U.S. patent application number 11/002842 was filed with the patent office on 2005-05-12 for methods for micronization of hydrophobic drugs.
This patent application is currently assigned to Brown University Research Foundation. Invention is credited to Liu, Zhi, Mathiowitz, Edith, Thanos, Christopher.
Application Number | 20050100595 11/002842 |
Document ID | / |
Family ID | 23205142 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100595 |
Kind Code |
A1 |
Mathiowitz, Edith ; et
al. |
May 12, 2005 |
Methods for micronization of hydrophobic drugs
Abstract
The invention involves methods and products related to the
micronization of hydrophobic drugs. A method of micronizing
hydrophobic drugs using a set of solutions including an aqueous
solution is provided. The invention also relates to products of
micronized hydrophobic drugs and related methods of use.
Inventors: |
Mathiowitz, Edith;
(Brookline, MA) ; Thanos, Christopher;
(Cumberland, RI) ; Liu, Zhi; (West Roxbury,
MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Assignee: |
Brown University Research
Foundation
Providence
RI
|
Family ID: |
23205142 |
Appl. No.: |
11/002842 |
Filed: |
November 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11002842 |
Nov 30, 2004 |
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10758990 |
Jan 16, 2004 |
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6824791 |
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10758990 |
Jan 16, 2004 |
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10215208 |
Aug 8, 2002 |
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6746635 |
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60311043 |
Aug 8, 2001 |
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Current U.S.
Class: |
424/458 |
Current CPC
Class: |
A61K 9/146 20130101;
A61P 31/12 20180101; A61P 37/02 20180101; A61P 25/18 20180101; A61P
35/00 20180101; Y10T 428/2985 20150115; A61P 19/02 20180101; Y10T
428/31696 20150401; Y10T 428/2989 20150115; A61P 31/10 20180101;
A61P 31/00 20180101; A61K 9/5146 20130101; A61P 9/12 20180101; Y10T
428/2982 20150115; A61K 9/1688 20130101; A61K 9/14 20130101; Y10T
428/31685 20150401; A61P 3/10 20180101; A61K 9/5138 20130101; A61K
9/5192 20130101; A61P 25/08 20180101; B01J 13/06 20130101; A61K
9/1694 20130101; B01D 9/00 20130101 |
Class at
Publication: |
424/458 |
International
Class: |
A61K 009/54 |
Goverment Interests
[0002] Aspects of the invention were made with Government support
under Grant/Contract No. 2R01GM47636-05 awarded by the National
Institute of Health. The Government may retain certain rights in
this invention.
Claims
We claim:
1-49. (canceled)
50. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, wherein the first solvent is free of polymer, wherein the
hydrophobic agent and the solvent form a mixture having a
continuous phase, introducing a second solvent into the mixture,
and introducing an aqueous solution into the mixture wherein the
aqueous solution causes precipitation of the hydrophobic agent to
produce a composition of micronized hydrophobic agent having an
average particle size of 1 micron or less, and wherein the
hydrophobic agent is selected from the group consisting of
anticonvulsant, antidiabetic, antifungal, antihypertensive,
anti-infective, antimicrobial, antimycotic, antineoplastic,
anti-cancer, anti-epileptic, antiparasitic, antiproliferative,
antiviral, blood glucose regulator, diuretic, hormone,
hypoglycemic, hypotensive, immunomodulator, immunoregulator,
immunosuppressant, mood regulator, psychotropic, dyes, and imaging
agents.
51. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, wherein the first solvent is free of polymer, wherein the
hydrophobic agent and the solvent form a mixture having a
continuous phase, introducing a second solvent into the mixture,
and introducing an aqueous solution into the mixture wherein the
aqueous solution causes precipitation of the hydrophobic agent to
produce a composition of micronized hydrophobic agent having an
average particle size of 1 micron or less, and wherein the
hydrophobic agent is an antineoplastic, anti-cancer, or
antiproliferative agent.
52. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, wherein the first solvent is free of polymer, wherein the
hydrophobic agent and the solvent form a mixture having a
continuous phase, introducing a second solvent into the mixture,
and introducing an aqueous solution into the mixture wherein the
aqueous solution causes precipitation of the hydrophobic agent to
produce a composition of micronized hydrophobic agent having an
average particle size of 1 micron or less, and wherein the
hydrophobic agent is an antifungal or antimycotic agent.
53. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, wherein the first solvent is free of polymer, wherein the
hydrophobic agent and the solvent form a mixture having a
continuous phase, introducing a second solvent into the mixture,
and introducing an aqueous solution into the mixture wherein the
aqueous solution causes precipitation of the hydrophobic agent to
produce a composition of micronized hydrophobic agent having an
average particle size of 1 micron or less, and wherein the
hydrophobic agent is an anti-infective or antimicrobial agent.
54. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, wherein the first solvent is free of polymer, wherein the
hydrophobic agent and the solvent form a mixture having a
continuous phase, introducing a second solvent into the mixture,
and introducing an aqueous solution into the mixture wherein the
aqueous solution causes precipitation of the hydrophobic agent to
produce a composition of micronized hydrophobic agent having an
average particle size of 1 micron or less, and wherein the
hydrophobic agent is a dye or an imaging agent.
55. The method of claim 51, wherein the anti-cancer agent is
Paclitaxel.
56. The method of claim 50, further comprising preparing
microparticles by spray drying the micronized hydrophobic
agent.
57. The method of claim 50, further comprising preparing
microparticles by performing phase inversion nanoencapsulation
(PIN) on the micronized hydrophobic agent.
58. The method of claim 50, wherein the second solvent is an
alcohol.
59. The method of claim 50, wherein greater than 90% of the
micronized hydrophobic agent have a particle size less than 1
micron.
60. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, with a polymer, wherein the hydrophobic agent and the
first solvent form a mixture having a continuous phase, introducing
a second solvent into the mixture, and introducing an aqueous
solution into the mixture wherein the aqueous solution causes
precipitation of the hydrophobic agent to produce a composition of
micronized hydrophobic agent having an average particle size of 1
micron or less, and wherein the hydrophobic agent is selected from
the group consisting of anticonvulsant, antidiabetic, antifungal,
anti-infective, antimicrobial, antihypertensive, antimycotic,
antineoplastic, anti-cancer, anti-epileptic, antiparasitic,
antiproliferative, antiviral, blood glucose regulator, diuretic,
hormone, hypoglycemic, hypotensive, immunomodulator,
immunoregulator, immunosuppressant, mood regulator, psychotropic,
dyes, and imaging agents.
61. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, with a polymer, wherein the hydrophobic agent and the
first solvent form a mixture having a continuous phase, introducing
a second solvent into the mixture, and introducing an aqueous
solution into the mixture wherein the aqueous solution causes
precipitation of the hydrophobic agent to produce a composition of
micronized hydrophobic agent having an average particle size of 1
micron or less, and wherein the hydrophobic agent is an
antineoplastic, anti-cancer, or antiproliferative agent.
62. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, with a polymer, wherein the hydrophobic agent and the
first solvent form a mixture having a continuous phase, introducing
a second solvent into the mixture, and introducing an aqueous
solution into the mixture wherein the aqueous solution causes
precipitation of the hydrophobic agent to produce a composition of
micronized hydrophobic agent having an average particle size of 1
micron or less, and wherein the hydrophobic agent is an antifungal
or antimycotic agent.
63. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, with a polymer, wherein the hydrophobic agent and the
first solvent form a mixture having a continuous phase, introducing
a second solvent into the mixture, and introducing an aqueous
solution into the mixture wherein the aqueous solution causes
precipitation of the hydrophobic agent to produce a composition of
micronized hydrophobic agent having an average particle size of 1
micron or less, and wherein the hydrophobic agent is an
anti-infective or antimicrobial agent.
64. A method for micronizing a hydrophobic agent, comprising:
dissolving a hydrophobic agent in an effective amount of a first
solvent, with a polymer, wherein the hydrophobic agent and the
first solvent form a mixture having a continuous phase, introducing
a second solvent into the mixture, and introducing an aqueous
solution into the mixture wherein the aqueous solution causes
precipitation of the hydrophobic agent to produce a composition of
micronized hydrophobic agent having an average particle size of 1
micron or less, and wherein the hydrophobic agent is a dye or an
imaging agent.
65. The method of claim 61, wherein the anti-cancer agent is
Paclitaxel.
66. The method of claim 60, wherein the preparation contains less
than 5% polymer.
67. The method of claim 60, further comprising preparing
microparticles by performing phase inversion nanoencapsulation on
the micronized hydrophobic agent.
68. The method of claim 60, wherein the second solvent is an
alcohol.
69. The method of claim 60, wherein greater than 90% of the
micronized hydrophobic agent have a particle size less than 1
micron.
70. A preparation of micronized hydrophobic agent prepared
according to the method of claim 50.
71. A preparation of micronized hydrophobic agent prepared
according to the method of claim 60.
72. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is composed of less than 5% polymer
carrier and is free of surfactant, and wherein the hydrophobic
agent is selected from the group consisting of anticonvulsant,
antidiabetic, antifingal, antihypertensive, anti-infective,
antimicrobial, antimycotic, antineoplastic, anti-cancer,
anti-epileptic, antiparasitic, antiproliferative, antiviral, blood
glucose regulator, diuretic, hormone, hypoglycemic, hypotensive,
immunomodulator, immunoregulator, immunosuppressant, mood
regulator, psychotropic, dyes, and imaging agents.
73. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is composed of less than 5% polymer
carrier and is free of surfactant, and wherein the hydrophobic
agent is an antineoplastic, anti-cancer, or antiproliferative
agent.
74. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is composed of less than 5% polymer
carrier and is free of surfactant, and wherein the hydrophobic
agent is an antifungal or antimycotic agent.
75. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is composed of less than 5% polymer
carrier and is free of surfactant, and wherein the hydrophobic
agent is an anti-infective or antimicrobial agent.
76. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is composed of less than 5% polymer
carrier and is free of surfactant, and wherein the hydrophobic
agent is a dye or an imaging agent.
77. The composition of claim 73, wherein the anti-cancer agent is
Paclitaxel.
78. The composition of claim 72, wherein the wherein the
preparation is free of polymer carrier.
79. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is free of polymer carrier and
wherein the crystallinity of the micronized hydrophobic agent is at
least 50% of the crystallinity of the non-micronized hydrophobic
agent, and wherein the hydrophobic agent is selected from the group
consisting of anticonvulsant, antidiabetic, antifungal,
antihypertensive, anti-infective, antimicrobial, antimycotic,
antineoplastic, anti-cancer, anti-epileptic, antiparasitic,
antiproliferative, antiviral, blood glucose regulator, diuretic,
hormone, hypoglycemic, hypotensive, immunomodulator,
immunoregulator, immunosuppressant, mood regulator, psychotropic,
dyes, and imaging agents.
80. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is free of polymer carrier and
wherein the crystallinity of the micronized hydrophobic agent is at
least 50% of the crystallinity of the non-micronized hydrophobic
agent, and wherein the hydrophobic agent is an antineoplastic,
anti-cancer, or antiproliferative agent.
81. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is free of polymer carrier and
wherein the crystallinity of the micronized hydrophobic agent is at
least 50% of the crystallinity of the non-micronized hydrophobic
agent, and wherein the hydrophobic agent is an antifungal or
antimycotic agent.
82. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is free of polymer carrier and
wherein the crystallinity of the micronized hydrophobic agent is at
least 50% of the crystallinity of the non-micronized hydrophobic
agent, and wherein the hydrophobic agent is an anti-infective or
antimicrobial agent.
83. A composition, comprising a preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein the preparation is free of polymer carrier and
wherein the crystallinity of the micronized hydrophobic agent is at
least 50% of the crystallinity of the non-micronized hydrophobic
agent, and wherein the hydrophobic agent is a dye or an imaging
agent.
84. The composition of claim 80, wherein the anti-cancer agent is
Paclitaxel.
85. The composition of claim 79, wherein the crystallinity is at
least 75%.
86. The composition of claim 79, wherein the crystallinity is
greater than 90%.
87. A method for delivering an agent to a subject, comprising:
orally administering a solid preparation of micronized hydrophobic
agent having an average particle size of less than 1 micron,
wherein the preparation is composed of less than 5% polymer and is
free of surfactant, and wherein the hydrophobic agent is selected
from the group consisting of anticonvulsant, antidiabetic,
antifungal, antihypertensive, anti-infective, antimicrobial,
antimycotic, antineoplastic, anti-cancer, anti-epileptic,
antiparasitic, antiproliferative, antiviral, blood glucose
regulator, diuretic, hormone, hypoglycemic, hypotensive,
immunomodulator, immunoregulator, immunosuppressant, mood
regulator, psychotropic, dyes, and imaging agents.
88. A method for delivering an agent to a subject, comprising:
orally administering a solid preparation of micronized hydrophobic
agent having an average particle size of less than 1 micron,
wherein the preparation is composed of less than 5% polymer and is
free of surfactant, and wherein the hydrophobic agent is an
antineoplastic, anti-cancer, or antiproliferative agent.
89. A method for delivering an agent to a subject, comprising:
orally administering a solid preparation of micronized hydrophobic
agent having an average particle size of less than 1 micron,
wherein the preparation is composed of less than 5% polymer and is
free of surfactant, and wherein the hydrophobic agent is an
antifungal or antimycotic agent.
90. A method for delivering an agent to a subject, comprising:
orally administering a solid preparation of micronized hydrophobic
agent having an average particle size of less than 1 micron,
wherein the preparation is composed of less than 5% polymer and is
free of surfactant, and wherein the hydrophobic agent is an
anti-infective or antimicrobial agent.
