U.S. patent application number 10/763149 was filed with the patent office on 2004-08-05 for stable flowable protien and nucleic acd formulations using non-aqueous, anhydrous, aprotic, hydrophobic, non-polar vehicles with low reactivity.
This patent application is currently assigned to Alza Corporation. Invention is credited to Huang, Manley T., Maskiewicz, Victoria Knepp, Prestrelski, Steven Joseph, Smith, Jessica G..
Application Number | 20040151779 10/763149 |
Document ID | / |
Family ID | 26703379 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040151779 |
Kind Code |
A1 |
Maskiewicz, Victoria Knepp ;
et al. |
August 5, 2004 |
Stable flowable protien and nucleic acd formulations using
non-aqueous, anhydrous, aprotic, hydrophobic, non-polar vehicles
with low reactivity
Abstract
This invention relates to stable non-aqueous formulations which
are suspensions of proteinaceous substances or nucleic acids in
non-aqueous, anhydrous, aprotic, hydrophobic, non-polar vehicles
with low reactivity. More specifically, the present invention
relates to stable protein or nucleic acid formulations wherein the
compound remains in stable, dry powder form, yet the formulation is
flowable and, therefore amenable to delivery to an animal via
injection transdermal administration, oral delivery or using an
implantable device for sustained delivery. These stable
formulations may be stored at elevated temperatures (e.g.,
37.degree. C.) for long periods of time and are especially useful
as flowable formulations which can be shipped and/or stored at high
temperatures or in implantable delivery devices for long term
delivery (e.g., 1-12 months or longer) of drug.
Inventors: |
Maskiewicz, Victoria Knepp;
(Ridgefield, CT) ; Prestrelski, Steven Joseph;
(Mountain View, CA) ; Smith, Jessica G.;
(Sunnyvale, CA) ; Huang, Manley T.; (Palo Alto,
CA) |
Correspondence
Address: |
PHILIP S. JOHNSON
JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Assignee: |
Alza Corporation
|
Family ID: |
26703379 |
Appl. No.: |
10/763149 |
Filed: |
January 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10763149 |
Jan 22, 2004 |
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09849402 |
May 4, 2001 |
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6730328 |
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09849402 |
May 4, 2001 |
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09269685 |
Dec 14, 1999 |
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6264990 |
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09269685 |
Dec 14, 1999 |
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PCT/US97/18575 |
Oct 15, 1997 |
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60028167 |
Oct 16, 1996 |
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60052920 |
Jul 15, 1997 |
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Current U.S.
Class: |
424/499 |
Current CPC
Class: |
A61K 38/212 20130101;
A61K 9/10 20130101; A61K 47/06 20130101; A61P 7/02 20180101; A61K
9/0014 20130101; A61K 9/0026 20130101; A61K 9/0019 20130101; A61K
38/4826 20130101; A61K 48/00 20130101; A61K 38/38 20130101; A61K
9/0024 20130101; A61K 38/4846 20130101 |
Class at
Publication: |
424/499 |
International
Class: |
A61K 009/50 |
Claims
What is claimed is:
1. A stable non-aqueous composition of an active agent comprising:
a) an active agent containing powder wherein the active agent
hydration in said powder is less than about 10%; and b) at least
one anhydrous, aprotic, hydrophobic, non-polar, low-reactivity
vehicle, wherein said active agent is selected from the group
consisting of proteins, proteinaceous compounds, and nucleic
acids.
2. The composition of claim 1 wherein at least about 80% of the
active agent remains stable for at least one month at 37.degree.
C.
3. The composition of claim 1 wherein said active agent hydration
is less than about 5%.
4. The composition of claim 1 wherein said vehicle is selected from
the group consisting of perhalohydrocarbons, unsubstituted
saturated hydrocarbons, halogenated hydrocarbons, esters of
unsubstituted saturated or halogenated hydrocarbons and ethers of
unsubstituted saturated or halogenated hydrocarbons.
5. The composition of claim 1 wherein said vehicle is selected from
the group consisting of MO, PFD, MF, PTA and TD.
6. The composition of claim 1 wherein said powder comprises up to
about 30% (w/w) of said composition.
7. The composition of claim 1 wherein said active agent is a
protein selected from the group consisting of Factor IX, Factor
VIII, alpha-interferon, consensus interferon, beta-galactosidase,
lactate dehydrogenase, chymotrypsin, trypsinogen, an antibody, and
analogs thereof.
8. The composition of claim 1 wherein said active agent is a
nucleic acid selected from the group consisting of DNA, RNA and
oligonucleotides.
9. The composition of claim 8 wherein said nucleic acid is in the
form of at least one selected from the group consisting of a
nucleic acid/lipid complex, a nucleic acid-containing liposome, a
ribozyme, a viral vector, a virosome, nucleic acid-containing
dendrimers, nucleic acid-containing cationic polymers and
nucleic-acid-containing PLGA particles.
10. The composition of claim 1 wherein said active agent is
pharmaceutically useful.
11. A method for preparing the composition of claim 1 comprising
suspending an active agent-containing powder with active agent
hydration less than about 10% in at least one anhydrous, aprotic,
hydrophobic, non-polar, low-reactivity vehicle.
12. A method for treating a subject suffering from or susceptible
to a condition which may be alleviated or prevented by
administration of an active agent according to claim 1, said method
comprising administering to said subject an effective amount of the
composition of claim 1.
13. The method of claim 12 wherein said condition is hemophilia and
the active agent in said composition is selected from the group
consisting of Factor VIII, Factor IX, and analogs thereof.
14. The invention of any of claims 1 and 11 wherein said powder
comprises about 10 to about 30% (w/w) of said composition.
15. The invention of any of claims 1 and 12 wherein administration
of the composition is via a route selected from the group
consisting of parenteral, transdermal, mucosal, oral and
enteral.
16. The invention of any of claims 1 and 12 wherein administration
of the composition is via an implantable delivery device.
17. The invention of any of claims 1 and 12 wherein administration
of the composition is long-term continuous administration.
Description
FIELD OF THE INVENTION
[0001] This invention relates to stable non-aqueous formulations of
proteins and nucleic acids. The stable formulations of the present
invention are suspensions of particles containing proteins or
nucleic acids in non-aqueous, anhydrous, aprotic, hydrophobic,
non-polar vehicles with low reactivity.
BACKGROUND OF THE INVENTION
REFERENCES
[0002] The following references are referred to by numbers in
brackets ([ ]) at the relevant portion of the specification.
[0003] 1. Ahem and Manning, Eds., Stability of Protein
Pharmaceuticals, A: Chemical and Physical Pathways of Protein
Degradation, Plenum Press, New York, 1992.
[0004] 2. Wang et al., 1988, J. Parenteral Science and Technology
42: S4-S26
[0005] 3. Deetz et al., 1988, Trends in Biotechnol. 6: 15-19
[0006] 4. Chin et al., 1994, Biotechnol. Bioeng. 44: 140-145
[0007] 5. Klibanov, 1989, TIBS 14: 141-144
[0008] 6. Zaks et al., 1984, Science 224:1249-1251
[0009] 7. Affleck et al., 1992, Proc. Natl. Acad. Sci. USA
89:1100-1104
[0010] 8. Zaks et al., 1988, J. Biol. Chem. 263:8017-8021
[0011] 9. Volkin et al., 1991, Biotechnol. Bioeng. 37: 843-853
[0012] 10. Guagliardi et al., 1989, Chimicaoggi 31-36
[0013] 11. Paulaitis et al., 1992, Annals New York Acad. Sci.
