U.S. patent application number 10/284428 was filed with the patent office on 2003-11-13 for composition for sustained release of human growth hormone.
Invention is credited to Auer, Henry, Bernstein, Howard, Ganmukhi, Medha M., Johnson, Olufunmi Lily, Khan, M. Amin.
Application Number | 20030211153 10/284428 |
Document ID | / |
Family ID | 25530460 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211153 |
Kind Code |
A1 |
Johnson, Olufunmi Lily ; et
al. |
November 13, 2003 |
Composition for sustained release of human growth hormone
Abstract
A pharmaceutical composition for the sustained release of human
growth hormone from a polymer matrix is disclosed. The
pharmaceutical composition comprises a biocompatible polymer,
particles of metal cation-complexed human growth hormone wherein
said particles are dispersed within the biocompatible polymer and
an aqueous injection vehicle.
Inventors: |
Johnson, Olufunmi Lily;
(Cambridge, MA) ; Ganmukhi, Medha M.; (Carlisle,
MA) ; Bernstein, Howard; (Cambridge, MA) ;
Auer, Henry; (Chicago, IL) ; Khan, M. Amin;
(Downingtown, PA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
25530460 |
Appl. No.: |
10/284428 |
Filed: |
October 29, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10284428 |
Oct 29, 2002 |
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09505508 |
Feb 17, 2000 |
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6500448 |
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09505508 |
Feb 17, 2000 |
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09252746 |
Feb 19, 1999 |
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6051259 |
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09252746 |
Feb 19, 1999 |
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09033193 |
Mar 2, 1998 |
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5891478 |
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09033193 |
Mar 2, 1998 |
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08831604 |
Apr 10, 1997 |
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08831604 |
Apr 10, 1997 |
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08473544 |
Jun 7, 1995 |
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5654010 |
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08473544 |
Jun 7, 1995 |
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07984323 |
Dec 2, 1992 |
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Current U.S.
Class: |
424/469 ;
514/11.4; 514/8.6 |
Current CPC
Class: |
Y10S 514/839 20130101;
Y10S 530/839 20130101; A61K 9/1694 20130101; B82Y 5/00 20130101;
Y10T 428/2985 20150115; A61K 9/1611 20130101; A61K 9/1647 20130101;
A61K 38/27 20130101 |
Class at
Publication: |
424/469 ;
514/2 |
International
Class: |
A61K 009/26; A61K
038/27 |
Claims
What is claimed is:
1. A pharmaceutical composition for the sustained release of human
growth hormone from a polymer matrix, comprising: a) a
biocompatible polymer; b) particles of metal cation-complexed human
growth hormone, wherein said particles are dispersed within the
biocompatible polymer; and c) an aqueous injection vehicle.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 09/505,508, filed Feb. 17, 2000, which is a
Continuation of U.S. patent application Ser. No. 09/252,746, filed
Feb. 19, 1999 now U.S. Pat. No. 6,051,259 which is a Continuation
of U.S. patent application Ser. No. 09/033,193, filed Mar. 2, 1998,
now U.S. Pat. No. 5,891,478, which is a Continuation of U.S. patent
application Ser. No. 08/831,604, filed Apr. 10, 1997, now
abandoned, which is a Continuation of U.S. patent application Ser.
No. 08/473,544, filed Jun. 7, 1995, now U.S. Pat. No. 5,654,010,
which is a Continuation-in-Part of U.S. patent application Ser. No.
07/984,323, filed Dec. 2, 1992, now abandoned. All of the above are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Human growth hormone (hGH) is a protein secreted by the
pituitary gland and which can be produced by recombinant genetic
engineering. hGH will cause growth in all bodily tissues which are
capable of growth.
[0003] hGH is typically used to treat patients suffering from
hypopituitary dwarfism. Currently, aqueous hGH is administered 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 injections result in patient compliance
problems.
[0004] To resolve the problems associated with repetitive
injections of aqueous hGH, attempts have been made to formulate
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.
[0005] However, these controlled release devices often exhibited
high initial bursts of hGH release and minimal hGH release
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 aggregated hGH which is
immunogenic in vivo and likely has reduced biological activity.
