United States Patent [191
`Johnson et al.
`
`[54] COMPOSITION FOR SUSTAINED RELEASE
`OF HUMAN GROWTH HORMONE
`
`[75] Inventors: OluFunmi Lily Johnson, Cambridge,
`Mass.; Medha M. Ganmukhi,
`Wexford, Pa; Howard Bernstein,
`Cambridge; Henry Auer, Belmont, both
`of Mass.; M. Amin Khan, Dowington,
`Pa
`
`[73] Assignee: Alkermes, Inc., Cambridge, Mass.
`
`[21] Appl. N0.: 473,544
`[22] Filed:
`Jun. 7, 1995
`
`Related US. Application Data
`
`[63] Continuation-impart of Ser. No. 984,323, Dec. 2, 1992,
`abandoned.
`
`[51] Int. Cl.6 .............................. .. A61F 2/02; A61K 9/14;
`»
`A61K 9/50; A61K 38/24
`[52] US. Cl. ........................ .. 424/502; 424/423; 424/426;
`424/489; 424/499; 424/500; 424/501; 514/21;
`530/399; 530/839
`[58] Field of Search ................................... .. 424/423, 426,
`424/489, 499, 500, 501, 502; 514/21; 530/399,
`839
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`6/1987 Kent et a1. ............................ .. 424/490
`4,675,189
`8/1988 Hutchinson
`.... .. 424/426
`4,767,628
`8/1989 Yim et a1.
`424/85 7
`4,853,218
`1/1990 Langer et a1.
`424/428
`4,891,225
`1/1990 'Iioe et a1.
`424/422
`4,897,268
`3/1990 Langer et al.
`424/428
`4,906,474
`4,962,091 10/1990 Eppstein et a1. ..
`....... .. 514/2
`4,981,696
`l/1991 Loomis et a1.
`424/486
`
`4,985,404
`
`1/1991 Mitchell . . . . . . . . . . . .
`
`5/1991 Gombotz et al. .
`6/1992 Ron et a1. . . . . . . . . .
`
`. . . .. 514/2
`
`424/497
`. . . . . .. 424/80
`
`5,019,400
`5,122,367
`
`5,126,147
`5,176,907
`5,192,741
`
`424/497
`6/1992 Silvestri et al.
`424/78.08
`l/1993 Leong ............. ..
`3/1993 Orsolini et a1. ................... .. 514/4
`
`FOREIGN PATENT DOCUMENTS
`
`0330180A1 2/1989 European Pat. O?. .
`0537559Al 10/1992 European Pat. O?‘. .
`WO90/09166 8/1990 WIPO
`WO90/l3780 11/1990 WIPO
`WO92/l1844 7/1992 WIPO
`WO91/l2882 9/1992 WIPO
`WO93/027l2 2/1993 WIPO
`WO93/0786l 4/1993 WIPO
`WO93/17668 9/1993 WIPO
`WO93/25221 12/1993 WIPO
`WO94/l2158 6/1994 WIPO
`
`USOO565401OA
`[11] Patent Number:
`[45] Date of Patent:
`
`5,654,010
`Aug. 5, 1997
`
`WO95/29664 ll/1995 WIPO.
`WO9607399A1 3/1996 WIPO.
`OTHER PUBLICATIONS
`Cunningham, B.C., et al., “Dimerization of Human Growth
`Hormone by Zinc,” Science, 253: 545-548 (Aug. 2, 1991).
`Costantino, H.R., et al., “Solid-Phase Aggregation of Pro
`teins under Phannaceutically Relevant Conditions,” Journal
`of Pharmaceutical Sciences, 83(12): 1662-1669 (1994).
`Costantino, H.R., et al., “Moisture-Induced Aggregation of
`Lyophilized Insulin,” Pharmaceutical Research, 11(1):
`21-29 (1994).
`Costantino, H.R., et al., “Aggregation of a Lyophilized
`Pharmaceutical Protein, Recombinant Human Albumin:
`Effect of Moisture and Stabilization by Excipients,” Bio
`technology, 13: 493-496 (1995).