672:278-282
[0014] 12. Matsuura et al., 1993, J. Amer. Chem. Soc.
115:1261-1264
[0015] 13. Zaks et al., 1988, J. Biol. Chem. 263:3194-3201
[0016] 14. Prestrelski et al., 1993, Biophys. J. 65:661-671
[0017] 15. Zhang et al., 1995, Pharm. Res. 12, 1447-1452
[0018] 16. Singer et al., 1962, Adv. Prot. Chem. 1-68
[0019] 17. Volkin et al., 1991, Biotechnol. Bioeng. 37: 843-853
[0020] 18. Aldercreutz et al., 1987, Biocatalysis 1: 99-108
[0021] 19. Guinn et al., 1991, Biotechnol. Bioeng. 37: 303-308
[0022] 20. Desai et al. 1995, J. Am. Chem. Soc. 117: 3940-3945
[0023] 21. Yu et al., 1996, J. Pharm. Sci. 85: 396-401
[0024] 22. Burke et al., 1989, J. Am. Chem. Soc. 111: 8290-8291
[0025] 23. Kanerva et al., 1989, J. Am. Chem. Soc. 111:
6865-6866
[0026] 24. Desai et al., 1994, J. Am. Chem. Soc. 116:9420-9422
[0027] 25. Chang et al., January 1996, Pharm. Tech. 80-84
[0028] 26. Manning et al., 1989, Pharm. Res. 6: 903-918
[0029] 27. Hageman, 1988, Drug Dev. Ind. Pharm. 14:2047-2070
[0030] 28. Bell et al., 1995, Biopolymers 35: 201-209
[0031] 29. Meadows, 1996, U.S. Pat. No. 5,480,914
[0032] 30. Meadows, 1996, U.S. Pat. No. 5,518,731
[0033] 31. Hageman, 1994, International Publication No.
WO94/06452
[0034] 32. Hofland et al., 1996, Proc. Natl. Acad. Sci.
93:7305-7309
[0035] 33. Sullivan, 1996, BioPharm September: 50-51 and 65-66.
[0036] 34. Huang et al., 1996, International Publication No.
WO96/27393.
[0037] 35. Debs et al., 1993, International Publication No.
WO93/25673.
[0038] 36. Lemoine and Cooper, Ed., Gene Therapy, Bios Scientific
Publishers, Oxford, UK, 1996.
[0039] 37. Debs et al., 1993, International Publication No.
WO93/24640.
[0040] 38. Gibco technical report.
[0041] 39. Boehringer Mannheim technical report.
[0042] 40. Avanti polar lipid technical report.
[0043] 41. Szoka et al., 1996, International Publication No.
WO96/41873.
[0044] 42. Huang et al., 1990, Nucl. Acids Res. 18(4): 937-947.
[0045] The disclosure of each of the above publications, patents or
patent applications is hereby incorporated by reference in its
entirety to the same extent as if the language of each individual
publication, patent and patent application were specifically and
individually incorporated by reference.
BACKGROUND OF THE INVENTION
[0046] Peptides, polypeptides, proteins and other proteinaceous
substances (e.g., viruses, antibodies), collectively referred to
herein as proteins, have great utility as pharmaceuticals in the
prevention, treatment and diagnosis of disease. Proteins are
naturally active in aqueous environments, thus the preferred
formulations of proteins have been in aqueous solutions. However,
proteins are only marginally stable in aqueous solutions. Thus,
protein pharmaceuticals often require refrigeration or have short
shelf-lives under ambient conditions. Further, many proteins have
only limited solubility in aqueous solutions. Even when they are
soluble at high concentrations, they are prone to aggregation and
precipitation.
[0047] Proteins can degrade via a number of chemical mechanisms,
including deamidation of asparagine and glutamine; oxidation of
methionine and, to a lesser degree, tryptophan, tyrosine and
histidine; hydrolysis of peptide bonds; disulfide interchange; and
racemization of chiral amino acid residues [1, 2 and 24-28]. Water
is a reactant in nearly all of these degradation pathways. Further,
water acts as a plasticizer which facilitates unfolding and
irreversible aggregation of proteins. Since water is a participant
in almost all protein degradation pathways, reduction of the
aqueous protein solution to a dry powder provides an alternative
formulation methodology to enhance the stability of protein
pharmaceuticals. Proteins can be dried using various techniques,
including freeze-drying, spray-drying and dessication. Aqueous
solutions of proteins are thus dried and stored as dry powders
until their use is required.
[0048] A serious drawback to drying of proteins is that often one
would like to use proteins in some sort of liquid form. Parenteral
injection and the use of drug delivery devices for sustained
delivery of drug are two examples of applications where one would
like to use proteins in a liquid form. For injection, dried
proteins must be reconstituted, adding additional steps which are
time-consuming and where contamination may occur, and exposing the
protein to potentially destabilizing conditions [15].
[0049] The sustained parenteral delivery of drugs, in particular
proteins and nucleic acids, provides many advantages. The use of
implantable devices for sustained delivery of a wide variety of
drugs or other beneficial agents is well known in the art. Typical
devices are described, for example, in U.S. Pat. Nos. 5,034,229;
5,057,318; and 5,110,596. The disclosure of each of these patents
is incorporated herein by reference.
[0050] Proteins are only marginally soluble in non-aqueous
solvents, and such solvents typically unfold and denature proteins
[4, 16]. Solubilization of native proteins in non-aqueous solvents
typically requires derivatization or complexation of the protein
[12]. In attempting to achieve enzymatic catalysis in organic
media, Klibanov and others have shown that certain catalytic
enzymes can be suspended in non-aqueous vehicles as powders,
typically in hydrophilic organic solvents including alcohol ketones
and esters [3, 5-11, 13 and 18-23]. With enzyme hydration levels
.gtoreq.10% and/or the addition of low molecular weight protic
compounds, these enzymes can have enough conformational mobility to
exhibit appreciable enzymatic activity. Optimal activity levels are
apparently achieved at enzyme hydration of approximately 30%. At a
minimum, such enzymatic activity requires a level of "essential
water" hydrating the protein. However, hydration levels (generally
10-40% w/w water/piotein) and/or protic solvents, such as those
used in these studies, typically result in unacceptable stability
of proteins for pharmaceutical purposes. A further requirement for
catalysis in non-aqueous solvents is that the enzyme be dried from
a solution having a pH near the optimal pH for the enzymatic
activity. This pH limitation is detrimental to storage of protein
pharmaceuticals, because most protein degradation mechanisms are pH
dependent, and it is often the case that proteins are most stable
when dried at pH values far from the value where they exhibit
bioactivity [1]. Further, such catalytic enzyme systems are not
amenable to the addition of protein stabilizers, particularly those
that function by hydrogen bonding to the protein and reducing
enzyme hydration (e.g. carbohydrates) [14].
[0051] The use of perfluorocarbons as components of drug delivery
vehicles for certain ophthalmic compositions has been disclosed
[29, 30]. Similarly, suspensions of growth hormone in triacetin or
polyethylene glycol has been published [31].