91. A method for delivering an agent to a subject, comprising:
orally administering a solid preparation of micronized hydrophobic
agent having an average particle size of less than 1 micron,
wherein the preparation is composed of less than 5% polymer and is
free of surfactant, and wherein the hydrophobic agent is a dye or
an imaging agent.
92. The method of claim 88, wherein the anti-cancer agent is
Paclitaxel.
93. The method of claim 87, wherein the bioactivity of the
hydrophobic agent is retained.
94. The method of claim 87, wherein there is at least a 5% increase
in the relative bioavailability of the micronized hydrophobic agent
as compared to the non-micronized hydrophobic agent.
95. The method of claim 87, wherein the preparation is free of
polymer.
96. The method of claim 87, wherein the micronized hydrophobic
agent is microencapsulated by phase inversion
nanoencapsulation.
97. A method for delivering an agent to a subject, comprising:
administering microparticles of a micronized hydrophobic agent
encapsulated by phase inversion nanoencapsulation having an average
particle size of less than 1 micron, wherein the preparation is
composed of less than 5% polymer and is free of surfactant, and
wherein the hydrophobic agent is selected from the group consisting
of anticonvulsant, antidiabetic, antifungal, antihypertensive,
anti-infective, antimicrobial, antimycotic, antineoplastic,
anti-cancer, anti-epileptic, antiparasitic, antiproliferative,
antiviral, blood glucose regulator, diuretic, hormone,
hypoglycemic, hypotensive, immunomodulator, immunoregulator,
immunosuppressant, mood regulator, psychotropic, dyes, and imaging
agents.
98. A method for delivering an agent to a subject, comprising:
administering microparticles of a micronized hydrophobic agent
encapsulated by phase inversion nanoencapsulation having an average
particle size of less than 1 micron, wherein the preparation is
composed of less than 5% polymer and is free of surfactant, and
wherein the hydrophobic agent is an antineoplastic, anti-cancer, or
antiproliferative agent.
99. A method for delivering an agent to a subject, comprising:
administering microparticles of a micronized hydrophobic agent
encapsulated by phase inversion nanoencapsulation having an average
particle size of less than 1 micron, wherein the preparation is
composed of less than 5% polymer and is free of surfactant, and
wherein the hydrophobic agent is an antifungal or antimycotic
agent.
100. A method for delivering an agent to a subject, comprising:
administering microparticles of a micronized hydrophobic agent
encapsulated by phase inversion nanoencapsulation having an average
particle size of less than 1 micron, wherein the preparation is
composed of less than 5% polymer and is free of surfactant, and
wherein the hydrophobic agent is an anti-infective or antimicrobial
agent.
101. A method for delivering an agent to a subject, comprising:
administering microparticles of a micronized hydrophobic agent
encapsulated by phase inversion nanoencapsulation having an average
particle size of less than 1 micron, wherein the preparation is
composed of less than 5% polymer and is free of surfactant, and
wherein the hydrophobic agent is a dye or an imaging agent.
102. The method of claim 98, wherein the anti-cancer agent is
Paclitaxel.
103. The method of claim 97, wherein the microparticles are
administered orally.
104. The method of claim 97, wherein the bioactivity of the
hydrophobic agent is retained.
105. The method of claim 97, wherein there is at least a 5%
increase in the relative bioavailability of the micronized
hydrophobic agent as compared to the non-encapsulated micronized
hydrophobic agent.
106. The method of claim 97, wherein the preparation is free of
polymer.
107. A method for achieving 100% bioactivity comprising: orally
administering to the subject a solid preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein 100% of the orally administered agent is bioactive,
and wherein the hydrophobic agent is selected from the group
consisting of anticonvulsant, antidiabetic, antifingal,
antihypertensive, anti-infective, antimicrobial, antimycotic,
antineoplastic, anti-cancer, anti-epileptic, antiparasitic,
antiproliferative, antiviral, blood glucose regulator, diuretic,
hormone, hypoglycemic, hypotensive, immunomodulator,
immunoregulator, immunosuppressant, mood regulator, psychotropic,
dyes, and imaging agents.
108. A method for achieving 100% bioactivity comprising: orally
administering to the subject a solid preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein 100% of the orally administered agent is bioactive,
and wherein the hydrophobic agent is an antineoplastic,
anti-cancer, or antiproliferative agent.
109. A method for achieving 100% bioactivity comprising: orally
administering to the subject a solid preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein 100% of the orally administered agent is bioactive,
and wherein the hydrophobic agent is an antifungal or antimycotic
agent.
110. A method for achieving 100% bioactivity comprising: orally
administering to the subject a solid preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein 100% of the orally administered agent is bioactive,
and wherein the hydrophobic agent is an anti-infective or
antimicrobial agent.
111. A method for achieving 100% bioactivity comprising: orally
administering to the subject a solid preparation of micronized
hydrophobic agent having an average particle size of less than 1
micron, wherein 100% of the orally administered agent is bioactive,
and wherein the hydrophobic agent is a dye or an imaging agent.
112. The method of claim 108, wherein the anti-cancer agent is
Paclitaxel.
113. The method of claim 107, wherein the preparation is composed
of less than 5% polymer and is free of surfactant.
114. The method of claim 107, wherein the preparation is free of
polymer.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to U.S. provisional application Ser. No. 60/311,043, filed Aug. 8,
2001, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to methods of micronizing hydrophobic
drugs and related products and methods of using the micronized
hydrophobic drugs.
BACKGROUND OF THE INVENTION
[0004] Many hydrophobic agents, both active and non-active have
utility in a variety of in vivo settings. Although techniques exist
for preparing and formulating hydrophobic agents, these techniques
are limited. Some methods of formulation cause a loss of
bioactivity. Other methods produce large drug particles or
particles of inconsistent sizes that lead to problems in drug
delivery.
[0005] One method for formulating hydrophobic agents involves the
generation of microparticles. Microparticles, microcapsules and
microspheres (hereinafter "microparticles") have important
applications in the pharmaceutical, agricultural, textile and
cosmetics industry as delivery vehicles. In these fields of
application, a drug, protein, hormone, peptide, fertilizer,
pesticide, herbicide, dye, fragrance or other agent is encapsulated
in a polymer matrix and delivered to a site either instantaneously
or in a controlled manner in response to some external impetus
(i.e., pH, heat, water, radiation, pressure, concentration
gradients, etc.). Microparticle size can be an important factor in
determining the release rate of the encapsulated material.
[0006] Many microencapsulation techniques exist which can produce a
variety of particle types and sizes under various conditions.
Methods typically involve solidifying emulsified liquid polymer
droplets by changing temperature, evaporating solvent, or adding
chemical cross-linking agents. Physical and chemical properties of
the encapsulant and the material to be encapsulated can sometimes
dictate the suitable methods of encapsulation, making only certain
methodologies useful in certain circumstances. Factors such as
hydrophobicity, molecular weight, chemical stability, and thermal
stability affect encapsulation. Significant losses are frequently
associated with multiple processing steps. These parameters can be
particularly important in respect of encapsulating bioactive agents
because losses in the bioactivity of the material due to the
processing steps or low yields can be extremely undesirable.
SUMMARY OF THE INVENTION
[0007] In some aspects, the invention involves a method of
micronizing hydrophobic drugs. The micronized drugs prepared by
these methods have a variety of properties that are advantageous in
the field of drug delivery. For instance, the methods of the
invention allow for the formation of particles that have an average
particle size of less than 1 micron. The micronized drugs also
exhibit enhanced crystallinity and may be used to prepare particles
which result in improved relative bioavailability when administered
to a subject. Several of these surprising properties are
demonstrated in the examples section below.
[0008] According to one aspect of the invention, a method for
micronizing a hydrophobic agent is provided. A hydrophobic agent is
dissolved in an effective amount of a first solvent that is free of
polymer. The hydrophobic agent and the solvent form a mixture
having a continuous phase. A second solvent and then an aqueous
solution are introduced into the mixture. The introduction of the
aqueous solution causes precipitation of the hydrophobic agent and
produces a composition of micronized hydrophobic agent having an
average particle size of 1 micron or less.
[0009] According to another aspect of the invention, a method for
micronizing a hydrophobic agent is provided. A hydrophobic agent is
dissolved in an effective amount of a first solvent with a polymer.
The hydrophobic agent and the first solvent form a mixture having a
continuous phase. A second solvent and then an aqueous solution is
introduced into the mixture. The introduction of the aqueous
solution causes precipitation of the hydrophobic agent to produce a
composition of micronized hydrophobic agent having an average
particle size of 1 micron or less. In one embodiment, the final
preparation contains less than 5% polymer. In yet another
embodiment, the polymer is removed by the aqueous solution.
[0010] The hydrophobic agent may be dissolved in the first solvent
in a variety of ways depending on the agent. Such methods include,
but are not limited to, heating, sonicating, high shearing, or high
stirring the hydrophobic agent in the first solvent.
[0011] The second solvent is optionally an alcohol selected from
the group consisting of: methanol (methyl alcohol), ethanol, (ethyl
alcohol), 1-propanol (n-propyl alcohol), 2-propanol (isopropyl
alcohol), 1-butanol (n-butyl alcohol), 2-butanol (sec-butyl
alcohol), 2-methyl-1-propanol (isobutyl alcohol),
2-methyl-2-propanol (t-butyl alcohol), 1-pentanol (n-pentyl
alcohol), 3-methyl-1-butanol (isopentyl alcohol),
2,2-dimethyl-1-propanol (neopentyl alcohol), cyclopentanol
(cyclopentyl alcohol), 1-hexanol (n-hexanol), cyclohexanol
(cyclohexyl alcohol), 1-heptanol (n-heptyl alcohol), 1-octanol
(n-octyl alcohol), 1-nonanol (n-nonyl alcohol), 1-decanol (n-decyl
alcohol), 2-propen-1-ol (allyl alcohol), phenylmethanol (benzyl
alcohol), diphenylmethanol (diphenylcarbinol), triphenylmethanol
(triphenylcarbinol), glycerin, phenol, 2-methoxyethanol,
2-ethoxyethanol, 3-ethoxy-1,2-propanediol, Di(ethylene
glycol)methyl ether, 1,2-propanediol, 1,3-propanediol,
1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,
1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol,
2,4-pentanediol, 2,5-pentanediol, 3,4-pentanediol, and
3,5-pentanediol. A preferred alcohol is isopropanol. The second
solvent may also be a mixture of alcohols selected from the
aforementioned group.
[0012] Microparticles of the micronized hydrophobic agent may be
prepared by a variety of methods including, for example, spray
drying, interfacial polymerization, hot melt encapsulation, phase
separation encapsulation, spontaneous emulsion, solvent evaporation
microencapsulation, solvent removal microencapsulation,
coacervation, and low temperature microsphere formation. One
preferred method of preparing microparticles of the hydrophobic
agent is by performing phase inversion nanoencapsulation (PIN).
[0013] According to another aspect of the invention, microparticles
are provided. The microparticles may be produced by the processes
described above. The microencapsulated product may be composed of
particles having various sizes. In one embodiment, more than 90% of
the particles have a size less than 1 micron.
[0014] According to still another aspect of the invention, a
composition comprising a preparation of micronized hydrophobic
agent having an average particle size of less than 1 micron is
provided. The preparation is composed of less than 5% polymer
carrier and is free of surfactant. In one embodiment, the
preparation is free of polymer carrier.
[0015] The invention, also provides in some aspects a composition
comprising a preparation of micronized hydrophobic agent having an
average particle size of less than 1 micron, wherein the
preparation is free of polymer carrier and wherein the
crystallinity of the micronized hydrophobic agent is at least 50%
of the crystallinity of the non-micronized hydrophobic agent. In
one embodiment, the crystallinity is at least 75%. In another
embodiment, the crystallinity is greater than 90%.
[0016] The invention also encompasses methods for delivering a
hydrophobic agent to a subject, by administering an encapsulated
product including the agent, to the subject. The solid preparation
of the micronized hydrophobic agent having an average particle size
of less than 1 micron, composed of less than 5% polymer and free of
surfactant is administered orally. In one embodiment, the
bioactivity of the hydrophobic agent is retained. In another
embodiment, the micronized hydrophobic agent has at least a 5%
increase in relative bioavailability compared to the non-micronized
hydrophobic agent. In some embodiments, the preparation is free of
polymer.
[0017] In other aspects, the invention provides methods for
delivering an agent to a subject by administering microparticles of
a micronized hydrophobic agent encapsulated by phase inversion
nanoencapsulation. The average microparticle size is less than 1
micron and the preparation, is composed of less than 5% polymer and
is free of surfactant. The microencapsulated micronized hydrophobic
agent may be administered orally. In one embodiment, the
bioactivity of the hydrophobic agent is retained. In another
embodiment, the micronized hydrophobic agent has at least a 5%
increase in relative bioavailability compared to the non-micronized
hydrophobic agent. In some embodiments, the preparation is free of
polymer.
[0018] The invention also provides a method for achieving 100%
bioactivity. The method comprises orally administering to the
subject a solid preparation of micronized hydrophobic agent having
an average particle size of less than 1 micron wherein 100% of the
orally administered agent is bioactive. In an embodiment, the
preparation is composed of less than 5% polymer and is free of
surfactant. In some embodiments, the preparation is free of
polymer.
[0019] The foregoing aspects of the invention as well as various
objects, features, and advantages are discussed in greater detail
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. SEM micrographs of dicumarol formulations. (A) Stock
Dicumarol, (B) Spray-Dried Dicumarol, (C) Micronized Dicumarol, and
(D) Dicumarol in pFA:SA.