`Cleland, J.L., et al., “One Month Continuous Release
`Recombinant Human Growth Hormone-PLGA Formula
`tions,” Proceed. Intern. Symp. Control. Rel. Bioact. Mater,
`22: 149-150 (1995).
`Cleland, J.L., et al., “Characterization of Recombinant
`Human Growth Hormone-PLGA Formulations in Animals,”
`Proceed. Intern. Symp. Control. Rel. Bioact. Maten, 22:
`143-144 (1995).
`Primary Examiner-Carlos Azpuru
`Attorney, Agent, or Finn-Hamilton, Brook, Smith &
`Reynolds, RC.
`ABSTRACT
`[57]
`A composition, and methods of forming and using said
`composition, for the sustained release of biologically active,
`stabilized human growth hormone (hGH). The sustained
`release composition of this invention comprises a polymeric
`matrix of a biocompan'ble polymer and particles of biologi
`cally active, stabilized hGH, wherein said particles are
`dispersed within the biocompatible polymer. The method of
`the invention for producing a composition for the sustained
`release of biologically active hGH, includes dissolving a
`biocompatible polymer in a polymer solvent to form a
`polymer solution, dispersing particles of biologically active,
`stabilized hGH in the polymer solution, and then solidifying
`the polymer to form a polymeric matrix containing a dis
`persion of said hGH particles. The method for using a
`composition of the invention is a method for providing a
`therapeutically effective blood level of biologically active,
`non-aggregated hGH in a subject for a sustained period In
`this method, a subject is administered an e?ective dose of
`the sustained release composition of the present invention.
`The method of using the sustained release composition of
`the present invention comprises providing a therapeutically
`eifective blood level of biologically active, non-aggregated
`human growth hormone in a subject for a sustained period
`by administering to the subject a dose of said sustained
`release composition.
`
`11 Claims, No Drawings
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`LUYE1009
`IPR of Patent No. 6,667,061
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`5,654,010
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`1
`COMPOSITION FOR SUSTAINED RELEASE
`OF HUMAN GROWTH HORMONE
`
`RELATED APPLICATIONS
`This application is a Continuation-in-Part of US. patent
`application Ser. No. 07/984,323, ?led Dec. 2, 1992, now
`abandoned, which is incorporated herein by reference in its
`entirety.
`
`2
`The advantages also include increased patient compliance
`and acceptance by reducing the required number of injec
`tions. The advantages further include the ability to use
`smaller amounts of hGH compared to bolus injection regi
`men because serum hGH levels are maintained closer to
`therapeutical thresholds.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The human growth hormone (hGH) used in this invention
`is biologically active hGH in its molecular (monomeric or
`non-aggregated) form. Molecular hGH is typically nou
`immunogenic.
`Aggregated hGH may induce an immune response result
`ing in antibodies formed against hGH. This may compro
`mise the e?icacy of long-term hGH therapy. Additionally,
`aggregated hGH may stimulate an auto-immune response to
`endogenous hGH.
`A sustained release of biologically active, non-aggregated
`human growth hormone is a release which results in mea
`surable serum levels of biologically active, monomeric hGH
`over a period longer than that obtained following direct
`administration of aqueous hGH. It is preferred that a sus
`tained release be a release of hGH for a period of about a
`week or more, and more preferably for a period of about two
`weeks or more.
`A sustained release of biologically active, non-aggregated
`hGH from a polymeric matrix can be continuous or non
`continuous release with relatively constant or varying rates
`of release. The continuity of hGH released and level of hGH
`released can be established by using, inter-alia, one or more
`types of polymer compositions, hGH loadings, and/or selec
`tion of excipients to produce the desired eifect.
`Stabilized (hGH) comprises biologically active, non
`aggregated hGH which is complexed with at least one type
`of multivalent metal cation, having a valency of +2 or more,
`from a metal cation component. Stabilized hGH in the
`sustained release composition of the present invention is in
`particulate form.
