U.S. patent application number 10/715867 was filed with the patent office on 2004-10-07 for biodegradable macromers for the controlled release of biologically active substances.
Invention is credited to Hubbell, Jeffrey A., Kieras, Mark T., Ron, Eyal S., Rowe, Stephen C..
Application Number | 20040197369 10/715867 |
Document ID | / |
Family ID | 21981471 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197369 |
Kind Code |
A1 |
Hubbell, Jeffrey A. ; et
al. |
October 7, 2004 |
Biodegradable macromers for the controlled release of biologically
active substances
Abstract
A method for delivering a biologically active substance
including the steps of: (a) combining said biologically active
substance with a macromer; (b) forming a mixture of the combination
formed in step (a); (c) polymerizing said mixture to form articles;
and (d) administering said articles, or a portion thereof, to a
mammal, where step (c) takes place in the absence of a
polymerizable monovinyl monomer, is disclosed.
Inventors: |
Hubbell, Jeffrey A.;
(Zumikon, CH) ; Kieras, Mark T.; (Newburyport,
MA) ; Ron, Eyal S.; (Lexington, MA) ; Rowe,
Stephen C.; (Wellesley, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
21981471 |
Appl. No.: |
10/715867 |
Filed: |
November 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10715867 |
Nov 17, 2003 |
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09689575 |
Oct 12, 2000 |
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6703037 |
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09689575 |
Oct 12, 2000 |
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09118242 |
Jul 17, 1998 |
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6153211 |
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60053029 |
Jul 18, 1997 |
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Current U.S.
Class: |
424/423 ;
424/486 |
Current CPC
Class: |
B01J 13/02 20130101;
A61K 9/1641 20130101; A61K 9/1647 20130101; A61K 9/1635
20130101 |
Class at
Publication: |
424/423 ;
424/486 |
International
Class: |
A61F 002/00; A61K
009/14 |
Claims
What is claimed is:
1-58. (Canceled)
59. A composition comprising a protein within a biodegradable
polymerized macromer, said macromer comprising at least one water
soluble region, at least one degradable region which is
hydrolyzable under in vivoconditions, and polymerized end groups,
wherein said polymerized end groups are separated by at least one
degradable region and wherein said composition contains at least 5%
by weight of said protein.
60. The composition of claim 59, wherein said water soluble region
comprises poly(ethylene glycol).
61. The composition of claim 59, wherein said said degradable
region comprises a polyester.
62. The composition of claim 59, wherein said protein is bovine
somatotropin, human growth hormone, granulocyte macrophage colony
stimulating factor, insulin-like growth factor-I, insulin-like
growth factor-II, an interferon, an antibody, or an
immunoglobulin.
63. The composition of claim 62, wherein said antibody is selected
from anti-HER, anti-VEGF, and anti-Tac antibodies.
64. The composition of claim 62, wherein said immunoglobulin is
selected from immunoglobulin A, immunoglobulin D, immunoglobulin E,
immunoglobulin G, and immunoglobulin M.
65. The composition of claim 62, wherein said interferon is
selected from interferon alpha and interferon beta.
66. A composition comprising a peptide within a biodegradable
polymerized macromer, said macromer comprising at least one water
soluble region, at least one degradable region which is
hydrolyzable under in vivoconditions, and polymerized end groups,
wherein said polymerized end groups are separated by at least one
degradable region and wherein said composition contains at least 5%
by weight of said peptide.
67. The composition of claim 66, wherein said water soluble region
comprises poly(ethylene glycol).
68. The composition of claim 66, wherein said degradable region
comprises a polyester.
69. The composition of claim 66, wherein said peptide is LHRH,
glucagon like peptide-1, insulin, calcitonin, somatostatin, or
GLP-1 amylin.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. S No. 60/053,029,
filed Jul. 18, 1997, entitled "Biodegradable Hydrogels for Drug
Delivery," having as inventor Stephen C. Rowe.
BACKGROUND OF THE INVENTION
[0002] The invention relates to methods for administering
biologically active substances, and biodegradable compositions for
administering these substances.
[0003] The rapid advances in the fields of genetic engineering and
biotechnology have led to the development of an increasing number
of proteins and peptides that are useful as pharmaceutical agents.
The development of methods for administering these new
pharmaceutical agents is thus gaining increasing importance. In
particular, the local or systemic administration of biologically
active substances, such as proteins, is a current concern.
[0004] The delivery of proteins can be complicated, as proteins
will degrade in many of the carriers that have traditionally been
used for the administration of small molecules. In many cases, the
active forms of proteins are difficult to formulate in
biodegradable polymers. Synthetic materials, such as biodegradable
hydrogels, can be used to deliver proteins. In many methods,
however, the delivery of the protein to the systemic and local
circulation is relatively rapid, and is determined primarily by the
rate of dissolution of the protein particles. These methods can be
of limited utility, as drug release can occur in an initial "burst"
rather than at a sustained, controlled rate.
SUMMARY OF THE INVENTION
[0005] In a first aspect, the invention features a method for
delivering a biologically active substance including the steps of:
(a) combining the active substance with a macromer; (b) forming a
mixture of the combination formed in step (a); (c) polymerizing the
mixture to form articles; and (d) administering the articles, or a
portion thereof, to a mammal, where step (c) takes place in the
absence of a polymerizable monovinyl monomer.
[0006] In a second aspect, the invention features a method for
delivering a biologically active substance including the steps of:
(a) combining the active substance with a macromer; (b) forming a
mixture of the combination formed in step (a); (c) polymerizing the
mixture to form articles; and (d) administering the articles, or a
portion thereof, to a mammal, where step (c) takes place in the
absence of a water soluble polymerizable monovinyl monomer.
[0007] In a third aspect, the invention features a method for
delivering a biologically active substance including the steps of:
(a) combining the active substance with a macromer; (b) forming a
mixture of the combination formed in step (a); (c) polymerizing the
mixture to form articles; and (d) administering the articles, or a
portion thereof, to a mammal, where step (c) takes place in the
absence of a vinyl pyrrolidone monomer. The invention also features
compositions formed by these methods.