[0006] 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.
SUMMARY OF THE INVENTION
[0007] This invention relates to 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 biocompatible polymer and particles of
biologically active, metal cation-stabilized hGH, wherein said
particles are dispersed within the biocompatible polymer.
[0008] 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 dispersion of said hGH particles.
[0009] The method of using the sustained release composition of the
present invention comprises providing a therapeutically effective
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.
[0010] The advantages of this sustained release formulation for hGH
include longer, more consistent in vivo blood levels of hGH, lower
initial bursts of hGH, and increased therapeutic benefits by
eliminating fluctuations in serum hGH levels. The advantages also
include increased patient compliance and acceptance by reducing the
required number of injections. The advantages further include the
ability to use smaller amounts of hGH compared to bolus injection
regimen because serum hGH levels are maintained closer to
therapeutical thresholds.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention.
[0012] 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
non-immunogenic.
[0013] Aggregated hGH may induce an immune response resulting in
antibodies formed against hGH. This may compromise the efficacy of
long-term hGH therapy. Additionally, aggregated hGH may stimulate
an auto-immune response to endogenous hGH.
[0014] A sustained release of biologically active, non-aggregated
human growth hormone is a release which results in measurable 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 sustained 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.
[0015] 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 selection of excipients to
produce the desired effect.
[0016] 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.
[0017] Suitable multivalent metal cations include metal cations
contained in biocompatible metal cation components. A metal cation
component is biocompatible if the cation component is non-toxic to
the recipient, in the quantities used, and also presents no
significant deleterious or untoward effects on the recipient's
body, such as an immunological reaction at the injection site.
[0018] Typically, the molar ratio of metal cation component to hGH,
for the metal cation stabilizing the hGH, is between about 4:1 to
about 100:1 and more typically about 4:1 to about 10:1.
[0019] A preferred metal cation used to stabilize hGH is Zn.sup.+2.
In a more preferred embodiment, the molar ratio of metal cation
component, containing Zn.sup.+2 cations, to hGH is about 6:1.
[0020] The suitability of a metal cation for stabilizing hGH can be
determined by one of ordinary skill in the art by performing 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 determine 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
characteristics of a sustained release composition, is reduced by
complexing at least one type of metal cation with hGH prior to
contacting the hGH with a polymer solution.
[0021] Stabilized hGH is typically stabilized against significant
aggregation in vivo over the sustained release period. Significant
aggregation is defined as an amount of aggregation resulting in
aggregation of about 15% or more of the initial amount of
encapsulated hGH monomer. Preferably, aggregation is maintained
below about 5% of the initial dose of hGH monomer. More preferably,
aggregation is maintained below about 2% of the initial dose.
[0022] 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 buffers to stabilize the hGH during
lyophilization.
[0023] Bulking agents typically comprise inert materials. Suitable
bulking agents are known to those skilled in the art.
[0024] A polymer, or polymeric matrix, suitable for the sustained
release composition of the present invention, must be
biocompatible. A polymer is biocompatible if the polymer, and any
degradation products of the polymer, are non-toxic to the recipient
and also present no significant deleterious or untoward effects on
the recipient's body, such as an immunological reaction at the
injection site.
[0025] The polymer of the hGH sustained release composition must
also be biodegradable. Biodegradable, as defined 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.
[0026] 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, polyesteramides, polyanhydrides, poly(amino acids),
polyorthoesters, polycyanoacrylates, poly(p-dioxanone),
poly(alkylene oxalate)s, biodegradable polyurethanes, blends and
copolymers thereof.
[0027] Further, the terminal functionalities of the polymer can be
modified. For example, polyesters can be blocked, unblocked or a
blend of blocked and unblocked polymers. A blocked polymer is as
classically defined in the art, specifically having blocked
carboxyl end groups. Generally, the blocking group is derived from
the initiator of the polymerization and is typically an alkyl
group. An unblocked polymer is as classically defined in the art,
specifically having free carboxyl end groups.
[0028] 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 polymer 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.
