OA17703A

OA17703A - A heat stable oxygen carrier-containing pharmaceutical composition for different treatment applications.

Info

Publication number: OA17703A
Application number: OA1201400009
Authority: OA
Inventors: Bing Lou Wong; Sui Yi Kwok; Sze Hang Lau
Original assignee: Billion King International Limited
Priority date: 2011-07-11
Filing date: 2012-07-11
Publication date: 2017-10-11

Abstract

A highly purified and heat stable cross-linked nonpolymeric 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, uncross-linked tetrameric hemoglobin, and plasma protein impurities effectively. Addition of N-acetyl cysteine after heat treatment and optionally before heat treatment 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. The inventive tetrameric hemoglobin can also be used to prevent tumor metastasis and recurrence following surgical tumor excision. Further the inventive tetrameric hemoglobin can be administered to patients prior to chemotherapy and radiation treatment.

Description

This application is an international patent application which daims priority from U.S. Continuation-in-Part Application No. 13/179,590 filed on 11 July, 2011, now Patent No. 8,048,856, the disclosure of which is incorporated by reference

Copyright Notice/Permission

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 ail copyright rights whatsoever. The following notice applies to the processes, experiments, and data as described below and in the drawings attached hereto: Copyright © 2010, Billion King International Limited, Ail Rights Reserved.

Technical Field [0001] The présent invention relates to a method for the préparation of a heat stable oxygencarrier-containing pharmaceutical composition and the composition made by the process. The présent invention also relates to the use of the heat stable oxygen camer-containing pharmaceutical composition for cancer treatment, oxygen-deprivation disorders and organ préservation for humans and other animais.

Background of Invention [0002] Hemoglobin plays an important rôle 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.

[0003] Naturally-occurring hemoglobin is a tetramer which is generally stable when présent within red blood cells. However, when naturally-occurring hemoglobin is removed from red blood cells, it becomes unstable in plasma and splits into two α-β dimers. Each of these dimers is approximately 32 kDa in molecular weight. These dimers may cause substantial rénal 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.

[0004] In order to solve the problem, recent developments in hemoglobin processing hâve 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 présent inventors, polymeric hemoglobin more readily converts to met-hemoglobin in blood circulation. Methemoglobin cannot bind oxygen and therefore cannot oxygenate tissue. Therefore, the crosslinking 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.

[0005] 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 rénal injury in a mammalian body; this rénal 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.

[0006] Another problem with prior art hemoglobin products is a sudden increase in blood pressure following administration. In the past, vasoconstriction events hâve been recorded from older génération of hemoglobin based oxygen carriers. For instance, the Hemopure® 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 hâve 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 préparé hemoglobin which will not cause vasoconstriction and high blood pressure when applied to a mammal.

[0007] Further problems with prior art attempts to create stable hemoglobin include the presence of protein impurities such as immunoglobin G that can cause allergie effects in mammals. Therefore, there is a need in the art for a process which can produce stable tetrameric hemoglobin without protein impurities.

[0008] 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 [0009] The présent invention provides a method for processing a nonpolymeric, heat stable purified cross-linked tetrameric hemoglobin suitable for use in mammals without causing severe rénal injury, vascular detrimental effects and severe adverse events including death. The présent invention removes the dimeric form of hemoglobin, uncross-linked tetrameric hemoglobin, phospholipids and protein impurities. Additionally, the présent invention uses (1) an instant cytolysis apparatus for précisé and controlled hypotonie lysis, (2) a flowthrough column chromatography, (3) a high température short time (HTST) apparatus for heat processing the hemoglobin solution in the purification process to remove the undesirable nonstabilized dimers of hemoglobin and to remove the protein impurities, for example immunoglobin-G, so that rénal 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.

[0010] 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 hypotonie 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.

[0011] 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.

[0012] At least the cc-cc subunits of the purified hemoglobin are cross-linked by bis-3,5dibromosalicyl fumarate to form heat stable cross-linked hemoglobin without the formation of polymeric hemoglobin such that the molecular weight of the résultant nonpolymeric crosslinked tetrameric hemoglobin is 60 - 70 kDa. The expression “nonpolymeric” as used herein, refers to tetrameric hemoglobin that is not intermolecularly cross-linked with other hemoglobin molécules or any other non-hemoglobin molécules such as PEG. A suitable physiological buffer such as phosphate buffered saline (PBS), lactated Ringer's solution, acetated Ringer's solution, or Tris buffer is exchanged for the cross-linked tetrameric hemoglobin. Any residual chemicals are removed using tangential-flow filtration.

[0013] 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. Prior to the heat treatment N-acetyl cysteine is optionally added at a concentration of approximately 0.2% to the cross-linked tetrameric hemoglobin to prevent formation of met-hemoglobin. Immediately following heat treatment and cooling, N-acetyl cysteine is added at a concentration of approximately 0.2 % to 0.4% to further prevent formation of met-hemoglobin. The heat treatment is preferably a high température short time treatment conducted at approximately 70°C to 95°C for 30 seconds to 3 hours with subséquent cooling to 25° C. Any précipitâtes formed during the heat treatment are removed by centrifugation or a filtration apparatus to form a clear solution thereafter.

[0014] The dimer-free, phospholipid-free, protein impurities-free, heat stable, nonpolymeric 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 oxygénation of tumors in a hypoxie condition, thereby enhancing the sensitivity towards radiation and chemotherapeutic agents. The heat stable cross-linked tetrameric hemoglobin is also used for préservation of organ tissue during transplant or for préservation of the heart in situations where there is a lack of oxygen supply in vivo, such as in an oxygen-deprived heart.

[0017] Moreover, the heat stable cross-linked tetrameric hemoglobin produced by the above method is used for reducing cancerous tumor récurrence and minimizing tumor cell metastasis. Said hemoglobin is administered prior to ischemia for a tumor removal surgery and during reestablishment of blood supply (reperfusion) upon removal of tumor. Said hemoglobin can also be used to increase oxygénation of cancerous tissues and reducing size of a tumor.

Brief Description of the Drawings [0018] FIG. 1 is a flow-chart depicting an overview of the process of the présent invention.

[0019] FIG. 2 schematically depicts an instant cytolysis apparatus used in the process of the présent invention.

[0020] 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°C for 45 seconds to 2 minutes or 80°C for 30 minutes.

[0021] FIG. 4 depicts electrospray ionization mass spectrometry (ESI-MS) analysis for the heat stable cross-linked tetrameric hemoglobin.

[0022] FIG. 5 shows a circular dichroism spectroscopy analysis for (a) purified hemoglobin solution and (b) heat stable cross-linked tetrameric hemoglobin.

