises a novel method for removing volatile solvents from a pharmaceutical matrix. The method involves contacting the pharmaceutical matrix with a suitable moistened gas under conditions that entrain residual solvent in the gas, and removing the solvent / gas mixture. Preferably, the wetting gas is passed through a fluidized bed of the pharmaceutical matrix.

Pharmaceutical matrices include dry powder microspheres, such as gelatin microspheres, prepared according to the methods described above, and oil-based microspheres, such as liposomes, known in the art. Including, for example, beads, polymer or non-polymer processed materials, drug-related raw materials, excipients, and end products including biopharmaceuticals,
Without limitation, it can be any pharmaceutical preparation. The present invention is particularly suitable for use with matrices that tend to physically trap the entrained solvent such that methods such as heat or vacuum drying do not effectively remove the solvent.

The solvent can be any residual volatile solvent remaining after the basic preparation method.
Examples of solvents commonly used in preparing pharmaceutical preparations are water, acetone, and aliphatic alcohols (eg, methanol, ethanol, isopropyl alcohol)
, DMSO, chloroform, methylene chloride and the like.

The gas does not react with the pharmaceutical preparation and can be any suitable gas capable of removing and transporting the solvent. Examples of suitable gases are air, nitrogen, argon, and carbon dioxide.

The relative humidity in the gas must be between about 85 and about 96%. The purpose of humidity is to keep enough moisture in the pharmaceutical matrix. For matrices that tend to trap the solvent, a low water content prevents solvent escape. When the solvent is water, a lower relative humidity must be used. 0-50% humidity is preferred, depending on the moisture sorption and desorption properties of the matrix.

To increase the water content of the pharmaceutical matrix, in order to obtain a particularly high humidity, the matrix could simply be placed in an airtight chamber with a suitable salt solution. However, the evaporated solvent cannot exit the closed chamber and will therefore equilibrate with the solvent adsorbed in the matrix. Further, non-flowing dry matrices are prone to agglomeration under high relative humidity. Wetting gas can be passed over the matrix to continuously remove the evaporated solvent, but there are still three problems: (1) the matrix, such as powder, is difficult to retain (2) the dry matrix is retentive and may form aggregates;
And (3) the evaporated solvent can be dangerous and difficult to control.

To overcome these problems, fluid beds were prepared according to methods known to those skilled in the art. For example, Chemical Engineers' Handbook, 5th ed., RHPerry and CHChil
Porter, .HF, McCormick, PY, in ton, eds. (McGraw-Hill, New York, 1973).
See Lucas, RL, and Wells, DF, "Gas-Solid Systems,". Conveniently, a column such as a chromatography column can be used. The column containing the residual solvent is packed into the column, and the moistened gas is passed to the bottom of the column and through the pharmaceutical matrix to fluidize the matrix bed. The advantages of using such a column are: (1) By uniformly exposing the pharmaceutical matrix to a gas stream, the moistened gas can be distributed evenly over the entire area of the bottom filter support. And (2) the column can be easily removed for sample loading, removal and washing. Due to the gas flow, the pharmaceutical matrix is completely fluidized in the column. Continuous fluidization reduces agglomeration of the dry matrix.

The residual water is then removed by passing the dry gas through the matrix and removing the wetting gas. "Dry" for this purpose means having a relative humidity of less than 50%.

Devices suitable for use in the present invention include, for example, Fluid Air Inc., Aurora, IL;
Commercially available from ro Inc., Columbia, MD. Other changes will be apparent to those skilled in the art, for example, how to change the equipment and methods required for different matrices and batch sizes.

When used in combination with the above process for the preparation of drug / gelatin microspheres using acetone as a solvent, a humidity of 92% at room temperature can reduce the residual acetone concentration to less than 200 ppm within 8 hours. . It is believed that the acetone is absorbed and strongly bound into the gelatin matrix by hydrogen bonds that prevent its removal from the polymer, even under relatively high vacuum and / or at temperatures above its boiling point. By increasing the water content of the gelatin, hydrogen-bonded acetone molecules are quickly replaced by water molecules. The reduced glass transition temperature of gelatin at higher moisture content also increases the rate of acetone diffusion from the gelatin matrix.

The result is a dry powder with a pharmaceutically acceptable concentration of residual solvent.

III. Product The resulting product is a free flowing dry powder comprising gelatin microspheres containing the desired drug. In particular, the following parameters are preferred.

Average volume particle diameter 20-80 μm Span * <1.0>% of particles with 100 μm volume particle diameter <10% <% of particles with 10 μm volume particle diameter <1% (laser beam scattering
Performance of released drug) Activity Process scale (based on dry weight)> 100 g Preferred residual concentration when solvent is acetone <250 ppm [* Span is an index of particle size distribution calculated from percentile.

[0051]

(Equation 1)

Here, D (v, 0.9), D (v, 0.1) and D (v, 0.5) are 90/100, 10/100 and 100 minutes of the microsphere, respectively. The drug microspheres of the present invention may be administered as prepared as described above, but the microspheres of such a drug may still contain one or more of the active ingredients or active ingredients. It may be incorporated into a pharmaceutical preparation comprising it or mixed with microspheres containing other agents. Drug-containing gelatin microspheres can be mixed with drug-free placebo gelatin microspheres to achieve a mixture with a lower concentration of drug per unit volume or weight.

Suitable pharmaceutical preparations include, for example, ointments, gels, pastes, creams, sprays (including aerosols), lotions, powders of the active ingredient in suitable excipients,
It can take the form of suspensions, solutions and emulsions. Examples of suitable excipients are pharmaceutically acceptable fillers and extenders, binders, wetting agents, dissolution retarders, disintegrants, resorption accelerators, surfactants, adsorbent carriers, and lubricants. including. Excipient (s)
It should be understood that must be selected to preserve the microsphere integrity and activity of the drug and be compatible with the chosen route of administration.

Pharmaceutical preparations, which are powders and sprays, contain, for example, suitable diluents, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder or a mixture of these substances. Can be. Aerosol sprays can, for example, contain the usual propellants, for example chlorofluorohydrocarbons.

Pharmaceutical compositions which are ointments, pastes, creams and gels are suitable for example with suitable diluents, such as animal and vegetable fats, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite , Silicic acid, talc and zinc oxide or mixtures thereof.

Pharmaceutical compositions which are solutions and emulsions are, for example, suitable diluents and emulsifiers known to those skilled in the art, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Glycol, 1,3-butylene glycol, dimethylformamide, oils (for example,
Peanut oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbital or mixtures thereof. Pharmaceutical preparations, which are suspensions, may contain suitable diluents (eg, ethyl alcohol, propylene glycol), surfactants (eg, ethoxylated isostearyl alcohol, polyoxyethylene sorbate and sorbitan esters), microcrystals It can include cellulose, aluminum metahydroxide, bentonite, agar and tragacanth or mixtures thereof.

The pharmaceutical preparations of the present invention can also include coloring and preservative agents, as well as flavoring agents. The pharmaceutical preparations according to the invention comprise from 0.1 to 99.5% by weight, preferably from 0.5 to 95% by weight of drug-containing microspheres in the total composition.

The preparation of the above-mentioned pharmaceutical compositions is carried out by any suitable method known to the person skilled in the art.

There are a number of viruses that infect warm-blooded animals via mucosal surfaces, such as rhinoviruses and adenoviruses. Suitable antiviral agents for the treatment of these infections are known, but are increased on such mucosal surfaces, especially the nasal route, which is one of the major routes of infection for such viruses. It is desirable to provide a preparation for delivery.

Since the primary route of infection by rhinovirus is the nasal and ocular mucosal surfaces, it is desirable to protect such mucosal surfaces by topical administration of appropriate anti-rhinovirus agents.

Thus, the major human rhinovirus receptor (ICAM), which can remain in the nasal route for an extended period of time to provide a protective effect against rhinovirus infection in a safe and economically effective manner It is desirable to have a preparation of -1) or a fragment thereof.

[0062] The gelatin microspheres of the present invention are particularly suitable for intranasal administration. "In the nasal cavity" and "
Intranasal administration], dry powders, intranasal drops, intranasal sprays, and creams,
By nasal administration is meant a form intended to reside in the nasal cavity, including gels or other preparations suitable for topical administration to the nasal cavity. However, the pharmaceutical gelatin / drug microspheres of the invention can also be used for topical administration, for example, in the eye, mouth, ear,
For topical administration to the skin, they can be prepared, for example, as creams, lotions, ointments, salves and the like. Oral preparations may also include lozenges and mucoadhesive buccal tablets,
Etc. can be included.

The present invention will be described in further detail with reference to the following examples directed to the administration of anti-rhinovirus agents, particularly ICAM-1 and fragments thereof, wherein the gelatin / drug microspheres of the present invention Also, other water-soluble pharmaceutical chemical and biological agents, such as small compounds such as antibiotics, proteins, peptides, and polypeptides (eg, vaccines, antigens, antibodies, lymphokines, and any of the foregoing) Fragment), nucleotides (eg, gene, DNA, RNA, and fragments of any of the foregoing), hydrocarbons, and Enviroxine, Pirodavir, interferon-alpha, sialidase inhibitors, acyclovir, adenosine, arabinoside, interferon And local administration of antiviral agents such as interferon-inducing agents Suitable are.

