U.S. patent application number 15/183077 was filed with the patent office on 2017-01-05 for methods for purifying polysaccharides and pharmaceutical compositions and medical devices containing the same.
The applicant listed for this patent is Aurelien Forget, Venkatram Prasad Shastri. Invention is credited to Aurelien Forget, Venkatram Prasad Shastri, Daniel Vonwil.
Application Number | 20170002099 15/183077 |
Document ID | / |
Family ID | 53797515 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170002099 |
Kind Code |
A1 |
Forget; Aurelien ; et
al. |
January 5, 2017 |
Methods for Purifying Polysaccharides and Pharmaceutical
Compositions and Medical Devices Containing the Same
Abstract
Methods for removing endotoxin from naturally occurring
materials, such as polysaccharides (e.g., agarose and/or
carrageenan) are described herein. Polysaccharides that are
substantially free of endotoxins and uses thereof are also
described. The polysaccharide materials can be isolated from
microorganisms, multicellular organisms, such as, algae, plants,
seaweed, etc. The method involves the use of acidic and basic
solutions to hydrolyze the lipid-polysaccharide bond in endotoxins.
Cleaving the fatty acid from the polysaccharide reduces the
water-solubility of the fatty acid and enables its removal with an
organic solvent such as ethanol. The polysaccharide component can
also undergo acidic or basic hydrolysis due to the weak glycosidic
bond between the sugar rings.
Inventors: |
Forget; Aurelien; (Paris,
FR) ; Vonwil; Daniel; (Badenwurtemberg, DE) ;
Shastri; Venkatram Prasad; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Forget; Aurelien
Shastri; Venkatram Prasad |
Paris |
|
FR
US |
|
|
Family ID: |
53797515 |
Appl. No.: |
15/183077 |
Filed: |
June 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14624294 |
Feb 17, 2015 |
9388252 |
|
|
15183077 |
|
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61940800 |
Feb 17, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08B 37/0039 20130101;
A61K 6/898 20200101; A61K 6/898 20200101; A61K 6/898 20200101; A61L
27/20 20130101; A61K 6/898 20200101; A61L 27/52 20130101; A61K
6/898 20200101; A61L 31/145 20130101; A61L 26/0023 20130101; A61K
31/729 20130101; C08L 5/12 20130101; A61L 27/20 20130101; C08L 1/00
20130101; C08L 5/12 20130101; C08L 5/12 20130101; C08L 1/00
20130101; A61L 15/28 20130101; C08L 5/12 20130101; C08B 37/0003
20130101; C08L 5/12 20130101; C08J 3/075 20130101; A61L 31/042
20130101; A61K 31/7016 20130101; A61L 2400/04 20130101; C08J
2305/12 20130101; A61L 15/60 20130101; A61K 9/06 20130101; A61K
9/0019 20130101; A61K 47/36 20130101; A61K 38/18 20130101; A61L
15/60 20130101 |
International
Class: |
C08B 37/00 20060101
C08B037/00; A61K 9/06 20060101 A61K009/06; A61K 38/18 20060101
A61K038/18; A61K 6/097 20060101 A61K006/097; C08J 3/075 20060101
C08J003/075; A61L 26/00 20060101 A61L026/00; A61L 27/20 20060101
A61L027/20; A61L 27/52 20060101 A61L027/52; A61L 31/14 20060101
A61L031/14; A61L 31/04 20060101 A61L031/04; A61K 47/36 20060101
A61K047/36; A61L 15/28 20060101 A61L015/28 |
Claims
1. A method for isolating and purifying a naturally occurring
agarose or derivative thereof produced from a biological source,
the method comprising: (i) dispersing the agarose in one or more
aliphatic alcohols to disrupt the bacterial wall to solubilize the
lipid portion of endotoxins; (ii) removing the aliphatic alcohol to
remove the lipid portion of the endotoxins and obtain the agarose
or derivative thereof in solid form; (iii) dispersing the solid
agarose or derivative thereof in a basic solution to hydrolyze
lipid-inner core bonds of the endotoxins and solubilize the
polysaccharide component of the endotoxins; (iv) washing the
solution from step (iii) with an aliphatic alcohol to remove free
lipids; (v) removing the basic solution in step (iii) to obtain the
agarose or derivative thereof in solid form; (vi) dispersing the
solid agarose or derivative thereof in an acidic solution to
hydrolyze lipid-inner core bonds of the endotoxins not cleaved in
step (iii); (vii) removing the acidic solution in step (vi) to
obtain the agarose or derivative thereof in solid form; (viii)
dispersing the solid agarose or derivative thereof from step (vii)
in a second basic solution to further cleave lipid-inner core bonds
and to neutralize the acid from step (vi); (ix) removing the basic
solution in step (viii) to obtain the agarose or derivative thereof
in solid form; and (x) dispersing or suspending or dissolving the
solid agarose or derivative thereof from step (ix) in sterile water
to solubilize the polysaccharide component of the endotoxin and to
neutralize any residual acid and base; and (xi) removing the
sterile water in step (x) to obtain the agarose or derivative
thereof in solid form, wherein the agarose or derivative thereof is
substantially free of endotoxin.
2. The method of claim 1, wherein aliphatic alcohol in step (i)
and/or (iv) is selected from the group consisting of ethanol,
n-propanol, isopropanol, glycerol, and combinations thereof.
3. The method of claim 1, wherein the acidic solution is any
aqueous solution of an acid selected from the group consisting of
hydrochloric acid, sulfuric acid, citric acid, acetic acid, and
formic acid.
4. The method of claim 3, wherein the concentration of the acidic
solution is from about 0.05 to about 2M.
5. The method of claim 3, wherein the acidic solution is 0.5 M
HCl.
6. The method of claim 1, wherein the basic solution in step (iii)
and/or (viii) is an aqueous solution of a base selected from the
group consisting of sodium hydroxide, potassium hydroxide, and
calcium hydroxide.
7. The method of claim 6, wherein the concentration of the base is
from about 0.01 m to about 2M, preferably from about 0.1M to about
0.5M.
8. The method of claim 1, wherein the basic solution is 0.25 M
NaOH.
9. The method of claim 1, wherein the solid agarose or derivative
thereof in step (xi) is dissolved in an autoclavable physiological
buffer solution.
10. The method of claim 9, wherein the buffer is Ringer Buffer.
11. The method of claim 9, wherein the buffer is removed to obtain
the agarose or derivative thereof in solid form.
12. The method of claim 1, wherein the agarose or derivative
thereof is sterilized using a technique selected from the group
consisting of steam and gamma irradiation.
13. The method of claim 1, wherein the agarose or derivative
thereof is a hydrogel-forming material.
14. A pharmaceutical composition comprising a purified agarose or
derivative thereof, wherein the agarose or derivative thereof is
substantially free of endotoxins, and one or more pharmaceutically
acceptable carriers.
15. The pharmaceutical composition of claim 14, further comprising
one or more therapeutic and prophylactic agents selected from the
group consisting of proteins, peptides, growth factors, small
molecule drugs, and combinations thereof.
