CA2778010C - A novel blood substitute with complete red blood cell functions - Google Patents
A novel blood substitute with complete red blood cell functions Download PDFInfo
- Publication number
- CA2778010C CA2778010C CA2778010A CA2778010A CA2778010C CA 2778010 C CA2778010 C CA 2778010C CA 2778010 A CA2778010 A CA 2778010A CA 2778010 A CA2778010 A CA 2778010A CA 2778010 C CA2778010 C CA 2778010C
- Authority
- CA
- Canada
- Prior art keywords
- hemoglobin
- blood
- soluble complex
- sod
- cat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present document describes a soluble complex which comprises a hemoglobin cross-linked with an antioxidant enzyme, and acid-base and carbon dioxide transport enzyme. Cross-linking is enhancing in vivo circulation time of the soluble complex, and prevents harmful in vivo effects that could be sustained by an individual subjected to the individual free components of the soluble complex.
Description
File No. P1754CA00 Title: A NOVEL BLOOD SUBSTITUTE WITH COMPLETE RED BLOOD CELL
FUNCTIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of US provisional patent application 61/490,304, filed on May 26, 2012.
BACKGROUND
(a) Field The subject matter disclosed generally relates to artificial blood substitutes and method of using and making the same. Particularly, the artificial blood substitutes comprises soluble complexes made by crosslinking stroma free hemoglobin (SFHb) or hemoglobin (Hb) with antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] and an enzyme with acid-base and carbon dioxide transport (carbonic anhydrase, CA) to form soluble complexes of Poly-[SFHb-SOD-CAT-CA]
(b) Related Prior Art
FUNCTIONS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of US provisional patent application 61/490,304, filed on May 26, 2012.
BACKGROUND
(a) Field The subject matter disclosed generally relates to artificial blood substitutes and method of using and making the same. Particularly, the artificial blood substitutes comprises soluble complexes made by crosslinking stroma free hemoglobin (SFHb) or hemoglobin (Hb) with antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] and an enzyme with acid-base and carbon dioxide transport (carbonic anhydrase, CA) to form soluble complexes of Poly-[SFHb-SOD-CAT-CA]
(b) Related Prior Art
[0002] Increased demand for blood transfusions in emergency and surgical settings has led to urgent need for red blood cell (RBC) substitutes.
Different types of oxygen-carrying blood substitutes have been investigated.
One of such products is the nanobiotechnology based polyhemoglobin (polyHb).
Crosslinking haemoglobin (Hb) with bifunctional agents such as diacid or glutaraldehyde results in the formation of polyHb. These complex of 4-5 hemoglobin (Hb) molecules have been shown to replace blood loss by maintaining Hb levels in a safe range during surgery and perform adequate oxygen transport. Two of these nanobiotechnological products had reached Phase III clinical trials [Jahr JS et at, J Trauma 64: 1484-97 (2008) and Moore EE et al, J Am Coll Surg. 208: 1-13 (2009)], but the US FDA did not approve these products because of side effects. These side effects include a 3%
incidence of myocardial infarction in ambulance trauma patients infused with polyhemoglobin, as compared to only 0.6% in patients receiving only saline File No. P1754CA00 solution. This is most likely due to ischemia-reperfusion injury especially in those patients with prior ischemic heart conditions. Furthermore, in all cases of hemorrhagic shock there is also accumulation of carbon dioxide and acid base changes [Moore EE et al, J Am Coll Surg. 208: 1-13 (2009)].
Different types of oxygen-carrying blood substitutes have been investigated.
One of such products is the nanobiotechnology based polyhemoglobin (polyHb).