[0052] The field of gene therapy or gene transfer is advancing both
experimentally and clinically. Nucleic acids have been transferred
into cells using viral vectors such as adenovirus, retrovirus,
adeno-associated virus, vaccinia virus, and sindbis virus, among
others. Non-viral methods have also been used, including calcium
phosphate precipitation, DEAE dextran, injection of naked DNA,
electroporation, cochleates, cationic lipid complexes, liposomes,
polymers (such as dendrimers and PLGA), virosomes, and the
like.
[0053] DNA complexed with cationic lipids and/or liposomes has been
shown to be an efficient means of transfecting a variety of
mammalian cells. Such complexes are simple to prepare and may be
used with a wide variety of DNA's and RNA's with little restriction
to the size of nucleic acid. They have the ability to transfect
many different cell types with efficiency and are not immunogenic
[32, 33, 35, 36]. Current nucleic acid formulations, including
DNA/liposome and RNA/liposome complexes, must be mixed shortly
before administration, resulting in inconvenience in manufacture,
shipping, storage and administration [35, 37-40]. Frequently, these
two-part formulations are not very highly concentrated, requiring
the administration of large volumes of solution. Dry powder
formulations containing lyophilized nucleic acid/liposome complexes
have also been used [34, 41], but they require reconstitution with
suitable aqueous solution just prior to administration. Aqueous
complexes are inherently unstable and lose most, if not all, of
their transfection activity within hours or a few days [41].
[0054] Consequently, there is a need for pharmaceutical
compositions that can overcome these limitations of the prior art.
Such a composition should maintain the stability of the active
compound, preferably at both room and body temperature (25 and
37.degree. C.), and exist in at least a flowable state for
injection, incorporation into delivery systems designed for
immediate, delayed, or long term administration or other means of
administration.
SUMMARY OF THE INVENTION
[0055] The present invention provides stable non-aqueous
formulations which are suspensions of peptides, polypeptides,
proteins and other proteinaceous substances ("proteins" or
"proteinaceous substances") or DNA- and RNA-containing compositions
("nucleic acids") in anhydrous, aprotic, hydrophobic, non-polar
vehicles with low reactivity. More specifically, the present
invention relates to stable formulations wherein the proteinaceous
substance or nucleic acid remains in stable, dry powder form, yet
the formulation is flowable and, therefore amenable to delivery to
an animal via for example, injection, ambulatory infusion or an
implantable device for sustained delivery. These stable
formulations may be stored at elevated temperatures (e.g.,
37.degree. C.) for long periods of time and are especially useful
as flowable formulations which can be shipped and/or stored at high
temperatures or in implantable delivery devices for long term
delivery (e.g., 1-12 months or longer) of drug.
[0056] In one aspect, the invention provides stable protein
compositions comprising a proteinaceous powder wherein the protein
hydration in said powder is less than about 10%; and at least one
anhydrous, aprotic, hydrophobic, non-polar, low-reactivity vehicle.
In a preferred embodiment, up to about 30% (w/w) proteinaceous
powder may be used in these flowable compositions.
[0057] In another aspect, the invention provides methods for
preparing stable protein compositions, said methods comprising
suspending a proteinaceous powder with protein hydration less than
about 10% in at least one anhydrous, aprotic, hydrophobic,
non-polar, low-reactivity vehicle.
[0058] In a further aspect, the invention provides methods for
treating a subject suffering from or susceptible to a condition
which may be alleviated or prevented by administration of a
proteinaceous compound, said methods comprising administering to
said subject an effective amount of a stable flowable protein
composition comprising a proteinaceous powder wherein the protein
hydration in said powder is less than about 10%; and at least one
anhydrous, non-polar, aprotic, hydrophobic, low-reactivity
vehicle.
[0059] In yet a further aspect, the invention provides stable
nucleic acid compositions comprising a nucleic acid-containing
powder wherein the nucleic acid hydration in said powder is less
than about 10%; and at least one anhydrous, non-polar, aprotic,
hydrophobic, low-reactivity vehicle.
[0060] In yet still another aspect, the invention provides methods
for preparing stable nucleic acid compositions, said methods
comprising suspending a nucleic acid-containing powder with nucleic
acid hydration less than about 10% in at least one anhydrous,
non-polar, aprotic, hydrophobic, low-reactivity vehicle.
[0061] In yet still a further aspect, the invention provides
methods for treating a subject suffering from or susceptible to a
condition which may be alleviated or prevented by administration of
a nucleic acid-containing compound, said methods comprising
administering to said subject an effective amount of a stable
nucleic acid composition comprising a nucleic acid-containing
powder wherein the nucleic acid hydration in said powder is less
than about 10%; and at least one anhydrous, non-polar, aprotic,
hydrophobic, low-reactivity vehicle.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention is drawn to the unexpected discovery
that suspending dry protein- or nucleic acid-containing particles
in anhydrous, aprotic, hydrophobic, non-polar vehicles of low
reactivity results in stable flowable non-aqueous formulations.
Previously known formulations of proteinaceous compounds, which are
dilute buffered aqueous solutions containing excipients such as
EDTA or polysorbate 80 which must be stored at low temperatures
(4-25.degree. C.), or lyophilized powders or particles which must
often be stored at low temperature and must then be reconstituted
before administration, form degradation products using degradation
pathways such as acid/base catalyzed hydrolysis, deamidation,
racemization and oxidation. Similarly, previously known
formulations of nucleic acids, even those prepared from lyophilized
powders, are administered as dilute aqueous solutions which are not
stable for long periods of time and which must be stored at low
temperatures. In contrast, the presently claimed formulations
stabilize proteins and nucleic acid compounds at elevated
temperatures (e.g., 37.degree. C.) and at high concentrations
(i.e., up to about 30%).
[0063] Standard peptide and protein formulations consist of dilute
aqueous solutions. Drug stability is usually achieved by varying
one or more of the following: pH, buffer type, ionic strength,
excipients (EDTA, polysorbate 80, etc). For these formulations,
degradation pathways requiring water (hydrolysis, deamidation,
racemization) cannot be fully stabilized. In contrast, in the
present invention, proteinaceous compounds formulated in
non-aqueous, anhydrous, aprotic, hydrophobic, non-polar vehicles
with low-reactivity, such as mineral oil (MO), perfluorodecalin
(PFD), methoxyflurane (MF), perfluorotributylamine (PTA) and
tetradecane (TD), were shown to be chemically and physically more
stable than those formulated in aqueous solution. MO, PFD, MF, PTA
and TD are considered anhydrous, aprotic, hydrophobic, non-polar
vehicles of low reactivity. Such vehicles decrease the rate of
degradation since they isolate the proteinaceous compounds from
water and they lack the ability to contribute protons or other
reactive moieties to degradation reactions.
[0064] The invention consists of using anhydrous, aprotic,
non-polar, hydrophobic vehicles with low reactivity such as MO,
PFD, MF, PTA or TD to stabilize protein formulations against both
chemical and physical degradation. The discovery consists of the
realization that use of MO, PFD, MF, PTA or TD improves the overall
stability of proteins in a wide range of formulation conditions,
including high concentrations and elevated temperatures, thus
making possible shipping and/or storage of protein formulations at
ambient temperature and the delivery of proteins in long term
implantable devices that would not otherwise be feasible. The
present invention provides flowable pharmaceutical formulations of
proteinaceous substances that exhibit the requisite protein
stability. These non-aqueous formulations comprise two components:
1) a protein in a stabilized powder formulation of low protein
hydration; and 2) an anhydrous, hydrophobic, aprotic, non-polar
vehicle of low reactivity and solubility power towards protein
compounds. Optionally, the dry powder form of the protein may
contain stabilizers and other excipients. Such stabilizers and
excipients are those that further reduce protein hydration or
protect from interfacial tension or other dehydration
process-specific destabilization known to those skilled in the
art.