The present invention can be better understood with consideration of the following examples. As used hereinbefore and hereinafter, unless stated to the contrary, all temperatures and temperature ranges refer to the degree Celsius system, the word percent or (%) means percent by weight, and mol and The moles represent gram moles.

[0065]

【Example】

Example 1 Production of tlCAM (453) Rhinovirus is a member of the genus Picornavirus and is responsible for 50% of human colds in one year. However, during the season (mid-September to mid-October)
Rhinoviruses cause up to 75% of all colds. About 90% of all rhinoviruses bind to the major human rhinovirus receptor (HRR). An antiviral agent comprising the major human rhinovirus receptor or a fragment thereof,
It is an effective inhibitor of rhinovirus infection of susceptible cells. The major human rhinovirus receptor is identical to the protein known as intercellular adhesion molecule-1 (ICAM-1) except for G> A at nucleotide 1462, and has amino acid position 442.
Results in the amino acid substitution Glu> Lys. ICAM-1 is 45-50k
A glycoprotein having a molecular weight of D (excluding hydrocarbons) and eight possible sites for N-linked hydrocarbon bonds, and glycosylated proteins have a molecular weight of 82-88 kD.
ICAM-1 has 507 amino acids and has a cytoplasmic region, a membrane permeable region, and five extracellular immunoglobulin-like regions (amino acids 1-88, 89-185, 18
6-284, 285-385, and 386-453). A presently preferred embodiment for antiviral purposes is to use a complete H that retains rhinovirus binding activity.
This is a fragment consisting of the first 453 amino acids of the RR sequence.

Recombinant tlCAM (453) was obtained from Chinese hamster ovary (CHO) cells containing the cDNA code for tlCAM (453) maintained by continuous perfusion in custom media without any plasma-derived components. Purified from fermentation broth of continuous cell culture. “TlCAM (453)” is the first 453 of ICAM-1.
Means amino acids (regions 1 to 5). The sequence of ICAM-1 is tlCAM (45
A method for preparing the cDNA code for 3) is known to those skilled in the art. For example, Staunton, DE, SD Marlin, C. Stratowa, MLDustin, and TA Spr
inger, “Primary Structure of ICAM-1 Demonstrates Interaction Between Me
mbers of the Immunogulobulin and Integrin Supergene Families ", Cell 52: 9
25-933 (1988), EP-A-362,531. The tlCAM (453) obtained from the clarified and concentrated fermentation broth is obtained by hydrophobic chromatography, metal ion chromatography, precipitation at low pH, filtration, anion exchange chromatography, hydroxyapatite adsorption, size Purified by exclusion chromatography and a second HAP adsorption step. Virus inactivation was shown to reduce control virus titration by low pH precipitation (down to 36 and 22 logs, respectively, to reduce MuLV and reovirus titrations) and pasteurization (up to another 5 logs). ). filtering the tlCAM (453) -containing solution to produce a liquid drug substance;
It was stored at -35 <0> C until it was prepared into a gelatin based microsphere powder. Some lots are in phosphate buffered saline (PBS or 0.15 M NaCl, 0
. Lyophilized in 01 M Na ₂ HPO ₄ , adjusted to pH 7.0) and reconstituted before use. Enrich tlCAM (453) to> 50 g / L and add 10% of low ionic strength.
Diafiltered in mM histidine buffer, pH 7.0.

Example 2 Preparation of Gelatin Microspheres A) Material tlCAM (453) : tlCAM (453) was prepared as in Example 1. Albumin : 25% albumin (human) USP (Bayer Corporation, Elkhart
, IN) Gelatin grade : Hormel Foo, an acid-type gelatin extracted from porcine skin
ds (Austin, MN). Gelatin is usually graded by its bloom strength, which is an indicator of gel strength under certain conditions (see, for example, US Pat.
540, 979). These studies are based on 225 (Hormel grade P-
7) Performed on gelatins having bloom strengths of 250 (grade P-8) and 275 (grade P-9). Oil : Various oils were used. Corn oil of food grade was obtained from the grocery store (for ^{example, Mazola (R), CPC International} , Englewood Cliffs, NJ). Pharmaceutical or NF grades were supplied by Ruger Chemical (Irvington, NJ). Ru soybean oil
It was either food grade or NF grade from Ger Chemical Company. Surfactants: Span 80 ^{(R) (sorbitan} monooleate), Span 85 ^(R) (trioleate), and Arlacel 186 ^TM (glyceryl monooleate) is, ICI America
s (Wilmington, DE). Pluronic L-1011 ^TM is based on BASF (Mt.Olive,
NJ). Lecithin was supplied by Central Soya (Ft. WAyne, IN). Solvent : Acetone and n-heptane were from JT Baker (Phillipsbur, NJ), EMScien
Reagent grade purchased from ce (Gibbstown, NJ) and Mallinkrodt (Chesterfield, MO).

B) Method: Since the supply amount of tlCAM (453) was restricted, tlCAM (4
In the absence of 53), small-scale range finding studies were performed and then the method
The effect of adding AM (453) was evaluated, and finally, the raw material was saved by scaling up the resulting method.

An emulsion volume of 200 mL was chosen for these range finding experiments.
This scale allowed the performance of several experiments per day, providing sufficient material from each experiment for characterization. The results of each experiment were initially evaluated by a visual test of the flowability of the resulting powder. Viscous or highly agglomerated material was rejected without further analysis. Acceptable materials were analyzed for particle size distribution by laser beam diffusion by visual inspection.

After determining the method on the 200-mL scale (8-10 g based on dry weight), evaluate its effect on the method and evaluate the effect of the method on molecular binding and biological activity. For this, tlCAM (453) was added.

Finally, the method established in range finding experiments was extended to the 150-g level to establish commercial utility.

C) The overall planned range finding experiment of the process was performed in a 400-mL beaker (7.3 cm diameter) with a total volume of 2
Performed by emulsifying 00 mL. A 1.5 "radial flow impeller (Product R100, Lightnin Equipment, Milwaukee, WI) or a 2" high efficiency axial flow impeller (Product A310) was used. In the first experiment, the gelatin solution was added to the oil phase while stirring at 700 rpm. Then, after 10 minutes, the stirring speed was increased to 400
rpm. The design of the impeller is shown in FIG. In other experiments, the stirring speed was kept constant.

The initial temperature of the emulsification was at least 45 ° C. and was allowed to cool by natural convection. Two 100-mL washes with heptane, followed by two 100-mL washes with acetone.
Washing was performed using a mL wash. For each wash, the resulting suspension was agitated by hand or using a motor-driven mixer. After each wash, the solids were allowed to settle by gravity and the supernatant was decanted. The final powder is # 3 Whatman (
Collected by vacuum filtration on a Buchner funnel using filter paper from Fairfield, NJ). It was dried at 34-40 ° C under vacuum overnight.

D) Powder characterization: The suitability of the powder was evaluated step by step. Initial evaluations required that the powder be free flowing, without the viscosity due to residual oil. Any lump was removed by passing the powder through a 60- or 70-mesh sieve. The goal,
Most powders had either a sieve with a cut-off diameter of 250 and 212 μm, respectively, as it was to have a powder that fell within the particle size range 10-100 μm, and preferably 20-80 μm. It must pass easily.

E) Scaling and scaling studies: The range finding experiments in section C above served to determine the appropriate composition for the emulsions to be prepared, ie the choice of oils and surfactants, concentrations, etc. The reproducibility of the method was tested on a 2.5-g scale (50 mL emulsion). This was considered the minimum scale at which the method could be performed without changing important parameters.

F) Calculation of tlCAM (453) and albumin loading : The theoretical loading of tlCAM (453) and albumin was calculated based on the following basis. i) The water content of the raw materials of gelatin and the final product was assumed to be equivalent. Therefore, the water content could be neglected. ii) Only the polypeptide part (and not the hydrocarbon part) of the tlCAM (453) molecule was taken into account.

[0077] These assumptions do not pose difficulties when used permanently in calculating the addition amount. These assumptions do not apply when calculating the yield of dry weight powder and must be corrected.

The theoretical addition rate is

[0079]

(Equation 2)

Is given by This formula is based on the mass balance for the preparation of a gelatin solution containing tlCAM (453) (or albumin). Where f _i is the mass fraction (or addition rate) of ICAM (453) (or albumin) and c _i is the concentration of g / L of tlCAM (453) (or albumin) in the initial solution in and, [rho _i is, TlCAM (453) (or albumin) is the density of g / L of solution and m _i is the mass of TlCAM used (453) (or albumin) solution. The salt concentration of the buffer relative to the tlCAM (453) (or albumin) solution is given by c _b (g / L), which means that the tlCAM (453) (or albumin) concentration is low and the buffer is If it is saline, it can be important. For low salt buffers, the correction given by c _b is small. The total weight of gelatin used was such that the gelatin solution was tlCAM (453
A G _g m _g for if) (or albumin) is mixed with a solution. _mg represents the mass of the gelatin solution, and G _g represents the mass fraction (g / g) of the gelatin before the addition of tlCAM (453) (or albumin). The density of the solution was measured and found to be 1020 g / L for tlCAM (453) at 50 g / L and 1070 g / L for albumin at 250 g / L.