[0029] The amount of hGH, which is contained in a dose of sustained
release microparticles, or in an alternate sustained release
device, containing biologically active, stabilized hGH particles is
a therapeutically or prophylactically effective 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.
[0030] In one embodiment, an hGH sustained release composition
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 effect 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. Amore 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% (w/w).
[0031] In another embodiment, a hGH sustained release composition
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.
[0032] 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 reservoir of metal
cations to further lengthen the period of time over which the hGH
is stabilized by a metal cation to enhance the stability of hGH in
the composition.
[0033] A metal cation component used in modulating release
typically contains at least one type of multivalent metal cation.
Examples of second metal cation components suitable to modulate hGH
release, include, or contain, for instance, Mg(OH).sub.2,
MgCO.sub.3 (such as 4MgCO.sup.3.Mg(OH).sub.2.5H.sub.2O), ZnCO.sub.3
(such as 3Zn(OH).sub.2.2ZnCO.sub.3), CaCO.sub.3,
Zn.sub.3(C.sub.6H.sub.5O.sub.7).s- ub.2, Mg(OAc).sub.2, MgSO.sub.4,
Zn(OAc).sub.2, ZnSO.sub.4, ZnCl.sub.2, MgCl.sub.2 and
Mg.sub.3(C.sub.6H.sub.5O.sub.7).sub.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.
[0034] 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 U.S. Pat. No.
5,656,297 to Bernstein et al., and co-pending PCT Patent
Application PCT/US95/05511, the teachings of which are incorporated
herein by reference in their entirety.
[0035] The hGH sustained release composition of this invention can
be formed into many shapes such as a film, a pellet, a cylinder, a
disc or a microparticle. A microparticle, as defined 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
microsphere. 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.
[0036] 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 biologically active,
stabilized hGH are dispersed in a polymer solution.
[0037] 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 polymer 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.
[0038] A suitable polymer solvent, as defined herein, is solvent in
which the polymer is soluble but in which the stabilized hGH
particles are substantially insoluble and non-reactuve. Examples of
suitable polymer solvents include polar organic liquids, such as
methylene chloride, chloroform, ethyl acetate and acetone.
[0039] 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 suitable for
forming a complex of metal cation and hGH. 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 even more preferred embodiment, hGH is complexed with
Zn.sup.+2.
[0040] Suitable pH conditions to form a complex of hGH typically
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.
[0041] 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 buffer. For aqueous solvents, it is
preferred that water used be either deionized water or
water-for-injection (WFI).
[0042] 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.
[0043] 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 even more preferred
embodiment, hGH is complexed with Zn.sup.+2.
[0044] 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
complexed hGH mixture include those known in the art.
[0045] 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, filed 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.
[0046] In another embodiment, a second metal cation component,
which is not contained in the stabilized hGH particles, is also
dispersed within the polymer solution.
[0047] 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 second
metal cation component and stabilized hGH and can be mixed into a
polymer solvent sequentially, in reverse order, intermittently,
separately or through concurrent additions.
[0048] The method for forming a composition for modulating the
release of a biologically active agent from a biodegradable polymer
is further described in U.S. Pat. No. 5,656,297 to Bernstein et
al.
[0049] In this method, the polymer solvent is then solidified to
form a polymeric matrix containing a dispersion of stabilized hGH
particles.
[0050] 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. 3,523,906, issued to Vranchen et al., U.S. Pat.
No. 3,691,090, issued to Kitajima et al., or U.S. Pat. No.
4,389,330, issued to Tice et al. Solvent evaporation is typically
used as a method to form hGH sustained release microparticles.
[0051] 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. Emulsifiers 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.
[0052] 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, filed 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 sustained release composition with a specific
hGH content.
[0053] In this method, the polymer solution, containing the
stabilized hGH particle dispersion, is processed to create
droplets, wherein at least a significant 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 polymer 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.
[0054] Means suitable for freezing droplets to form microparticles
include directing the droplets into or near a liquified 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.
[0055] 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-glycolid- e) polymers.
[0056] A wide range of sizes of hGH sustained release
microparticles 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 can be produced by this
process, for example microparticles ranging from greater than about
1000 to about 1 micrometers in diameter.