`Suitable multivalent metal cations include metal cations
`contained in biocompatible metal cation components. A
`metal cation component is biocompatible if the cation com
`ponent is non-toxic to the recipient, in the quanti?es used,
`and also presents no signi?cant deleterious or untoward
`elfects on the recipient’s body, such as an immunological
`reaction at the injection site.
`Typically, the molar ratio of metal cation component to
`hGH, for the metal cation stabilizing the hGH, is between
`about 4:1 to about 10:1.
`A preferred metal cation used to stabilize hGH is Zn”. In
`a more preferred embodiment, the molar ratio of metal
`cation component, containing Zn+2 cations, to hGH is about
`6:1.
`The suitability of a metal cation for stabilizing hGH can
`be determined by one of ordinary skill in the art by per
`forming a variety of stability indicating techniques such as
`polyacrylamide gel electrophoresis, isoelectric focusing,
`reverse phase chromatography, HPLC and potency tests on
`hGH lyophilized particles containing metal cations to deter~
`mine the potency of the hGH after lyophilization and for the
`duration of release from microparticles. In stabilized hGH,
`the tendency of hGH to aggregate within a microparticle
`during hydration in vivo and/or to lose biological activity or
`potency due to hydration or due to the process of forming a
`sustained release composition, or due to the chemical char
`
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`
`BACKGROUND OF THE INVENTION
`Human growth hormone (hGH) is a protein secreted by
`the pituitary gland and which can be produced by recombi
`nant genetic engineering. hGH will cause growth in all
`bodily tissues which are capable of growth.
`hGH is typically used to treat patients suffering from
`hypopituitary dwar?sm. Currently, aqueous hGH is admin
`istered as a subcutaneous bolus three times a week or once
`daily to patients to maintain suitable serum levels of hGH.
`For patients chronically receiving hGH, the frequent injec
`tions result in patient compliance problems.
`To resolve the problems associated with repetitive injec
`tions of aqueous hGH, attempts have been made to formu
`late controlled release devices containing higher doses of
`hGH than a bolus injection, encapsulated within a polymeric
`matrix wherein the hGH would be released in vivo over a
`period of about a week or more.
`However, these controlled release devices often exhibited
`high initial bursts of hGH release and minimal hGH release
`30
`thereafter. Further, due to the high concentration of hGH
`within these controlled release devices, the hGH molecules
`have tended to aggregate after several days to form aggre
`gated hGH which is immunogenic in vivo and likely has
`reduced biological activity.
`Therefore, a need exists for a means for sustaining the
`release of biologically active hGH in vivo without causing
`an immune system response over the release period of the
`hGH.
`
`35
`
`SUMMARY OF THE INVENTION
`
`This invention relates to a composition, and methods of
`forming and using said composition, for the sustained
`release of biologically active, stabilized human growth hor
`mone (hGH). The sustained release composition of this
`invention comprises a polymeric matrix of a biocompatible
`polymer and particles of biologically active, stabilized hGH,
`wherein said particles are dispersed within the biocompat
`ible polymer.
`The method of the invention for forming a composition
`for the sustained release of hGH, includes dissolving a
`biocompatible polymer in a polymer solvent to form a
`polymer solution, dispersing particles of biologically active,
`stabilized hGH in the polymer solution, and then solidifying
`the polymer to form a polymeric matrix containing a dis
`persion of said hGH particles.
`The method of using the sustained release composition of
`the present invention comprises providing a therapeutically
`elfective blood level of biologically active, non-aggregated
`human growth hormone in a subject for a sustained period
`by administering to the subject a dose of said sustained
`release composition.
`The advantages of this sustained release formulation for
`hGH include longer, more consistent in vivo blood levels of
`hGH, lower
`bursts of hGH, and increased therapeutic
`bene?ts by eliminating ?uctuations in serum hGH levels.
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`3
`acteristics of a controlled release composition, is reduced by
`complexing at least one type of metal cation with hGH prior
`contacting the hGH with a polymer solution.
`Stabilized hGH is typically stabilized against signi?cant
`aggregation in vivo over the sustained release period.