Crosslinking haemoglobin (Hb) with bifunctional agents such as diacid or glutaraldehyde results in the formation of polyHb. These complex of 4-5 hemoglobin (Hb) molecules have been shown to replace blood loss by maintaining Hb levels in a safe range during surgery and perform adequate oxygen transport. Two of these nanobiotechnological products had reached Phase III clinical trials [Jahr JS et at, J Trauma 64: 1484-97 (2008) and Moore EE et al, J Am Coll Surg. 208: 1-13 (2009)], but the US FDA did not approve these products because of side effects. These side effects include a 3%
incidence of myocardial infarction in ambulance trauma patients infused with polyhemoglobin, as compared to only 0.6% in patients receiving only saline File No. P1754CA00 solution. This is most likely due to ischemia-reperfusion injury especially in those patients with prior ischemic heart conditions. Furthermore, in all cases of hemorrhagic shock there is also accumulation of carbon dioxide and acid base changes [Moore EE et al, J Am Coll Surg. 208: 1-13 (2009)].
[0003] The advantages of hemoglobin-based blood substitutes such as polyHb compared to donor blood are:
[0004] (1) They do not have blood group antigens. Thus, they can be transfused immediately on the spot without the need for the patients to be transported to the hospital for cross-matching and typing as in the case of donor blood. This is particularly important in emergency or during natural or man made disasters where transportation is difficult.
= [0005] (2) Unlike RBC, polyHb can be sterilized and therefore do not have the potential of infective agents in case of epidemics (earlier examples of AIDS
and the resulting HIV contaminated donor blood can now be avoided in future epidemics of other types of infective agents), [0006] (3) Donor blood has to be stored at 4 C and is only good for about 42 days. PolyHb can be stored at room temperature for more than a year. This allows for PolyHb in plastic bags to be kept anywhere that has potential need for this product for at least 1 year and longer if kept in lower temperature.
[0007] However, polyHb only has only one of the three important functions of RBC. The three important functions of RBC are: (1) transport and supply oxygen, (2) transport and remove carbon dioxide, and (3) remove harmful oxygen radicals. At present, industrial RBC substitutes in the form of polyHb only transport oxygen, and they were not approved by the US FDA because of side effects. Furthermore, the antioxidant enzyme activities of SOD and CAT of RBC
extracts are not sufficient to prevent ischemia-reperfusion injury in severe hemorrhagic shock, stroke, myocardial infarction and other conditions. SOD and CAT have been complexed with polyHb to form polyHb-SOD-CAT (US patent File No. P1754CA00 No. 5,606,025 to D'Agnillo and Chang. However, this complex still lacks one of the important function of RBC in that it does not transport and remove carbon dioxide from blood and will therefore cause side effects. Therefore, there is a need for more complete RBC substitutes with all 3 functions of RBC to treat blood loss.
[0008] Furthermore, there is a need for more complete RBC substitutes to prevent ischemia-reperfusion injuries.
SUMMARY
[0009] According to an embodiment, there is provided a soluble complex comprising hemoglobin cross-linked with an antioxidant enzyme for reduction of reactive oxygen species and an acid-base and carbon dioxide transport enzyme for CO2 and 02 transport.
[0010] The antioxidant enzyme may be chosen from super oxide dismutase (SOD), catalase (CAT), or combinations thereof.
[0011] The antioxidant enzyme may be a combination of super oxide dismutase (SOD) and catalase (CAT).
[0012] The ratio of hemoglobin to super oxide dismutase (SOD) may be from about 1 g : 4 000 U to about 1 g : 25 000 U.
[0013] The ratio of hemoglobin to super oxide dismutase (SOD) may be about 1 g : 18 000 U.
[0014] The ratio of hemoglobin to catalase (CAT) may be from about 1 g:
25,000 U to about 1 g : 310,000 U.
[0015] The ratio of hemoglobin to catalase (CAT) may be about 1 g :
310 000 U.
[0016] The acid-base and carbon dioxide transport enzyme may comprise carbonic anhydrase (CA).
File No. P1754CA00 [0017] The ratio of hemoglobin to carbonic anhydrase (CA) may be from about 1 g : 80 000 U to about 1 g : 250 000 U.
[0018] The ratio of hemoglobin to carbonic anhydrase (CA) may be about 1 g : 130 000 U.
[0019] The hemoglobin may comprise stroma free hemoglobin (SFHb) or hemoglobin (Hb).