16. The pharmaceutical composition of claim 15, wherein the
therapeutic or prophylactic agent is a growth factor.
17. The pharmaceutical composition of claim 14, wherein the agarose
or agarose derivative thereof and one or more pharmaceutically
acceptable carriers are in the form of a drug delivery system.
18. A medical device comprising a purified agarose or derivative
thereof, wherein the agarose or derivative thereof is substantially
free of endotoxins.
19. The device of claim 18, wherein the device is selected from the
group consisting of wound dressings, hemostatic materials (dressing
or powder), dermal, bone or teeth fillers, implantable gel,
implantable devices, and cell carriers.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 14/624,294, filed Feb. 17, 2015, which claims priority to U.S.
Provisional Application No. 61/940,800, filed Feb. 17, 2014, the
disclosures of which are incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention is in the field of methods for the
purification of naturally occurring materials having clinical
importance, particularly the removal of endoxtoxins.
BACKGROUND OF THE INVENTION
[0003] Medical devices, such as dermal fillers or implants made
from naturally occurring materials including polysaccharides, are
in direct contact with organ tissue and therefore must be made from
biocompatible materials. Such materials have to (1) be inert, (2)
induce little or no immune response, and (3) in some cases be
degradable by the organism. Unfortunately, medical devices can be
contaminated during their manufacturing, transportation and/or
storage.
[0004] Contamination such as by microorganisms (e.g., bacteria,
virus or spores) or chemicals can induce inflammatory responses,
which can lead to fever, hypotension, systemic shock and even
death. Moreover, many of these materials are isolated from
naturally occurring sources, such as microorganisms, and typically
contain unacceptable levels of endotoxins. Therefore, the
successful translation of medical devices to the clinic requires
ensuring their safety until implantation.
[0005] One of the most serious contaminations that can occur in
medical devices prepared from naturally occurring materials, such
as polysaccharides, is endotoxins. Endotoxins are integral
components of Gram-negative bacterial cell walls. Endotoxins are
exogenous pyrogenic substances, i.e. they can induce fever.
Endotoxins typically contain a polysaccharide chain, which is water
soluble, bound to a hydrophobic or water-insoluble lipid strand. A
representative structure for an endotoxin is shown in FIG. 6:
[0006] Due to their amphiphilicity, endotoxins are difficult to
remove from commercially useful materials.
[0007] Several sterilization processes are commonly used to prevent
contamination including filtration, gamma irradiation, ethylene
oxide, steam, and chemical sterilization. These procedures are
able, to varying degrees, to kill microorganisms (bacteria and
mammalian cells), destroy viruses (protein carriers of genetic
information such as DNA or RNA), or degrade endotoxins
(lipopolysaccharides originating from bacteria wall). Autoclaving,
which is a common technique used for sterilization, cannot remove
endotoxins. Moreover, the removal of endotoxins from commercially
useful materials, such as agarose or carrageenan, is challenging
since these materials themselves are polysaccharides. Moreover,
these techniques can also generate degradation products of the
material, which can be toxic, or causes changes to its mechanical
properties, affecting the proper operation of the device. A summary
of the drawbacks of various sterilization techniques is shown in
Table 1.
TABLE-US-00001 TABLE 1 Advantages and drawbacks of the different
sterilization methods Material mechanical Technique Sterility*
Endotoxin properties preservation Cost Filtration +/+/- - + $
Irradiation +/+/+ - - $$$ Ethylene Oxide +/+/+ - - $$$ Steam +/+/-
- - ++ $$ Ethanol +/-/- + +++ $ *Bacteria/Virus/Spores
[0008] Irradiation and EO are two techniques that are fast and
quite efficient for removal of viruses and endotoxins.
Unfortunately they cannot be used for the sterilization of
polysaccharide, such as agarose, as they modify the polysaccharide
backbone. Therefore specific considerations of the sterilization
method have to be taken into account for obtaining a material that
meets the requirements of regulatory agencies. Table 2 shows some
of the requirements of the Food and Drug Administration (FDA) in
the United States for clinical grade materials.
TABLE-US-00002 TABLE 2 Some of the requirement for clinical grade
materials Level Regulation Source Ref. Ethanol Content <0.5% v/v
FDA (1) Microbiology N.D. FDA Endotoxins <0.05 EU/ml FDA (2) (1)
Serdons, et al. Journal of Nuclear Medicine: Official Publication,
Society of Nuclear Medicine 2008; 49: 2071 (2) FDA
Bacterial/Endotoxins Pyrogens. Available at
http://www.fda.gov/ICECI/InspectionGuides/InspectionTechnicalGuides/ucm07-
2918.htm#.Ug0qTDAoFos.email
[0009] Dialysis of naturally occurring materials material against
pyrogen free water has been explored to overcome the limitations of
the techniques discussed above. Dialysis, however, is an expensive
and time consuming process.
[0010] There exists a need for improved methods for removing
endotoxin from naturally occurring materials, such as
polysaccharides.
[0011] Therefore, it is an object of the invention to provide for
improved methods for removing endotoxin from naturally occurring
materials, such as polysaccharides.
[0012] It is also an object of the invention to provide for
improved methods for removing endotoxin from naturally occurring
materials, such as polysaccharides, which are cost effective and
can be performed over a relatively short period of time.
[0013] It is also an object of the invention to provide for
improved methods for removing endotoxin from naturally occurring
materials, such as polysaccharides, which are cost effective and
can be performed over a relatively short period of time, and which
do not significantly alter the chemical, physical, and/or
mechanical properties of the material.
SUMMARY OF THE INVENTION
[0014] Methods for removing endotoxin from naturally occurring
materials, such as polysaccharides (e.g., agarose and/or
carrageenan) are described herein. The materials can be isolated
from microorganisms, multicellular organisms, such as, algae,
plants, seaweed, etc. The method involves the use of acidic and
basic solutions to hydrolyze the lipid-polysaccharide bond in
endotoxins. Cleaving the fatty acid from the polysaccharide reduces
the water-solubility of the fatty acid and enables its removal with
an organic solvent such as ethanol. The polysaccharide component
can also undergo acidic or basic hydrolysis due to the weak
glycosidic bond between the sugar rings.
[0015] The acceptable endotoxin level unit concentration (EU/ml)
established by the Food and Drug Administration (FDA) in the United
States for medical devices is 0.05 EU/ml or 20 EU/device. The
endotoxin level of commercially available agarose after steam
sterilization has an EU level above FDA standards. However, after
the endotoxin removal procedure described herein and autoclaving,
the endotoxins level was about 0.02 EU/ml.
