U.S. patent application number 13/855738 was filed with the patent office on 2013-08-29 for method for the preparation of a heat stable oxygen carrier-containing pharmaceutical composition and the use thereof.
This patent application is currently assigned to Billion King International Limited. The applicant listed for this patent is Billion King International Limited. Invention is credited to Sui Yi KWOK, Bing Lou WONG.
Application Number | 20130219829 13/855738 |
Document ID | / |
Family ID | 45353226 |
Filed Date | 2013-08-29 |
United States Patent
Application |
20130219829 |
Kind Code |
A1 |
WONG; Bing Lou ; et
al. |
August 29, 2013 |
METHOD FOR THE PREPARATION OF A HEAT STABLE OXYGEN
CARRIER-CONTAINING PHARMACEUTICAL COMPOSITION AND THE USE
THEREOF
Abstract
A highly purified and heat stable cross-linked tetrameric
hemoglobin suitable for use in mammals without causing renal injury
and vasoconstriction is provided. A high temperature and short time
(HTST) heat processing step is performed to remove undesired
dimeric form of hemoglobin and the plasma proteins impurities
effectively. Addition of N-acetyl cysteine to the heat stable
cross-linked tetrameric hemoglobin maintains a low level of
met-hemoglobin. The heat stable cross-linked tetrameric hemoglobin
can improve and prolong oxygenation in normal and hypoxic tissue.
In another aspect, the product is used in the treatment of various
types of cancer such as leukemia, colorectal cancer, lung cancer,
breast cancer, liver cancer, nasopharyngeal carcinoma and
esophageal cancer. Another application is heart preservation in
situations where there is a lack of oxygen supply in vivo, such as
in heart transplant or oxygen-deprived heart. The presently claimed
invention also includes a method for preparing a packaged
cross-linked tetrameric hemoglobin composition.
Inventors: |
WONG; Bing Lou; (Irvine,
CA) ; KWOK; Sui Yi; (Hong Kong, HK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Billion King International Limited; |
Hong Kong |
|
HK |
|
|
Assignee: |
Billion King International
Limited
Hongkong
HK
|
Family ID: |
45353226 |
Appl. No.: |
13/855738 |
Filed: |
April 3, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12821214 |
Jun 23, 2010 |
|
|
|
13855738 |
|
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Current U.S.
Class: |
53/440 ;
206/524.1 |
Current CPC
Class: |
A61P 7/00 20180101; B65B
1/00 20130101; A61K 38/42 20130101 |
Class at
Publication: |
53/440 ;
206/524.1 |
International
Class: |
B65B 1/00 20060101
B65B001/00 |
Claims
1. A method for the preparation of a highly purified and heat
stable oxygen carrier-containing pharmaceutical composition
packaged in a multilayer, flexible infusion package, the oxygen
carrier-containing pharmaceutical composition including hemoglobin,
the hemoglobin consisting essentially of nonpolymeric cross-linked
tetrameric hemoglobin, the method comprising: a) providing
mammalian whole blood including at least red blood cells and
plasma; b) separating the red blood cells from the plasma in the
mammalian whole blood; c) filtering the red blood cells that were
separated from the plasma to obtain a filtered red blood cell
fraction; d) washing the filtered red blood cell fraction to remove
plasma protein impurities, resulting in washed red blood cells; e)
disrupting the washed red blood cells by a precise and controlled
hypotonic lysis for 2 to 30 seconds or a time otherwise sufficient
to lyse the red blood cells in an instant cytolysis apparatus to
create a solution comprising a lysate of disrupted red blood cells
at a flow rate of 50-1000 L/hr; f) performing filtration to remove
at least a portion of the waste retentate from the lysate; g)
extracting a first hemoglobin solution from the lysate; h)
performing a first ultrafiltration process, using an
ultrafiltration filter configured to remove impurities having a
higher molecular weight than hemoglobin to further remove any
viruses and residual waste retentate from the first hemoglobin
solution to obtain a second hemoglobin solution; i) performing
flowthrough column chromatography on the purified hemoglobin
solution to remove protein impurities; j) performing a second
ultrafiltration process using an ultrafiltration filter configured
to remove impurities and to concentrate the purified hemoglobin
solution; k) cross-linking the hemoglobin in a
bis-3,5-dibromosalicyl fumarate cross-linking solution to form
cross-linked hemoglobin in a deoxygenated environment wherein the
cross-linked hemoglobin is nonpolymeric cross-linked tetrameric
hemoglobin; l) exchanging a suitable physiological buffer for the
cross-linking solution; m) removing any residual chemicals by
tangential flow filtration; n) heat treating the cross-linked
hemoglobin in a deoxygenated environment to denature and
precipitate any residual non-reacted hemoglobin, non-stabilized
hemoglobin (dimer) and any other protein impurities such that the
resulting heat stable cross-linked tetrameric hemoglobin has an
undetectable concentration of dimer and consists essentially of
nonpolymeric cross-linked tetrameric hemoglobin; o) adding N-acetyl
cysteine immediately following heat treating the cross-linked
tetrameric hemoglobin to maintain a low level of met-hemoglobin; p)
removing precipitate by a centrifugation or a filtration apparatus
to form a clear solution; q) adding the purified and heat stable
cross-linked tetrameric hemoglobin to a pharmaceutically acceptable
carrier and r) introducing the purified and heat stable
cross-linked tetrameric hemoglobin and pharmaceutically acceptable
carrier to a multilayer, flexible infusion package.
2. The method of claim 1 wherein said multilayer, flexible infusion
package comprises a multilayer aluminum/polyethylene terephthalate
(PET)/nylon/polyethylene (PE) overwrap with a thickness of 0.14 mm
and an oxygen transmission rate of 0.006 cm.sup.3 per 100 square
inches per 24 hours per atmosphere at room temperature.
3. The method of claim 2 wherein said multilayer, flexible infusion
package further comprises five layers of ethylene-vinyl-acetate
(EVA)/ethylene-vinyl alcohol (EVOH) material with a thickness of
0.4 mm that has an oxygen permeability of 0.006-0.132 cm.sup.3 per
100 square inches per 24 hours per atmosphere at room
temperature.
4. The method of claim 1 further comprising storage at ambient
conditions for a period of time up to two years.
5. A packaged oxygen carrier-containing pharmaceutical composition
formed according to the method of claim 1.
6. A packaged oxygen carrier-containing pharmaceutical composition
formed according to the method of claim 2.
7. A packaged oxygen carrier-containing pharmaceutical composition
formed according to the method of claim 3.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of the
U.S. non-provisional patent application Ser. No. 12/821,214 filed
Jun. 23, 2010, and the disclosure of which is incorporated herein
by reference and its entirety.
COPYRIGHT NOTICE/PERMISSION
[0002] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever. The following notice
applies to the processes, experiments, and data as described below
and in the drawings attached hereto: Copyright.COPYRGT. 2013,
Billion King International Limited, All Rights Reserved.
TECHNICAL FIELD
[0003] The present invention relates to a method for the
preparation of a heat stable oxygen-carrier-containing
pharmaceutical composition and the composition made by the process.
The present invention also relates to the use of the heat stable
oxygen carrier-containing pharmaceutical composition for cancer
treatment, oxygen-deprivation disorders and organ preservation for
humans and other animals.
BACKGROUND OF INVENTION
[0004] Hemoglobin plays an important role in most vertebrates for
gaseous exchange between the vascular system and tissue. It is
responsible for carrying oxygen from the respiratory system to the
body cells via blood circulation and also carrying the metabolic
waste product carbon dioxide away from body cells to the
respiratory system, where the carbon dioxide is exhaled. Since
hemoglobin has this oxygen transport feature, it can be used as a
potent oxygen supplier if it can be stabilized ex vivo and used in
vivo.