[0020] The soluble complex may have a molecular weight of more than 100 kDa.
[0021] The soluble complex may have a molecular weight from about more than 100 kDa to more than 450 kDa.
[0022] According to another embodiment, there is provided an artificial blood substitute comprising a soluble complex according to the present invention, in a suitable pharmaceutical fluid.
[0023] According to another embodiment, there is provided a method of treating a medical condition comprising administering a therapeutically effective amount of a soluble complex according to the present invention, or an artificial blood substitute according to the present invention to a patient in need thereof.
[0024] The medical condition may be chosen from a blood loss, a condition with accumulation of carbon dioxide, an ischemia-reperfusion injury, and a diver's disease.
[0025] The ischemia-reperfusion injury may comprise a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
[0026] According to another embodiment, there is provided a method for removing carbon dioxide from a fluid comprising treating the fluid with an effective amount of a soluble complex according to the present invention, or an artificial blood substitute according to the present invention.
File No. P1754CA00 [0027] The fluid may be chosen from blood and air. The fluid may be blood in a dialysis. The fluid may be blood in an artificial lung. The fluid may be air undergoing environmental removal of CO2.
[0028] According to another embodiment, there is provided a use of a soluble complex according to the present invention for the preparation of a medicament for the treatment of a medical condition.
[0029] According to another embodiment, there is provided a use of a soluble complex according to the present invention for the treatment of a medical condition.
[0030] According to another embodiment, there is provided a use of an artificial blood substitute according to the present invention for the preparation of a medicament for the treatment of a medical condition.
[0031] According to another embodiment, there is provided a use of an artificial blood substitute according to the present invention for the treatment of a medical condition.
[0032] The condition may be chosen from a blood loss, a condition with accumulation of carbon dioxide, an ischemia-reperfusion injury, a diver's disease.
[0033] The ischemia-reperfusion injury may comprise a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
[0034] According to another embodiment, there is provided a use of a soluble complex according to the present invention or an artificial blood substitute according to the present invention for treating a fluid for removing carbon dioxide therefrom.
[0035] The fluid may be chosen from blood and air. The fluid may be blood in an artificial lung. The fluid may be blood in a dialysis.
File No. P1754CA00 [0036] According to another embodiment, there is provided a method of preparing a soluble complex comprising the steps of:
a) cross-linking a mixture comprising = a stroma-free hemoglobin or hemoglobin;
= at least one antioxidant enzyme;
= an acid-base and carbon dioxide transport enzyme, and = lysine b) purifying and concentrating said mixture to obtain said soluble complex.
[0037] The molar ratio of lysine to the stroma-free hemoglobin may be from about 7 : 1 to about 12 : 1. Preferably the molar ratio of lysine to the stroma-free hemoglobin may be 7:1.
[0038] The step a) may be performed with glutaraldehyde.
[0039] The molar ratio of glutaraldehyde to stroma-free hemoglobin may be from about 8:1 to about 32:1. Preferably, the molar ratio of glutaraldehyde to the stroma-free hemoglobin may be 16:1.
[0040] The step of cross-linking may be performed for 24h at 4 C.
[0041] The step of cross-linking may be stopped with lysine at molar ratio to the stroma-free hemoglobin of 200:1.
[0042] The step of purifying and concentrating may be performed by filtering the mixture, dialyzing the mixture, microconcentrating the mixture, or combinations thereof.
[0043] The following terms are defined below.
[0044] The term "pharmaceutically acceptable fluid" is intended to mean a preservative solution, a saline solution, an isotonic (about 0.9%) saline solution, or about a 5% albumin solution, suspension, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic File No. P1754CA00 substances suitable for delivery to a patient may be included in the compositions of the present invention. The compositions may be solutions, suspensions or any appropriate formulation suitable for administration, and are typically sterile and free of undesirable particulate matter. The compositions may be sterilized by conventional sterilization techniques.
