U.S. patent application number 11/212223 was filed with the patent office on 2006-01-05 for slow release protein polymers.
Invention is credited to Durga Annavajula, Jeffrey A. Hubbell, Beadle P. Retnarajan, Stephen C. Rowe, Kalvin Yim.
Application Number | 20060003009 11/212223 |
Document ID | / |
Family ID | 22654167 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003009 |
Kind Code |
A1 |
Rowe; Stephen C. ; et
al. |
January 5, 2006 |
Slow release protein polymers
Abstract
The invention features articles for delivery of a biologically
active substance, methods for making such articles, and methods for
treating an animal using the articles.
Inventors: |
Rowe; Stephen C.;
(Wellesley, MA) ; Yim; Kalvin; (North Andover,
MA) ; Retnarajan; Beadle P.; (Beverty, MA) ;
Hubbell; Jeffrey A.; (Zumikon, CH) ; Annavajula;
Durga; (Acton, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
22654167 |
Appl. No.: |
11/212223 |
Filed: |
August 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10650115 |
Aug 26, 2003 |
6939557 |
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11212223 |
Aug 26, 2005 |
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09772174 |
Jan 29, 2001 |
6699504 |
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10650115 |
Aug 26, 2003 |
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60178852 |
Jan 28, 2000 |
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Current U.S.
Class: |
424/486 ;
514/11.4; 514/5.9 |
Current CPC
Class: |
A61K 9/0053 20130101;
A61K 9/1635 20130101; A61K 38/1816 20130101; A61K 9/5146 20130101;
A61K 38/193 20130101; A61K 9/1641 20130101; A61K 38/28 20130101;
A61K 38/09 20130101; A61K 38/21 20130101; A61K 9/1647 20130101;
A61K 9/0043 20130101; A61K 38/27 20130101; A61K 9/0019 20130101;
A61K 9/5138 20130101; A61K 9/0073 20130101 |
Class at
Publication: |
424/486 ;
514/002 |
International
Class: |
A61K 38/29 20060101
A61K038/29; A61K 38/27 20060101 A61K038/27; A61K 9/14 20060101
A61K009/14 |
Claims
1-10. (canceled)
11. A biocompatible therapeutic article comprising, a macromer
having polymerized end groups, precipitated insulin, and a molecule
or mixture of molecules which preferentially excludes proteins,
wherein said molecule or mixture of molecules is present in an
amount sufficient to reduce the solubility of said insulin in said
article to less than 10 mg/ml.
12. The biocompatible therapeutic article of claim 1, wherein said
molecule which preferentially excludes proteins is selected from
the group consisting of a macromer, poly(ethylene glycol),
hyaluronic acid, and poly(vinylpyrrolidone).
13. The biocompatible therapeutic article of claim 1, wherein said
macromer comprises: (a) a region forming a central core; (b) at
least two degradable regions attached to said core; and (c) at
least two polymerized end groups, wherein said polymerized end
groups are attached to said degradable regions.
14. The biocompatible therapeutic article of claim 3, wherein said
central core comprises a polymer selected from the group consisting
of poly(ethylene glycol), poly(ethylene oxide), poly(vinyl
alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline),
poly(ethylene oxide)-co-poly(propylene oxide) block copolymers,
polysaccharides, carbohydrates, proteins, and combinations
thereof.
15. The biocompatible therapeutic article of claim 3, wherein said
degradable regions comprise a polymer selected from the group
consisting of poly(.alpha.-hydroxy acids), poly(lactones),
poly(amino acids), poly(anhydrides), poly(orthoesters),
poly(orthocarbonates), and poly(phosphoesters).
16. The biocompatible therapeutic article of claim 1, wherein said
article comprises at least 5% insulin by dry weight.
17. The biocompatible therapeutic article of claim 1, wherein said
article comprises at least 10% insulin by dry weight.
18. The biocompatible therapeutic article of claim 1, wherein said
molecule or mixture of molecules is present in an amount sufficient
to reduce the solubility of said insulin in said article to less
than 1 mg/ml.
19. The biocompatible therapeutic article of claim 1, wherein the
time at which 5% of the releasable insulin is released from the
article is greater than 1/16 of t.sub.50.
20. The biocompatible therapeutic article of claim 1, wherein said
insulin is released from said article such that t.sub.50 is greater
than or equal to 5/8 of t.sub.80.
21. The biocompatible therapeutic article of claim 1, wherein said
articles release at least 80% of the insulin at a time 11/4 times
greater than t.sub.50.
22. The biocompatible therapeutic article of claim 1, wherein said
article has a particle size of less than about 75 microns.
23. The biocompatible therapeutic article of claim 1, wherein said
macromer has a water soluble region comprising poly(ethylene
glycol) of about 500 to 20,000 daltons.
24. The biocompatible therapeutic article of claim 1, wherein said
mixture of molecules comprises a positively charged ion-carrying
reagent.
25. The biocompatible therapeutic article of claim 1, wherein said
mixture of molecules comprises a negatively charged ion-carrying
reagent.
26. The biocompatible therapeutic article of claim 1, wherein said
mixture of molecules comprises a surfactant.
27. The biocompatible therapeutic article of claim 1, further
comprising a pharmaceutically acceptable excipient.
28. The biocompatible therapeutic article of claim 27, wherein said
excipient is a carbohydrate.
29. The biocompatible therapeutic article of claim 28, wherein said
carbohydrate is selected from mannitol, sucrose, trehalose,
galactose, and lactose.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority
from U.S. Ser. No. 09/772,174, filed Jan. 29, 2001; which claims
benefit of U.S. Ser. No. 60/178,852, filed Jan. 28, 2000, entitled
"Slow Release Protein Polymers," having as inventors Steven C.
Rowe, Kalvin Yim, Beadle P. Retnarajan, and Jeffrey A. Hubbell.
BACKGROUND OF THE INVENTION
[0002] The invention relates to biodegradable compositions for
sustained-release drug delivery and methods for administering a
biologically active substance via these compositions.
[0003] Rapid advances in the fields of genetic engineering and
biotechnology have led to the development of an increasing number
of proteins and polypeptides that are useful as pharmaceutical
agents. The development of methods for administering these new
pharmaceutical agents is thus becoming increasingly important.
[0004] Most proteins have relatively short half-lives, requiring
frequent administration to achieve efficacious blood levels. To
increase patient convenience and to improve efficacy and safety by
keeping blood levels within the therapeutic range, smoothly
releasing injectable depot formulations of protein drugs are highly
desirable.
[0005] Recent polymer developments have improved the ability to
deliver proteins and peptides by allowing for slower and steadier
release of the molecule in the patient's system. However, in many
cases, the active form of the protein is difficult to formulate in
biodegradable polymers. Synthetic materials, such as biodegradable
hydrogels, have also been developed for use in delivering proteins.
Despite the advances provided by the available polymers and
hydrogels, the delivery of protein to the systemic and local
circulation is still relatively rapid, in some cases too rapid to
allow this route of administration to be used.
SUMMARY OF THE INVENTION
[0006] The present invention features articles for delivery of a
biologically active substance (hereafter "BAS"), and methods for
making such articles. The articles of the invention improve the
bioavailability of the BAS by formulating the BAS in an insoluble
form. The invention also features methods of treating an animal
using the articles for delivery of a BAS.
[0007] Accordingly, in a first aspect the invention features a
biocompatible therapeutic article for delivery of a BAS, comprising
a macromer, a molecule or mixture of molecules which preferentially
excludes proteins, and the BAS, wherein the BAS is in an insoluble
format upon completion of the formulation of the article comprising
the macromer, molecule, or mixture of molecules which
preferentially excludes proteins, and BAS.
[0008] In a preferred embodiment of the first aspect of the
invention, the biocompatible therapeutic article has at least one
of the following properties: the BAS is less than 15% aggregated;
the article contains at least 10% macromer and at least 5% BAS, as
measured by dry weight; the time at which 5% of the releasable BAS
is released from the article is greater than 1/16 of t.sub.50; or
the t.sub.50 is greater than or equal to 5/8 of t.sub.80. More
preferably the biocompatible therapeutic article has at least two
of the above properties. Most preferably, the biocompatible
therapeutic article has all of the above properties.
[0009] In another embodiment of the first aspect of the invention,
the molecule which preferentially excludes proteins is a macromer,
poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone).
In yet another embodiment, the macromer is a hydrogel. In still
another embodiment, the solubility of a protein in the article
comprising the macromer, molecule that preferentially excludes
proteins, and BAS is less than 5-10 mg/ml, and more preferably is
less than 1 mg/ml.
[0010] In another embodiment of the first aspect of the invention,
the mixture of molecules comprises a positively charged
ion-carrying reagent, for example, triethanolamine or Tris, when
the pH is such that the protein is negatively charged. In still
another embodiment, the mixture of molecules comprises a negatively
charged ion-carrying reagent, such as sodium dodecyl sulfate, when
the pH is such that the protein is positively charged. In yet
another embodiment, the mixture of molecules comprises a
surfactant, for example, Tween 20, Tween 80, or poloxamer F68. In a
second aspect, the invention features a method for making a
therapeutic article for delivery of a BAS, involving (a) combining
the BAS with a molecule or mixture of molecules which
preferentially excludes proteins; (b) combining the mixture formed
in step (a) with a macromer, wherein the BAS is in an insoluble
form and remains insoluble upon combining with the molecule or
mixture of molecules which preferentially excludes proteins and the
macromer; (c) forming a mixture of the combination formed in step
(b); and (d) polymerizing the mixture to form an article.
[0011] In one embodiment of the second aspect of the invention,
steps (a) and (b) are combined into a single combination step.
[0012] In a preferred embodiment of the second aspect of the
invention, the biocompatible therapeutic article has at least one
of the following properties: the BAS is less than 15% aggregated;
the article contains at least 10% macromer and at least 5% BAS, as
measured by dry weight; the time at which 5% of the releasable BAS
is released from the article is greater than 1/16 of t.sub.50; or
the t.sub.50 is greater than or equal to 5/8 of t.sub.80. More
preferably the biocompatible therapeutic article has at least two
of the above properties. Most preferably, the biocompatible
therapeutic article has all of the above properties.
[0013] In another embodiment of the second aspect of the invention,
the molecule which preferentially excludes proteins is a macromer,
poly(ethylene glycol), hyaluronic acid, or poly(vinylpyrrolidone).
In yet another embodiment, the macromer is a hydrogel. In yet
another embodiment, the macromer is a hydrogel. In still another
embodiment, the solubility of a protein in the article comprising
the macromer, molecule that preferentially excludes proteins, and
BAS is less than 5-10 mg/ml, and more preferably is less than 1
mg/ml.
[0014] In another embodiment of the second aspect of the invention,
the mixture of molecules comprises a positively charged
ion-carrying reagent, for example, triethanolamine, when the pH is
such that the protein is negatively charged. In still another
embodiment, the mixture of molecules comprises a negatively charged
ion-carrying reagent, such as sodium dodecyl sulfate, when the pH
is such that the protein is positively charged. In yet another
embodiment, the mixture comprises a surfactant, for example, Tween
20, Tween 80, or poloxamer F68.