[0005] Naturally-occurring hemoglobin is a tetramer which is
generally stable when present within red blood cells. However, when
naturally-occurring hemoglobin is removed from red blood cells, it
becomes unstable in plasma and splits into two .alpha.-.beta.
dimers. Each of these dimers is approximately 32 kDa in molecular
weight. These dimers may cause substantial renal injury when
filtered through the kidneys and excreted. The breakdown of the
tetramer linkage also negatively impacts the sustainability of the
functional hemoglobin in circulation.
[0006] In order to solve the problem, recent developments in
hemoglobin processing have incorporated various cross-linking
techniques to create intramolecular bonds within the tetramer as
well as intermolecular bonds between the tetramers to form
polymeric hemoglobin. The prior art teaches that polymeric
hemoglobin is the preferred form in order to increase circulatory
half-life of the hemoglobin. However, as determined by the present
inventors, polymeric hemoglobin more readily converts to
met-hemoglobin in blood circulation. Met-hemoglobin cannot bind
oxygen and therefore cannot oxygenate tissue. Therefore, the
cross-linking taught by the prior art that causes the formation of
polymeric hemoglobin is a problem. There is a need in the art for a
technique that permits intramolecular crosslinking to create stable
tetramers without the simultaneous formation of polymeric
hemoglobin.
[0007] Further problems with the prior art attempts to stabilize
hemoglobin include production of tetrameric hemoglobin that
includes an unacceptably high percentage of dimer units; the
presence of dimers makes the hemoglobin composition unsatisfactory
for administration to mammals. The dimeric form of the hemoglobin
can cause severe renal injury in a mammalian body; this renal
injury can be severe enough to cause death. Therefore, there is a
need in the art to create stable tetrameric hemoglobin with
undetectable dimeric form in the final product.
[0008] Another problem with prior art hemoglobin products is a
sudden increase in blood pressure following administration. In the
past, vasoconstriction events have been recorded from older
generation of hemoglobin based oxygen carriers. For instance, the
Hemopure.RTM. product (Biopure Co., USA) resulted in higher mean
arterial pressure (124.+-.9 mmHg) or 30% higher when compared to
the baseline (96.+-.10 mmHg) as disclosed by Katz et al., 2010.
Prior attempts to solve this problem have relied on sulfhydryl
reagents to react with hemoglobin sulfhydryl groups, allegedly to
prevent endothelium-derived relaxing factor from binding to the
sulfhydryl groups. However, the use of sulfhydryl treatment adds
processing steps, resulting in added cost and impurities which must
be later removed from the hemoglobin composition. Thus there is a
need in the art for a process to prepare hemoglobin which will not
cause vasoconstriction and high blood pressure when applied to a
mammal.
[0009] Further problems with prior art attempts to create stable
hemoglobin include the presence of protein impurities such as
immunoglobin G that can cause allergic effects in mammals.
Therefore, there is a need in the art for a process which can
produce stable tetrameric hemoglobin without protein
impurities.
[0010] In addition to the above problems, there is a need in the
art for a stabilized tetrameric hemoglobin that is dimer free,
phospholipid free and capable of production on an industrial
scale.
SUMMARY OF INVENTION
[0011] The present invention provides a method for processing a
heat stable purified tetrameric hemoglobin suitable for use in
mammals without causing severe renal injury, vascular detrimental
effects and severe adverse events including death. The present
invention removes the dimeric form of hemoglobin, phospholipids and
protein impurities. Additionally, the present invention uses (1) an
instant cytolysis apparatus for precise and controlled hypotonic
lysis, (2) a flowthrough column chromatography, (3) a high
temperature short time (HTST) apparatus for heat processing the
hemoglobin solution in the purification process to remove the
undesirable non-stabilized dimers of hemoglobin and to remove the
protein impurities, for example immunoglobin-G, so that renal
injury, vascular detrimental effects and other toxicity reactions
can be avoided, and (4) an air-tight infusion bag packaging to
avoid oxygen intrusion into the product.
[0012] The method includes a starting material of mammalian whole
blood including at least red blood cells and plasma. Red blood
cells are separated from the plasma in the mammalian whole blood
followed by filtering to obtain a filtered red blood cell fraction.
The filtered red blood cell fraction is washed to remove plasma
protein impurities. The washed red blood cells are disrupted by a
controlled hypotonic lysis for a time sufficient to lyse red blood
cells without lysing white blood cells in an instant cytolysis
apparatus at a flow rate of 50-1000 liters/hr. Filtration is
performed to remove at least a portion of the waste retentate from
the lysate. A first hemoglobin solution is extracted from the
lysate.
[0013] A first ultrafiltration process is performed using an
ultrafiltration filter configured to remove impurities having a
higher molecular weight than tetrameric hemoglobin and to further
remove any viruses and residual waste retentate from the first
hemoglobin solution to obtain a second hemoglobin solution.
Flowthrough column chromatography is performed on the second
hemoglobin solution to remove protein impurities, dimeric
hemoglobin and phospholipids to form a phospholipid-free hemoglobin
solution. A second ultrafiltration process is performed on the
phospholipid-free hemoglobin solution using a filter configured to
remove impurities resulting in a concentrated purified
phospholipid-free hemoglobin solution.
[0014] Any of the .alpha.-.alpha., .alpha.-.beta., or .beta.-.beta.
subunits of the purified hemoglobin are cross-linked by
bis-3,5-dibromosalicyl fumarate to form heat stable cross-linked
hemoglobin without the formation of polymeric hemoglobin such that
the molecular weight of the resultant cross-linked tetrameric
hemoglobin is 60-70 kDa. Any residual chemicals are removed by
using tangential-flow ultrafiltration. Following this procedure,
the cross-linked hemoglobin is heat-treated to remove any residual
non-cross-linked tetrameric hemoglobin and any non-stabilized
hemoglobin, for example the dimeric form of hemoglobin, and any
other protein impurities. N-acetyl cysteine is added at a
concentration of 0.2-0.4% to the heat stable cross-linked
tetrameric hemoglobin to maintain a low level of met-hemoglobin,
i.e. below 5%, or preferably less than 2%. A precipitate is formed
and then removed by a centrifugation or a depth filtration
apparatus to form a clear solution thereafter. The dimer-free,
phospholipid-free, protein impurities-free, heat stable
cross-linked tetrameric hemoglobin is then added to a
pharmaceutically acceptable carrier.
[0015] Thereafter, the heat stable cross-linked tetrameric
hemoglobin is formulated and packaged in a custom-made and
air-tight polyethylene, ethylene-vinyl-acetate, ethylene-vinyl
alcohol (PE, EVA, EVOH) infusion bag. The packaging prevents oxygen
contamination which results in the formation of inactive
met-hemoglobin.
[0016] The heat stable cross-linked tetrameric hemoglobin produced
by the above method is used for the treatment of various cancers
such as leukemia, colorectal cancer, lung cancer, breast cancer,
liver cancer, nasopharyngeal cancer and esophageal cancer. The
mechanism for destroying cancer cells is to improve oxygenation of
tumors in a hypoxic condition, thereby enhancing the sensitivity
towards radiation and chemotherapeutic agents. The heat stable
cross-linked tetrameric hemoglobin is also used for preservation of
organ tissue during transplant or for preservation of the heart in
situations where there is a lack of oxygen supply in vivo, such as
in an oxygen-deprived heart.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a flow-chart depicting an overview of the process
of the present invention.
[0018] FIG. 2 schematically depicts an instant cytolysis apparatus
used in the process of the present invention.
[0019] FIG. 3 depicts high performance liquid chromatography
analysis for (a) non-heat treated cross-linked tetrameric
hemoglobin, and (b) heat stable cross-linked tetrameric hemoglobin
which has undergone a heat treatment at 90.degree. C. for 2 minutes
or 80.degree. C. for 30 minutes.
[0020] FIG. 4 depicts electrospray ionization mass spectrometry
(ESI-MS) analysis for the heat stable cross-linked tetrameric
hemoglobin.