[0016] The mechanical properties of the hydrogel after the
procedure described herein were assessed. The entire process,
purification and packaging, was performed manually, and therefore
this assessment also took into account the operator error leading
to variations in syringe loading. The mechanical testing reveals
that there were no significant changes in the hydrogel properties
between the untreated and the purified product. This suggests that
the use of acidic and basic solutions and steam sterilization does
not alter the agarose backbone. The hemolytic properties of the
agarose was compared to silicone and medical (surgical steel) using
the 24 h lysis test available from HaemoScan. The amount of lysis
induced by agarose was 3.9%/cm.sup.2, which is considered
acceptable according to ISO 10993-4 (<5%).
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a flow chart describing one embodiment of the
isolation and purification procedure described herein.
[0018] FIG. 2 is a graph showing the concentration of endotoxin
(EU/ml) in commercial agarose after autoclaving (i.e., before
endotoxin removal) and agarose after the purification process
described herein (e.g., after endotoxin removal).
[0019] FIG. 3 is a graph showing the concentration of residual
ethanol (% v/v) in commercial agarose after autoclaving (i.e.,
before endotoxin removal) and agarose after the purification
procedure described herein (e.g., after endotoxin removal) compared
to endotoxin free water.
[0020] FIG. 4 is a graph showing the shear modulus G' (Pa) for
commercial agarose after autoclaving (i.e., before endotoxin
removal) and agarose after the purification procedure described
herein.
[0021] FIG. 5 is a graph showing percent lysis per cm.sup.2 as a
function of material.
[0022] FIG. 6 is a representative structure of an endotoxin, having
a water soluble polysaccharide chain bound to a hydrophobic lipid
strand.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0023] "Polysaccharide derivative" as used herein refers to a
polysaccharide that possesses the same core as the parent compound,
but differs from the parent compound, with one or more substituents
attached to the core, which may include one or more atoms,
functional groups, or substructures. In general, a derivative can
be imagined to be formed, at least theoretically, from the parent
compound via chemical and/or physical processes. Typically in a
polysaccharide derivative the constituent saccharide moieties are
substituted by one or more substituents that differ from those of
the parent polysaccharide. Exemplary substituents include but are
not limited to carboxylate, phosphate, sulfate, and sulfonate
groups, and combinations of such groups.
[0024] "Agarose derivative," as used herein refers to a molecule
that has substantially the same basic structure as agarose (i.e.,
alternating D-galactose and 3,6-anhydro-L-galactopyranose linked by
.alpha.-(1.fwdarw.3) and .beta.-(1.fwdarw.4) glycosidic bonds)
wherein the constituent saccharide moieties are substituted by one
or more substituents that differ from those of agarose. Exemplary
substituents include but are not limited to carboxylate, phosphate,
sulfate, and sulfonate groups, and combinations of such groups.
[0025] "Substantially free of endotoxin", as described herein,
means that in endotoxin free water placed on top of an equivalent
volume of a 2% polysaccharide sample (2 day exposure) less than
0.05 Endotoxin Units can be found per milliliter.
[0026] The term "targeting moiety" as used herein refers to a
moiety that localizes to or away from a specific locale. The moiety
may be, for example, a protein, nucleic acid, nucleic acid analog,
carbohydrate, or small molecule. The entity may be, for example, a
therapeutic compound such as a small molecule, or a diagnostic
entity such as a detectable label. The locale may be a tissue, a
particular cell type, or a subcellular compartment. In one
embodiment, the targeting moiety directs the localization of an
active entity. The active entity may be a small molecule, protein,
polymer, or metal. The active entity may be useful for therapeutic,
prophylactic, or diagnostic purposes.
[0027] "Adhesion site", as used herein, refers to a peptide
sequence to which a molecule, for example, an adhesion-promoting
receptor on the surface of a cell, binds. Examples of adhesions
sites include, but are not limited to, the RGD sequence from
fibronectin, and the YIGSR sequence from laminin. Preferably
adhesion sites are incorporated into the biomaterial of the present
invention
[0028] "Growth factor", as used herein refers to endogenous or
synthetic peptides or proteins that impact status of mammalian
cells, such as cell cycle, proliferation, differentiation, and/or
cell death. Exemplary growth factors include but are not limited to
heparin-binding growth factors (HBGFs). HBGFs are a large class of
growth factors that include the 23 fibroblast growth factors
identified to date (FGFs 1-23), HBBM (heparin-binding brain
mitogen), HB-GAF (heparin-binding growth associated factor), HB-EGF
(heparin-binding EGF-like factor) HB-GAM (heparin-binding growth
associated molecule), TGF-.alpha. (transforming growth
factor-.alpha.), TGF-.beta.s (transforming growth factor-.beta.s),
PDGF (platelet-derived growth factor), EGF (epidermal growth
factor), VEGF (vascular endothelial growth factor), IGF-1
(insulin-like growth factor-1), IGF-2 (insulin-like growth
factor-2), HGF (hepatocyte growth factor), IL-1 (interleukin-1),
IL-2 (interleukin-2), IFN-.alpha. (interferon-.alpha.), IFN-.gamma.
(interferon-.gamma.), TNF-.alpha. (tumor necrosis factor-.alpha.),
SDGF (Schwannoma-derived growth factor) and many other growth
factors such as, cytokines, lymphokines and chemokines that have an
affinity for heparin.
[0029] "Growth factor binding site", as used herein, refers to a
peptide sequence to which a growth factor, or a molecule(s) which
binds a growth factor binds. For example, the growth factor binding
site may include a heparin binding site. This site will bind
heparin, which will in turn, bind heparin-binding growth factors,
for example, bFGF, VEGF, BMP, or TGF.beta..
[0030] "Protease binding site", as used herein, refers to a peptide
sequence which is a substrate for an enzyme.
II. Methods for Removal of Endotoxin
[0031] Methods for removing endotoxin from naturally occurring
materials, such as polysaccharides (e.g., agarose and/or
carrageenan) are described herein. The materials can be isolated
from microorganisms, multicellular organisms, such as, algae,
plants, seaweed, etc. The method involves the use of acidic and
basic solutions to hydrolyze the lipid-polysaccharide bond in
endotoxins. Cleaving the fatty acid from the polysaccharide reduces
the water-solubility of the fatty acid and enables its removal with
an organic solvent such as ethanol. The polysaccharide component of
the endotoxin can also undergo acidic or basic hydrolysis due to
the weak glycosidic bond between the sugar rings.
[0032] In some embodiments, the method is as follows. Step 1
involves disruption of the bacteria to free the endotoxin and
solubilize the lipid portion of the endotoxin (i.e. Lipid A in FIG.
6). In some embodiments, the material to be purified, such as a
naturally occurring polysaccharide, is dissolved in an organic
solvent. The solvent should be biocompatible and volatile so that
it is easily removed. In some embodiments, the organic solvent is
an aliphatic alcohol, such as ethanol, n-propanol, isopropanol, or
glycerol. Non-alcoholic solvents, such as acetone, may also be
used. In some embodiments, the aliphatic alcohol is ethanol. The
solution of the material to be purified can be agitated, such as by
stirring, shaker tray, or sonication. Once this step is complete,
the material is isolated, typically as a solid, using techniques
known in the art, such as centrifugation and removal of the
supernatant.