[0015] In a third aspect the invention features a method of
treating an animal, involving administering the biocompatible
therapeutic article of the first aspect of the invention to a
mammal. Preferably the mammal is a rodent, and most preferably the
mammal is a human.
[0016] In yet other preferred embodiments, the articles are
administered to the lung of the mammal, or are administered
intravenously, subcutaneously, intramuscularly, orally, or
nasally.
[0017] In a preferred embodiment of any of the above aspects of the
invention, the macromer comprises: (a) a region forming a central
core; (b) at least two degradable regions attached to the core; and
(c) at least two polymerizable end groups, where the polymerizable
end groups are attached to the degradable regions. In preferred
embodiments, the region forming a central core is a water soluble
region. The water soluble region may be poly(ethylene glycol),
poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone),
poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide)
block copolymers, polysaccharides, carbohydrates, proteins, and
combinations thereof. The degradable region is selected from the
group consisting of poly(.alpha.-hydroxy acids), poly(lactones),
poly(amino acids), poly(anhydrides), poly(orthoesters),
poly(orthocarbonates), and poly(phosphoesters). Preferably, the
poly(.alpha.-hydroxy acid) is poly(glycolic acid), poly(DL-lactic
acid), or poly(L-lactic acid), and the poly(lactone) is
poly(.epsilon.-caprolactone), poly(.delta.-valerolactone), or
poly(.gamma.-butyrolactone). In another preferred embodiment, the
degradable region comprises poly(caprolactone). In yet another
embodiment, the polymerizable end groups contain a carbon-carbon
double bond capable of polymerizing the macromer.
[0018] In other embodiments of the above aspects of the invention,
the macromer includes: (a) a water soluble region comprising a
three-armed poly(ethylene glycol) with a molecular weight of 3,000
to 6,000 daltons; (b) lactate groups attached to the region in (a);
and (c) acrylate groups capping the region in (b). The macromer may
alternatively include: (a) a water soluble region comprising
poly(ethylene glycol) with a molecular weight of either 2,000 or
3,400 daltons; (b) lactate groups on either side of the region in
(a); and (c) acrylate groups capping either side of the region in
(b). In another alternative, the macromer may include (a) a water
soluble region comprising poly(ethylene glycol) with a molecular
weight of 3,400 daltons; (b) caprolactone groups on either side of
region in (a); and (c) acrylate groups capping either side of the
region in (b).
[0019] In still other embodiments of any of the above aspects of
the invention, the article includes at least 5%, more preferably
10%, and most preferably 20-30% active substance by dry weight. In
still another embodiment, the article is biodegradable.
[0020] In a more preferred embodiment of any of the above aspects
of the invention, the macromer includes a water soluble region
consisting of a three-armed PEG with a molecular weight of 4,200 to
5,400 daltons; lactate groups one end of each arm of the PEG; and
acrylate groups capping the lactate groups.
[0021] In another more preferred embodiment of the above aspects of
the invention, the macromer is made of a triad ABA block copolymer
of acrylate-poly(lactic acid)-PEG-acrylate-poly(lactic
acid)-acrylate. The PEG has a MW of 3,400 daltons; the poly(lactic
acids) on both sides had an average of about five lactate units per
side; and the macromer is therefore referred to herein as "3.4kL5."
In another more preferred embodiment, a lower molecular weight PEG,
such as MW 2,000 daltons PEG is used in place of the MW 3,400 PEG,
and the resulting macromer is abbreviated as "2kL5."
[0022] In yet another more preferred embodiment of the above
aspects of then invention, the macromer is an
acrylate-PCL-PEG-PCL-acrylate macromer. The PEG has a MW of 3,400
daltons and has polycaprolactone on both sides, with an average of
about 6 caproyl units per side. This macromer is referred to herein
as "3.4kC6."
[0023] In other preferred embodiments, the BAS is a protein or
peptide. More preferably the protein is chosen from a group
consisting of hormones, antibodies, differentiation factors,
angiogenic factors, enzymes, cytokines, chemokines, interferons,
colony-stimulating factors, and growth factors. Most preferably,
the protein is a hormone, such as human growth hormone, or a
peptide, such as LHRH.
[0024] In still other embodiments of the second and third aspects
of the invention, the therapeutic articles release at least 80% of
the BAS at a time 11/4 times greater than t.sub.50. At least 80% of
the therapeutic articles may have a particle size of less than
about 80 microns. The water soluble region may consist essentially
of PEG having a molecular weight of about 500 to 20,000 daltons,
and more preferably, between 1,000 and 10,000 daltons. The
degradable region may comprise a blend of at least two different
polymers. In addition, the macromer may be non-degradable.
[0025] In still other embodiments of the second and third aspects
of the invention, the therapeutic article is capable of releasing
the BAS for at for a period of time at least 2 times greater than
t.sub.50. The article is also capable of delivering a therapeutic
dose of the BAS for at for a period of time at least 11/4 times
greater than t.sub.50.
[0026] By "macromer" is meant a polymer with three components: (1)
a biocompatible, water soluble region; (2) a
biodegradable/hydrolyzable region, and (3) at least two
polymerizable regions.
[0027] By "biologically active substance" or "BAS" is meant a
compound, be it naturally-occurring or artificially-derived, that
is incorporated into an article and which may be released and
delivered to a site. Biologically active substances may include,
for example, peptides, polypeptide, proteins, synthetic organic
molecules, naturally occurring organic molecules, nucleic acid
molecules, and components thereof.
[0028] By "a molecule or mixture of molecules that preferentially
excludes proteins" is meant a molecule or mixture of molecules, be
it naturally-occurring or artificially-derived, that, when added to
a solution, confers a lower level of solubility of the protein or
polypeptide in said solution. Preferably, protein solubility will
be decreased 50-fold; more preferably, 100-fold and most preferably
about 200-fold. Preferably the solubility of a protein in a
solution that includes said molecule or mixture of molecules that
preferentially excludes proteins is less than 5-10 mg/ml, and more
preferably is less than 1 mg/ml.
[0029] By "substantially pure polypeptide" or "protein" is meant a
polypeptide or protein that has been separated from the components
that naturally accompany it. The terms polypeptide and protein may
be used interchangeably. Typically, the polypeptide is
substantially pure when it is at least 60%, by weight, free from
the proteins and naturally-occurring organic molecules with which
it is naturally associated. A substantially pure polypeptide may be
obtained, for example, by extraction from a natural source (e.g., a
cell expressing the desired polypeptide), by expression of a
recombinant nucleic acid encoding a desired polypeptide, or by
chemically synthesizing the polypeptide. Purity can be assayed by
any appropriate method, e.g., by column, chromatography,
polyacrylamide gel electrophoresis, agarose gel electrophoresis,
optical density, or HPLC analysis.
[0030] A protein is substantially free of naturally associated
components when it is separated from those contaminants which
accompany it in its natural state. Thus, a protein which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those derived
from eukaryotic organisms but synthesized in E. coli or other
prokaryotes.
[0031] By "purified nucleic acid" is meant a nucleic acid that is
free of the genes which, in the naturally-occurring genome of the
organism from which the nucleic acid of the invention is derived,
flank the gene. The term therefore includes, for example, a
recombinant DNA which is incorporated into a vector; into an
autonomously replicating plasmid or virus; or into the genomic DNA
of a prokaryote or eukaryote; or which exists as a separate
molecule (e.g., a cDNA or a genomic or cDNA fragment produced by
PCR or restriction endonuclease digestion) independent of other
sequences. It also includes recombinant DNA which is part of a
hybrid gene encoding additional polypeptide sequence.
[0032] By "biocompatible" is meant that any compound or substance
which is administered to a subject, cell, or tissue is used to
treat, replace, or augment a function of the subject, cell or
tissue, and is not harmful to said function.
[0033] By "insoluble" is meant that the solubility of a compound is
less than 1 g/100 ml in a solution. The solution may be an aqueous
solution, an organic solvent, such as dimethylsulfoxide, or a
mixture of aqueous and organic solvents. As used herein, a BAS is
in an insoluble format upon completion of the formulation for a
therapeutic article for delivery of the BAS. The BAS remains in an
insoluble format upon delivery of the therapeutic article to a
patient, and is then slowly released at a controlled rate for
localized or systemic delivery to the patient. As used herein, by
"aggregated" is meant that a BAS is releasable as individual
molecules. The percent of a BAS in an article which is aggregated
can be determined, for example, by SEC-HPLC.
[0034] By "therapeutic dose," when referring to a BAS, is meant a
plasma level between the minimum effective level and the toxic
level.
[0035] By a "mixture" is meant a composition in which all of the
compounds contained in the composition are evenly distributed.
[0036] As used herein, by "pore size" is meant the dimensions of a
space in the intact polymer through which a macromer, component of
a macromer, or a BAS potentially can pass. Pore sizes which are
utilized as part of the invention are those smaller than the BAS as
it is present in the particular embodiment (e.g., a protein
molecule, or aggregate thereof).
[0037] As used herein, by "period of release" is meant the length
of time it takes for a specified percent of the BAS to be released
from an article. The period of release may be assessed, for
example, by measuring the time it takes for 50% or 80% of the BAS
to be released from the article.
[0038] By "low burst effect" is meant that the amount of BAS
released from an article is released relatively steadily over time,
rather than at an initial fast rate, followed by a slower rate. For
example, a BAS has a low burst effect (e.g., less than or equal to
20% burst) upon release from an article when the period of release
for 5% of the releasable BAS is greater than 1/16 of .sub.0, or
when the t.sub.50 is greater than or equal to 5/8 of t.sub.80. In
contrast to a low burst article, a high burst article (e.g., one
which rapidly releases 30% of the BAS) might release 5% of its
releasable BAS in less than 1/18 of 50 and have a to equal to 1/2
of t.sub.80.
[0039] A specific example of a low burst product of the present
invention is one in which less than 20% of the BAS comes out in the
first day for a product designed to release a BAS for 10 days.
[0040] By "t.sub.50" is meant the time at which 50% of the original
load of BAS has been released. As used herein, preferably 5% of the
releasable BAS is released at a time which is greater than 1/16 of
t.sub.50, or the t.sub.50 is greater than or equal to 5/8 of the
t.sub.80.
[0041] By "t.sub.80" is meant the time at which 80% of the original
load of BAS has been released. As used herein, preferably 5% of the
releasable BAS is released at a time which is greater than 1/16 of
t.sub.50, or the t.sub.50 is greater than or equal to 5/8 of
thet.sub.80.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 is the release profile of microspheres made with
precipitated hGH.
[0043] FIG. 2 is the release profile of hGH from 2kL5
microspheres.
[0044] FIG. 3 is a graph depicting the release of spray-dried bSA
from 4.2kL3-A3 microspheres.
[0045] FIG. 4 is a graph of the effects of biologically active
particle size on the release of bovine serum albumen (bSA) from 30%
3.4kL5.