[0021] FIG. 5 shows a circular dichroism spectroscopy analysis for
(a) purified hemoglobin solution and (b) heat stable cross-linked
tetrameric hemoglobin.
[0022] FIG. 6 shows an improvement of oxygenation in normal tissue.
Injection of 0.2 g/kg heat stable cross-linked tetrameric
hemoglobin solution results in a significant increase in (A) plasma
hemoglobin concentration and (B) oxygen delivery to muscle. A
significant increase in oxygenation is observed for a longer period
of time compared with the plasma hemoglobin level.
[0023] FIG. 7 shows an improvement of oxygenation in hypoxic tumor
tissue. Injection of 0.2 g/kg heat stable cross-linked tetrameric
hemoglobin solution results in a significant increase in oxygen
delivery to the head and neck squamous cell carcinoma (HNSCC)
xenograft.
[0024] FIG. 8 shows partial tumor shrinkage in rodent models of (A)
nasopharyngeal carcinoma (NPC) and (B) liver tumor.
[0025] FIG. 9 demonstrates the mean arterial pressure changes in a
rat model of severe hemorrhagic shock after the treatment with the
heat stable cross-linked tetrameric hemoglobin.
[0026] FIG. 10 is an elution profile for flowthrough column
chromatography; the hemoglobin solution is in the flowthrough
fraction.
[0027] FIG. 11 schematically depicts a flowthrough CM column
chromatography system with ultrafiltration for an industrial scale
operation.
[0028] FIG. 12 is a schematic depiction of an apparatus used for
HTST heat treatment processing step.
[0029] FIG. 13 demonstrates the temperature profile in the HTST
processing apparatus and the time taken to remove unstabilized
tetramer (dimer) in the system at 85.degree. C. and 90.degree. C.
of the present invention.
[0030] FIG. 14 demonstrates the rate of met-hemoglobin formation in
the system at 85.degree. C. and 90.degree. C. in the HTST
processing apparatus of FIG. 12.
[0031] FIG. 15 is a schematic depiction of an infusion bag for the
heat stable cross-linked tetrameric hemoglobin of the present
invention.
DETAILED DESCRIPTION OF INVENTION
[0032] Hemoglobin is an iron-containing oxygen-transport protein in
red blood cells of the blood of mammals and other animals.
Hemoglobin exhibits characteristics of both the tertiary and
quaternary structures of proteins. Most of the amino acids in
hemoglobin form alpha helices connected by short non-helical
segments. Hydrogen bonds stabilize the helical sections inside the
hemoglobin causing attractions within the molecule thereto folding
each polypeptide chain into a specific shape. A hemoglobin molecule
is assembled from four globular protein subunits. Each subunit is
composed of a polypeptide chain arranged into a set of
.alpha.-helix structural segments connected in a "myoglobin fold"
arrangement with an embedded heme group.
[0033] The heme group consists of an iron atom held in a
heterocyclic ring, known as a porphyrin. The iron atom binds
equally to all four nitrogen atoms in the center of the ring which
lie in one plane. Oxygen is then able to bind to the iron center
perpendicular to the plane of the porphyrin ring. Thus a single
hemoglobin molecule has the capacity to combine with four molecules
of oxygen.
[0034] In adult humans, the most common type of hemoglobin is a
tetramer called hemoglobin A consisting of two .alpha. and two
.beta. non-covalently bound subunits designated as .alpha.2.beta.2,
each made of 141 and 146 amino acid residues respectively. The size
and structure of .alpha. and .beta. subunits are very similar to
each other. Each subunit has a molecular weight of about 16 kDa for
a total molecular weight of the tetramer of about 65 kDa. The four
polypeptide chains are bound to each other by salt bridges,
hydrogen bonds and hydrophobic interaction. The structure of bovine
hemoglobin is similar to human hemoglobin (90.14% identity in a
chain; 84.35% identity in .beta. chain). The difference is the two
sulfhydryl groups in the bovine hemoglobin positioned at .beta. Cys
93, while the sulfhydryls in human hemoglobin are at positioned at
a Cys 104, .beta. Cys 93 and .beta. Cys 112 respectively.
[0035] In naturally-occurring hemoglobin inside the red blood
cells, the association of an .alpha. chain with its corresponding
.beta. chain is very strong and does not disassociate under
physiological conditions. However, the association of one
.alpha..beta. dimer with another .alpha..beta. dimer is fairly weak
outside red blood cells. The bond has a tendency to split into two
.alpha..beta. dimers each approximately 32 kDa. These undesired
dimers are small enough to be filtered by the kidneys and be
excreted, with the result being potential renal injury and
substantially decreased intravascular retention time.
[0036] Therefore, it is necessary to stabilize any hemoglobin that
is used outside of red blood cells both for efficacy and safety.
The process for producing the stabilized hemoglobin is outlined
below; an overview of the process of the present invention is
presented in the flow chart of FIG. 1.
[0037] Initially, a whole blood source is selected as a source of
hemoglobin from red blood cells. Mammalian whole blood is selected
including, but not limited to, human, bovine, porcine, equine, and
canine whole blood. The red blood cells are separated from the
plasma, filtered, and washed to remove plasma protein
impurities.
[0038] In order to release the hemoglobin from the red blood cells,
the cell membrane is lysed. Although various techniques can be used
to lyse red blood cells, the present invention uses lysis under
hypotonic conditions in a manner which can be precisely controlled
at volumes suitable for industrial-scale production. To this end,
an instant cytolysis apparatus as seen in FIG. 2 is used to lyse
the red blood cells. Hypotonic lysis creates a solution of lysate
including hemoglobin and a waste retentate. To enable
industrial-scale production, the lysis is carefully controlled such
that only red blood cells are lysed without lysing white blood
cells or other cells. In one embodiment, the size of the instant
cytolysis apparatus is selected such that the red blood cells
traverse the apparatus in 2 to 30 seconds or otherwise a time
sufficient to lyse the red blood cells and preferably, 30 seconds.
The instant cytolysis apparatus includes a static mixer. Deionized
and distilled water is used as a hypotonic solution. Of course it
is understood that the use of other hypotonic solutions having
different saline concentrations would result in different time
periods for red blood cell lysis. Because the controlled lysis
procedure lyses the red blood cells only, not white blood cells or
cellular matter, it minimizes the release of toxic proteins,
phospholipids or DNA from white blood cells and other cellular
matter. A hypertonic solution is added immediately after 30
seconds, that is, after the red blood-cell containing solution has
traversed the static mixer portion of the instant cytolysis
apparatus. The resultant hemoglobin has a higher purity and lower
levels of contaminants such as undesired DNA and phospholipids than
hemoglobin resulted from using other lysis techniques. Undesired
nucleic acids from white blood cells and phospholipids impurities
are not detected in the hemoglobin solution by polymerase chain
reaction (detection limit=64 pg) and high performance liquid
chromatography (HPLC, detection limit=1 .mu.g/ml) method
respectively.
[0039] Two ultrafiltration processes are performed: one which
removes impurities having molecular weights greater than hemoglobin
before flowthrough column chromatography, and another which removes
impurities having molecular weights less than hemoglobin after
flowthrough column chromatography. The latter ultrafiltration
process concentrates the hemoglobin. In some embodiments, a 100 kDa
filter is used for the first ultrafiltration, while a 30 kDa filter
is used for the second ultrafiltration.
[0040] Flowthrough column chromatography is used to remove protein
impurities in the purified hemoglobin solution such as
immunoglobin-G, albumin and carbonic anhydrase. In some
embodiments, column chromatography is carried out by using one or a
combination of commercially available ion exchange columns such as
a DEAE column, CM column, hydroxyapatite column, etc. The pH for
column chromatography is typically from 6 to 8.5. In one
embodiment, a flowthrough CM column chromatography step is used to
remove protein impurities at pH 8.0. Enzyme-linked immunosorbent
assay (ELISA) is performed to detect the protein impurities and
phospholipids remaining in the sample after elution from the column
chromatography. This unique flowthrough column chromatography
separation enables a continuous separation scheme for
industrial-scale production. The ELISA result shows that the amount
of these impurities are substantially low in the eluted hemoglobin
(immunoglobin-G: 44.3 ng/ml; albumin: 20.37 ng/ml; carbonic
anhydrase: 81.2 .mu.g/ml). The protein impurities removal results
using different kinds of column with different pH values are shown
in Table 1 below.
