U.S. patent application number 10/494068 was filed with the patent office on 2004-12-09 for proteins stabilized with polysaccharide gums.
Invention is credited to Alavattam, Sreedhara, Brody, Richard, Jones, Randy L.
Application Number | 20040247684 10/494068 |
Document ID | / |
Family ID | 21756111 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040247684 |
Kind Code |
A1 |
Brody, Richard ; et
al. |
December 9, 2004 |
Proteins stabilized with polysaccharide gums
Abstract
Described are heat stable aqueous solutions or gels comprising a
biologically effective amount of a protein and an effective
stabilizing amount of a polysaccharide gum as well as heat stable
solutions or gels suitable for use in an implantable drug delivery
device at body temperature. Also disclosed are lyophilized
compositions having biologically activity, where such lyophilized
compositions are formed by lyophilozing the stabilized solutions or
gels of the invention. Such lyophilized powders can be used after
reconstitution with an amount of aqueous that provide an effective
stabilizing concentration of polysaccharide and a pharmaceutically
acceptable amount of therapeutic protein particularly against
thermal and oxidative stress.
Inventors: |
Brody, Richard;
(Worthington, OH) ; Alavattam, Sreedhara;
(Columbus, OH) ; Jones, Randy L; (Delaware,
OH) |
Correspondence
Address: |
FROST BROWN TODD, LLC
2200 PNC CENTER
201 E. FIFTH STREET
CINCINNATI
OH
45202
US
|
Family ID: |
21756111 |
Appl. No.: |
10/494068 |
Filed: |
July 21, 2004 |
PCT Filed: |
October 30, 2002 |
PCT NO: |
PCT/US02/34752 |
Current U.S.
Class: |
424/488 ;
424/85.1; 424/94.1; 514/54; 514/7.6; 514/9.7 |
Current CPC
Class: |
A61K 9/0024 20130101;
A61K 47/36 20130101; A61K 9/19 20130101 |
Class at
Publication: |
424/488 ;
424/085.1; 424/094.1; 514/002; 514/054 |
International
Class: |
A61K 038/48; A61K
038/19; A61K 038/22; A61K 009/14; A61K 038/43 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 30, 2001 |
US |
10012.667 |
Claims
What is claimed is:
1. A heat stable aqueous solution or gel comprising a biologically
effective amount of a protein and a stabilizing effective amount of
a polysaccharide gum material.
2. A heat stable aqueous solution or gel according to claim 1
containing one or more minor amounts of a pharmaceutically
acceptable excipient.
3. A heat stable aqueous solution or gel according to claim 1
wherein said protein comprises an enzyme, an antibody, a hormone, a
growth factor, and a cytokine.
4. A heat stable aqueous solution or gel according to claim 3
wherein said protein is a hormone.
5. A heat stable aqueous solution or gel according to claim 3
wherein said protein is a cytokine.
6. A heat stable aqueous solution or gel according to claim 1
wherein said gum comprises gum arabic, guar gum, xanthan gum,
locust bean gum, tragacanth gum, gum karaya, and gum ghatti.
7. A heat stable aqueous solution or gel according to claim 6
wherein said gum is gum arabic.
8. A heat stable aqueous solution or gel according to claim 6
wherein said gum is present at from about 5%(w/v) to about 50%(w/v)
or the gum's solubility limit, and wherein when gum Arabic is
selected, the concentration is over 10%(w/v).
9. A heat stable aqueous solution or gel according to claim 8
wherein said gum is present above 10%(w/v) to about 50%(w/v), or
the gum's solubility limit.
10. A heat stable aqueous solution or gel according to claim 9
wherein said gum is present at from about 20% (w/v) to about 50%
(w/v), or the gum's solubility limit.
11. A heat stable aqueous solution or gel according to claim 2
wherein said pharmaceutically acceptable excipient is selected from
the group consisting of antioxidants, preservatives and surface
active agents.
12. A heat stable aqueous solution or gel for use in an implantable
drug delivery device comprising a pharmaceutically effective amount
of a protein and a stabilizing effective amount of a polysaccharide
gum material.
13. A heat stable solution or gel according to claim 12 wherein
said stabilized solution or gel contains one or more minor amounts
of a pharmaceutically acceptable excipient.
14. A heat stable aqueous solution or gel according to claim 13
wherein said pharmaceutically acceptable excipient is selected from
the group consisting of antioxidants, preservatives and surface
active agents.
15. A heat stable aqueous solution or gel according to claim 12
wherein said protein is selected from the group consisting of an
antibody, a hormone, a growth factor, and a cytokine.
16. A heat stable aqueous solution or gel according to claim 15
wherein said protein is a hormone or a growth factor.
17. A heat stable aqueous solution or gel according to claim 15
wherein said protein is a cytokine.
18. A heat stable aqueous solution or gel according to claim 12
wherein said gum is selected from the group consisting of gum
arabic, guar gum, xanthan gum, locust bean gum, tragacanth gum, gum
karaya, and gum ghatti.
19. A heat stable aqueous solution or gel according to claim 18
wherein said gum is gum arabic.
20. A heat stable aqueous solution or gel according to claim 18
wherein said gum is present at from about 5% (w/v) to about 50%
(w/v) or the gum's solubility limit.
21. A heat stable aqueous solution or gel according to claim 20
wherein said gum is present at from about 10% (w/v) to about 50%
(w/v) or the gum's solubility limit.
22. A heat stable aqueous solution or gel according to claim 21
wherein said gum is present at from about 20% (w/v) to about 50%
(w/v) or the gum's solubility limit
23. A lyophilized composition having biological activity, wherein
said lyophilized composition is formed by lyophilizing a heat
stable solution or gel comprising a biologically effective amount
of a protein and a stabilizing effective amount of a polysaccharide
gum material.
24. The lyophilized composition according to claim 23, wherein the
lyophilized polysaccharide and protein dry particles are
reconstituted in an aqueous buffer in a manner so as to get high
concentration of gum that stabilizes the protein.
25. A lyophilized composition according to claim 23, wherein the
said lyophilized powder is added to an implantable device that
controls the release of therapeutic protein.
26. A lyophilized composition according to claim 23, wherein the
lyophilized powder is reconstituted in an aqueous buffer to provide
high concentrations of the polysaccharide that will stabilize the
said therapeutic protein, and this reconstituted aqueous gum gel or
solution is further added to an implantable device.
27. A lyophilized composition according to claim 23 wherein said
protein is selected from the group consisting of an enzyme, an
antibody, a hormone, a growth factor, and a cytokine.
28. A lyophilized composition according to claim 23 wherein said
gum is selected from the group consisting of gum arabic, guar gum,
xanthan gum, locust bean gum, tragacanth gum, gum karaya, and gum
ghatti.
29. A lyophilized composition according to claim 28 wherein said
gum is gum arabic.
30. A lyophilized composition according to claim 28 wherein said
gum is present at from about 5% to about 50% (w/v) after
reconstitution in aqueous buffer, or the solubility limit of the
gum, and wherein when gum arabic is selected said gum is present at
greater than 10% (w/v).
31. A lyophilized composition according to claim 30 wherein said
gum is present at from about 10% (w/v) to from about 50% (w/v)
after reconstitution in aqueous buffer.