[0023] FIG. 6 shows an improvement of oxygénation in normal tissue. Injection of 0.2g/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 oxygénation is observed for a longer period of time compared with the plasma hemoglobin level.

[0024] FIG. 7 shows an improvement of oxygénation in hypoxie tumor tissue. Injection of 0.2g/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.

[0025] FIG. 8 shows partial tumor shrinkage in rodent models of (A) nasopharyngeal carcinoma (NPC) and (B) liver tumor.

[0026] FIG. 9 demonstrates the mean arterial pressure changes in a rat model of severe hémorrhagie shock after the treatment with the heat stable cross-linked tetrameric hemoglobin. [0027] FIG. 10 is an elution profile for flowthrough column chromatography; the hemoglobin solution is in the flowthrough fraction.

[0028] FIG. 11 schematically depicts a flowthrough CM column chromatography system with ultrafiltration for an industrial scale operation.

[0029] FIG. 12 is a schematic depiction of an apparatus used for HTST heat treatment processing step.

[0030] FIG. 13 demonstrates the température profile in the HTST processing apparatus and the time taken to remove unstabilized tetramer (dimer) in the system at 85°C and 90°C of the présent invention.

[0031] FIG. 14 demonstrates the rate of met-hemoglobin formation in the system at 85°C and 90°C in the HTST processing apparatus of FIG. 12.

[0032] FIG. 15 is a schematic depiction of an infusion bag for the heat stable cross-linked tetrameric hemoglobin of the présent invention.

[0033] FIG. 16 shows a schematic drawing summarizing the surgical and hemoglobin product administration procedures during liver resection.

[0034] FIG. 17 shows représentative examples of intra-hepatic liver cancer récurrence and metastasis and distant lung metastasis induced in the rats of the IR injury group after hepatectomy and ischemia/reperfusion procedures and its protection using the inventive heat stable cross-linked tetrameric hemoglobin.

[0035] FIG. 18 shows the histological examination in experimental and control groups at four weeks after liver resection and ER injury procedures.

[0036] FIG. 19A shows the volume (cm³) of recurred liver tumor found in rats of the IR injury group (Control group) after hepatectomy and IR procedures and rats having treated with the inventive heat stable cross-linked tetrameric hemoglobin (Hb Treatment group).

[0037] FIG. 19B. shows the liver récurrence rate (left) and the average recurred tumor size (right) of the IR injury rats after hepatectomy and IR procedures (Control group) and rats having treated with the inventive heat stable cross-linked tetrameric hemoglobin (Hb group).

[0038] FIG. 20 shows représentative examples of intra-hepatic liver cancer récurrence and metastasis and distant lung metastasis induced in the rats of the IR injury group after hepatectomy and ischemia/reperfusion procedures (control group: C10 & Cl3) and rats treated with the inventive heat stable cross-linked tetrameric hemoglobin (Hb treatment group: Y9, Y10&Y11).

[0039] FIG. 21 shows the représentative examples of liver partial oxygen pressure (mmHg) from the first administration of the subject inventive hemoglobin product or RA buffer (control) throughout the hepatic surgery and reperfusion.

[0040] FIG. 22 shows a comparison between levels of circulating endothélial progenitor cells (EPC) in peripheral blood of rats with or without treatment of the subject hemoglobin product 28 days post-hepatic surgery.

Detailed Description of Invention [0041] Hemoglobin is an iron-containing oxygen-transport protein in red blood cells of the blood of mammals and other animais. Hemoglobin exhibits characteristics of both the tertiary and quatemary 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 molécule thereto folding each polypeptide chain into a spécifie shape. A hemoglobin molécule is assembled from four globular protein subunits. Each subunit is composed of a polypeptide chain arranged into a set of oc-helix structural segments connected in a “myoglobin fold” arrangement with an embedded heme group.

[0042] The heme group consists of an iron atom held in a heterocyclic ring, known as a porphyrin. The iron atom binds equally to ail 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 molécule has the capacity to combine with four molécules of oxygen.

[0043] In adult humans, the most common type of hemoglobin is a tetramer called hemoglobin A consisting of two a and two β non-covalently bound subunits designated as α2β2, each made of 141 and 146 amino acid residues respectively. The size and structure of a and β 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 sait bridges, hydrogen bonds and hydrophobie interaction. The structure of bovine hemoglobin is similar to human hemoglobin (90.14% identity in a chain; 84.35% identity in β chain). The différence is the two sulfhydryl groups in the bovine hemoglobin positioned at β Cys 93, while the sulfhydryls in human hemoglobin are at positioned at a Cys 104, β Cys 93 and β Cys 112 respectively.

[0044] In naturally-occurring hemoglobin inside the red blood cells, the association of an a chain with its corresponding β chain is very strong and does not disassociate under physiological conditions. However, the association of one αβ dimer with another αβ dimer is fairly weak outside red blood cells. The bond has a tendency to split into two αβ dimers each approximately 32 kDa. These undesired dimers are small enough to be filtered by the kidneys and be excreted, with the resuit being potential rénal injury and substantially decreased intravascular rétention time.

[0045] 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 présent invention is presented in the flow chart of FIG. 1.

[0046] 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.

[0047] 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 présent invention uses lysis under hypotonie 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. Hypotonie lysis créâtes 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 hypotonie solution. Of course it is understood that the use of other hypotonie solutions having different saline concentrations would resuit 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 hypertonie 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 résultant 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 (détection limit = 64 pg) and high performance liquid chromatography (HPLC, détection limit = 1 pg/ml) method respectively.

[0048] 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 concentrâtes 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.

[0049] 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 séparation enables a continuous séparation scheme for industrial-scale production. The ELISA resuit 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 qg/ml). The protein impurities removal results using different kinds of column with different pH values are shown in Table 1 below.

[0050] Table 1

Removal percentage (%)

Column (pH condition)

	Carbonic anhydrase	Albumin	Immunoglobin-G
DEAE(atpH 7.5)	—	68	29.8
DEAE(atpH 7.8)	—	60	50.9
CM (atpH 6.2)	—	32	21.8
CM(atpH8.0)	5.6	53.2	66.4
Hydroxyapatite (at pH 7.5)	4.5	23.5	22.8

[0051] Following the column chromatographie process, the hemoglobin is subjected to crosslinking by bis-3, 5-dibromosalicyl fumarate (DBSF). In order to prevent formation of polymeric hemoglobin, the reaction is carefully controlled in a deoxygenated environment (preferably less than 0.1 ppm dissolved oxygen level) with a molar ratio of hemoglobin to DBSF between 1:2.5 to 1:4.0 for a period of time from 3 to 16 hours at ambient température (15-25°C), preferably at a pH of around 8-9, such that the résultant cross-linked hemoglobin is tetrameric hemoglobin having a molecular weight of 60-70 kDa, demonstrating that polymeric hemoglobin is not présent. The yield of the DBSF reaction is high, > 99% and the dimer concentration in the final product is low. Optionally, the présent process does not require sulfhydryl treatment reagents such as iodoacetamide to react with the hemoglobin before cross-linking as used in various prior art processes.