[0008] In a fourth aspect, the invention features a method for
delivering a biologically active substance including the steps of:
(a) combining the active substance with a macromer; (b) forming a
mixture of the combination formed in step (a); (c) polymerizing the
mixture to form articles; and (d) administering the articles, or a
portion thereof, to a mammal, where the articles release at least
80% of the active substance at a time 2.5 times greater than
t.sub.50.
[0009] In a fifth aspect, the invention features a method for
delivering a biologically active substance including the steps of:
(a) combining the active substance with a macromer; (b) forming a
mixture of the combination formed in step (a); (c) polymerizing the
mixture to form articles; and (d) administering the articles, or a
portion thereof, to a mammal, where the articles release a
therapeutic dose of the active substance for a period of time at
least 2.5 times greater than t.sub.50.
[0010] In a sixth aspect, the invention features a composition for
delivering a biologically active substance, the composition
including particles including a hydrogel and a biologically active
substance, where the release kinetics of the particles are
independent of particle size, where the particles have a mass mean
diameter of about 50 nm to about 1 mm.
[0011] In a seventh aspect, the invention features a method for
making articles for the controlled release of a biologically active
substance including the steps of: (a) combining the active
substance with a biodegradable, polymerizable macromer, the
macromer including at least one water soluble region, at least one
degradable region which is hydrolyzable under in vivo conditions,
and polymerizable end groups having the capacity to form additional
covalent bonds resulting in macromer polymerization, where the
polymerizable end groups are separated by at least one degradable
region, in the presence of an initiator; (b) polymerizing the
macromer in the absence of light to form a hydrogel and to
incorporate the active substance into the hydrogel; and (c) forming
the hydrogel into articles capable of controlled release of the
active substance. The initiator may be a radical initiator or an
ionic initiator.
[0012] In an eighth aspect, the invention features a method for
making a polymerized hydrogel, the method including the steps of:
(a) combining a hydrophobic, water insoluble macromer, an
initiator, and water; (b) allowing the macromer to swell; (c)
mixing the macromer to form a homogenous mixture; and (d)
polymerizing the macromer to form a hydrogel. Preferably, the
method further includes adding a biologically active substance to
the mixture before step (d).
[0013] In a ninth aspect, the invention features a method for
making a polymerized hydrogel including the steps of: (a) combining
a hydrophilic macromer and a hydrophobic, water insoluble macromer;
(b) heating and stirring the combination formed in step (a) to form
a homogenous mixture; (c) cooling the mixture to room temperature
(d) adding water and an initiator to the mixture and allowing the
mixture to swell; and (e) polymerizing the macromer to form a
hydrogel. Preferably, the method further includes adding a
biologically active substance to the mixture before step (e).
[0014] In a tenth aspect, the invention features a method for
delivering a protein including the steps of: (a) combining the
protein with a polymerizable hydrophilic polymer; (b) forming a
mixture of the combination formed in step (a); (c) polymerizing the
mixture to form articles; and (d) administering the articles, or a
portion thereof, to a mammal, where the protein remains intact, and
where at least 70% of the protein is released from the
articles.
[0015] In an eleventh aspect, the invention features a method for
delivering a biologically active substance, the method including
the steps of: (a) combining the active substance with a
biodegradable, polymerizable macromer in an aqueous solution, in
the presence of a free radical initiator; (b) dispersing the
solution to form fine droplets including the macromer and the
biologically active substance; (c) polymerizing the macromer in the
droplets, thereby forming hydrogel particles having the
biologically active substance incorporated therein, where the
particles are capable of controlled release of the biologically
active agent; and (d) administering the articles, or a portion
thereof, to a mammal, where step (c) takes place in the absence of
a vinyl pyrrolidone monomer. Preferably, at least 80% of the
particles have a particle size of smaller than about 5 .mu.m.
[0016] In a twelfth aspect, the invention features a composition
including a biologically active substance enclosed within a
biodegradable, polymerizable macromer, the macromer including at
least one water soluble region, at least one degradable region
which is hydrolyzable under in vivo conditions, and polymerizable
end groups having the capacity to form additional covalent bonds
resulting in macromer polymerization, where the polymerizable end
groups are separated by at least one degradable region, where the
composition contains at least 5% by weight of the active
substance.
[0017] In a thirteenth aspect, the invention features an insoluble
macromer including at least one water soluble region, at least one
degradable region which is hydrolyzable under in vivo conditions,
and polymerizable end groups having the capacity to form additional
covalent bonds resulting in macromer polymerization, where the
polymerizable end groups are separated by at least one degradable
region.
[0018] In a fourteenth aspect, the invention features composition
for the sustained delivery of a protein, where the composition
includes an insoluble macromer with at least one water soluble
region, at least one degradable region which is hydrolyzable under
in vivo conditions, and polymerizable end groups having the
capacity to form additional covalent bonds resulting in macromer
polymerization, where the polymerizable end groups are separated by
at least one degradable region.
[0019] In a fifteenth aspect, the invention features a macromer
including at least one water soluble region, at least one
degradable region which is hydrolyzable under in vivo conditions,
and polymerizable end groups having the capacity to form additional
covalent bonds resulting in macromer polymerization, where the
polymerizable end groups are separated by at least one degradable
region, where the degradable region consists essentially of
poly(trimethylene carbonate).
[0020] In a sixteenth aspect, the invention features a composition
for the subcutaneous administration of LHRH, where the composition
includes a core of poly(ethylene glycol) having a molecular weight
of about 1000 daltons, and a degradable region consisting of
poly(caprolactone), where the composition is capable of delivering
a therapeutic dose of LHRH for more than 30 days.
[0021] In a seventeenth aspect, the invention features a
composition comprising glucacon like peptide-1 and a macromer that
includes at least one water soluble region, at least one degradable
region which is hydrolyzable under in vivo conditions, and
polymerizable end groups having the capacity to form additional
covalent bonds resulting in macromer polymerization, where the
polymerizable end groups are separated by at least one degradable
region.