[0057] Yet another method of forming an hGH sustained release
composition, from a polymer solution, includes film casting, such
as in a mold, to form a film 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 known in the art, or the temperature of the polymer solution
is reduced, until a film or shape, with a consistent dry weight, is
obtained. Film casting of a polymer solution, containing a
biologically active agent, is further described in U.S. Pat. No.
5,656,297 to Bernstein et al., the teachings of which are
incorporated herein by reference in their entirety.
[0058] It is believed that the release of the hGH can occur by two
different mechanisms. The hGH can be released by diffusion through
aqueous filled 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.
[0059] 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 influence the rate of hydration of
the polymer. These properties include, for instance, the ratio of
different monomers, such as lactide and glycolide, comprising 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
hydration and which can alter the rate of erosion of the
polymer.
[0060] By altering the properties of the polymer, the contributions
of diffusion and/or polymer degradation to hGH release can be
controlled. For example, increasing the glycolide 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.
[0061] 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.
[0062] 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), administration 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.
[0063] The invention will now be further and specifically described
by the following examples.
EXAMPLE 1
[0064] Formation of Zn.sup.+2-Stabilized hGH
[0065] 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. The human growth hormone was stabilized by forming
insoluble complexes with zinc.
[0066] The hGH was dissolved in samples of a 4 mM sodium
bicarbonate buffer (pH 7.2) to form hGH solutions with a
concentrations between 0.1 and 0.5 mM hGH. A 0.9 mM Zn.sup.+2
solution was prepared from deionized water and zinc acetate
dihydrate and then was added to the hGH solutions to form
Zn.sup.+2-hGH complex. The pH of the Zn.sup.+2-hGH complex was then
adjusted to between 7.0 and 7.4 by adding 1% acetic acid. A cloudy
suspended precipitate, comprising Zn.sup.+2-stabilized hGH
formed.
[0067] The suspension of Zn.sup.+2-stabilized hGH was then
micronized using an ultrasonic nozzle (Type V1A; Sonics and
Materials, Danbury, Conn.) and sprayed into a polypropylene tub (17
cm diameter and 8 cm deep) containing liquid nitrogen to form
frozen particles. The polypropylene tub was then placed into a -80
.degree. C. freezer until the liquid nitrogen evaporated. The
frozen particles, which contained Zn.sup.-2-stabilized hGH, were
then lyophilized to form Zn.sup.+2-stabilized hGH particles.
EXAMPLE 2
[0068] Preparation of PLGA Microspheres Containing Biologically
Active, Aggregation-Stabilized hGH
[0069] Microspheres containing Zn.sup.+2-stabilized human growth
hormone (hGH) were prepared from hydrophilic
poly(lactide-co-glycolide) polymer RG502H having free carboxyl 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.).
[0070] 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 suspension. 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.degree. C. to extract the methylene
chloride and then freeze-dried to give a free-flowing powder.
EXAMPLE 3
[0071] Analysis of Encapsulated hGH Protein
[0072] 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 buffer containing 10 mM EDTA. Appropriate controls were
run to ensure that the extraction process did not affect the
integrity of the protein.
[0073] The integrity of the encapsulated hGH was analyzed by
measuring the percent of hGH monomer contained in the hGH after
encapsulation through size exclusion chromatography (SEC).
[0074] The results of SEC analyses of the hGH integrity of hGH
sustained release microspheres are provided below.
1 Formulation (polymer; % Zinc Carbonate) % Monomer (SEC) 31 K
unblocked; 6% ZnCO3 98.6 31 K unblocked; 6% ZnCO3 99.2 31 K
unblocked; 3% ZnCO3 97.7 31 K unblocked; 3% ZnCO3 97.8 31 K
unblocked; 1% ZnCO3 97.6 31 K unblocked; 0% ZnCO3 97.8 31 K
unblocked; 0% ZnCO3 97.1 10 K blocked; 1% ZnCO3 98.2 10 K blocked;
1% ZnCO3 98.4 8 K unblocked; 0% ZnCO3 98.5 10 K blocked; 1% ZnCO3
98.4
[0075] The results showed that the encapsulation process did not
cause aggregation of the protein. The yield percent protein
recovered by the extraction procedure (relative to the amount
measured by nitrogen content of the microspheres) ranged from about
40 to 98%. The variability is thought to be associated with loss of
material during the transfer steps in the procedure and the
extraction procedure is being modified to increase protein
recovery.