32. A lyophilized composition according to claim 31 wherein said
gum is present at from about 20% (w/v) to from about 50% (w/v)
after reconstitution in aqueous buffer.
33. A lyophilized composition according to claim 23 optionally
containing one or more pharmaceutically acceptable excipients.
34. An implantable drug delivery device containing a heat stable
aqueous solution or gel comprising a pharmaceutically effective
amount of a protein and a stabilizing effective amount of a
polysaccharide gum material.
35. An implantable drug delivery device containing a lyophilized
powder according to claim 23.
36. An implantable drug delivery device that is filled with a
lyophilized powder according to claim 23 that has been
reconstituted with an aqueous buffer to provide a thermally
stabilizing amount of polysaccharide and pharmaceutically
acceptable amount of therapeutic protein.
37. An implantable drug delivery device according to claim 32
wherein said stabilized solution or gel contains one or more minor
amounts of a pharmaceutically acceptable excipient.
38. An implantable drug delivery device according to claim 33
wherein said pharmaceutically acceptable excipient is selected from
the group consisting of antioxidants, preservatives and surface
active agents.
39. An implantable drug delivery device according to claim 32
wherein said protein is selected from the group consisting of an
antibody, a hormone, a growth factor, and a cytokine.
40. An implantable drug delivery device according to claim 35
wherein said protein is a hormone or a growth factor.
41. An implantable drug delivery device according to claim 32
wherein said gum is selected from the group consisting of gum
arabic, guar gum, xanthan gum, locust bean gum, tragacanth gum, gum
karaya, and gum ghatti.
42. An implantable drug delivery device according to claim 37
wherein said gum is gum arabic.
43. An implantable drug delivery device according to claim 37
wherein said gum is present at from about 5% (w/v) to from about
50% (w/v) or the gum's solubility limit.
44. An implantable drug delivery device according to claim 39
wherein said gum is present at from about 10% (w/v) to about 50%
(w/v) or the gum's solubility limit.
45. An implantable drug delivery device according to claim 40
wherein said gum is present at from about 20% (w/v) to about 50%
(w/v) or the gum's solubility limit.
46. An implantable drug delivery device according to claim 40
wherein said gum is present at from about 30% (w/v) to about 50%
(w/v) or the gum's solubility limit.
47. An implantable drug delivery device according to claim 40
wherein said gum is present at from about 40% (w/v) to about 50%
(w/v) or the gum's solubility limit.
48. An aqueous solution or gel stable against metal catalized
oxidation reactions comprising a biologically effective amount of a
protein and a stabilizing effective amount of a polysaccharide gum
material.
49. The oxidation stable solution or gel according to claim 48,
wherein when gum arabic is selected said gum is present above about
30%(w/v) to the gums solubility limit.
Description
[0001] This application claims the benefit of U.S. application Ser.
No. 10/012,667 filed Oct. 30, 2001, the content of which is
incorporated herein by reference as if completely rewritten
herein.
TECHNICAL FIELD
[0002] The present invention relates to a heat stable aqueous
solution or gel comprising a biologically effective amount of a
protein and an effective stabilizing amount of a polysaccharide gum
as well as heat stable solutions or gels suitable for use in an
implantable drug delivery device. This invention also relates to
lyophilized compositions having biological activity, where such
lyophilized compositions are formed by lyophilizing the stabilized
solutions or gels of the invention as well as the lyophilized
compositions that are reconstituted in an aqueous buffer so as to
provide a high concentration of polysaccharides that will stabilize
proteins under physiological conditions.
BACKGROUND OF THE INVENTION
[0003] The commercial market for recombinant protein
biopharmaceuticals is expanding rapidly as various biotechnology
and pharmaceutical companies develop and test biologically active
proteins. The emerging field of proteomics will likely provide
protein targets useful for drug development, thereby enabling the
market for recombinant protein biopharmaceuticals to continue its
expansion.
[0004] Currently, proteins are utilized in a variety of diagnostic
and therapeutic applications. For example, one protein used in a
diagnostic application is the enzyme glucose oxidase, which is used
in glucose assays. The hormone insulin is an example of a protein
utilized in therapeutic applications. However, proteins are
particularly sensitive to certain environmental conditions and may
not be stable at elevated temperatures, including physiological
temperature of 37.degree. C., in non-optimal aqueous solvent
systems, or in organic solvent systems. Protein stability may also
be affected by pH and buffer conditions and exposure to shear
forces or other physical forces.
[0005] The stability of a protein refers to both its conformational
stability, which is reflected in the protein's three-dimensional
structure, and its chemical stability, which refers to the chemical
composition of the protein's constituent amino acids. Protein
instability can result in a marked decrease or complete loss of a
protein's biological activity. Deleterious stresses such as organic
solvents, extremes of pH, high temperatures, and/or dehydration
(drying) can affect both the conformational and chemical stability
of a protein. Chemical instability can result from processes such
as (a) deamidation of the amino acids residues asparagine or
glutamine, (b) oxidation of cysteine or methionine amino acid
residues, or (c) cleavage at any of the peptide amide linkages of
the protein. Examples of conformational instability include
aggregation (fibrillation), precipitation, and subunit
dissociation.
[0006] Because an inactive protein is useless, and in some cases
deleterious, for most diagnostic and therapeutic applications,
there is a need for a means by which proteins can be stabilized in
solution at elevated temperatures (e.g. at and above room
temperature, at body temperature or higher. It is known in the art
that proteins can be stabilized in solution by the addition of
soluble excipients that stabilize the monomeric, correctly folded
protein conformation. Disaccharides such as trehalose, sucrose, or
lactose, and surface active agents such as phospholipids, Tween,
and Triton are examples of excipients useful for stabilizing
proteins. These stabilizers must be used in non-toxic levels
because in the case of therapeutic proteins, the stabilizers are
necessarily administered to the patient with the protein.
[0007] U.S. Pat. No. 5,834,273 issued to Futatsugi et al. on Nov.
10, 1998 provides a heat and protease resistant enzyme with
improved storage stability. This enzyme is modified with a
polysaccharide, polyamino acid, or synthetic polymer having a
plurality of carboxyl groups by means of a crosslinking agent
capable of binding both carboxyl groups and amino groups.
[0008] U.S. Pat. No. 5,736,625 issued to Callstrom et al. on Apr.
7, 1998 discloses a method for preparing water soluble,
saccharide-linked protein polymer conjugates that stabilize the
protein in a hostile environment. The claimed method includes
covalently binding the polymer to the protein through at least
three linkers, each linker having three or more hydroxyl groups.
The protein is conjugated at lysines or arginines.
[0009] U.S. Pat. No. 5,691,154 issued to Callstrom et al. on Nov.
25, 1997 provides an enzyme linked immunoassay in which the enzyme
is in the form of a water soluble polymer saccharide conjugate
which is stable in hostile environments. The conjugate includes the
enzyme which is linked to the polymer at multiple points through
saccharide linker groups.
[0010] U.S. Pat. No. 5,612,053 issued to Baichwal et al. on Mar.
18, 1997 discloses an inhalable powder formulation which includes
cohesive composites of particles containing a medicament and a
controlled release carrier which includes one or more
polysaccharide gums of natural origin.