[0022] In an eighteenth aspect, the invention features a hydrogel
composition for the sustained release of a biologically active
substance, where the composition includes particles having a tap
density of less than 0.4 g/cm.sup.3, where at least 50% of the
particles have a mass mean diameter of less than about 5 .mu.m, and
where the composition is formulated for pulmonary
administration.
[0023] In a nineteenth aspect, the invention features a composition
for the sustained release of a biologically active substance, where
the composition includes particles having a tap density of more
than 0.4 g/cm.sup.3.
[0024] In the aspects of the invention described above, preferred
embodiments are as follows. The time at which 10% of the releasable
active substance is released is greater than {fraction (1/10)} of
t.sub.50. Articles and macromer compositions include at least 2.5%
active substance by weight, and preferably includes at least 5%,
10%, 25%, or 40% active substance by weight. Macromers include: (a)
a water soluble 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.
[0025] The water soluble region includes 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. The water soluble region may include at least
2 arms.
[0026] The degradable region includes 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). For example, the
degradable region may include poly(trimethylene carbonate) or
poly(caprolactone). Alternatively, the degradable region may
contain a poly(.alpha.-hydroxy acid) selected from the group
consisting of poly(glycolic acid), poly(DL-lactic acid) and
poly(L-lactic acid). The degradable region may alternatively
include a poly(lactone) selected from the group consisting of
poly(.epsilon.-caprolactone), poly(.delta.-valerolactone), and
poly(.gamma.-butyrolactone). The degradable region may include a
copolymer of at least two different monomers or a blend of at least
two different monomers.
[0027] The polymerizable end groups contain a carbon-carbon double
bond capable of polymerizing the macromers.
[0028] The articles are administered to the lung of the mammal.
Alternatively, the articles are administered intravenously,
subcutaneously, intramuscularly, orally, or nasally. Preferably,
the articles are administered to humans, and the biologically
active substance is preferably a protein.
[0029] By "therapeutic dose," when referreing to a biologically
active substance, is meant a plasma level between the minimum
effective level and the toxic level.
[0030] By "release kinetics" is meant the rate at which a drug is
released from its device/dosage form.
[0031] 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.
[0032] By "intact," when used in the context of a protein or
peptide, is meant that the protein or peptide is in its
biologically active form, and is not degraded or aggregated.
[0033] By "insoluble in water" or "water insoluble" is meant that
the solubility of a compound is less than 1 g/100 ml in aqueous
solution or in aqueous solution containing up to 5% of an organic
solvent, such as dimethylsulfoxide.
[0034] The methods and compositions of the invention provide for
the controlled release of relatively large quantities of
biologically active agents, such as proteins. The macromers used to
deliver the proteins both protect the proteins from degrading and
also allow for adjusting the release rate of the proteins.
[0035] Proteins can be delivered over a period of hours, or over a
period of months. In addition, the methods and compositions of the
invention provide a relatively constant dose of the active
substance, rather than a burst of the substance.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a diagram showing particles in which protein
particles are unevenly dispersed in the carrier medium, and
particles in which protein particles are evenly dispersed in the
medium.
[0037] FIG. 2 is a graph showing the release profile of a substance
from a macromer composition.
[0038] FIG. 3 is a graph showing the release profile of bST from a
blend of 3.4KL4 and PEGDA.
[0039] FIG. 4 is a graph showing the release profile of insulin
from 3.4KL5.
[0040] FIG. 5 is a graph showing the daily and cumulative release
of ZnbST from a 50:50 blend of 3.4 5KC6 and 3.4 5KL6.
[0041] FIG. 6 is a graph showing the daily and cumulative release
of ZnbST from a 75:25 blend of 3.4KL5 and 3.4KC6.
[0042] FIG. 7 is a graph showing the daily release of ZnbST
monomer, dimer, and solubilizable monomer from a 75:25 blend of
3.4KL5 and 3.4KC6.
[0043] FIG. 8 is a graph showing the effect of bST injections and a
sustained delivery bST formulation on the growth of hyphysectomized
rats.
[0044] FIG. 9 is a graph showing the initial release of bST from
cylindrical hydrogel devices with small and large diameters.
[0045] FIG. 10 is a graph showing the effect of EPO injections and
a sustained delivery EPO formulation on the percentage of
reticulocytes.
[0046] FIG. 11 is a graph showing the effect of subcutaneous
insulin injections and a subcutaneous sustained release hydrogel
insulin formulation on blood glucose levels of diabetic rats.
[0047] FIG. 12 is a graph showing the effect of pulmonary sustained
release hydrogel formulation insulin on blood glucose levels of
diabetic rats.
[0048] FIG. 13 is a graph showing the in vitro release rate of EPO
from 3.4KL5.
[0049] FIG. 14 is a graph showing in vitro release of insulin from
3.4KL5 particles.
DETAILED DESCRIPTION
[0050] The invention provides methods and compositions for the
administration of biologically active substances. These methods and
compositions provide for the controlled, sustained delivery of
relatively large quantities of these substances.
[0051] In one embodiment, a biologically active substance is
combined with a biodegradable, polymerizable macromer in the
presence of a polymerization initiator. The macromer is polymerized
to form a hydrogel and to incorporate the substance within the
resulting hydrogel. The resulting hydrogel, containing the active
substance, is formed into articles capable of controlled release of
the substance.
[0052] Macromers
[0053] The macromers of the invention have at least one
water-soluble region, at least one degradable (e.g., hydrolyzable)
region, and at least one polymerizable region. The macromers may be
water-soluble or water insoluble. These macromers are polymerized
to form hydrogels, which are useful for delivering incorporated
substances at a controlled rate. 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.
[0054] The ratio between the water-soluble 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.
[0055] There are several variations of these macromers. 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.
[0056] In another embodiment, the 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
attached to each water soluble region.
[0057] 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 is formed of a degradable
backbone having water-soluble regions as branches or grafts
attached to the degradable backbone.
[0058] Two or more polymerizable regions are attached to the water
soluble branches or grafts.