`Stabilized hGH is typically stabilized against signi?cant
`aggregation in vivo over the sustained release period. Sig
`ni?cant aggregation is de?ned as an amount of aggregation
`resulting in aggregation of about 15% or more of the initial
`amount of encapsulated hGH monomer. Preferably, aggre
`gation is maintained below about 5% of the initial dose of
`hGH monomer. More preferably, aggregation is maintained
`below about 2% of the initial dose.
`The hGH in a hGH sustained release composition can also
`be mixed with other excipients, such as bulking agents or
`additional stabilizing agents, such as buifers to stabilize the
`hGH during lyophilization.
`Bulking agents typically comprise inert materials. Suit
`able bulking agents are known to those skilled in the art.
`A polymer, or polymeric matrix, suitable for the sustained
`release composition of the present invention must be bio
`compatible. A polymer is biocompatible if the polymer, and
`any degradation products of the polymer, are non-toxic to
`the recipient and also present no signi?cant deleterious or
`untoward e?iects on the recipient’s body, such as an immu
`nological reaction at the injection site.
`The polymer of the hGH sustained release composition
`must also be bidegradable. Biodegradable as de?ned herein,
`means the composition will degrade or erode in vivo to form
`smaller chemical species. Degradation can result, for
`example, by enzymatic, chemical and physical processes.
`Suitable biocompatible, biodegradable polymers include, for
`example, poly(lactides), poly(glycolides), poly(lactide-co
`glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly
`(lactic acid-co-glycolic acid)s, polycaprolactone,
`polycarbonates, polyesterarnides, polyanhydrides, poly
`(amino acids), polyorthoesters, polycyanoacrylates, poly(p
`dioxanone), poly(alkylene oxalate)s, biodegradable
`polyurethanes, blends and copolymers thereof.
`Further, the terminal functionalities of the polymer can be
`modi?ed. For example, polyesters can be blocked,
`unblocked or a blend of blocked and unblocked polymers. A
`blocked polymer is as classically de?ned in the art, speci?
`cally having'blocked carboxyl end groups. Generally, the
`blocking group is derived from the initiator of the polymer
`ization and is typically an alkyl group. An unblocked poly
`mer is as classically de?ned in the art, speci?cally having
`free carboxyl end groups.
`Acceptable molecular weights for polymers used in this
`invention can be determined by a person of ordinary skill in
`the art taking into consideration factors such as the desired
`polymer degradation rate, physical properties such as
`mechanical strength, and rate of dissolution of polymer in
`solvent. Typically, an acceptable range of molecular weights
`is of about 2,000 Daltons to about 2,000,000 Daltons. In a
`preferred embodiment, the polymer is a biodegradable poly
`mer or copolymer. In a more preferred embodiment, the
`polymer is a poly(lactide-co-glycolide) (hereinafter
`“PLGA”) with a lactide:glycolide ratio of about 1:1 and a
`molecular weight of about 5,000 Daltons to about 70,000
`Daltons. In an even more preferred embodiment, the
`molecular weight of the PLGA used in the present invention
`has a molecular weight of about 6,000 to about 31,000
`Daltons.
`The amount of hGH, which is contained in a dose of
`sustained release microparticles, or in an alternate sustained
`
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`4
`release device, containing biologically active, stabilized
`hGH particles is a therapeutically or prophylactically e?‘ec
`tive amount, which can be determined by a person of
`ordinary skill in the art taking into consideration factors such
`as body weight, condition to be treated, type of polymer
`used, and release rate from the polymer.
`In one embodiment, an hGH sustained release composi
`tion contains from about 0.01% (w/w) to about 50% (w/w)
`of biologically active, stabilized hGH particles. The amount
`of such hGH particles used will vary depending upon the
`desired eifect of the hGH, the planned release levels, the
`times at which hGH should be released, and the time span
`over which the hGH will be released. A preferred range of
`hGH particle loading is between about 0.1% (w/w) to about
`30% (w/w) hGH particles. A more preferred range of hGH
`particle loading is between about 0.1% (w/w) to about 20%
`(w/w) hGH particles. The most preferred loading of the
`biologically active, stabilized hGH particles is about 15%
`(WW). ’
`In another embodiment, a hGH sustained release compo
`sition also contains a second metal cation component, which
`is not contained in the stabilized hGH particles, and which
`is dispersed within the polymer. The second metal cation
`component preferably contains the same species of metal
`cation, as is contained in the stabilized hGH. Alternately, the
`second metal cation component can contain one or more
`different species of metal cation.