[0011] U.S. Pat. No. 5,492,821 issued to Callstrom et al. on Feb.
20, 1996 discloses water soluble protein polymer conjugates in
which proteins linked to an acrylic polymer at multiple points by
means of saccharide linker groups. These conjugates are also stable
in hostile environments.
[0012] U.S. Pat. No. 5,128,143 issued to Baichwal et al. on Jul. 7,
1992 provides, for oral delivery, a slow release pharmaceutical
excipient of an inert diluent and a hydrophilic material including
xanthan gum and a galactomannan gum capable of cross-linking the
xanthan gum in the presence of aqueous solutions.
[0013] Ispas-Szabo et al. demonstrated that the ability of starch
tablets to swell and release low molecular weight drugs could be
controlled by the degree that the starch was cross-linked. No data
related to protein stabilization was presented. Carbohydrate
Research 323, 163-175 (2000).
[0014] Artursson et al. demonstrated that proteins could be
incorporated into polyacryl starch microparticles. One incorporated
protein, the enzyme carbonic anhydrase, retained a low amount of
activity at temperatures where the free protein had no activity
(e.g., >70.degree. C.). At lower temperatures (e.g.,
<65.degree. C.), however, the free enzyme was more stable than
the enzyme incorporated into the microparticles. Journal of
Pharmaceutical Sciences 73, 1507-1513 (1984).
[0015] Gliko-Kabir et al. demonstrated that the swelling of
lyophilized guar gum powder in gastric or intestinal buffer could
be reduced from approximately 100 fold to approximately 5 fold if
the guar was crosslinked with glutaraldehyde. No data concerning
protein stabilization was presented. Pharmaceutical Research 15,
1019-1025(1998).
[0016] Bauman et al. demonstrated that carrageenan gum stabilized
the enzyme cholinesterase against heat when the enzyme was dried on
a urethane foam sheet with 8% starch. Analytical Biochemistry 19,
587-592, (1967).
[0017] U.S. Pat. No. 6,391,296 Bl issued to Toray Industries, Inc.
on May 21, 2002 and European Patent Application No. EP 0 950 663 A1
submitted by the same company and published on Oct. 20, 1999
disclose the use of gum arabic as a protein stabilizer. Aqueous
solutions containing 0.2%-2% gum arabic were shown in these
documents to stabilize proteins to storage at 4.degree. C. and to
freeze drying. There was no data and no discussion concerning the
ability of gum arabic to stabilize proteins at room temperature or
higher temperatures. While the examples cite the use of gum arabic
at 0.2%-2%, the US patent claims the use of gum arabic from 0.2% to
10%. No mention is made of possible benefits to using gum arabic as
a stabilizer at concentrations greater than 10% or at physiological
temperature, pH, or salt concentration.
[0018] Many of the methods that are known to stabilize proteins,
require that the protein be covalently attached to a solid support
or covalently substituted with a stabilizing molecule. Covalent
modification is not always practical for proteins in solutions and
can change the biological effectiveness of a therapeutic protein.,
Thus there is a need for a protein stabilization system that does
not require covalent modification of the protein.
[0019] The typical method of administering therapeutic proteins to
a patient or test subject is by means of needle-based injections.
Currently, many pharmaceutical and drug delivery companies are
seeking to develop alternative systems for the delivery of
therapeutic proteins. These alternative systems are expected to
require fewer dosings and to allow for more effective control over
the rate of protein release in the body.
[0020] One alternative drug delivery system known in the art
includes the formulation of the protein in a biodegradable, water
insoluble, polymer matrix. The polymer (e.g.,
poly(lactic-co-glycolic acid)) can be formulated as an injectable
or respirable microparticle. Alternately, the protein can be
formulated in a temperature sensitive polymer that is liquid at
room temperature but solidifies at 37.degree. C. after injection
into a patient. A third alternative is for the polymer to be
dissolved in a non-toxic water miscible solvent that dissolves in
plasma after injection leading to precipitation of the polymer. In
all cases, the polymer systems are developed for sustained release
of protein over time; however, the stability of the protein during
the release period is difficult to maintain and generally less than
50% of the total protein load can be delivered. Additionally, the
delivery of the protein is not uniform, but rather occurs with a
rapid initial burst which is followed by a much slower rate of
sustained protein release.
[0021] A second type of known delivery system includes an implanted
pump such as an osmotic pump. In this system, a suspension of
protein in a water miscible organic solvent is continuously
delivered to the patient or test subject through an orifice in the
osmotic pump implant. However, use of this system may prove
problematic because it is often difficult to suspend a high protein
load in the organic solvent, and only some proteins are stable to
prolonged incubation under the required non-aqueous or mixed
organic-aqueous conditions.
[0022] Thus, given the current state of the art, there is a need
for compositions and methods that effectively stabilize a variety
of proteins in various chemical and physical environments, and that
are compatible with a variety of drug delivery systems.
SUMMARY OF THE INVENTION
[0023] The present invention is directed to stable aqueous
solutions and gels of biologically active proteins wherein the
protein solutions and gels are stabilized by high molecular weight
polysaccharide gums. The stable protein solutions and gels may be
used in drug delivery systems and are protected against stresses
such as high temperatures, oxidation, organic solvents, extremes of
pH, drying, freezing, and agitation. Preferably, in the solutions
and gels of the invention, the polysaccharide gums are not bound to
the protein.
[0024] According to a preferred embodiment, the aqueous solutions
or gels of the invention include at least one biologically active
protein, wherein the protein may be an enzyme, antibody, hormone,
growth factor, or cytokine and at least one polysaccharide gum for
stabilizing the protein, wherein the polysaccharide gum may be, for
example, gum arabic, guar gum, xanthan gum, locust bean gum, gum
ghatti, gum karaya, tragacanth gum or a related polysaccharide.
[0025] Drug delivery systems compatible with the present invention
include implanted subcutaneous delivery systems and intravenous
drug delivery systems that can actively or passively deliver the
biologically active proteins.
[0026] In one embodiment of the present invention, high molecular
weight polysaccharide gums are used to stabilize therapeutic
proteins delivered by means of implanted drug delivery devices such
as a capsule, wherein the capsule includes a molecular weight
cut-off membrane with uniform pore size. The polysaccharide gum
stabilizes the protein contained by the capsule and the release of
the protein can be controlled by the membrane which is permeable to
the therapeutic protein but impermeable to the higher molecular
weight gum. This embodiment, therefore, would not necessarily be
compatible with small molecular weight stabilizers that would
diffuse out of the capsule faster than the protein. The membrane
retains the polysaccharide gum in the capsule and the capsule
prevents the gum from swelling and decreasing in concentration.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 shows the ability of gum arabic to stabilize
interferon-.gamma. under physiological conditions over 4 weeks.
[0028] FIG. 2 shows the ability of gum arabic to stabilize lactate
dehydrogenase (LDH) against oxidative stress by copper and ascorbic
acid.
[0029] FIG. 3 shows the ability of gum arabic to stabilize lactate
dehydrogenase against oxidative stress by copper, ascorbic acid,
and hydrogen peroxide.