TABLE-US-00001 TABLE 1 Removal percentage (%) Carbonic Column (pH
condition) anhydrase Albumin Immunoglobin-G DEAE (at pH 7.5) -- 68
29.8 DEAE (at pH 7.8) -- 60 50.9 CM (at pH 6.2) -- 32 21.8 CM (at
pH 8.0) 5.6 53.2 66.4 Hydroxyapatite (at pH 7.5) 4.5 23.5 22.8
[0041] Following the column chromatographic process, the hemoglobin
is subjected to cross-linking by bis-3, 5-dibromosalicyl fumarate
(DBSF). In order to prevent formation of polymeric hemoglobin, the
reaction is carefully controlled in a deoxygenated environment with
a molar ratio of hemoglobin to DBSF between 1:2.5 to 1:4.0 such
that the resultant cross-linked hemoglobin is tetrameric hemoglobin
having a molecular weight of 60-70 kDa, demonstrating that
polymeric hemoglobin is not present. The yield of the DBSF reaction
is high, >99% and the dimer concentration in the final product
is low.
[0042] Following the process of cross-linking subunits of the
hemoglobin by DBSF under a deoxygenated condition, the present
invention describes a heat processing step of the cross-linked
tetrameric hemoglobin solution in a deoxygenated environment. After
the heat processing step, N-acetyl cysteine (0.2-0.4%) is added
immediately to maintain a low level of met-hemoglobin (inactive
hemoglobin). In some embodiments, the cross-linked tetrameric
hemoglobin solution is heated under a range of temperatures from
50.degree. C. to 95.degree. C. for durations from 0.5 minutes to 10
hours. In some embodiments, the cross-linked tetrameric hemoglobin
solution is heated under a range of temperatures from 75.degree. C.
to 90.degree. C. and for durations from 1 or 3 minutes to 3 hours.
In some preferred embodiments, the cross-linked tetrameric
hemoglobin solution is heated under 80.degree. C. for 30 minutes.
And yet in other preferred embodiments, the cross-linked hemoglobin
solution is heated under 90.degree. C. for 2 minutes, then cooled
down to room temperature, and N-acetyl cysteine is added at a
concentration of 0.2-0.4%. A very low amount of met-hemoglobin
results, for example, less than 5%. High performance liquid
chromatography (HPLC), electrospray ionization mass spectrometry
(ESI-MS), circular dichroism (CD) spectroscopy and Hemox Analyzer
for p50 measurement are used thereafter to analyze and characterize
the heat stable cross-linked tetrameric hemoglobin. For a bovine
blood source originated hemoglobin, FIG. 3 shows that the dimeric
form of hemoglobin is undetectable in a HPLC system (detection
limit: 2.6 .mu.g/ml or 0.043%) for hemoglobin which has undergone a
heat treatment at 90.degree. C. for 2 minutes or 80.degree. C. for
30 minutes. The cross-linked tetrameric hemoglobin is found as heat
stable at 80 or 90.degree. C. for a period of time. The heat
process (High Temperature Short Time, HTST) step is a powerful step
to denature the natural unreacted tetrameric form and dimeric form
of hemoglobin. N-acetyl cysteine is added at a concentration of
0.2-0.4% to the heat stable cross-linked tetrameric hemoglobin to
maintain the low level of met-hemoglobin.
[0043] To analyze the outcome of this HTST step, a HPLC analytical
method is used to detect the amount of dimer after this heat
process step. The mobile phase for HPLC analysis contains magnesium
chloride (0.75M) which can separate dimer (non-stabilized tetramer)
and heat stable cross-linked tetrameric hemoglobin. For promoting
hemoglobin dissociation into dimers, magnesium chloride is
approximately 30 times more effective than sodium chloride at the
same ionic strength. The heat processing step also acts as a
denaturation step to dramatically remove those unwanted protein
impurities in the cross-linked tetrameric hemoglobin (undetectable
in immunoglobin-G; undetectable in albumin; 99.99% decrease in
carbonic anhydrase). Enzyme-linked immunosorbent assay (ELISA) is
performed to detect the protein impurities in the sample. Thus the
purified, heat stable cross-linked tetrameric hemoglobin solution
has an undetectable level of dimer (below detection limit: 0.043%),
and immunoglobin-G, and a very low amount of albumin (0.02
.mu.g/ml) and carbonic anhydrase (0.014 .mu.g/ml). Table 2 shows
the experimental results regarding the protein impurities and dimer
removal by HTST heat processing step. This HTST heat step enables
the selective separation of heat stable cross-linked tetramer from
unstable tetramer and dimer.
TABLE-US-00002 TABLE 2 Protein impurities (By ELISA) Carbonic By
HPLC p50 at Sample Immunoglobin-G Albumin anhydrase Tetramer Dimer
37.degree. C. condition (.mu.g/ml) (.mu.g/ml) (.mu.g/ml) (%) (%)
(mmHg) No heat 0.36 0.57 355.41 90.1 5.4 38 treatment 80.degree. C.
for 10 min Not 0.33 0.032 92.7 3.4 No data detectable 80.degree. C.
for 15 min Not 0.14 0.022 93.3 2.9 No data detectable 80.degree. C.
for 30 min Not 0.03 0.014 96.6 Not 32 detectable detectable No heat
0.29 0.52 261.80 91.8 5.3 38 treatment 90.degree. C. for 1.0 min
Not 0.21 >0.063 93.4 2.0 29 detectable 90.degree. C. for 1.5 min
Not 0.04 0.022 94.9 0.6 31 detectable 90.degree. C. for 2.0 min Not
0.02 0.016 96.1 Not 31 detectable detectable
[0044] Following the heat processing step on cross-linked
hemoglobin under a deoxygenated condition, the heat stable
cross-linked tetrameric hemoglobin is formulated and ready for
packaging. The present invention describes an air-tight packaging
step of the heat stable cross-linked tetrameric hemoglobin solution
in a deoxygenated environment. Heat stable cross-linked tetrameric
hemoglobin in the present invention is stable under deoxygenated
condition for more than two years.
[0045] In this invention, the oxygen carrier-containing
pharmaceutical composition is primarily intended for intravenous
injection application. Traditionally, prior products use
conventional PVC blood bag or Stericon blood bag which has high
oxygen permeability which will eventually shorten the life span of
the product since it turns into inactive met-hemoglobin rapidly
(within a few days) under oxygenated conditions.
[0046] The packaging used in the present invention results in the
heat stable cross-linked tetrameric hemoglobin being stable for
more than two years. A multi-layer package of EVA/EVOH material is
used to minimize the gas permeability and to avoid the formation of
inactive met-hemoglobin. A 100 ml infusion bag designed for use
with the purified and heat stable cross-linked tetrameric
hemoglobin of the present invention is made from a five layers
EVA/EVOH laminated material with a thickness of 0.4 mm that has an
oxygen permeability of 0.006-0.132 cm.sup.3 per 100 square inches
per 24 hours per atmosphere at room temperature. This material is a
Class VI plastic (as defined in USP<88>), which meets the
in-vivo Biological Reactivity Tests and the Physico-Chemical Test
and is suitable for fabricating an infusion bag for intravenous
injection purpose. This primary bag is particularly useful to
protect the heat stable cross-linked tetrameric hemoglobin solution
from long term oxygen exposure that cause its instability and
eventually affects its therapeutic properties.