[0045] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
[0047] Fig. 1 illustrates the carbonic anhydrase activity (CA): (1) SFHb ¨
content of red blood cells (SFHb) with CA activity; (2) PolySFHb ¨ content of red blood cells crosslinked into polySFHb with very low CA activity;(3) SFHb + SOD
+ CAT + CA ¨ enzymes added to extracts of red blood cell content without crosslinking, and (4) Poly-[Hb-SOD-CAT-CA] represent the previous components crosslinked into Poly-[Hb-SOD-CAT-CA] according to an embodiment of the present invention with resulting good CA activity. SFHb: stroma free hemoglobin;
PolySFHb: Crosslinked SFHb; SFHb+SOD+CAT+CA: add 15000 U SOD, 300,000 U CAT and 15,300 U CA to each gram of SFHb; Poly-[SFHb-SOD-CAT-CA] : crosslinked SFHb+SOD+CAT+CA.
[0048] Fig. 2 illustrates the superoxide dismutase (SOD): SFHb ¨ content of red blood cells (SFHb) with very low SOD activity; PolySFHb ¨ content of red File No. P1754CA00 blood cells crosslinked into polySFHb with very low SOD activity; SFHb + SOD +
CAT + CA ¨ enzymes added to extracts of red blood cell content without crosslinking, and Poly-[SFHb-SOD-CAT-CA] represent the previous components crosslinked into Poly-[SFHb-SOD-CAT-CA] according to an embodiment of the present invention with resulting greatly enhanced SOD activity. SFHb: stroma free hemoglobin; PolySFHb: Crosslinked SFHb; SFHb+SOD+CAT+CA : Add 1500 U SOD, 300,000 U CAT and 15,300 U CA to each gram of SFHb; Poly-[SFHb-SOD-CAT-CA]: crosslinked SFHb+SOD+CAT+CA.
[0049] Fig. 3 illustrates the catalase activity (CAT): SFHb ¨ content of red blood cells (SFHb) with very low SOD activity; PolySFHb ¨ content of red blood cells crosslinked into polySFHb with very low SOD activity; SFHb + SOD + CAT
+ CA ¨ enzymes added to extracts of red blood cell content without crosslinking, and Poly-[SFHb-SOD-CAT-CA] represent the previous components crosslinked into Poly-[SFHb-SOD-CAT-CA] according to an embodiment of the present invention with resulting markedly enhanced CAT activity. SFHb: stroma free hemoglobin; PolySFHb: Crosslinked SFHb; SFHb+SOD+CAT+CA : Add 1500 U
SOD, 300,000 U CAT and 15,300 U CA to each gram of SFHb; Poly-[SFHb-SOD-CAT-CA] : crosslinked SFHb+SOD+CAT+CA.
[0050] Fig. 4 illustrates an in vivo trial of the soluble complex according to an embodiment of the present invention. RBC: whole blood, PHcsc PolyHb with low enzyme, PHCSC: PolyHb with high enzyme = PolyHbCAT-SOD-CA.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] In embodiments there is disclosed a soluble complex formed by crosslinking hemoglobin with antioxidant enzyme and acid-base and carbon dioxide enzyme. Extracts from red blood cell contains hemoglobin contain very low antioxidant enzymes activity (e.g. SOD and CAT) and an acid-base and carbon dioxide transport activity (CA). However, when direct cross-linking of these enzymes is performed, it significantly decreases the SOD, CAT and CA
enzyme activity to levels much below the optimal levels. Furthermore, the File No. P1754CA00 antioxidant enzyme (e.g. of SOD and CAT) present in red blood cell extracts are insufficient to prevent ischemia-reperfusion injuries that occur in severe hemorrhagic shock, stroke, myocardial infarction and other conditions.
Furthermore, after crosslinking, the CA level is no longer sufficient for acid-base and carbon dioxide transport functions, especially in severe ischemia.