[0065] Among other factors, it has been surprisingly discovered
that when dispersed in certain vehicles, protein powders can
display significantly greater stability than that observed for the
dry powder alone. Such vehicles include long-chain alkanes, most
preferably perfluorinated forms of alkanes. The present invention
is especially advantageous because it provides the capacity to
store proteins under ambient conditions for extended periods or to
deliver proteins from implantable pumps for extended durations.
[0066] Lipid/DNA and lipid/RNA complexes facilitate nucleic acid
uptake into cells both in-vitro and in-vivo. However, such
complexes are inherently unstable in solution, losing most, if not
all, of their activity after only a few days at ambient
temperatures. This feature severely limits their applicability for
use in such devices as implantable pumps, depot injection or other
sustained release delivery systems where prolonged residence at
37.degree. C. is needed. Lyophilization of these complexes results
in more stable compositions, but such powders require
reconstitution prior to administration to render them flowable, and
the reconstituted solutions are not stable. The present invention
provides flowable pharmaceutical formulations of nucleic acids that
exhibit the requisite stability. These non-aqueous formulations
comprise two components: 1) a nucleic acid in a stabilized powder
formulation of low hydration; and 2) an anhydrous, hydrophobic,
aprotic, non-polar vehicle of low reactivity and solubility power
towards nucleic acids. Optionally, the dry powder form of the
nucleic acid may contain the nucleic acid in the form of lipid/DNA
complexes, liposomes ribozymes, viral vectors, virosomes,
dendrimers, cationic polymers, PLGA particles or the like, and/or
may optionally contain stabilizers and other excipients. Such
stabilizers and excipients are those that further reduce hydration
or protect from interfacial tension or other process-specific
destabilization known to those skilled in the art.
[0067] The flowable formulations of the present invention are
useful in a variety of delivery systems, including, but not limited
to, various pumping devices (syringe pumps, implantable pumps,
etc.), transdermal reservoir systems, liquid fill capsules,
injectable depot compositions and the like. An additional advantage
of the present invention over the prior art is that the
formulations of the present invention prevent back diffusion of
water vapor (and subsequent hydrolytic degradation) because the
hydrophobic vehicle of the formulation acts as a barrier to water
vapor. This is especially important when the formulations are used
in implantable devices which must remain in an aqueous environment
at elevated temperatures for long periods of time.
[0068] A further advantage of the present invention is that it
allows for the formulation of proteins or nucleic acids in a
flowable state at extremely high concentrations (up to about 30%
w/w). Because the protein or nucleic acid is in a dry state, it is
not subject to the degradation processes (e.g., aggregation,
precipitation or fragmentation) observed for high concentration
aqueous solutions.
[0069] A. Definitions:
[0070] As used herein, the following terms have the following
meanings:
[0071] The term "chemical stability" means that an acceptable
percentage of degradation products produced by chemical pathways
such as oxidation, hydrolysis or enzymatic action is formed and/or
that acceptable biological activity is retained. In particular, a
formulation is considered chemically stable if no more than about
40% breakdown products are formed and/or at least 40% biological
activity is retained after one week at 37.degree. C.
[0072] The term "physical stability" means that an acceptable
percentage of aggregates (e.g., dimers, trimers and larger forms)
and/or cleavage products is formed. In particular, a formulation is
considered physically stable if no more that about 10% aggregates
and/or clevage products are formed after one week at 37.degree.
C.
[0073] The term "stable formulation" means that at least about 50%
chemically and physically stable protein or nucleic acid compound
remains after one week at 37.degree. C. Particularly preferred
formulations are those which retain at least about 65%, and most
particularly, at least about 80% chemically and physically stable
compound under these conditions. Especially preferred stable
formulations include those which remain flowable at high protein or
nucleic acid loading (e.g., at or above 1%).
[0074] The terms "protein" and/or "proteinaceous compound" and/or
"proteinaceous substance" mean peptides, polypeptides, proteins,
viruses, antibodies, etc. which comprise polymers of amino acid
residues bound together by amide (CONH) linkages. Both
naturally-derived or purified and recombinantly produced moieties
are included in these terms. These terms also include lipoproteins
and post-translationally modified forms, e.g., glycosylated
proteins. Analogs, derivatives, agonists, antagonists and
pharmaceutically acceptable salts of any of these are included in
these terms. The terms also include proteins and/or protein
compounds and/or protein substances which have D-amino acids,
modified, derivatized or non-naturally occurring amino acids in the
D- or L-configuration and/or peptomimetic units as part of their
structure.
[0075] The term "excipient" means a more or less inert component
which is added to the finished formulation other than the
therapeutic ingredient.
[0076] The term "non-polar vehicle" means a vehicle which has a
dielectric constant of less than or equal to about 15.
[0077] The term "aprotic vehicle" means a vehicle which does not
contain acidic hydrogen (i.e., a hydrogen attached to an oxygen or
nitrogen).
[0078] The term "anhydrous vehicle" means a vehicle which does not
contain water, including water adsorbed on its surface or combined
as water of crystallization.
[0079] The terms "vehicle with low reactivity" and/or
"low-reactivity vehicle" mean a vehicle which generally does not
solubilize or otherwise react with proteinaceous compounds and/or
nucleic acids. Low-reactivity vehicles are non-polar and have a
Hildebrandt number of less than about 8.0. Examples of vehicles
with low reactivity include: a) saturated hydrocarbons, b)
halogenated saturated or unsaturated hydrocarbons, and c) esters
and ethers of a) or b).
[0080] The terms "proteinaceous particle" and/or "proteinaceous
powder" mean particles which contain proteins, proteinaceous
compounds or proteinaceous substances. The proteinaceous particles
of the present invention may, optionally, contain excipients, as
defined above. Such excipients may include carbohydrates, non-ionic
surfactants, buffers, salts, carrier proteins, preservatives and
the like. However, the proteinaceous powders of the present
invention do not contain polymers, nor are they encapsulated by
polymeric materials (i.e., they are not microparticles or
microcapsules as defined, for example, in U.S. Pat. No.
5,518,731).
[0081] The term "hydration" means water molecules associated with
either the protein or nucleic acid, excipients or carriers.
[0082] The term "hydrophobic" means incapable of dissolving to any
appreciable extent in water.
[0083] The term "nucleic acid" means linear unbranched (linear or
circular) chains of nucleotides in which the 5' phosphoric group of
each nucleotide is esterified with the 3' hydroxyl of the adjoining
nucleotide. The term includes ribonucleic acid (RNA) and
deoxyribonucleic acid (DNA) constructs. The term nucleic acid
includes single and double stranded molecules, oligonucleotides,
gene expression constructs, mRNA molecules, ribozymes, and the
like. Naturally-derived or purified, synthetically produced and
recombinantly produced moieties are included in the term. The term
also includes analogs, derivatives, and constructs that include
promoter, leader, signal, polyadenylation or intron sequences,
locus control regions, markers, and the like. Nucleic acids
containing modified, derivatized or non-naturally occurring
nucleotide units as part of their structure are also included in
the term.