EXAMPLE 4
[0076] Determination of the Effect of Zinc Carbonate on In vitro
Release Kinetics
[0077] The microspheres were formed as described in Example 2 and
contained 15% w/w hGH (6:1 Zn:hGH protein complex); 0%, 1%, 6%, 10%
or 20% w/w zinc carbonate; and poly(lactide-co-glycolide)
polymer.
[0078] In vitro release kinetics of the hGH sustained release
microsphere formulations containing various concentrations of zinc
carbonate were determined by suspending an aliquot (10 mg) of each
type of microsphere in different 1.5 ml samples of HEPES buffer (50
mM Hepes, 10 mM KCl, 0.1% NaN3) pH 7.2 and then incubating at
37.degree. C. The amount of protein released was quantitated by
sampling the buffer at 1, 3, 7, 10, 14, 21, 28 days after
incubation and replenishing with fresh buffer after each
sampling.
[0079] A curve of cumulative percent released (relative to initial
hGH content in the starting mass of microspheres) versus time was
plotted. Released protein samples from each time point were assayed
for hGH monomer content by size exclusion chromatography.
[0080] Zinc carbonate is thought to act as a reservoir of zinc ions
so that the formation of the Zn-hGH complex is favored and
dissociation into soluble hGH disfavored. Because the aqueous
solubility of zinc carbonate is low, the release of zinc ions from
the reservoir is slow thus modulating the solubility of the
protein.
[0081] The analysis found that in the absence of zinc carbonate,
the rate of release of the encapsulated hGH was very rapid and all
the protein was released in a very short period.
EXAMPLE 5
[0082] Assay for hGH After in Vivo Degradation of Blocked-PLGA
Zn.sup.+2-Stabilized hGH Microspheres
[0083] Microspheres of blocked-PLGA, containing 15% w/w
Zn.sup.+2-stabilized hGH and 0%, 6%, 10% or 20% ZnCO.sub.3 were
formed by the method of Example 2. Groups of test rats were
injected subcutaneously with 50 mg samples of the different hGH
microspheres. The rats were sacrificed after 60 days and the skin
sample were excised from the injection sites. The excised skin
samples were placed in 10% Neutral Buffered Formalin for at least
24 hours. They were then trimmed with a razor blade to remove
excess skin and placed in PBS.
[0084] Tissue samples were processed by Pathology Associates, Inc.
(Frederick, Md.). The skin samples were embedded in
glycomethacrylate, sectioned and assayed for the presence of hGH
using a HistoScan/LymphoScan Staining Kit (Product #24-408M;
Accurate Chemical & Scientific Corp., Westbury, N.Y.) according
to the manufacturer's instructions. Tissue samples were scored for
the presence or absence of staining which was indicative of the
presence or absence of hGH in the sample.
[0085] All skin samples, associated with hGH microsphere
injections, tested positive for the presence of hGH thus indicating
that the blocked-PLGA microspheres still contained hGH after 60
days in vivo.
[0086] The method described in Example 2 was used to form
microspheres by encapsulating 0% or 15% w/w hGH, in the form of
Zn:hGH complex, and also 0%, 1% or 6% w/w ZnCO.sub.3 salt, within
blocked-PLGA and within unblocked-PLGA.
[0087] In vivo degradation of unblocked-PLGA microspheres versus
blocked-PLGA microspheres were compared by injecting samples of
microspheres into rats and then analyzing the microspheres
remaining at the injection site at various times post-injection.
Three rats were assayed at each time point for each microsphere
sample. On the day of administration of the microspheres, 750 .mu.l
of vehicle (3% carboxymethyl cellulose (low viscosity) and 1%
Tween-20 in saline) was added to vials containing 50.+-.1 mg of
microspheres. Immediately, the vials were shaken vigorously to form
a suspension which was then aspirated into a 1.0 cc syringe without
a needle.