G) Results Selection of Surfactants Five oil-soluble surfactants with different chemical compositions were tested. They are,
Span 80 and 85, Pluronic L-1011, ARlacel 186, and soy lecithin. Each of these was tested in a 1% strength corn oil in a 200-mL model system under the conditions shown in Table I.

[0082]

[Table 1]

These experiments showed that Span 80 and L-1011 were superior to other surfactants (data not shown for L-1011). L-1011 is practical (
Compendial (USP or NF) grade, so Span 80 was selected for use in further studies.

Concentration of Span 80 The concentration of surfactant was 0.1, 1, and 2% (w / v) and 20
At a 0 mL volume scale, studies were performed at a gelatin: oil ratio of 1: 2 to 1: 4 (Table II).

[0085]

[Table 2]

[0086]

[Table 3]

The table shows that, at a ratio of 1: 4, the particles decreased in size by increasing the concentration of Span 80 to 1%, whereas increasing the surfactant concentration above 1% had little effect. It was not shown. This effect was even more pronounced when the amount of gelatin was increased relative to the oil, representing more difficult emulsification conditions. Based on these experiments, 1% Span 80 was used for all further experiments.

Gelatin-oil ratio This ratio is a 20% (w / w) solution of porcine 25 Bloom type A gelatin:
It was determined as the volume ratio of corn oil containing 1% Span 80. A ratio of 1: 4 to 1: 1 was tested. Higher ratios are preferred because on some scales oil and solvent consumption and processing capacity are reduced. At the same time, a higher proportion of the solution of gelatin in the emulsion increases the likelihood that the droplets or beads will collide with each other, leading to an increased occurrence of coalescence or agglomeration.

In fact, attempts to use the 1: 1 ratio have failed due to emulsion inversion, difficulty in cleaning the oil, and reasons including unacceptably large (> 200 μm) microspheres.

In experiments on various scales, microspheres of the desired particle size were obtained in a ratio of 1: 3. The difference between the 1: 3 and 1: 2 ratios is slight, and on a 200-mL scale, five experiments at a 1: 3 ratio result in a particle size of 62.8 ± 14.6 μm, while 1 : 2 resulted in 77.0 μm microspheres (FIG. 2). Mixing was used at 600/700 RPM (start / end) at 1.5
"R100 (radial flow) Implemented using an impeller.

Oil Types Both corn and soybean oil were evaluated for a period of time. The results of these comparisons under two different conditions are shown in Table III.

[0092]

[Table 4]

The table implies that there is no significant difference in the results between normally refined corn and soybean oil, as can be expected from their similar viscosities.

Selection of processing conditions, agitation speed, type of impeller, and suitable surfactant, type of oil, etc., for the cleaning method can be applied on a larger scale. Appropriate mixing conditions during the emulsification period are more dependent on scale. However, the above experiments show the relative importance of these variables. Table II and Table II
As shown in I, the choice of the impeller was hardly related to the final particle diameter at the 200-mL scale. This may be because the two impellers used on this scale were almost similar in size to the container itself. The ratio of the impeller to the diameter of the vessel was 0.52 and 0.70.

The mixing and washing conditions at the 200 mL scale were more relevant to the final particle size. Initially, washing was performed by occasionally stirring the emulsion in a solvent with a spatula. This was changed to continuous mixing using a propeller mixer.
At the same time, the emulsification method was changed. 700 rpm for 10 minutes and then 400 rpm
Instead of stirring, the stirring speed was kept constant. Table IV summarizes the results of these changes.

[0096]

[Table 5]

The table shows that before these changes were made, the particles were large and varied in size from batch to batch. It also shows that mixing conditions during the emulsion cooling period affect the final diameter.

Two experiments were performed using two mixing speeds, but with continuous agitation during the wash phase. In these two experiments, there was a decrease in both the average volume particle diameter and the variability measured by the standard deviation between experiments.

A number of additional experiments were performed in which both changes were performed. These experiments (25
In), there was also a decrease in both the average volume particle diameter and the standard deviation to the initial preparation conditions using either the A310 or R100 impeller.

H) Summary The results of the above experiments allowed the definition of a "standard method" before studying the effect of incorporating the drug. The choice of surfactant-Span 80-, the concentration was set to 1% (w / v), as higher concentrations provide little additional benefit. Corn and soybean oil are similar in many respects, the particle size distribution does not differ significantly, both are available in practical grades, and both are commonly used as pharmaceutical excipients. Corn oil was chosen because its price is cheaper than soybean oil.

The gelatin solution was set at 20% (w / w) solids. At higher concentrations, it is difficult to remove entrained air or inject the gelatin solution. Lower concentrations do not appear to provide much benefit and will 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 mentioned above, reducing the oil 1: 2 has made it more difficult to obtain the desired particle size distribution.

Rapid cooling or longer agitation reduced the average volume particle diameter. Since gelation of gelatin solutions is a kinetic phenomenon, gel strength is a function of both time and temperature.

Example 3 Incorporation of tlCAM (453) tlCAM (453) was used to assess the effect of protein on particle size, ie, measure protein loading and evaluate the effect of processing conditions on biological activity. Incorporated into gelatin microspheres produced by the method of Example 2. The emulsification conditions were a 1: 3 volume ratio of 20% (w / w) swine 250 Bloom type A gelatin in water: 1% Span 80 in corn oil.

The first experiment was performed as shown in Table V.

Table V Parameters for tlCAM (453) on 240-mL Scale D [4,3] 72 μm Total Solids 20% tlCAM (453) Biological Activity Starting Material 0.43 μg / mL Release from Preparation 0.43 (Control) 0.29 Theoretical addition rate 8.9% Final addition rate 7.8% (87% of theory) Control lot (2) D [4,3] = 53, 60 μm tlCAM (453) The results of an experiment that incorporates
At the bottom are the results of two control experiments. The tlCAM (453) additive material had a slightly higher average volume particle diameter than the control. Tl released from the preparation
CAM (453) had comparable biological activity to the starting material. The measured addition rate is 7
. 8%, which was 87% of the expected loading based on the composition of the original gelatin solution.

Example 4 Reproducibility of the Method The reproducibility of the method was determined on a 50-60-mL scale (2.5-3 g) under the same conditions as in Example 3, as shown in Table VI. It was studied in several experiments.

[0108]

[Table 6]

The plaque measurement was performed according to the method of Greve et al., J. Virol. 65: 6015-23 (1991) and the results are shown below.

[0110]

[Table 7]

The intensity results are expressed as the concentration in μg / mL required to suppress viral infection of susceptible cells. Thus, higher values reflect lower intensity.

These experiments had several purposes: (1) show that the preparation method of Example 3 could be performed reproducibly; (2) tlCAM (453) Indicating that it will maintain activity once released from
) To determine the appropriate initial loading required to obtain 10% tlCAM (453) in the final preparation.

First, two experiments were performed to determine what the final addition of tlCAM (453), expressed as a percentage of the initial theoretical addition. These are summarized in Experiments 4 and 5 in Table VI. These two experiments, together with the results shown in Table V, showed that the tlCAM (453) addition rate was 8% of the expected value from the gelatin solution.
Between 5 and 87%. Thus, for a 10% addition, the initial tlCAM (453) content must be 11.8%.

Experiments 1, 2, and 3 in Table VI were performed to show constant tlCAM (453) loading, particle size distribution, and yield. Furthermore, the tlCAM released from the powder (
The biological activity of 453) was measured and found to be comparable to the starting material.

Interestingly, the yield of tlCAM (453) was <100%. tl
Nephelometric measurements of CAM (453) show both active and inactive tlCAM (453).
And thus the loss of material is not relevant for the measurement. tlCAM (453)
The most likely point of loss is in the first acetone wash. If all the water present is extracted into the acetone phase, it will contain 17% water. This may be sufficient to dissolve tlCAM (453) and possibly also gelatin.

The biological activity of tlCAM (453) is not affected by the conditions of the method.

Example 5 Scaling of the Method The parameters for the scaling of the method were studied. In an initial, larger scale experiment, it was assumed that only the mixing conditions needed to be changed, and that the parameters of the other methods would be able to be kept constant.

A target size of 150 g was chosen. At this scale (3 L emulsion), cooling under ambient conditions is too slow, so that
Dipped in a 冷凍 55 ° C. frozen water bath. The gelatin solution was added to the warmed oil and the freezer started immediately. It took about 70 minutes for the emulsion to reach its final temperature of 10 ° C.