[0052] At this point phosphate buffered saline (PBS), a physiological buffer, is exchanged for the cross-linking solution and any residual chemicals are removed by tangential flow filtration. [0053] Following the process of cross-linking of the hemoglobin by DBSF under a deoxygenated condition, the présent invention provides a heat processing step for the crosslinked tetrameric hemoglobin solution in a deoxygenated environment. Prior to heat treatment, N-acetyl cysteine is optionally added to prevent formation of met-hemoglobin (inactive hemoglobin). After the heat processing step, the solution is cooled and N-acetyl cysteine is immediately added to maintain a low level of met-hemoglobin. If N-acetyl cysteine is added before and after heat treatment, the amount added before heat treatment is approximately 0.2%, while the amount added after heat treatment is approximately 0.2 to 0.4%. However, if N-acetyl cysteine is added only after heat treatment, then the amount added is 0.4%.

[0054] In some embodiments, the cross-linked tetrameric hemoglobin solution is heated in a deoxygenated environment (less than 0.1 ppm dissolved oxygen level) under a range of températures from 50°C to 95°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 températures from 70°C to 95°C and for durations from 30 seconds to 3 hours. In some preferred embodiments, the cross-linked tetrameric hemoglobin solution is heated under 80°C for 30 minutes. And yet in other preferred embodiments, the linked hemoglobin solution is heated to 90°C for 30 seconds to 3 minutes, then rapidly cooled down to approximately 25° C in approximately 15 to 30 seconds, and the N-acetyl cysteine is added as set forth above. A very low amount of met-hemoglobin results, for example, less than 3%. Without the use of N-acetyl cysteine, the amount of met-hemoglobin formed is approximately 16%, an unacceptably high percentage for pharmaceutical applications.

[0055] 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 (détection limit: 2.6 pg/ml or 0.043%) for hemoglobin which has undergone a heat treatment at 90°C for 45 seconds to 2 minutes or 80°C for 30 minutes. The cross-linked nonpolymeric tetrameric hemoglobin is found as heat stable at 80 or 90 °C for a period of time. The heat process (High Température Short Time, HTST) step is a powerful step to dénaturé the naturel unreacted tetrameric form and dimeric form of hemoglobin.

[0056] 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 magnésium chloride (0.75M) which can separate dimer (non-stabilized tetramer) and heat stable cross-linked tetrameric hemoglobin. For promoting hemoglobin dissociation into dimers, magnésium chloride is approximately 30 times more effective than sodium chloride at the same ionic strength. The heat processing step also acts as a dénaturation 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 détection limit: 0.043%), and immunoglobin-G, and a very low amount of albumin (0.02 pg/ml) and carbonic anhydrase (0.014 pg/ml). Table 2 shows the experimental results regarding the protein impurities and dimer removal by the HTST heat processing step. This HTST heat step enables the sélective séparation of heat stable cross-linked tetramer from unstable tetramer and dimer.

[0057] Table 2

Sample condition	Protein impurities (By ELISA)	ByHPLC	p50 at 37°C (mmHg)
Immunoglobin- G (pg/ml)	Albumin (pg/ml)	Carbonic anhydrase (pg/ml)	Tetramer (%)	Dimer (%)
No heat treatment	0.36	0.57	355.41	90.1	5.4	38
80°C for 10min	Not détectable	0.33	0.032	92.7	3.4	No data
80°C for 15min	Not détectable	0.14	0.022	93.3	2.9	No data

80°C for 30min	Not détectable	0.03	0.014	96.6	Not détectable	32
No heat treatment	0.29	0.52	261.80	91.8	5.3	38
90°C for 1.0min	Not détectable	0.21	>0.063	93.4	2.0	29
90°C for 1.5min	Not détectable	0.04	0.022	94.9	0.6	31
90°C for 2.0min	Not détectable	0.02	0.016	96.1	Not détectable	31

[0058] Following the heat processing step for the cross-linked hemoglobin under a deoxygenated condition, the heat stable cross-linked tetrameric hemoglobin is ready for pharmaceutical formulation and packaging. The présent 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 présent invention is stable under deoxygenated condition for more than two years.

[0059] 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 tums into inactive methemoglobin rapidly (within a few days) under oxygenated conditions.

[0060] The packaging used in the présent 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 présent 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 cm³ per 100 square inches per 24 hours per atmosphère at room température. 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.

[0061] 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 présent invention uses as secondary packaging an aluminum overwrap pouch which prevents oxygénation and also prevents light exposure. The composition of the overwrap pouch includes 0.012mm of polyethylene terephthalate (PET), 0.007mm of aluminum (Al), 0.015mm of nylon (NY) and 0.1mm of polyethylene (PE). The overwrap film has a thickness of 0.14mm and an oxygen transmission rate of 0.006 cm³ per 100 square inches per 24 hours per atmosphère at room température. This secondary packaging lengthens the stability time for the hemoglobin, extending the product shelf-life.

[0062] The hemoglobin of the présent invention is analyzed by various techniques, including ESI-MS. ESI-MS enables the analysis of very large molécules. 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 resuit indicates that the size of heat stable cross-linked tetrameric hemoglobin is 65 kDa (nonpolymeric hemoglobin tetramers). 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°C. The CD resuit shows that heat stable cross-linked tetrameric hemoglobin has around 42 % of alpha-helix, 38 % of beta-sheet, 2.5 % of beta-tum and 16 % of random coil. It further confirms that the cross-linked tetrameric hemoglobin is heat stable.

[0063] The process in this invention is applicable to large scale industrial production of the heat stable cross-linked tetrameric hemoglobin. In addition, the heat stable cross-linked tetrameric hemoglobin in combination with a pharmaceutical carrier (e.g. water, physiological buffer, in capsule form) is suitable for mammalian use.

[0064] The présent invention further discloses the uses of the oxygen carrier-containing pharmaceutical composition in improving tissue oxygénation, in cancer treatment, in the treatment of oxygen-deprivation disorders such as hémorrhagie shock, and in heart préservation under a low oxygen content environment (e.g. heart transplant). The dosage is selected to hâve a concentration range of approximately 0.2-1.3g/kg with an infusion rate of less than 10 ml/hour/kg body weight.