[0088] Rats (Sprague-Dawley males) were anesthetized with a
halothane and oxygen mixture. The injection sites (intrascapular
region) were shaven and marked with a permanent tatoo to provide
for the precise excision of skin at the sampling time points. Each
rat was injected with an entire vial of microspheres using 18 to 21
gauge needles.
[0089] On designated days (days 15, 30, 59 and 90 post-injection
for animals receiving blocked-PLGA microspheres, or days 7, 14, 21,
28 and 45 post-injection for animals receiving unblocked-PLGA
microspheres) the rats were sacrificed by asphyxiation with
CO.sub.2 gas and the skin at the injection sites (including
microspheres) was excised. Since the microspheres tended to clump
at the injection sites, the presence or absence of microspheres was
determined visually.
[0090] The visual inspections found that the unblocked-PLGA
microspheres degraded substantially faster than the blocked-PLGA
microspheres, and that the addition of ZnCO.sub.3 to the
blocked-PLGA substantially slowed polymeric degradation. For
example, in the rats injected with unblocked-PLGA microspheres
containing 0% hGH and 0% or 1% ZnCO.sub.3, no microspheres were
visible on day 21. In addition, for rats injected with blocked-PLGA
microspheres containing 0% hGH and 0% ZnCO.sub.3, a few
microspheres were visible on day 60 and none were visible on day
90. Furthermore, for rats injected with blocked-PLGA microspheres
containing 0% or 15% hGH and 6% ZnCO.sub.3, microspheres were
visible on day 90.
EXAMPLE 6
[0091] In Vivo Pharmnacokinetic Studies of hGH Sustained Release
Microspheres in Rats
[0092] Studies were conducted in rats to screen various hGH
microsphere formulations, determine pharmacokinetic parameters
following intravenous (IV), subcutaneous (SC) and SC osmotic pump
(Alzet) administration of hGH, and to evaluate serum profiles and
in vivo release-rate of various hGH microsphere formulations.
[0093] Sprague-Dawley rats were divided into groups of three each,
randomized by body weight, and one hGH microsphere formulation was
administered to each group. Rats were injected subcutaneously with
approximately 7.5 mg of hGH in 50 mg of one type of the different
microspheres, suspended in 0.75 ml of an aqueous injection vehicle.
The vehicle composition was 3% CMC (low viscosity), 1% Polysorbate
20, in 0.9% NaCl. The microsphere dose delivered was determined
indirectly by weighing the residual dose in the injection vial and
correcting for residual injection vehicle. The hGH dose was then
computed from the protein loading of the microspheres determined by
nitrogen analysis.
[0094] Blood samples were collected at pre-determined intervals for
up to 30 days after injection. Blood samples of 250 .mu.l were
collected during the first 24 hours and at least 400 .mu.l at time
points after 24 hours. Blood samples were clotted and hGH
concentrations in serum were determined using a radio-immuno assay.
A radio-immunoassay (RIA) kit from ICN was validated and used to
determine the hGH levels in rat serum.
[0095] For the determination of pharmacokinetic parameters, hGH in
saline was administered to rats by subcutaneous bolus injection,
intravenously or delivered via an osmotic pump (Alzet Model 2ML4)
which was implanted subcutaneously.
[0096] Three groups of rats received single subcutaneous injections
of hGH in 0.9% NaCl at 0.5 or 7.5 mg/kg at a dose volume of 1.0
ml/kg and two groups received single intravenous bolus injections
of hGH in 0.9% NaCl solution at about 1.0 mg and 5.0 mg of hGH per
kg rat with a dose volume of 1.0 ml/kg. For the Alzet pump study,
rats were divided into four groups of three rats each, randomized
by body weight and dosed with about 20 mg/ml and 40 mg/ml hGH in
0.9% saline solution loaded into pumps (Alzet Model 2002, 200
.mu.l, 14 days release), and with about 4 mg/ml and 12 mg/ml hGH in
0.9% saline solution loaded into pumps (Alzet Model 2ML4, 2ml, 28
days release). Expected release rates from the pumps correspond to
about 2% and 4 to 6% of the ProLease hGH dose (about 15 mg/kg) per
day, respectively. The Alzet pumps were implanted subcutaneously in
the inter-scapular region after soaking for 1-2 minutes in sterile
saline.