[0047] For secondary protection of blood products, it has been
known to use aluminum overwrap to protect against potential air
leakage and to maintain the product in a deoxygenated state.
However, there is a potential of pin holes in the aluminum overwrap
that compromise its air tightness and make the product unstable.
Therefore the present invention uses as secondary packaging an
aluminum overwrap pouch which prevents oxygenation and also
prevents light exposure. The composition of the overwrap pouch
includes 0.012 mm of polyethylene terephthalate (PET), 0.007 mm of
aluminum (Al), 0.015 mm of nylon (NY) and 0.1 mm of polyethylene
(PE). The overwrap film has a thickness of 0.14 mm and an oxygen
transmission rate of 0.006 cm.sup.3 per 100 square inches per 24
hours per atmosphere at room temperature. This secondary packaging
lengthens the stability time for the hemoglobin, extending the
product shelf-life.
[0048] The hemoglobin of the present invention is analyzed by
various techniques, including ESI-MS. ESI-MS enables the analysis
of very large molecules. It is an ionization technique that
analyzes the high molecular weight compound by ionizing the
protein, and then separating the ionized protein based on
mass/charge ratio. Therefore, the molecular weight and the protein
interactions can be determined accurately. In FIG. 4, ESI-MS
analysis result indicates that the size of heat stable cross-linked
tetrameric hemoglobin is 65 kDa. The far UV CD spectra from 190 to
240 nm reveal the secondary structures of globin portion of the
hemoglobin. In FIG. 5, the consistency of the spectra of purified
hemoglobin solution and heat stable cross-linked tetrameric
hemoglobin reveals that the hemoglobin chains are properly folded
even after the heat treatment at 90.degree. C. for 2 minutes. The
CD result shows that heat stable cross-linked tetrameric hemoglobin
has around 42% of alpha-helix, 38% of beta-sheet, 2.5% of beta-turn
and 16% of random coil. It further confirms that cross-linked
tetrameric hemoglobin is heat stable.
[0049] The process in this invention is applicable to large scale
industrial production of the said heat stable cross-linked
tetrameric hemoglobin. In addition, the said heat stable
cross-linked tetrameric hemoglobin in combination with a
pharmaceutical carrier (e.g. water, physiological buffer, in
capsule) is suitable for mammalian use.
[0050] The present invention further discloses the uses of the
oxygen carrier-containing pharmaceutical composition in improving
tissue oxygenation, in cancer treatment, in the treatment of
oxygen-deprivation disorders such as hemorrhagic shock, and in
heart preservation under a low oxygen content environment (e.g.
heart transplant). The dosage is selected to have a concentration
range of approximately 0.2-1.3 g/kg with an infusion rate of less
than 10 ml/hour/kg body weight.
[0051] For uses in cancer treatment, the oxygen carrier-containing
pharmaceutical composition of the present invention serves as a
tissue oxygenation agent to improve the oxygenation in tumor
tissues, thereby enhancing chemosensitivity and radiation
sensitivity.
[0052] In addition, the ability of the heat stable cross-linked
tetrameric hemoglobin to improve oxygenation in normal tissues
(FIG. 6) and in extremely hypoxic tumor (FIG. 7), human
nasopharyngeal carcinoma (using CNE2 cell line) is demonstrated in
this invention. The representative oxygen profile along the tissue
track of a human CNE2 xenograft is showed in FIG. 7. Oxygen partial
pressure (pO.sub.2) within the tumor mass is directly monitored by
a fibreoptic oxygen sensor (Oxford Optronix Limited) coupled with a
micro-positioning system (DTI Limited). After intravenous injection
of 0.2 g/kg of the heat stable cross-linked tetrameric hemoglobin,
the median pO.sub.2 value rises from baseline to about two-fold of
relative mean oxygen partial pressure within 15 minutes and extends
to 6 hours. Further, the oxygen level on average still maintains a
level of 25% to 30% above the baseline value 24 to 48 hours post
infusion. No commercial products or existing technologies show as
high an efficacy when compared to the oxygen carrier-containing
pharmaceutical composition prepared in this invention.
[0053] For tumor tissue oxygenation, a representative oxygen
profile of a human head and neck squamous cell carcinoma (HNSCC)
xenograft (FaDu) is shown in FIG. 7. After intravenous injection of
0.2 g/kg of the heat stable cross-linked tetrameric hemoglobin, a
significant increase in the mean pO.sub.2 of more than 6.5-fold and
5-fold is observed at 3 and 6 hours, respectively (FIG. 7).
[0054] For applications in cancer treatment, the oxygen
carrier-containing pharmaceutical composition of the present
invention serves as a tissue oxygenation agent to improve the
oxygenation in tumor tissues, thereby enhancing chemo- and
radiation sensitivity. In conjunction with X-ray irradiation and
the heat stable cross-linked tetrameric hemoglobin, tumor growth is
delayed. In FIG. 8A, the representative curves show significant
tumor shrinkage in rodent models of nasopharyngeal carcinoma. Nude
mice bearing CNE2 xenografts are treated with X-ray alone (2Gy) or
in combination with the heat stable cross-linked tetrameric
hemoglobin (2Gy+Hb). 1.2 g/kg of the heat stable cross-linked
tetrameric hemoglobin is injected intravenously into the mouse
approximately 3 to 6 hours before X-ray irradiation and results in
a partial shrinkage of nasopharyngeal carcinoma xenograft.
[0055] In one embodiment, significant liver tumor shrinkage is
observed after injecting the composition, in conjunction with a
chemotherapeutic agent. In FIG. 8B, the representative chart shows
significant tumor shrinkage in a rat orthotopic liver cancer model.
Buffalo rats bearing a liver tumor orthograft (CRL1601 cell line)
are treated with 3 mg/kg cisplatin alone, or in combination with
0.4 g/kg of the heat stable cross-linked tetrameric hemoglobin
(Cisplatin+Hb). Administration of the heat stable cross-linked
tetrameric hemoglobin before cisplatin injection results in a
partial shrinkage of the liver tumor.
[0056] For the use in the treatment of oxygen-deprivation disorders
and for heart preservation, the oxygen carrier-containing
pharmaceutical composition of the present invention serves as a
blood substitute providing oxygen to a target organ.
[0057] The mean arterial pressure changes in a rat model of severe
hemorrhagic shock after treatment with 0.5 g/kg of the heat stable
cross-linked tetrameric hemoglobin are shown in FIG. 9. In a rat
model of severe hemorrhagic shock, the mean arterial pressure is
returned back to a safe and stable level and maintained at or about
the baseline after treatment with the heat stable cross-linked
tetrameric hemoglobin. Following treatment with the heat stable
cross-linked tetrameric hemoglobin, the time required for the mean
arterial pressure to return to normal is even shorter than
administrating autologous rat's blood which serves as a positive
control. The results indicate that a vasoconstriction event does
not occur after the transfusion of the heat stable cross-linked
tetrameric hemoglobin.
EXAMPLES
[0058] The following examples are provided by way of describing
specific embodiments of this invention without intending to limit
the scope of this invention in any way.
Example 1
Overall Process
[0059] A schematic flow diagram of the process of the present
invention is illustrated in FIG. 1. Bovine whole blood is collected
into an enclosed sterile container/bag containing 3.8% (w/v)
tri-sodium citrate solution as anti-coagulant. Blood is then
immediately mixed well with tri-sodium citrate solution to inhibit
blood clotting. Red blood cells (RBC) are isolated and collected
from plasma and other smaller blood cells by an apheresis
mechanism. A "cell washer" is used for this procedure with gamma
sterilized disposable centrifuge bowl. RBC are washed with an equal
volume of 0.9% (w/v sodium chloride) saline.
[0060] Washed RBC are lysed to release hemoglobin content by
manipulating hypotonic shock to the RBC cell membrane. A
specialized instant cytolysis apparatus for RBC lysis device
depicted in FIG. 2 is used for this purpose. Following RBC lysis,
hemoglobin molecules are isolated from other proteins by
tangential-flow ultrafiltration using a 100 kDa membrane.