[0046] FIG. 5 is a graph of the effect of biologically active
particle size and protein loading on the release of fine-ground bSA
(10% loaded) and various crystalline particles (1-10% loaded) from
3.4kL5.
[0047] FIG. 6 is a graph of the effects of biologically active
particle size on the release of human growth hormone (hGH) from 30%
3.4kL5.
[0048] FIG. 7 is a graph of the effect of microsphere pore size on
release of micronized hGH from articles.
DETAILED DESCRIPTION
[0049] The invention provides methods and compositions for the
administration of a biologically active substance (BAS) in an
insoluble format. The compositions of the invention improve the
bioavailability of the BAS by formulating the BAS in an insoluble
format. These methods and compositions provide for the controlled,
sustained delivery of relatively large quantities of these
substances, with a low burst effect.
Macromers
[0050] The macromers of the present invention have at least one
region forming a central core, at least one degradable (e.g.,
hydrolyzable) region, and at least one polymerizable region. The
macromers may be water-soluble or water insoluble. Preferably, the
region forming a central core is water soluble. If desired, the
macromers may be polymerized to form hydrogels, which are useful
for delivering incorporated substances at a controlled rate.
Methods to formulate macromers and shape them into articles are
described, for example in WO 99/03454, hereby incorporated by
reference. An important aspect of the macromers is that the
polymerizable regions are separated by at least one degradable
region. This separation facilitates uniform degradation in
vivo.
[0051] The ratio between the central core region and the
hydrolyzable region of the macromer determines many of the general
properties of the macromer. For example, the water solubility of
the macromers can be controlled by varying the percentage of the
macromer that consists of hydrophobic degradable groups.
[0052] There are several variations of the macromers of the present
invention. For example, the polymerizable regions can be attached
directly to the degradable regions; alternatively, they can be
attached indirectly via water-soluble, nondegradable regions, with
the polymerizable regions separated by a degradable region. For
example, if the macromer contains a single water-soluble region
coupled to a degradable region, one polymerizable region can be
attached to the water-soluble region, and the other to the
degradable region.
[0053] In another embodiment, a water-soluble region forms the
central core of the macromer and has at least two degradable
regions attached to it. At least two polymerizable regions are
attached to the degradable regions so that, upon degradation, the
polymerizable regions, particularly in the polymerized gel form,
are separated. Alternatively, if the central core of the macromer
is formed by a degradable region, at least two water soluble
regions can be attached to the core, and polymerizable regions are
attached to each water soluble region.
[0054] In still another embodiment, the macromer has a
water-soluble backbone region, with a degradable region attached to
the macromer backbone. At least two polymerizable regions are
attached to the degradable regions, such that they are separated
upon degradation, resulting in gel product dissolution. In a
further embodiment, the macromer backbone region is formed of a
degradable backbone region having water-soluble regions as branches
or grafts attached to the degradable backbone. Two or more
polymerizable regions are attached to the water soluble branches or
grafts.
[0055] In another variation, the macromer backbone may have
multiple arms; e.g., it may be star-shaped or comb-shaped. The
backbone may include a water-soluble region, a biodegradable
region, or a water-soluble, biodegradable region. The polymerizable
regions are attached to this backbone. Again, the polymerizable
regions must be separated at some point by a degradable region.
[0056] Throughout the specification, the following abbreviations
are, sometimes used to describe the specific macromers of the
invention. In three particular examples, a macromer having a water
soluble region consisting of PEG with a molecular weight of 4,000
daltons, with 5 lactate groups on either side of this region,
capped on either side with acrylate groups, is referred to as
"4kL5." Similarly, a macromer having a water soluble region
consisting of PEG with a molecular weight of 3,400 daltons, with 6
caprolactone groups on either side of this region, capped on either
side with acrylate groups, is referred to as "3.4kC6." Likewise, a
macromer having a water soluble region consisting of PEG having a
molecular weight of 5,400 daltons and 3 arms, each arm containing 3
lactate groups, extending from this region, capped on either side
with acrylate groups, is referred to as "4.2kL3-A3."
Water-Soluble Region
[0057] In preferred embodiments, the central core is a water
soluble region. This water soluble region of the macromer may
include poly(ethylene glycol), poly(ethylene oxide), poly(vinyl
alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline),
poly(ethylene oxide)-co-poly(propylene oxide) block copolymers,
polysaccharides, carbohydrates, or proteins, or combinations
thereof.
[0058] The macromer preferably comprises a water soluble core
region comprising PEG, as PEG has high hydrophilicity and water
solubility, as well as good biocompatibility. The PEG region
preferably has a molecular weight of about 400 to about 40,000
daltons, and more preferably has a molecular weight of about 1,000
to about 30,000 daltons, about 1,000 to about 20,000 daltons, or
about 2,000 to about 10,000 daltons.
Degradable Region
[0059] The degradable region of the macromer may contain, for
example, poly(a-hydroxy acids), poly(lactones), poly(amino acids),
poly(anhydrides), poly(orthoesters), poly(orthocarbonates) or
poly(phosphoesters), or blends or copolymers of these polymers.
[0060] Exemplary poly(.alpha.-hydroxy acids) include poly(glycolic
acid), poly(DL-lactic acid), and poly(L-lactic acid). Exemplary
poly(lactones) include poly(.epsilon.-caprolactone),
poly(.delta.-valerolactone), poly(.gamma.-butyrolactone),
poly(1,5-dioxepan-2-one), and poly(trimethylene carbonate).
[0061] The degradable region may comprise a blend of at least two
different polymers. Examples of copolymers include a copolymer of
caprolactone and glycolic acid; and a copolymer of caprolactone and
lactic acid.
Polymerizable Region
[0062] The polymerizable regions of the macromer preferably contain
carbon-carbon double bonds capable of polymerizing the macromers.
The choice of an appropriate polymerizable group permits rapid
polymerization and gelation. Polymerizable regions containing
acrylates are preferred because they can be polymerized using
several initiating systems, as discussed below. Examples of
acrylates include acrylate, methacrylate, and methyl
methacrylate.
Polymerization of Macromers
[0063] If desired, the macromers of the present invention may be
polymerized using polymerization initiators under the influence of
long wavelength ultraviolet light, visible light, thermal energy,
or a redox system. The polymerization can be conducted at room
temperature or at lower temperatures, for example, temperatures
less than 20.degree. C. During polymerization, substances such as
proteins are physically incorporated into the resulting polymer
network of the hydrogel.
[0064] Polymerization of the macromers may be initiated in situ by
light having a wavelength of 320 nm or longer. When the
polymerizable region contains acrylate groups, the initiator may be
any of a number of suitable dyes, such as xanthine dyes, acridine
dyes, thiazine dyes, phenazine dyes, camphorquinone dyes,
acetophenone dyes, or eosin dyes with triethanolamine,
2,2-dimethyl-2-phenyl acetophenone, and 2-methoxy-2-phenyl
acetophenone.
[0065] The polymerization may also take place in the absence of
light. For example, the polymerization can be initiated with a
redox system, using techniques known to those of skill in the art.
In some cases it is advantageous to polymerize macromers using the
redox system of the invention, as radical initiator production
occurs at reasonable rates over a wide range of temperatures.
[0066] Initiators that can be used in the redox system include,
without limitation, peroxides such as acetyl, benzoyl, cumyl and
t-butyl; hydroperoxides such as t-butyl and cumyl, peresters such
as t-butyl perbenzoate; acyl alkylsulfonyl peroxides, dialkyl
peroxydicarbonates, diperoxyketals, ketone peroxide, azo compounds
such as 2,2'-azo(bis)isobutyronitrile (AIBN), disulfides, and
tetrazenes.
Shaping of Articles
[0067] The articles of the present invention may be formed in any
shape desired. For example, the articles may be shaped to fit into
a specific body cavity. They may also be formed into thin, flat
disks or particles, such as microspheres. Alternatively, the
articles may be shaped, then processed into the desired shape
before use, or ground into fine particles. The desired shape of the
article will depend on the specific application.
[0068] Macromer particles may be prepared using techniques known in
the art, including single and double emulsion solvent evaporation,
spray drying, and solvent extraction. As used herein, the term
"particles" includes, but is not limited to, microspheres. In a
microsphere, a BAS is dispersed throughout the particle. The
particles may have a smooth or irregular surface, and may be solid
or porous. Methods for making microspheres are described in the
literature, for example, in U.S. Pat. No. 4,272,398, Mathiowitz and
Langer (J. Controlled Release 5:13-22 (1987)); Mathiowitz et al.
(Reactive Polymers 6:275-283 (1987)); Mathiowitz et al. (J. Appl.
Polymer Sci. 35:755-774 (1988)); Mathiowitz et al. (Scanning
Microscopy 4:329-340 (1990)); Mathiowitz et al. (J. Appl. Polymer
Sci., 45:125-134 (1992)); and Benita et al. (J. Pharm. Sci.
73:1721-1724 (1984)), hereby incorporated by reference. In one
preferred embodiment of the present invention, the microspheres are
formed into hydrogel droplets.
[0069] In solvent evaporation, described, for example, in
Mathiowitz, et al., (1990), Benita et al. (1984), and U.S. Pat. No.
4,272,398, a polymer is dissolved in a volatile organic solvent,
such as methylene chloride. An agent to be incorporated, either in
soluble form or dispersed as fine particles, is optionally added to
the polymer solution, and the mixture is suspended in an aqueous
phase that contains a surface active agent such as poly(vinyl
alcohol). The resulting emulsion is stirred until most of the
organic solvent evaporates, leaving solid microspheres, which may
be washed with water and dried overnight in a lyophilizer.
[0070] In solvent removal, as described, for example, by Park et
al. (J. Controlled Release 55:181-191 (1998)), a therapeutic or
diagnostic agent is dispersed or dissolved in a solution of a
selected polymer in a volatile organic solvent such as methylene
chloride. The mixture can then be suspended in oil, such as silicon
oil, by stirring, to form an emulsion. As the solvent diffuses into
the oil phase, the emulsion droplets harden into solid polymer
microspheres.
[0071] Processes for preparing ultrafine particles of biological
molecules by atomizing liquid solutions of the macromolecules,
drying the droplets formed in the atomization step, and collecting
the particles are described in PCT WO 97/41833, hereby incorporated
by reference.
[0072] Spray drying is implemented by passing a homogenous mixture
of a BAS, such as a therapeutic agent, and the polymerizable
macromer used to form a hydrogel through a nozzle, spinning disk,
or equivalent device to atomize the mixture to form fine droplets.
The substance and the polymerizable mactomer may be provided in a
solution or suspension, such as an aqueous solution. The fine
droplets are exposed to light to cause polymerization of the
macromer and formation of the hydrogel droplets incorporating the
substance. Hydrogels may be formed according to the methods
described in U.S. Pat. No. 5,410,016, hereby incorporated by
reference, or other techniques known in the art of polymer
chemistry.