[0097] The formulations of hGH sustained release microspheres,
synthesized as described in Example 2 contained 15% w/w hGH
complexed with Zn in a ratio of 6:1 Zn:hGH; 0%, 1%, 3% or 6% w/w
zinc carbonate; and 8K unblocked PLGA, 10K blocked PLGA or 31K
unblocked PLGA.
[0098] To evaluate the various hGH sustained release formulations,
Cmax, Cd5 and Cmax/Cd5 were the in vivo indices used, where Cmax is
the maximum serum concentration observed, and Cd5 is the serum
concentration at day 5 which should approximate the steady state
concentration. The results were as follows:
2 Burst % Monomer C day 5 Formulation in vitro (%) Day 7 Cmax
(ng/ml) (ng/ml) Cmax/Css 8 K PLGA 22.0 .+-. 0.9 99.3* 323.3 .+-.
98.6 20.4 .+-. 14.2 19.5 .+-. 10.6 unblocked 0% ZnCO3 8 K PLGA 16.4
.+-. 1.6 97.3* 309.0 .+-. 67.1 20.4 .+-. 14.2 39.5 .+-. 17.7
unblocked 1% ZnCO3 8 K PLGA 15.9 .+-. 6.9 98.7 670.5 .+-. 244.4 9.0
.+-. 4.2 44.8 .+-. 22.6 unblocked 3% ZnCO3 8 K PLGA 17.6 .+-. 2.7
99.3 358.0 .+-. 58.9 18.8 .+-. 14.7 42.4 .+-. 6.8 unblocked 6%
ZnCO3 31 K PLGA 12.3 .+-. 1.1 98.2 592 .+-. 318.2 4.5 .+-. 1.5
132.5 .+-. 47.9 unblocked 0% ZnCO3 31 K PLGA 11.4 .+-. 1.3 98.8
432.7 .+-. 91.6 5.1 .+-. 0.3 84.1 .+-. 14.9 unblocked 3% ZnCO3 31 K
PLGA 7.9 .+-. 1.9 99.4 643.6 .+-. 203.9 8.0 .+-. 2.6 93.3 .+-. 62.0
unblocked 3% ZnCO3 31 K PLGA 15.8 .+-. 0.5 99.8 1691.8 .+-. 340.0
6.6 .+-. 0.8 262.2 .+-. 83.5 unblocked 6% ZnCO3 10 K PLGA 12.7 .+-.
0.1 99.3 615.9 .+-. 384.3 4.5 .+-. 1.0 155.0 .+-. 126.8 blocked 1%
ZnCO3 10 K PLGA 18.1 .+-. 3.2 99.6 1053.2 .+-. 293.3 3.6 .+-. 0.8
291.7 .+-. 71.1 blocked 3% ZnCO3 10 K PLGA 9.9 .+-. 1.4 99.0 1743.5
.+-. 428.4 4.9 .+-. 2.7 516.1 .+-. 361.6 blocked 6% ZnCO3 *Value
obtained from duplicate batch of the same formulation.
[0099] The results of the screening showed that the two unblocked
polymers (8K and 31K) had different in vivo release kinetics
compared to the original formulation, which used blocked 10K PLGA
and 6% w/w zinc carbonate. Cmax values were generally lower with
the unblocked polymer formulations than with the lead formulation
which suggested that the in vivo burst may be lower with the
unblocked polymer formulations. The burst was defined as the
percent of hGH released in the first 24 hours after injection. The
in vitro burst values were between 8-22%. The zinc carbonate
content of the formulations did not appear to have an effect on the
burst or the in vitro release profile.