[0030] FIG. 4 is a schematic diagram of a capsule with a
semi-permeable membrane.
[0031] FIG. 5 is a tabular presentation of chymotrypsin release in
48 hours through a 100K membrane.
[0032] FIG. 6 is a schematic diagram of the apparatus used to
generate the chymotrypsin data for FIG. 5.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention is directed to a heat stable aqueous
solution or gel comprising a biologically effective amount of a
protein and a stabilizing effective amount of a polysaccharide gum
material. The invention is further directed to a heat stable
aqueous solution or gel comprising a biologically effective amount
of a protein and a stabilizing effective amount of a polysaccharide
gum material wherein said protein is selected from the group
consisting of an enzyme, an antibody, a hormone, a growth factor,
and a cytokine wherein said gum is selected from the group
consisting of gum arabic, guar gum, xanthan gum, locust bean gum,
gum karaya, gum ghatti, and tragacanth gum.
[0034] Another embodiment of the invention relates to a heat stable
solution or gel comprising a pharmaceutically effective amount of a
protein and a stabilizing effective amount of a gum material
wherein said stabilized solution or gel is contained in an
implantable drug delivery device.
[0035] A further embodiment of the invention is directed to a
lyophilized composition having biological activity, wherein said
lyophilized composition is formed by lyophilizing a heat stable
solution or gel comprising a biologically effective amount of a
protein and a stabilizing effective amount of a gum material. In a
preferred embodiment, the lyophilized dry powder is reconstituted
in an aqueous buffer to give a high concentration of gum, wherein
the gum preferentially stabilizes the protein to various stresses
under physiological conditions. As used herein the term
"biologically active protein" includes proteins and polypeptides
that are administered to patients as the active drug substance for
prevention of or treatment of a disease or condition as well as
proteins and polypeptides that are used for diagnostic purposes,
such as enzymes used in diagnostic tests or in in vitro assays as
well as proteins that are administered to a patient to prevent a
disease such as a vaccine. Contemplated for use in the compositions
of the invention, but not limited to, are therapeutic proteins and
polypeptides such as enzyrmes, e.g., glucocerebrosidase, adenosine
deaminase; antibodies, e.g., Herceptin.RTM. (trastuzumab),
Orthoclone OKT.RTM.3 (muromonab-CD3); hormones, e.g., insulin and
human growth hormone (HGH); growth factors, e.g., fibroblast growth
factor (FGF), nerve growth factor (NGF), human growth hormone
releasing factor (HGHRF), and cytokines, e.g., leukemia inhibitory
factor (LIF), granulocyte-colony stimulating factor (G-CSF),
granulocytemacrophage-colony stimulating factor (GM-CSF),
interleukin-6 (IL-6), interleukin-11 (IL-11), interleukin-9 (IL-9),
oncostatin-M (OSM), ciliaryneurotrophic factor (CNTF),
interferon-(x, and interferon-.gamma.. As used herein, the
Polysaccharide Solubility Limit is the concentration of
polysaccharide obtained after an aqueous buffer, typically a
phosphate buffered saline (PBS) is slowly added to a solid
polysaccharide, with thorough mixing, until all of the solid
material has either dissolved or has hydrated to form a gel.
Depending on the polysaccharide used, the solubility limit can be
in the vicinity of 10% or can be higher than 50%. Physiological
condition as pertained to this invention is typically human body
temperature under normal conditions, that is, 37.degree. C. a
neutral pH of around 7+1, and a physiological concentration of
saline (0.9%).
[0036] The term "pharmaceutically effective amount" refers to that
amount of a therapeutic protein having a therapeutically relevant
effect on a disease or condition to be treated. A therapeutically
relevant effect relieves to some extent one or more symptoms of a
disease or condition in a patient or returns to normal either
partially or completely one or more physiological or biochemical
parameters associated with or causative of the disease or
condition. Specific details of the dosage of a particular active
protein drug may be found in the drug labeling, i.e., the package
insert (see 21 CFR .sctn. 201.56 & 201.57) approved by the
United States Food and Drug Administration.
[0037] Polysaccharide gums are natural products extracted from
various plants, trees and bacteria, such as Cyamopsis
tetragonolobus (guar gum) and Ceratonia siliqua (locust bean or
carob gum) and Astragalus gummifer (tragacanth) from plants of the
Leguminosae family; gum arabic and tamarind gum from respectively
the Acacia senegal tree and Tamarindus indica tree; xanthan gum
from the bacterial genus Xanthamonas campestris; gum ghatti from
Anogeissus latifolia and gum karaya from Sterculia urens. Many
grades and forms of polysaccharide gums are commercially
available.
[0038] The gum Arabic used in the solutions of the invention has a
highly branched galactose core with linkages to other sugars and
contains .about.1% glycoprotein; the locust gum used herein has a
mannan chain (1->4) with galactose substituted at the 6-position
of .about.20% of the mannose units; the guar gum used herein has a
mannan chain (1->4) with galactose substituted at the 6-position
of -40% of the mannose units; and the xanthan gum used herein has a
glucan (1->4) chain with trisaccharides substituted on every
other glucose.
[0039] According to the preferred embodiment of the present
invention, increasing concentrations of high molecular weight
polysaccharide gums (i.e., greater than 200 kilodaltons) are
utilized for effective protein stabilization. The polysaccharide
gums described herein are more effective protein stabilizers than
commonly used small molecule protein stabilizers such as
monosaccharides, disaccharides, and detergents. High molecular
weight, branched chain or substituted polysaccharides such as gum
arabic, guar gum, xanthan gum, and locust bean gum are more
effective protein stabilizers than linear chain polysaccharides
such as cellulose, agarose, xylan, konjak, or chitosan. The
polysaccharide gums used herein are typically used at
concentrations of gum (% w/v) that are near or at the upper limit
of the solubility of the particular gum in aqueous solutions. The
gums used herein will form either a viscous solution in water or
will form a gel. In general, from about 0.5% to from about 35%
weight to volume ("w/v") will be used depending on the particular
polysaccharide. Gum arabic has exceptional solubility in aqueous
solution and up to about 50% formulations have been made. Preferred
are gum arabic concentrations above 10%, concentrations above 20%
give are more preferred, while concentrations above 30% are yet
more preferred, and concentrations above 40% are the most preferred
as these have resulted in the greatest stability in many of the
tests herein. In a preferred embodiment, stabilizing gum
concentrations should provide stabilities above 70% protein
activity under physiological conditions (at 37.degree. C.) for at
least about a two weeks and most preferably, for patient ease of
use, at least 1 month or more. Recent tests have shown that gum
arabic concentrations of about 50% provide about full
interferon-gamma activity after 1 month and that 45% activity was
still available after two months at physiogical condtions (refer to
Table 9).
[0040] Polysaccharide gums are hydrogels that can absorb many times
their weight of water. Therefore, it is preferable to restrict the
tendency of the gums to swell in order to maintain the high
polysaccharide concentrations that effectively stabilize proteins.
The high gum concentration can be maintained by enclosing the gels
in a capsule with a molecular membrane that is permeable to the
protein but impermeable to the higher molecular weight gum. The
capsules can be implanted into a patient or test subject for the
controlled release of stabilized protein over extended periods.