[0059] In another variation, the 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.
[0060] Throughout the specification, the following abbreviations
are sometimes used to describe the specific macromers of the
invention. In two particular examples, a macromer having a water
soluble region consisting of poly(ethylene glycol) with a molecular
weight of 4000 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 poly(ethylene glycol 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."
[0061] Water-Soluble Region
[0062] The water soluble region 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.
[0063] The macromer preferably comprises a water soluble core
region comprising poly(ethylene glycol) (PEG), as PEG has high
hydrophilicity and water solubility, as well as good
biocompatibility. The poly(ethylene glycol) region preferably has a
molecular weight of about 400 to about 40,000 Da, and more
preferably has a molecular weight of about 1,000 to about 30,000
Da, about 1,000 to about 20,000 Da, or about 2,000 to about 10,000
Da.
[0064] Degradable Region
[0065] The degradable region may contain, for example,
poly(.alpha.-hydroxy acids), poly(lactones), poly(amino acids),
poly(anhydrides), poly(orthoesters), poly(orthocarbonates) or
poly(phosphoesters), or blends or copolymers of these polymers.
[0066] 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).
[0067] Examples of copolymers include a copolymer of caprolactone
and glycolic acid; and a copolymer of caprolactone and lactic
acid.
[0068] Polymerizable Region
[0069] The polymerizable regions preferably contain carbon-carbon
double bonds capable of polymerizing the macromers. The choice of
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.
[0070] Polymerization Step
[0071] The macromers are 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 gel.
[0072] The polymerization 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.
[0073] The polymerization may also take place in the absence of
light. For example, the polymerization can be initiated with a
redox system, as described in more detail in the Examples. In some
cases it is advantageous to be able to polymerize using the redox
system of the invention, as radical initiator production occurs at
reasonable rates over a wide range of temperatures.
[0074] 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.
[0075] Properties of the Macromers
[0076] The articles of the 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.
[0077] More importantly, the articles are capable of degrading
under in vivo conditions at rates which permit the controlled
release of incorporated substances. Release may occur by diffusion
of the material 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.
[0078] The rate of release depends, in part, on the composition of
the water soluble region, such as the molecular weight of the
components in the water soluble region. The rate of release of the
biologically active agent also may be dependent upon the degree of
polymerization of the macromer, as well as on other factors.
[0079] The rate of release of the substance 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.
[0080] The precise rate of release can be further modified by
altering the ratio of hydrophilic and hydrophobic components. For
example, a very soluble macromer will yield, after polymerizing, 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 that is in between the release rate of a hydrogel containing
only lactic acid and a hydrogel containing only caprolactone as the
primary degradable group.
[0081] In addition, the rate of release of a given article depends
on the quantity of the loaded substance, as a percent of the final
product formulation; the solubility of the active substance; the
hydrophilicity of the active substance (hydrophilic active
substances will generally be released faster than hydrophobic
ones); and, in the case of suspensions, particle size. 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.
[0082] As shown in FIG. 1, 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 release.
[0083] Generally, water-soluble substances will yield homogenous
systems when incorporated into the macromers of the invention.
Substances that do not solubilize in water within the time it takes
to form the macromers of the invention will yield heterogenous
systems. The amount of burst in the heterogenous systems can be
minimized by using a particulate suspension with small
particles.
[0084] A release profile of a substance is shown in FIG. 2. The
horizontal axis shows the time after administration, and the
vertical axis represents the amount of material released. As shown
in FIG. 2, time t.sub.50 is the time at which 50% of the releasable
material has been released. Time t.sub.10 is, correspondingly, the
time at which 10% of the releasable material has been released. The
amount of releasable active substance is the amount that is
released from an article in a period of time 10 times greater than
the period of time it takes for 10% of the incorporated active
substance to be released.
[0085] When the release curve is perfectly linear, t.sub.10=1/5 of
t.sub.50. When there is an initial burst, t.sub.10 is much less
than 1/5 of t.sub.50. In the methods and compositions of the
invention, t.sub.10 is preferably greater than {fraction (1/10)} of
t.sub.50. In other words, there is no, or very little, initial
"burst" of release of the material.
[0086] The 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, or 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.
[0087] 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. The water soluble region
preferably has 4, 6, or 8 arms and a molecular weight of 10,000 to
40,000 daltons.
[0088] High Load Characteristics
[0089] Therapeutic agents may be readily incorporated in high yield
into the articles described herein. For example, articles may be
prepared containing at least 2.5% active substance by weight.
Preferably, the articles contain at least 5, 10, 25, or 40% by
weight.
[0090] The amount of loaded active substance may be measured by
dissolving pieces of the articles into an appropriate solvent and
assaying the amount of active substance present by means available
in the art, such as spectrophotometry.
[0091] Shaping of Articles
[0092] The articles formed using the procedures described above 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 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.
[0093] 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 therapeutic or other agent substantially 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 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).
[0094] 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.
[0095] In solvent removal, 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 he 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.
[0096] 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.
[0097] Spray drying is implemented by passing a homogenous mixture
of a substance, such as a therapeutic agent, and the polymerizable
macromer used to form the hydrogel through a nozzle, spinning disk
or equivalent device to atomize the mixture to form fine droplets.
The substance and the polymerizable macromer 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.
[0098] 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 polymeriz and
polymerize the macromers to form hydrogel particles incorporating
the substance, such as a biologically active agent. Typically,
polymerization may be conducted at room temperature.
[0099] The microspheres prepared using the techniques described
above are freeze dried, so they have a long shelf life (without
biodegradation) and the drug 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, the freeze dried or reconstituted particles may
be used.
[0100] Biologically Active Substances
[0101] Biologically active substances that can be incorporated into
the compositions 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, peptides, 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.
[0102] Exemplary therapeutic agents include calcitonin, granulocyte
macrophage colony stimulating factor (GMCSF), ciliary neurotrophic
factor, parathyroid hormone, and the cystic fibrosis transmembrane
regulator gene.