`The second metal cation component acts to modulate the
`release of the hGH from the polymeric matrix of the
`sustained release composition, such as by acting as a reser
`voir of metal cations to further lengthen the period of time
`over which the hGH is stabilized by a matal cation to
`enhance the stability of hGH in the composition.
`A metal cation component used in modulating release
`typically contains at least one type of multivalent metal
`cation. Examples of second metal cation components suit
`able to modulate hGH release, include, or contain, for
`instance, Mg(OH)2, MgCO3 (such as 4MgCO3.Mg(OH)
`2.5H2O), ZnCOs (such as 3Zn(OH)2.2ZnCO3), CaCO3, Zn3
`(C6H5O7)2’ Mg(OAC)2, Mgsom Zn(OAc)2’ 211804, ZnCl-Zs
`M gCl2 and Mg3(C6H5O7)2. A suitable ratio of second metal
`cation component-to-polymer is between about 1:99 to
`about 1:2 by weight. The optimum ratio depends upon the
`polymer and the second metal cation component utilized.
`A polymeric matrix containing a dispersed metal cation
`component to modulate the release of a biologically active
`agent from the polymeric matrix is further described in
`co-pending US. patent application Ser. No. 08/237,057,
`?led May 3, 1994, and co-pending PCI‘ Patent Application
`PCT/US95/05511, the teachings of which are incorporated
`herein by reference in their entirety.
`The hGH sustained release composition of this invention
`can be formed into many shapes such as a ?lm, a pellet, a
`cylinder, a disc or a microparticle. A microparticle, as
`de?ned herein, comprises a polymeric component having a
`diameter of less than about one millimeter and having
`stabilized hGH particles dispersed therein. A microparticle
`can have a spherical, non-spherical or irregular shape. It is
`preferred that a microparticle be a rnicrosphere. Typically,
`the microparticle will be of a size suitable for injection. A
`preferred size range for microparticles is from about 1 to
`about 180 microns in diameter.
`In the method of this invention for forming a composition
`for the sustained release of biologically active, non
`aggregated hGH, a suitable amount of particles of biologi
`cally active, stabilized hGH are dispersed in a polymer
`solution.
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`A suitable polymer solution contains between about 1%
`(w/w) and about 30% (w/w) of a suitable biocompatible
`polymer, wherein the biocompatible polymer is typically
`dissolved in a suitable polymer solvent. Preferably, a poly
`mer solution contains about 2% (w/v) to about 20% (w/v)
`polymer. A polymer solution containing 5% to about 10%
`(w/w) polymer is most preferred.
`A suitable polymer solvent, as de?ned herein, is solvent in
`which the polymer is soluble but in which the stabilized
`hGH particles are substantially insoluble and non-reactive.
`Examples of suitable polymer solvents include polar organic
`liquids, such as methylene chloride, chloroform, ethyl
`acetate and acetone.
`To prepare biologically active, stabilized hGH particles,
`hGH is mixed in a suitable aqueous solvent with at least one
`suitable metal cation component under pH conditions suit
`able for forming a complex of metal cation and hGH.
`Suitable pH conditions to form a complex of hGH typi
`cally include pH values between about 7.0 and about 7.4.
`Suitable pH conditions are typically achieved through use of
`an aqueous buffer, such as sodium bicarbonate, as the
`solvent.
`Suitable solvents are those in which the hGH and the
`metal cation component are each at least slightly soluble,
`such as in an aqueous sodium bicarbonate bu?er. For
`aqueous solvents, it is preferred that water used be either
`deionized water or water-for-injection (WFI).