[0065] For uses in cancer treatment, the oxygen carrier-containing pharmaceutical composition of the présent invention serves as a tissue oxygénation agent to improve the oxygénation in tumor tissues, thereby enhancing chemosensitivity and radiation sensitivity.

[0066] In addition, the ability of the heat stable cross-linked tetrameric hemoglobin to improve oxygénation in normal tissues (FIG. 6) and in extremely hypoxie tumors (FIG. 7), human nasopharyngeal carcinoma (using CNE2 cell line) is demonstrated in this invention. The représentative oxygen profile along the tissue track of a human CNE2 xenograft is showed in FIG. 7. Oxygen partial pressure (pO₂) 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 0.2g/kg of the heat stable cross-linked tetrameric hemoglobin, the médian pO₂ 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.

[0067] For tumor tissue oxygénation, a représentative oxygen profile of a human head and neck squamous cell carcinoma (HNSCC) xenograft (FaDu) is shown in FIG. 7. After intravenous injection of 0.2g/kg of the heat stable cross-linked tetrameric hemoglobin, a significant increase in the mean pO₂ of more than 6.5-fold and 5-fold is observed at 3 and 6 hours, respectively (FIG. 7).

[0068] For applications in cancer treatment, the oxygen carrier-containing pharmaceutical composition of the présent invention serves as a tissue oxygénation agent to improve the oxygénation 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 représentative 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 crosslinked tetrameric hemoglobin (2Gy+Hb). 1.2g/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.

[0069] In one embodiment, significant liver tumor shrinkage is observed after injecting the composition, in conjunction with a chemotherapeutic agent. In FIG. 8B, the représentative 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 3mg/kg cisplatin alone, or in combination with 0.4g/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.

[0070] For the use in the treatment of oxygen-deprivation disorders and for heart préservation, the oxygen carrier-containing pharmaceutical composition of the présent invention serves as a blood substitute providing oxygen to a target organ.

[0071] The mean arterial pressure changes in a rat model of severe hémorrhagie shock after treatment with 0.5g/kg of the heat stable cross-linked tetrameric hemoglobin are shown in FIG. 9. In a rat model of severe hémorrhagie shock, the mean arterial pressure is retumed 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 retum 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 [0072] The following examples are provided by way of describing spécifie embodiments of this invention without intending to limit the scope of this invention in any way.

[0073] Example 1 [0074] Process Overview [0075] A schematic flow diagram of the process of the présent invention is illustrated in FIG.

1. Bovine whole blood is collected into an enclosed stérile 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.

[0076] Washed RBC are lysed to release hemoglobin content by manipulating hypotonie 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 molécules 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 12-14g/dL by a 30 kDa membrane. Column chromatography is carried out to remove the protein impurities.

[0077] The concentrated hemoglobin solution is first reacted with DBSF to form heat stable cross-linked tetrameric hemoglobin molécules under a deoxygenated condition. A heat treatment step is then performed under deoxygenated conditions at 90°C for 30 seconds to three minutes before final formulation and packaging.

[0078] Example 2 [0079] Time & Controlled Hypotonie lysis and filtration [0080] Bovine whole blood is freshly collected and transported under a cool condition (2 to 10°C). The red blood cells are separated from the plasma via a cell washer and subsequently with a 0.65 pm filtration. After washing the red blood cells (RBC) filtrate with 0.9% saline, the filtrate is disrupted by hypotonie lysis. The hypotonie 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 (12-14g/dL) is mixed with 4 volumes of purified water to generate a hypotonie shock to RBC cell membranes. The period of hypotonie shock is controlled to avoid unwanted lysis of white blood cells and platelets. The hypotonie 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 hypertonie buffer as it exits the static mixer. The hypertonie 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.

[0081] Following the RBC lysis, the lysate of red blood cells is filtered by a 0.22 pm 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 (détection limit = 64 pg) and HPLC (détection limit = 1 pg/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.

[0082] Example 3 [0083] Viral clearance study on stroma-free hemoglobin solution [0084] In order to demonstrate the safety of the product from this invention, the virus removal abilities of (1) 0.65 pm 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 résistance to physical and chemical agents or treatments.

[0085] Table 3

Target Virus	Model Virus	Taxonomy	Genome	Structure	Size [nm]	Stability*
Hepatitis C virus (HCV)	Bovine viral diarrhea virus (BVDV)	Flaviviridae	ssRNA	enveloped	40-60	low
-	Encephalomyocarditis virus (EMCV)	Picomavirus	ssRNA	non- enveloped	25-30	medium
Parvovirus B19	Bovine parvovirus (BPV)	Parvoviridae	ssDNA	non- enveloped	18-26	very high
Hepatitis B virus (HBV)	Pseudorabies virus (PRV)	Herpesviridae	dsDNA	enveloped	120- 200	Low to medium

[0086] The validation scheme is briefly shown in the following Table 4.

[0087] Table 4

Diafiltration	Ultrafiltration
Cell Washing	Virus spiking I
Virus spiking I	! Ultrafiltration
Y Diafiltration I	1 Virus tests
Virus tests

[0088] The summary of the log réduction results of the 4 viruses in (1) 0.65 μιη diafiltration and (2) 100 kDa ultrafiltration is shown in the following Table 5. Ail four viruses, BVDV, BPV, EMCV and PRV, are effectively removed by 0.65 μιη diafiltration and 100 kDa ultrafiltration.

[0089] Table 5

Viruses	BVDV	BPV	EMCV	PRV
Run	1	2	1	2	1	2	1	2
0.65pm Diafîltration	2.69	3.20	3.73	3.53	3.25	>3.90	2.67	2.63
lOOkDa Ultrafiltration	>4.68	>4.38	5.87	5.92	3.60	3.43	>6.05	3.27
Cumulative maximum	>7.88	9.65	>7.50	>8.72
Cumulative minimum	>7.07	9.40	6.68	5.90

Annotation:

> no residual infectivity determined [0090] Example 4 [0091] Flowthrough column chromatography [0092] A CM column (commercially available from GE healthcare) is used to further remove any protein impurities. The starting buffer is 20mM sodium acetate (pH 8.0), and the elution buffer is 20mM sodium acetate, 2M NaCl (pH 8.0). After the équilibration of the CM column 10 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 ELIS A. The results are indicated in the following Table 6.

[0093] Table 6

	Protein impurities
Immunoglobin-G	Carbonic anhydrase	Albumin
Before CM column	1320 ng/ml	860.3 pg/ml	435.2 ng/ml
Flowthrough (containing hemoglobin)	44.3 ng/ml	81.2 pg/ml	20.4 ng/ml

[0094] 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.