[0073] In another embodiment, hydrogel particles are prepared by a
water-in-oil emulsion process, wherein the polymerizable macromers
and the substance to be incorporated are suspended in a
water-in-oil emulsion and exposed to light to polymerize the
macromers to form hydrogel particles incorporating the substance,
such as a BAS. Typically, polymerization may be conducted at room
temperature.
[0074] The microspheres prepared using the techniques described
above are freeze dried, so they have a long shelf life (without
biodegradation) and the BAS remains biologically active. Prior to
use for injectable formulations, the microspheres are reconstituted
in a suitable solution, such as saline or other liquids. For
pulmonary delivery, either freeze dried or reconstituted particles
may be used.
Properties of the Macromers
[0075] The articles of the present invention are biodegradable.
Biodegradation occurs at the linkages within the extension
oligomers and results in fragments which are non-toxic and easily
removed from the body and/or are normal, safe chemical
intermediates in the body. These materials are particularly useful
for the delivery of hydrophilic materials, since the water soluble
regions of the polymer allow water to access the materials trapped
within the polymer.
Use of the Macromers
[0076] Macromers can be shaped into articles, for example,
microspheres, and these articles are capable of degrading under in
vivo conditions at rates which permit the controlled release of
incorporated substances. Release of such a substance may occur by
diffusion of the substance from the polymer prior to degradation
and/or by diffusion of the material from the polymer as it
degrades. Degradation of the polymer facilitates eventual
controlled release of free macromolecules in vivo by gradual
hydrolysis of the terminal ester linkages. The burst effects that
are sometimes associated with other release systems are thus
avoided in a range of formulations.
[0077] The rate of release of a BAS depends on many factors, for
example, the composition of the water soluble region, the degree of
polymerization of the macromer. The rate of release of a BAS also
depends on the rate of degradation of the degradable region of the
macromer. For example, glycolic esters lead to very rapid
degradation, lactic esters to somewhat slower degradation, and
caprolactic esters to very slow degradation. When the degradable
region consists of polyglycolic acid, the release period is less
than one week. When the degradable region consists of poly(lactic
acid), the release period is about one week. When the degradable
region consists of a copolymer of caprolactone and lactic acid or a
copolymer of trimethylene carbonate and lactic acid, the release
period is two to four weeks. When the degradable region consists of
poly(trimethylene carbonate) or a copolymer of caprolactone and
trimethylene carbonate, the release period is about three to eight
weeks. When the degradable region consists of poly(trimethylene
carbonate) or poly(caprolactone), the release period is longer than
about five weeks.
[0078] The precise rate of release of a BAS from an article can be
further modified by altering the ratio of hydrophilic and
hydrophobic components of the article. For example, a very soluble
macromer will yield, after polymerization, a hydrophilic gel;
hydrophilic hydrogels have been shown to degrade more rapidly than
hydrophobic ones. A blend of a hydrophilic macromer (e.g., 4kL5)
with a hydrophobic water insoluble macromer (3.4kC6) is used to
form a polymerized hydrogel. This hydrogel will have a release rate
that is in between the release rate of a hydrogel containing only
lactic acid and a hydrogel containing only caprolactone. A macromer
in which the degradable region is a copolymer of caprolactone and
lactic acid will also have a release rate which is in between the
release rate of a hydrogel containing only lactic acid and a
hydrogel containing only caprolactone as the primary degradable
group. Similarly, hydrophilicity of the active substance also
affect the release rate of the BAS, with hydrophilic active
substances generally released faster than hydrophobic
substances.
[0079] The rate of release of a given BAS from a therapeutic
article depends on the quantity of the loaded substance, as a
percent of the final product formulation. For example, it is
generally thought in the polymer field that while a large amount of
BAS loading results in a longer period of therapeutic dose
delivery, it also results in a large burst effect. Therefore, an
article which is loaded with a high amount of a BAS, and which also
exhibits a low burst effect would be an optimal article. The
articles or the present invention exhibit these
characteristics.
[0080] Other factors which affect the release rate of a BAS from an
article are the aggregation and the solubility of the BAS. In order
for the articles of the present invention to have release profiles
which are optimal for delivering a BAS, the percent of the BAS
which is aggregated should be low. The articles of the present
invention contain BAS which are preferably less than 15%
aggregated. In preferred embodiments, the articles have this
characterization of low aggregation even when they and contain at
least 2.5% BAS by dry weight, more preferably at least 5%, and most
preferably 20 or 40% by dry weight.
[0081] As stated above, another factor which affects the rate of
release of a BAS from an article is the solubility of the BAS in
the article. In the field of polymer chemistry, it has generally
been thought that water-soluble substances, such as a BAS, will
yield homogenous systems when incorporated into the macromers of
the invention. It has also been thought that substances that do not
solubilize in water within the time it takes to form the macromers
of the invention will yield heterogenous systems. While the amount
of burst in the heterogenous systems can be minimized by using a
particulate suspension with small particles, it is generally
thought that substances should be in a water soluble format for
optimal delivery in a polymer delivery system. The articles of the
present invention contain a BAS in an insoluble format, and these
articles exhibit a low burst effect, an unexpected result.
[0082] Yet another factor that affects the release rate of a BAS
from an article is the particle size of the BAS. For example, the
articles of the present invention feature a BAS which has been
ground and sieved to isolate fine particles which are smaller than
approximately 75 microns in any dimension. These particles were
used to generate microspheres and the release of the BAS from the
microspheres was measured. This release rate was compared to the
release rate of the same BAS from the same microspheres, with the
exception that the BAS was not fine-ground. The results of these
studies indicated that a BAS which is fine-ground results in
release rates which are slower and have a low burst effect. By
adjusting the factors discussed above, degradation and controlled
release may be varied over very wide ranges. For example, release
may be designed to occur over hours, days, or months.
[0083] The methods of the invention can produce particles that
behave as homogenous drug delivery systems. Because of the
homogenous nature of the articles of the invention, there is no
initial burst of released substance. In addition, the uniform
consistency makes it possible to incorporate relatively high
amounts of protein, while still minimizing the burst effect.
[0084] The present invention also features insoluble macromers.
These macromers contain at least one water-soluble region, at least
one degradable (e.g., hydrolyzable) region, and at least one
polymerizable region. The degradable region contains polymers of
glycolic acid, lactic acid, caprolactone, trimethylene carbonate,
or blends or copolymers thereof. The degradable region must be
water insoluble. For example, a macromer having a degradable region
containing 15-20 lactide units can be prepared; this macromer will
provide a relatively fast release rate. A macromer with a
degradable region containing 6 caprolactone units will provide a
relatively slow release rate. A macromer with a degradable region
containing a copolymer of 6 caprolactone units, 4 lactide units,
and 4 glycolide units will provide a fast release rate, and a
macromer with a degradable region containing a copolymer of 3
lactide units and 7 trimethylene carbonate units will provide an
intermediate release rate.
[0085] The water soluble region of these macromers is preferably
PEG. The water soluble region can have multiple arms; for example,
it may be star-shaped or comb-shaped, as described, for example in
U.S. Pat. No. 5,410,016, incorporated herein by reference. The
water soluble region preferably has 3, 4, 6, or 8 arms and a
molecular weight of 500 to 20,000, preferably, 1,000 to 10,000
daltons.
Methods for Increasing Protein Precipitation
[0086] The articles of the present invention, can be made to
contain a BAS in an insoluble format, by combining the BAS with a
molecule, or mixture or molecules which preferentially excludes
proteins, and a macromer, forming a mixture of these reagents, and
polymerizing the mixture. A molecule or, mixture of molecules which
preferentially exclude proteins can be used in the formation of the
article to increase protein precipitation. Examples of molecules
which preferentially exclude proteins include, but are not limited
to, macromers, poly(ethylene glycol), hyaluronic acid, and
poly(vinylpyrrolidone). A reagent which carries a positive or
negative ion charge may be used in the formation of the articles of
the invention in order to increase the precipitation of the BAS in
the mixture which is then polymerized to form the article. The
optimal reagent to be used depends on the charge of the protein,
which is affected by the pH of the mixture. Examples of mixtures of
molecules which preferentially exclude proteins include, but are
not limited to, a mixture of molecules comprising a positively
charged ion-carrying reagent, for example, triethanolamine or Tris
(for example, when the pH is such that the protein is negatively
charged); or a mixture of molecules comprising a negatively charged
ion-carrying reagent, such as sodium dodecyl sulfate (for example,
when the pH is such that the protein is positively charged). A
mixture comprising a surfactant, for example, Tween 20, Tween 80,
or poloxamer F68, may also be used to increase the precipitation of
the protein.
High Load and Low Burst Characteristics
[0087] A therapeutic agent, for example, a BAS may be readily
incorporated in high yield into the articles described herein. For
example, articles may be prepared containing at least 5% active
substance by dry weight. Preferably, the articles contain at least
10, 25, or 40% by dry weight.
[0088] As discussed above, the BAS of the present invention is in
an insoluble format when combined with a macromer and formed into
an article. The combination of high load and the insoluble format
of the active substance in the article provides the article with a
slow release profile, with little initial burst. These results are
surprising given the view in the field of polymers that an article
containing an insoluble active substance will have large initial
burst of the active substance.
[0089] The BAS contained in the articles of the present invention
is insoluble. The formulation of articles containing an insoluble
BAS may be achieved, for example, by mixing the BAS with PEG, and
then combining these reagents with the desired macromer.
[0090] The amount of BAS loaded into a microsphere may be measured
by combining it with a macromer and shaping into articles. The
articles may then be placed into an appropriate solvent, for
example phosphate buffered release media (0.01% NaN.sub.3, 0.05 M
PBS, pH 7.4) and assayed for the amount of BAS present by means
available in the art, such as spectrophotometry.
Biologically Active Substances
[0091] A BAS that can be incorporated into the articles of the
invention include therapeutic, diagnostic, and prophylactic agents.
They can be naturally occurring compounds, synthetic organic
compounds, or inorganic compounds. Substances that can be
incorporated into the articles of the invention include proteins,
polypeptides, carbohydrates, inorganic materials, antibiotics,
antineoplastic agents, local anesthetics, antiangiogenic agents,
vasoactive agents, anticoagulants, immunomodulators, cytotoxic
agents, antiviral agents, antibodies, neurotransmitters,
psychoactive drugs, oligonucleotides, lipids, cells, tissues,
tissue or cell aggregates, and combinations thereof.
[0092] Exemplary therapeutic agents include growth hormone, for
example human growth hormone, calcitonin, granulocyte macrophage
colony stimulating factor (GMCSF), ciliary neurotrophic factor,
parathyroid hormone, and the cystic fibrosis transmembrane
regulator gene. Other specific therapeutic agents include
parathyroid hormone-related polypeptide, somatostatin,
testosterone, progesterone, estradiol, nicotine, fentanyl,
norethisterone, clonidine, scopolomine, salicylate, salmeterol,
formeterol, albeterol, and valium.