[0103] Other specific therapeutic agents include parathyroid
hormone-related peptide, somatostatin, testosterone, progesterone,
estradiol, nicotine, fentanyl, norethisterone, clonidine,
scopolomine, salicylate, salmeterol, formeterol, albeterol, and
valium.
[0104] Drugs for the treatment of pneumonia may be used, including
pentamidine isethiouate. Drugs for the treatment of pulmonary
conditions, such as asthma, may be used, including albuterol
sulfate, .beta.-agonists, metaproterenol sulfate, beclomethasone
diprepionate, triamcinolone acetamide, budesonide acetonide,
ipratropium bromide, flunisolide, cromolyn sodium, ergotamine
tartrate, and protein or peptide drugs such as TNF antagonists or
interleukin antagonists.
[0105] Other therapeutic agents include cancer chemotherapeutic
agents, such as cytokines, lymphokines, and DNA, and vaccines, such
as attenuated influenza virus. Nucleic acids that can be
incorporated include genes, 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.
[0106] 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.
[0107] 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, fibroblast
growth factor, follicle stimulating hormone, granulocyte colony
stimulating factor, glial fibrallary acidic protein, growth hormone
releasing factor, human alpha-1 antitrypsin, human atrial
natriuretic factor, human chorionic gonadotropin, human growth
hormone, 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-II, 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, A tumor necrosis factor,
macrophage inhibitory factor, Mullerian inhibiting substance,
megakaryocyte stimulating factor, melanocyte stimulating factor,
neutrophil chematactic 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, thrombopoietin, thyroid stimulating hormone,
urokinase-plasminogen activator, vascular endothelial growth
factor, and vasoactive intestinal peptide.
[0108] Exemplary diagnostic agents include gases and other
commercially available imaging agents that are used in positron
emission tomography (PET), computer assisted tomography (CAT),
single photon emission computerized tomography, X-ray, fluoroscopy,
and magnetic resonance imaging (MRI). Suitable materials for use as
contrast agents in MRI include gadolinium chelates, as well as
iron, magnesium, manganese, copper and chromium chelates. Examples
of materials useful for CAT and X-rays include iodine based
materials.
[0109] A preferred biologically active substance is a protein.
Proteins are defined as consisting of 100 amino acid residues or
more; peptides are less than 100 amino acid residues. Unless
otherwise stated, the term protein refers to both proteins and
peptides. 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, NSAIDs or
corticosteriods. Other exemplary proteins include enzymes, such as
DNase and proteases. Other proteins include cytokines, interferons,
including interferon alpha and interferon beta, poetins,
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).
[0110] Proteins are stable in the hydrogels of the 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 peptides can be further minimized by
co-incorporating peptidase-inhibitors.
[0111] Routes of Administration
[0112] Inhalation
[0113] 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
in U.S. Provisional Patent Application Serial No. 60/053,029, filed
Jul. 18, 1997.
[0114] 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.
[0115] 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.
[0116] The size and density of the particles can also be selected
to maximize the quantity of active substance 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
.mu.m), 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.
[0117] 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.
[0118] 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.).
[0119] 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 ppm 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 .mu.m to 0.5 mm.
[0120] 1.645 g of these particles was placed in a 10 mL graduated
cylinder. The graduated cylinder was mounted on top of Autotap
densimeter (Quantachrome). The sample was taped 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.
[0121] 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
.mu.m, 1 .mu.m, or 10 .mu.m thick.
[0122] 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, I.
"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.
[0123] 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.
[0124] Examples of drug that can be administered to the lung
include, without limitation, insulin, antitrypsin, calcitonin,
alpha interferon, beta interferon, GLP-1, and DNAse.
[0125] Nasal Delivery
[0126] The compositions can also be used to administer compounds
nasally.
[0127] For example, a vaccine containing freeze dried or
reconstituted microspheres can be administered nasally.
[0128] Intramuscular and Subcutaneous Administration
[0129] The articles of the invention can be used to administer
microspheres that degrade over several days to 3 months, by
intramuscular injection or by subcutaneous injection.
[0130] 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. For this application, particle sizes of up to 0.5 mm can be
used.
[0131] In other embodiments, the active agent is a vaccine, such as
tetanus vaccine, other proteins or peptides, 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.
[0132] Intravenous Administration
[0133] Hydrogel microspheres that contain a drug useful in treating
clotting disorders, such as Factor VIII or Factor IX for
hemophilia, can be administered by intravenous injection. The drug
is released over days to weeks. A therapeutic level of the drug is
maintained that results in a better clinical outcome. In addition,
potentially lower total doses of drugs can be administered, with a
corresponding economic benefit. These approaches help promote
patient compliance.
[0134] 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 .mu.m are
preferred.
[0135] 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 drug 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 DNAase as the
enzymatic drug).
[0136] 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 drug locally, either killing tumot 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 there have
been significant side effects.
[0137] The microspheres of the invention will 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.
[0138] Oral Administration
[0139] 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's found in the upper GI tract. Such an enteric
formulation could also be designed using several available
technologies to gradually expel drug-containing microspheres as the
enteric capsule traverses the gastrointestinal tract. This is
described in more detail in provisional application U.S. S No.
60/053,029 and in Mathiowitz et al., Nature 386 (6623): 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.
[0140] 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.
[0141] Targeting
[0142] 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, Vol. 279, 323-324
(1998).
[0143] Microspheres can be made with both a drug and a targeting
molecule.
[0144] Double microspheres can also be made, in which the inner
sphere contains drug and the outer PEG shell contains the targeting
molecule or reagent.
[0145] Excipients and Carriers
[0146] 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. 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, peptides, 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.
[0147] 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.
[0148] 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.
[0149] 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; 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 was used, the resulting macromer is abbreviated as
"2KL5."
[0150] In other examples an acrylate-PCL-PEG-PCL-acrylate macromer
was used. The PEG had a MW of 3,400 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."
[0151] All animals studies described herein were conducted with the
approval of the Institutional Animal Care and Use Committee.
EXAMPLE 1
General Preparation of a Macromer Solution
[0152] 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.