[0052] Therefore, according to an embodiment of the present invention, the soluble complex (e.g. Poly-[SFHb-SOD-CAT-CA]) of the present invention comprises the three functions of RBC: It is an oxygen carrier (PolySFHb) with antioxidant activity (SOD, CAT) and also the ability to facilitate the transport and acid-base functions (CA activity). It also comprises enhanced SOD, CAT and CA
activities to enhance the function of this novel red blood cell substitute.
The SOD
and CAT levels in this Poly-[SFHb-SOD-CAT-CA] cross-linked polyhemoglobin are much higher than those in RBC. Therefore, it is believed that it may also be used as a therapeutic agent for the treatment or prevention of ischemia-reperfusion injuries and to facilitate the removal of accumulated carbon dioxide in the case of severe hemorrhagic shock and other conditions.
[0053] According to an embodiment of the present invention, the hemoglobin component of the soluble complex acts as an oxygen carrier. The hemoglobin component of the soluble complex may be prepared from any suitable hemoglobin extracted from red blood cells, from human or animals (bovine, Porcine etc) or recombinant Hb or other types of organisms and even synthetic hemoglobin.
[0054] The soluble complex of the present invention also includes a therapeutically effective amount of an antioxidant enzyme. This antioxidant enzyme may be any suitable antioxidant enzymes. The antioxidant enzyme may be either super oxide dismutase (SOD), catalase (CAT), or combinations thereof.
Preferably, the antioxidant enzyme is a combination of super oxide dismutase (SOD) and catalase (CAT). These enzymes may be from sources exogenous from the hemoglobin. The SOD enzyme may be present in a ratio with the File No. P1754CA00 hemoglobin component corresponding to from about 1g of hemoglobin to 4 000 U to 25 000 U of SOD. Preferably, the optimal ratio of the-hemoglobin to the super oxide dismutase (SOD) is about 1 g :18 000 U. The CAT enzyme may be present in a ratio with the hemoglobin corresponding from about 1g of hemoglobin to 25 000 U to 310,000 U of CAT. Preferably, the ratio of hemoglobin preparation to said catalase (CAT) is about 1 g : 310 000 U.
[0055] The soluble complex of the present invention also includes a therapeutically effective amount of an acid-base and carbon dioxide transport enzyme. Preferably, the acid-base and carbon dioxide transport enzyme comprises carbonic anhydrase (CA), but synthetic enzymes (e.g. synthetic enzyme bearing the active sites necessary to provide CA activity) with the same activity may also be used. The CA enzyme may be from an exogenous source.
The CA enzyme may be present in a ratio with the hemoglobin component corresponding from about 1g of hemoglobin to 80 000 U to 250 000 U of CA.
Preferably, the ratio of hemoglobin to said carbonic anhydrase (CA) is about 1 g:
130 000 U.
[0056] The components of the soluble complex are chemically bonded because hemoglobin and the enzymes, when in the free form, are rapidly removed after infusion and they may also cause harmful effects. Especially sensitivity, allergic and immunological types of reactions. Thus, according to one embodiment of the present invention, the soluble complex may be Poly-[SFHb-SOD-CAT-CA], where the SFHb, SOD, CAT and CA are cross-linked to each other.
[0057] The SOD and CAT activity of the present invention is much higher than that normally present in the RBC and can therefore enhance the antioxidant activity. This enhanced ability to prevent the harmful effects of oxygen radicals is important in ischemia-reperfusion encountered in severe hemorrhagic shock, stroke, myocardial infarction. The CA activity is important because in any File No. P1754CA00 hemorrhagic shock or ischemic condition, there is also accumulation of carbon dioxide that also needs to be removed.
[0058] According to another embodiment, the soluble complex of Poly-[SFHb-SOD-CAT-CA] may have a molecular weight of at least 100 kDa.