[0021] FIG. 2. DSC thermogram of dicumarol formulations.
[0022] FIG. 3. In vitro dissolution curves. Micronized Dicumarol
(Circles), FA:SA Encapsulated Dicumarol (Triangles), Stock
Dicumarol (Diamonds), and Spray-Dried Dicumarol (Squares). Points
represent mean.+-.standard error.
[0023] FIG. 4. Pig and rat control curves. IV [pig] (Squares), IV
[rat] (Circles), Blank FA:SA Microspheres [pig] (Diamonds), and
Blank FA:SA Microspheres [rat] (Triangles). Points represent
mean.+-.standard error.
[0024] FIG. 5. Plasma curves in the rat after oral administration,
Micronized Dicumarol (Circles), Spray-Dried Dicumarol (Triangles),
FA:SA Dicumarol Nanospheres (Squares), and Stock Dicumarol
(Diamonds). Points represent mean.+-.standard error.
[0025] FIG. 6. Plasma curves in the pig after oral feeding of
Spray-Dried Dicumarol (Squares), Micronized Drug (Diamonds), and
FA:SA Dicumarol Nanospheres (Triangles). Points represent
mean.+-.standard error.
[0026] FIG. 7. SEM micrographs of dicumarol formulations. (A) Stock
dicumarol, (B) Micronized Dicumarol with FA:SA [MDAP], (C)
Spray-Dried [SD], (D) Phase-Inverted Spray-Dried with FA:SA [AM],
(E) Encapsulation of B with p(FA:SA) using phase inversion
microencapsulation FA:SA [AN], and (F) Phase-Inverted Spray-Dried
with PLA [NN].
[0027] FIG. 8. In vitro dissolution and release studies of
dicumarol formulations. The top inset shows the first 4 hours.
Micronized Dicumarol with p(FA:SA) (MDAP) (diamonds), Encapsulation
of MDAP with p(FA:SA) (AN) (triangles), Encapsulation of
Spray-Dried Dicumarol with p(FA:SA) (AM) (X), Spray-Dried Dicumarol
(SD) (squares), Encapsulation of MDAP with poly(lactic)acid
(circles), Encapsulation of SD in p(FA:SA) using Solvent Removal
Technique (-), Stock Dicumarol (+). Points represent
mean.+-.standard error.
[0028] FIG. 9. In vivo control curves in the pig. IV administration
(Diamonds), Blank p(FA:SA) microspheres (Squares).
[0029] FIG. 10. Plasma curves from study groups administered with
spray-dried formulations. SD (diamonds), AM (squares). Points
represent mean.+-.standard error.
[0030] FIG. 11. Plasma curves from pigs fed with micronized
formulations. Doses are as follows: (MDAP) 23.0 mg/kg, (AN) 18.2
mg/kg, and (NN) 30.6 mg/kg. MDAP (squares), (AN) diamonds, NN
(triangles). Points represent mean.+-.standard error.
[0031] FIG. 12. Dose escalation of AN formulation. AN 3.6 mg/kg
(triangles), AN 10.9 mg/kg (diamonds), AN 18.2 mg/kg (squares).
Points represent mean.+-.standard error.
[0032] FIG. 13. Dose escalation of MDAP formulation. MDAP 5 mg/kg
(triangles), MDAP 15 mg/kg (diamonds), MDAP 23 mg/kg (squares).
Points represent mean.+-.standard error.
DETAILED DESCRIPTION
[0033] The invention is based, in some aspects, on a new method for
micronizing hydrophobic agents. It was discovered according to the
invention that the method for micronizing hydrophobic agents
results in a product having enhanced properties which lead to
better plasma concentration in vivo administration. For instance,
the micronized agent has reduced particle size, increased
crystallinity and enhanced bioactivity and relative bioavailability
when administered to a subject. The dramatic increases in
bioactivity and relative bioavailability were unexpected.
[0034] The micronized agent prepared according to the methods of
the invention may be used directly, e.g., administered to a
subject, or it may be further manipulated into pharmaceutical
compositions, such as microparticles. As shown in the Examples
section, when the micronized agent was used to produce
microparticles and those microparticles were delivered to animals
the relative bioavailability increased dramatically compared to
non-micronized formulations. In Example 4 it was demonstrated that
these microparticles were capable of achieving close to and in some
instances more than 100% relative bioavailability when orally
administered (Table 4). Relative bioavailability as used herein
refers to the amount of a drug available for detection in the
systemic circulation as compared to an administered IV dose. The
relative bioavailability of a drug administered by a route other
than IV, is generally a function of the ability of the drug to
permeate and penetrate into the systemic circulation. Relative
bioavailability is affected by a variety of factors, most
importantly permeability and solubility of the drug. Although
absolute (100%) bioavailability might be achieved with IV
administration, difficulties and limitations may be encountered in
IV administration (such as drug precipitation/crystallinization in
the blood) which render bioavailabity relative rather than
absolute. If a drug administered by IV undergoes precipitation or
crystallization, then the same drug administered by a different
route may in some cases have a relative bioavailability greater
than 100%.
[0035] The method in some aspects involves the formation of a
continuous phase mixture or preparation of the hydrophobic agent to
be micronized and a first solvent. This mixture or preparation is
free of polymer. As used herein, a mixture or a preparation free of
polymer refers to a mixture or preparation that has no detectable
amount of polymer.
[0036] In some aspects of the invention, the mixture or preparation
is substantially free of polymer. As used herein, substantially
free of polymer is more than 97% free of polymer. In some
embodiments the mixture or preparation is more than 98%, or more
than 99%, or 100% free of polymer. In some embodiments, the mixture
or preparation is absolutely free of polymer. As used herein
absolutely free of polymer refers to a mixture or preparation that
is 100% free of polymer.
[0037] The method may be accomplished by dissolving or dispersing
the hydrophobic agent in an effective amount of the solvent. A
second solvent that is immiscible with the original solvent and an
aqueous solution are introduced into the mixture. The introduction
of the aqueous solution causes the precipitation of the hydrophobic
agent to produce a micronized drug.
[0038] The micronized hydrophobic agent may be used without further
manipulation. For instance, it may be administered directly to a
subject. The micronization procedure transforms the hydrophobic
agent from a compound which when delivered directly to a subject
has low relative bioavailability into one which, because of the
micronized properties, has a much higher relative bioavailability
when administered. Although it is not necessary, it is also
possible to further process the micronized agent to produce
microparticles or to incorporate the agent into other drug delivery
devices. Microparticles of the micronized hydrophobic agent can be
prepared by several common microencapsulation techniques. Suitable
methods of encapsulation may be selected to produce the desired
physical and chemical properties of the encapsulant and the
material to be encapsulated. These optional methods are described
in more detail below.
[0039] The methods are particularly useful for micronizing
hydrophobic agents. The hydrophobic agent may be any type of
hydrophobic compound including active agents and non-active agents.
The hydrophobic agent is an agent that does not adsorb or absorb
water. Hydrophobic active and non-active agents include, but are
not limited to, adhesives, gases, pesticides, herbicides,
fragrances, antifoulants, dies, salts, oils, inks, cosmetics,
catalysts, detergents, curing agents, flavors, foods, fuels,
metals, paints, photographic agents, biocides, pigments,
plasticizers, propellants and the like. The active agent also may
be a bioactive agent. The bioactive agent may be, for example,
adrenergic agent; adrenocortical steroid; adrenocortical
suppressant; aldosterone antagonist; amino acid; anabolic;
analeptic; analgesic; anesthetic; anorectic; anti-acne agent;
anti-adrenergic; anti-allergic; anti-amebic; anti-anemic;
anti-anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic;
antibacterial; anticholinergic; anticoagulant; anticonvulsant;
antidepressant; antidiabetic; antidiarrheal; antidiuretic;
anti-emetic; anti-epileptic; antifibrinolytic; antifungal;
antihemorrhagic; antihistamine; antihyperlipidemia;
antihypertensive; antihypotensive; anti-infective;
anti-inflammatory; antimicrobial; antimigraine; antimitotic;
antimycotic, antinauseant, antineoplastic, antineutropenic,
antiparasitic; antiproliferative; antipsychotic; antirheumatic;
antiseborrheic; antisecretory; antispasmodic; antithrombotic;
anti-ulcerative; antiviral; appetite suppressant; blood glucose
regulator; bone resorption inhibitor; bronchodilator;
cardiovascular agent; cholinergic; depressant; diagnostic aid;
diuretic; dopaminergic agent; estrogen receptor agonist;
fibrinolytic; fluorescent agent; free oxygen radical scavenger;
gastrointestinal motility effector, glucocorticoid; hair growth
stimulant; hemostatic; histamine H.sub.2 receptor antagonists;
hormone; hypocholesterolemic; hypoglycemic; hypolipidemic;
hypotensive; imaging agent; immunizing agent; immunomodulator;
immunoregulator; immunostimulant; immunosuppressant; keratolytic;
LHRH agonist; mood regulator; mucolytic; mydriatic; nasal
decongestant; neuromuscular blocking agent; neuroprotective; NMDA
antagonist; non-hormonal sterol derivative; plasminogen activator;
platelet activating factor antagonist; platelet aggregation
inhibitor; psychotropic; radioactive agent; scabicide; sclerosing
agent; sedative; sedative-hypnotic; selective adenosine Al
antagonist; serotonin antagonist; serotonin inhibitor; serotonin
receptor antagonist; steroid; thyroid hormone; thyroid inhibitor;
thyromimetic; tranquilizer; amyotrophic lateral sclerosis agent;
cerebral ischemia agent; Paget's disease agent; unstable angina
agent; vasoconstrictor; vasodilator; wound healing agent; xanthine
oxidase inhibitor; Anti-cancer, e.g. paclitaxel.
[0040] Bioactive agents also include immunological agents such as
allergens (e.g., cat dander, birch pollen, house dust, mite, grass
pollen, etc.) and antigens from pathogens such as viruses,
bacteria, fungi and parasites. These antigens may be in the form of
whole inactivated organisms, peptides, proteins, glycoproteins,
carbohydrates or combinations thereof. Specific examples of
pharmacological or immunological agents that fall within the
above-mentioned categories and that have been approved for human
use may be found in the published literature.
[0041] The hydrophobic agent is added to and dissolved in a first
solvent. The first solvent is any suitable solvent that is capable
of dissolving the hydrophobic agent. Typically the solvent will be
a common organic solvent such as a halogenated aliphatic
hydrocarbon such as methylene chloride, chloroform and the like; an
alcohol; an aromatic hydrocarbon such as toluene; a halogenated
aromatic hydrocarbon; an ether such as methyl t-butyl; a cyclic
ether such as tetrahydrofuran; ethyl acetate; diethylcarbonate;
acetone; or cyclohexane. The solvents may be used alone or in
combination as the first solvent. It is desirable that the solvent
be inert with respect to the agent being micronized. Those of skill
in the art will be able to select an appropriate first solvent for
the particular hydrophobic agent being micronized based on the
guidance provided herein.
[0042] In some embodiments the hydrophobic agent is dissolved in an
effective amount of the first solvent with a polymer. When a
polymer is present in the first solvent, the agent is added to the
solvent optionally after the polymer is dissolved in the solvent.
If a polymer is used, the solvent chosen should be capable of
dissolving the polymer, and it is desirable that the solvent be
inert with respect to the polymer. It is preferred that the final
product has a low concentration of polymer or no polymer at all.
This could be achieved by choosing a polymer that is slightly
soluble in water, or else a polymer that degrades in water. This
way some of the polymer will disappear from the final
formulation.
[0043] A "polymer" may be any suitable (micronizing) material
consisting of repeating units including, but not limited to,
nonbioerodible and bioerodible polymers. As used herein the term
"polymer" includes polymer carriers and surfactants. A "polymer
carrier" is a polymer that does not function as a surfactant. A
surfactant is a surface-active agent that modifies the nature of
surfaces, i.e., which may involve reducing the surface tension of
water. Surfactants include but are not limited to wetting agents,
detergents, emulsifiers, dispersing agents, penetrants, and
antifoams. Some surfactants are polymers and others are
non-polymeric. Thus only a subset of surfactants are polymeric
surfactants.
[0044] Such polymers have been described in great detail in the
prior art. They include, but are not limited to: polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes and copolymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, polymers of acrylic and
methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate),
poly(ethylmethacrylate), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate),
poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl
acetate), poly vinyl chloride polystyrene and
polyvinylpryrrolidone.
[0045] Examples of preferred non-biodegradable polymers include
ethylene vinyl acetate, poly(meth)acrylic acid, polyamides,
copolymers and mixtures thereof.
[0046] Examples of preferred biodegradable polymers include
synthetic polymers such as polymers of lactic acid and glycolic
acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic
acid), poly(valeric acid), poly(caprolactone),
poly(hydroxybutyrate), poly(lactide-co-glycolide) and
poly(lactide-co-caprolactone), and natural polymers such as
alginate and other polysaccharides including dextran and cellulose,
collagen, chemical derivatives thereof (substitutions, additions of
chemical groups, for example, alkyl, alkylene, hydroxylations,
oxidations, and other modifications routinely made by those skilled
in the art), albumin and other hydrophilic proteins, zein and other
prolamines and hydrophobic proteins, copolymers and mixtures
thereof. In general, these materials degrade either by enzymatic
hydrolysis or exposure to water in vivo, by surface or bulk
erosion. The foregoing materials may be used alone, as physical
mixtures (blends), or as co-polymers. Some preferred polymers are
polyesters, polyanhydrides, polystyrenes and blends thereof.