[0153] Macromer solutions having the following ingredients were
prepared.
1 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
Preparation of a Hydrogel from a Water Insoluble Macromer
[0154] 0.5 g of 3.4KC6 was added to a 20-cc scintillation vial. 0.5
mL of 200 mM TEOA, pH 6.95/PBS buffer was added, and the macromer
was allowed to swell. The macromer was then mixed until it formed a
homogeneous mixture. To this mixture were added 20 .mu.L of 1000
PPM eosin solution in PBS, 10 .mu.L of a 35% solution of n-VP, and
0.0845 g ZnbST.
[0155] The resulting solution was 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.
[0156] A light source (ILC Technology, Inc. Xenon Light Source with
Fiber Optics) was adjusted to about a 5-cm distance for
illumination from the light source to the glass slide, using clamps
and a stand. Both sides of the disc were illuminated for two
minutes each to form an opaque disc.
EXAMPLE 3
Preparation of a Hydrogel from a 50:50 Blend of Water Soluble and
Insoluble Macromer
[0157] 0.56 g 3.4KL5 and 0.56 g were placed in a scintillation
vial. The vial was placed in a 52.degree. C. oven; the mixture was
sporadically mixed until it formed a homogenous composition. It was
then cooled to room temperature. To 0.5 g of the above mixture were
added 0.5 mL of 200 mM TEOA and pH 6.95/PBS buffer. The resulting
macromer was allowed to swell.
[0158] Once swollen, the macromer was mixed until it formed an
homogeneous composition with a dough-like consistency. To this
composition were added 20 .mu.L 1000-PPM Eosin solution in PBS and
10 .mu.L 35% solution of n-VP and 0.0845 g of ZnbST. The resulting
solution was stirred, then 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.
[0159] A light source (ILC Technology, Inc. Xenon Light Source with
Fiber Optics) was adjusted to about a 5-cm distance. The center of
the disc was illuminated; both sides of the disc were illuminated
for two minutes each, to form an opaque disc.
EXAMPLE 4
Production of Microspheres Using a REDOX Initiating System
[0160] 300 mg of 3.4KL5 was dissolved in 1 mL of PBS containing
0.5% ammonium persulfate. 30 mL of silicone oil (100 cp) was
degassed with nitrogen. 0.25 mL of the aqueous media containing the
macromer was added to the oil and stirred at 2000 rpm using a
Silverson homogenizer equipped with 5/8" head. After the
combination was mixed thoroughly for 5 minutes, 0.5 mL of
tetramethylethylene diamine was added. The resulting emulsion was
stirred for 30 minutes. After 30 minutes, 20 mL of heptane was
added. The resulting suspension was centrifuged at 2000 rpm for 2
minutes and collected from the bottom of the centrifuge tube. The
resulting microspheres were analyzed by light microscopy @
400.times. using phase contrast. The average microsphere size was
found to be 2.5 .mu.m.
EXAMPLE 5
Long Term Release of bST
[0161] Device Preparation: A blend of a degradable macromer
(3.4KL5) and a non-degradable macromer (PEG-diacrylate, MW 3,400)
was used. The protein used was ZnbST (Monsanto/Protiva). The
protein was loaded at a loading of 20%, based on dry weight. 3
samples were prepared, as follows.
[0162] Sample preparation: 20 .mu.L of the bST-precursor solution
were prepared, as described in Example 1. The mixture was pipetted
using a positive displacement pipette with a silanized glass tip.
The solution was 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. A light source (ILC Technology,
Inc. Xenon Light Source with Fiber Optics) was adjusted to about a
5-cm distance from the glass slide using clamps and a stand. The
center of the disc was illuminated; both sides of the disc were
illuminated, for two minutes each.
[0163] The clips, the glass slide, and the spacers were carefully
removed. With a spatula and tweezers, the discs were removed and
weighed on a clean, tared silanized glass slide. The disc was
placed into a heat-sealed membrane bag, as described in more detail
below. One 20 .mu.L disc 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, 0.05M PBS, pH 7.4), placed on an orbital shaker
turning at 100 rpm, and incubated at 39.degree. C.
[0164] For each time point, the bag was placed into fresh 2.0 .mu.L
of PBS Release Media. Samples were collected for analysis every day
for as long as the bST was being released.
[0165] 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 disc was placed into the bag,
the last side was sealed using the same heat-sealing technique.
[0166] The samples were analyzed daily by SEC-HPLC. Monomers,
dimers, and soluble aggregates could be detected using this method.
The mobile phase used was 0.08M 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.mu. particle size,
300.times.7.8 mm ID, equipped with a guard column (Bio-Rad
Bio-Sil.RTM. SEC 250 Guard, 5.mu. 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.
[0167] The results are shown in FIG. 3. As shown there, bST was
released over 14 days. No detectable levels of dimers or soluble
aggregates were apparent in the release media. There was a minimal
initial release of 12% on each of the first two days, followed by a
moderate release rate.
EXAMPLE 6
Short Term Release of Insulin
[0168] Device Preparation: A degradable macromer (3.4KL5) was used.
The protein used was Zn-Insulin (purchased from Sigma). The protein
was loaded at a loading of 47%, based on dry weight. Three samples
were prepared.
[0169] The samples were prepared as described in Example 4. The
samples were analyzed by SEC-HPLC for the detection of monomers,
dimers and soluble aggregates, using the conditions described in
Example 5.
[0170] The results are shown in FIG. 4. Insulin was released over
24 hours; no dimers or soluble aggregates were detected. Complete
release (100%) was achieved within 24 hours.
EXAMPLE 7
Drug Release from Blends of Insoluble and Soluble Macromers
[0171] Devices were prepared as describe above. Macromers
containing a blend of a soluble macromer (3.4KL5) and an insoluble
macromer (3.4KC6) were used in a ratio of 50:50. The protein used
was ZnbST (Protiva/Monsanto); it was loaded at a loading of 25%,
based on dry weight. Six samples were prepared. The samples were
analyzed by SEC-HPLC, as described above. The samples were
monitored for the presence of monomers, dimers and soluble
aggregates.