[0093] Drugs for the treatment of pneumonia may be used, including
pentamidine isethionate. Drugs for the treatment of pulmonary
conditions, such as asthma, may be used, including albuterol
sulfate, .beta.-agonists, metaproterenol sulfate, beclomethasone
dipropionate, triamcinolone acetamide, budesonide acetonide,
ipratropium bromide, flunisolide, cromolyn sodium, ergotamine
tartrate, and protein or polypeptide drugs such as TNF aritagonists
or interleukin antagonists.
[0094] Other therapeutic agents include cancer chemotherapeutic
agents, such as cytokines, chemokines, lymphokines, and
substantially purified nucleic acids, and vaccines, such as
attenuated influenza virus. Substantially purified nucleic acids
that can be incorporated include genomic nucleic acid sequences,
cDNAs encoding proteins, expression vectors, antisense molecules
that bind to complementary nucleic acid sequences to inhibit
transcription or translation, and ribozymes. For example, genes for
the treatment of diseases such as cystic fibrosis can be
administered. Polysaccharides, such as heparin, can also be
administered.
[0095] Other therapeutic agents include tissue plasminogen
activator (t-PA), superoxide dismutase, catalase luteinizing
hormone releasing hormone (LHRH) antagonists, IL-11 platelet
factor, IL-4 receptor, enbrel, IL-1 receptor antagonists, TNF
receptor fusion proteins, megakaryocyte growth and development
factor (MGDF), stemgen, anti-HER-2 and anti-VEGF humanized
monoclonal antibody, anti-Tac antibody, GLP-1 amylin, and GLP-1
amylin analogues.
[0096] Additional therapeutic agents include atrial natriuretic
factor, atrial natriuretic peptide, beta-human chorionic
gonadotropin, basic fibroblast growth factor, bovine growth
hormone, bone morphogenetic protein, B cell stimulating factor-1, B
cell stimulating factor-2, bovine somatotropin, carcinobreaking
factor, cartilage induction factor, corticotropin releasing factor,
colony stimulating factor, differentiating factor-1, endothelial
cell growth factor, erythroid differentiation factor, elongation
factor 1-alpha, epidermal growth factor, erythropoietin,
thrombopoietin, thymopoietin, fibroblast growth factor, follicle
stimulating hormone, granulocyte colony stimulating factor, glial
fibrillary acidic protein, growth hormone releasing factor, human
alpha-1 antitrypsin, human atrial natriuretic factor, human
chorionic gonadotropin, human leukemia inhibitory factor,
hemopoietin-1, hepatocyte growth factor, human transforming growth
factor, human thyroid-stimulating hormone, interferon,
immunoglobulin A, immunoglobulin D, immunoglobulin E, insulin-like
growth factor-1, insulin-like growth factor-11, immunoglobulin G,
immunoglobulin M, interleukin-1, interleukin-2, interleukin-3,
interleukin-4, interleukin-5, interleukin-6, kidney plasminogen
activator, lectin cell adhesion molecule, luteinizing hormone,
leukemia inhibitor factor, monoclonal antibody, macrophage
activating factor, macrophage cytotoxic factor, macrophage colony
stimulating factor, megakaryocyte colony stimulating factor, tumor
necrosis factor, macrophage inhibitory factor, Mullerian inhibiting
substance, megakaryocyte stimulating factor, melanocyte stimulating
factor, neutrophil chemotactic factor, nerve growth factor, novel
plasminogen activator, nonsteroidal anti-inflammatory drug,
osteogenic factor extract, antitumor lymphokine, prostate-specific
antigen, anti-platelet activating factor, plasminogen activator
inhibitor, platelet-derived growth factor, platelet-derived wound
healing formula, plasmatic human interleukin inducing protein,
tumor angiogenesis factor, tissue control factor, T cell growth
factor, T cell modulatory peptide, transforming growth factor,
tumor growth inhibitor, tumor inhibiting factor, tissue inhibitor
of metalloproteinases, tumor necrosis factor, tissue plasminogen
activator, thyroid stimulating hormone, urokinase-plasminogen
activator, vascular endothelial growth factor, and vasoactive
intestinal peptide.
[0097] A preferred BAS is a substantially purified polypeptide or
protein. Proteins are generally defined as consisting of 100 amino
acid residues or more; polypeptides are less than 100 amino acid
residues. Unless otherwise stated, the term protein, as used
herein, refers to both proteins and polypeptides. The proteins may
be produced, for example, by isolation from natural sources or
recombinantly. Examples include insulin and other hormones,
including growth hormones, such as human growth hormone and bovine
growth hormone. Other exemplary proteins include Factor VIII,
Factor IX, Factor VIIa, and anti-inflammatory agents, such as
interleukins, including interleukin-4. Other exemplary proteins
include enzymes, such as DNase and proteases. Other proteins
include cytokines, interferons, including interferon alpha and
interferon beta, poetins, angiogenic factors, differentiation
factors, colony-stimulating factors, growth factors, ceredase,
gibberellins, auxins, and vitamins, and fragments thereof.
Exemplary growth factors include vascular endothelial growth factor
(VEGF), endothelial cell growth factor (ECGF), basic fibroblast
growth factor (bFGF), and platelet derived growth factor
(PDGF).
[0098] Proteins are stable in the hydrogels of the present
invention. For example, many of the proteins are protected from
dimerization or aggregation, as discussed below in the Examples.
The enzymatic degradation of proteins or polypeptides can be
further minimized by co-incorporating peptidase-inhibitors.
Treatment of an Animal Using Slow Release Protein Polymers
[0099] The polymer articles of the present invention may be used to
treat an animal, for example, a mouse, rat, or human, by delivering
a BAS to the animal. The articles may contain such a BAS as any of
those described above. Various routes of administration may be used
to deliver the articles of the present invention, as described
below.
[0100] The results of the treatment of an animal with therapeutic
articles containing a BAS, as described herein, will vary according
to the BAS being delivered. For example, if hGH is delivered
through the therapeutic articles of the present invention, one
would expect to observe an increase in growth as a result of such a
treatment. If erythropoietin is delivered through the therapeutic
articles, one would expect to observe an increase in reticulocytes
in the animal as a result of the treatment. If insulin is delivered
through the therapeutic articles, then the treatment should result
in a decrease in blood glucose levels.
[0101] The articles of the present invention provide optimal
delivery of a BAS, because they release the BAS in a controlled
manner with a low burst effect. The result of such a delivery rate
is that the drug is delivered steadily over a desired period of
time. A slower and steadier rate of delivery may in turn result in
a reduction in the frequency with which the BAS must be
administered to the animal. In addition, a low burst effect may be
highly desirable in some circumstances where the delivery of too
much BAS to a site is deleterious to the animal.
Routes of Administration of the Articles
Inhalation
[0102] The use of the hydrogel particles of the invention can
enhance the delivery of drugs to the lung. Administration to the
lung provides for the delivery of drugs that can be transported
across the lung tissue barriers and into circulation, as described
WO 99/03454.
[0103] A problem with the delivery of active substances to the lung
is that pulmonary macrophages can take up the materials, thus
preventing the material from entering into systemic and local
circulation. Uptake occurs when proteins adsorbed to the particles'
surfaces bind with receptors on the surfaces of the macrophages. To
prevent uptake, the invention provides nonionic hydrogels, e.g.,
formed with polymers based on polyethylene glycol. These hydrogels
adsorb low levels of proteins and thus bind poorly to cell
surfaces. Anionic hydrogels, e.g., formed with polyacrylic acid,
also adsorb relatively low levels of proteins and thus bind poorly
to cell surfaces.
[0104] In a further embodiment, biocompatible microcapsules may be
formed and the surface provided with water soluble non-ionic
polymers such as polyethylene oxide (PEO), to create resistance to
cell adhesion, as described in U.S. Pat. No. 5,380,536, hereby
incorporated by reference.
[0105] The size and density of the particles can also be selected
to maximize the quantity of BAS that is delivered to the lung. For
example, the macrophages will not take up large particles as
efficiently as they will take up small particles. However, large
particles are not delivered to the deep lung as well as small
particles are. To overcome these conflicting factors, the invention
provides small particles that can swell as they hydrate. The
particles are administered to the deep lung as small (i.e., 1-5
microns), dry, or slightly wet, particles; upon hydration, they
swell, and therefore become resistant to uptake by the pulmonary
macrophages. The swelling can occur when the particles are hydrated
from the dry state and when they are hydrated from one state of
hydration to another by a change in temperature, pH, salt
concentration, or the presence of other solvents, for example,
depending upon the chemical and physical nature of the hydrogel
polymer.
[0106] As used herein, the term "dry" means that the particles of
the powder have a moisture content such that the powder is readily
dispersible in an inhalation device to form an aerosol. Preferably,
the moisture content of the particles is below 10% by weight water,
more preferably below about 5%, or optionally below about 2%, or
lower.
[0107] The density of the particles is expressed in terms of tap
density. Tap density is a standard measure of the envelope mass
density. The envelope mass density of an isotropic particle is
defined as the mass of the particle divided by the minimum sphere
envelope volume within which it can be enclosed. The density of
particles can be measured using a GeoPyc (Micrometers Instrument
Corp., Norcross, Ga.) or a AutoTap (Quantachrome Corp., Boyton
Beach, Fla.).
[0108] For example, the density of 3.4kL5 particles was determined
as follows. 3.4kL5 (1.0025 g), 200 mM TEOA in PBS; pH 7 (1.0260 g),
and 1000 .mu.m Eosin (0.1028 g) were combined. 200 mg of this
solution was mixed with talc (0.1015 g). The resulting suspension
was placed in a 100 .mu.l glass pipet and polymerized by light for
15 seconds (ILC Technology, Inc. Xenon Light Source with Fiber
Optics). The rod was pushed out, placed on aluminum foil, and
further polymerized for 3.5 minutes. The hardened rod was
lyophilized (vacuum 15E-3 mbar, trap temp. -50.degree. C.) for 18
hours. The dry rod (water content<10%) was cut into small
pieces, placed in heptane, and minced using a homogenizer
(Silverson L4RT-A) at 5,000 rpm to small particles. The wet
particles were air-dried, followed by nitrogen gas flow. The
particles sizes ranged from 1 micron to 0.5 mm.
[0109] 1.645 g of these particles was placed in a 10 mL graduated
cylinder. The graduated cylinder was mounted on top of an Autotap
densimeter (Quantachrome). The sample was tapped 100 times and the
particles' volume was read. The process was repeated until no
change in volume was observed. The final volume was 2.8 ml. The tap
density of the particles was 1.6435 g/2.8 ml 0.5870 g/ml.
[0110] In addition to particles, the polymer may be provided in
other shapes suitable for delivery to the deep lung. For example,
PEG emulsion microspheres are subjected to high pressure and a
vacuum onto a flat plate to form very light very thin layers, for
example, having a snow flake consistency, that react differently to
fluidic wind forces. The resulting thin flakes can be, e.g., 0.01
micron, 1 micron, or 10 microns thick.