[0047] Bioadhesive polymers are also useful. A bioadhesive polymer
is one that binds to mucosal epithelium under normal physiological
conditions. Bioadhesion in the gastrointestinal tract proceeds in
two stages: (1) viscoelastic deformation at the point of contact of
the synthetic material into the mucus substrate, and (2) formation
of bonds between the adhesive synthetic material and the mucus or
the epithelial cells. In general, adhesion of polymers to tissues
may be achieved by (i) physical or mechanical bonds, (ii) primary
or covalent chemical bonds, and/or (iii) secondary chemical bonds
(i.e., ionic). These are particularly useful when the particles
formed by the methods of the invention are delivered orally or to
other mucosal tissue.
[0048] Physical or mechanical bonds can result from deposition and
inclusion of the adhesive material in the crevices of the mucus or
the folds of the mucosa. Secondary chemical bonds, contributing to
bioadhesive properties, consist of dispersive interactions (i.e.,
Van der Waals interactions) and stronger specific interactions,
which include hydrogen bonds. The hydrophilic functional groups
primarily responsible for forming hydrogen bonds are the hydroxyl
and the carboxylic groups. Numerous bioadhesive polymers are
discussed in WO 93/21906. Representative bioadhesive polymers of
particular interest include bioerodible hydrogels described by H.
S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules. 1993,
26:581-587, the teachings of which are incorporated herein,
polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,
polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),
poly(ethyl methacrylates), poly butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), and poly(octadecl acrylate). A preferred
polymer is poly(fumaric-co-sebacic)acid.
[0049] Polymers with enhanced bioadhesive properties can be
provided wherein anhydride monomers or oligomers are incorporated
into the polymer. The oligomer excipients can be blended or
incorporated into a wide range of hydrophilic and hydrophobic
polymers including proteins, polysaccharides and synthetic
biocompatible polymers. Anhydride oligomers may be combined with
metal oxide particles to improve bioadhesion even more than with
the organic additives alone. Organic dyes because of their
electronic charge and hydrophobicity/hydrophilicity can either
increase or decrease the bioadhesive properties of polymers when
incorporated into the polymers. The incorporation of oligomer
compounds into a wide range of different polymers that are not
normally bioadhesive dramatically increases their adherence to
tissue surfaces such as mucosal membranes.
[0050] As used herein, the term "anhydride oligomer" refers to a
diacid or polydiacids linked by anhydride bonds, and having carboxy
end groups linked to a monoacid such as acetic acid by anhydride
bonds. The anhydride oligomers have a molecular weight less than
about 5000, typically between about 100 and 5000 daltons, or are
defined as including between one to about 20 diacid units linked by
anhydride bonds. In one embodiment, the diacids are those normally
found in the Krebs glycolysis cycle. The anhydride oligomer
compounds have high chemical reactivity.
[0051] The oligomers can be formed in a reflux reaction of the
diacid with excess acetic anhydride. The excess acetic anhydride is
evaporated under vacuum, and the resulting oligomer, which is a
mixture of species which include between about one to twenty diacid
units linked by anhydride bonds, is purified by recrystallizing,
for example from toluene or other organic solvents. The oligomer is
collected by filtration, and washed, for example, in ethers. The
reaction produces anhydride oligomers of mono and poly acids with
terminal carboxylic acid groups linked to each other by anhydride
linkages.
[0052] The anhydride oligomer is hydrolytically labile. As analyzed
by gel permeation chromatography, the molecular weight may be, for
example, on the order of 200-400 for fumaric acid oligomer (FAPP)
and 2000-4000 for sebacic acid oligomer (SAPP). The anhydride bonds
can be detected by Fourier transform infrared spectroscopy by the
characteristic double peak at 1750 cm.sup.-1 and 1820 cm.sup.-1,
with a corresponding disappearance of the carboxylic acid peak
normally at 1700 cm.sup.-1.
[0053] In one embodiment, the oligomers may be made from diacids
described for example in U.S. Pat. No. 4,757,128 to Domb et al.,
U.S. Pat. No. 4,997,904 to Domb, and U.S. Pat. No. 5,175,235 to
Domb et al., the disclosures of which are incorporated herein by
reference. For example, monomers such as sebacic acid,
bis(p-carboxy-phenoxy)propane, isophathalic acid, fumaric acid,
maleic acid, adipic acid or dodecanedioic acid may be used.
[0054] Organic dyes, because of their electronic charge and
hydrophilicity/hydrophobicity, may alter the bioadhesive properties
of a variety of polymers when incorporated into the polymer matrix
or bound to the surface of the polymer. A partial listing of dyes
that affect bioadhesive properties include, but are not limited to:
acid fuchsin, alcian blue, alizarin red s, auramine o, azure a and
b, Bismarck brown y, brilliant cresyl blue ald, brilliant green,
carmine, cibacron blue 3GA, congo red, cresyl violet acetate,
crystal violet, eosin b, eosin y, erythrosin b, fast green fcf,
giemsa, hematoylin, indigo carmine, Janus green b, Jenner's stain,
malachite green oxalate, methyl blue, methylene blue, methyl green,
methyl violet 2b, neutral red, Nile blue a, orange II, orange G,
orcein, paraosaniline chloride, phloxine b, pyronin b and y,
reactive blue 4 and 72, reactive brown 10, reactive green 5 and 19,
reactive red 120, reactive yellow 2,3, 13 and 86, rose bengal,
safranin o, Sudan III and IV, Sudan black B and toluidine blue.
[0055] The working molecular weight range for the polymer is on the
order of 1 kDa-150,000 kDa, although the optimal range is 2 kDa-50
kDa. The working range of polymer concentration is 0.01-50%
(weight/volume), depending primarily upon the molecular weight of
the polymer and the resulting viscosity of the polymer solution. In
general, the low molecular weight polymers permit usage of a higher
concentration of polymer. A preferred concentration range according
to the invention may be on the order of 0.0%-10% (weight/volume),
while the optimal polymer concentration in the micronized product
typically will be below 5% and preferably be close to or equal to
0%. It has been found that polymer concentrations on the order of
0-5% are particularly useful according to the methods of the
invention.
[0056] The hydrophobic agent and the first solvent (with or without
a polymer) form a continuous mixture. The hydrophobic agent is
dissolved in the first solvent by any of a variety of methods,
depending on the type of agent. Preferred methods include heating,
sonicating, high shearing, or stirring the hydrophobic agent in the
first solvent.
[0057] A second solution is then introduced into the mixture. The
"second solvent" as used herein is a solvent that is immiscible
with the original solvent. The second solvent includes, for
example, any suitable alcohol or a combination of alcohols.
Typically the solvent will be a common alcohol. Preferred alcohols
are methanol (methyl alcohol), ethanol, (ethyl alcohol), 1-propanol
(n-propyl alcohol), 2-propanol (isopropyl alcohol), 1-butanol
(n-butyl alcohol), 2-butanol (sec-butyl alcohol),
2-methyl-1-propanol (isobutyl alcohol), 2-methyl-2-propanol
(t-butyl alcohol), 1-pentanol (n-pentyl alcohol),
3-methyl-1-butanol (isopentyl alcohol), 2,2-dimethyl-1-propanol
(neopentyl alcohol), cyclopentanol (cyclopentyl alcohol), 1-hexanol
(n-hexanol), cyclohexanol (cyclohexyl alcohol), 1-heptanol
(n-heptyl alcohol), 1-octanol (n-octyl alcohol), 1-nonanol (n-nonyl
alcohol), 1-decanol (n-decyl alcohol), 2-propen-1-ol (allyl
alcohol), phenylmethanol (benzyl alcohol), diphenylmethanol
(diphenylcarbinol), triphenylmethanol (triphenylcarbinol),
glycerin, phenol, 2-methoxyethanol, 2-ethoxyethanol,
3-ethoxy-1,2-propanediol, Di(ethylene glycol)methyl ether,
1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol,
1,4-butanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol,
1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 2,5-pentanediol,
3,4-pentanediol, and 3,5-pentanediol. The most preferred alcohol is
isopropanol. One or more of the alcohols can be used in
combination.
[0058] An aqueous solution is then introduced into the above
mixture. The addition of the aqueous solution causes precipitation
of the hydrophobic agent and produces a composition of micronized
agent. An "aqueous solution" as used herein is a solution in which
water is the only or main component. The first and second solvents
are selected such that when these two immiscible solvents are added
to the aqueous solution, the three solutions become miscible.
[0059] The micronized hydrophobic agent may be used without further
manipulation. For instance, it may be administered directly to a
subject. Alternatively, the micronized hydrophobic agent may be
further processed prior to use, e.g. it may be processed to produce
microparticles. Microparticles of the micronized hydrophobic agent
may be prepared using any one of several common microencapsulation
techniques. Different microencapsulation techniques produce a
variety of microparticles having different properties under various
conditions. Suitable methods of encapsulation may be selected to
produce the desired physical and chemical properties of the
encapsulant and the material to be encapsulated. As used herein the
terms "microparticle" and "microencapsulation" are used broadly to
refer to particles, spheres or capsules that have sizes on the
order of microns as well as nanometers. Thus, the terms
"microparticle" "microsphere", "nanoparticle, "nanosphere",
"nanocapsule" and "microcapsule" are used interchangeably.
[0060] Common microencapsulation techniques include but are not
limited to spray drying, interfacial polymerization, hot melt
encapsulation, phase separation encapsulation (solvent removal and
solvent evaporation), spontaneous emulsion, solvent evaporation
microencapsulation, solvent removal microencapsulation,
coacervation, and low temperature microsphere formation and phase
inversion nanoencapsulation (PIN). Each of these methods is well
known in the art. A brief summary of the methods is presented
below.
[0061] In spray drying, the core material to be encapsulated is
dispersed or dissolved in a solution. Typically, the solution is
aqueous and preferably the solution includes a polymer. The
solution or dispersion is pumped through a micronizing nozzle
driven by a flow of compressed gas, and the resulting aerosol is
suspended in a heated cyclone of air, allowing the solvent to
evaporate from the microdroplets. The solidified microparticles
pass into a second chamber and are trapped in a collection
flask.
[0062] Interfacial polycondensation is used to microencapsulate a
core material in the following manner. One monomer and the core
material are dissolved in a solvent. A second monomer is dissolved
in a second solvent (typically aqueous) which is immiscible with
the first. An emulsion is formed by suspending the first solution
through stirring in the second solution. Once the emulsion is
stabilized, an initiator is added to the aqueous phase causing
interfacial polymerization at the interface of each droplet of
emulsion.
[0063] In hot melt microencapsulation the core material (to be
encapsulated) is added to molten polymer. This mixture is suspended
as molten droplets in a nonsolvent for the polymer (often
oil-based) which has been heated to approximately 10.degree. C.
above the melting point of the polymer. The emulsion is maintained
through vigorous stirring while the nonsolvent bath is quickly
cooled below the glass transition of the polymer, causing the
molten droplets to solidify and entrap the core material.
[0064] In solvent evaporation microencapsulation, the polymer is
typically dissolved in a water immiscible organic solvent and the
material to be encapsulated is added to the polymer solution as a
suspension or solution in organic solvent. An emulsion is formed by
adding this suspension or solution to a beaker of vigorously
stirring water (often containing a surface active agent to
stabilize the emulsion). The organic solvent is evaporated while
continuing to stir. Evaporation results in precipitation of the
polymer, forming solid microcapsules containing core material.
[0065] The solvent evaporation process is designed to entrap a
liquid core material in PLA, PLA/PGA copolymer, or PLA/PCL
copolymer microcapsules. The PLA or copolymer is dissolved in a
miscible mixture of solvent and nonsolvent, at a nonsolvent
concentration which is immediately below the concentration which
would produce phase separation (i.e., cloud point). The liquid core
material is added to the solution while agitating to form an
emulsion and disperse the material as droplets. Solvent and
nonsolvent are vaporized, with the solvent being vaporized at a
faster rate, causing the PLA or copolymer to phase separate and
migrate towards the surface of the core material droplets. This
phase separated solution is then transferred into an agitated
volume of nonsolvent, causing any remaining dissolved PLA or
copolymer to precipitate and extracting any residual solvent from
the formed membrane. The result is a microcapsule composed of PLA
or copolymer shell with a core of liquid material.
[0066] In solvent removal microencapsulation, the polymer is
typically dissolved in an oil miscible organic solvent and the
material to be encapsulated is added to the polymer solution as a
suspension or solution in organic solvent. An emulsion is formed by
adding this suspension or solution to a beaker of vigorously
stirring oil, in which the oil is a nonsolvent for the polymer and
the polymer/solvent solution is immiscible in the oil. The organic
solvent is removed by diffusion into the oil phase while continuing
to stir. Solvent removal results in precipitation of the polymer,
forming solid microcapsules containing core material.
[0067] In phase separation microencapsulation, the material to be
encapsulated is dispersed in a polymer solution by stirring. While
continuing to uniformly suspend the material through stirring, a
nonsolvent for the polymer is slowly added to the solution to
decrease the polymer's solubility. Depending on the solubility of
the polymer in the solvent and nonsolvent, the polymer either
precipitates or phase separates into a polymer rich and a polymer
poor phase. Under proper conditions, the polymer in the polymer
rich phase will migrate to the interface with the continuous phase,
encapsulating the core material in a droplet with an outer polymer
shell.
[0068] Spontaneous emulsification involves solidifying emulsified
liquid polymer droplets by changing temperature, evaporating
solvent, or adding chemical cross-linking agents. Physical and
chemical properties of the encapsulant and the material to be
encapsulated dictates the suitable methods of encapsulation.