[0084] The terms "lipid/DNA complex" and "lipid/RNA complex" mean
complexes that form between nucleic acids and small, cationic
unilamellarvesicles held together by electrostatic interactions
rather than by encapsulation of the nucleic acids in liposomes. A
variety of topological arrangements can occur, such as DNA
condensation, liposome aggregation and fusion.
[0085] The term "liposome" means the multi- or unilamellar vesicles
formed from phospholipids which are used as carriers for drugs and
macromolecules, especially nucleic acids.
[0086] The terms "nucleic acid particle" and/or "nucleic acid
powder" mean particles which contain DNA or RNA. The nucleic acid
may optionally be complexed with lipids or in liposomes, ribozymes,
viral vectors, virosomes, dendrimers, cationic polymers, PLGA
particles, or the like. The nucleic acid particles of the present
invention may, optionally, contain excipients, as defined above.
Such excipients may include carbohydrates, non-ionic surfactants,
buffers, salts, carrier proteins, preservatives and the like.
[0087] B. Preparation of Formulations:
[0088] The present invention is drawn to non-aqueous formulations
of proteinaceous particles and nucleic acid particles with less
than about 10% hydration suspended in anhydrous, aprotic,
hydrophobic, non-polar vehicles with low reactivity, which
formulations are stable for prolonged periods of time, even at
elevated temperatures. Standard dilute aqueous peptide and protein
formulations require manipulation of buffer type, ionic strength,
pH and excipients (e.g., EDTA and ascorbic acid) to achieve
stability. Standard nucleic acid formulations require formulation
or reconstitution immediately prior to administration. In contrast,
the claimed formulations achieve stabilization of protein or
nucleic acid compounds by the use of dry particles and hydrophobic,
anhydrous, non-polar, aprotic low-reactivity vehicles. In
particular, stability and flowability of high concentrations (up to
about 30%, w/w) of compound has been provided by the formulations
of the present invention.
[0089] Examples of proteins and proteinaceous compounds which may
be formulated using the present invention include those proteins
which have biological activity or which may be used to treat a
disease or other pathological condition. They include, but are not
limited to growth hormone, Factor VIII, Factor IX and other
coagulation factors, chymotrypsin, trypsinogen, alpha-interferon,
beta-galactosidase, lactate dehydrogenase, growth factors, clotting
factors, enzymes, immune response stimulators, cytokines,
lymphokines, interferons, immunoglobulins, interleukins, peptides,
somatostatin, somatotropin analogues, somatomedin-C, Gonadotropic
releasing hormone, follicle stimulating hormone, luteinizing
hormone, LHRH, LHRH analogues such as leuprolide, nafarelin and
goserelin, LHRH agonists and antagonists, growth hormone releasing
factor, calcitonin, colchicine, gonadotropins such as chorionic
gonadotropin, oxytocin, octreotide, somatotropin plus an amino
acid, vasopressin, adrenocorticotrophic hormone, epidermal growth
factor, prolactin, somatotropin plus a protein, cosyntropin,
lypressin, polypeptides such as thyrotropin releasing hormone,
thyroid stimulation hormone, secretin, pancreozymin, enkephalin,
glucagon, endocrine agents secreted internally and distributed by
way of the bloodstream, and the like. Further agents that may be
delivered include .alpha..sub.1 antitrypsin, insulin and other
peptide hormones, adrenal cortical stimulating hormone, thyroid
stimulating hormone, and other pituitary hormones, interferon
.alpha., .beta., and .delta., erythropoietin, growth factors such
as GCSF, GM-CSF, insulin-like growth factor 1, tissue plasminogen
activator, CF4, dDAVP, tumor necrosis factor receptor, pancreatic
enzymes, lactase, interleukin-1 receptor antagonist, interleukin-2,
tumor suppresser proteins, cytotoxic proteins, retroviruses and
other viruses, viral proteins, antibodies, recombinant antibodies
and antibody fragments and the like.
[0090] Examples of nucleic acid compounds which may be formulated
using the present invention include those nucleic acids which code
for proteins which have biological activity or which may be used to
treat a disease or other pathological condition, such as the
protein compounds listed above. Nucleic acids, including sense or
antisense oligonucleotides, which block production of unwanted
proteins are also useful in the present invention. Also included in
nucleic acids which may be used in the present invention are those
which, either directly or by coding for a protein, stimulate an
animal to produce immunity against a disease condition (e.g.,
cancer) or infection by a pathogenic organism such as a bacteria,
virus or protozoa.
[0091] The above agents are useful for the treatment or prevention
of a variety of conditions including but not limited to hemophilia
and other blood disorders, growth disorders, diabetes, leukemia,
hepatitis, renal failure, HIV infection, hereditary diseases such
as cerebrosidase deficiency and adenosine deaminase deficiency,
hypertension, septic shock, autoimmune diseases such as multiple
sclerosis, Graves disease, systemic lupus erythematosus and
rheumatoid arthritis, shock and wasting disorders, cystic fibrosis,
lactose intolerance, Crohn's disease, inflammatory bowel disease,
gastrointestinal and other cancers. Analogs, derivatives,
antagonists, agonists and pharmaceutically acceptable salts of the
above may also be used.
[0092] The protein and nucleic acid compounds useful in the
formulations and methods of the present invention can be used in
the form of a salt, preferably a pharmaceutically acceptable salt.
Useful salts are known to those of skill in the art and include
salts with inorganic acids, organic acids, inorganic bases or
organic bases. Nucleic acids may also be complexed with lipids or
be presented as liposomes, ribozymes, viral vectors, virosomes,
dendrimers, cationic polymers, PLGA particles, or the like.
[0093] The proportion of protein or nucleic acid may vary depending
on the compound, the condition to be treated or prevented, the
expected dose and the duration of administration. (See, for
example, The Pharmacological Basis of Therapeutics, Gilman et al.,
7th ed. (1985) and Pharmaceutical Sciences, Remington, 18th ed.
(1990), the disclosures of which are incorporated herein by
reference.) Applicable routes include oral, enteral, transdermal,
percutaneous, parenteral, mucosal and the like, all of which are
known to those of skill in the art. The concentration of protein or
nucleic acid in high concentration formulations may range from at
least about 1% (w/w) up to about 30% while still maintaining
flowability. A preferred range is from about 10% to about 30%
(w/w).
[0094] The vehicles useful in the present invention are
non-aqueous, anhydrous, aprotic, non-polar, hydrophobic vehicles
with low reactivity. Such vehicles have a dielectric constant less
than or equal to about 15; do not contain acidic hydrogen, i.e.,
hydrogen attached to an oxygen or nitrogen; and generally do not
solubilize or otherwise react with proteinaceous compounds.
Preferred vehicles include: a) saturated hydrocarbons, b)
halogenated saturated or unsaturated hydrocarbons, and c) esters
and ethers of a) or b). Particularly preferred vehicles are
perhalohydrocarbons and unsubstituted saturated hydrocarbons. Most
preferred vehicles are biocompatible, such as perfluorodecalin,
perfluorotripropylamine, perfluoro-N-methyldecahydroquindine,
perfluoro-octohydro quinolidine, perfluoro-N-cyclohexylpyrilidine,
perfluoro-N,N-dimethylcyclohexyl methylamine,
perfluoro-dimethyl-adamanta- ne, perfluorotri-methylbicyclo
(3.3.1)nonane, bis(perfluorohexyl)ethene,
bis(perfluorobutyl)ethene, perfluoro-1-butyl-2-hexyl ethene,
tetradecane, methoxyflurane or mineral oil.