Hemoglobin in the filtrate is collected for flowthrough column
chromatography and further concentrated to 12-14 g/dL by a 30 kDa
membrane. Column chromatography is carried out to remove the
protein impurities.
[0061] The concentrated hemoglobin solution is first reacted with
DBSF to form heat stable cross-linked tetrameric hemoglobin
molecules under deoxygenated condition. A heat treatment step is
then performed at 90.degree. C. for 2 minutes before final
formulation and packaging.
Example 2
Time & Controlled Hypotonic Lysis and Filtration
[0062] Bovine whole blood is freshly collected and transported
under a cool condition (2 to 10.degree. C.). The red blood cells
are separated from the plasma via a cell washer and subsequently
with a 0.65 .mu.m filtration. After washing the red blood cells
(RBC) filtrate with 0.9% saline, the filtrate is disrupted by
hypotonic lysis. The hypotonic lysis is performed by using the
instant cytolysis apparatus depicted in FIG. 2. The instant
cytolysis apparatus includes a static mixer to assist in cell
lysis. A RBC suspension with controlled hemoglobin concentration
(12-14 g/dL) is mixed with 4 volumes of purified water to generate
a hypotonic shock to RBC cell membranes. The period of hypotonic
shock is controlled to avoid unwanted lysis of white blood cells
and platelets. The hypotonic solution passes through the static
mixer portion of the instant cytolysis apparatus for 2 to 30
seconds or otherwise a time sufficient to lyse the red blood cells
and preferably, 30 seconds. The shock is terminated after 30
seconds by mixing the lysate with 1/10 volume of hypertonic buffer
as it exits the static mixer. The hypertonic solution used is 0.1M
phosphate buffer, 7.4% NaCl, pH 7.4. The instant cytolysis
apparatus of FIG. 2 can process at 50 to 1000 liters of lysate per
hour and, preferably at least 300 liters per hour in a continuous
manner.
[0063] Following the RBC lysis, the lysate of red blood cells is
filtered by a 0.22 .mu.m filter to obtain a hemoglobin solution.
Nucleic acids from white blood cells and phospholipids impurities
are not detected in the hemoglobin solution by polymerase chain
reaction (detection limit=64 pg) and HPLC (detection limit=1
.mu.g/ml) method respectively. A first 100 kDa ultrafiltration is
performed to remove impurities having a higher molecular weight
than hemoglobin. A flowthrough column chromatography is followed to
further purify the hemoglobin solution. A second 30 kDa
ultrafiltration is then performed to remove impurities having a
lower molecular weight than hemoglobin and for concentration.
Example 3
Viral Clearance Study on Stroma-Free Hemoglobin Solution
[0064] In order to demonstrate the safety of the product from this
invention, the virus removal abilities of (1) 0.65 .mu.m
diafiltration step and (2) 100 kDa ultrafiltration step are
demonstrated by virus validation study. This is done by the
deliberate spiking of a down-scaled version of these two processes
with different model viruses (encephalomyocarditis virus,
pseudorabies virus, bovine viral diarrhea virus and bovine
parvovirus). In this study, four types of viruses (see Table 3) are
used. These viruses vary in their biophysical and structural
features and they display a variation in resistance to physical and
chemical agents or treatments.
TABLE-US-00003 TABLE 3 Target Size Virus Model Virus Taxonomy
Genome Structure [nm] Stability* Hepatitis Bovine viral diarrhea
Flaviviridae ssRNA enveloped 40-60 low C virus virus (BVDV) (HCV)
-- Encephalomyocarditis Picornavirus ssRNA non- 25-30 medium virus
(EMCV) enveloped Parvovirus Bovine parvovirus Parvoviridae ssDNA
non- 18-26 very high B19 (BPV) enveloped Hepatitis Pseudorabies
virus Herpesviridae dsDNA enveloped 120-200 Low to B virus (PRV)
medium (HBV)
[0065] The validation scheme is briefly shown in the following
Table 4.
TABLE-US-00004 TABLE 4 Diafiltration Ultrafiltration Cell Washing
Virus spiking .dwnarw. .dwnarw. Virus spiking Ultrafiltration
.dwnarw. .dwnarw. Diafiltration Virus tests .dwnarw. Virus
tests
[0066] The summary of the log reduction results of the 4 viruses in
(1) 0.65 .mu.m diafiltration and (2) 100 kDa ultrafiltration is
shown in the following Table 5. All four viruses, BVDV, BPV, EMCV
and PRV, are effectively removed by 0.65 .mu.m diafiltration and
100 kDa ultrafiltration.
TABLE-US-00005 TABLE 5 Viruses BVDV BPV EMCV PRV Run 1 2 1 2 1 2 1
2 0.65 .mu.m Diafiltration 2.69 3.20 3.73 3.53 3.25 .gtoreq.3.90
2.67 2.63 100 kDa Ultrafiltration .gtoreq.4.68 .gtoreq.4.38 5.87
5.92 3.60 3.43 .gtoreq.6.05 3.27 Cumulative maximum .gtoreq.7.88
9.65 .gtoreq.7.50 .gtoreq.8.72 Cumulative minimum .gtoreq.7.07 9.40
6.68 5.90 Annotation: .gtoreq. no residual infectivity
determined
Example 4
Flowthrough Column Chromatography
[0067] A CM column (commercially available from GE healthcare) is
used to further remove any protein impurities. The starting buffer
is 20 mM sodium acetate (pH 8.0), and the elution buffer is 20 mM
sodium acetate, 2M NaCl (pH 8.0). After the equilibration of the CM
column with starting buffer, the protein sample is loaded into the
column. The unbound protein impurities are washed with at least 5
column volume of starting buffer. The elution is performed using
25% elution buffer (0-0.5M NaCl) in 8 column volume. The elution
profile is shown in FIG. 10; the hemoglobin solution is in the
flowthrough fraction. The purity of the flowthrough fraction is
analyzed by ELISA. The results are indicated in the following Table
6.
TABLE-US-00006 TABLE 6 Protein impurities Carbonic Immunoglobin-G
anhydrase Albumin Before CM column 1320 ng/ml 860.3 .mu.g/ml 435.2
ng/ml Flowthrough 44.3 ng/ml 81.2 .mu.g/ml 20.4 ng/ml (containing
hemoglobin)
[0068] As the hemoglobin solution is in the flowthrough from the CM
column chromatography at pH 8 (not in the eluate), it is a good
approach for continuous industrial scale operation. The first
ultrafiltration set-up is connected directly to the flowthrough CM
column chromatography system, and the flowthrough tubing can be
connected to the second ultrafiltration set-up for industrial scale
operation. The schematic industrial process configuration is shown
in FIG. 11.
Example 5
Preparation of Heat Stable Cross-Linked Tetrameric Hemoglobin
[0069] (5a) Cross-Linking Reaction with DBSF
[0070] The cross-linking reaction is carried out in a deoxygenated
condition. DBSF is added to the hemoglobin solution to form
cross-linked tetrameric hemoglobin. In these embodiments, the molar
ratio of hemoglobin to DBSF is between 1:2.5 to 1:4.0. DBSF
stabilization procedure stabilizes the tetrameric form of
hemoglobin (65 kDa) and prevents dissociation into dimers (32 kDa)
which are excreted through the kidneys. This process is carried out
in an inert atmosphere of nitrogen to prevent oxidation of the
hemoglobin to form ferric met-hemoglobin which is physiologically
inactive. The completeness of DBSF reaction is monitored by
measuring the residual DBSF using HPLC. The yield of the DBSF
reaction is high, >99%.
[0071] (5b) HTST Heat Process Step
[0072] A High Temperature Short Time (HTST) processing apparatus is
shown in FIG. 12. A heating process using the HTST processing
apparatus is performed on the cross-linked tetrameric hemoglobin.