[0095] Example 5 [0096] Préparation of heat stable cross-linked tetrameric hemoglobin [0097] (5a) Cross-linking reaction with DBSF [0098] The cross-linking reaction is carried out in a deoxygenated condition. DBSF is added to the hemoglobin solution to form cross-linked tetrameric hemoglobin without formation of polymeric hemoglobin. DBSF stabilization procedure stabilizes the tetrameric form of hemoglobin (65 kDa) and prevents dissociation into dimers (32 kDa) which are excreted through the kidneys. In this embodiment, a molar ratio of hemoglobin to DBSF of 1:2.5 is used and the pH is 8.6. This process is carried out for a period of 3-16 hours at ambient température (15-25°C) in an inert atmosphère of nitrogen to prevent oxidation of the hemoglobin to form ferrie met-hemoglobin which is physiologically inactive (dissolved oxygen level maintained at less than 0.1 ppm). The completeness of DBSF reaction is monitored by measuring the residual DBSF using HPLC. The yield of the DBSF reaction is high, > 99%.

[0099] (5b) HTST heat process step [00100] A High Température 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°C for 30 seconds to 3 minutes, and preferably 45 to 60 seconds although other conditions can be selected as discussed above and the apparatus modified accordingly. A solution containing cross-linked hemoglobin optionally with 0.2% of N-acetyl cysteine added thereto is pumped into a HTST processing apparatus (first section of the HTST heat exchanger is pre-heated and maintained at 90°C) at a flow rate of 1.0 liter per minute, the résidence time of the first section of the apparatus is between 45 to 60 seconds, then the solution is passed through at the same flow rate into another section of the heat exchanger that is maintained at 25°C. The time required for cooling is between 15 to 30 seconds. After cooling down to 25°C, N-acetyl cysteine is immediately added at a concentration of 0.2% to 0.4%, preferably at 0.4%. This chemical addition after the HTST heating process is very important to maintain methemoglobin (inactive hemoglobin) at a low level. The set-up of the processing apparatus is easily controlled for industrial operation. A température profile with dimer content is shown in FIG. 13. 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 filtration apparatus to form a clear solution thereafter.

[00101] During the HTST heating process at 90°C, met-hemoglobin (inactive hemoglobin) is increased (shown in FIG. 14). After immédiate addition of N-acetyl cysteine, a low level of met-hemoglobin, approximately less than 3%, can be maintained.

[00102] 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°C.

[00103] Table 7

Sample condition	Protein impurities (by ELIS A)	ByHPLC	p50 at 37°C (mmHg)
Immunoglobin- G (pg/ml)	Albumin (pg/ml)	Carbonic anhydrase (pg/ml)	Tetramer (%)	Dimer (%)
No heat treatment	0.29	0.52	261.80	91.8	5.3	38
90°C for 2min	Not détectable	0.02	0.016	96.1	Not détectable	31
Removal (%)	100.0	96.15	99.99		100.0

[00104] Example 6 [00105] Packaging [00106] Because the product of the présent 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 o

of 0.006 to 0.132 cm per 100 square inches per 24 hours per atmosphère at room température. This spécifie 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: 0.012mm of Polyethylene terephthalate (PET), 0.007mm of Aluminum (Al), 0.015mm of Nylon (NY) and 0.1mm of Polyethylene (PE). The overwrap film has a thickness o

of 0.14mm and oxygen transmission rate of 0.006 cm per 100 square inches per 24 hours per atmosphère at room température. A schematic depiction of the infusion bag is depicted in FIG. 15. The overall oxygen permeability for each infusion bag according to the présent invention is 0.0025 cm³ per 24 hours per atmosphère at room température.

[00107] Example 7 [00108] Improvement of oxygénation [00109] (7a) Improvement of oxygénation in normal tissue [00110] Some studies for the normal tissue oxygénation by heat stable cross-linked tetrameric hemoglobin are carried out (shown in FIG. 6). A comparative pharmacokinetic and pharmacodynamie study is conducted in buffalo rats. Male inbred buffalo rats are individually administered with 0.2g/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™ 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 (pO₂) is directly measured by the Oxylab™ tissue oxygénation and température monitor (Oxford Optronix Limited) in hind leg muscle of buffalo rats. Rats are anesthetized by intra-peritoneal injection of 30-50mg/kg pentobarbitone solution followed by insertion of oxygen sensor into the muscle. Ail pO₂ readings are recorded by Datatrax2 data acquisition system (World Précision Instrument) in a real-time manner. Results demonstrate that after an intravenous injection of 0.2g/kg of the heat stable cross-linked tetrameric hemoglobin, the mean pO₂ 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).

[00111] (7b) Significant improvement of oxygénation in extremely hypoxie tumor area

Improvement of oxygénation in an extremely hypoxie 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 x 10⁶ 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 (pO₂) within the tumor mass is directly monitored by the Oxylab™ tissue oxygénation and température monitor (Oxford Optronix

Limited). Ail pO₂ readings are recorded by the Datatrax2 data acquisition system (World Précision Instrument) in a real-time manner. When the pO₂ reading is stabilized, 0.2g/kg heat stable cross-linked tetrameric hemoglobin solution is injected intravenously through the tail vein of the mice and the tissue oxygénation is measured. Results demonstrate that after intravenous injection of 0.2g/kg of the said heat stable cross-linked tetrameric hemoglobin, a significant increase in the mean pO₂ of more than 6.5-fold and 5-fold is observed in 3 and 6 hours, respectively (FIG. 7).

[0107] Example 8 [0108] Cancer treatment studies: A significant tumor shrinkage in Nasopharyngeal Carcinoma [0109] A significant tumor shrinkage is observed after administration of heat stable crosslinked tetrameric hemoglobin solution in combination with X-ray irradiation (FIG. 8A). A human nasopharyngeal carcinoma xenograft model is employed. Approximately 1 x 10⁶cancer 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:

[0110] Group 1: Ringer’s acetate buffer (Ctrl) [0111] Group 2: Ringer’s acetate buffer + X-ray irradiation (2Gy) [0112] Group 3: Heat stable cross-linked tetrameric hemoglobin + X-ray irradiation (2Gy+Hb) [0113] 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 50mg/kg pentobarbitone solution. 2 Grays of X-ray is delivered to the xenograft of tumorbearing mice by a linear accelerator system (Varian Medical Systems). For Group 3, 1.2g/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 altemate day starting with the first day of treatment. Tumor weights are calculated using the équation 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).