`It is understood that the hGH can be in a solid or a
`dissolved state, prior to being contacted with the metal
`cation component. It is also understood that the metal cation
`component can be in a solid or a dissolved state, prior to
`being contacted with the hGH. In a preferred embodiment,
`a buffered aqueous solution of hGH is mixed with an
`aqueous solution of the metal cation component
`Typically, the complexed hGH will be in the form of a
`cloudy precipitate, which is suspended in the solvent.
`However, the complexed hGH can also be in solution. In an
`ever; more preferred embodiment, hGH is complexed with
`Zn+ .
`The complexed hGH is then dried, such as by
`lyophilization, to form a particulate of stabilized hGH. The
`complexed hGH, which is suspended or in solution, can be
`bulk lyophilized or can be divided into smaller volumes
`which are then lyophilized. In a preferred embodiment, the
`complexed hGH suspension is micronized, such as by use of
`an ultrasonic nozzle, and then lyophilized to form stabilized
`hGH particles. Acceptable means to lyophilize the com
`plexed hGH mixture include those known in the art.
`Preferably, particles of stabilized hGH are between about
`1 to about 6 micrometers in diameter. The hGH particles can
`be fragmented separately, as described in co-pending U.S.
`patent application Ser. No. 08/006,682, ?led Jan. 21, 1993,
`which describes a process for producing small particles of
`biologically active agents, which is incorporated herein in its
`entirety by reference. Alternately, the hGH particles can be
`fragmented after being added to a polymer solution, such as
`by means of an ultrasonic probe or ultrasonic nozzle. In
`another embodiment, a second metal cation component,
`which is not contained in the stabilized hGH particles, is also
`dispersed within the polymer solution.
`It is understood that a second metal cation component and
`stabilized hGH can be dispersed into a polymer solution
`sequentially, in reverse order, intermittently, separately or
`through concurrent additions. Alternately, a polymer, a sec
`ond metal cation component and stabilized hGH and can be
`mixed into a polymer solvent sequentially, in reverse order,
`intermittently, separately or through vconcurrent additions.
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`6
`The method for forming a composition for modulating the
`release of a biologically active agent from a biodegradable
`polymer is further described in co-pending U.S. patent
`application Ser. No. 08l237,057.
`In this method, the polymer solvent is then solidi?ed to
`form a polymeric matrix containing a dispersion of stabi
`lized hGH particles.
`One suitable method for forming an hGH sustained
`release composition from a polymer solution is the solvent
`evaporation method described in U.S. Pat. No. 3,737,337,
`issued to Schnoring et al., U.S. Pat. No. 3,529,906, issued to
`Vranchen et al., U.S. Pat. No. 3,691,090, issued to Kitajima
`et al., or U.S. Pat N 0. 4,389,330, issued to Tice et al. Solvent
`evaporation is typically used as a method to form hGH
`sustained release microparticles.
`In the solvent evaporation method, a polymer solution
`containing a stabilized hGH particle dispersion, is mixed in
`or agitated with a continuous phase, in which the polymer
`solvent is partially miscible, to form an emulsion. The
`continuous phase is usually an aqueous solvent. Emulsi?ers
`are often included in the continuous phase to stabilize the
`emulsion. The polymer solvent is then evaporated over a
`period of several hours or more, thereby solidifying the
`polymer to form a polymeric matrix having a dispersion of
`stabilized hGH particles contained therein.
`A preferred method for forming hGH sustained release
`microparticles from a polymer solution is described in U.S.
`Pat. No. 5,019,400, issued to Gombotz et al., and co-pending
`U.S. patent application Ser. No. 08/443,726, ?led May 18,
`1995, the teachings of which are incorporated herein by
`reference in their entirety. This method of microsphere
`formation, as compared to other methods, such as phase
`separation, additionally reduces the amount of hGH required
`to produce a controlled release composition with a speci?c
`hGH content.
`In this method, the polymer solution, containing the
`stabilized hGH particle dispersion, is processed to create
`droplets, wherein at least a signi?cant portion of the droplets
`contain polymer solution and the stabilized hGH particles.
`These droplets are then frozen by means suitable to form
`microparticles. Examples of means for processing the poly
`mer solution dispersion to form droplets include directing
`the dispersion through an ultrasonic nozzle, pressure nozzle,
`Rayleigh jet, or by other known means for creating droplets
`from a solution.