Factors affecting particle size, in which the emulsion composition was kept constant, were: i) the size and type of the impeller ii) the position of the impeller iii) the dimensions of the tank iv) the temperature profile with time v) tlCAM (453) was expected (maintaining 20% solids).

It is important to apply the required degree of agitation without entrainment of air. The stirring speed is limited by the formation of swirls in the emulsion. Since swirling is undesirable, even when the impeller was optimally positioned, it would not have been possible to obtain particles of the desired diameter with constant agitation. As the emulsion was cooled, the viscosity of the emulsion increased, making it possible to increase the stirring speed without increasing swirling during the cooling process. When this was performed, particles of the desired size were achieved.

Thus, agitation is of two things: first, it provides enough shear to break the gelatin solution into sufficiently small droplets, and second, it stagnates near the wall. It is important to provide sufficient flow of liquid so that no area is formed. The latter consideration seemed to be more important as the emulsion appeared to weaken as the emulsion cooled, especially at a gelatin-oil ratio of 1: 3. Under such conditions, a small impeller would provide a high degree of spring and could only flow in the area surrounding the impeller.

[0122] Usually, if there is a problem with swirling, it can be solved by adding deflectors. In fact, the deflector enabled an increase in the mixing speed compared to the configuration without the deflector. At the same time, the gap between the deflector and the tank wall is 1/8 "(3
cm) alone, can accumulate the formation of cooled, slow-moving substances. Care must be taken to avoid this problem when using baffles.

To scale up, two other methods were used, each with its advantages. In the first method, the mixing shaft was located along the axis of the beaker. The impeller itself was large enough to fit easily into the container and was located near the bottom. In this configuration, a large deep spiral was formed, exposing the center of the impeller, so that only the tip of the blade was immersed in the solution. The emulsion was cooled by filling an ice bag around the container. With this configuration, experiments with and without tlCAM (453) were performed on a 3-L scale. The results are shown in Table VII.

[0124]

[Table 8]

The table shows that to produce microspheres with an average diameter of 50-70 μm, 850 rpm
Indicates that a 31/2 "(8.9 cm) marine propeller with a shallow pitch (゜ 10 °) when driven with a tlCAM (453%) was useful.
) Had no significant effect on particle size.

This method has several disadvantages. The propeller causes the oil phase to bounce, especially during the first phase of the experiment, and air can be entrained in the emulsion, causing tlCAM (453) denaturation or foaming, which is Will hinder the stage. Therefore, methods were also sought in which swirls and air entrainment were minimized.

On larger scales (greater than 120 g), favorable rates of cooling add to the complexity of the scale-up. Since the cooling is performed from the vessel wall, the boundary layer of the cooling device,
And thus a more viscous emulsion is present proximal to the tank wall. This layer is kept thin by the mixing action. At higher cooling rates, the thermal (and thus hydrodynamic) boundary layer becomes thicker, and stronger mixing is needed to offset this effect.

At larger scales and lower cooling rates, it was not clear whether extra stirring time was needed to cure. A one hour maintenance phase was included in the process to avoid potential problems.

A study was performed to determine the appropriate impeller for evaluation. A 3-L emulsion containing 750 mL gelatin solution was placed in a beaker having a diameter of 20.3 cm at 50 ° C.
Maintained. The impellers listed below are installed in three different positions, vertically, and in the center,
Vertically, off-center, off-center, 10 to 10
Placed at a 15 ° angle and tested (see FIG. 3).

The maximum speed at which each impeller was able to rotate was checked and the mixing dynamics at that speed were qualitatively tested. The results are shown in Table VIII.

[0131]

[Table 9]

Direct comparison of different impeller designs has been difficult because the available impeller diameters are not all the same. However, the most effective design was a radial flow impeller, which appeared to have to be located off-center.

As shown in FIG. 3, it was determined that the impeller was installed to be inclined at one quarter of the “upper left” of the mixing vessel. This arrangement minimizes swirl formation. A number of experiments arranged in increasing order, but incorporating various impellers, vessel diameters, and mixing speeds are summarized in Table IX.

[0134]

[Table 10]

Table IX shows that the particles obtained at the 300-g scale were unacceptably large. One specific form at 150-g, 4.5-L (17.1
cm diameter) beaker, 3 "(7.6 cm) radial flow (R10
0) Since the impeller was successful, no laborious experiments were performed.

Slight adjustments in conditions may be necessary to achieve the desired particle size distribution, which are within the skill of the art. For example, the impeller to tank diameter ratio actually increased because a vessel was used whose diameter increased from 7.3 cm to 20 cm. This may be related to the thixotropic nature of the cooling emulsion, which tends to constrain the area of active fluid motion relative to the area around the impeller. On substantially higher scales (e.g.,> 1000 g, where the emulsion volume would be> 25 L), the method can be impractical, and other methods may be used to enhance the level of mixing. Must be replaced with a method. One alternative is a semi-batch form of operation where the mixing is performed in-line. Minor changes in scale will be implemented with changes in overall processing time.

Example 6 Modified Delivery to Avoid Precipitation of Drugs If the drug to be delivered is a protein having an isoelectric point that tends to precipitate during the methods of Examples 3 and 5, Adjustments can be easily made to avoid excessive precipitation. For example, a 20% by weight gelatin solution has a pH value of about 5, close to the isoelectric point of albumin, so that when added to a warm gelatin solution,
Albumin precipitates. Precipitation was avoided by adjusting the pH of the gelatin solution to 6.0-6.1 or by first dissolving the gelatin in 0.019 M NaOH.

Similar adjustments to avoid such problems with other drugs are expected to occur to those skilled in the art.

Example 7 General Preparation of Pharmaceutical Grade tlCAM (453) / Gelatin Microspheres Gelatin / tlCAM (453) microspheres were prepared using tlCAM (corn oil) in corn oil.
453) and a solution of gelatin, prepared by mixing the solutions to form a water-in-oil emulsion. This emulsification method uses corn oil, gelatin, Span 80, water for injection, and tlCAM (453) in L-histidine buffer as raw materials. Next, the wet gelatin beads are washed with acetone to remove oils and surfactants, and the gelatin beads are dehydrated to dry microsphere powder.
The residual water content is fixed by removing the residual acetone and fluidizing the bed of microspheres with a stream of air at a controlled humidity.

Table X lists the quantitative composition of the tlCAM (453) / gelatin microspheres and placebo (gelatin microspheres only) bulk powder preparations prepared by this method.

[0141]

[Table 11]

Example 8 General Preparation of Pharmaceutical Grade tlCAM (453) / Gelatin Microspheres The following is a standard general method used repeatedly in the manufacture of tlCAM (453) / gelatin microspheres. 1. Concentrate tlCAM (453) to> 50 g / L, low ionic strength, pH
Diafiltered into 7.0 10 mM histidine buffer. Ultrafiltration / diafiltration is replaced by histidine diafiltration.
Model S10Y30 (30,000 MWC) with at least 7 volumes of buffer
Perform using O) tangential flow ultrafiltration cartridges (Amicon, Beverly, MA). 2. The final UF / DF preparation tlCAM (453) bulk is passed through a 0.2-micron filter through a sterile polyethylene terephthalate copolymer (PETG).
), Filter sterilized into bottles and store below -30 ° C. TlCA by immunoturbidimetry
Aliquots of sterile prepared bulk are used to quantify M (453) content, tlCAM (453) integrity by SDS-PAGE, biological activity by measuring cell-based plaque, and microbial loading. Immunoturbidimetry measures the concentration of a particular protein in solution based on a measurement of the intensity of light scattered from a precipitate formed by mixing the protein of interest with an antibody reactive therewith. It is a method of measuring. Automated instruments for performing this measurement are, for example, Behring Diagnost
ics, Inc. (Somerville, NJ). 3. During preparation, by placing the bottle in a water bath set at 40 ° C. or less, t
Thaw lCAM (453). 4. 104.7 g gelatin in histidine buffer from step 1, tlCA
A solution composition of 14.9 g of M (453) and water for injection sufficient for a total weight of 600 g is prepared in a 1 L glass beaker. This tlCAM (453) gelatin solution comprises 20% (w / w) tlCAM (453) comprising 12.4% total solids.
Has a total solids content of The mixture is stirred while covered in a 50 ° C. water bath for no more than 30 minutes. In another 4.5 L stainless steel beaker, add corn oil 220
8g of (2400 mL) and Span 80 (= 1%) 24g , lightnin (R) mixer (
Mix at 200 rpm in a 50 ° C water bath for no more than 30 minutes using a 3-inch radial flow impeller mounted on a Lightnin Equipment Co., Milwaukee, WI). 5. To produce a water-in-oil emulsion, the speed of the mixer in the oil solution should be 30
Increase to 0 rpm and gently inject the warm tlCAM (453) gelatin solution into the oil / surfactant solution for about 1 minute. Next, the mixer speed was set to 425.
rpm to form droplets of gelatin-water in the oil / surfactant phase. Lower the set point of the water bath to 10 ° C and mix the emulsion until the temperature reaches 15 ° C. During this period, the speed of the mixer is increased at 10 minute intervals to the maximum achievable without causing entrainment of air. This step is performed using gelatin-tlCAM (
The water droplet of 453) is cooled and solidified into a gel bead. When the emulsion temperature reaches 15 ° C., the emulsion is mixed at a mixing speed of 640 rpm or less within 60 minutes. This step hardens and stabilizes the gelatin / tlCAM (453) bead. 6. The gel bead is separated from the oil and surfactant by transferring the cooled emulsion to a 7.6 L stainless steel beaker and then washing the emulsion with acetone. Six washes with acetone remove the oil / surfactant phase from the emulsion and dehydrate the tlCAM (453) / gelatin to the target particle size microspheres. 7. A 3.0 micron Teflon®-type FS filter (Millipore, Bedford, MA ⁾ in a Buchner funnel set on a filtration flask connected to a vacuum line
3.) Collect microspheres on top. The microspheres are dried to a free flowing powder by applying a vacuum until no more filtrate comes out of the funnel. Transfer all microspheres from the Buchner funnel to a 60-mesh stainless steel sieve and sift on a collection pan. This powder is tlCAM (45) which is matched in the previous stage to the next production stage.
3) / one of two sub-batches of bulk powder batch of gelatin microspheres. Powder Teflon ^(R) - lined lid transferred into bottles Furintogara scan type III, which is capped with, to remove acetone as shown in the following Example 15, 2
Store at ~ 8 ° C.