[0111] The particles can be administered to the respiratory system
alone, or in any appropriate pharmaceutically acceptable excipient,
such as a liquid, for example, saline, or a powder. Aerosol
dosages, formulations and delivery systems may be selected for a
particular therapeutic application, as described, for example, in
Gonda ("Aerosols for delivery of therapeutic and diagnostic agents
to the respiratory tract," in Critical Reviews in Therapeutic Drug
Carrier Systems, 6:273-313, 1990); and in Moren ("Aerosol dosage
forms and formulations," in: Aerosols in Medicine. Principles,
Diagnosis and Therapy, Moren, et al., Eds., Elsevier, Amsterdam,
1985).
[0112] Pulmonary drug delivery may be achieved using devices such
as liquid nebulizers, aerosol-based metered dose inhalers, and dry
powder dispersion devices. For the use of dry powder dispersion
devices, the polymer particle incorporating the therapeutic agent
is formulated as a dry powder, for example, by lyophilization or
spray-drying. Methods for preparing spray-dried,
pharmaceutical-based dry powders including a pharmaceutically
acceptable amount of a therapeutic agent and a carrier are
described in PCT WO 96/32149, hereby incorporated by reference.
[0113] Examples of a BAS that can be administered to the lung
include, without limitation, insulin, antitrypsin, calcitonin,
alpha interferon, beta interferon, GLP-1, and DNAse.
Nasal Delivery
[0114] The articles of the present invention can also be used to
administer compounds nasally. For example, a vaccine containing
freeze dried or reconstituted microspheres can be administered
nasally. Intramuscular and Subcutaneous Administration The articles
of the present invention can be used to administer microspheres
that degrade over several days to 3 months, by intramuscular
injection or by subcutaneous injection.
[0115] For example, growth hormone can be administered
subcutaneously; the hormone leaves the microspheres at the site of
injection as they degrade. Growth hormone enters the systemic
circulation, where, in turn, it exerts its effects directly, and
indirectly through induction of somatomedin production in the liver
and in other tissues. For this application, particle sizes of up to
0.5 mm can be used.
[0116] In other embodiments, the active agent is a vaccine, such as
tetanus vaccine, other proteins or polypeptides, or more complex
immunogens. The vaccine is released over time, from one week to
many weeks, resulting in an improved immune response to the
vaccine, compared to a bolus injection followed by one or more
booster shots with the same total dose of immunogen. Mixtures of
different types of microspheres can result in initial and booster
shot-type immunization as well.
Intravenous Administration
[0117] Articles that contain a BAS useful in treating clotting
disorders, such as Factor VIII or Factor IX for hemophilia, can be
administered by intravenous injection. The BAS is released over
days to weeks. A therapeutic level of the BAS is maintained that
results in a better clinical outcome. In addition, potentially
lower total doses of a BAS can be administered, with a
corresponding economic benefit. These approaches help promote
patient compliance.
[0118] In the case of intravenous injection, it is important to
formulate the microspheres in acceptable agents so the microspheres
do not aggregate and clog blood vessels. The microspheres must be
appropriately sized, so that they don't lodge in capillaries. For
this application, particle sizes of 0.2-0.5 microns are
preferred.
[0119] In a number of inflammatory conditions, as part of the
inflammatory process that is mediated by selectin and ICAM
expression binding with neutrophil intravisation, blood vessels
become leaky at the site of inflammation. Hydrogel microspheres may
be administered; these microspheres will leak out of blood vessels
at the site of inflammation, and then release their BAS payload
locally over a period of time. Disease conditions where this
approach may be useful could include, but are not limited to,
inflammatory bowel diseases, asthma, rheumatoid arthritis,
osteoarthritis, emphysema, and cystic fibrosis (with DNase as the
enzymatic drug).
[0120] Hydrogel microspheres that contain cytokines, lymphokines,
or other compounds to treat cancer can be administered by
intravenous injection. Blood vessels within large solid tumors are
generally leaky, and the blood flow within them is often slow.
Thus, microspheres could lodge within solid tumors and release
their anticancer BAS locally, either killing tumor cells directly
or by activating the immune system locally. This approach could be
used, for example, with compounds such as interleukin 2, where the
systemic and local toxicity has been dose limiting and where the
resulting side effects are significant.
[0121] The microspheres of the present invention may be cleared
relatively slowly from the circulation. Alternatively, the
microspheres can be targeted to exit the circulatory system through
leaky blood vessels or through more active targeting mechanisms,
e.g., receptor mediated targeting mechanisms.
Oral Administration
[0122] In some portions of the gastrointestinal tract, there is
relatively good transport of proteins across the intestinal mucosa
into the systemic and local circulation. The compositions of the
invention, for example, freeze dried microspheres containing
protein (with very small particle sizes), can therefore be
administered orally in an appropriate enteric formulation that
protects the drug-containing microspheres from enzymatic attack and
the low pH found in the upper GI tract. Such an enteric formulation
could also be designed using several available technologies to
gradually expel BAS-containing microspheres as the enteric capsule
traverses the gastrointestinal tract. This is described in more
detail in WO 99/63454 and in Mathiowitz et al. (Nature 386: 410-414
(1997)). It is anticipated that this approach will have a number of
advantages over other approaches for delivering proteins and other
molecules, even small molecules, orally. First, PEG and proteins
are compatible, so the major manufacturing and stability problems
found with other drug delivery approaches can be avoided. Secondly,
dried hydrogels are very adhesive to wet tissue. The microparticles
will bind well to the GI tract and will be transported into the
system via the gastrointestinal circulation or release their
contents on the intestinal mucosa; in turn, the drug will enter the
systemic and gastrointestinal circulation. Chemical enhancers, or
formulations containing compositions that utilize specific and
non-specific biological transport mechanisms to facilitate
transport across the GI tract into the systemic circulation, can be
included as well.
Targeting
[0123] Targeting ligands can be attached to the particles via
reactive functional groups on the particles. Targeting ligands
permit binding interactions of the particle with specific receptor
sites, such as those within the lungs or those on endothelial cells
specific to different regions in the body's microvasculature. A
targeting ligand is selected which specifically or non-specifically
binds to particular targets. Exemplary targeting ligands include
antibodies and fragments thereof including antibody variable
regions, lectins, hormones, or other organic molecules capable of
specific binding to receptors on the surfaces of the target cells.
Other ligands are described in Science (279:323-324 (1998)), hereby
incorporated by reference.
[0124] Microspheres can be made with both a BAS and a targeting
molecule. Double microspheres can also be made, in which the inner
sphere contains drug and the outer PEG shell contains the targeting
molecule or reagent.
Excipients and Carriers
[0125] The particles incorporating a therapeutic agent or
diagnostic agent may be provided in combination with one or more
pharmaceutically acceptable excipients available in the art, as
described, for example, in PCT WO 95/31479, hereby incorporated by
reference. Excipients may be selected that can, in some
applications, enhance stability, dispersability, consistency, and
bulking to ensure uniform pulmonary delivery. The excipient may be,
e.g., human serum albumin (HSA), bulking agents such as
carbohydrates, amino acids, polypeptides, pH adjusters or buffers,
and salts. Additional excipients include zinc, ascorbic acid,
mannitol, sucrose, trehalose, cyclodextrans, polyethylene glycol,
and other commonly used pharmaceutical excipients, including those
described in The United States Pharmacopeia, published by the
United States Pharmacopeia Convention, Inc., 1995 (see, e.g., pp.
2205-2207). Exemplary carbohydrates include monosaccharides, such
as galactose, and disaccharides such as lactose. Excipients that
stabilize proteins are especially useful.
[0126] In some cases, the excipients are used as carriers; i.e.,
they are used to modulate the release rate of the active
substances. For example, mannitol can be used to accelerate or
delay release.
[0127] There now follow particular examples that describe the
preparation of compositions of the invention, and the methods of
the invention. These examples are provided for the purpose of
illustrating the invention, and should not be construed as
limiting.
[0128] In some of the following Examples a macromer made of a triad
ABA block copolymer of acrylate-PLA-PEG-PLA-acrylate was used. The
PEG had a MW of 3,400 daltons; the poly(lactic acids) on both sides
had an average of about five lactate units per side; they are
therefore referred to herein as "3.4kL5." When a lower molecular
weight PEG, such as 2,000 daltons was used, the resulting macromer
is abbreviated as "2kL5."
[0129] In other Examples an acrylate-PCL-PEG-PCL-acrylate macromer
was used. The PEG had a MW of 3,400 daltons and had
polycaprolactone on both sides, with an average of about 6 caproyl
units per side. The polymer is referred to herein as "3.4kC6."
[0130] In yet other Examples a 3-arm macromer was used. This
macromer consisted of a PEG core with 3 arms, 3 lactate groups
attached to each arm of the PEG. The PEG had a MW of 4,200 daltons
and the polymer is referred to herein as "4.2kL3-A3."
EXAMPLE 1
General Preparation of a Macromer Solution
[0131] The protein was weighed out, and the following components
were added to the protein: (i) 90 mM TEOA/PBS, pH 8.0; (ii) 35%
n-vinyl pyrrolidinone (n-VP); and (iii) 1000 ppm Eosin. The
resulting mixture was stirred well using a spatula. The solution
was kept in the dark for about 10 minutes, or until the macromer
had absorbed all of the solution, or until the solution was
homogenous.
[0132] Macromer solutions having the following ingredients were
prepared. TABLE-US-00001 Amount Amount Amount 1000 Amount 90 mM 35%
ppm Amount Amount Total Protein TEOA n-VP Eosin 3.4 kL5 2 kL5
amount 15 mg 57 mg 15 mg 3 mg 45 mg 0 mg 135 mg 15 mg 57 mg 15 mg 3
mg 0 mg 45 mg 135 mg
EXAMPLE 2
Precipitation of hGH and Formulating into Hydrogel Microspheres
[0133] To a 100 mg/ml hGH solution in 5 mM ammonium hydrogen
carbonate buffer 77 .mu.l of a 1300 mM triethanolamine, pH 8,
solution was added. Upon the addition of 400 mg of PEG 2K to the
above solution, a fine precipitate of hGH was formed. The sample
was centrifuged at 4000 rpm for several minutes and 0.9 ml of the
supernatant was removed. To the precipitated mixture, 1 g of
4.2kL5-A3 macromer was added, followed by the addition of 0.1 ml of
a 10 mM Eosin Y solution. The mixture was then emulsified in an oil
phase to form microspheres which were polymerized using an argon
laser. The in vitro release characteristics of this formulation are
shown in FIG. 1. No burst was observed and release continued for at
least 5 days.
EXAMPLE 3
[0134] Micronization of Freeze-Dried Human Growth Hormone and
Formulation Into Hydrogel Microspheres
[0135] A 10 mg/ml solution of hGH ammonium acetate was frozen and
freeze-dried, resulting in a dry cake of pure hGH. The following
macromer solution was prepared: 1 g 2kL5, 1.2 g phosphate buffered
saline (pH 7), 0.4 g of a solution of 25% trehalose and 0.4% F-68
in water, 0.24 g of a 10% solution of 2,2-dimethoxy 2
phenyl-acetophenone in tetrahydrofuran. To this solution was added
0.2 g of the freeze-dried hGH. Following mixing to disperse the
hGH, the hGH in suspension was further micronized by 20 passages
through a 1.5 inch 20 g needle. This suspension was then emulsified
in an oil phase to form microspheres which were polymerized by
exposure to UV light (365 nm) for 3 min. The in vitro release
characteristics of this micropheres are shown in FIG. 2.