In this example, the condition for heat treatment is 90.degree. C.
for 1 to 3 minutes, and preferably 2 minutes although other
conditions can be selected as discussed above and the apparatus
modified accordingly. A solution containing cross-linked hemoglobin
is pumping from a first agitator 1201 into a HTST processing
apparatus (first section of the HTST heat exchanger 1202 is
pre-heated and maintained at 90.degree. C.) at a flow rate of 1.0
liter per minute, the residence time of the first section of the
apparatus is between 75 to 150 seconds (preferably 120 seconds),
then the solution is passed through at the same flow rate into
another section of the heat exchanger 1203 that is maintained at
25.degree. C. The time required for cooling is between 30 to 120
seconds (preferably 90 seconds). After that, the HTST processed
solution containing cross-linked hemoglobin is pumped into a second
agitator 1204 for cooling down to 25.degree. C., N-acetyl cysteine
is immediately added at a concentration of 0.2% to 0.4%, preferably
at 0.2%. This chemical addition after the HTST heating process is
very important to maintain met-hemoglobin (inactive hemoglobin) at
a low level. The set-up of the processing apparatus is easily
controlled for industrial operation. A temperature profile with
dimer content is shown in FIG. 13. In FIG. 13, two solid lines
represent the change of temperature from 25 to 85.degree. C. (solid
line with rhombus) and from 25 to 90.degree. C. (solid line with
triangle) against time, respectively; two dashed lines represent
the percentage change of dimer from 25.degree. C. to 85.degree. C.
(dashed line with rhombus) and the percentage change of dimer from
25.degree. C. to 90.degree. C. (dashed line with triangle) against
time. If the hemoglobin is not cross-linked, it is not heat stable
and forms a precipitate after the heat step. The precipitate is
then removed by a centrifugation or a depth filtration apparatus to
form a clear solution thereafter.
[0073] During the HTST heating process at 90.degree. C.,
met-hemoglobin (inactive hemoglobin) is slightly increased or not
increased at all (shown in FIG. 14). In FIG. 14, the solid line
represents the percentage change of met-hemoglobin against time at
85.degree. C. while the dashed line represents the percentage
change of met-hemoglobin against time at 90.degree. C. After
immediate addition of N-acetyl cysteine, a low level of
met-hemoglobin can be maintained.
[0074] The following Table 7 shows that protein impurities such as
immunoglobin-G, albumin, carbonic anhydrase and undesirable
non-stabilized tetramer or dimers are removed after the heat
treatment step. The amount of immunoglobin-G, albumin and carbonic
anhydrase are measured using an ELISA method, while the amount of
dimer is determined by an HPLC method. The purity of heat stable
cross-linked tetrameric hemoglobin is extremely high after the HTST
heating processing step, in the range of 98.0 to 99.9%. The p50
value, oxygen partial pressure (at which the hemoglobin solution is
half (50%) saturated) measured by a Hemox Analyzer, is maintained
at around 30 to 40 mmHg throughout the HTST heating processing step
and therefore, the heat treated cross-linked tetrameric hemoglobin
is stable at 90.degree. C.
TABLE-US-00007 TABLE 7 Protein impurities (by ELISA) Carbonic By
HPLC p50 at Sample Immunoglobin-G Albumin anhydrase Tetramer Dimer
37.degree. C. condition (.mu.g/ml) (.mu.g/ml) (.mu.g/ml) (%) (%)
(mmHg) No heat 0.29 0.52 261.80 91.8 5.3 38 treatment 90.degree. C.
for 2 min Not 0.02 0.016 96.1 Not 31 detectable detectable Removal
(%) 100.0 96.15 99.99 -- 100.0 --
Example 6
Packaging
[0075] Because the product of the present invention is stable under
deoxygenated conditions, the packaging for the product is important
to minimize gas permeability. For intravenous application, a custom
designed, 100 ml infusion bag is made from a five-layer EVA/EVOH
laminated material with a thickness of 0.4 mm that has an oxygen
permeability of 0.006 to 0.132 cm.sup.3 per 100 square inches per
24 hours per atmosphere at room temperature. This specific material
is a Class VI plastic (as defined in USP<88>), which meets
the in-vivo biological reactivity tests and the physico-chemical
test and are suitable for fabricating containers for intravenous
injection purpose (note that other forms of packaging can be made
from this material as well depending upon the desired application).
A secondary packaging aluminum overwrap pouch is also applied to
the primary packaging infusion bag that provides an additional
barrier, minimizing light exposure and oxygen diffusion. The layers
of the pouch comprise: 0.012 mm of Polyethylene terephthalate
(PET), 0.007 mm of Aluminum (Al), 0.015 mm of Nylon (NY) and 0.1 mm
of Polyethylene (PE). The overwrap film has a thickness of 0.14 mm
and oxygen transmission rate of 0.006 cm.sup.3 per 100 square
inches per 24 hours per atmosphere at room temperature. A schematic
depiction of the infusion bag is depicted in FIG. 15. The infusion
bag 1500 is designed with 100 mL nominal filling capacity with a 10
mm diameter eyelet 1503 at the bottom to facilitate the bag in
hanging for infusion. The infusion bag 1500 is designed with an
inverted V shape to maximize drainage of the product solution to
the IV port 1504 location. The IV port 1604 is made of EVA material
(USP Class VI) and it allows connection to an infusion set when it
is twisted off at the twist-off by the end user at a later time. To
minimize the amount of air bubble potentially entrapped inside the
IV port 1504, the port is designed with its port membrane located
at the lowest possible point in the port tube. The infusion bag
1500 is filled via a co-extruded 8.times.6.times.150 mm filling
tube 1501 made of EVA/polyvinyl chloride (PVC) tube (USP Class VI).
The distal end of the filling tube 1501 has a rotating male luer
lock 1502 (USP Class VI) attached with a guard 1506 and a white
click clamp 1505 for protection from contamination during handling
and filling. Once the infusion bag 1500 has been filled with the
claimed composition, the filling tube 1601 is removed by welding
the infusion bag 1500 under the filling tube 1501 to give the bag
its final outlook. The numbers with two decimal places represent
the dimension of items in millimeters (mm) scale as shown in FIG.
15. For example, "15.00" represents 15.00 mm. The letter "R"
represents the radius and the symbol "O" represents diameter. The
overall oxygen permeability for each infusion bag according to the
present invention is 0.0025 cm.sup.3 per 24 hours per atmosphere at
room temperature.
Example 7
Improvement of Oxygenation
[0076] (7a) Improvement of Oxygenation in Normal Tissue
[0077] Some studies for the normal tissue oxygenation by heat
stable cross-linked tetrameric hemoglobin are carried out (shown in
FIG. 6). A comparative pharmacokinetic and pharmacodynamic study is
conducted in buffalo rats. Male inbred buffalo rats are
individually administered with 0.2 g/kg heat stable cross-linked
tetrameric hemoglobin solution or ringer's acetate buffer (control
group), through the penile vein of the rats by bolus injection. The
concentration-time profile of plasma hemoglobin is determined by
Hemocue.TM. photometer at 1, 6, 24, 48 hours and compared with the
baseline reading. The methods are based on photometric measurement
of hemoglobin where the concentration of hemoglobin is directly
read out as g/dL. Oxygen partial pressure (pO.sub.2) is directly
measured by the Oxylab.TM. tissue oxygenation and temperature
monitor (Oxford Optronix Limited) in hind leg muscle of buffalo
rats. Rats are anesthetized by intra-peritoneal injection of 30-50
mg/kg pentobarbitone solution followed by insertion of oxygen
sensor into the muscle. All pO.sub.2 readings are recorded by
Datatrax2 data acquisition system (World Precision Instrument) in a
real-time manner Results demonstrate that after an intravenous
injection of 0.2 g/kg of the heat stable cross-linked tetrameric
hemoglobin, the mean pO.sub.2 value rises from baseline to about
two-fold of the relative mean oxygen partial pressure within 15
minutes and extends to 6 hours. Further, the oxygen level on
average is still maintained at 25% to 30% above the baseline value
24 to 48 hours post injection (FIG. 6B).