The tlCAM (453) / gelatin microspheres produced by this method are approximately 10
% TlCAM (453) content. The particles produced in this way are shown in FIG.

Example 9 General Procedure for Preparation of Placebo The following is a standard general procedure used repeatedly for the manufacture of tlCAM (453) / gelatin microspheres.

Placebo gelatin microspheres (without drug addition) are prepared by the method according to steps 4 to 7 of the method of Example 8 above, omitting the addition of tlCAM (453). t
The process steps used for the production of both lCAM (453) -loaded microspheres and placebo microspheres were compared to placebo gelatin microspheres to obtain similar particle sizes.
In the method of emulsifying microspheres with tlCAM (453), slightly different gelatin:
Identical except using the oil ratio. 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 both emulsion volumes constant. You.

Example 10 General Procedure for the Preparation of Albumin / Gelatin Microspheres The following is a standard general procedure used repeatedly for the production of albumin / gelatin microspheres.

Albumin / gelatin microspheres are prepared according to the method of Example 8, with the following changes to the preparation of the gelatin solution. 0.019 M NaOH 436.3 at 50 ° C.
A solution of gelatin and albumin is prepared by dissolving 104.8 g of 250-bloom gelatin in g. 25% albumin (human) USP (Bayer Co
(rp, Elkhardt, IN) 58.7 g are added. This corresponds to a solids concentration of 20% and an albumin loading of 12.3%, resulting in an albumin loading of 10% in the final product. The presence of NaOH in the albumin / gelatin solution is necessary to prevent isoelectric precipitation of albumin. Otherwise, this concentration of gelatin solution is p
H4-5. The albumin / gelatin solution is emulsified in corn oil and processed as in Example 8, steps 4-7.

The powders recovered from the two experiments, prepared according to the method described above, were combined after vacuum filtration and treated by the acetone removal method described in Example 13 and the water removal method of Example 14 to give a volume Gelatin microspheres having an average volume particle diameter of 43 μm without detectable particles of diameter <10 μm or> 100 μm were obtained. The final albumin addition was 10.6% by weight.

Example 11 Adjustment of Particle Size A comparison was made between placebo (gelatin only) microspheres prepared by the method of Example 9 and albumin-loaded microspheres prepared by the method of Example 10.
The results are shown in Table XI.

[0150]

[Table 12]

[0151] This clearly shows that the presence of albumin increased the average volume particle diameter.

[0152] tlCAM (453) also increased the average volume particle diameter. To study this, experiments were performed on a medium scale, i.e. using an 800 mL emulsion volume in a 12.5-cm container and a 2 "R100 impeller (40 g dry weight). At the scale (Table XII) and with a 1: 3 gelatin: oil ratio, the albumin-loaded microspheres were only slightly larger than the placebo microspheres (73 to 62 μm), at a 1: 4 gelatin: oil ratio. The albumin-loaded microspheres had an average volume particle diameter of 57 μm compared to 47 μm for placebo.

[0153]

[Table 13]

However, the average volume particle diameter of tlCAM (453) -loaded microspheres was much larger than placebo (92 and 165 μm versus 62 μm, respectively). These experiments showed that tlCAM (453) caused an increase in average volume particles.

The effect of added protein on particle size was easily minimized by changing the gelatin: oil ratio from 1: 3 to 1: 4, while keeping the volume of the emulsion constant. This was first shown on an 800-mL scale (Table XII). At an oil: gelatin ratio of 1: 4, both albumin and tlCAM (453) -loaded microspheres were larger than placebo microspheres, but more importantly, albumin-loaded and t-CAM.
Both lCAM (453) added microspheres showed a pharmaceutically acceptable particle size distribution.

Preparation of tlCAM (453) -Added Gelatin Microspheres in Larger Batches Example 12 To prepare larger batches, the methods used above in Table XII (3000 mL) were used and are further shown below. Have been. Materials 250-bloom gelatin 105 g tlCAM (453) in histidine buffer 15 g (12.4% of theoretical) corn oil (ρ = 0.92) 2200 g Span 80 24 g emulsified gelatin-tlCAM (453) solution Weight 600 g Diameter of beaker 17.3cm impeller, 76mmR100, off-center position, shaft, 10-15 ° from vertical speed 400, increase to 640rpm during cooling period Temperature first 45-50 ° C Cool down to 15 ° C over 70 minutes <15 ° C 1 hour cure wash 7 washes with acetone, with 10 min stirring and 10 min settling time, washing volume, 13 L 2-52 L 6-71 1 L (5 min stirring and sedimentation) Filtration in Buchner funnel 3. Use 3.0-μm Millipore Fluoropore filter.

The results are shown in Table XIII below.

[0158]

[Table 14]

[0159] The following general methods of removal of Example 13 the residual acetone is a standard general method repeatedly used for removal of residual acetone from a pharmaceutical composition comprising gelatin microspheres.

The product of Example 8 (240 g theoretical weight) was applied to a 13-cm Amicon Vantage® S2 column (Amicon, Beverly ⁾ connected to a sterile air supply and two gas scrubbing bottles.
, MA). 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 moistened while passing through the first bottle at a flow rate of 15 L / min. The relative humidity is adjusted to 90 by adjusting the temperature of the water bath.
Set to ~ 95%. At 15 L / min, the powder fluidizes the powder bed to an average. The treatment is carried out for about 11 hours, by which time the residual acetone concentration in the powder has been reduced to less than 250 ppm.

[0161] The following general methods for Examples 14 moisture reduction is repeatedly used and standardized general method for water loss pharmaceutical composition comprising gelatin microspheres.

Using a device similar to that in Example 12, reduce the water content of the microspheres. In this case, the relative humidity adjusted by setting the water bath temperature at 2-4 ° C. is 35-40%, and the target range of the final moisture content in the microspheres is 12-18%. Remove the bulk tlCAM (453) microspheres from the column and at 2-8 ° C,
Teflorn ^(R) - stored in bottles of flint glass with a backing lid type -III.

Example 15 Preparation of 400 g Gelatin / tlCAM (453) Microspheres Porcine gelatin (type A) was purchased from Hormel Foods (Austin, MN). Corn oil and Span 80 (sorbitan monooleate, NF grade) are available from Ruger Chemi
Supplied by cal Co. Inc. (Irvington, NJ). Acetone (reagent grade) EM Sci
ence (Gibbstown, NJ). Purified ICAM was prepared according to Example 1 in 10 mL histidine buffer pH 7.0 (〜50 mg / ml). Human serum albumin (HAS lot # 684PO67A, commercial material) was purchased from Bayer
Corporation, Biological Products, Clayton NC. The vessels, impellers, shafts and baffles used in these experiments were made from stainless steel (SS316).

From the 150 g scale of Examples 8, 9 and 10 to larger scales, the strategy for the geometric expansion of the manufacturing process was based on the principle of constant mixing power per unit volume. The geometric scaling method from 150 g to 400 g batch size would involve a proportional increase in container size while maintaining a constant height-to-diameter ratio (H / D) of the emulsion floor.

Based on the parameters of the known method at the smaller 150 g scale, to determine the equations needed to calculate the geometrically scaled method parameters for the larger batch size, the following derivative is Was used.