`Means suitable for freezing droplets to form micropar
`ticles include directing the droplets into or near a liqui?ed
`gas, such as liquid argon and liquid nitrogen to form frozen
`microdroplets which are then separated from the liquid gas.
`The frozen microdroplets are then exposed to a liquid
`non-solvent, such as ethanol, or ethanol mixed with hexane
`or pentane.
`The solvent in the frozen microdroplets is extracted as a
`solid and/or liquid into the non-solvent to form stabilized
`hGH containing microparticles. Mixing ethanol with other
`non-solvents, such as hexane or pentane, can increase the
`rate of solvent extraction, above that achieved by ethanol
`alone, from certain polymers, such as poly(lactide—co
`glycolide) polymers.
`A wide range of sizes of hGH sustained release micro
`particles can be made by varying the droplet size, for
`example, by changing the ultrasonic nozzle diameter. If very
`large microparticles are desired, the microparticles can be
`extruded through a syringe directly into the cold liquid
`Increasing the viscosity of the polymer solution can also
`increase microparticle size. The size of the microparticles
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`can be produced by this process. for example microparticles
`ranging from greater than about 1000 to about 1 microme
`ters in diameter.
`Yet another method of forming an hGH sustained release
`composition, from a polymer solution, includes ?lm casting,
`such as in a mold, to form a ?lm or a shape. For instance,
`after putting the polymer solution containing a dispersion of
`stabilized hGH particles into a mold, the polymer solvent is
`then removed by means lmown in the art, or the temperature
`of the polymer solution is reduced, until a ?lm or shape, with
`a consistent dry weight, is obtained. Film casting of a
`polymer solution, containing a biologically active agent, is
`further described in co-pending U.S. patent application Ser.
`No. 08/237,057, the teachings of which are incorporated
`herein by reference in their entirety.
`It is believed that the release of the hGH can occur by two
`diiferent mechanisms. The hGH can be released by diifusion
`through aqueous ?lled channels generated in the polymeric
`matrix, such as by the dissolution of the hGH or by voids
`created by the removal of the polymer’s solvent during the
`synthesis of the sustained release composition. A second
`mechanism is the release of hGH due to degradation of the
`polymer.
`The rate of degradation can be controlled by changing
`polymer properties that in?uence the rate of hydration of the
`polymer. These properties include, for instance, the ratio of
`ditferent monomers, such as lactide and glycolide, compris
`ing a polymer; the use of the L-isomer of a monomer instead
`of a racemic mixture; and the molecular weight of the
`polymer. These properties can affect hydrophilicity and
`crystallinity, which control the rate of hydration of the
`polymer. Hydrophilic excipients such as salts, carbohydrates
`and surfactants can also be incorporated to increase hydra
`tion and which can alter the rate of erosion of the polymer.
`By altering the properties of the polymer, the contribu
`tions of di?usion and/or polymer degradation to hGH
`release can be controlled. For example, increasing the gly
`colide content of a poly(lactide-co-glycolide) polymer and
`decreasing the molecular weight of the polymer can enhance
`the hydrolysis of the polymer and thus, provides an
`increased hGH release from polymer erosion.
`In addition, the rate of polymer hydrolysis is increased in
`non-neutral pH’s. Therefore, an acidic or a basic excipient
`can be added to the polymer solution, used to form the
`microsphere, to alter the polymer erosion rate.
`The composition of this invention can be administered to
`a human, or other animal, by injection, implantation (e.g,
`subcutaneously, intramuscularly, intraperitoneally,
`intracranially, intravaginally and intradermally), administra
`tion to mucosal membranes (e.g., intranasally or by means
`of a suppository), or in situ delivery (e.g. by enema or
`aerosol spray) to provide the desired dosage of hGH based
`on the known parameters for treatment with hGH of the
`various medical conditions.
`The invention will now be further and speci?cally
`described by the following examples.