Factors such as hydrophobicity, molecular weight, chemical
stability, and thermal stability affect encapsulation.
[0069] In solvent evaporation microencapsulation, the polymer is
typically dissolved in a water immiscible organic solvent and the
material to be encapsulated is added to the polymer solution as a
suspension or solution in organic solvent. An emulsion is formed by
adding this suspension or solution to a beaker of vigorously
stirring water (often containing a surface active agent to
stabilize the emulsion). The organic solvent is evaporated while
continuing to stir. Evaporation results in precipitation of the
polymer, forming solid microcapsules containing core material,
containing the micronized hydrophobic agent.
[0070] Some solvent evaporation processes are specifically designed
to entrap a liquid core material in PLA, PLA/PGA copolymer, or
PLA/PCL copolymer microcapsules. The PLA or copolymer is dissolved
in a miscible mixture of solvent and nonsolvent, at a nonsolvent
concentration which is immediately below the concentration which
would produce phase separation (i.e., cloud point). The liquid core
material is added to the solution while agitating to form an
emulsion and disperse the material as droplets. Solvent and
nonsolvent are vaporized, with the solvent being vaporized at a
faster rate, causing the PLA or copolymer to phase separate and
migrate towards the surface of the core material droplets. This
phase separated solution is then transferred into an agitated
volume of nonsolvent, causing any remaining dissolved PLA or
copolymer to precipitate and extracting any residual solvent from
the formed membrane. The result is a microcapsule composed of PLA
or copolymer shell with a core of liquid material containing
micronized hydrophobic agent.
[0071] In solvent removal microencapsulation, the polymer is
typically dissolved in an oil miscible organic solvent and the
material to be encapsulated is added to the polymer solution as a
suspension or solution in organic solvent. An emulsion is formed by
adding this suspension or solution to a beaker of oil with vigorous
stirring, in which the oil is a nonsolvent for the polymer and the
polymer/solvent solution is immiscible in the oil. The organic
solvent is removed by diffusion into the oil phase while continuing
to stir. Solvent removal results in precipitation of the polymer,
forming solid microcapsules containing core material.
[0072] Encapsulation procedures for various substances using
coacervation techniques have been described in the prior art, for
example, in GB-B-929 406; GB-B-929 401; U.S. Pat. Nos. 3,266,987;
4,794,000 and 4,460,563. Coacervation is a process involving
separation of colloidal solutions into two or more immiscible
liquid layers (Ref. Dowben, R. General Physiology, Harper &
Row, New York, 1969, pp. 142-143.). Through the process of
coacervation compositions comprised of two or more phases and known
as coacervates may be produced. The ingredients that comprise the
two phase coacervate system are present in both phases; however,
the colloid rich phase has a greater concentration of the
components than the colloid poor phase.
[0073] Components that may be used to formulate the coacervate
system comprise anionic, cationic, amphoteric, and non-ionic
surfactants. Anionic surfactants include di-(2 ethylhexyl) sodium
sulfosuccinate; non-ionic surfactants include the fatty acids and
the esters thereof; surfactants in the amphoteric group include (1)
substances classified as simple, conjugated and derived proteins
such as the albumins, gelatins, and glycoproteins, and (2)
substances contained within the phospholipid classification, for
example lecithin. The amine salts and the quaternary ammonium salts
within the cationic group also comprise useful surfactants. Other
surfactant compounds useful to form coacervates include
compositions within the groups known as the polysaccharides and
their derivatives, the mucopolysaccharides and the polysorbates and
their derivatives. Synthetic polymers that may be used as
surfactants include compositions such as polyethylene glycol and
polypropylene glycol. Further examples of suitable compounds that
may be utilized to prepare coacervate systems include
glycoproteins, glycolipids, galactose, gelatins, modified fluid
gelatins and galacturonic acid.
[0074] In addition, substances that are not intrinsically surface
active may be used to prepare coacervates provided that they can be
made so by chemical or other means. Fatty acids are not considered
to be surface active compounds. However, when fatty acids are
reacted with an alkaline chemical entity the resulting products
will have surface-active properties.
[0075] Low temperature microsphere formation has been described,
see, e.g., U.S. Pat. No. 5,019,400. The method is a process for
preparing microspheres which involves the use of very cold
temperatures to freeze polymer-biologically active agent mixtures
into polymeric microspheres. The polymer is generally dissolved in
a solvent together with an active agent that can be either
dissolved in the solvent or dispersed in the solvent in the form of
microparticles. The polymer/active agent mixture is atomized into a
vessel containing a liquid non-solvent, alone or frozen and
overlayed with a liquefied gas, at a temperature below the freezing
point of the polymer/active agent solution. The cold liquefied gas
or liquid immediately freezes the polymer droplets. As the droplets
and non-solvent for the polymer is warmed, the solvent in the
droplets thaws and is extracted into the non-solvent, resulting in
hardened microspheres.
[0076] Phase separation microencapsulation proceeds more rapidly
than the procedures described in the preceding paragraphs. A
polymer is dissolved in the solvent. An agent to be encapsulated
then is dissolved or dispersed in that solvent. The mixture then is
combined with an excess of nonsolvent and is emulsified and
stabilized, whereby the polymer solvent no longer is the continuous
phase. Aggressive emulsification conditions are applied in order to
produce microdroplets of the polymer solvent. After emulsification,
the stable emulsion is introduced into a large volume of nonsolvent
to extract the polymer solvent and form microparticles. The size of
the microparticles is determined by the size of the microdroplets
of polymer solvent.
[0077] One method for microencapsulating the micronized hydrophobic
agent is by phase inversion nanoencapsulation (PIN). In PIN, a
polymer is dissolved in an effective amount of a solvent. The agent
to be encapsulated is also dissolved or dispersed in the effective
amount of the solvent. The polymer, the agent and the solvent
together form a mixture having a continuous phase, wherein the
solvent is the continuous phase. The mixture is introduced into an
effective amount of a nonsolvent to cause the spontaneous formation
of the microencapsulated product, wherein the solvent and the
nonsolvent are miscible. PIN has been described by Mathiowitz et
al. in U.S. Pat. No. 6,131,211 and U.S. Pat. No. 6,235,224 that are
incorporated herein by reference.
[0078] Thus, the invention also provides compositions of the
micronized hydrophobic agent or microparticles produced using the
micronized hydrophobic agent by the processes described above as
well as other processes known in the art. The microencapsulated
product or micronized hydrophobic agent consist of particles having
various sizes. In some embodiments the particles have an average
particle size of less than one micron. In other embodiments more
than 90% of the particles have a size less than 1 micron.
[0079] The compositions of the invention have some properties which
are advantageous over prior art products. For instance, the
preparation of micronized hydrophobic agent has enhanced
crystallinity over that of similar prior art preparations. The
micronized hydrophobic agent of the invention may have at least 50%
of the crystallinity of a non-micronized hydrophobic agent. In some
embodiments it has at least 75%, 80%, 85%, 90%, or 95% of the
crystallinity of a non-micronized hydrophobic agent. As used herein
the term crystallinity refers to a property of a preparation having
a body that is formed by the solidification of a chemical element,
a compound, or a mixture and has a regularly repeating internal
arrangement of its atoms and often external plane faces. The
crystallinity of a preparation may be determined by methods known
in the art. Some of the methods of measuring crystallinity are
described in Introduction to Polymers, 2nd Edition; Young RJ,
Lovell Pa. 1991, Chapman and Hall Publishing, London, UK. One of
the methods to measure crystallinity is thermal analysis. An
example of this type of analysis is presented in Example 1 below.
The magnitude of thermal change is proportional to the amount of
crystalline component. A compound having enhanced crystallinity has
improved release properties when administered in vivo.
[0080] As demonstrated in the Examples below, the compositions of
the invention also allow for enhanced bioactivity and relative
bioavailability of the hydrophobic agent. Many drug processing
techniques result in a loss of bioactivity of the drug. The
micronization process described herein is sufficient to allow the
drug to retain its bioactivity. As a result, when the drug is
delivered to a subject the drug is active. As used herein
bioactivity refers to the normal function of a known drug. It
refers to the presence or absence of a function and can be used to
describe relative changes in levels of function rather than an
absolute value.
[0081] The micronized hydrophobic agent also exhibits at least a 5%
increase in relative bioavailability compared to the non-micronized
hydrophobic agent. The dramatic difference in relative
bioavailability of in vivo administered micronized agent versus
non-micronized agent is demonstrated in the Examples. When
administered through different routes, such as orally, the
micronized hydrophobic agent exhibited dramatically increased
relative bioavailability.
[0082] The compositions may include a physiologically or
pharmaceutically acceptable carrier, excipient, or stabilizer mixed
with the micronized hydrophobic agent. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredients. The term "pharmaceutically-acceptable carrier" means
one or more compatible solid or liquid filler, dilutants or
encapsulating substances which are suitable for administration to a
human or other vertebrate animal. The term "carrier" denotes an
organic or inorganic ingredient, natural or synthetic, with which
the active ingredient is combined to facilitate the application.
The components of the pharmaceutical compositions also are capable
of being commingled with the compounds of the present invention,
and with each other, in a manner such that there is no interaction
which would substantially impair the desired pharmaceutical
efficiency.
[0083] The micronized hydrophobic agent may be administered per se
or in the form of a pharmaceutically acceptable salt. When used in
medicine the salts may be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically acceptable salts thereof. Such salts
include, but are not limited to, those prepared from the following
acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric,
maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric,
methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0084] Suitable buffering agents include: acetic acid and a salt
(1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a
salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
Suitable preservatives include benzalkonium chloride (0.003-0.03%
w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and
thimerosal (0.004-0.02% w/v).
[0085] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the micronized hydrophobic agent in
water-soluble form. Additionally, suspensions of the micronized
hydrophobic agent may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions.
[0086] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0087] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0088] The micronized hydrophobic agent may be administered by any
ordinary route for administering medications. Depending upon the
type of disorder to be treated, the micronized hydrophobic agent of
the invention may be inhaled, ingested or administered by systemic
routes. Systemic routes include oral and parenteral. For use in
therapy, an effective amount of the micronized hydrophobic agent
can be administered to a subject by any mode that delivers the
nucleic acid to the organ or tissue being treated or monitored.
Preferred routes of administration include but are not limited to
oral, parenteral, intramuscular, intranasal, intratracheal,
intrathecal, intravenous, inhalation, transdermal,
intratracheobronchial (including intrapulmonary), ocular, vaginal,
and rectal.
[0089] For oral administration, the compounds can be formulated
readily by combining the active compound(s) with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a subject to be treated.
Pharmaceutical preparations for oral use can be obtained as solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Optionally the oral formulations may also be formulated
in saline or buffers for neutralizing internal acid conditions or
may be administered without any carriers.
[0090] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of compound doses.
[0091] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres, as described above,
formulated for oral administration may also be used. All
formulations for oral administration should be in dosages suitable
for such administration.
[0092] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0093] For administration by inhalation, the compounds for use
according to the present invention may be conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch. Those of skill in
the art can readily determine the various parameters and conditions
for producing aerosols without resort to undue experimentation.
Inhaled medications are preferred in some embodiments because of
the direct delivery to the lung. Several types of metered dose
inhalers are regularly used for administration by inhalation. These
types of devices include metered dose inhalers (MDI),
breath-actuated MDI, dry powder inhaler (DPI), spacer/holding
chambers in combination with MDI, and nebulizers. Techniques for
preparing aerosol delivery systems are well known to those of skill
in the art. Generally, such systems should utilize components which
will not significantly impair the biological properties of the
agent (see, for example, Sciarra and Cutie, "Aerosols," in
Remington's Pharmaceutical Sciences, 18th edition, 1990, pp.
1694-1712; incorporated by reference).
[0094] The compounds, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0095] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0096] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be formulated with suitable hydrophobic
materials (for example as an emulsion in an acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives, for example,
as a sparingly soluble salt.
[0097] The compositions are administered to a subject. A "subject"
as used herein shall mean a human or vertebrate mammal including
but not limited to a dog, cat, horse, cow, pig, sheep, goat, or
primate, e.g., monkey. The compositions are administered in
effective amounts. An effective amount of a particular agent will
depend on factors such as the type of agent, the purpose for
administration, the severity of disease if a disease is being
treated etc. Those of skill in the art will be able to determine
effective amounts.
[0098] The invention will be further more fully understood by
reference to the following Examples. These Examples, however, are
merely intended to illustrate the embodiments of the invention and
are not to be construed to limit the scope of the invention.
EXAMPLES
Example 1
Effect of Micronization on Dissolution of Dicumarol--In vitro
Effects
[0099] Materials and Methods
[0100] Dicumarol and reagent source: Dicumarol was purchased from
Sigma-Aldrich (St. Louis, Mo.) and was stored at room temperature.
Coulter Particle Analysis showed that the mean particle diameter
was 18.5 .mu.m based on volume statistics. All reagents and
solvents used throughout were purchased from either Fisher
(Pittsburg, Pa.) or Mallinckrodt (Phillipsburg, N.J.) and were of
the highest grade available.