Volume of the geometrically scaled emulsion bed = πR ² H, where R is the radius of the bucket and H is the height of the emulsion. By emulsion capacity in less than 150g scale (πR _S ² H _S), the capacity of the emulsion floor in a larger geometrical scale batch size (πR _L ² H _L) Ru split,

[0167]

(Equation 3)

Comes out. If the radius is replaced by the bucket diameter (R = D / 2),

[0169]

(Equation 4)

When the numerator and denominator are X and に よ り by D _L and D _S respectively,

[0171]

(Equation 5)

Is obtained. Because of the geometric similarity, H / D is constant at all scales, the above equation becomes

[0173]

(Equation 6)

[Here, the diameter of the vessel used for the 150 g scale smaller than D _S − (6.7 ″) The capacity of the emulsion at the 150 g scale smaller than V _S − (3.0 L) D _L −greater than 150 g Diameter of containers of any size _VL- volume of emulsions of any size greater than 150 g].

Thus, by knowing the emulsion volume and container diameter at the smaller 150 g scale and the emulsion volume for the larger scale (fixed to a certain scale), the container diameter to be used for that larger scale (D _L ) can be calculated.

Power per unit capacity (P / v)

[0177]

(Equation 7)

[Where N-mixing speed (rpm) d-impeller diameter (P / v) _S- mixing power per unit volume generated on a small 150 g scale (P / v) _L- any greater than 150 g Mixing force per unit capacity generated on a scale].

The force-per-capacity equation accounts for dimensional and kinetic similarities and covers a wide range of Reynolds numbers [Rushton, JH, EWCostich, HJEveret
t, Chem. Eng. Progress 46 (8): 395-404 (1950)], where the number of forces is applicable to turbulent regimes that are constant for a given impeller design.

Equations 1 and 2 were used to calculate geometric scaling parameters for various large scales, ranging from 300 to 1000 g. These values are shown in Table XI
V, which is responsible for the proportional increase in emulsion volume and impeller diameter to generate mixing power per unit volume in the emulsion, similar to that generated on a 150 g scale.

[0181]

[Table 15]

In addition, emulsifying systems are more complex than simple mixing methods, and the increased shear stress is important in addition to the mixing power. Mixing times were not kept constant as they depended on batch size and cooling rate.

Emulsifier for Scale-Up Method at 400g Scale The relative amounts of gelatin, oil and Span 80 used in the scale-up method at 400g scale are outlined in Table XV. Contrary to the 150 g scale method, this method
The same gelatin to oil ratio is used for the production of both gelatin microspheres and ICAM / gelatin microspheres.

[0184]

[Table 16]

The emulsification method on a 400 g scale (8 L emulsion volume) requires a 9.3 "diameter vessel with a 4.0" radius impeller at an initial mixing speed of 343 rpm, as seen in Table XIV. did. The gelatin solution was prepared using a 3 "radius impeller in a 3L stainless steel container placed in a water bath maintained at 50 ° C.
It was prepared at a mixing speed of rpm. The mixture of corn oil and Span 80 was mixed at 250 rpm using a 4 "radius impeller in a 12L stainless steel container placed in a water bath maintained at 50 ° C. Dissolved gelatin The solution was added to the oil / Span mixture and emulsification was performed under various experimental conditions (with and without baffle) and cooling rates (slow, medium and fast) to achieve the desired 50 μm particle size range. Microspheres were generated.To evaluate the effect of various method parameters on the emulsion droplets produced and the particle size of the microspheres, a camera accessory (Pola) was used.
Photomicrographs of the emulsion at various points during the emulsification process were taken with a light microscope (Zeiss, Germany) with an Android Microcam, UK). The microspheres were washed with acetone to remove oil and moisture traces from the microsphere preparation. The acetone washed microspheres were then vacuum filtered using a Buchner funnel and sieved using a 75 μm mesh sieve to remove large particles and aggregates from the microsphere preparation. This is followed by humidified air (RH ~ 90%) in a chromatographic column for ~ 16 hours.
Followed by an acetone removal step by fluidizing the powder bed. After removing the acetone, the water concentration of the microspheres was adjusted to 13-15% by circulating clean air (RH-35%) through the column. The dried microspheres were stored in a glass container.

Emulsification without baffles (similar to a 150 g scale process) The manufacturing method used was similar to that used for the smaller 150 g scale. A 4.0 "radius impeller was placed in the upper left quarter and placed off center in a 9.25" diameter vessel at a 12 degree angle to normal.

Emulsion emulsion volume using a baffle plate (8 L), bucket diameter (9.25 ″) and impeller size (
4.0 ") was similar to that used in the experimental apparatus without baffles. In addition, four stainless steel baffles (10 inches high and 3/4" wide) were vertically oriented. And equidistant from each other (90 ° away) and 3/8 "from the side of the container
Was placed. The impeller was positioned in the center of the container (straight, without tilt) as seen in FIG. 3b.

Cooling Methods Three cooling methods were evaluated as follows.

Slow cooling The cooling method used was similar to that used on a smaller 150 g scale. The gelatin solution (50 ° C.) was added to the vessel containing the oil / Span mixture (in a 50 ° C. water bath) and mixed at a preset initial mixing speed. Immediately after the addition, the set temperature of the water bath was changed from 50 ° C to 10 ° C, and the mixing speed was increased as the emulsion was gradually cooled to 10 ° C.

Slow cooling rates were evaluated for emulsification experiments with and without baffles. For emulsification experiments performed without using baffles, three initial mixing speeds, 343,
375 and 415 rpm were used. Experiments using baffles were evaluated at two initial mixing speeds, 425 and 475 rpm.

Medium Cooling Cooling rates higher than those achieved on a 150 g scale (~ 0.47 degrees / minute) (~ 1
. The experiment was performed in an attempt to achieve 0 ° / min). The gelatin solution (50 ° C.) was added to the oil / Span mixture (50 ° C.) and mixed at a preset initial mixing speed.
Immediately after the addition, the water bath was turned off and ice was slowly added to the warm water bath at intervals to maintain a cooling rate of 1.0 degrees / minute. Medium speed cooling was performed by the baffle and evaluated against two initial mixing speeds, 625 and 640 rpm.

Rapid Cooling The rapid cooling rate (〜2.3 degrees / minute) was achieved as follows. 50 ℃ oil / Sp
Immediately after the addition of the gelatin solution to the mixture, the initial mixing speed was increased to a predetermined rpm. Emulsion at 50 ° C for a fixed time (20-40 minutes)
Mixing at this speed produced droplets in the 50 μm size range. The emulsion was then rapidly cooled to 15 ° C. by draining warm water from the water bath and replacing it with a slurry of ice water. The bath temperature was set at 0.1 ° C. Emulsion is 50 ℃
The mixing speed was increased when rapidly cooled to -15 ° C. The emulsion is 15 ° C
When the temperature of was reached, the bath temperature was increased to 10 ° C., the microspheres were maintained at this temperature, and
The mixture was stirred at 5 rpm for 1 hour.

The rapid cooling method with baffles was used to produce gelatin microspheres, gelatin microspheres with albumin, and gelatin microspheres with ICAM under the following two experimental conditions.

The high mixing speed impeller was positioned 1.5 "above the bottom of the vessel and evaluated for three initial mixing speeds of 600, 625, and 650 rpm with a final mixing speed of 780 rpm.
In addition, initial mixing times of 20, 30, and 40 minutes at 650 rpm were tested.

Lower Mixing Speed In order to reduce the mixing speed and still produce emulsion droplets of the desired particle size range (５０50 μm), the impeller is raised 2.5 ″ from the bottom and the final An initial mixing speed of 425 rpm with 605 rpm was evaluated.

Study of ICAM Release from Gelatin Microspheres Release studies of ICAM / gelatin microspheres were performed as follows. Weigh 20 mg of ICAM / gelatin microspheres into 12 × 75 mm polypropylene test tubes,
2.0 ml of phosphate buffered saline (pH 7.2) was added to the tube. The microspheres were briefly swirled into a tube and suspended in a 40 ° C. water bath to suspend. The tube was swirled at 5 minute intervals for a total of 1.0 hour. Transfer the resulting solution to a syringe filter (
0.2 μm, 25 mm Gelman Acrodisk (Fisher Scientific, Norcross, GA),
(Low protein binding membrane) and provided in duplicate for ICAM determination by immunoturbidimetry.

Emulsifier for scale-up process at 400 g scale Under several experimental conditions (with and without baffle) using several cooling methods (slow, medium and fast cooling) At the initially set mixing speed, 4
By the emulsification method carried out in a 9.25 "diameter vessel with a radius impeller,
Gelatin microspheres and ICAM / gelatin microspheres were prepared. The results obtained under these various conditions are summarized below.

Emulsification without deflector (slow cooling) Three emulsification experiments with initial mixing speeds of 343, 375 and 415 rpm were evaluated. The maximum possible initial mixing speed without causing excessive splashing and air entrainment is 41
It was 5 rpm. A minimum mixing speed of 343 rpm with a 4 "radius impeller was determined by geometric scaling calculations (values listed in Table XIV) and provided mixing conditions similar to those obtained with a 150 g batch (6). 0.7 "diameter vessel, 3.0" impeller and initial mixing speed of 425 rpm).