[0033] Once the lipid portion of the endotoxin has been removed,
the lipid inner core bond must be hydrolyzed to release and
solubilize the polysaccharide component of the endotoxin. This
hydrolysis can be achieved by a series of base- and acid-catalyzed
hydrolyses. In one embodiment, the solid from step 1 is dispersed
or suspended in a basic solution. Any base can be used. In some
embodiments, the base is an inorganic base, such as sodium
hydroxide, potassium hydroxide, or calcium hydroxide. In some
embodiments, the base is sodium hydroxide.
[0034] The strength of the base solution can vary. However, it is
preferable that the strength is sufficiently low that it does not
degrade the material to be purified, e.g., the polysaccharide
backbone (such as, for example, the agarose backbone). In some
embodiments, the strength of the basic solution is from about 0.01
to about 2M, preferably from about 0.1M to about 0.5M. In some
embodiments, the strength is 0.25M. The free lipids are removed by
washing with an organic solvent, such as an aliphatic alcohol
(e.g., ethanol). Once this step is complete, the material is
isolated, typically as a solid, using techniques known in the art,
such as centrifugation and removal of the supernatant.
[0035] Once the first base hydrolysis step is complete,
acid-hydrolysis is performed to cleave the lipid/inner core bond
that was not cleaved under basic conditions. Any acid can be used.
In some embodiments, the acid is a mineral acid, such as
hydrochloric acid, sulfuric acid, citric acid, acetic acid, or
formic acid. In some embodiments, the acid is hydrochloric
acid.
[0036] The strength of the acid solution can vary. However, it is
preferable that the strength is sufficiently low that it does not
degrade the material to be purified, e.g., the polysaccharide
backbone. In some embodiment, the strength of the acid solution is
from about 0.01 to about 2M, preferably from about 0.1 M to about
1M. In some embodiments, the strength is 0.5M. Optionally, free
lipids may be removed by washing with an organic solvent, such as
an aliphatic alcohol (e.g., ethanol). Once this step is complete,
the material is isolated, typically as a solid, using techniques
known in the art, such as centrifugation and removal of the
supernatant.
[0037] One the acid hydrolysis step is completed, a second base
hydrolysis step is performed. The second base hydrolysis step is
performed as described above to complete the hydrolysis of the
lipid/inner core bond and to neutralize any residual acid from the
previous step. The base can be the same base as above or a
different base and the strength can be the same or different. Once
this step is complete, the material is isolated, typically as a
solid, using techniques known in the art, such as centrifugation
and removal of the supernatant.
[0038] Once the acid/base hydrolysis steps are complete, the solid
material to be purified is dispersed or suspended in sterile water
to remove polysaccharide component of the endotoxin and neutralize
residual acid and/or base. Once this step is complete, the material
is isolated, typically as a solid, using techniques known in the
art, such as centrifugation and removal of the supernatant. The
material to be purified can be dissolved in an autoclavable
physiological buffer, such as Ringer Buffer) for additional
neutralization of residual acid and/or base. Once this step is
complete, the material is isolated, typically as a solid, using
techniques known in the art, such as centrifugation and removal of
the supernatant.
[0039] The purified material can be assayed for endotoxin levels,
residual solvent levels, presence of microbiological organisms, and
the mechanical properties of the material (e.g., hydrogel-forming
materials).
[0040] The endotoxin level unit concentration (EU/ml) established
by the Federal Drug Agency (FDA) is 0.05 EU/ml. The endotoxin level
of commercially available agarose after steam sterilization (but
not purified as described herein) exhibited an EU level above FDA
standards. However, after endotoxin removal using the procedure
described herein and autoclaving, the endotoxins level was about
0.02 EU/ml. The assay for residual solvent showed levels less than
0.2% v/v, which is less than the 0.5% v/v established by the FDA.
Agarose that was purified as described herein and autoclaved showed
no growth of bacterial colonies after incubation for up to one week
in Luria Broth (LB).
[0041] When the purified material is to be used for structural
applications, such as artificial tissue, to mimic extracellular
matrices, etc., the mechanical properties of the material are
critical. Mechanical testing revealed that there were no
significant changes in the hydrogel properties between the
commercial untreated agarose and the agarose purified as described
herein. This suggests that procedure described herein did not alter
the agarose backbone.
[0042] After the purification described above, the material can be
stored until needed. However, if the material is to be used
immediately, the material can be dissolved in a suitable solvent,
such as sterilized water, the pH adjusted if necessary to 7, and
the solution filtered through a filter (e.g., 40 micron pore size)
to remove any insoluble submicron impurities. The volume of the
solution can be adjusted to obtain the desired concentration of
material, e.g., 2% w/v, and loaded in autoclavable devices, such as
syringes. The syringes are autoclaved to sterilize the syringe and
solubilize the material. Upon cooling, the material, if it is
hydrogel-forming, will gel which is a convenient phase for
storage.
[0043] The procedure described above can be used to remove
endotoxin for a variety of naturally occurring materials. In some
embodiments, the material is a hydrogel-forming material. In some
embodiments, the naturally occurring material is a polysaccharide,
such as hydrogel-forming polysaccharides. In some embodiments, the
polysaccharide is agarose or carrageenan. The structures of agarose
and carrageenan are shown below:
##STR00001##
[0044] An agarose is a polysaccharide polymer material, generally
extracted from seaweed. Agarose is a linear polymer made up of the
repeating unit of agarobiose, which is a disaccharide made up of
D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is one of
the two principal components of agar, and is purified from agar by
removing agar's other component, agaropectin.
[0045] Agarose is a linear polymer with a molecular weight of about
120,000, consisting of alternating D-galactose and
3,6-anhydro-L-galactopyranose linked by .alpha.-(1.fwdarw.3) and
.beta.-(1.fwdarw.4) glycosidic bonds. The
3,6-anhydro-L-galactopyranose is an L-galactose with an anhydro
bridge between the 3 and 6 positions, although some L-galactose
units in the polymer may not contain the bridge. Some D-galactose
and L-galactose units can be methylated, and pyruvate and sulfate
are also found in small quantities.
[0046] Each agarose chain contains .about.800 molecules of
galactose, and the agarose polymer chains form helical fibers that
aggregate into supercoiled structure with a radius of 20-30 nm. The
fibers are quasi-rigid, and have a wide range of length depending
on the agarose concentration. When solidified, the fibers form a
three-dimensional mesh of channels of diameter ranging from 50 nm
to >200 nm depending on the concentration of agarose used. The
3-D structure is held together with hydrogen bonds and can
therefore be disrupted by heating back to a liquid state.
[0047] Agarose is available as a white powder which dissolves in
near-boiling water, and forms a gel when it cools. Agarose gels and
melts at different temperatures, and the gelling and melting
temperature vary depending on the type of agarose. Standard
agaroses derived from Gelidium have a gelling temperature of
35-38.degree. C. and a melting temperature of 90-95.degree. C.,
while those derived from Gracilaria have a gelling temperature of
40-42.degree. C. and a melting temperature of 85-90.degree. C. The
melting and gelling temperature may be dependent on the
concentration of the gel, particularly at low gel concentration of
less than 1%. The gelling and melting temperature is therefore
given at a specified concentration.
