Link:  Pharm/Biotech Resources

Title:  Sustained release ionic conjugate

United States Patent:  6,911,218

Issued:  June 28, 2005

Inventors:  Ignatious; Francis Xavier (Millville, MA); Loughman; Thomas Ciaran (Dublin, IE); Shalaby; Shalaby Wahba (Pendleton, SC); Touraud; Franck Jean-Claude (Vernon, FR)

Assignee:  Ipsen Manufacturing Ireland Limited (Dublin, IE)

Appl. No.:  171740

Filed:  April 22, 1997

PCT Filed:  April 22, 1997

PCT NO:  PCT/IE97/00030

371 Date:  April 20, 1999

102(e) Date:  April 20, 1999

PCT PUB.NO.:  WO97/39738

PCT PUB. Date:  October 30, 1997

A method of spherifying a sustained release ionic conjugate which contains a free carboxyl group-containing biodegradable polymer and a free amino group-containing drug which are ionically bonded to each other.

TECHNICAL FIELD

This invention relates to sustained release drug delivery systems and, in particular, to a method of making microparticles of a sustained release ionic conjugate.

BACKGROUND ART

Biodegradable polymeric drug delivery formulations have been developed and utilized for the controlled in vivo release of drugs. See e.g., U.S. Pat. Nos. 3,773,919 and 4,767,628. Such biodegradable polymeric formulations are designed to allow an entrapped drug to slowly diffuse through a polymer matrix or coating when the biodegradable polymer is depolymerized.

International Publication No. WO 94/15587 describes sustained release ionic molecular conjugates of polyesters and drugs. Since polyester degradation is a key step in the release process, the surface area of the conjugate particles can control the release profile of the drug from the conjugate. Thus, the conjugate particles should be of similar size and shape to insure both the minimum and reproducible surface area, e.g., microspheres.

DISCLOSURE OF INVENTION

In one aspect, this invention features a method of making microparticles of a sustained release ionic conjugate containing a free carboxyl group-containing biodegradable polymer (a polyester made of monomers such as lactic acid, e-caprolic acid, glycolic acid, trimethylene carbonate, or p-dioxanone; or a copolymer thereof; the monomers can be optically isomers or racemates) and a free amino group-containing drug (e.g., a peptide drug such as somatostatin or LHRH) which are ionically bonded to each other. The method includes the steps of (1) obtaining a first solution in which the conjugate is dissolved; (2) mixing the first solution (added as small droplets, e.g., through an atomizing nozzle such as sonic nozzle, pneumatic nozzle, rotary atomizer, or pressure nozzle) with a first liquid to form a first dispersion, wherein the first liquid is miscible with the first solution, and the conjugate is not soluble in the first liquid and precipitates out of the first dispersion; and (3) isolating the conjugate from the first dispersion.

In one embodiment, the drug is soluble in the first liquid, which can be an alcohol (e.g., ethanol or isopropyl alcohol), hexane, or water; or a mixture thereof. When ethanol is used as the first liquid, it can be maintained between about 0° C. and -;30° C. when it is being used. When isopropyl alcohol is used, it can be maintained between about 0° C. and -;70° C., e.g., cooled by the addition of dry ice.

The first solution, which may contain acetone, dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran, or glyme, or mixtures thereof can be obtained by (1) dissolving the biodegradable polymer in a second liquid (e.g., acetone, tetrahydrofuran, glycone, ethyl acetate, methyl acetate, acetonitrile, ethyl formate, or glyme; or a mixture thereof) to form a second solution; (2) dissolving the drug in a third liquid (e.g., water or acetone; or a mixture thereof) to form a third solution, wherein the third liquid is miscible with the first liquid and the second liquid; and (3) mixing the second solution and the third solution to form the first solution, wherein the mixing causes the drug to ionically bond to the biodegradable polymer and form the conjugate in the first solution. The first solution may comprise up to 40% by weight of the conjugate (e.g., between 25 and 35 percent by weight of the conjugate). In one example, a base, e.g., NaOH or KOH, can be added to the second solution prior to mixing the second solution and the third solution. Neutralization of the carboxyl groups of the biodegradable polymer with the base facilitates the formation of the ionic conjugate.

Alternatively, the first solution is obtained by dissolving the biodegradable polymer and the drug in a second liquid (e.g., acetone or a mixture of acetone and water) to form the first solution, thereby forming the conjugate in the first solution. According to this method, the biodegradable polymer can be first dissolved in the second liquid, a base is then added to the second solution, and the drug is subsequently dissolved in the second liquid. Also, if desired, the first solution can be partially or completely evaporated from the first dispersion prior to isolation of the conjugate. The processed conjugate can be conveniently isolated by centrifuging or filtering the first dispersion, and the isolated conjugate can be mixed with an aqueous mannitol solution prior to vacuum drying (e.g., lyophilization). The isolated conjugate can be further shaped into a film or a rod. The isolated conjugate can also be spherified into microspheres of average diameter of 5 to 200 μm, e.g., as described herein. By "spherification" or "spherifying" is meant the processing of a microparticle into a shape close to a sphere.