The results of the three experiments are outlined in Table XVI.

[0200]

[Table 17]

Slow cooling rate-0.25 degrees / minute The average particle size of the microspheres generated by all three experiments is large (> 70 μm)
, Much larger than the acceptable product specification of ５０50 μm. Larger emulsion volumes (8 L) on a 400 g scale required ~ 2.5 hours to slowly cool from 50 ° C to 15 ° C. This corresponded to a cooling rate of 0.25 ° C./min, much slower than the 0.47 ° C./min observed on a scale of 150 g (3 L emulsion capacity). During this slow cooling period, the three mixing rates evaluated were insufficient to provide the necessary soap to create and maintain small emulsion droplets. These droplets aggregated into large droplets, which resulted in large particles on cooling. In addition, the high percentage (10-20%) of microspheres was above 100 μm, which was outside the <10% tolerance. Microspheres smaller than 10 μm were not generated by this method.

The principle of geometric scaling and the constant mixing power per unit volume assume that the mixing method represents the particle size of the microspheres. However, experiments performed to date indicate that droplet agglomeration on cooling (due to low shear rate) and low cooling rate are important factors affecting particle size. These two cases, as described below,
It was studied using baffles and rapid cooling rates.

Emulsification Using Baffles High shear rates associated with high mixing speeds could not be achieved without secondary sources of turbulence and increased shear in the system. A baffle was used to increase the mixing speed and the system's mixing speed during the emulsification step to produce smaller droplets and consequently smaller microspheres. A schematic representation of the batch emulsification method using baffles can be seen in FIG. By using deflectors, higher initial mixing speeds up to 650 μm with minimal splashing and entrainment of air were achieved.

[0204] The emulsification method using baffles was evaluated using three different cooling methods.

The slowly cooled (deflected) prepared microspheres had an average diameter of> 65 μm (Table 3 XVIb). Pictures of the emulsion droplets and the resulting microspheres at various stages in the process can be seen in FIG. Emulsion droplets of the desired particle size range (５０50 μm) were achieved during the emulsification process with higher shear rates achieved with baffles. However, during a long cooling period, the emulsion droplets aggregated to form larger emulsion droplets, which upon cooling resulted in large microspheres (> 65 μm).

The above experiments showed that a slow cooling rate of ０．２0.25 degrees / minute was not effective in producing 50 μm microspheres. The following experiments (slow and fast cooling) studied higher cooling rates.

Medium Cooling A medium cooling rate of 1.0 degrees / minute also results in droplet agglomeration and large particles (> 13
5 μm) and particles above 100 μm. These results are tabulated
Summarized in XVII.

[0208]

[Table 18]

Medium Cooling Rate to 1.0 ° / min FIG. 7 shows the appearance of emulsion droplets and solid microspheres at various stages during the medium cooling process. Emulsion droplets within the desired particle size range (〜50 μm) were achieved during the emulsification stage at a higher mixing speed of 625 rpm. However, during cooling from 50 ° C. to 15 ° C. (35 minutes), small emulsion droplets aggregated to form large microspheres. Higher initial mixing speeds could result in thermodynamically unstable, smaller droplets that resulted in a higher degree of agglomeration. this is,
Medium-speed cooling methods (Table XVI) were better than those observed using slow cooling methods (Table XVI).
The higher particle size observed in XVII) and the higher percentage of particles above 100 μm could be explained.

The rapid cooling method was evaluated in an attempt to rapidly gel 〜50 m emulsion droplets into 50 μm microspheres and prevent them from agglomeration.

Under the two different experimental conditions described before rapid cooling (high mixing and low mixing),
A rapid cooling rate of 2.3 degrees / minute was achieved. The emulsion was cooled from 50C to 15C in ~ 15 minutes.

Three initial emulsification rates at high mixing speeds of 600, 625 and 650 rpm were evaluated. An emulsification time of 20 minutes at the mixing speed described above produces droplets in the size range of 50 μm, which when cooled rapidly have an average particle volume diameter of 57 μm, 60, 69 and 62 μm, without fines and above Produced less than 10% of the particles. These results are summarized in Table XVIII.

[0213]

[Table 19]

Rapid Cooling Rate-2.3 Degrees / min The method transferred to the pilot plant required a range of specified initial emulsification times, rather than a fixed 20 minute mixing time. The minimum emulsification time required to produce a 50 μm emulsion droplet before rapid cooling was 20 minutes. This method, 4
Repeated for an emulsification time of 0 minutes, followed by a rapid cooling which produced particles having a particle size of 63 μm (Table XVIIIa).

The method was effective at 20 and 40 minutes, so 30 ± 10 at 650 rpm
The initial emulsification time in minutes could be specified. Three repetitions, performed with an emulsification time of 30 minutes followed by rapid cooling, resulted in an average particle size of ５５55 μm. These results are summarized in Table XVIIIb.

The method can be summarized below. Time Emulsion temperature (° C.) Add gelatin to RPM 50 375 oil 0 50 650 10 50 650 20 50 650 30 50 650 The resulting emulsion was rapidly cooled as previously described. During the cooling process,
The rpm was increased at the following set temperatures. Emulsification temperature (° C.) RPM 40 700 35 715 30 730 28 750 23 765 18 780 15 780 Next, the microspheres were washed with acetone, filtered and dried.

The albumin / gelatin microspheres produced by the method described above are 61, 58 and 70μ.
The ICAM / gelatin microspheres had an average particle size of m and a volume diameter of 66 μm. The results are summarized in Table XIX and shown in FIG.

[0218]

[Table 20]

Rapid cooling rate to 2.3 degrees / minute Low mixing rate The high mixing rate method bounced during the emulsification process and resulted in entrainment of air during the maintenance phase (1 hour at 15 ° C, 780 rpm). To minimize the bounce,
The impeller was raised 2.5 "from the bottom of the vessel and used at a lower mixing speed. The initial mixing speed evaluated was 42 with a final rpm of 605 during the cooling process.
It was 5 rpm.

The methods evaluated were as follows. Time (minutes) Emulsion temperature (° C) RMP 50 375 Add gelatin to oil 0 50 425 10 50 425 20 50 425 30 50 425 The resulting emulsion was rapidly cooled as previously described. As the emulsion cooled, the rpm was increased to the set temperature as described below.

Emulsion temperature (° C.) RMP 45 475 40 525 35 550 30 575 25 590 20 605 15 605 Next, the microspheres were washed with acetone, filtered and dried.

Gelatin microspheres, albumin / gelatin microspheres and ICAM / gelatin microspheres (various loadings) prepared by the low mixing speed method have a particle size range of 50 μm with a Span of around 0.8. There was no fine powder below 10 μm, and less than 3.5% above 100 μm. These results are summarized in Table XX and Table XXI and are shown in FIG.

[0223]

[Table 21]

[0224]

[Table 22]

Typical particle sizes of the microspheres obtained by this process are shown in Table XXII and FIG.

[0226]

[Table 23]

As can be seen from the particle size table, the average particle size [D (4,3)] is 0.7
56.36 μm with a Span of 8 and without particles with a volume particle diameter of <10 μm, but with ≒ 3% of particles with a volume particle diameter greater than 100 μm.

Study on Release of ICAM from Gelatin Microspheres 12.5, 10.5, 10.0, 5.0, 2.5 and 1.5% were prepared with various theoretical additives. The resulting ICAM / gelatin microspheres were dissolved in this example as described above and measured for ICAM by immunoturbidimetry. All batches showed an actual loading of 量 100%, within the measurement variability. The ICAM / gelatin microsphere preparation was lysed and the sample was bioassayed. The ICAM / gelatin microsphere preparation was comparable to the ICAM standard (103% of the standard). ED ₅₀ is 0.30 against standard
It was 0.298 compared to 3. These results indicated that ICAM was still potent after the preparation and washing method.

Conclusions The strategy for extending the production of microspheres to a scale of 400 g has been
It was a geometric scale expansion with constant mixing force per unit volume, based on the water-in-oil emulsification method developed at the 0 g scale. Several experimental conditions (with and without baffles) and various cooling methods (slow, medium and fast) were evaluated. The slow cooling method without baffles required ~ 2.5 hours to cool from 50 ° C to 15 ° C. The evaluated mixing speeds (343, 375, and 415 rpm) produced small emulsion droplets, did not provide the necessary shear to maintain, and produced large microspheres upon cooling. The mixing speed can be reduced to 4 without causing significant bounce and entrainment of air.
It could not be increased above 15 rpm. Deflectors have been introduced into the system to increase the mixing speed and the shear stress of the system. The emulsification rate is 65
Increased to 0 rpm, resulting in emulsion droplets in the 50 μm particle size range.
However, during the slow (cooling time 2.5 hours) and medium speed cooling (cooling time 35 minutes) phases, these 50 μm emulsion droplets agglomerated, which resulted in very large particles on cooling. . The rapid cooling method requires a 50 μm
Was developed to gel the emulsion droplets. The method was evaluated under two conditions, high mixing speed with low impeller height and low mixing speed at higher impeller height. Both methods consistently produced microspheres in the 50 μm particle size range. Three factors have proven to be important in achieving the desired 50 μm particle size range of microspheres. They include the initial emulsification time (〜30 minutes), the appropriate container geometry and mixing speed, and the rapid cooling rate (〜2.3 degrees / minute).