[0048] In some embodiments, the constituent saccharide moieties of
agarose may be substituted by one or more substituents that differ
from those of naturally occurring agarose to form a chemically
modified agarose derivative. Exemplary substituents include but are
not limited to carboxylate, phosphate, sulfate, or sulfonate
groups, or combinations such groups.
[0049] Natural agarose contains uncharged methyl groups. The extent
of methylation is directly proportional to the gelling temperature.
Synthetic methylation, however, has the reverse effect, whereby
increased methylation lowers the gelling temperature. A variety of
chemically modified agarose derivatives with different melting and
gelling temperatures are available; these are often made by
hydroxyethylation of agarose. In certain embodiments, a
carboxylated agarose derivative, depending on the degree of
carboxylation, may have a variable gelation temperature as low as
10.degree. C. (see Forget, et al. Proceedings of the National
Academy of Sciences, 2013; 110(32):12877-12892). An agarose gel or
chemically modified agarose derivative gel can also have high gel
strength at a low concentration, such as in the range of 0.5-2.0%.
Gel strength, refers to the force (Pascals) that must be applied to
fracture a gel, such as an agarose gel, of a standard
concentration.
[0050] Agarose can be used for medical applications in different
forms, including an injectable viscous suspension for dermal
filling or as a hydrogel patch for a variety of applications, such
as cartilage repair. Agarose can also be used to mimic
extracellular matrices. Agarose, due to its insolubility in water
at room temperature, undergoes hydrolysis only at high temperature.
Therefore use of low concentrated basic or acidic solution at room
temperature or lower as described above should not degrade agarose.
Moreover, endotoxins are of low molecular weight (e.g., 1-10 kDa),
are partially soluble in water, and can be efficiently separated
from the higher molecular weight, immiscible in cold water, agarose
(e.g., 120 kDa) by filtration.
[0051] Carrageenans are a family of linear sulfated polysaccharides
that are extracted from red edible seaweeds. They are widely used
in the food industry, for their gelling, thickening, and
stabilizing properties. All carrageenans are high-molecular-weight
polysaccharides made up of repeating galactose units and
3,6-anhydrogalactose (3,6-AG), both sulfated and nonsulfated. The
units are joined by alternating alpha 1-3 and beta 1-4 glycosidic
linkages. Carrageenans are large, highly flexible molecules that
curl forming helical structures. This gives them the ability to
form a variety of different gels at room temperature.
[0052] There are three main varieties of carrageenan, which differ
in their degree of sulfation. The primary differences that
influence the properties of kappa, iota, and lambda carrageenan are
the number and position of the ester sulfate groups on the
repeating galactose units. Kappa-carrageenan has one sulfate per
disaccharide. Iota-carrageenan has two sulfates per disaccharide.
Lambda carrageenan has three sulfates per disaccharide. Higher
levels of ester sulfate lower the solubility temperature of the
carrageenan and produce lower strength gels, or contribute to gel
inhibition (lambda carrageenan). All are soluble in hot water, but
in cold water, only the lambda form (and the sodium salts of the
other two) are soluble. Kappa forms strong, rigid gels in the
presence of potassium ions. Iota forms soft gels in the presence of
calcium ions. Lambda does not gel, and is used as a thickening
agent.
III. Applications
[0053] The purified polysaccharides described herein can be used
for a variety of applications, particularly applications where
endotoxin levels must be below regulatory standards, such as
consumable products (e.g., foods, beverages, etc.), pharmaceutical
compositions for drug delivery. In certain embodiments, the
purified polysaccharides and one or more pharmaceutically
acceptable carriers form a drug carrier to deliver therapeutic
and/or prophylactic agents. In other embodiments, the purified
polysaccharides are used in a variety of medical devices, such as
but not limited to wound dressings, hemostatic materials, dermal,
bone, or teeth fillers, implantable gels, devices for cell
delivery, and other implantable devices.
[0054] In some embodiments, the purified polysaccharide or
derivative forms a hydrogel in vivo, such as when the hydrogel is
administered to a patient, such as when it is placed subdermally
(below the skin), adjacent to bone, adjacent to cartilage, or
placed intramuscularly (within muscle). In other embodiments, the
purified polysaccharide or derivative forms a hydrogel prior to
administration to a patient. For example, the polysaccharide
hydrogel may be loaded with one or more cells or therapeutic or
prophylactic agents to form an implantable drug delivery device and
implanted in a patient. Optionally, the hydrogel may be implanted
in a patient subdermally (below the skin), adjacent to bone,
adjacent to cartilage, or placed intramuscularly (within muscle).
In some embodiments, the hydrogel implanted in the patient can be
used to induce angiogenesis and/or vascular formation.
[0055] A. Pharmaceutical Compositions
[0056] In some embodiments, the polysaccharides are used as
controlled release materials to provide sustained and/or delayed
release of one or more therapeutic, prophylactic, and/or diagnostic
agents. The agents can be encapsulated in or dispersed with the
polysaccharide, particular hydrogel forming polysaccharides, and/or
the agent can be covalently or non-covalently bound or associated
with the polysaccharide. For example, the polysaccharide can be
chemically modified to introduce one or more reactive functional
groups to which can be bound or associate one or more active
agents, targeting moieties, and/or other groups.
[0057] The compositions can be formulated as microparticles or
nanoparticles which form a hydrogel upon contact with biological
fluids. The particles can be administered by any route of
administration, such as enteral (e.g., oral) or parenteral.
[0058] Exemplary classes of agents include, but are not limited to,
anti-analgesics, anti-inflammatory drugs, antipyretics,
antidepressants, antiepileptics, antiopsychotic agents,
neuroprotective agents, anti-proliferatives, such as anti-cancer
agents (e.g., taxanes, such as paclitaxel and docetaxel; cisplatin,
doxorubicin, methotrexate, etc.), growth factors, anti-infectious
agents, such as antibacterial agents and antifungal agents,
antihistamines, antimigraine drugs, antimuscarinics, anxioltyics,
sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma
drugs, cardiovascular drugs, corticosteroids, dopaminergics,
electrolytes, gastro-intestinal drugs, muscle relaxants,
nutritional agents, vitamins, parasympathomimetics, stimulants,
anorectics and anti-narcoleptics. Nutraceuticals can also be
incorporated. These may be vitamins, supplements such as calcium or
biotin, or natural ingredients such as plant extracts or
phytohormones.
[0059] The agents can be small molecules, i.e., organic, inorganic,
or organometallic agents having a molecule weight less than 2000,
1500, 1200, 1000, 750, or 500 amu, biomolecules or macromolecules
(e.g., having MW greater than 2000), or combinations thereof.