Over time, the protein is steadily released from the capsule thus,
decreasing the concentration of protein inside the capsule while
the concentration of the stabilizing gum within the capsule remains
constant.
[0041] The present invention includes polysaccharide gums that are
incorporated into drug delivery devices for the purposes of (i)
stabilizing proteins and (ii) controlling the rate at which the
proteins diffuse from the delivery device. The polysaccharide gums
of the present invention stabilize native protein conformations,
even at high protein concentrations. Thus, the delivery device can
be loaded with a protein/gum composition that contains a high
concentration of protein, or with a mixture in the solid form,
thereby increasing the drug load of the device.
[0042] In various embodiments, the compositions of the present
invention are utilized for the stabilization of proteins during
membrane-controlled release from capsules or other devices
implanted into a patient or test subject. In this case, the
delivery device is designed to prevent the polysaccharide from
swelling so that the stabilizing effects of high polysaccharide
concentrations are maintained inside the capsule. Since it is
unnecessary for the polysaccharide gums described herein to bind to
proteins to effect protein stabilization, proteins can be released
from the solution or gel by diffusion. Additionally, the polymeric
properties of polysaccharide gums provide an additional mechanism
for stabilizing proteins by restricting a protein's molecular
mobility.
[0043] The physical state of the polysaccharide gums of the present
invention depends on the conditions used to prepare the gum
solutions. Viscosities can vary several fold for each gum depending
on factors such as the mixing rate used to prepare the hydrated gum
and whether the gum was heat treated or freeze-thawed. Manipulating
the viscosity of the gums will permit the rate at which proteins
are released from the gums to be controlled, as the rate of
diffusion is inversely proportional to the solution viscosity.
[0044] The stabilized protein solutions and gels of the invention
may contain minor amounts (from about 0.5% to about 5.0% w/v) of
auxiliaries and/or excipients, such as N-acetyl-dl-tryptophan,
caprylate, acetate, citrate, glucose and electrolytes, such as the
chlorides, phosphates and bicarbonates of sodium, potassium,
calcium and magnesium. They can furthermore contain: acids, bases
or buffer substances for adjusting the pH, salts, sugars or
polyhydric alcohols for isotonicity and adjustment, preservatives,
such as benzyl alcohol or chlorobutanol, and antioxidants, such as
sulphites, acetylcysteine, Vitamin E or ascorbic acid.
[0045] Suitable tonicity adjustment agents may be, for instance,
physiologically acceptable inorganic chlorides, e.g. sodium
chloride; sugars such as dextrose; lactose; mannitol; sorbitol and
the like. Preservatives suitable for physiological administration
may be, for instance, esters of parahydroxybenzoic acid (e.g.,
methyl, ethyl, propyl and butyl esters, or mixtures of them),
chlorocresol and the like.
[0046] The pH of the solution can be adjusted using a
physiologically acceptable acid e.g. an inorganic mineral acid such
as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric and the
like, or an organic acid such as acetic, succinic, tartaric,
ascorbic, citric, glutamic, benzoic, methanesulphonic,
ethanesulfonic and the like, or a physiologically acceptable base,
such as sodium hydroxide, potassium hydroxide, calcium hydroxide,
magnesium hydroxide and the like, an physiologically acceptable
buffer solution, e.g. a chloride buffer, an acetate buffer, a
phosphate buffer and the like.
[0047] In another embodiment of the present invention, the drug
delivery device is a capsule that is filled with multiple layers of
polysaccharide gums of varying viscosities. This capsule includes a
molecular membrane capable of retaining the gum, but which is
permeable to various therapeutic proteins. In this embodiment, a
layer of viscous gum (e.g., guar gum) adjacent to the polymer
membrane controls the release of the protein, while a layer of less
viscous gum (e.g., gum arabic) that has been formulated with the
protein provides a stable reservoir of protein.
[0048] In still another embodiment, hollow fibers with specifically
defined molecular weight cutoffs are filled with solutions or gels
of gum and protein. Hollow fibers with controlled pore sizes are
useful for rapidly dialyzing proteins. Preferably, the fibers are
made of biocompatible materials that can be implanted in a patient
or test subject. The fibers may be filled with solutions of protein
formulated with guar gum or with locust gum. The gums control the
rate of protein release as well as providing protein stabilization
during release. Because the hollow fibers have a very large surface
area to volume ratio, this approach is most useful for gum/protein
gels with slow protein diffusion rates. A positive attribute of an
implant that contains multiple hollow fibers is that all the
therapeutic protein will not be in the same capsule, thereby
lessening the possibility of a capsule failure, which might release
a toxic dose of the protein.
[0049] An alternate embodiment of hollow fibers includes the steps
of filling the hollow fibers with a gum arabic/protein solution and
imbedding multiple fibers in a matrix of guar or locust gums. The
guar or locust matrix is then enclosed in a dialysis membrane or a
membrane enclosed capsule. This embodiment has many of the
stabilization and diffusion properties of the multi-layered capsule
approach, but in this case the main drug load is not in a single
capsule and is, therefore, less vulnerable to a single capsule
failure.
[0050] According to the present invention, a preferred method for
stabilizing a therapeutic protein in a drug delivery system
comprises the steps of (a) providing a protein as an aqueous
solution; (b) adding a polysaccharide gum to the protein; and (c)
adding the gum/protein solution or gel to a capsule that contains a
molecular membrane. Alternatively, the protein/polysaccharide gum
can be dried by lyophilization or spray drying and added to the
capsule. In this method, the capsule is preferably fabricated from
a biocompatible material and capable of containing the
polysaccharide gum in a fixed volume to prevent the gum from
swelling upon exposure to an aqueous environment. The capsules
comprise a single large protein reservoir or may be comprised of a
plurality of hollow fibers. The membrane is fabricated from silica
or a polymer and has pore sizes, which permit the membrane to be
permeable to the protein but impermeable to the higher molecular
weight polysaccharide gum. The polysaccharide gum is selected based
on its ability to both stabilize the protein and control the
protein's rate of release. In this method, multiple gums and
multiple layers of gums can be used in the capsule.
[0051] The following examples illustrate the effectiveness of the
compositions and methods of the present invention in stabilizing
proteins under different environmental conditions and in different
model delivery systems.