As set forth in the illustrative embodiments above, many modifications and variations of the present invention are expected to occur to those skilled in the art, and consequently, as set forth in the appended claims. Only the constraints must be provided for them.

It is therefore intended in the appended claims to cover all equivalent modifications that fall within the scope of the claimed invention.

[Brief description of the drawings]

FIG. 1 shows an impeller suitable for use in the present invention. (A) A310 is a high efficiency axial flow impeller, (B) A200 is a four blade axial flow impeller, and (C) R100 is a radial flow impeller. Everything is Lightnin Equip
ment.

FIG. 2 shows the results of the gelatin: oil ratio study of Example 2.

FIG. 3 shows the preferred position of the impeller in the emulsification stage for (A) less than 3L and (B) more than 3L. (1) 3 "radius impeller (upper left 1/4, 12 degrees to perpendicular), (2) 4" radius impeller, (3) deflector.

FIG. 4 shows a schematic representation of a batch emulsification process suitable for preparing microspheres. (1
) Gelatin solution (50 ° C), (2) oil / Span mixture (50 ° C), (3) water-in-oil emulsion, (4) acetone, (5) solid microspheres suspended in oil, (6)
Acetone wash, (7) oil / acetone, (8) acetone, (9) microspheres, (10
) Vacuum, (11) 75 μm mesh sieve, (12) dried microspheres.

FIG. 5 is a scanning electron micrograph showing the structure of tlCAM (453) / gelatin microspheres prepared by the method of Example 8 (Panels A and B) and by the method of Illum et al. (Panels C and D). Is shown. The average volume particle diameter of the preparations of panels A and B is 46.4 μm, with 0.5% of the particles having a volume particle diameter of> 100 μm;
Undetectable concentrations of particles have a volume particle diameter of <10 μm. In contrast, the average volume particle diameter of panels C and D is 123 μm, 39%> 100 μm,
33% are <10 μm.

FIG. 6 shows the appearance of emulsion droplets and microspheres at various stages during the emulsification process of the batch of Example 15, using baffles, slow cooling, and high mixing speeds; 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 the appearance of emulsion droplets and microspheres at various stages during the emulsification process of the batch of Example 15, using baffles, medium speed cooling, and high mixing speeds; (A) Emulsion droplet at 625 rpm (1 minute), (B) 625r
emulsion droplets at pm (20 minutes), (C) cooled emulsion (<
15C), and (D) microspheres (washed and dried).

FIG. 8 shows the appearance of droplets and microspheres of the emulsion at various stages during the emulsification process of the batch of Example 15, using baffles, rapid cooling, and high mixing speeds; A) Emulsion droplets immediately after gelatin addition at 375 rpm, (B) 2
Emulsion droplet after mixing at 625 rpm for 0 minute, (C) 15 ° C. at 625 rpm
Gelatin microspheres suspended in oil after mixing for less than 1 hour, and (D) gelatin microspheres after washing, filtration and vacuum drying.

FIG. 9 shows the appearance of emulsion droplets and microspheres at various stages during the low mixing speed method of Example 15 using baffles and rapid cooling, (A) 425 rpm
(30 minutes) emulsion, (B) emulsion cooled to 15 ° C,
And (C) microspheres (washed and dried).

FIG. 10: Typical obtained by laser particle sizing of microspheres produced by the rapid cooling method of Example 15 using baffles, higher impeller height, and lower mixing speed. A typical particle size distribution is shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) B01J 2/08 A61K 37/02 13/04 B01J 13/02 A (31) Priority claim number 08/942, 403 (32) Priority date October 1, 1997 (Oct. 1, 1997) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM ), AL, A , AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Wang, Way California, United States 94502 A Lamenda Lailask Street 923 (72) Inventor Cordle, Margaret 94501 Alameda Greenbrier Road 400, Calif. 94433 Lunia Airfield Homestead Court 2402 (72) Inventor, Concescio, Neville M. 94105 San Francisco Beale Street 570, Suite 119, Calif., USA U.S.F. DD63 EE23 EE41 EE42 EE53 GG02 GG05 GG16 4C084 AA02 AA03 BA22 BA34 BA42 DA39 MA43 MA59 NA06 NA08 NA12 NA13 ZB332 4G005 AA01 AB15 BA11 BB06 BB13 BB26 DB02W DB06Z DB22X DC02W DC12W DC32W EA03

Claims (29)

[Claims] 1. A method of preparing a delivery system for an intranasal drug comprising gelatin microspheres in combination with an agent intended for intranasal delivery, comprising: (a) heating the gelatin at a temperature above the gelation temperature of the gelatin; And preparing a low salt or salt free aqueous solution of the drug to be delivered; (b) preparing a solution of a suitable oil and a suitable surfactant; (c) the solution of step (a) and Emulsifying the mixture of solutions of step (b)
Producing a water-in-oil emulsion with a volume ratio of 1: 2 and 1:10, (d) continuing emulsification until the target droplet size is achieved, (e) ) To reduce the temperature of the emulsion to below the gelation temperature of the gelatin at a controlled fast rate to achieve gelation prior to coalescence of the droplets, thereby binding the drug at the desired particle size Wherein the emulsions in steps (c, d) and (e) above are mixed at a maximum velocity consistent with a low or no vortex circulation pattern, (F) separating drug-containing microspheres from the oil / surfactant phase; and (g) dehydrating the drug-containing microspheres to produce a dry powder. 2. The method of claim 1, wherein said gelatin of step (1a) has a bloom strength of about 250. 3. The method of claim 1, wherein said oil of step (1b) is a vegetable oil. 4. The method of claim 1, wherein the surfactant of step (1b) has a hydrophilic-lipophilic balance of 4-6. 5. The method of claim 1, wherein said volume ratio of step (1c) is between 1: 3 and 1: 4.
The method of claim 1. 6. The method of claim 1, wherein said droplet size in step (1d) is between 20 and 80 μm. 7. The method of claim 6, wherein said droplet size in step (1d) is between 40 and 60 μm. 8. The method according to claim 1, wherein the temperature of step (1e) is reduced at a rate of 2.0 to 2.5 ° C./min. 9. The method of claim 1, wherein the drug-containing microspheres are removed from the oil / surfactant phase by washing with a water-miscible solvent. 10. The method of claim 1, wherein the drug-containing microspheres are removed from the oil / surfactant phase by washing with a hydrocarbon solvent, followed by washing with a water-miscible solvent. 11. The method of claim 1, wherein said agent is ICAM-1 or a fragment, analog, or derivative thereof. 12. The method according to claim 1, wherein said method is a batch method. 13. The method of claim 1, wherein said method is a continuous method. 14. A residual solvent from the pharmaceutical matrix, comprising contacting the pharmaceutical matrix with a wetting gas under conditions that entrain the residual solvent in the gas, and removing the gas. Removal method. 15. The pharmaceutical matrix according to claim 14, wherein the pharmaceutical matrix is a dry powder.
The described method. 16. The method of claim 15, wherein the humidified gas is passed through the dry powder under conditions that create a fluidized bed in the dry powder. 17. The method of claim 16, wherein the fluidized bed is contained in a column with filters at both ends that is permeable to the humidifying gas but not to the dry powder. 18. The dry powder of claim 16, wherein the dry powder comprises gelatin microspheres.
The described method. 19. The method of claim 18, wherein said gelatin microspheres encapsulate a drug. 20. The method of claim 19, wherein said agent is ICAM-1 or a fragment, analog or derivative thereof. 21. The method of claim 1, wherein residual solvent is removed from the generated microspheres by the method of claim 14. 22. The method of claim 21, wherein said agent is ICAM-1 or a fragment, analog or derivative thereof. 23. The product of the method of claim 1, wherein said agent is ICAM-1 or a fragment, analog, or derivative thereof. 24. The product of the method of claim 14, wherein said agent is ICAM-1 or a fragment, analog, or derivative thereof. 25. The product of the method of claim 21, wherein said agent is ICAM-1 or a fragment, analog, or derivative thereof. 26. The majority of the microspheres are present as non-aggregated individual particles.
Gelatin microspheres having an average volume particle diameter between 20-80 μm. 27. The gelatin microsphere of claim 26, wherein the percentage of particles having a volume particle diameter of> 100 μm is less than 10% as measured by laser light scattering analysis. 28. The percentage of particles having a volume particle diameter of <10 μm
28. The gelatin microspheres of claim 27, wherein the microspheres are <1% as measured by laser light scattering analysis. 29. An intranasal drug delivery system comprising the gelatin microspheres of claim 26 conjugated to ICAM-1 or a fragment, analog, or derivative thereof.