[0114] Example 9 [0115] Cancer treatment studies: a significant shrinkage in liver tumor [0116] 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 x 10⁶ 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 pièces and orthotopically implanted into the left liver lobe of a second group of rats. Rats bearing liver tumor are randomized into three groups as foliows:

[0117] Group 1: Ringer’s acetate buffer (Control) [0118] Group 2: Ringer’s acetate buffer + cisplatin (Cisplatin) [0119] Group 3: Heat stable cross-linked tetrameric hemoglobin+ cisplatin (Cisplatin+Hb) [0120] Rats implanted with liver tumor tissue are treated with 3mg/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 portai vein. For Group 3, 0.4g/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).

[0121] Example 10 [0122] Treatment of Acute Severe Hémorrhagie Shock in Rats [0123] Heat stable cross-linked tetrameric hemoglobin is also used as a resuscitation agent in a model of Acute Severe Hémorrhagie Shock in rats. 50 Sprague-Dawley rats are randomly divided into 3 groups according to resuscitation agents, 16 to 18 rats in each group.

[0124] Group 1: Lactate Ringer’s solution (Négative Control, 16 rats) [0125] Group 2: Animal autologous blood (Positive Control, 16 rats) [0126] Group 3: Heat stable cross-linked tetrameric hemoglobin treatment group (0.5 g Hb /kg of body weight, 18 rats) [0127] Acute severe hémorrhagie shock is established by withdrawing 50% of animal whole blood, which is estimated as 7.4% of body weight. After hémorrhagie 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 infosed into the animais. The infusion rate of heat stable cross-linked tetrameric hemoglobin is set at 5 ml/h, thereafter, ail experimental animais are observed for 24 hours. A panel of parameters is observed and analyzed during study period including survival, hemodynamics, myocardial mechanics, cardiac output, cardiac fonction, blood gas, tissue oxygen delivery & consumption, tissue perfusion & oxygen tension (liver, kidney and brain), liver & rénal fonction, hemorheology (blood viscosity), and mitochondrial respiratory control rate (liver, kidney and brain). Above ail, 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 négative control group and the autologous blood group (shown in the following Table 8).

[0128] Table 8

Groups	Survival no. after 24-hour	24-hour survival rate (%)
Négative control	3 in 16 rats	18.8
Rat’s Autologous Blood	10 in 16 rats	62.5
0.5 g Hb/kg	13 in 18 rats	72.0

*Hb= heat stable cross-linked tetrameric hemoglobin [0129] Example 11: Method of Preventing Post-operative Liver Tumor Récurrence and Metastasis [0130] Surgical resection of liver tumors is a frontline treatment of liver cancer. However, post-operative récurrence and metastasis of cancer remains a major attribute of unfavorable prognosis in these patients. For instance, previous studies reported that hepatic resection is associated with a 5-year survival rate of 50% but also a 70% récurrence rate. Follow-up studies on hepatocellular carcinoma (HCC) patients also reveal that extrahepatic métastasés from primary HCC were detected in approximately 15% of HCC patients with the lungs being the most frequent site of extrahepatic métastasés. It has been suggested that surgical stress, especially ischemia/reperfusion (IR) injury introduced during liver surgery is a major cause of tumor progression. Conventionally, hepatic vascular control is commonly used by surgeons to prevent massive hemorrhage during hepatectomy. For example, inflow occlusion by clamping of the portai triad (Pringle maneuver) has been used to minimize blood loss and reduce the requirement of perioperative transfusions. A recent Japanese study shows that 25% surgeons apply a Pringle maneuver on a routine basis. However, Pringle maneuver induces various degrees of ischémie injury in the remnant liver and is associated with cancer récurrence and metastasis.

[0131] Association of IR injury and tumor progression is also supported by previous animal studies. Firstly, the effect of IR injury and hepatic resection on liver cancer récurrence and metastasis was demonstrated in a recent study with an orthotopic liver cancer model. Hepatic IR injury and hepatectomy resulted in prominent récurrence and metastasis of liver tumors. Similar results were obtained in a colorectal liver metastasis mouse model where introduction of IR injury accelerates the outgrowth of colorectal liver metastasis.

[0132] Previously, several protective strategies hâve been studied for use to reduce IR injury during resection. For example, the application of a short period of ischemia before prolonged clamping, known as ischémie preconditioning (IP), was suggested to trigger hepatocellular defense mechanisms and has been used to reduce IR injury during liver resection. Others apply intermittent clamping (IC) procedures which allows cycles of inflow occlusion followed by reperfusion. Both methods were suggested to be effective in protecting against postoperative liver injury in non-cirrhotic patients undergoing major liver surgery. However, in a tumor setting, animal studies also show that IP failed to protect the liver against accelerated tumor growth induced by IR injury. In addition, some groups attempt to use antioxidants such as α,-tocopherol and ascorbic acid to protect the liver from IR injury, thereby preventing liver metastasis. However, both anti-oxidants failed to restrict intrahepatic tumor growth stimulated by IR.

[0133] Mechanistically, different lines of evidence suggest hypoxia is associated with tumor récurrence and metastasis for a number of reasons: (1) studies show that hypoxie tumor is more résistant to radiation- and a chemo- therapy, tumor cells that survive the treatment are prone to recur; clinical evidence also suggests that patients with more hypoxie tumor areas hâve higher rates of métastasés; (2) under hypoxie condition, cancer cells become more aggressive through the activation of hypoxia inducible factor-1 (HIF-1) pathway. This in tum triggers complementary responses involving pro-angiogenic factor vascular endothélial growth factor (VEGF) and receptors such as c-Met and CXCR4, which enhanced cell motility and homing to spécifie, distant organs; (3) recent studies also demonstrated that circulating cancer cells (CTCs) become more aggressive under hypoxie condition. Circulating tumor cells detected in the peripheral blood of cancer patients was shown to be an index of disease aggression in patients with distant metastasis, while hypoxia enabled those cells a more aggressive phenotype and diminished apoptotic potential. In particular, cancer stem cell population, which is more radio-resistant were enriched under reduced oxygen level in brain tumor.

[0134] Therefore, in view of the above observations and studies, the nonpolymeric crosslinked tetrameric hemoglobin of the présent invention is used to prevent post-operative liver tumor récurrence and metastasis following hepatic resection. A rat orthotopic liver cancer model is established. Hepatocellular carcinoma cell line (McA-RH7777 cells) is used to establish the orthotopic liver cancer model in Buffalo rats (Male, 300-350g). FIG. 16 shows a schematic drawing summarizing the surgical and hemoglobin product administration procedures. McA-RH7777 cells (3x105/100 μΐ) are injected into the hepatic capsule of buffalo rat to induce solid tumor growth. Two weeks later (when the tumor volume reaches about 10x10mm), tumor tissue is collected and eut into 1-2 mm cubes and implanted into the left liver lobes of a new group of buffalo rats. Two weeks after orthotopic liver tumor implantation, the rats undergo liver resection (left lobe bearing liver tumor) and partial hepatic IR injury (30 minutes of ischemia on right lobe).