EXAMPLE 1
[0052] A 1 mg/ml solution of chymotrypsin was made by dissolving 20
mgs of chymotrypsin in 20 mL of phosphate buffered saline (PBS, pH
7.4). Solutions of the gums containing chymotrypsin were made as
follows: 1.66 ml of 1 mg/mL chymotrypsin solution in PBS was added
to 0.83 g of gum arabic and homogenized to give a 33% gum arabic
solution by weight, using the assumption that 1 mL of the
chymotrypsin/PBS solution will approximately be equal tol gram. A
similar procedure was repeated for 33% sorbitol. To 0.5 g of
tragacanth gum, gum guar or xanthan gum, 2 ml of 1 mg/mL
chymotrypsin in PBS was added and homogenized to give a 20%
solution. To 0.625 g of gum karaya and gum ghatti, 1.875 mL of 1
mg/mL chymotrypsin in PBS was added and homogenized to give a 25%
solution. Similarly, to 0.35 g of locust bean gum, 2.15 mL of 1
mg/mL chymotrypsin in PBS was added and homogenized to give a 14%
solution. The pH of all the solutions, except 33% sorbitol, was
adjusted to 7.4 using 1 M NaOH. All the samples (33% gum arabic,
33% sorbitol, 20% tragacanth gum, 20% gum guar, 20% xanthan gum and
14% locust bean gum) were prepared in 50 mL centrifuge tubes and
incubated at 60.degree. C. for 7.5 min in a water bath. The samples
were cooled on ice and diluted 20-fold in order to give a final
concentration of 0.05 mg/mL chymotrypsin. Dilutions were performed
by adding 31.73 mL PBS to the 33% gum solutions, 35.625 mL for the
25% gum solutions, 38 ml PBS for the 20% gum solutions 5 and 40.85
mL PBS to the 14% gum solution. These solutions were homogenized
before assaying them for chymotrypsin activity using N-benzoyl
L-tyrosine ethyl ester as the enzyme substrate according to
published literature (J. Biotech, 1994, v35, p9-18). The results
are summarized in Table 1.
1TABLE 1 The Effect of Polysaccharides on the Stability of the
Enzyme Chymotrypsin Incubated at 60.degree. C. for 7.5 Minutes
Concentration of Stabilizer % Recovery % Recovery Stabilizer (%
w/v) 60.degree. C. Room Temp. None -- 0% 100% Sorbitol 33%
.about.27% .about.90% Gum Arabic 33% .about.98% .about.100% Locust
Gum 14% .about.85% .about.100% Guar Gum 20% .about.92% .about.100%
Xanthan Gum 20% .about.50% .about.100% Tragacanth gum 20%
.about.68% .about.100% Gum Karaya 25% .about.30% .about.75% Gum
Ghatti 25% .about.30% .about.100%
[0053] As shown in Table 1, an accelerated aging study performed at
60.degree. C., gum arabic, guar gum, xanthan gum, locust bean gum
and tragacanth gum all stabilized the activity of the enzyme
chymotrypsin over 50%. Recoveries are significantly higher than
those obtained by incubation with sorbitol, a monosaccharide shown
in the literature to stabilize chymotrypsin. Other gums such as gum
karaya and gum ghatti, stabilized chymotrypsin activity around
30%.
[0054] As indicated in Example 1, chymotrypsin is stabilized at
60.degree. C. by high concentrations of gum arabic, guar gum,
xanthan gum, tragacanth, gum karaya, gum ghatti and locust gum. The
stabilizing effects of gum arabic, xanthan gum, locust gum, and
guar gum were found to decrease as the gum concentration decreased,
as shown in Table 2.
2TABLE 2 The Effect of Different Polysaccharide Concentrations on
the Stability of the Enzyme Chymotrypsin Incubated at 60.degree. C.
for 7.5 Minutes % Recovery of Chymotrypsin Activity.sup.a 30% 20%
15% 10% 5% 2.5% 1% Gum Gum Gum Gum Gum Gum Gum Gum Arabic 92% ND
38% ND ND ND ND Guar ND 58% 33% 27% 12% 4% 0% Xanthan ND 46% 31%
26% 14% 3% 0% Locust ND ND 70%.sup.b 48% 26% 24% 14% .sup.aND means
that the experiment under those conditions was not performed.
.sup.bLocust gum was tested at 14% rather than 15%.
[0055] Following the procedure of Example 1, other polysaccharides
and two surfactants were tested. None of the materials listed in
Table 3 were effective to stabilize aqueous solutions of
chymotrypsin against elevated temperatures.
3TABLE 3 Polysaccharides and Surfactants that do not Stabilize
Chymotrypsin at 60.degree. C. Concentration of Sugar or Surfactant
Additive (% w/v) Structure Cellulose 25% Linear .beta.1,4-glucose
chain Agarose 14% Linear chain of galactose and anhydro-galactose
Beechwood 50% Linear xylose chain Xylan Barley Beta 17%
.beta.1,3-glucan chain Glucan Konjak 25% Linear chain of glucose +
mannose Glucomannan Chitosan 17% Linear chain of anhydro-N-acetyl
glucosamine Amylopectin 33% Branched glucose chain Untreated 33%
Branched amylopectin + linear Starch amylose Hydroxyethylstarch 33%
Starch chemically modified with hydroxyethyl groups Dextran 33%
.alpha. 1,6-anhydro-D-glucose chain Tween 20 1%
Polyoxyethylene(20)sorbita- n monolaurate Tween 80 1%
Polyoxyethylene(20)sorbitan monooleate
[0056] Following the procedures described in Example 1, the
concentration of chymotrypsin the solution was varied and the
concentration of gum Arabic was held at 33% (w/v) in the solution.
The results are summarized in Table 4.
4TABLE 4 Stabilization of Increasing Concentrations Of Chymotrypsin
by Gum Arabic Gum Arabic Chymotrypsin Concentration Concentration %
Activity (% w/v) (mg/ml) 7.5 Min., 60.degree. C. 0 1.0 0 0 3.3 0 0
10. 0 0 33. 0 0 100 0 33% 1 97 .+-. 17%.sup.a 33% 3.3 62 .+-.
6%.sup.a 33% 10 57 .+-. 5%.sup.a 33% 33 41%.sup.b 33% 100 53%.sup.b
.sup.aAverage result of two experiments .sup.bResult of a single
experiment
EXAMPLE 2
[0057] Gum arabic (33%) chymotrypsin solutions were prepared as
described in Example 1, with the exception that PBS was made with
0.1% sodium azide. A solution containing 1 mg/ml chymotrypsin and
no gum arabic was prepared as the control. Aliquots of the test and
control solutions were added to centrifuge tubes and these tubes
were incubated at 37.degree. C. for a period of time. The results
are summarized in Table 5.
5TABLE 5 Chymotrypsin Stabilization at 37.degree. C. by 33% (w/v)
Gum Arabic Time Stabilizer Weeks % Activity Control (None) 0 100
Control 1 15 Control 2 0 Control 4 0 Gum Arabic 1 130 Gum Arabic 2
80 Gum Arabic 4 82 Gum Arabic 8 58
[0058] As shown in Table 5, gum arabic was tested for its ability
to stabilize chymotrypsin to long-term incubation at 37.degree. C.
in aqueous buffer, pH 7.4 (physiological conditions). The results
shown in Table 5 indicate that gum arabic protects chymotrypsin and
only approximately 40% of the activity of chymotrypsin is lost
after incubation at 37.degree. C. for eight weeks. In contrast, 85%
of the activity is lost after one week and all the activity is lost
after the second week at 37.degree. C. in the absence of
stabilizer.
[0059] It is important that the concentration of the gum stabilizer
in the aqueous solution be high enough to effectively stabilize the
protein. The stabilization of the particular protein is dependent
on the concentration of polysaccharide gum in the solution. As
shown in Table 6, the activity of chymotrypsin exposed to heat
stress depends on the concentration of gum, with higher
concentrations giving better stability.