[0172] The results are shown in FIG. 5. A release of ZnbST over 20
days was observed; very low concentrations (less than 2%) of dimers
or soluble aggregates were detected. In addition, no initial burst
release was observed.
EXAMPLE 8
Drug Release from Blends of Insoluble and Soluble Macromers
[0173] Devices were prepared as describe above. A blend of a
soluble macromer (3.4KL5) and an insoluble macromer (3.4KC6) were
used, in a ratio of 75:25. The protein ZnbST (Protiva/Monsanto) was
loaded at a loading of 25%, based on dry weight. Six samples were
prepared. The samples were analyzed by SEC-HPLC to detect monomers,
dimers, and soluble aggregates, as described above.
[0174] The results are shown in FIGS. 6 and 7. A long release of
ZnbST over 17 days was observed; within 13 days of release, 90% of
the incorporated ZnbST was released. Very little dimer or aggregate
was released.
EXAMPLE 9
Controlled Release of Bovine Somatotropin in Hypophysectomized
Rats{tc .backslash.l4 "Example 9: Controlled Release of Bovine
Somatotropin in Hypophysectomized Rats}
[0175] 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
lack of growth. On day 1 of the study the rats weighed 118.+-.1.5
grams (mean.+-.sem, n=18). The rats were 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 PEGDA (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.
[0176] The results are shown in FIG. 8. The untreated control group
did not grow during the study, and after 11 days weighed an average
of 119.+-.2.9 grams. The rats of Group 3, which received 100 .mu.g
bST daily during the study, exhibited continued growth and weighed
151.+-.4 grams after 11 days of treatment. The rats of Group 2 grew
at a rate similar to the rats of Group 3, and weighed 145.+-.3.7 g
after 11 days (p=0.32 for the comparison with Group 3, t-test).
EXAMPLE 10
Release of bST
[0177] A macromer mixture containing approximately 30% (w/w) of bST
was prepared using the methods described above. The
macromer/protein mixture was put in a glass cylinder having an
internal diameter of either 1.12 mm or 0.61 mm. The system was
exposed to light for 20 seconds, removed from the glass cylinder,
placed on aluminum foil, and exposed to light for an additional 3.5
minutes. The resulting hydrogel cylinders were placed in 1 mL of
release media (PBS, pH 7.4), and the released bST was monitored by
HPLC. Initial data indicated that the release from the larger
diameter cylinder closely trailed the release from the small
diameter cylinder. In addition, the characteristics of the bST
release indicated degradation/swelling of a controlled system. The
system showed the following fraction release M/M.sub..infin. as a
power function of time t for a short time-period:
M/M.sub..infin.=k't.sup.n, where k' is a constant characteristic of
the system and n is an exponent characteristic of the mode of
transport. For n=0.5, the drug release follows a Fickian-diffusion
mechanism. For n>0.5, non-Fickian behavior was observed.
[0178] When the data presented in FIG. 9 was analyzed for
erosion/diffusion release mechanisms. The large cylinder had a
value of M/M.sub..infin.=1E-06t.sup.2, and the smaller cylinder had
a value of M/M.sub..infin.=3E-05t.sup.2. Therefore when n=2,
non-Fickian behavior was observed.
[0179] In a different analysis based only on diffusion, the flux
from the cylinder was analyzed using the following Fickian
equation:
[0180] J=D*A*.DELTA.C/.DELTA.X, where J is the flux; D is the
diffusion constant; A is the surface area; C is the concentration
in the cylinder; and S is the distance from the center. In this
analysis the flux should differ dramatically whether the release
occurred from either a large or a small diameter cylinder.
Theoretical analysis predicted that under Fickian diffusion, when
the smaller diameter cylinder released 20%, the larger diameter
cylinder would release 7% of the incorporated drug. It was
observed, however, that when the smaller diameter cylinder released
20%, the larger diameter cylinder released 16%. Therefore,
non-Fickian behavior was observed.
[0181] In these hydrogel systems, the initial release phase
involved water uptake (swelling); as a result, the homogeneous drug
concentration profile within the matrix became sigmoidal. A high
drug concentration exists in the center of the cylinder, and very
little or no drug is available at the circumference of the device.
Such cylindrical systems yield release kinetics independent of the
radius of the cylinder. A detailed description of this phenomenon
can be found in Ping I. Lee, "Diffusion Controlled Matrix Systems,"
in Treatise on Controlled Drug Delivery, Kydonieus, A., ed. pp.
155-197 (1992).
EXAMPLE 11
Controlled Release of Erythropoeitin in Rats
[0182] The controlled delivery of active human erythropoeitin (EPO)
was confirmed in male Sprague-Dawley rats purchased from Taconic
Labs (Germantown, N.Y.). Hydrogel devices were manufactured to
contain 3000 Units per device, as described in Example 14. These
devices were prepared in the absence of vinyl pyrrolidone, and
other polymerizable monovinyl monomers. One of these devices was
implanted in each of 3 rats. Three other rats received a
subcutaneous injection of EPO (1000 Units) daily for 3 days. A
control group of 3 rats received no treatment.
[0183] On day 5 after implantation of the device or 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.
[0184] The results are shown in FIG. 10. As shown there, the rats
in the control group had about 2.5% reticulocytes. The rats with
the implants had about 12% reticulocytes, and the rats that
received injections had about 19% reticulocytes after five
days.
EXAMPLE 12
Controlled Release of Insulin in Diabetic Rats
[0185] 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 anesthesia 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. The
devices were prepared in the absence of vinyl pyrrolidone, and
other polymerizable monovinyl monomers. Blood samples were taken at
15, 30, 60, 120, and 180 minutes after implantation of the device
and were used to determine blood glucose levels.
[0186] The results are shown in FIG. 11. As shown there, the blood
glucose level dropped. This demonstrates that the devices are
capable of releasing insulin in its active form.