[0100] The serum concentrations between days 4 and 6 were
maintained at a fairly constant level above baseline (or the
pre-bleed levels) with the unblocked polymer formulations, while
serum concentrations with the blocked formulations, at the same
time points were close to the baseline levels. The in vitro release
data for up to 7 days showed that the released hGH protein was
monomeric. Useful data could not be obtained beyond day 6 because
of anti-hGH antibody formulation in the rats.
EXAMPLE 7
[0101] Rhesus Monkey Pharmacokinetics Study
[0102] The objective of this primate study was to evaluate the
pharmacokinetic profiles of different hGH sustained release
formulations as compared to more traditional methods of
administering hGH (e.g., bolus sc injections, daily sc injections
and sc injection combined with the use of an osmotic pump) and to
determine which hGH sustained release formulation gave the optimal
hGH blood concentration profile.
[0103] The formulations for the hGH sustained release microspheres
tested were 1) 15% hGH (complexed with Zn at a 6:1 Zn:hGH ratio),
6% w/w zinc carbonate and 10K blocked PLGA; 2) 15% hGH (complexed
with Zn at a 6:1 Zn:hGH ratio), 1% w/w zinc carbonate and 8K
unblocked PLGA ("RG502H" PLGA polymer); and 3) 15% hGH (complexed
with Zn at a 6:1 Zn:hGH ratio), 1% w/w zinc carbonate and 31K
unblocked PLGA ("RG503H" PLGA polymer).
[0104] There were four monkeys per group and each animal received a
single subcutaneous injection into the dorsal cervical region on
Day 1. A dose of 160 mg of hGH sustained release microspheres (24
mg of hGH) was administered to each monkey in 1.2 ml of injection
vehicle through a 20 gauge needle. The injection vehicle was an
aqueous vehicle containing 3% w/v Carboxymethyl Cellulose (sodium
salt), 1% v/v Tween 20 (Polysorbate 20) and 0.9% sodium
chloride.
[0105] The hGH dose was intended to provide measurable hGH serum
concentrations for pharmacokinetic analysis. To obtain
pharmacokinetic parameters additional study groups of four monkeys
each were included, specifically 1) a single subcutaneous injection
(24 mg hGH), 2) daily subcutaneous injections (24 mg/28 days=0.86
mg hGH/day), 3) a subcutaneous injection (3.6 mg hGH) combined with
an Alzet osmotic pump (20.4 mg hGH)(total dose of 24 mg hGH), and
4) a subcutaneous injection of the injection vehicle as a control
(only used 3 monkeys for the vehicle control group).
[0106] Blood samples were collected at the following times for hGH,
IGF1, IGFBP3 and anti-hGH antibody analyses: -7, -5, -3, pre-dose
and, 0.5, 1, 2, 3, 5, 8, 10, 12, 24, 28,32 and 48 hours, 5, 4, 6,
8, 11, 14, 17, 20, 23, 26, 29, 32, 25, 28, 41, 44, 47, 50, 53, 56
days post-dose.
[0107] The concentrations of IGF-1 and hGH in the serum were then
measured. An IRMA kit from RADIM (distributed by: Wein
Laboratories, P.O. Box 227, Succasunna, N.J.) was used to quantify
hGH in monkey serum. The IRMA assay had a limit of quantification
in PBS buffer of 0.1 ng/mL and in pooled juvenile rhesus monkey
serum of 1.5 ng/mL with a basal GH level of about 4 ng/mL.
[0108] The IRMA assay was validated over the concentration range
1.5-75 ng/mL for pooled juvenile rhesus monkey serum. The
measurement precision and accuracy are within the range of
.+-.10%.
[0109] The results showed that the hGH sustained release
microspheres were releasing significant, sustained levels of hGH
over a one month period while the subcutaneous injections were not
able to maintain the same serum levels.
[0110] The IGF-1 serum profile showed that serum IGF-1
concentrations were elevated above the baseline values between days
2 and 29 after administering the microparticles. This shows that
enough hGH was being released from the hGH sustained release
microspheres to cause a pharmacodynamic effect. This also indicates
that the hGH released was biologically active which suggest that
the encapsulation process had not adversely affected the biopotency
of hGH.
[0111] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
* * * * *