`
`45
`
`50
`
`55
`
`5,654,010
`
`10
`
`20
`
`30
`
`35
`
`8
`concentrations between 0.1 and 0.5 mM hGH. A 0.9 mM
`Zn+2 solution was prepared from deionized water and zinc
`acetate dihydrate and then was added to the hGH solutions
`to form a Zn+2-hGH complex. The pH of the Zn+2-hGH
`solution was then adjusted to between 7.0 and 7.4 by adding
`1% acetic acid. A cloudy suspended precipitate, comprising
`Zia-stabilized hGH formed.
`The suspension of Zn+2-stabilized hGH was then micron
`ized using an ultrasonic nozzle (Type VlA; Sonics and
`Materials, Danbury, Conn.) and sprayed into a polypropy
`lene tub (17 cm diameter and 8 cm deep) containing liquid
`nitrogen to form frozen particles. The polypropylene tub was
`then placed into a —80° C. freezer until the liquid nitrogen
`evaporated. The frozen particles, which contained Zn”
`stabilized hGH, were then lyophilized to form Zn”
`stabilized hGH particles.
`
`EXAMPLE 2
`
`Preparation of PLGA Microspheres Containing
`Biologically Active, Zia-Stabilized hGH
`Microspheres containing Zia-stabilized human growth
`hormone (hGH), were prepared from hydrophilic poly
`(lactice-co-glycolide) polymer RG502H having free car
`boxyl end groups (hereinafter “unblocked-PLGA”) (50:50
`PLGA, 9,300 Daltons; Boehringer Ingelheim Chemicals,
`Inc.) or a more hydrophobic PLGA polymer having blocked
`carboxyl end groups (hereinafter “blocked-PLGA”) (50:50
`PLGA, 10,000 Daltons; Lot #115-56-1, Birmingham
`Polymers, Inc., Birmingham, Ala.).
`The polymer was dissolved in methylene chloride at room
`temperature. The lyophilized hGH particles were added to
`the polymer solution and zinc carbonate was also added. The
`mixture was then sonicated to give a homogeneous suspen
`sion. The suspension was atomized through a sonicating
`nozzle on to a bed of frozen ethanol, overlaid with liquid
`nitrogen. The vessel containing the microspheres was stored
`at —80° C. to extract the methylene chloride and then
`freeze-dried to give a free-?owing powder.
`
`EXAMPLE 3
`
`Analysis of Encapsulated hGH Protein
`The integrity of encapsulated hGH was determined by
`dissolving unhydrated microspheres into methylene chloride
`and acetone, collecting the protein, freeze-drying and
`re-constituting in HEPES buifer containing 10 mM EDTA.
`Appropriate controls were run to ensure that the extraction
`process did not a?’ect the integrity of the protein.
`The integrity of the encapsulated hGH was analyzed by
`measuring the percent of hGH monomer contained in the
`hGH sample after encapsulation by size exclusion chroma
`tography (SEC).
`The results of SEC analyses of the hGH integrity of hGH
`sustained release microspheres are provided below.
`
`EXANEPLE 1
`
`Formation of Zia-Stabilized hGH
`Human growth hormone (hGH), whose DNA sequence is
`described in U.S. Pat. No. 4,898,830, issued to Goeddel et
`al. was used in this Example. Human growth hormone was
`stabilized by forming an insoluble complexes with zinc.
`The hGH was dissolved in Samples of a 4 mM sodium
`bicarbonate buifer (pH 7.2) to form hGH solutions with
`
`65
`
`Formulation (polymer; % Zinc Carbonate)
`
`% Monomer (SEC)
`
`31K unblocked; 6% ZnCO3
`31K unblocked; 6% ZnCO3
`31K unblocked; 3% ZnCO3
`31K unblocked; 3% ZnCO3
`31K unblocked; 1% ZnCO3
`31K unblocked; 0% ZnCO3
`31K unblocked; 0% ZnCO3 '
`
`98.6
`99.2
`97.7
`97.8
`97.6
`97.8
`97.1
`
`LUYE1009
`IPR of Patent No. 6,667,061
`
`

`
`-continued
`
`Formulation (polymer;