[0060] Examples of small molecule therapeutic agents include, but
are not limited to, acyclovir, amikacin, anecortane acetate,
anthracenedione, anthracycline, an azole, amphotericin B,
bevacizumab, camptothecin, cefuroxime, chloramphenicol,
chlorhexidine, chlorhexidine digluconate, clortrimazole, a
clotrimazole cephalosporin, corticosteroids, dexamethasone,
desamethazone, econazole, eftazidime, epipodophyllotoxin,
fluconazole, flucytosine, fluoropyrimidines, fluoroquinolines,
gatifloxacin, glycopeptides, imidazoles, itraconazole, ivermectin,
ketoconazole, levofloxacin, macrolides, miconazole, miconazole
nitrate, moxifloxacin, natamycin, neomycin, nystatin, ofloxacin,
polyhexamethylene biguanide, prednisolone, prednisolone acetate,
pegaptanib, platinum analogues, polymicin B, propamidine
isethionate, pyrimidine nucleoside, ranibizumab, squalamine
lactate, sulfonamides, triamcinolone, triamcinolone acetonide,
triazoles, vancomycin, anti-vascular endothelial growth factor
(VEGF) agents, VEGF antibodies, VEGF antibody fragments, vinca
alkaloid, timolol, betaxolol, travoprost, latanoprost, bimatoprost,
brimonidine, dorzolamide, acetazolamide, pilocarpine,
ciprofloxacin, azithromycin, gentamycin, tobramycin, cefazolin,
voriconazole, gancyclovir, cidofovir, foscarnet, diclofenac,
nepafenac, ketorolac, ibuprofen, indomethacin, fluoromethalone,
rimexolone, anecortave, cyclosporine, methotrexate, tacrolimus and
combinations thereof.
[0061] In one embodiment, the particles/liposomes contain an
anti-tumor agent. Classes of antitumor agents include, but are not
limited to, angiogenesis inhibitors, DNA
intercalators/crosslinkers, DNA synthesis inhibitors, DNA-RNA
transcription regulators, enzyme inhibitors, gene regulators,
microtubule inhibitors, and other antitumor agents.
[0062] Examples of angiogenesis inhibitors include, but are not
limited to, Angiostatin K1-3, DL-.alpha.-Difluoromethyl-ornithine,
Endostatin, Fumagillin, Genistein, Minocycline, Staurosporine,
(.+-.)-Thalidomide, revlimid, and analogs and derivatives
thereof.
[0063] Examples of DNA intercalators/cross-linkers include, but are
not limited to, Bleomycin, Carboplatin, Carmustine, Chlorambucil,
Cyclophosphamide, cis-Diammineplatinum(II) dichloride (Cisplatin),
Melphalan, Mitoxantrone, Oxaliplatin, analogs and derivatives
thereof.
[0064] Examples of DNA-RNA transcription regulators include, but
are not limited to, Actinomycin D, Daunorubicin, Doxorubicin,
Homoharringtonine, Idarubicin, and analogs and derivatives
thereof.
[0065] Examples of enzyme inhibitors include, but are not limited
to, S(+)-Camptothecin, Curcumin, (-)-Deguelin,
5,6-Dichlorobenz-imidazole 1-.beta.-D-ribofuranoside, Etoposide,
Formestane, Fostriecin, Hispidin, 2-Imino-1-imidazoli-dineacetic
acid (Cyclocreatine), Mevinolin, Trichostatin A, Tyrphostin AG 34,
Tyrphostin AG 879, and analogs and derivatives thereof.
[0066] Examples of gene regulators include, but are not limited to,
5-Aza-2'-deoxycytidine, 5-Azacytidine, Cholecalciferol (Vitamin
D3), Hydroxytamoxifen, Melatonin, Mifepristone, Raloxifene, all
trans-Retinal (Vitamin A aldehyde), Retinoic acid, all trans
(Vitamin A acid), 9-cis-Retinoic Acid, 13-cis-Retinoic acid,
Retinol (Vitamin A), Tamoxifen, Troglitazone, and analogs and
derivative thereof.
[0067] Examples of microtubule inhibitors include, but are not
limited to, Colchicine, Dolastatin 15, Nocodazole, Paclitaxel,
docetaxel, Podophyllotoxin, Rhizoxin, Vinblastine, Vincristine,
Vinorelbine (Navelbine), and analogs and derivatives thereof.
[0068] Examples of other antitumor agents include, but are not
limited to, 17-(Allylamino)-17-demethoxygeldanamycin,
4-Amino-1,8-naphthalimide, Apigenin, Brefeldin A, Cimetidine,
Dichloromethylene-diphosphonic acid, Leuprolide (Leuprorelin),
Luteinizing Hormone-Releasing Hormone, Pifithrin-.alpha.,
Rapamycin, Sex hormone-binding globulin, Thapsigargin, Urinary
trypsin inhibitor fragment (Bikunin), and analogs and derivatives
thereof.
[0069] In other embodiments, the agent is a biomolecule, such as a
nucleic acid, protein, peptide, growth factor, etc.
[0070] Examples of classes of growth factors and growth-factor like
peptides include, but are not limited to, FGF, TGF .beta., BMPs,
IGFs, VEGFs, NGF, BDNF, HGH and PDGFs. Examples of specific growth
factors or growth factor-like peptides include, but are not limited
to, BMP 2, BMP 7, TGF .beta.1, TGF .beta.3, FGF-2, NGF, IGF 1, IGF
2 PDGF AB, human growth releasing factor, PTH 1-84, PTH 1-34 and
PTH 1-25.
[0071] The nucleic acid can alter, correct, or replace an
endogenous nucleic acid sequence. The nucleic acid is used to treat
cancers, correct defects in genes in other pulmonary diseases and
metabolic diseases affecting lung function, genes such as those for
the treatment of Parkinson's and ALS where the genes reach the
brain through nasal delivery.
[0072] Gene therapy is a technique for correcting defective genes
responsible for disease development. Researchers may use one of
several approaches for correcting faulty genes: A normal gene may
be inserted into a nonspecific location within the genome to
replace a nonfunctional gene. An abnormal gene can be swapped for a
normal gene through homologous recombination. The abnormal gene can
be repaired through selective reverse mutation, which returns the
gene to its normal function. The regulation (the degree to which a
gene is turned on or off) of a particular gene can be altered.
[0073] The nucleic acid can be a DNA, RNA, a chemically modified
nucleic acid, or combinations thereof. For example, methods for
increasing stability of nucleic acid half-life and resistance to
enzymatic cleavage are known in the art, and can include one or
more modifications or substitutions to the nucleobases, sugars, or
linkages of the polynucleotide. The nucleic acid can be custom
synthesized to contain properties that are tailored to fit a
desired use. Common modifications include, but are not limited to
use of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs),
morpholinos, peptide nucleic acids (PNA), phosphorothioate
linkages, phosphonoacetate linkages, propyne analogs, 2'-O-methyl
RNA, 5-Me-dC, 2'-5' linked phosphodiester linkage, Chimeric
Linkages (Mixed phosphorothioate and phosphodiester linkages and
modifications), conjugation with lipid and peptides, and
combinations thereof.