[0135] Two groups of rats with implanted tumor tissue are used for comparison of tumor récurrence and métastasés. In group 1, rats are anesthetized with pentobarbital and administered intravenously with 0.2g/kg of the nonpolymeric heat stable cross-linked tetrameric hemoglobin of the présent invention 1 hour before ischemia. Ischemia is introduced in the right lobe of the liver by clamping of right branches of hepatic portai vein and hepatic artery with a bulldog clamp. Subsequently, ligation is performed in the left liver lobe followed by resection of the left liver lobe bearing the liver tumor. At 30 minutes after ischemia, an additional 0.2g/kg of the heat stable cross-linked tetrameric hemoglobin is injected through the inferior vena cava followed by reperfusion. In group 2, ringer’s acetate buffer is injected as a vehicle control with the same procedure. Ail rats are sacrificed 4 weeks after the hepatectomy procedures.

[0136] To examine tumor growth and metastasis, the liver and lungs of Buffalo rats are sampled at 4 weeks after Ischemia/reperfusion and hepatectomy procedures for morphological examination. Tissue is harvested, parafilm-embedded and sectioned followed by Hematoxylin and Eosin (H&E) staining. Local recurrence/metastasis (intrahepatic) and distant metastasis (lungs) are confirmed by histological examination. Table 9 summarizes the observations.

[0137] Table 9: Comparison of tumor récurrence / metastasis at four weeks after liver resection and IR injury in a rat orthotopic liver cancer model.

	Control (n=6)	Treatment (n=5)
Intrahepatic	4 (66.7%)	2 (40%)
metastasis/recurrence
Lung metastasis	4 (66.7%)	2 (40%)

[0138] To examine the protective effects of nonpolymeric heat stable cross-linked tetrameric hemoglobin on liver tumor récurrence and metastasis, ail rats are sacrificed 4 weeks after the hepatectomy and IR procedures. Lungs and liver tissues are harvested; hepatic tumor recurrence/metastasis and distant metastasis in the lungs are compared in both groups. Results show that the hemoglobin treatment decreases occurrence of récurrence and metastasis in both organs.

[0139] FIG. 17 shows représentative examples of intra-hepatic liver cancer récurrence and metastasis and distant lung metastasis induced in the rats of the IR injury group after hepatectomy and ischemia/reperfusion procedures and its protection using the inventive heat stable cross-linked tetrameric hemoglobin. In FIG. 17A, extensive intrahepatic liver cancer recurrence/metastasis is observed in the IR injury group. Distant lung metastasis is also occurred in the same rat (indicated by a solid arrow). In FIG. 17B, intrahepatic liver cancer recurrence/metastasis is observed in another case in the IR injury group (indicated by a dotted arrow). Extensive lung metastasis is observed in the same case (indicated by solid arrows). In contrast, FIG. 17C shows a représentative example of protection from intrahepatic liver cancer recurrence/metastasis and distant lung metastasis in the inventive heat stable crosslinked tetrameric hemoglobin treated rat.

[0140] FIG. 18 shows the histological examination in both groups at four weeks after liver resection and IR injury procedures. Histological examination (H&E staining) of liver and lung tissues in both the IR injury and hemoglobin treatment groups is perfoimed to confirm the identity of the tumor nodules. Représentative fields showing intrahepatic recurrence/metastasis in the hemoglobin treatment (T3) and IR injury groups (Tl and T2) are shown. Histological examination showing a normal liver architecture in the treatment group is included for comparison (NI). In addition, distant metastasis in the lungs is found in the same rat in IR injury group (M). Lung tissue without metastasis is shown in the treatment group (N2) for comparison.

[0141] To further confirm the protective effects of non-polymeric heat stable cross-linked tetrameric hemoglobin on tumor récurrence and metastasis, récurrence rate of tumor and size of the recurred tumor post-ischemia/reperfusion and hepatectomy procedures are investigated. Again, rats with implanted tumor tissue prepared by injection of McA-RH7777 cells as described above are treated intravenously with either approximately 0.2-0.4g/kg of the non polymeric heat stable cross-linked tetrameric hemoglobin of the présent invention or Ringer's acetate (RA) buffer as a négative control prior to ischemia and at reperfusion upon hepatic resection procedure as described in FIG. 16. A total of 24 rats are tested, where 11 rats are treated with the subject hemoglobin and 13 are négative control rats which are merely treated with RA buffers. Ail rats are sacrificed 4 weeks after the hepatectomy and IR procedures, livers and lungs of the test rats are examined for tumor récurrence/ metastasis and the relative size of the recurred tumors are measured.

[0142] FIG. 19A shows liver tumor récurrence in test rats and the volume of individual recurred tumors. Liver tumor recurred/metastasis in 9 of the 13 non- treated control rats, whereas only 4 of the 11 treated rats experienced tumor recurrences/metastasis. It is also évident that where tumor récurrence is seen, the sizes of the recurred tumors of rats having treated with the subject hemoglobin are significantly smaller than those untreated. The results show that tumor récurrence rate is greatly reduced and recurred tumor size is significantly reduced with treatment of the subject invention, as summarized in FIG. 19B.

[0143] FIG. 20 illustrâtes représentative examples of liver and lung tissues harvested 4 weeks post hepatectomy and IR procedures of rats having treated with the subject inventive nonpolymeric heat stable cross-linked tetrameric hemoglobin and the IR injury (négative control) group. As seen in représentative examples of the untreated négative control group, rats C10 and 13, extensive intrahepatic liver cancer récurrence/ metastasis and distant lung metastasis are observed (circled). On the other hand, intrahepatic liver cancer récurrence/ metastasis and distant lung metastasis are prevented by the treatment of the subject inventive hemoglobin, as seen in rats Y9, Y10 and Y11.