[0101] Poly(fumaric-co-sebacic) anhydride synthesis: The polymer
used was the polyanhydride poly(fumaric-co-sebacic) anhydride
[p(FA:SA)] and was synthesized using melt polycondensation. Fumaric
acid and sebacic acid monomers were purchased from Aldrich,
purified in boiling ethanol, acetylated, and polymerized using melt
polycondensation at 180.degree.. A Bruker DPX300 NMR was used for
1D proton NMR analysis. The polymer in deuterated chloroform was
analyzed using peak ratios of the olefinic protons of the fumaric
acid monomer (.delta.=6.91 and 6.97) and the internal aliphatic
protons of the sebacic acid monomer (.delta.=1.32). The normalized
molar ratio was determined to be FA:SA 17:83. For analysis of
molecular weight, a 5% solution of p(FA:SA) in chloroform was
analyzed on a Perkin Elmer LC pump model 250 gel permeation
chromatography system composed of an isocratic LC pump model 250,
an LC column oven model 101, an LC-30 RI detector, and a 900 series
interface computer. Samples were eluted through a PL gel 5 .mu.m
mixed column and a 5 .mu.m/50 .ANG. column connected in series at a
flow rate of 1.0 mL/min and a temperature of 40.degree. C. The
system was calibrated with a series of monodisperse polystyrene
standards (MW: 600-200,000) in chloroform, and the molecular weight
of p(FA:SA) was found to be 12 kDa. The polymer was stored under a
nitrogen purge at -20.degree. C. until use.
[0102] Scanning Electron Microscopy: All samples were
sputter-coated with an Au--Pd target for 3.5 minutes and spread out
on a carbon-backed adhesive disc on top of the SEM stub. The
Hitachi 2700 was used to visualize the samples at an accelerating
voltage of 8 kV.
[0103] DSC: A Pyris 1 DSC with an Intercooler 2P Cooling System was
used to thermally characterize the formulations. Following a
baseline run at a heating and cooling rate of 10.degree. C./min
from 0 to 320.degree. C., 5 mg samples were hermetically sealed in
aluminum pans and run under a nitrogen purge using the same
parameters.
[0104] Formulating Dicumarol: Particulates of dicumarol were
produced through two techniques yielding different size
distributions. One method was used to produce sub-micron particles,
and the other produced particles with a median diameter of
approximately 3 .mu.m.
[0105] Spray-drying was used to create the 3 .mu.m formulation. 20
g of dicumarol was dissolved in 8 L methylene chloride to make a
0.25% (w/v) solution. This solution was spray-dried in a Lab Plant
SD-04 Laboratory Spraydrier using a pressure pot at a pressure of
68 psi, an atomizer pressure of 65 psi, and a solvent flow rate of
30 mL/min. The drying temperature of the inlet and outlet were
45.degree. C. and 24.degree. C. respectively. The spray-dried
microparticles (SD) were collected off of the walls of the device,
lyophilized, and stored at -20.degree. C. until further use.
[0106] The sub-micron particulates were produced using a novel
technique. 330 mg dicumarol was dissolved in 30 mL
dimethylsulfoxide with a micro magnetic stir-bar rotating at 900
rpm. The temperature of the solution was raised until dissolution
occurred, which was typically around 100.degree. C. The entire
volume of this solution was dispersed in 500 mL isopropyl alcohol,
creating a two-phase system. After vigorous stirring, 600 mL
distilled water was added in a stream resulting in a colloidal
dispersion of a milky precipitate. Using a cylindrical pressure
filtration apparatus, the nanoparticles were collected on 100 nm
filter paper composed of mixed cellulose esters. The powder was
then frozen and lyophilized for 48 hours.
[0107] To study the effect of encapsulation on the micronized drug,
the polymer p(FA:SA) 17:83 was employed as a carrier and the phase
inversion procedure was used to produce nanospheres. 100 mg of
sub-micron dicumarol particulates was probe sonicated for 3 minutes
at amplitude 35% in 20 mL methylene chloride, causing complete
dissolution of the drug. 100 mg p(FA:SA) 17:83 was dissolved in
this solution by sonicating for an additional 30 s. The resultant
solution was dispersed into 1.0 L petroleum ether and the
precipitate was collected using a 100 nm filter composed of
mixed-cellulose esters. The microsphere formulation was then frozen
and lyophilized for 24 hours. The dicumarol loading was determined
in this formulation by a simple extraction protocol. Microspheres
were incubated overnight in 2.5 N NaOH at 37.degree. C. Upon
dissolution, an aliquot of approximately 15 .mu.g based on
theoretical loading was added to 2.5 N NaOH to make a total volume
of 800 .mu.L. This mixture was agitated for 2 minutes and
centrifuged for 2 minutes at 11,269 g. The supernatant was removed
and analyzed on the Shimadzu UV-2501 spectrophotometer and compared
to a linear standard curve of dicumarol in NaOH.
[0108] Particle Sizing: All microspheres and micronized
formulations were sized using laser-light diffractometry via the
Coulter Particle Size Analyzer LS 230. A 250 .mu.g/mL suspension of
microspheres in 1% pluronic F127 [poly(ethylene
oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)]/1%
hydroxypropylmethylcellulose (HPMC) was introduced into the small
volume fluid module. Only the Coulter output based on volume
measurements was used for analysis.
[0109] In vitro Release Study: We generated release profiles for
all of the formulations by incubating them in PBS Buffer (pH=7.2)
at 37.degree. C. Sink conditions were maintained by keeping the
total concentration of dicumarol in water below the solubility
limit, 28 .mu.g/mL. All release studies were scaled up to 5 mg
dicumarol in 180 mL PBS buffer, and each group consisted of n=4
samples. At different time points, 120 .mu.L supernatant was
obtained from each sample, placed into an Amicon Ultrafree-MC
centrifugal filter device with a nominal molecular weight cutoff of
5 kDa, and centrifuged for 5 minutes at 11,269 g to remove any
residual crystallized dicumarol. 100 .mu.L of the supernatant was
removed and stored at 4.degree. C. until it was analyzed. 120 .mu.L
of fresh buffer was added back to each sample after the
timepoints.
[0110] Results:
[0111] Characterization of Dicumarol Formulations: SEM micrographs
of the dicumarol formulations are displayed in FIG. 1. FIG. 1A
shows the stock dicumarol as supplied by Sigma (St. Louis, Mo.).
The particulates were primarily in the range of 10-20 microns and
have a cuboidal appearance. Spray-dried dicumarol, shown in FIG.
1B, has a round appearance and appears to be hollow. These
particles were roughly 3 microns in diameter. The micronized drug
is shown in FIG. 1C. This formulation in non-spherical and most of
the population is in the 300 nm to 1 micron range. FIG. 1D shows
the FA:SA nanospheres fabricated from the micronized formulation.
Particles in this formulation were generally 1 micron in size. The
loading determination of the FA:SA formulation along with the
Coulter particle size data is presented in Table 1.
1TABLE 1 Coulter particle size analysis of dicumarol formulations.
Data reported were generated from Coulter volume calculations.
Coulter Particle Analysis Formulation 25% < 50% < 75% <
Loading Stock 12.98 .mu.m 18.95 .mu.m 24.82 .mu.m 100% Spray-Dried
1.474 .mu.m 3.053 .mu.m 5.319 .mu.m 100% Micronized 0.433 .mu.m
0.535 .mu.m 0.701 .mu.m 100% Encapsulated in 0.782 .mu.m 1.414
.mu.m 2.312 .mu.m 31% FASA
[0112] Thermal analysis using DSC revealed the presence of a solid
solution of drug in the FA:SA formulation. FIG. 2 shows the DSC
thermograms of the four formulations, and for comparison, the FA:SA
polymer alone. AH is shown on the left of each melt. The bottom
curve for the blank FA:SA polyanhydride shows a variety of peaks.
Between 60.degree. C. and 90.degree. C., the trimodal peak of the
polymer melt is seen. At 275.degree. C., the polymer and its
components begin to degrade, which continues beyond 300.degree. C.
The stock dicumarol shows a distinct melt at approximately
290.degree. C., which is consistent with the values previously
reported. In the FA:SA/Dicumarol nanosphere formulation, the peak
due to the melt is completely gone, indicating that a true
molecular dispersion was obtained. The spray-dried and micronized
formulations both show the same drug melt at about 290.degree. C.,
but they have a lower AH than the stock dicumarol. Because the
magnitude of .DELTA.H is proportional to the relative amount of the
crystalline component, the lower .DELTA.H is probably due to the
quenching of the drug solution during both of those processes,
which could lead to decreased crystal growth. This process affected
the crystallinity of the spray-dried formulation the most, as the
.DELTA.H was reduced by 32%. It must be noted that the melting peak
in the DSC trace for the spray-dried formulation was very small
because of a very small sample size. The decrease in crystallinity
was not proportional to the peak area as presented, but was a
function of the .DELTA.H.
[0113] The results of in vitro dissolution studies are shown in
FIG. 3. Because the dissolution studies were done in a static
environment, where only a small amount and not the total amount of
buffer was replaced, the dissolved dicumarol was allowed to
recrystallize. This led to a polymorphic state in which different
populations of dicumarol were dissolved and recrystallized at
different rates. In vitro, this can be seen by the increase in
concentration followed by a decrease, and sometimes again followed
by an increase. In vivo, however, we would not have expected to see
this behavior because the dissolved dicumarol would have readily
bound to plasma proteins and became metabolized.
[0114] The micronized formulation showed the most rapid
dissolution, reaching a concentration of 36.9 .mu.g/mL after only
24 hours. The dicumarol dissolved in FA:SA showed the next highest
amount of dissolution, with concentrations reaching 28.8 .mu.L
after 72 hours. The time lag evident in this formulation can be
attributed to the polymer coating, which degrades by surface
erosion and has been used in many controlled release delivery
systems. The stock dicumarol showed more of a depot effect and does
not appear to recrystallize after 600 hours. The spray-dried
formulation showed the lowest amount of dissolution, reaching only
13.6 .mu.g/mL after 288 hours.
Example 2
Effect of Micronization on Relative Bioavailability of
Dicumarol--In vivo Effects
[0115] Materials and Methods
[0116] Animal Models: The following animal work was performed in
accordance with the Principles of Laboratory Animal Care (NIH
publication #85-23, revised 1985). Both female Yorkshire pigs and
male Sprague-Dawley rats were used. Pig starting weights ranged
from 15 to 20 kg, and were divided into groups of n=3, 4, or 5. The
groups were kept for 12-14 weeks and were administered each
formulation throughout the study. Male CD Rats weighing
approximately 250 g were also used, and were divided into groups of
between 8 and 12 for each study group. The rat groups were each
used for only one study, and were sacrificed after the last time
point.
[0117] After a fasting period of 12 hours, animals were orally
gavaged with microsphere formulations suspended in a solution of 1%
HPMC and 1% pluronic F 127. The microsphere dose was suspended
immediately prior to administration using bath sonication for 3
minutes and was administered to the stomach through a gavage tube.
The suspension concentration was kept constant at 25 mg/mL, and
several flushes of vehicle were administered following the dose.
During administration, pigs were sedated with a combination of
ketamine and medetomidine, and immediately following the procedure,
the medetomidine-antagonist atipamezole was given for reversal of
anesthesia. Rats were anesthetized using isoflurane gas.
[0118] A control group in each species was gavaged with blank
p(FA:SA) 17:83 microspheres suspended in 1% F 127/1% HPMC in order
to generate a baseline for the experiment. Additionally, each
species had an IV group to which we administered 25 mg/kg dicumarol
dissolved in a mixture of 50% propylene glycol, 10% ethanol, and
40% 100 mM Tris at a pH of 9.0. The dicumarol was dissolved in the
vehicle at a concentration of 20 mg/mL and administered through a
chronic catheter placed in the external jugular vein of the pig.
The IV dose was administered through the dorsal penile vein of the
rats via a 23-gauge needle.
[0119] At specific time points, generally 0, 1, 2, 3, 5, 8, 11, 14,
25, 29, 36, 48, 60, 72, 84, and 96 hours, blood was collected from
each animal. In the pig, the heparin block was removed, 1 cc of
fresh blood was collected, and another 1.5 cc heparin solution was
added to the catheter. 300 .mu.L of rat blood was sampled from the
tail vein. The blood samples were collected in heparinized 1.5 mL
siliconized microfuge tubes and centrifuged for 5 minutes at 11,269
g. Approximately 200 .mu.L plasma was removed and stored at
4.degree. C. prior to being analyzed.
[0120] Dicumarol Quantification: A double extraction technique with
slight modifications was employed. A 50 .mu.L sample of the plasma
in a 15 mL Falcon tube was first acidified with 300 .mu.L of a
citrate/phosphate buffer with pH=3.0 by shaking and allowing the
mixture to interact for 5 minutes. Next, the dicumarol was
extracted from the plasma by adding 3 mL heptane and rotating each
sample end over end for 10 minutes. The tubes were centrifuged for
5 minutes at 3000 rpm and the top heptane layer was separated and
put into a new tube. Next, 1 mL of 2.5 N NaOH was added to each
tube and the mixture was again rotated end over end for 10 minutes.
Following centrifugation at 3000 rpm for 5 minutes, the aqueous
phase was removed and the absorbance was read at 315 nm on a
Shimadzu UV-2501 spectrophotometer. A standard curve for this assay
was obtained by doping plasma obtained from control animals with
known amounts of dicumarol in sodium hydroxide.
[0121] Bioactivity: Plasma samples taken at the T.sub.max were
tested for drug activity using the prothrombin time test (PTT)
performed by IDEXX Veterinary Services in North Grafton, Mass.
Plasma was collected and submitted for testing in tubes coated with
citrate.
[0122] Statistics and Pharmacokinetic Analysis: Standard errors
were calculated and t-tests were performed using Microsoft Excel.
T-tests were employed to compare the plasma curves generated from
the different formulations, and p was calculated using AUCs
normalized by dose. AUC, C.sub.max, and T.sub.max were all
calculated from the Graphpad Prism Software. Non-compartmental
pharmacokinetics were assumed.