[0074] In some embodiments, the nucleic acid includes
internucleotide linkage modifications such as phosphate analogs
having achiral and uncharged intersubunit linkages (e.g., Sterchak,
E. P. et al., Organic Chem., 52:4202, (1987)), or uncharged
morpholino-based polymers having achiral intersubunit linkages
(see, e.g., U.S. Pat. No. 5,034,506). Some internucleotide linkage
analogs include morpholidate, acetal, and polyamide-linked
heterocycles. Other backbone and linkage modifications include, but
are not limited to, phosphorothioates, peptide nucleic acids,
tricyclo-DNA, decoy oligonucleotide, ribozymes, spiegelmers
(containing L nucleic acids, an apatamer with high binding
affinity), or CpG oligomers.
[0075] Phosphorothioates (or S-oligos) are a variant of normal DNA
in which one of the nonbridging oxygens is replaced by a sulfur.
The sulfurization of the internucleotide bond dramatically reduces
the action of endo- and exonucleases including 5' to 3' and 3' to
5' DNA POL 1 exonuclease, nucleases S1 and P1, RNases, serum
nucleases and snake venom phosphodiesterase. In addition, the
potential for crossing the lipid bilayer increases. Because of
these important improvements, phosphorothioates have found
increasing application in cell regulation. Phosphorothioates are
made by two principal routes: by the action of a solution of
elemental sulfur in carbon disulfide on a hydrogen phosphonate, or
by the more recent method of sulfurizing phosphite triesters with
either tetraethylthiuram disulfide (TETD) or
3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD). The latter methods
avoid the problem of elemental sulfur's insolubility in most
organic solvents and the toxicity of carbon disulfide. The TETD and
BDTD methods also yield higher purity phosphorothioates.
[0076] Peptide nucleic acids (PNA) are molecules in which the
phosphate backbone of oligonucleotides is replaced in its entirety
by repeating N-(2-aminoethyl)-glycine units and phosphodiester
bonds are replaced by peptide bonds. The various heterocyclic bases
are linked to the backbone by methylene carbonyl bonds. PNAs
maintain spacing of heterocyclic bases that is similar to
oligonucleotides, but are achiral and neutrally charged molecules.
Peptide nucleic acids are typically comprised of peptide nucleic
acid monomers. The heterocyclic bases can be any of the standard
bases (uracil, thymine, cytosine, adenine and guanine) or any of
the modified heterocyclic bases described below. A PNA can also
have one or more peptide or amino acid variations and
modifications. Thus, the backbone constituents of PNAs may be
peptide linkages, or alternatively, they may be non-peptide
linkages. Examples include acetyl caps, amino spacers such as
8-amino-3,6-dioxaoctanoic acid (referred to herein as 0-linkers),
and the like. Methods for the chemical assembly of PNAs are well
known.
[0077] In some embodiments, the nucleic acid includes one or more
chemically-modified heterocyclic bases including, but are not
limited to, inosine, 5-(1-propynyl) uracil (pU), 5-(1-propynyl)
cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine,
pseudoisocytosine, 5 and
2-amino-5-(2'-deoxy-.beta.-D-ribofuranosyl)pyridine
(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine
derivatives, 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine,
aziridinylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methyl guanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,
4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid
methylester, 2,6-diaminopurine, and 2'-modified analogs such as,
but not limited to O-methyl, amino-, and fluoro-modified analogs.
Inhibitory RNAs modified with 2'-flouro (2'-F) pyrimidines appear
to have favorable properties in vitro.
[0078] In some embodiments the nucleic acid includes one or more
sugar moiety modifications, including, but are not limited to,
2'-O-aminoethoxy, 2'-O-amonioethyl (2'-OAE), 2'-O-methoxy,
2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-0,4'-C-methylene (LNA),
2'-O-(methoxyethyl) (2'-OME) and 2'-O--(N-(methyl)acetamido)
(2'-OMA).
[0079] Methods of gene therapy typically rely on the introduction
into the cell of a nucleic acid molecule that alters the genotype
of the cell. Introduction of the nucleic acid molecule can correct,
replace, or otherwise alters the endogenous gene via genetic
recombination. Methods can include introduction of an entire
replacement copy of a defective gene, a heterologous gene, or a
small nucleic acid molecule such as an oligonucleotide. This
approach typically requires delivery systems to introduce the
replacement gene into the cell, such as genetically engineered
viral vectors.
[0080] Methods to construct expression vectors containing genetic
sequences and appropriate transcriptional and translational control
elements are well known in the art. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Expression vectors generally contain
regulatory sequences necessary elements for the translation and/or
transcription of the inserted coding sequence. For example, the
coding sequence is preferably operably linked to a promoter and/or
enhancer to help control the expression of the desired gene
product. Promoters used in biotechnology are of different types
according to the intended type of control of gene expression. They
can be generally divided into constitutive promoters,
tissue-specific or development-stage-specific promoters, inducible
promoters, and synthetic promoters.
[0081] Viral vectors include adenovirus, adeno-associated virus,
herpes virus, vaccinia virus, polio virus, AIDS virus, neuronal
trophic virus, Sindbis and other RNA viruses, including these
viruses with the HIV backbone. Also useful are any viral families
which share the properties of these viruses which make them
suitable for use as vectors. Typically, viral vectors contain,
nonstructural early genes, structural late genes, an RNA polymerase
III transcript, inverted terminal repeats necessary for replication
and encapsidation, and promoters to control the transcription and
replication of the viral genome. When engineered as vectors,
viruses typically have one or more of the early genes removed and a
gene or gene/promoter cassette is inserted into the viral genome in
place of the removed viral DNA.
[0082] Gene targeting via target recombination, such as homologous
recombination (HR), is another strategy for gene correction. Gene
correction at a target locus can be mediated by donor DNA fragments
homologous to the target gene (Hu, et al., Mol. Biotech.,
29:197-210 (2005); Olsen, et al., J. Gene Med., 7:1534-1544
(2005)). One method of targeted recombination includes the use of
triplex-forming oligonucleotides (TFOs) which bind as third strands
to homopurine/homopyrimidine sites in duplex DNA in a
sequence-specific manner. Triplex forming oligonucleotides can
interact with either double-stranded or single-stranded nucleic
acids. When triplex molecules interact with a target region, a
structure called a triplex is formed, in which there are three
strands of DNA forming a complex dependent on both Watson-Crick and
Hoogsteen base-pairing. Triplex molecules are preferred because
they can bind target regions with high affinity and specificity. It
is preferred that the triplex forming molecules bind the target
molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12. Methods
for targeted gene therapy using triplex-forming oligonucleotides
(TFO's) and peptide nucleic acids (PNAs) are described in U.S.