[0144] Examples 12: Treatment with nonpolymeric heat stable cross-linked tetrameric hemoglobin reduces ischemia [0145] As demonstrated in Example 7, intravenous injection of the subject nonpolymeric heat stable cross-linked tetrameric hemoglobin to hypoxie tumor significantly improves the oxygénation therein. Accordingly, the oxygénation effect of the subject hemoglobin product during tumor resection and JR procedure is investigated. Rats with implanted liver tumor tissue prepared by injection of McA-RH7777 cells as described in Example 11 are used and are subjected to surgery and 0.2-0.4g/kg of the subject hemoglobin product or RA buffer administration procedures as outline in FIG. 16. Partial oxygen pressure of liver is measured from the time the subject hemoglobin product/ RA buffer is first administered to the hepatic tumor and throughout the IR procedure, hepatic tumor resection and after reperfusion. Results (FIG. 21) shows that increased oxygénation with the subject hemoglobin treatment is observed after introduction of ischemia. In addition, as seen in FIG. 21, the liver having treated with the subject hemoglobin has approximately 3-fold higher partial oxygen pressure than without treatment after reperfusion. It is confirmed that the treatment of the subject hemoglobin prior to ischemia and at reperfusion upon tumor resection significantly improves the oxygénation of the liver tissue as compared to non-treatment. In view of the strong corrélation between hypoxie tumor and the increased likelihood of tumor recurrences/metastasis suggested in the art, the profound oxygénation effects of the présent hemoglobin product and the use thereof during tumor resection procedure as demonstrated in this example, the usefulness of the présent hemoglobin product to reduce tumor récurrence and metastasis are evidently confîrmed.

[0146] Examples 13: Treatment with nonpolymeric heat stable cross-linked tetrameric hemoglobin reduces circulating endothélial progenitor cell levels [0147] Different lines of study hâve demonstrated the significance of cancer stem cells (CSCs) and/ or progenitor cell populations in the progression of liver cancer. Importantly, previous studies show that a significantly higher level of circulating endothélial progenitor cells (EPCs) is found in BICC patients, including those undergoing hepatectomy.

[0148] Accordingly, the level of circulating EPCs is evaluated by expression of surface molécules such as CD133, CD34 and VEGFR2. The circulating endothélial progenitor cell levels post- hepatic resection surgery and IR procedure with or without the treatment of the subject hemoglobin product is investigated. Two groups of rats with implanted hepatic tumor are subjected to treatment of the subject hemoglobin or RA buffer (control), respectively prior to ischemia and at reperfusion upon hepatic resection as described in above Example 11 and FIG. 16. Number of circulating EPC of the two group of rats are then measured at 0, 3, 7 14, 21 and 28 days after hepatic resection and IR procedures. Results (FIG. 22) shows that while EPC levels of the treated and non-treated groups are comparable during day 0- day 3 postsurgery, EPC levels of the hemoglobin treated group are profoundly lower than those RA buffer treated group. These results are consistent with the results of Example 11 where the protection effects of the subject hemoglobin to reduce and minimize tumor récurrence/ metastasis are verified.

[0149] As a resuit of the above investigations, it is concluded that treatment with the nonpolymeric heat stable cross-linked tetrameric hemoglobin of the présent invention has a preventative effect on both the récurrence of hepatic tumors and on metastasis in other organs. [0150] 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.

Claims

What is claimed:

1. Use of a non-polymeric, highly purified and heat stable oxygen carrier containing composition in the manufacture of a médicament for reducing cancerous tumor récurrence and/or minimizing tumor cell metastasis in a mammal, wherein said composition comprising a cross-linked tetrameric hemoglobin with an undetectable dimer concentration.
2. The use of claim 1, wherein said composition is administered to said mammal prior to disruption of blood supply and during re-establishment of blood supply during a surgical removal of a tumor.
3. The use of claim 2, wherein said composition is administered in a range of approximately 0.2g/kg-1.2g/kg body weight of the mammal.
4. The use of claim 1, wherein said cancerous tumor and tumor cell are hepatic or nasopharyngeal.
5. The use of claim 1, wherein said cancerous tumor and tumor cell are hypoxie.
6. The use of claim 1, wherein said cross-linked tetrameric hemoglobin has a molecular weight of 60-70kDa and N-acetyl cysteine at a concentration of 0.2-0.4%.
7. The use of claim 6, wherein said composition is free of vasoconstricting impurities and protein impurities, non-pyrogenic, endotoxin-free, phospholipid-free, stroma-free and a met-hemoglobin level of less than 5%.
8. Use of a non-polymeric, highly purified and heat stable oxygen carrier containing composition in the manufacture of a médicament for treating cancerous tissues, wherein said composition comprising a cross-linked tetrameric hemoglobin with an undetectable dimer concentration.
9. The use of claim 8, wherein the cancerous tissues are treated by radiation or chemotherapy treatment.
10. The use of claim 9, wherein said composition is administered to cancerous tissues prior to the radiation or chemotherapy treatment of the cancerous tissues.
11. The use of claim 8, wherein the cancerous tissues are nasopharyngeal carcinoma or a liver tumor.
12. The use of claim 8, wherein the cancerous tissues are hypoxie.
13. The use of claim 8, wherein said cross-linked tetrameric hemoglobin has a molecular weight of 60-70kDa and N-acetyl cysteine at a concentration of 0.2-0.4%.
14. The use of claim 13, wherein said composition is free of vasoconstricting impurities and protein impurities, non-pyrogenic, endotoxin-free, phospholipid-free, stroma-free and a met-hemoglobin level of less than 5%.
15. The use of claim 10, wherein said composition is administered by infusion in a range of approximately 0.2-1.2g/kg body weight and at a rate of less than lOml/hour/kg body weight.
16. Use of a non-polymeric, highly purified and heat stable oxygen carrier containing composition in the manufacture of a médicament for increasing oxygénation of cancerous tissues, wherein said composition comprising a cross-linked tetrameric hemoglobin with an undetectable dimer concentration.
17. Use of a non-polymeric, highly purified and heat stable oxygen carrier containing composition in the manufacture of a médicament for reducing size of a tumor, wherein said composition comprising a cross-linked tetrameric hemoglobin with an undetectable dimer concentration.
18. A non-polymeric, highly purified and heat stable oxygen carrier containing composition for reducing cancerous tumor récurrence and/or minimizing tumor cell metastasis in a mammal, wherein said composition comprising a cross-linked tetrameric hemoglobin with an undetectable dimer concentration.
19. The composition of claim 18, wherein said composition is administered to said mammal prior to disruption of blood supply and during re-establishment of blood supply during a surgical removal of a tumor.

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20. The composition of claim 19, wherein said composition is administered in a range of approximately 0.2g/kg-1.2g/kg body weight of the mammal.
21. The composition of claim 18, wherein said cross-linked tetrameric hemoglobin has a molecular weight of 60-70kDa and N-acetyl cysteine at a concentration of 0.2-0.4%.
22. The composition of claim 18, wherein said composition is free of vasoconstricting impurities and protein impurities, non-pyrogenic, endotoxin-free, phospholipid-free, stromafree and a met-hemoglobin level of less than 5%.