[0095] The proteinaceous or nucleic acid powders useful in the
present invention are solid particles wherein the hydration of the
particle is less than about 10% (w/w water/compound). In contrast
to previous protein formulations, where hydration and flexibility
were required in order to maintain enzymatic activity, the proteins
of the particles used in the present invention have minimal
flexibility and minimal exposure to the degradative effects of
moisture since protein hydration is minimized. In contrast to
previous nucleic acid formulations, which required hydration in
order to administer the formulation, the present formulations
reduce hydration and degradation of the nucleic acid compounds
while providing a flowable formulation suitable for administration.
The powders may be produced by milling, spray drying, spray
freeze-drying, lyophilization, precipitation, and the like. These
protected powder particulates are preferably prepared using solid
processing. They may optionally include protecting agents such as
carbohydrates, sucrose, trehalose, sorbitol, raffinose, dextrans or
cyclodextrins which may, for example, hydrogen bond to the proteins
to reduce their effective hydration. They may also contain bulking
agents such as glycine or mannitol that modify the morphology
and/or processing characteristics of the proteins or nucleic acids,
buffers that modify the pH, and non-ionic surfactants which protect
from surface absorption and solubilize the protein or nucleic
acids. The formulation of dry protein or nucleic acid powders is
well known to those skilled in the art.
[0096] Here, protein or nucleic acid hydration refers to the
fraction of the total moisture in a powder formulation associated
with the protein or nucleic acid. Certain excipients (e.g.,
carbohydrates) reduce the amount of water associated with proteins
[14] or nucleic acids. For purposes of this application, protein or
nucleic acid hydration will be equal to the moisture content of the
powder (determined, for example, by Karl Fischer analysis),
expressed as a percentage, multiplied by the fractional weight of
protein or nucleic acid in the powder.
[0097] Generally, the stable formulations of the present invention
may be prepared by simply suspending the desired amount, which may
be a therapeutically effective amount, of the desired proteinaceous
or nucleic acid powder in the selected vehicle. Preferred vehicles
include MO, PFD, MF, PTA and TD.
[0098] C. Methodology:
[0099] We have found that stable non-aqueous formulations of
protein or nucleic acid compounds may be prepared by suspending dry
(less than about 10% hydration) particles containing the protein or
nucleic acid compound to be formulated in anhydrous, aprotic,
hydrophobic, low-reactivity vehicle.
[0100] We have tested these formulations for stability by
subjecting them to aging at elevated temperature (37.degree. C.)
and measuring the chemical and/or physical stability of the
formulations. Results of these studies (shown, for example, in
Examples 1, 2 and 3) demonstrate that these formulations were
stable for at least one month at 37.degree. C.
[0101] A major aspect of the invention is that the flowable
non-aqueous formulations of the present invention are chemically
and physically stable at high temperatures for long periods of
time. Such formulations are stable even when high concentrations
are used. Thus, these formulations are advantageous in that they
may be shipped and stored at temperatures at or above room
temperature for long periods of time. They are also suitable for
use in implantable delivery devices.
DISCLOSURE OF EXAMPLES OF THE INVENTION
[0102] The following method was used to perform the studies in the
Examples that follow.
[0103] Karl Fischer Moisture Analysis: Vials and stoppers were
dried overnight in a vacuum oven at 80.degree. C. Approximately 6
mg of sample was weighed into a dry vial and the vial was
stoppered. Control vials were prepared by simply stoppering an
empty dry vial (i.e., a vial containing no sample). Subsequently,
150 .mu.L aliquots of dry methanol was added to sample and control
vials via a 250 .mu.L Hamilton Syringe (Hamilton Co., Reno, Nev.)
which had been previously washed three times with dry methanol. The
vials were then sonicated at room temperature until all solids were
dispersed, centrifuged, and 100 .mu.L of the supernatant methanol
was injected into an Aquatest 10 Coulometric Moisture Analyzer
(SeraDyn Inc., Indianapolis, Ind.). The resultant readings were
recorded, and water content of the sample calculated by subtracting
the control reading from that of the sample.
[0104] The following reagents were used to perform the studies in
the Examples that follow.
[0105] Perfluorodecalin, perfluorotributylamine and tetrdecane were
purchased from Aldrich Chemical Company (Milwaukee, Wis.).
Methoxyflurane was purchased from Abbott Laboratories (North
Chicago, Ill.). Light Mineral Oil USP was purchased from Spectrum
Chemical Corp. (Gardena, Calif.).
[0106] The following examples are offered to illustrate this
invention and are not meant to be construed in any way as limiting
the scope of this invention.
EXAMPLE 1
Alpha-Inteferon Formulations Stability of alpha Interferon
(.alpha.-IFN) Suspensions
[0107] Human recombinant Interferon-.alpha.-2a (.alpha.-IFN)
(Scitech Genetics Ltd., lot#036R2801) was formulated as a 5 mg/mL
solution containing 5 mM citrate, 0.5% sucrose, and 0.005% Tween
80, pH 4.5. Aliquots of 200 .mu.L of this solution were then
dispensed into 1 mL glass lyophilization vials, partially covered
with lyophilization stoppers, and lyophilized using an FTS systems
lyophilizer according to the following cycle:
[0108] Pre-cool shelves to 5.degree. C.;
[0109] Load vials;
[0110] Freeze to -50.degree. C. at 2.5.degree. C./min;
[0111] When product is at -30.degree. C. set vacuum to 125 mT;
[0112] Hold at -50.degree. C. for 30 min;
[0113] Ramp to 0.degree. C. at 0.5.degree. C./min;
[0114] Hold at 0.degree. C. for 120 min;
[0115] Ramp to 20.degree. C. at 1.degree. C./min;
[0116] Hold at 20.degree. C. for 120 min;
[0117] Ramp to 30.degree. C. at 1.degree. C./min;
[0118] Hold at 20.degree. C. for 1000 min; and
[0119] Stopper vials.
[0120] The resultant powder had a moisture content of approximately
5% (w/w) as determined by Karl Fischer analysis and a protein
hydration of about 2.5%. Suspensions were prepared by adding 100
.mu.L of either perfluorodecalin (PFD), methoxyflurane (MF), or
mineral oil (MO) to the vials containing the .alpha.-IFN powder,
and the vials incubated at 37.degree. C. Samples were pulled at 2
and 4 weeks, and the .alpha.-IFN extracted from the non-aqueous
phase by adding 700 .mu.L of buffer (containing 5 mM citrate, 0.5%
sucrose, and 0.005% Tween 80, pH 4.5) and gently inverting the
vials. After 15 minutes, an aliquot of the aqueous phase was
removed and analyzed for stability by reverse phase HPLC and
reduced and non-reduced SDS-PAGE electrophoresis.
[0121] The formulations remained chemically stable as determined by
reverse phase HPLC (Table 1). In addition, no aggregation or
cleavage products were observed on reduced or non-reduced SDS-PAGE
gels.
1TABLE 1 Stability of .alpha.-IFN suspensions at 37.degree. C. as
measured by reverse phase chromatography % Recovery % Recovery %
Recovery Time PFD susp MF susp MO susp (weeks) 37.degree. C.