[0187] To test the pulmonary delivery system, the neck was opened
with a midline incision and the trachea 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
was 3 Units insulin) was instilled into the lung and the tube
removed. Blood samples were taken and analyzed as described above
for the subcutaneous device.
[0188] The results are shown in FIG. 12. Glucose levels dropped
significantly within 30 minutes and remained low (below 150 mg/dl)
for at least 180 minutes.
EXAMPLE 13
Controlled Release of Human Growth Hormone in Hypophysectomized
Rats
[0189] The controlled delivery of active human growth hormone (hGH,
MW 20 Kd) was confirmed in the hypophysectomized rat model.
Hypophysectomized female rats purchased from Taconic Labs
(Germantown, N.Y.) were weighed each morning. Prior to the
initiation of the study the rats were held 7 days to confirm lack
of growth. The rats were divided into 3 groups of equal mean
weights. Group 1 remained untreated and served as a negative
control. Group 2 received an implant of hGH in a hydrogel made of a
3:1 blend of 3.4KL5 and 3.4KC6 (each device contained approximately
1 mg of hGH). The rats in Group 3 were injected with 100 .mu.g hGH
subcutaneously each day for the duration of the study.
[0190] Initial results indicate that the previous results obtained
with bST were reproducible using hGH. The untreated control group
did not grow during the study. The rats of Group 3, which received
100 .mu.g hGH daily during the study, exhibited continued growth.
The rats of Group 2 grew at a rate similar to the rats of Group
3.
EXAMPLE 14
Release of EPO from Macromers
[0191] To a sterile 20 mL vial were added: 0.0330 g of TEOA (neat),
1.0076 g of 3.4KL5, 0.0598 g of 1000 ppm eosin (in PBS, pH 7.0),
and 2.32 g solution of EPO (10,000 units/mL). No vinyl pyrrolidone,
or other polymerizable monovinyl monomer was added. The resulting
mixture was mixed and polymerized by light (ILC Technology, Inc.
Xenon Light Source with Fiber Optics).
[0192] The rate of in vitro release was conducted by averaging the
release from 3 discs containing an average of 2500 units per disc.
The release was conducted in 4 mL of PBS (pH 7.4) at 39.degree. C.
The release media was exchanged daily. Analysis was done by size
exclusion chromatography. (HPLC: model 2690 by waters, Column: SEC
250 by BioRad, mobile phase: 0.8M TFA in 60% acetonitrile @ 1.5
mL/min, detector wavelength: 220 nm).
[0193] The results are shown in FIG. 13. As shown there, EPO was
released for at least 120 hours. After 120 hours, over 500 units of
EPO were still being released.
EXAMPLE 15
Release of Insulin from Macromer Particles
[0194] To a sterile 20 mL vial were 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 of insulin (Sigma). No
vinyl pyrrolidone, or other polymerizable monovinyl monomer was
added. The resulting mixture was mixed and placed into 10 mL glass
tubes. The tubes were exposed to xenon light (ILC Technology, Inc.
Xenon Light Source with Fiber Optics) for 10 seconds. The
semi-cured hydrogel was pushed out of the glass tube and further
polymerized for 3.5 minutes. The cured hydrogel rods were placed in
15 mL of heptane and ground using a homogenizer (Silverson L4RT-A)
for 30 seconds @ 5000 rpm, followed by 30 seconds @ 3000 rpm. The
heptane was decanted, and the powder was dried under nitrogen. The
resulting particles had a size distribution from 2 mm to 500
mm.
[0195] Particles (16 mg) were placed in a porous (0.8 mm)
"release-bag" (described in Example 5). The in vitro release was
calculated by averaging the release from 2 release bags. The
release-bag was placed into 2 mL of PBS (pH 7.4) at 39.degree. C.
The release media was exchanged every 15 minutes for the first 2
hours and every 30 minutes thereafter. Analysis was done by size
exclusion chromatography. (HPLC: model 2690 by waters, Column: SEC
250 by BioRad, mobile phase: 0.8M TFA in 60% acetonitrile @ 1.5
mL/min, detector wavelength: 220 nm).
[0196] The results are shown in FIG. 14. As shown there, insulin
was released over 90 minutes. After 90 minutes, 100 .mu.g of
insulin was still being released.
EXAMPLE 16
Release of Luteinizing Hormone Releasing Hormone (LHRH)
[0197] 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 1KC3, 0.0206 g of 1000 PPM eosin
(in PBS, pH 7.0) and 0.0615 g of LHRH (Sigma). No vinyl
pyrrolidone, or other polymerizable monovinyl monomer is added. The
resulting mixture is placed between two glass sheets and
polymerized by xenon light (ILC Technology, Inc. Xenon Light Source
with Fiber Optics) for 2 minutes on each side. The final hydrogel
sheet is cryo-milled to produce an injectable powder.
EXAMPLE 17
Pulmonary Devices Containing Human Growth Hormone (hGH)
[0198] 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.). No vinyl
pyrrolidone, or other polymerizable monovinyl monomer is added. 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 @
5000 rpm, followed by 30 seconds @ 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 18
Release of GLP-1
[0199] GLP-1 (glucacon like peptide-1) is a peptide drug that has
shown promise in the treatment of Type II diabetics. 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 1KC3, 0.0206 g of 1000 PPM eosin (in PBS, pH 7.0) and
0.0615 g of GLP-1. The resulting mixture is placed between two
glass sheets and polymerized by xenon light (ILC Technology, Inc.
Xenon Light Source with Fiber Optics) for 2 minutes on each side.
The final hydrogel sheet is cryo-milled to produce an injectable
powder.
EXAMPLE 19
Oral Formulation for Release of Proteins
[0200] Using the procedure of Example 15, one of insulin, human
growth hormone, human alpha interferon, or erythropoietin is
incorporated into macromer particles. Using cryomilling or the
milling procedure of Example 15, 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.
[0201] 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).
[0202] For other drugs, normal rats are used and blood drug levels
are measured at these same time points using RIA or ELIZA
techniques.
[0203] 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.
OTHER EMBODIMENTS
[0204] 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.
[0205] 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.
* * * * *