[0078] (7b) Significant Improvement of Oxygenation in Extremely
Hypoxic Tumor Area
[0079] Improvement of oxygenation in an extremely hypoxic tumor
area is evaluated by a human head and neck squamous cell carcinoma
(HNSCC) xenograft model. A hypopharyngeal squamous cell carcinoma
(FaDu cell line) is obtained from the American Type Culture
Collection. Approximately 1.times.10.sup.6 cancer cells are
injected subcutaneously into four to six week-old inbred BALB/c
AnN-nu (nude) mice. When the tumor xenograft reaches a diameter of
8-10 mm, oxygen partial pressure (pO.sub.2) within the tumor mass
is directly monitored by the Oxylab.TM. tissue oxygenation and
temperature monitor (Oxford Optronix Limited). All pO.sub.2
readings are recorded by the Datatrax2 data acquisition system
(World Precision Instrument) in a real-time manner. When the
pO.sub.2 reading is stabilized, 0.2 g/kg heat stable cross-linked
tetrameric hemoglobin solution is injected intravenously through
the tail vein of the mice and the tissue oxygenation is measured.
Results demonstrate that after intravenous injection of 0.2 g/kg of
the said heat stable cross-linked tetrameric hemoglobin, a
significant increase in the mean pO.sub.2 of more than 6.5-fold and
5-fold is observed in 3 and 6 hours, respectively (FIG. 7).
Example 8
Cancer Treatment Studies
A Significant Tumor Shrinkage in Nasopharyngeal Carcinoma
[0080] A significant tumor shrinkage is observed after
administration of heat stable cross-linked tetrameric hemoglobin
solution in combination with X-ray irradiation (FIG. 8A). A human
nasopharyngeal carcinoma xenograft model is employed. Approximately
1.times.10.sup.6 cancer cells (CNE2 cell line) are injected
subcutaneously into four to six week-old inbred BALB/c AnN-nu
(nude) mice. When the tumor xenograft reaches a diameter of 8-10
mm, tumor-bearing mice are randomized into three groups as
follows:
[0081] Group 1: Ringer's acetate buffer (Ctrl)
[0082] Group 2: Ringer's acetate buffer+X-ray irradiation (2Gy)
[0083] Group 3: Heat stable cross-linked tetrameric
hemoglobin+X-ray irradiation (2Gy+Hb)
[0084] Nude mice bearing CNE2 xenografts are irradiated with
X-irradiation alone (Group 2) or in combination with heat stable
cross-linked tetrameric hemoglobin (Group 3). For X-ray irradiation
(Groups 2 and 3), mice are anesthetized by an intra-peritoneal
injection of 50 mg/kg pentobarbitone solution. 2 Grays of X-ray is
delivered to the xenograft of tumor-bearing mice by a linear
accelerator system (Varian Medical Systems). For Group 3, 1.2 g/kg
heat stable cross-linked tetrameric hemoglobin is injected
intravenously through the tail vein into the mouse before X-ray
treatment. Tumor dimensions and body weights are recorded every
alternate day starting with the first day of treatment. Tumor
weights are calculated using the equation 1/2LW2, where L and W
represent the length and width of the tumor mass, measured by a
digital caliper (Mitutoyo Co, Tokyo, Japan) at each measurement.
Group 1 is the non-treatment control group. Results (shown in FIG.
8) demonstrate that significant shrinkage of the CNE2 xenograft is
observed in mice treated with the heat stable cross-linked
tetrameric hemoglobin solution in conjunction with X-irradiation
(Group 3, FIG. 8A).
Example 9
Cancer Treatment Studies
A Significant Shrinkage in Liver Tumor
[0085] In addition, significant tumor shrinkage is observed after
administration of heat stable cross-linked tetrameric hemoglobin
solution in combination with cisplatin (FIG. 8B). A rat orthotopic
liver cancer model is employed. Approximately 2.times.10.sup.6 rat
liver tumor cells labeled with luciferase gene (CRL1601-Luc) are
injected into the left lobe of the liver in a buffalo rat. Tumor
growth is monitored by a Xenogen in vivo imaging system. Two to
three weeks after injection, the tumor tissue is harvested,
dissected into small pieces and orthotopically implanted into the
left liver lobe of a second group of rats. Rats bearing liver tumor
are randomized into three groups as follows:
[0086] Group 1: Ringer's acetate buffer (Control)
[0087] Group 2: Ringer's acetate buffer+cisplatin (Cisplatin)
[0088] Group 3: Heat stable cross-linked tetrameric
hemoglobin+cisplatin (Cisplatin+Hb)
[0089] Rats implanted with liver tumor tissue are treated with 3
mg/kg of cisplatin alone (Group 2) or in conjunction with heat
stable cross-linked tetrameric hemoglobin (Group 3). For groups 2
and 3, rats are anesthetized by an intra-peritoneal injection of
30-50 mg/kg pentobarbitone solution and cisplatin are administered
via the left portal vein. For Group 3, 0.4 g/kg heat stable
cross-linked tetrameric hemoglobin is injected intravenously
through the penile vein of the rat before cisplatin treatment.
Group 1 is the non-treatment control group. Importantly, a
significant shrinkage of liver tumor is observed 3 weeks after
treatment (FIG. 8B).
Example 10
Treatment of Acute Severe Hemorrhagic Shock in Rats
[0090] Heat stable cross-linked tetrameric hemoglobin is also used
as a resuscitation agent in a model of Acute Severe Hemorrhagic
Shock in rats. 50 Sprague-Dawley rats are randomly divided into 3
groups according to resuscitation agents, 16 to 18 rats in each
group.
[0091] Group 1: Lactate Ringer's solution (Negative Control, 16
rats)
[0092] Group 2: Animal autologous blood (Positive Control, 16
rats)
[0093] Group 3: Heat stable cross-linked tetrameric hemoglobin
treatment group (0.5 g Hb/kg of body weight, 18 rats)
[0094] Acute severe hemorrhagic shock is established by withdrawing
50% of animal whole blood, which is estimated as 7.4% of body
weight. After hemorrhagic shock is established for 10 minutes,
Lactate Ringer's solution, animal autologous blood, or 0.5 g Hb/kg
of heat stable cross-linked tetrameric hemoglobin are infused into
the animals. The infusion rate of heat stablecross-linked
tetrameric hemoglobin is set at 5 ml/h, thereafter, all
experimental animals are observed for 24 hours. A panel of
parameters is observed and analyzed during study period including
survival, hemodynamics, myocardial mechanics, cardiac output,
cardiac function, blood gas, tissue oxygen delivery &
consumption, tissue perfusion & oxygen tension (liver, kidney
and brain), liver & renal function, hemorheology (blood
viscosity), and mitochondrial respiratory control rate (liver,
kidney and brain). Above all, survival is the primary end point.
After 24 hours of observation, the heat stable cross-linked
tetrameric hemoglobin treatment group has a much higher survival
rate compared with the Lactate Ringer's solution or negative
control group and the autologous blood group (shown in the
following Table 8).
TABLE-US-00008 TABLE 8 Groups Survival no. after 24-hour 24-hour
survival rate (%) Negative control 3 in 16 rats 18.8 Rat's
Autologous 10 in 16 rats 62.5 Blood 0.5 g Hb/kg 13 in 18 rats 72.0
*Hb = heat stable cross-linked tetrameric hemoglobin
[0095] While the foregoing invention has been described with
respect to various embodiments, such embodiments are not limiting.
Numerous variations and modifications would be understood by those
of ordinary skill in the art. Such variations and modifications are
considered to be included within the scope of the following
claims.
* * * * *