In another aspect, this invention features a method of spherifying a sustained release ionic conjugate as described above. The method includes the steps of (1) mixing the conjugate with a first liquid (e.g., an oil such as silicon oil, mineral oil, sesame oil, or a vegetable oil) to form a first dispersion, wherein the conjugate has the shape of a microparticle and is not soluble in the first liquid; (2) heating the first dispersion to a temperature greater than the Tg or Tm of the conjugate; (3) cooling the first dispersion below the Tg or Tm of the conjugate; (4) mixing the first dispersion with a second liquid (e.g., hexane, heptane, isopropyl myristate, or an alcohol such as ethanol or isopropyl alcohol) to form a second dispersion, wherein the second liquid is miscible with the first liquid and the conjugate is not soluble in the second liquid; and (5) isolating the conjugate from the second dispersion. The conjugate may have the shape of a microcapsule with an average diameter of between 5 μm to 200 μm prior to mixing with the first liquid, and the first dispersion thus formed is vigorously stirred while being heated to aid in the separation of the particles. Once the conjugate has been isolated, it can be rinsed with the second liquid and then vacuum dried. Optionally, it can also be mixed with an aqueous mannitol solution prior to vacuum drying.

A third aspect of this invention features a method of spherifying the above-described sustained release ionic conjugate (e.g., a microcapsule having an average diameter of between 5 μm to 200 μm). The method includes the steps of (1) mixing the conjugate in a first liquid (e.g., water) to form a first dispersion, wherein the conjugate is in the shape of a microparticle and the conjugate is not soluble in the first liquid; (2) stirring the first dispersion; (3) mixing the stirred dispersion with a second liquid (e.g., dichloromethane or chloroform) in such an amount so that it is absorbed by the conjugate but does not solubilize the conjugate, wherein the second liquid is miscible with the first liquid; (4) evaporating the second liquid from the first dispersion; and (5) isolating the precipitated conjugate from the first dispersion. If necessary, the method may further include the step of adding a surfactant (e.g., lecithin, Tween 20, polysorbate, or lauryl sulfate) to the first dispersion to aid in the stabilization of the first dispersion, and the isolated conjugate can be rinsed with the first liquid and vacuum dried. Again, the isolated conjugate can be mixed with an aqueous mannitol solution prior to vacuum drying.

In a further aspect of this invention, this invention features a method of spherifying the above-described sustained release ionic conjugate. The method includes the steps of (1) dissolving the conjugate in a first liquid (e.g., acetonitrile) to form a first solution; (2) stirring the first solution with a second liquid (e.g., an oil) to form a first dispersion, wherein the second liquid is immiscible with the first solution; (3) evaporating the first liquid from the first dispersion to precipitate the conjugate from the first dispersion; and (4) isolating the precipitate conjugate from the first dispersion. In the stirring step, the first solution can be added to the second liquid as small droplets.

The above method can further include the step of rinsing the isolated conjugate with a third liquid (e.g., hexane, heptane, or octane) which is miscible with the second liquid and not a solvent for the isolated conjugate. If desired, the isolated conjugate can be mixed with an aqueous mannitol solution prior to vacuum drying.

The biodegradable polymer in the above-described conjugate may contain at least one free carboxyl group (e.g., two to ten free carboxyl groups per polymer chain). Examples of carboxylic acid containing biodegradable polymers include polyesters containing units of lactic acid, e-caprolic acid, p-dioxanone, e-caprionic acid, substituted and unsubstituted trimethylene carbonate, 1,5-dioxepan-2-one, 1,4-dioxepan-2-one, glycolic acid, alkylene oxylate, cycloalkylene, cycloalkylene oxylate, alkylene succinate, or 3-hydroxy butyrate in optically active forms or as racemates; or copolymers of any of the above. Additional free carboxylic acid groups can be incorporated into the biodegradable polyester by reaction, e.g., ring opening polymerization or polycondensation, with polycarboxylic acids such as malic acid, tartaric acid, pamoic acid, citric acid, succinic anhydride, and glutaric anhydride. Thus, the biodegradable polymer can be a water insoluble polyester including lactic acid units with or without glycolic acid units. Other biodegradable polymers such as polyorthoesters, polyorthocarbonates, and polyantals may also be used. The biodegradable polymer may have an average degree of polymerization, e.g., average number of monomers per polymer chain, between 10 and 300.