37.degree. C. 37.degree. C. 0 98 .+-. 3 92 .+-. 6 101 .+-. 1 2 103
.+-. 2 81 .+-. 3 94 .+-. 3 4 98 .+-. 1 81 .+-. 1 84 .+-. 2 *Numbers
represent mean .+-. standard deviation of 2-3 samples
[0122] RP-HPLC
[0123] Instrument: Hewlett Packard HP-1090
[0124] Flow Rate: 0.3 mL/min
[0125] Detection: 210 nm
[0126] Column: Waters Delta-Pak, c18, 150.times.2 mm, 300
.ANG..
[0127] Mobile Phase: A=30/70/0.2 Acetonitrile[Water/TFA B=80/20/0.2
Acetonitrile/Water/TFA
[0128] Gradient:
2 Time % B 0 23 45 35 55 52 60 90 65 90 68 23
[0129] SDS-PAGE
[0130] Apparatus: Life Technologies Vertical Gel Electrophoresis
system.
[0131] Gel: 15% discontinuous, 15.times.17 cm, 0.8 mm thick.
[0132] Running Conditions: 200 v, 50 mA, approximately 3 hrs.
[0133] Staining: Coomassie Blue R-250
[0134] Gel Analysis: Bio-Rad GS-700 image analyzer with Molecular
Analyst software.
EXAMPLE 2
Stability of Chymotrypsin Formulations
[0135] Formulations were prepared containing 2% chymotrypsin
(Worthington Biochemical Corp., 1.times. Crystallized, Lot#
H5B7405L), determined by Karl Fischer analysis to have a water
content and protein hydration of approximately 7% (w/w), either
dissolved in 0.1M borate buffer, pH 8.0, or suspended (as a dry
powder) in either perfluorodecalin or Light Mineral Oil, U.S.P.
Samples were stored at 37.degree. C. for 10 weeks, and assayed for
chymotrypsin activity using casein as a substrate.
[0136] The results are shown in Table 2 and demonstrate stability
of the formulations.
[0137] Chymotrypsin Bioactivity Assay
[0138] Samples were diluted in 0.1 M borate buffer, pH 8.0, such
that the final chymotrypsin concentration for assay was
approximately 2-50 .mu.g/mL. A casein substrate solution was
prepared by suspending 1 gm of casein in 95 mL borate buffer, pH
8.0 and heating in a boiling water bath until the casein had
dissolved (approximately 10 minutes), then adding 1.1 mL 5%
CaCl.sub.2 and diluting the solution to 100 mL with 0.1M borate
buffer solution at pH 8.0. The substrate solution (1.0 mL) was
prewarmed at 37.degree. C. in a heating block, and to it was added
1.0 mL of the sample. The solutions were mixed and incubated at
37.degree. C. for exactly 20 minutes. Subsequently, 3.0 mL of 5%
trichloroacetic acid was added, and the resultant mixture was
allowed to stand at room temperature for 30 minutes, then
centrifuged for 20 minutes at 3,000 g. The absorbance of the
supernatant was read in a UV spectrophotometer at 280 nm and the
activity (in Units/mg) calculated by the following equation. 1
Activity = A t ( C ) ( t )
[0139] where: A.sub.t=absorbance of supernatant (at 280 nm) at time
t of the reaction (in this case, 20 minutes); C=concentration of
chymotrypsin in sample; and t=time of reaction (20 minutes).
3TABLE 2 Activity of chymotrypsin formulations when stored at
37.degree. C. Time % Remaining % Remaining % Remaining (weeks) PFD*
MO* Buffer* 0 103 .+-. 5 100 .+-. 5 100 .+-. 4 1 97 .+-. 2 86 .+-.
1 23 .+-. 2 3 102 .+-. 3 96 .+-. 3 19 .+-. 2 6 102 .+-. 2 89 .+-. 1
22 .+-. 4 10 102 .+-. 3 92 .+-. 2 *Numbers represent mean .+-..+-.
standard deviation of 6 samples
EXAMPLE 3
Stability of Plasma Protein Suspensions
[0140] Formulations of a post-translationally modified plasma
protein of 55 kilodalton molecular weight were prepared containing
1 mg/mL protein and approximately 30 mg/mL excipients, buffered to
a neutral pH. One mL aliquots of the above solutions were pipetted
into 3 mL glass vials, covered with lyophilization stoppers, loaded
into a freeze dry chamber (FTS Systems Inc.), and lyophilized.
[0141] The resultant powder had a final moisture content of about
0.25% (w/w) water, as determined by Karl Fischer analysis and
protein hydration of about 0.008%. Suspensions were prepared by
adding 1 mL of either perfluorodecalin (PFD) or methoxyflurane (MF)
to the vials containing the dry protein powder, and the vials were
then incubated at 37.degree. C. Control samples of the lyophilized
powder were stored at -80.degree. C. Samples were pulled at 0, 4.5,
6.5, 8.5 and 12.5 weeks and analyzed for activity using a
bioactivity assay, and for chemical stability by size exclusion
chromatography.
[0142] The results are summarized in Tables 3 and 4 and show that
the formulations remained chemically (as determined by biological
activity) and physically (as determined by SEC) stable.
[0143] Size Exclusion Chromatography
[0144] Column: TSD G3000 swx1 column, 7.8.times.300 mm, 5 .mu.m
(ToSoHaas TO8541 or equivalent)
[0145] Mobile Phase: 50 mM Na.sub.2HPO.sub.4, 150 mM NaCl, pH
7.0
[0146] Flow Rate: 1.0 mL/min
[0147] Detector: 214 nm
[0148] Injection Volume: 50 .mu.L
4TABLE 3 Stability of plasma protein suspensions at 37.degree. C.
as measured by bioactivity assay % LS.sup..dagger. %
LS.sup..dagger. % LS.sup..dagger. Time Lyo. Powder PFD susp. MF
susp (weeks) -80.degree. C. 37.degree. C. 37.degree. C. 0 92 .+-.
14 84 .+-. 14 92 .+-. 4 1.5 98 .+-. 12 109 .+-. 9 107 .+-. 16 4.5
89 .+-. 2 86 .+-. 4 61 .+-. 20 6.5 94 .+-. 7 101 .+-. 0 68 .+-. 15
8.5 110 .+-. 2 97 .+-. 2 62 .+-. 5 12.5 111 .+-. 7 105 .+-. 11 2
.dagger. % LS = % Label Strength = [ protein ] test [ protein ]
control * Numbers represent mean .+-..+-. standard deviation of 3
samples
[0149]
5TABLE 3 Stability of plasma protein suspensions at 37.degree. C.
as measured by size exclusion chromatography. % LS.sup..dagger. %
LS.sup..dagger. % LS.sup..dagger. Time Lyo. Powder PFD susp. MF
susp (weeks) -80.degree. C.* 37.degree. C.* 37.degree. C.* 0 92
.+-. 0 96 .+-. 1 84 .+-. 7 1.5 107 .+-. 3 106 .+-. 2 104 .+-. 4 4.5
108 .+-. 2 96 .+-. 1 67 .+-. 35 6.5 113 .+-. 2 101 .+-. 2 79 .+-.
12 8.5 105 .+-. 1 95 .+-. 4 57 .+-. 5 12.5 100 .+-. 3 98 .+-. 1 3
.dagger. % LS = % Label Strength = [ protein ] test [ protein ]
control *Numbers represent mean .+-..+-. standard deviation of 3
samples
EXAMPLE 4
High Concentration Flowable Formulations
[0150] Solutions were prepared containing either Albumin (Sigma,
Lot 129FO1431), Lysozyme (Sigma Lot 65H7025) or Trypsinogen
(Worthington Lot# 38E273N) and sucrose in a 1:1 (w/w) ratio. The
solutions were spray dried on a Yamato ADL 31 Spray Dryer (Yamato
Corp., NY) with the following parameters: inlet temp 120.degree.