[0123] Results
[0124] In vivo Studies: Bioactivity tested positive using the
prothrombin time test. Samples taken from plasma corresponding to
the C.sub.max all showed clotting times longer than 90 seconds,
compared to 12 to 17 seconds in normal animals.
[0125] FIG. 4 shows the control curves, including the IV bolus
injection and oral delivery of blank FA:SA nanospheres to both the
rat and pig. The IV curves both peaked very rapidly and showed only
a downward slope indicating elimination. The rats received 24.0
mg/kg while the pigs received 24.4 mg/kg. The IV dose was
administered in a vehicle, which consisted of 50% propylene glycol,
40% Tris base, and 10% ethanol. This mixture, although used
frequently as an IV vehicle, has the potential to disrupt normal
physiology due to its hyperviscosity and pH. The blank microsphere
curves serve as proof for the negligible interference of p(FA:SA)
with the detection of dicumarol, as both curves for the rat and pig
were extremely low and fluctuated within about 5 mg/mL of the
baseline.
[0126] Drug formulations were administered to animals at a dose of
25 mg/kg. The p(FA:SA) nanospheres, however, were fed at a lower
dose due to difficulty determining dicumarol loading early in the
experiment. Because of this, the amplitude of the plasma curves
cannot be compared directly with the other formulations in FIG. 5;
only the profile can be analyzed, including T.sub.max. The only
comparisons that can be made were based on relative bioavailability
calculated from area under the curve and dose administered which is
presented later in table 2.
[0127] FIG. 5 shows the plasma curves generated from the rat
experiments. All doses were 25 mg/kg except for the p(FA:SA)
formulation, which is 18.2 mg/kg. The micronized drug showed the
highest absorption, reaching 120 .mu.g/mL after only 3 hours,
followed by a continuous decrease until 60 hours. The next highest
concentration was achieved by the spray-dried dicumarol particles,
which reached 90 .mu.g/mL after 3 hours and declined very rapidly
within the next 30 hours. The FA:SA nanosphere formulation showed
excellent absorption, with relatively high concentrations in the
blood until 60 hours. This formulation reached 88 .mu.g/mL after 6
hours, decreasing to 47 .mu.g/mL after 24 hours, where it stayed
for an additional 12 hours. The stock dicumarol showed the lowest
levels of absorption, reaching 64 .mu.g/mL after 3 hours and
decreasing significantly after 15 hours.
[0128] Results in the pig were very similar to those from the rat
(FIG. 6). Again, all doses were 25 mg/kg except for the FA:SA
formulation, which was 18.2 mg/kg. The micronized drug shows very
good absorption, reaching 112 .mu.g/mL after 5 hours, and showing a
second peak at 30 hours. The FA:SA formulation showed a more
prolonged absorption, with high concentrations extending to 30
hours. The spray-dried dicumarol peaked at 2 hours at a
concentration of 86 .mu.g/mL and rapidly decreased by 24 hours. In
both rats and pigs, it was clear that the micronized drug and FA:SA
formulation offered an advantage over the slightly amorphous
spray-dried formulation and the stock dicumarol. Pharmacokinetic
analysis was used to more accurately compare the formulations and
to draw conclusions based on both the animal model as well as the
characteristics of the formulation.
[0129] Pharmacokinetic calculations are presented in Table 2. In
both cases, the FA:SA nanosphere formulation showed the highest
relative bioavailability, with 132% in the rat and 114% in the pig.
The polymer's ability to control the release and absorption was
also reflected by these results. Within each species, C.sub.max was
among the lowest and T.sub.max was the highest in the p(FA:SA)
formulation. Because this system was a solid solution, the
dissolution of the drug was totally dependent upon the degradation
of the polymer, which in this case was a relatively slow surface
degrading mechanism. The micronized drug also showed improved
relative bioavailability over other formulations, with 100% in the
rat and 101% in the pig. But control of dissolution afforded by the
polymer was lost, evidenced by the increase in C.sub.max and
decrease in T.sub.max in both species. The semi-amorphous
spray-dried dicumarol formulation showed the worst absorption, with
only 85% in the rat and 58% in the pig. C.sub.max and T.sub.max
were intermediate between the micronized and FA:SA formulations in
both the rat and pig.
2TABLE 2 Pharmacokinetic calculations. Dose C.sub.max T.sub.max AUC
Relative Formulation (mg/kg) (.mu.g/mL) (hours) (.mu.g-hour/ml)
Bioavailability RAT MODEL IV Bolus 25 129 .+-. 10.0 3 .+-. 0.0 2625
.+-. 143 100 .+-. 0.0% Stock 25 73 .+-. 6.0 14 .+-. 2.0 2666 .+-.
206 90.1 .+-. 7.3% Spray-Dried 25 100 .+-. 6.0 7 .+-. 1.0 2238 .+-.
109 85.3 .+-. 4.2% Micronized Drug 25 144 .+-. 13.0 4.2 .+-. 0.7
2624 .+-. 233 100.0 .+-. 9.1% FA:SA Nanospheres 18.2 75 .+-. 7.7
21.1 .+-. 7.5 2535 .+-. 157 132.6 .+-. 10.7% PIG MODEL IV Bolus 24
178 .+-. 16.5 1.0 .+-. 0.0 2116 .+-. 155 100 .+-. 0.0% Spray-Dried
25 91 .+-. 5.0 2.8 .+-. 0.8 1283 .+-. 102 58.2 .+-. 7.5% Micronized
Drug 25 117 .+-. 13.1 6.5 .+-. 0.9 2366 .+-. 407 100.9 .+-. 8.9%
FA:SA Nanospheres 18.2 67 .+-. 3.4 10.4 .+-. 0.6 1848 .+-. 164
113.8 .+-. 14.6%
[0130] Statistical analysis of the pharmacokinetic data was
performed in order to compare the spray-dried, micronized, and
FA:SA nanosphere dicumarol formulations. Two-tailed t-tests were
used to compare AUCs, and in the case of the FA:SA formulation,
AUCs were normalized by dose to account for the difference. In all
cases where sample populations differ, unequal variance was
assumed. In the rat model, the FA:SA nanosphere formulation was
statistically different from the other formulations with p<0.03
in all cases. In the pig both the FA:SA formulation and micronized
drug were significantly different from the spray dried dicumarol,
with p<0.05 in both cases.
Example 3
Effect of Micronization and Incorporation of a Bioadhesive Polymer
on the Dissolution of Dicumarol--In vitro Effects
[0131] Materials and Methods
[0132] Dicumarol and Reagent Source and methods for
Poly(fumaric-co-sebacic) anhydride synthesis, Scanning Electron
Microscopy and formulating dicumarol were described in Example
1.
[0133] The dicumarol formulation will be referred to throughout
this Example and Example 4 as micronized drug with adhesive
polymer, or MDAP. P(FA:SA) was used in this process because it was
found to prevent aggregation, both in the micronization process as
well as in suspension.
[0134] NMR: A Bruker DPX300 NMR was used for ID proton NMR
analysis. The polymer in deuterated chloroform was analyzed using
peak ratios of the olefinic protons of the fumaric acid monomer
(.delta.=6.91 and 6.97) and the internal aliphatic protons of the
sebacic acid monomer (.delta.=1.32). The normalized molar ratio is
determined to be FA:SA 17:83.
[0135] GPC: For analysis of molecular weight, a 5% solution of
p(FA:SA) in chloroform was analyzed on a Perkin Elmer LC pump model
250 gel permeation chromatography system composed of an isocratic
LC pump model 250, an LC column oven model 101, an LC-30 RI
detector, and a 900 series interface computer. Samples were eluted
through a PL gel 5 .mu.m mixed column and a 5 .mu.m/50 .ANG. column
connected in series at a flow rate of 1.0 mL/min and a temperature
of 40.degree. C. The system was calibrated with a series of
monodisperse polystyrene standards (MW: 600-200,000) in chloroform,
and the molecular weight of p(FA:SA) was 12 kDa. The polymer was
stored under a nitrogen purge at -20.degree. C. until use.
[0136] Microencapsulation: Using different fabrication techniques,
microsphere characteristics were varied based on formulation
parameters including size and bioadhesiveness. These two parameters
were key to the enhancement of relative bioavailability, and it is
our aim to fabricate enough formulations to differentiate their
relative importance. All formulations were frozen, lyophilized for
at least 24 hours, and kept at -20.degree. C. until use.
[0137] The micronized formulations discussed previously were fed by
themselves orally to pigs. Additionally, these formulations were
encapsulated in different size distributions and polymer
compositions using the phase inversion technique. This resulted in
solid solutions within their respective polymer carriers.
[0138] The phase inversion procedure was used to produce nano- and
microspheres. 666 mg spray-dried dicumarol was probe sonicated for
3 minutes at amplitude 35% in 25 mL methylene chloride. 1.0 g
p(FA:SA) 17:83 was added to this and sonicated for an additional 30
s. The resultant solution (4% w/v p(FA:SA)) containing dicumarol
microparticulates was dispersed into 1.0 L petroleum ether and the
precipitate was collected using either a 100 or 220 nm filter. The
microsphere formulation was then frozen and lyophilized for 24
hours. We will refer to this formulation as adhesive microspheres
(AM). Another formulation was fabricated using the same technique
but encapsulating the MDAP micronized drug. We will refer to this
formulation as adhesive nanospheres (AN).
[0139] A non-adhesive formulation was made from poly(lactic acid)
(PLA) with a molecular weight of 24 kDa using the same technique
and encapsulating the MDAP micronized drug. This formulation will
be referred to as nonadhesive nanospheres (NN).
[0140] The dicumarol loading was determined in all formulations by
a simple extraction protocol. Microspheres were incubated overnight
in 2.5 N NaOH at 37.degree. C. Upon dissolution, an aliquot of
approximately 15 .mu.g based on theoretical loading was added to
2.5 N NaOH to make a total volume of 800 .mu.L. This mixture was
agitated for 2 minutes and centrifuged for 2 minutes at 11,269 g.
The supernatant was removed and analyzed on the Shimadzu UV-2501
spectrophotometer and compared to a linear standard curve of
dicumarol in NaOH.
[0141] Due to the size of the animal model, we scaled up our
fabrication process. Because the initial solvent ratios were
crucial for reproducibility, we maintained the conditions for this
step as previously described. 10 batches were fabricated
simultaneously, and they were combined into a pressure pot for
filtration. The precipitate in petroleum ether was passed through a
293 mm wide plate filter made by Millipore.RTM. with a pore size of
either 100 or 220 nm depending on the formulation.
[0142] Particle Sizing: All microspheres and micronized
formulations were sized using laser-light scattering via the
Coulter Particle Size Analyzer LS 230. A 250 .mu.g/mL suspension of
microspheres in 1% pluronic F127 [poly(ethylene
oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)]/1%
hydroxypropylmethylcellulose (HPMC) was introduced into the small
volume fluid module. Only the Coulter output based on volume
measurements was used for analysis.
[0143] In vitro Release Study: We generated release profiles for
all of the formulations by incubating them in PBS Buffer (pH=7.2)
at 37.degree. C. Sink conditions were maintained by keeping the
total concentration of dicumarol in water below the solubility
limit, 28 .mu.g/ml. All release studies were scaled up to 5 mg
dicumarol in 180 mL PBS buffer, and each group consisted of n=4
samples. At different time points, 120 .mu.L supernatant was
obtained from each sample, placed into an Amicon Ultrafree-MC
centrifugal filter device with a nominal molecular weight cutoff of
5 kDa, and centrifuged for 5 minutes at 11,269 g to remove any
residual crystallized dicumarol. 100 .mu.L of the supernatant was
removed and stored at 4.degree. C. until it was analyzed. This
amount was replenished with fresh buffer.
[0144] Results
[0145] Formulation Characterization: SEM analysis revealed very
different morphologies of the various formulations (FIG. 7). The
stock dicumarol is shown in FIG. 7A to illustrate the dissimilar
appearance compared to the formulations prepared for these
experiments. It has a very blocky structure with smooth surfaces,
and is approximately 25 .mu.m in size. The spray-dried formulation
(SD) show a morphology resembling hollow and solid spheres with a
small amount of nanoprecipitate on the surface (FIG. 7B). It is
much smaller than the stock dicumarol. The micronized drug with 7%
p(FA:SA) (MDAP) is much smaller in size than the spray-dried and
stock dicumarol, and retained the somewhat blocky appearance of the
original drug particles (FIG. 7C). This population of particles
also appeared to be very monodisperse. The microspheres obtained by
encapsulating the spray-dried drug in p(FA:SA) (AM) showed a very
porous structure, were somewhat uniform in size, and were not
aggregated (FIG. 7D). The p(FA:SA) microspheres encapsulating the
dissolved MDAP formulation (AN) had a much more spherical shape
than the micronized formulation from which it was made and were
just slightly larger in size (FIG. 7E). The polymer coating also
appears to have a porous structure. The non-adhesive poly(lactic
acid) formulation (NN) fabricated with the MDAP formulation were
extremely small and showed evidence of encapsulation based on the
uniform morphology of the microsphere population (FIG. 7F).
[0146] Coulter particle size analysis revealed quantitatively the
differences seen in the SEM micrographs. The results are presented
in Table 3, along with a description of each formulation and the
abbreviation we were using to describe it. The volume statistics
can be found corresponding to each formulation, and the data are
displayed as the percent of the population less than the size
listed. Analysis of the data for the 50 percentile showed that the
MDAP micronized drug made from the novel process described earlier
and the formulations incorporating it, AN and NN, were much smaller
than the spray-dried formulations.