Published Application No. 20070219122 and their use for treating
infectious diseases such as HIV are described in U.S. Published
Application No. 2008050920. The triplex-forming molecules can also
be tail clamp peptide nucleic acids (tcPNAs), such as those
described in U.S. Published Application No. 2011/0262406.
[0083] Double duplex-forming molecules, such as a pair of
pseudocomplementary oligonucleotides, can also induce recombination
with a donor oligonucleotide at a chromosomal site. Use of
pseudocomplementary oligonucleotides in targeted gene therapy is
described in U.S. Published Application No. 2011/0262406.
[0084] B. Devices
[0085] In some embodiments, the purified polysaccharides can be
used to prepare or form medical devices, particular devices for
tissue repair, strengthening, and/or reinforcement. The materials
can also be used to mimic extracellular matrices.
Examples
Example 1
Purification of Polysaccharide and Removal of Bacterial
Lipopolysaccharide
[0086] The procedure was performed under a laminar flow hood that
was previously cleaned with an antibacterial/antiviral solution
followed by a 1 hour UV light exposure. The operator wore gloves
and protective gear required for chemical and biological work to
avoid product contamination and to ensure personal safety. All the
solutions used were pharmaceutical grade and all tools, which were
in direct contact with the product or the reagents were sterile and
specified endotoxin free. A flow chart of the isolation and
purification process is shown in FIG. 1.
[0087] The procedure for isolating and purifying agarose was
conducted as follows:
[0088] Step 1: In order to disrupt the bacteria wall and solubilize
the lipid part of the endotoxin, the agarose was incubated under
agitation for two minutes in ethanol. Agarose powder was recovered
by centrifugation and removal of the supernatant.
[0089] Step 2: A 0.25 M solution of sodium hydroxide was used to
hydrolyze the lipid-inner core bond and solubilize the
polysaccharide. The free lipids were removed with an ethanol wash.
The agarose was recovered by centrifugation and removal of the
supernatant.
[0090] Step 3: A 0.5 M solution of hydrochloride acid was used to
hydrolyze the lipid/inner core bond that was not cleaved under
basic conditions and to solubilize the polysaccharide. Optionally,
an ethanol wash may be performed to remove free lipids. The agarose
was recovered by centrifugation and removal of the supernatant.
[0091] Step 4: A second wash with a 0.25 M solution of sodium
hydroxide was performed for further cleavage of the lipid/inner
core bound and to neutralize the acid from the previous step. The
agarose was recovered by centrifugation and removal of the
supernatant.
[0092] Step 5: A solution of sterile water was used for final
removal of the polysaccharide and neutralization of residual acid
and/or base. The agarose was recovered by centrifugation and the
supernatant was removed.
[0093] Step 6: An autoclavable physiological buffer solution
(Ringer Buffer) was used for final solubilization of agarose and
neutralization of the acidic and basic solutions. The agarose was
recovered by centrifugation and removal of the supernatant.
[0094] Step 7: The pH of the agarose suspension was adjusted to 7
and filtrated through a 40 .mu.m pore size filter that retained the
agarose but removed soluble submicron impurities.
[0095] Step 8: The volume was adjusted to 2% w/v agarose hydrogel
and loaded into autoclavable syringes, which were packed in
autoclave pouches prior to steam sterilization. This last step is
used to sterilize the syringes and to solubilize the agarose in the
syringe. After cooling, the agarose gelled, which is a convenient
phase for storage. The resulting agarose loaded syringes were
analyzed to assess the efficiency of the procedure.
[0096] The characterization of the agarose after the sterilization
protocol involved four steps: (a) endotoxin removal was quantified;
(b) the presence of microbiological organisms was assessed, (c) the
amount of remaining solvent was measured and (d) the hydrogel
mechanical properties were characterized.
[0097] Measurement of endotoxins levels was used to assess the
effectiveness of the procedure. The acceptable endotoxin level unit
concentration (EU/ml) established by the Food and Drug
Administration (FDA) in the United States for medical devices is
0.05 EU/ml. The endotoxin level of commercially available agarose
after steam sterilization has an EU level above FDA standards.
Whereas, after the endotoxin removal procedure described above and
autoclaving, the endotoxins level was about 0.02 EU/ml (n=5, see
FIG. 2).
[0098] Solvents, such as ethanol, can cause tissue damage via
dehydration of cells and contact with nerves (e.g., neurolysis).
Ethanol is classified as a class 3 solvent with low potential
toxicity by the FDA. The maximum allowed concentration is 0.5% v/v.
Since ethanol is used to sterilize agarose powder, the amount
remaining in the hydrogel has to be assessed. Gas Chromatography
(GC) measurement showed a concentration of 0.16% v/v (see FIG.
3).
[0099] As discussed above, the elimination of bacterial organisms
can be performed using techniques known in the art, including
chemical or thermal treatment as well as sterile filtration or
irradiation. In order to assess if the protocol describe herein was
as efficient as the industry standards, the presence of microbial
contamination was monitored. After the sequential washes and
autoclaving, incubation of agarose for up to a week with Luria
Broth (LB) media did not show any bacterial colonies.
[0100] Optionally, sterilization using steam or gamma irradiation
may be applied to the purified polysaccharide or polysaccharide
derivative (i.e., agarose or agarose derivative thereof) such that
the sterilization process when applied does not substantially alter
the properties (i.e., gelation temperature or shear modulus) by
more than 10% as compared to the properties of the polysaccharide
or polysaccharide derivative prior to purification. Sterilization
techniques, such as steam or gamma irradiation are well-known in
the art.
[0101] The mechanical properties of the hydrogel after the
procedure described herein were assessed. The entire process,
purification and packaging, was performed manually, and therefore
this assessment also took into account the operator error leading
to variations in syringe loading. In certain embodiments, the
gelation temperature and/or mechanical properties (i.e., shear
modulus) of the purified polysaccharide or polysaccharide
derivative (e.g., agarose or agarose derivative thereof) are the
same or are substantially the same as the gelation temperature
and/or mechanical properties of the polysaccharide or
polysaccharide derivative prior to purification. As used herein
"substantially the same" means about 10% or less relative change to
the gelation temperature and/or mechanical property values of the
polysaccharide or polysaccharide derivative prior to purification.
Methods for determining the physical properties of polysaccharide
hydrogels are known in the art. Exemplary methods are provided in
Forget, et al. Proceedings of the National Academy of Sciences,
2013; 110(32):12877-12892, the disclosure of which is incorporated
herein by reference.
[0102] The mechanical testing reveals that there were no
significant changes in the hydrogel properties between the
untreated and the purified product (n=3, see FIG. 4). This suggests
that the use of acidic and basic solutions and steam sterilization
does not alter the agarose backbone.
[0103] The hemolytic properties of the agarose were compared to
silicone and medical (surgical steel) using the 24 h lysis test
available from HaemoScan. The results are shown in FIG. 5. The
amount of lysis induced by agarose was 3.9%/cm.sup.2, which is
considered acceptable according to ISO 10993-4 (<5%).
[0104] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0105] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *
References