C., outlet temperature 65.degree. C., atomizer 1.2 kg/cm.sup.2. The
powders were then transferred to a vacuum oven and allowed to
further dry at 30.degree. C. overnight under full vacuum. The
moisture content of the powders studied was approximately 4.5 %
(w/w) as determined by Karl Fischer analysis with a protein
hydration of about 2.25%.
[0151] Pastes were formulated by mixing 700 mg of each powder with
1.0 mL of perfluorodecalin (approximately 28% w/w). The pastes were
loaded into 1.0 cc syringes fitted with 30 Gauge needles (Becton
Dickinson), and extruded. All pastes extruded evenly and completely
with little effort.
EXAMPLE 5
Stability of Factor IX Suspensions
[0152] Coagulation Factor IX (FIX) from human serum
(Calbiochem-Novabiochem, La Jolla, Calif.) was formulated as a 0.5
mg/mL solution containing 60 mg/mL sucrose, 60 mg/mL mannitol, 1
mg/mL polysorbate 80 and 1.6 mg/mL histidine buffer buffered to a
pH of approximately 7. One mL aliquots of this solution were
lyophilized according to the cycle above. The resultant powder had
a moisture content of 1%, as determined by Karl Fischer
analysis.
[0153] Suspensions were prepared by adding 1 mL of perfluorodecalin
(PFD), perfluorotributylamine (PTA) or tetradecane (TD) to the
vials containing the dry FIX powder. The vials were incubated at
37.degree. C. Control samples of the lyophilized powder were stored
at -80.degree. C. Samples were pulled at 0 and 2 weeks and analyzed
for FIX activity by clotting bioactivity assay, and for chemical
stability by size exclusion chromatography.
[0154] The results (Tables 5 and 6) showed that the formulations
remained chemically (as determined by biological activity) and
physically (as determined by SEC) stable, even after
irradiation.
6TABLE 5 Stability of Factor IX Suspensions at 37.degree. C. as
Measured by Bioactivity Assay % LS.sup..dagger. % LS.sup..dagger. %
LS.sup..dagger. Time PFD susp. PTA susp. TD susp (weeks) 37.degree.
C. 37.degree. C. 37.degree. C. 0 97 .+-. 2 89 .+-. 3 95 .+-. 3 2 98
.+-. 2 96 .+-. 1 96 .+-. 1 4 .dagger. % LS = % Label Strength = [
protein ] test [ protein ] control * Numbers represent mean .+-.
standard deviation of 3 samples
[0155]
7TABLE 6 Stability of Factor IX Suspensions at 37.degree. C. as
Measured by Size Exclusion Chromatography % LS.sup..dagger. %
LS.sup..dagger. % LS.sup..dagger. Time PFD susp. PTA susp. TD susp
(weeks) 37.degree. C. 37.degree. C. 37.degree. C. 0 94 .+-. 1 93
.+-. 1 97 .+-. 1 2 94 .+-. 2 95 .+-. 2 96 .+-. 1 5 .dagger. % LS =
% Label Strength = [ protein ] test [ protein ] control * Numbers
represent mean .+-. standard deviation of 3 samples
EXAMPLE 6
Stability of Nucleic Acid Suspensions
[0156] Plasmid pCIN.CAT was made by cloning the coding sequence for
bacterial chloramphenicol acetyltransferase (CAT) into the
expression plasmid pCLneo (Promega). The CAT coding region was
isolated by PCR amplification from plasmid pSIS.CAT [42] by
standard techniques (PCR Technology, 1989, H. A. Erlich, ed.
Stockton Press, incorporated herein by reference). These primers
produced a unique Xhol restriction site at the 5'-end and a unique
Notl restriction site at the 3'end. This fragment was subcloned
into the Xhol and Notl sites of pCLneo by standard techniques
(Molecular Cloning, second edition. 1989. Sambrook, J., Fritsch, E.
F., and Maniatis, T., incorporated herein by reference.) Plasmid
DNA was grown in bacterial culture and isolated (Qiagen, GmbH).
[0157] Formulations were prepared containing 100 mg/ml sucrose, 100
mg/ml mannitol, 10 .mu.g/ml pCIN-CAT DNA, 50 .mu.g/ml of a 1:1
formulation of DOTMA (n-[1-(2,3
dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride) and DOPE
(dioleoyl phosphotidylethanolamine) (Lipofection, GIBCO BRL) in 10
mM Tris buffer at pH 7.1. Aliquots of 200 .mu.l of the above
formulation were pipetted into 1 ml glass vials and lyophilized
using the following protocol:
[0158] Precool shelf temperature to 5.degree. C.;
[0159] Load vials;
[0160] Freeze to -40.degree. C. at 0.4.degree. C./min and hold at
-40.degree. C. for 120 minutes;
[0161] Ramp to -10.degree. C. at 0.4.degree. C./min and hold for
240 minutes;
[0162] Ramp to -45.degree. C. at 0.4.degree. C./min and hold for
120 minutes;
[0163] Set vacuum to 100 mT;
[0164] Hold at -45.degree. C. for 360 minutes with vacuum at 100
mT;
[0165] Ramp to -25.degree. C. at 0.04.degree. C./min with vacuum at
100 mT;
[0166] Hold at 25.degree. C. for 1500 minutes with vacuum at 100
mT.
[0167] The subsequent dry powder had a moisture content of
approximately 2% as mastered by Karl Fischer analysis. Suspensions
were prepared by adding 300 .mu.l of perfluorodecalin (PFD) to the
vials in a glove box under dry nitrogen. Suspension, dry powder and
solution samples were incubated at 37.degree. C. for 1, 4 and 7
days, and subsequently monitored for biological activity by
monitoring gene transfer efficiency as measured by CAT expression
in HEK293 cells. Transfection of HEF293 cells with lipid/DNA
complexes was performed as described by the Manufacturer (GIBCO
BRL).
[0168] The results are shown in Table 7, and demonstrate that when
lipid/DNA complexes were formulated in aqueous solution,
essentially all activity was lost when the solution was stored at
37.degree. C. for 1 week. In contrast, both the lyophilized dry
nucleic acid powder and the nucleic acid powder suspended in PFD
retained essentially all their biological activity (within the
experimental variability of the assay) when stored for 1 week at
37.degree. C.
8TABLE 7 Transfection Activity of Lipid/DNA Constructs After
Incubation at 37.degree. C. (Numbers are mean .+-. standard
deviation of 12 replicates.) Average ng CAT Protein Expressed Per
mg Total Cellular Protein PFD Time Solution Dry Powder Suspension
(days) Formulation Formulation Formulation 0 478 .+-. 254 219 .+-.
114 n.d. 1 115 .+-. 46 628 .+-. 192 273 .+-. 122 4 13 .+-. 12 255
.+-. 137 284 .+-. 267 7 6 .+-. 3 377 .+-. 202 339 .+-. 151
[0169] Modification of the above-described modes of carrying out
various embodiments of this invention will be apparent to those of
skill in the art following the teachings of this invention as set
forth herein. The examples described above are not limiting, but
are merely exemplary of this invention, the scope of which is
defined by the following claims.
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