6TABLE 6 High Gum Concentrations are Required for Optimal Protein
Stabilization Concentration (% w/v) % Activity Recovered Of Gum
Arabic 1 Week, 37.degree. C. None 0 33 73 20 85 10 62 5 43 2.5 21
1.0 17
[0060] As indicated in Table 6, gum arabic is a stabilizer of
chymotrypsin from 1% to 33%. guar gum has been found to provide
stabilization to chymotrypsin at a concentration of about 2.5 to
20% (w/v), xanthan gum and tragacanth gum provide stabilization at
a concentrations of about 1.5 to 20% (w/v), gum karaya and gum
ghatti are effective in the concentration range of 1 to 25% and
locust gum is effective at a concentration of about 1 to 14% (w/v).
However, concentrations of gum arabic typically above 10% (w/v) are
required for effective use in medical and industrial settings.
Typically from one to several weeks, or months of stability are
preferred.
[0061] The polysaccharide gum stabilizers described herein, not
only are able to stabilize proteins in solution but are also able
to protect such proteins through conventional lyophilization and
subsequent reconstitution. In a preferred application, the
reconstitution of a lyophilized mixture of gum and protein is
carried out in a manner as to maintain the high concentrations of
the gum. Example 3, describes the preparation of an aqueous
solution of the enzyme lactate dehydrogenase (LDH), an enzyme that
loses its activity when lyophilized and then reconstituted.
EXAMPLE 3
[0062] A 1 mg/mL solution of lactate dehydrogenase (LDH) was
prepared by dissolving 5 mgs LDH in 5 mL of PBS, pH 7.4. A 10
.mu.g/mL stock solution of LDH was prepared by dissolving 200 .mu.L
of 1 mg/mL LDH solution in 19.8 mL PBS. The gum solutions with LDH
were prepared as follows: to 0.25 g of each gum or sorbitol, 2.25
mL of 10 ptg/mL LDH was added and homogenized. Aliquots of 1 mL in
plastic Eppendorf tubes were frozen at -70.degree. C. for 30 min
and lyophilized overnight using a Labconco model 77530 lyophilizer.
To each tube of the LDH lyophilizate, 2.25 mL water was added to
give a reconstituted solution of 10 pg/ml enzyme. The reconstituted
lactate dehydrogenase was assayed for activity using the published
literature method of Lovell and Winzor (Biochemistry, 1974, v13,
3527). The results are summarized in Table 7.
7TABLE 7 Stabilization of Lyophilized Lactate Dehydrogenase
(LDH).sup.a Concentration Before Stabilizer Lyophilization %
Activity Recovered None 10 20 Sorbitol 10 20 Gum Arabic 10 64
Xanthan gum 10 37 Locust Bean Gum 10 93 Agarose 10 59 Guar Gum 10
78 .sup.athe "% Activity" was not corrected for the extraction
yield of LDH from the gum solution.
[0063] As shown in Table 7, polysaccharide gums were tested for
their ability to stabilize lactate dehydrogenase, an enzyme that
loses activity when lyophilized. Both the gums that were effective
thermal stabilizers (i.e., gum arabic, guar gum, xanthan gum,
locust bean gum) and a gum that provided no thermal stabilization
(i.e., agarose) were effective stabilizers of lactate dehydrogenase
during the lyophilization process. This result indicates that
polysaccharide gums are effective stabilizers of a lyophilized
protein and shows that the gums can be used to protect therapeutic
proteins that are lyophilized prior to their addition to a drug
delivery device. The protein/polysaccharide powder may be added to
the delivery device dry, as a solution, as a gel, or as slurry. The
lyophilized protein-polysaccharide gums powder will be useful for
long term shelf storage of therapeutic proteins as well. In another
embodiment, the lyophilized powder will be stable in aqueous buffer
solution when it is reconstituted if a high enough concentration of
polysaccharide gum is present in the lyophilized powder or if an
additional amount is added later. Preferably the lyophilized powder
will contain a sufficient concentration of gum to stabilize the
material before and after reconstitution.
Example 4
[0064] Following the procedure described in Example 1, solutions of
lysozyme (1 mg/mL), lactate dehydrogenase (50 .mu.g/mL), and
glucose-6-phosphate dehydrogenase (50 .mu.g/mL) were subjected to
accelerated aging at respectively 90.degree. C., 60.degree. C., and
50.degree. C., and for 10 minutes each. The activity of lactate
dehydrogenase was assayed in accordance with the procedure of
Lovell and Winzor (Biochemistry, 1974, v13, 3527) described in
Example 3. The activity of glucose-6-phosphate dehydrogenase was
assayed using the method published in Arch. Biochem. Biophys, 1998,
v360, p10-14. The activity of lysozyme was assayed according to the
method published in Biochimica et Biophysica Acta, 1952, v8, p
302-309. The results are summarized in Table 8.
8TABLE 8 Thermal Stabilization of Lactate Dehydrogenase,
Glucose-6-Phosphate Dehydrogenase, and Lysozyme By Polysaccharide
Gums Conc. of Accelerated % Conc. of Stabilizer Aging Acti- Protein
Protein Stabilizer (% w/v) Conditions vity Lysozyme 1 mg/mL None 0
90.degree. C.; 5 10 min Lysozyme 1 mg/mL Gum 33 90.degree. C.; 85
Arabic 10 min Lactate 50 .mu.g/mL None 0 60.degree. C.; 2
Dehydrogenase 10 min Lactate 50 .mu.g/mL Trehalose 20 60.degree.
C.; 2 Dehydrogenase 10 min Lactate 50 .mu.g/mL Gum 33 60.degree.
C.; 23 Dehydrogenase Arabic 10 min Glucose-6- 50 .mu.g/mL None 0
50.degree. C.; 0 phosphate 10 min dehydrogenase Glucose-6- 50
.mu.g/mL Gum 33 50.degree. C.; 5 phosphate Arabic 10 min
dehydrogenase Glucose-6- 50 .mu.g/mL Guar 20 50.degree. C.; 60
phosphate Gum 10 min dehydrogenase Glucose-6- 50 .mu.g/mL Trehalose
20 50.degree. C.; 20 phosphate 10 min dehydrogenase
[0065] As shown in Table 8, various proteins are stabilized against
accelerated heat stress by different polysaccharide gums. In a
specific example, while glucose-6-phosphate dehydrogenase is not
stabilized significantly by gum Arabic, another polysaccharide, gum
guar provides good stability. Thus it is possible to stabilize
different proteins using different polysaccharides. Thus, in a
general embodiment, the stabilization of various proteins by
polysaccharides is not limited to the ones given in the above
examples.
Example 5
[0066] Commercial gum arabic was purified by a sequence of
dialysis, filtration, and lyophilization. Dialysis was performed by
first making a 9% solution of gum arabic in deionized water,
homogenizing the sample, and then adjusting the pH to 7.4 with 1 M
sodium hydroxide. The solution was put into 10,000 molecular weight
cut-off dialysis tubing and dialyzed at 4.degree. C. twice against
PBS and twice against deionized water. The sample was then
centrifuged and the supernatant filtered through a 0.22 .mu.m
filter, and lyophilized.
Example 6
[0067] Following the procedures described in Example 1, aliquots of
interferon-.gamma. (0.02 mg/mL) were incubated at 37.degree. C. in
PBS with 0.1% azide that contained no stabilizers, 33% purified gum
arabic, and 50% purified gum arabic (described in Example 5).