EXAMPLE 4
Formation of Articles
[0136] The microspheres, in the form of a hydrogel, were placed
onto a silanized glass slide. Using pieces of plastic sheets with
thicknesses of about 0.4 .+-.0.2 mm as spacers, another silanized
glass slide was placed on top and held firmly in place using binder
clips.
[0137] A light source (ILC Technology, Inc. Xenon Light Source with
Fiber Optics) was adjusted to about a 5-cm distance. The center of
the disk was illuminated; both sides of the disk were illuminated
for two minutes each, to form an opaque disk.
EXAMPLE 5
Preparation of Articles Containing 4.2kL3-A3 Macromers and bSA
[0138] To 1 ml of 50% ethanol/water solvent, 1 g of 4.2kL3-A3
macromer was added with 7.8 mg of
2,2-dimethoxy-2-phenyl-acetophenone (DMPA). On complete dissolution
of the macromer and DMPA, 250 mg of spray dried bSA was added and
stirred until the mixture was uniform. The entire mixture was then
emulsified in 200 g of a 0.5% lecithin in heavy white mineral oil
solution stirred at 600 rpm. Shortly afterwards, the dispersed
droplets were photo-polymerized using a Black-Ray B 100AP UV lamp
for a period of 15 minutes. The in vitro release profile of this
formulation, shown in FIG. 3. indicates no burst.
[0139] A degradable macromer (4.2kL3-A3) was combined with bSA. The
protein was loaded at a loading of 20%, based on dry weight. An
emulsion was formed using white heavy mineral oil. Polymerization
of the macromer into a hydrogel then occurred through spray drying
and UV polymerization techniques.
EXAMPLE 6
Analyses of Biological Active Substance Release from a Macromer
[0140] After formation of the articles, as described, for example,
in Example 4, the disks were removed and weighed on a clean, tared
silanized glass slide. The disk was placed into a heat-sealed
membrane bag, as described in more detail below. One 20 .mu.l disk
was placed in each bag. The bag was heat-sealed, placed in 2.0 ml
of phosphate buffer release media (0.01% NaN.sub.3, 0.05 M PBS; pH
7.4), placed on an orbital shaker turning at 100 rpm, and incubated
at 39.degree. C.
[0141] For each time point, the bag was placed into fresh 2.0 ml of
PBS Release Media. Samples were collected for analysis every day
for as long as the BAS was being released.
[0142] Membrane bags were prepared as follows. Membrane sheets were
cut into pieces of approximately 7.times.2.5 cm. The sheets were
folded in half. Using a Bunsen burner or a propane torch, a spatula
was heated until it became red. The edges of the sheets were
aligned, and the side of the membrane was cut with the red-hot
tweezer to seal the sides. Once the disk was placed into the bag,
the last side was sealed using the same heat-sealing technique.
[0143] The samples were analyzed daily by SEC-HPLC. Monomers,
dimers, and soluble aggregates could be detected using this method.
The mobile phase used was 0.08 M TFA in 60/40% CH.sub.3CN/H.sub.2O,
adjusted to pH 2.0, isocratic, with a flow rate of 1.5 ml/min. The
signals were detection at a wavelength of 220 nm. The column used
was a Bio-Rad Bio-Sil.RTM. SEC 250, 5 micron particle size,
300.times.7.8 mm ID, equipped with a guard column (Bio-Rad
Bio-Sil.RTM. SEC 250 Guard, 5 micron particle size, 80.times.7.8 mm
ID). The injection volume was 10 .mu.l. The standard calibration
curves were 0, 0.1, 0.25, 0.5, 0.75, and 1 mg/ml bST in the mobile
phase.
EXAMPLE 7
[0144] Production of Microspheres with Efficient Protein Loading
and Low Burst Effects
[0145] FIG. 3 shows an example of the high protein loading and low
burst characteristics of the therapeutic articles of the present
invention. The articles contain 4.2kL3-A3 macromers combined with
bSA (in either a monomer or dimer form) and formed into
microspheres using spray drying techniques. The bSA was loaded at a
calculated loading of 20%. Release of bSA from the microspheres was
assayed as described above. The release occurred at a slow steady
rate, and no burst effect was exhibited. After a period of 9 days,
less than 30% of the bSA was released from macromers containing
bSA. These results demonstrate that the therapeutic articles of the
present invention can provide slow release of a BAS, with little or
no burst effect.
EXAMPLE 8
Effect of the Particle Size of a BAS on Release of bSA from 30%
3.4kL5
[0146] The effect of the particle size of a BAS on its release from
an article, for example, a microsphere was also determined. The bSA
used to form the microspheres was either ground under liquid
nitrogen into fine particles of less than approximately 75 microns,
or were left unground. Microspheres containing 30% 3.4kL5 and
either the fine-ground or unground bSA were formed using the
methods described above, and were assayed for the release of bSA,
loaded at 25%, based on dry weight. FIG. 4 illustrates the results
of these studies. Compared to the microspheres containing unground
bSA, the microspheres containing fine-ground bSA released its bSA
over a longer period of time. In addition, the microspheres
containing the fine-ground bSA exhibited steady rate of release
(releasing less than 20% of the total bSA loaded within the first
24 hours), with no burst effect, while the microspheres containing
the unground bSA exhibited a burst effect (releasing approximately
50% of the total bSA loaded within 24 hours). These results
demonstrate that microspheres containing a BAS which has a small
particle size provide slower release profiles and low burst
effects.
EXAMPLE 9
Effects of BAS Particle Size and Protein Loading on the Release of
Fine-ground bSA (10% Loaded) and Various Crystalline Particles
(1-10% Loaded) from 3.4 kL5
[0147] The effects of the particle size of a BAS and protein
loading on the release of bSA from an article, was also examined
(FIG. 5). The bSA used to form the microspheres was either ground
under liquid nitrogen into fine particles of less than
approximately 75 microns, or were left unground. The fine-ground
bSA was combined with 3.4kL5 to form 30% 3.4kL5 microspheres loaded
with 10% bSA, using the methods described above. The unground bSA,
containing various particle sizes was combined with 3.4kL5 to form
30% 3.4kL5 microspheres loaded with 1-10% bSA. The microspheres
were then assayed for the release of bSA. FIG. 5 illustrates the
results of these studies. Compared to the microspheres containing
unground bSA, the microspheres containing fine-ground bSA, loaded
at 10%, released its bSA over a longer period of time. In addition,
the microspheres containing the fine-ground bSA exhibited a lower
burst effect (releasing approximately 20% of the total bSA loaded
within the first 24 hours) than its unground counterpart (releasing
almost 50% of the total bSA loaded within the first 24 hours).
These results demonstrate that microspheres containing a BAS which
has a small particle size and is highly loaded provide a desirable
BAS release profile compared to microspheres containing various
particle sizes and a lower load of BAS.
EXAMPLE 10
Effect of Protein Particle Size on Release of hGH from 30%
3.4kL5
[0148] The effect of the particle size of a BAS on its release from
microspheres was further examined using microspheres containing
either fine-ground or unground hGH. The hGH used to form the
microspheres was either ground under liquid nitrogen into fine
particles of less than approximately 75 microns, or were left
unground. Microspheres containing 30% 3.4kL5 and either the
fine-ground or unground hGH were formed using the methods described
above, and were assayed for the release of hGH, loaded at 25%,
based on dry weight, and 10% as manufacturing conditions. FIG. 6
illustrates the results of these studies. Compared to the
microspheres containing unground hGH, the microspheres containing
fine-ground hGH released its hGH over a longer period of time. In
addition, the microspheres containing the fine-ground hGH exhibited
a steady rate of release (releasing less than 40% of the total hGH
loaded within the first 24 hours), with no burst effect, while the
microspheres containing the unground hGH exhibited a high burst
effect (releasing approximately 70% of the total hGH loaded within
24 hours). These results demonstrate that microspheres containing a
BAS which has a small particle size provide the characteristics
which are highly suitable for delivery of agents for therapeutic
use: slow protein release and low burst effects.
EXAMPLE 1
Effect of Microsphere Pore Size on Release of hGH from
Macromers
[0149] The effect of the pore size of the microspheres containing
fine-ground hGH on the release rate of the hGH was also examined
(FIG. 7). Microspheres containing hGH, loaded at 25%, based on dry
weight, and 10% as manufacturing conditions, and either 30% 2kL5 or
30% 3.4kL5 were formed using the techniques described above. The
microspheres containing 2kL5 had a smaller pore size than the
microspheres containing 3.4kL5. The release rate of hGH from these
microspheres was then assessed using techniques described above.
These studies showed that fine-ground hGH was release from
2kL5-containing microspheres at a slower rate than it was released
from 3.4kL5-containing microspheres. These results suggest that
macromers which result in a smaller microsphere pore size release a
BAS at a slower rate than those which result in a larger
microsphere pore size.
EXAMPLE 12
Controlled Release of Bovine Somatotropin in Hypophysectomized
Rats
[0150] The controlled delivery of active bovine somatotropin (MW 20
Kd) was confirmed in the hypophysectomized rat model.
Hypophysectomized female rats were purchased from Taconic Labs
(Germantown, N.Y.). The rats were weighed each morning. Prior to
the initiation of the study, the rats were held 7 days to confirm a
lack of significant growth. On day 1 of the study the rats were
weighed. The rats were then divided into 3 groups of equal mean
weights. Group 1 remained untreated and served as a-negative
control. Group 2 received an implant of bST in a hydrogel made of a
blend of 3:1 of 3.4KL5 and poly(ethylene glycol) diacrylate (each
device contained-0.9 to 1.1 mg of bST). The rats in Group 3 were
injected with 100 .mu.g bST subcutaneously each day for the
duration of the study.
[0151] At the end of the 12 day treatment period, the rats were
analyzed for their growth over the period of treatment. The rats of
Groups 1 did not grow significantly, while the rats of Groups 2 and
3 grew at rates faster than Group 1 and approximately equal to one
another.
EXAMPLE 13
Controlled Release of Erythropoietin in Rats
[0152] The controlled delivery of active human erythropoietin (EPO)
was confirmed in male Sprague-Dawley rats purchased from Taconic
Labs (Germantown, N.Y.). Hydrogel devices were manufactured to
contain 3000 Units of EPO per device. One of these devices was
implanted in each of a number of rats (Group 1). Another group of
an equal number of rats (Group 2) received a subcutaneous injection
of EPO (1000 Units) daily for 3 days. A third group of rats (Group
3) received no treatment.