[0147] Drug loading determined by NaOH extraction was also
presented in Table 3. The encapsulated formulations containing
polymer ranged from 31% to 40% loading, which is a function of the
relative solubility of the polymer and drug within the
encapsulation process. MDAP was found to be composed of 93%
dicumarol, so this formulation will be considered a micronized drug
with a small amount of bioadhesive enhancer.
3TABLE 3 Formulation parameters and size information. Coulter data
is based on volume measurements. Formulation Coulter Sizing
Information Drug Name Composition 25% < 50% < 75% <
Loading SD Spray-Dried microparticulates 1.474 .mu.m 3.053 .mu.m
5.319 .mu.m 100% MDAP Micronized dicumarol with 0.415 .mu.m 0.525
.mu.m 0.612 .mu.m 93% 7% adhesive polymer AM Adhesive microapheres
1.421 .mu.m 3.533 .mu.m 8.208 .mu.m 40% (p(FA:SA) 17:83
microspheres with dicumarol particulates) AN Adhesive nanospheres
0.782 .mu.m 1.414 .mu.m 2.312 .mu.m 31% (p(FA:SA) 17:83 nanospheres
with homogenous drug dispersion) NN Nonadhesive nanospheres 0.478
.mu.m 0.552 .mu.m 0.650 .mu.m 39% (PLA nanospheres with homogenous
drug dispersion)
[0148] The general nature of the in vitro dissolution and release
curves for each formulation was one of sporadic dissolution and
recrystallization (FIG. 8). This was seen by the increasing
concentration followed by a lower concentration, sometimes again
followed by another increase. This dynamic process is due to the
hydrophobicity of the drug and other thermodynamic considerations.
Presumably, as the micronized drug was dissolved, a certain
population in solution was already recrystallizing into another
population of solid particles, which were larger than the original
formulation because of the slower rate at which crystallization
occurs at 37.degree. C. in buffer compared to the conditions of the
fabrication process. These large particles then underwent another
round of dissolution, which was much slower and was occurring
simultaneously with the dissolution of another population of
micronized particles from the original formulation. Only the first
100 hours of the in vitro dissolution curves are presented here in
order to compare these curves to the in vivo plasma curves, which
terminate at 96 hours.
[0149] Overall, the particles made from MDAP showed a much more
pronounced dissolution than the formulations made with the
spray-dried dicumarol. Both MDAP and AN showed a very nice
correlation between the amount of drug in solution and the amount
of p(FA:SA) in the formulation. The curves were staggered based on
drug loading, with the MDAP formulation with 93% dicumarol loading
showing the highest amount of dissolution, and AN with 31% loading
showing slightly less. The NN microspheres showed a much slower and
controlled dissolution, probably because of the hydrophobic nature
of the highly crystalline 24 kDa poly(lactic acid) used to
encapsulate the drug. The larger spray-dried formulations, AM and
SD, both showed extremely low levels of dissolution, and in both
cases, concentrations did not rise to comparable levels until 8
hours. This resembled more of a depot effect than the encapsulated
micronized formulations.
Example 4
Effect of Micronization and Incorporation of a Bioadhesive Polymer
on the Relative Bioavailability of Dicumarol--In vivo Effects
[0150] Animal Model: The following animal work was performed in
accordance with the Principles of Laboratory Animal Care (NIH
publication #85-23, revised 1985). Female Yorkshire pigs were used
throughout the study, with starting weights ranging from 15 to 20
kg, and were divided into groups of n=3, 4, or 5 depending on
availability. The groups were kept for 12-14 weeks and were
administered each formulation throughout the study.
[0151] Following a 2-week acclimation period, catheters were
surgically implanted into the external jugular vein of each of the
pigs. Under isoflurane anesthesia, an incision from just cranial to
the sternum to a point just caudal of the angle of the jaw was
made. Using blunt dissection, a section of the external jugular
vein was isolated. A Swan Ganz catheter was then fed through a
subcutaneous tunnel starting between the shoulders to the point of
the isolated jugular vein. A small incision was made in the vein
and the catheter was advanced towards the atrium until arrhythmia.
At this point, the catheter was retracted 3 cm distally and was
anchored to the surrounding tissues. The catheter was tested for
patency and flow, blocked with heparin, and the wound was closed.
The length of the catheter exiting from between the shoulders was
coiled up and stored within a protective jacket placed on each
animal.
[0152] After a fasting period of 12 hours, animals were orally
gavaged with microsphere formulations suspended in a solution of 1%
HPMC and 1% pluronic F 127. The microsphere dose was suspended
immediately prior to administration using bath sonication for 3
minutes and was administered to the stomach through a gavage tube
placed with the aid of a laryngoscope. The suspension concentration
was kept constant at 50 mg/mL, and several flushes of vehicle were
administered following the dose. For most groups, the dose given
was approximately 25 mg/kg, and several study groups were given
either 5 mg/kg or 15 mg/kg dicumarol to investigate the dose
response of 2 of the micronized formulations containing polymer.
During administration, animals were sedated with a combination of
ketamine and medetomidine, and immediately following the procedure,
the medetomidine-antagonist atipamezole was given for reversal of
anesthesia.
[0153] A control group was gavaged with blank p(FA:SA) 17:83
microspheres suspended in 1% F127/1% HPMC in order to generate a
baseline for the experiment. Additionally studied was an IV group
to which we administered 25 mg/kg dicumarol dissolved in a mixture
of 50% propylene glycol, 10% ethanol, and 40% 100 mM Tris at a pH
of 9.0. The dicumarol was dissolved in the vehicle at a
concentration of 20 mg/mL and administered through the catheter
over a period of 5 minutes.
[0154] At specific time points, generally 0, 1, 2, 3, 5, 8, 11, 14,
25, 29, 36, 48, 60, 72, 84, and 96 hours, blood was collected from
each animal. The heparin block was removed, 1 cc of fresh blood was
collected, and another 1.5 cc heparin solution was added to the
catheter. The blood samples were collected in heparinized 1.5 mL
siliconized microfuge tubes and centrifuged for 5 minutes at 11,269
g. Approximately 500 .mu.L plasma was removed and stored at
4.degree. C. prior to being analyzed.
[0155] Methods for Dicumarol Quantification, Bioactivity Assays,
and Statistics and Pharmacokinetic Analysis were described in
Example 2.
[0156] Results of In vivo Studies
[0157] Control Curves: The IV curve was as expected, with a very
sharp peak followed by a rapid decline in plasma levels (FIG. 9).
The majority of the drug was absorbed in the first 24 hours.
[0158] Spray-dried Formulations: Both SD and AM formulations were
fed at a dose of 25 mg/kg dicumarol, thus the dose for the AM
formulation and for all other formulations containing polymer are
scaled up because the loading was less than 100%. The plasma curves
are shown in FIG. 10. Both of the formulations showed an obvious
decrease in the concentration achieved compared to the IV curve in
FIG. 9. They offered a small amount of elongation of absorption,
but were overall much less pronounced. Between the two
formulations, SD showed an improvement over the encapsulated
formulation, which also declined to zero just after 30 hours.
[0159] Micronized Formulations: We attempted to maintain a standard
dose of 25 mg/kg dicumarol throughout these experiments. However,
due to the difficulty in determining the loading at the onset of
the study, three of the formulations were fed a different dose,
with AN receiving 18.2 mg/kg, NN receiving 30.6 mg/kg, and MDAP
receiving 23.0 mg/kg. For this reason, the plasma curves for these
three formulations could not be directly compared to each other or
any of the other curves. They can, however, be compared when
calculating relative bioavailability, because it is normalized by
dose. Additionally, statistical analysis using a t-test also
necessitated normalization by dose received.
[0160] The plasma curves from the studies using the formulations
with micronized drug are shown in FIG. 11. Compared to the
absorption of the spray-dried formulations, the overall magnitude
of absorption for these curves was elevated. The curves in FIG. 11
were very similar in shape, except for the NN formulation, which
showed an early peak with very low levels of absorption in the
later time points. Because the doses given were not the same, only
the curve profiles can be compared, and not the magnitude. All of
the micronized formulations showed relatively high concentrations
in the blood up until 60 hours, compared to only 30 hours with the
spray-dried formulations. The dose escalation curves were shown in
FIGS. 12 and 13. Doses for the AN formulation were 3.6 mg/kg, 10.9
mg/kg, and 18.2 mg/kg. Animals dosed with MDAP were fed doses of 5
mg/kg, 15 mg/kg, and 23 mg/kg. The AN curves in FIG. 12 show
elevated plasma levels for each increase in dose, and the time at
which the concentration is the highest, T.sub.max, is also greater
for each increasing dose. MDAP curves show similar results, with
each dose increasing the overall plasma levels significantly,
except that the T.sub.max shows no distinctive pattern.
[0161] Pharmacokinetic Analysis: The effect of the formulations in
this study can be compared by calculating the pharmacokinetic
parameters: relative bioavailability (BA), C.sub.max, and
T.sub.max. Table 4 displays this data, and we will continue to
refer to it throughout this section.
[0162] As seen in the plasma curves, the formulations containing
spray-dried drug showed much lower plasma levels than the
formulations with micronized drug. Within the spray-dried group,
relative bioavailability was 40% for the SD formulation and 31% for
the AM formulation. T-tests performed on AUC values normalized by
dose show that the absorption of SD and AM were statistically
different, with p=0.03. Additionally, the C.sub.man of the SD
formulation was almost double that of the encapsulated version,
which illustrates the degree of control that the polymer can offer
to reduce absorptive bursts. T.sub.max was also prolonged in the AM
formulation, again probably because of the modulation of the
polymer coating.
4TABLE 4 Pharmacokinetic Analysis Drug Dose Formu- (mg/ AUC
Relative C.sub.max T.sub.max lation kg) (.mu.g-hour/ml)
Bioavailability (.mu.g/mL) (hours) IV 24 2116 .+-. 155 100 .+-.
0.0% 178 .+-. 16.5 1.0 .+-. 0.0 SD 25 1283 .+-. 102 58 .+-. 7.5% 91
.+-. 5.0 2.8 .+-. 0.8 AM 25 916 .+-. 85 41.0 .+-. 7.0% 48.5 .+-.
10.4 5.8 .+-. 1.4 MDAP 5 576 .+-. 148 131.2 .+-. 33.8% 35.0 .+-.
7.0 5.5 .+-. 2.5 MDAP 15 1175 .+-. 239 89.6 .+-. 18.8% 60.0 .+-.
8.5 2.3 .+-. 0.3 MDAP 23 2247 .+-. 197 110.5 .+-. 6.7% 95.8 .+-.
6.5 7.4 .+-. 1.1 AN 3.6 480 .+-. 87 110.8 .+-. 4.6% 24.3 .+-. 4.8
5.3 .+-. 1.4 AN 10.9 1095 .+-. 140 149.5 .+-. 27.6% 38 .+-. 5.7 7.0
.+-. 1.0 AN 18.2 1848 .+-. 164 113.8 .+-. 14.6% 67 .+-. 3.4 10.4
.+-. 0.6 NN 30.6 1430 .+-. 128 25.3 .+-. 3.3% 97 .+-. 9.5 3.7 .+-.
0.7
[0163] Most of the formulations containing the ultra-micronized
drug were significantly higher in relative bioavailability than the
spray-dried formulations. T-tests comparing the normalized AUCs of
MDAP and AN to both SD and AM showed statistical significance
between the two groups of formulations, with p values ranging from
0.001 to 0.05. The nonadhesive NN formulation was also considerably
lower than MDAP and AN using the micronized drug, and p<0.05
from t-tests comparing NN to these other formulations.
[0164] The micronized drug with 7% FA:SA, MDAP, improved the
relative bioavailability of dicumarol to 76.5%. C.sub.max was
relatively high, at 95.8 .mu.g/mL, and peak concentrations were
seen in the plasma early in the experiment, with a T.sub.max of 7.4
hours. The addition of a higher content of FA:SA further improved
these parameters. The drug fully encapsulated in p(FA:SA), AN,
showed a reduction in C.sub.max to 67 .mu.g/mL and T.sub.max is
increased to 10.4 hours. The pronounced modulation of both
C.sub.max and T.sub.max can be attributed to the bioadhesion
afforded by FA:SA.
[0165] NN, containing the non-adhesive polymer coating,
significantly decreased the relative bioavailability to 37%.
Additionally, t-tests using normalized AUCs revealed p<0.05
between NN and all other formulations containing micronized drug.
The other two virtues of the bioadhesive coating, control of
C.sub.max and prolongation of T.sub.max, also disappeared compared
to the adhesive AN formulation, as C.sub.max increased from 67
.mu.g/mL to 97 .mu.g/mL and T.sub.max dropped from 10.4 hours to
3.7 hours.
[0166] The dose escalation studies showed that with both AN and
MDAP, the lowest dose was absorbed most efficiently, and relative
bioavailability reached 100% and 91% respectively. As the dose was
increased, relative bioavailability decreased, especially in the
MDAP group, where it fell to 62% at a dose of 15 mg/kg. Levels
improved to a more reasonable figure of 77% at 23 mg/kg. AN also
showed a decrease in relative bioavailability as the dose was
increased, but it leveled off at around 79% in this case. C.sub.max
rose with each increasing dose in both cases, and T.sub.max rose
proportionally only in the AN formulation.
[0167] Bioactivity: All of the formulations presented tested
positive for drug activity, with PTT times of samples taken at the
time point corresponding to the T.sub.max all exceeding 90 seconds
compared to the normal range of 12 to 17 seconds.
[0168] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the
invention.
[0169] All references, patents and patent publications that are
recited in this application are incorporated in their entirety
herein by reference.
* * * * *