The drug has one or more (e.g., one to ten) free amine groups. In one embodiment, the drug is an acid-stable peptide. Examples of suitable acid-stable peptides include growth hormone releasing peptide (GHRP), luteinizing hormone-releasing hormone (LHRH), adrenomedullin, growth hormone, somatostatin, bombesin, gastrin releasing peptide (GRP), calcitonin, bradykinin, galanin, melanocyte stimulating hormone (MSH), growth hormone releasing factor (GRF), amylin, adrenomedullin, tachykinins, secretin, parathyroid hormone (PTH), enkephalin, endothelin, calcitonin gene releasing peptide (CGRP), neuromedins, parathyroid hormone related protein (PTHrP), glucagon, neurotensin, adrenocorticotrophic hormone (ACTH), peptide YY (PYY), glucagon releasing peptide (GLP), vasoactive intestinal peptide (VIP), pituitary adenylated cyclase activating peptide (PACAP), motilin, substance P, neuropeptide Y (NPY), TSH, and analogs and fragments thereof. The drug may be soluble (e.g., greater than 0.1 mg/ml; preferably, greater than 1.0 mg/ml) in the first liquid.

Other features and advantages of the present invention will be apparent from the detailed description and from the claims.

It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

BEST MODES FOR CARRYING OUT THE INVENTION

EXAMPLE 1

18.0 g of a 6,000 g/mol 66/32/2 poly-L-lactic-co-glycolic-co-D,L-malic acid copolymer (66 percent L-lactic acid, 32 percent glycolic, and 2 percent malic acid; acid number of 0.373 milliequivalents/g) was dissolved in 180 g of acetone (solution of 10% copolymer by weight). 14.4 ml of 0.5 N aqueous NaOH was added to form the sodium carboxylate of the polymer. 4.28 g of the acetate salt of the peptide Lanreotide™ (Kinerton, Dublin, Ireland; D-Nal-c[Cys-Tyr-D-Trp-Val-Cys]-Thr-NH₂; acetate content=9.60 percent by weight) was separately dissolved in a mixture of 10 g of acetone and 10 g of deionized water. The amount of peptide dissolved corresponded to the stoichiometric ratio of acid groups from the copolymer (e.g., one) and the free amino groups for the peptide (e.g., two). The peptide solution was then added dropwise to the copolymer solution, and the resulting solution was stirred for two hours to allow for salt exchange and the resulting formation of the polymer/peptide ionic conjugate (PPIC).

EXAMPLE 2

In a temperature controlled jacketed reactor (Schott Glass AGB, Dublin, Ireland), a two liter bath of deionized water was precooled to 0° C. and was vigorously stirred. The above PPIC solution of Example 1 was then slowly added to the reactor using a Masterflex pump (Bioblock Scientific, Illkvch, France) which produced a flow rate of 10-15 ml/min through a silicone tubing fitted with a 19 gauge needle at its tip. The PPIC solution was fed through the needle that was positioned above a water bath at 0° C. The PPIC precipitated in the bath as small, solid particles. The solid particles were then separated from the supernatant by centrifugation (30 minutes at 5000 rpm and 0-5° C.), rinsed with fresh deionized water, resuspended water, recentrifuged, and then lyophilized. The isolated conjugate was filtered through a 100 μm sieve to remove any large particles which would not be capable of being injected through a 21 gauge needle. An analysis of the resulting particle sizes is described in Table I.

EXAMPLE 3

The PPIC solution of Example 1 was also precipitated as described above in Example 2, except that a bath of ethanol at a temperature of -;20° C. was used instead of a bath of water at 0° C. An analysis of the resulting particle sizes is described in Table I.

EXAMPLE 4

The PPIC solution of Example 1 was also dispersed at a controlled flow rate of 4 ml/min through an atomizing nozzle containing a hollow tip (Bioblock; 50 Watts, 20 kHz) over a bath of ethanol at -;10° C. in a temperature controlled jacket reactor. In this nebulization process, the copolymer solution was released from the probe as a fine mist of small droplets. The small droplets fell into the ethanol bath, causing the deionized water and acetone to be extracted from the droplets. As a result, the copolymer droplets hardened as small, solid particles. The particles were then recovered by centrifugation and lyophilized. An analysis of the resulting particle sizes is listed in Table I. What is meant by Diameter -;10 (i.e., D0.1), Diameter -;50 (i.e., D0.5), or Diameter -;90 (i.e., D0.9) is the smallest diameter which is greater than 10%, 50%, and 90% of the total particles, respectively. What is meant by specific area is the average specific area of the resulting particles.

Claim 1 of 20 Claims

1. A method of making microparticles of a sustained release ionic conjugate containing a free carboxyl group-containing biodegradable polymer and a free amino group-containing drug which are ionically bonded to each other, the method comprising:

obtaining a first solution in which said conjugate is dissolved, wherein said solution comprises at least one part acetone, acetonitrile, ethyl acetate, tetrahydrofuran, glyme or any combination thereof;

adding said first solution through an atomizing nozzle to a ethanol or isopropyl alcohol to form a dispersion, wherein said conjugate precipitates out of said dispersion; and

isolating said conjugate from said dispersion.

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