[0153] On day 5 after implantation of the device and the start of
the subcutaneous injections, venous blood samples were obtained
from each rat and stored in EDTA. The fraction of reticulocytes
(immature red blood cells) was determined after staining with
Acridine Orange by automated flow cytometry. The rats in Group 1
had 18% reticulocytes, the rats of Group 2 had 15% reticulocytes,
and the rats in Group 3 had 4% reticulocytes. !
EXAMPLE 14
Controlled Release of Insulin in Diabetic Rats
[0154] Sprague-Dawley rats were purchased from Taconic Labs
(Germantown, N.Y.). Diabetes was induced by treatment with
streptozotocin (65 mg/kg, i.v.) and confirmed 48 hours later by
elevation of blood glucose (>300 mg/dl). Following
anesthetization of the rat with pentobarbital (35 mg/kg), a
catheter was placed in a jugular vein. After a baseline blood
sample was taken for the determination of blood glucose
concentration, a hydrogel device containing 1 Unit of insulin was
implanted subcutaneously. Blood samples were taken at 15, 30, 60,
120, and 180 minutes after implantation of the device and used to
determine blood glucose levels.
[0155] The blood glucose level of the rat implanted with the
hydrogel device decreased, demonstrating that the devices was
capable of releasing insulin in its active form.
[0156] To test the pulmonary delivery system for insulin-containing
hydrogel particles, the neck of the rat was opened with a midline
incision and the trachea was exposed by blunt dissection. A slit
was cut into the trachea, and a small polyethylene tube was
advanced distally into the lung. A small volume of
insulin-containing hydrogel microparticles (total dose of 3 Units
of insulin) was instilled into the lung and the tube was removed.
Blood samples were taken and analyzed as described above for the
subcutaneous device.
[0157] The blood glucose levels dropped significantly within 30
minutes and remained low (below 150 mg/dl) for at least 180
minutes.
EXAMPLE 15
Controlled Release of Human Growth Hormone in Hypophysectomized
Rats
[0158] The controlled delivery of active human growth hormone (hGH,
MW 20 Kd) is confirmed in the hypophysectomized rat model.
Hypophysectomized female rats purchased from Taconic Labs
(Germantown, N.Y.) are weighed each morning. Prior to the
initiation of the study the rats are held 7 days to confirm lack of
growth. The rats are divided into 3 groups of equal mean weights.
Group 1 remains untreated and serves as a negative control. Group 2
receives an implant of hGH in a hydrogel made of a 3:1 blend of
3.4kL5 and 3.4kC6 (each device containing approximately 1 mg of
hGH). The rats in Group 3 are injected with 100 .mu.g hGH
subcutaneously each day for the duration of the study.
[0159] It is expected that the untreated control group will not
grow during the study, and that the rats of Groups 2 and 3,
receiving the hGH hydrogel implant and 100 .mu.g hGH injections
daily during the study, respectively, will exhibit continued
growth.
EXAMPLE 16
Pulmonary Devices Containing Human Growth Hormone (hGH)
[0160] To a 20 ml vial are added: 0.2559 g of 200 mM of TEOA (in
PBS buffer; pH 7.0), 0.2548 g of 3.4KL5, 0.0206 g of 1000 PPM eosin
(in PBS; pH 7.0), and 0.0615 g hGH (Genentech's hGH injectable
formulation, purified by a Millipore Centricon.TM.). The resulting
mixture is stirred and placed into 10 ml glass tubes. The tubes are
exposed to xenon light (ILC Technology, Inc. Xenon Light Source
with Fiber Optics) for 10 seconds. The semi-cured hydrogel is
pushed out of the glass tube and further polymerized for 3.5
minutes. The cured hydrogel rods are put into 15 ml of heptane and
are ground using a homogenizer (Silverson L4RT-A) for 30 seconds at
5000 rpm, followed by 30 seconds at 3000 rpm. The heptane is
decanted, and the powder is dried under nitrogen. The powder is
used for pulmonary, oral, or subcutaneous sustained delivery of
hGH.
EXAMPLE 17
Oral Formulation for Release of Proteins
[0161] Using the techniques described above, insulin, human growth
hormone, human alpha interferon, or erythropoietin is incorporated
into macromer particles. Using cryomilling or other milling
procedures known in the art, very small microparticles are
produced, preferably of an average size of less than about 500
nanometers. Such nanoparticles are then, introduced into the rat GI
tract surgically, using catheter infusion into the upper GI tract.
The dosing of such nanoparticles is based upon the assumption that
about 0.5% of the drug in the nanoparticles will be detectable in
the blood of such rats, e.g., by RIA, with the specific
pharmacology of each drug taken into account.
[0162] In the case of insulin, blood samples are taken at time
t=-15, 0, 30, 60, 90, 120, and 180 minutes, and monitored for
insulin by RIA and for blood glucose by glucometer (when insulin is
being administered, diabetic rats are utilized).
[0163] For other drugs, normal rats are used and blood drug levels
are measured at these same time points using RIA or ELISA
techniques.
[0164] In addition to the above procedures, the above
drug-containing microspheres can be modified to enhance their
absorption in the small intestine, colon, and other appropriate
areas of the GI tract. Such modifications can include precipitating
lipid bilayers around the microcapsules so they appear as fat-like
particles from digested food, linking molecules such as ferritin to
the particles, or putting a charged layer on the outside of the
microparticles.
EXAMPLE 18
Evaluation by Reverse Phase HPLC
[0165] Microspheres were prepared by first adding 0.154 ml of 3M
triethanolamine solution at pH 8.0 (TEOA) to approximately 2 ml of
100 mg/ml solution of hGH in ammonium bicarbonate and then mixing
well. Next, about 800 mg of solid PEG 2 k was added and mixed with
a spatula, resulting in a very small amount of precipitated hGH in
the solution. Samples were then centrifuged at 4000 rpm for thirty
minutes and about 1.8 g of supernatant was removed. About 1 g of
macromer. (4.4 k PEG tris(lactate).sub.3 triacrylate) was added to
each centrifuge tube and stirred. Next, about 0.1 to 0.15 mL of
TEOA was added, followed by 0.05 mL of 40 mM Eosin Y. The samples
were then centrifuged for three minutes at 4000 rpm. Samples were
then polymerized by forming a disc upon exposure to light as
described in Example 4 or by first making a microemulsion by mixing
with oil (PPG 2 k) and then exposing to light as described in
Example 4.
[0166] The resulting microspheres were analyzed by Reverse Phase
HPLC (RP-HPLC). Samples were prepared for RP-HPLC by first
extracting hGH from the microspheres with NaOH. Briefly, 10 mg of
microspheres was added to 1 mL of NaOH(1N)/Tris (50 mM, pH 7.5)
(1:9, v/v) solution and incubated at ambient temperature for 5
minutes. 5N HCl was used to titrate the solution to a final pH of
7.5. The sample was then microcentrifuged for 2 minutes and
filtered through a 0.45 micron filter. 100 .mu.L of the hGH
solution was injected onto a Vydac C4 column (214TP54) equilibrated
and run under isocratic conditions at 0.5 mL/min. employing
n-propanol/Tris (50 mM, pH 7.5)(29:71, v/v) as a solvent.
Separation was performed at column temperature of 45.degree. C.
over 50 minutes with UV detection at 220 nm. hGH eluted at a
retention time of 33.+-.3 minutes. Results are shown in Table I.
The term "% RP" refers to the percentage of protein (hGH) that is
not 1.0 found in the monomer peak, which may include forms of hGH
that are normally found in commercially marketed hGH preparations
and that are active and safe, such as oxidized and deamidated
forms. TABLE-US-00002 TABLE I Sample No. % RP 18-86-1 41 318-1 48
318-2 43
[0167] Other batches of microspheres were prepared by first adding
0.154 ml of 3M Tris buffer at pH 6.0 to about 2 ml of 100 mg/ml hGH
solution in ammonium bicarbonate and mixing well. To this solution,
about 800 mg of PEG 10 k was added as solid to obtain precipitated
protein. Samples were then centrifuged at 4000 rpm for thirty
minutes and about 1.8 g of supernatant was removed. About 1 g of
macromer (4.4 k PEG tris(lactate).sub.3 triacrylate) was added to
each centrifuge tube and stirred. Next, about 0.1 to 0.15 mL of
TEOA was added, followed by 0.05 mL of 40 mM Eosin Y. The samples
were then centrifuged for three minutes at 4000 rpm. Samples were
then polymerized by forming a disc upon exposure to light as
described in Example 4 or by first making a microemulsion by mixing
with oil (PPG 2 k) and then exposing to light as described in
Example 4. One sample, Sample No. 27-50, was prepared using PEG 20
k.
[0168] The resulting microspheres were analyzed by Reverse Phase
HPLC. Samples were prepared for RP-HPLC either by the NaOH
extraction method described above or by the following cryogrinding
method. Results are shown in Table II, with the sample preparation
method indicated. Approximately 10 mg of microsphere sample was
weighed in a microcentrifuge tube. A pestle was placed in the tube
and then the tube was immersed in a liquid nitrogen bath for
approximately one minute. With the tube still in the liquid
nitrogen bath, the sample was ground for approximately five
minutes. The tube was then removed and allowed to stand for
approximately two minutes at ambient temperature. The ground sample
was then suspended in about 1 mL of 25 mM potassium phosphate
buffer, pH 6.5. The pestle was removed, and the sample incubated
for about ten minutes at ambient temperature. The sample was then
centrifuged at 15000 rpm for about five minutes to obtain a clear
aqueous phase. The supernatant was the filtered through a 0.45
micron filter. RP-HPLC was performed by injecting 100 .mu.L of
sample onto a Vydac C-18 column (218TP54) equilibrated and run
under isocratic conditions at 1 mL/min, using n-propanol/potassium
phosphate (25 mM, pH 6.5) (27:73, v/v) as a solvent. Separation was
performed at a column temperature of 55.degree. C. over 30 minutes
with UV detection at 220 nm. TABLE-US-00003 TABLE II Sample No. %
RP 27-32 26 (NaOH) 27-50 (Tris, PEG 20k) 19 (NaOH) 320-4 20 (NaOH)
502 15.2 (NaOH) 507 17 (NaOH) 508 15 (NaOH) 530 16 (NaOH) 548 10.4
(Cryogrind) 556 7.7 (Cryogrind) 557 8.7 (Cryogrind) 561 8.5
(Cryogrind) 570 6.2 (Cryogrind) 575 7.2 (Cryogrind)
[0169] By comparison, microspheres made without a molecule that
preferentially excludes protiens ("Control" in Table III), yield %
RP values of 53% using the NaOH extraction method, and 23% using
the cryogrind method. Also presented in Table III is a camparison
of the two sample preparation methods. TABLE-US-00004 TABLE III
Sample No. % RP NaOH Method % RP Cryogrind Method Control 53 23 361
30.2 15 362 28 15
Other Embodiments
[0170] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0171] All publications and patents mentioned in this specification
are herein incorporated by reference to the same extent as if each
individual publication or patent was specifically and individually
indicated to be incorporated by reference.
* * * * *