Following incubation, samples were diluted in PBS that contained
0.5% BSA and 0.05% Tween 20. The activity of the interferon-.gamma.
in the sample was then measured using an enzyme linked
immunosorbent assay that has been shown to correspond to the
bioactivity (Mechanisms of Ageing and Development, 121, 47-58
(2000). The data is presented in Table 9 and FIG. 1.
9TABLE 9 Thermal Stabilization of Interferon-.gamma. By Gum Arabic
at 37.degree. C. and pH 7.4 Activity Activity Activity Protein +
Gum Arabic (GA) 1 Week 2 Weeks 4 Weeks Interferon-.gamma. + 33%
GA.sup.a 106 .+-. 17% 62 .+-. 15% 40 .+-. 13% Interferon-.gamma. +
50% GA.sup.b 133% 97% 133% Interferon-.gamma. Control.sup.b 1% 1%
9% (no stabilizers) .sup.aThese results are from the average of
three separate experiments, each using duplicate samples.
.sup.bThese results are from a single experiment using duplicate
samples.
Example 7
[0068] This example illustrates the ability of gum arabic to
stabilize proteins against metal catalyzed oxidation (MCO)
reactions. Lactate dehydrogenase is a tetrameric enzyme that
catalyzes the conversion of pyruvate to lactate in the presence of
NADH. The enzymatic activity of LDH is affected in the presence of
oxidants and is catalyzed by the presence of trace amounts of metal
ions in buffers. LDH, 10.sup.-5 mM or 1.4 .mu.g/ml, was prepared in
80 mM phosphate buffer containing 0.2M potassium chloride. MCO
reactions were carried out with 0.04 mM Cupric Sulfate, 2 mM
ascorbic acid (Asc) in the presence or absence of 2 mM hydrogen
peroxide (H.sub.2O.sub.2). All reactions were initiated by the
addition of the reductant (ascorbic acid) or oxidant (hydrogen
peroxide). The reaction mixture was incubated at room temperature
for 15 minutes and assayed using the following protocol for a
96-well plate reader. A 2.64 mM NADH and 4 mM Sodium pyruvate stock
solutions were made in 0.1M phosphate buffer, pH 7.3. All LDH
solutions were diluted to 0.5 ug/ml from the MCO reactions. Two
hundred .mu.l LDH and 25 .mu.l NADH were mixed in a microtiter
plate. The reaction was initiated by adding 751 of sodium pyruvate
and decrease in absorbance at 340 nm was monitored for 10 minutes.
The results are tabulated in Table 10 and shown in FIGS. 2 and 3.
FIG. 2 illustrates the stabilization of lactate dehydrogenase (LDH)
against oxidative stress caused by copper (Cu.sup.2+)+ascorbic acid
(Asc) with different concentrations of gum arabic (GA). FIG. 3
illustrates the stabilization of lactate dehydrogenase (LDH)
against oxidative stress caused by copper (Cu.sup.2+)+ascorbic acid
(Asc)+hydrogen peroxide (H.sub.2O.sub.2) with different
concentrations of gum arabic (GA).
10TABLE 10 Stabilizing Lactate dehydrogenase (LDH) against
Oxidative Stress. Reaction Conditions % Activity LDH 100 LDH +
Cu.sup.2+ + Ascorbic Acid 2 LDH + Cu.sup.2+ + Ascorbic Acid + 50%
GA 121 LDH + Cu.sup.2+ + Ascorbic Acid + 33% GA 75 LDH + Cu.sup.2+
+ Ascorbic Acid + 10% GA 37 LDH + Cu.sup.2+ + Ascorbic Acid + 2% GA
1 LDH + Cu.sup.2+ + Ascorbic Acid + H.sub.2O.sub.2 1 LDH +
Cu.sup.2+ + Ascorbic Acid + H.sub.2O.sub.2 + 50% GA 102 LDH +
Cu.sup.2+ + Ascorbic Acid + H.sub.2O.sub.2 + 33% GA 48 LDH +
Cu.sup.2+ + Ascorbic Acid + H.sub.2O.sub.2 + 10% GA 2 LDH +
Cu.sup.2+ + Ascorbic Acid + H.sub.2O.sub.2 + 2% GA 1
[0069] As seen in Table 10, gum arabic at 50% is an effective
stabilizer against oxidative degradation of chymotrypsin. The
stabilization decreases as the concentration of gum arabic
decreases.
[0070] Referring now to FIG. 4, a preferred method for stabilizing
a protein used in a drug delivery system includes the steps of
providing a protein as an aqueous solution; adding at least one
polysaccharide gum to the protein to form an aqueous solution or
gel and adding the solution or the gel to a delivery system 40. The
delivery system 40 typically comprises a capsule 41, wherein the
capsule 41 further comprises a porous semipermeable or molecular
membrane 43. The capsule 41 is fabricated from a biocompatible
material, and typically contains the protein 45 and polysaccharide
gum 47 in a fixed volume, preventing the gum 47 from swelling when
exposed to an aqueous environment. The membrane 43 of the capsule
41 is fabricated from silica or a polymer and comprises pores 49 of
a size that make the membrane 43 permeable to the proteinaceous
materials but impermeable to the larger polysaccharide gum. The
rate at which the protein diffuses from the capsule 41 can be
controlled by the viscosity of the gum as well as by the
permeability of the membrane 43. Further advantages of the present
invention will become apparent to those of ordinary skill in the
art upon reading and understanding the detailed description herein
of the preferred embodiments.
EXAMPLE 8
[0071] This example illustrates chymotrypsin release through a 100K
membrane using a 30% gum arabic, 5% gum guar or 5% locust bean gum
solution. The release of chymotrypsin from the polysaccharide gums
was evaluated because release of proteinaceous materials from the
microcapsule is an essential requirement for the gums to be used
for protein delivery, Referring now to FIGS. 5 and 6, chymotrypsin
was dissolved in a solution containing 30% gum arabic and added to
capsules 63 that contained 100K molecular weight cut off dialysis
membranes. The protein diffuses out of the viscous polysaccharide,
through the membrane 67, and into the exchange buffer solution 68
at approximately 85% of the rate at which chymotrypsin diffuses
from aqueous buffer through the membrane. The rates at which
chymotrypsin diffuses through guar gum and locust bean gum through
the membrane 67 are approximately 20% of the rate obtained with an
aqueous control. This indicates that guar and locust bean gums can
be used to significantly reduce the rate of protein delivery
through a membrane device, in addition to providing stabilization
for the protein. Additionally, mixtures of various gums can
effectively be used to stabilize and control the release of
therapeutic proteins from implantable capsules.
[0072] FIG. 6 illustrates the system 60 used for evaluating used
chymotrypsin release. A falcon tube 61 was fitted with a capsule 63
containing the chymotrypsin and gum materials 64. A cap 65 having a
hole fitted with membrane 67 allowed for diffusion of chymotrypsin
into an exchange buffer 68. A magnetic stir bar 69 provided for
thorough mixing of the exchange buffer 68.
[0073] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one skilled in
the art are intended to be included within the scope of the
following claims.
* * * * *