Preferably, the soluble complex of Poly-[SFHb-SOD-CAT-CA] has a molecular weight from about 100 kDa to more than 450 kDa, and most preferably, the soluble complex of Poly-[SFHb-SOD-CAT-CA] has a molecular weight of more than 450 kDa. Table I below, shows the molecular distribution of Poly-[SFHb-SOD-CAT-CA] and enzyme activities. Poly-[SFHb-SOD-CAT-CA] shows three molecular components: (1) low (<100 kDa), (3) intermediate (100-450 kDa), and (3) high molecular weight (>450kDa). The sample contains about 86%
components with molecular weight higher than 100 kDa. The low molecular weight fraction (<100 kDa) is discarded. Most of the Hb, SOD, CAT and CA
activity (70%, 82%, 90%, and 84% respectively) are in the Poly-[SFHb-SOD-CAT-CA] fraction with a molecular weight of greater than 450 kDa. The fraction with molecular weight between 100 kDa and 450 kDa contains Hb, SOD, CAT
and CA activities of 16%, 11%, 9%, and 13% respectively. In the fraction with molecular weight of less than 100 kDa the activities of SOD, CAT and CA
activities are even lower (14%, 5%, 0.5%, and 0.05%) ¨ this fraction contains the uncrosslinked Hb and enzymes and is discarded.
Table I. Molecular Distribution of PolySFHb-SOD-CAT-CA and enzyme activities Molecular MW Component SOD Activity CAT Activity CA Activity Weight (%) (%) (%) (%) >450 kDa 70 82 + 3 90 1 84 2 100-450 kDa 16 11 1 9 1 13 2 <100kDa 14 5 1 0.5 10. 05 3 1 File No. P1754CA00 [0059] According to another embodiment of the present invention, there are disclosed methods of treating medical conditions by administering a therapeutically effective amount of a soluble complex according to the present invention.
[0060] According to some embodiment the medical condition may be blood loss, conditions with accumulation of carbon dioxide, ischemia-reperfusion injuries, and diver's diseases.
[0061] The ischemia-reperfusion injury may comprise a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
[0062] According to another embodiment, there are disclosed methods of removing carbon dioxide from a fluid with an effective amount of a soluble complex according to the present invention, or with an artificial blood substitute according to the present invention.
[0063] The fluid may be for example blood and air, but could be any fluid from which carbon dioxide may be advantageously be removed. For example, the fluid may be blood in a dialysis or in artificial lung. According to another embodiment, the fluid may be air undergoing environmental removal of 002.
[0064] According to another embodiment of the present invention, there is disclosed a method of preparing a soluble complex according to the present invention. The method comprises the steps of cross-linking a mixture which comprises stroma-free hemoglobin, antioxidant enzymes, an acid-base and carbon dioxide transport enzyme, and lysine.
[0065] The molar ratio lysine to the stroma-free hemoglobin may be from about 7: 1 to about 12 : 1, or from about 8:1 to about 12:1, or from about 9:1 to about 12:1, or from about 10:1 to about 12:1 or from about 11:1 to about 12:1, or from about 7:1 to about 8:1, or from about 7:1 to about 9:1, or from about 7:1 to about 10:1, or from about 7:1 to about 11:1, or from about 8:1 to about 9:1, or from about 8:1 to about 10:1, or from about 8:1 to about 11:1, or from about 8:1 File No. P1754CA00 to about 12:1, or from about 9:1 to about 10:1, or from about 9:1 to about 11:1, or from about 9:1 to about 12:1, or from about 10:1 to about 11:1, or from about 11:
to about 12:1. Preferably, the molar ratio of lysine to the stroma-free hemoglobin is 7:1.
[0066] According to an embodiment, the cross-linking is performed with glutaraldehyde. The molar ratio of glutaraldehyde to the stroma-free hemoglobin may be from about 8:1 to about 32:1, or from about 8:1 to about 12:1, or from about 8:1 to about 16:1, or from about 8:1 to about 20:1, or from about 8:1 to about 24:1, or from about 8:1 to about 28:1, or from about 12:1 to about 16:1, or from about 12:1 to about 20:1, or from about 12:1 to about 24:1, or from about 12:1 to about 28:1, or from about 12:1 to about 32:1, or from about 16:1 to about 20:1, or from about 16:1 to about 24:1, or from about 16:1 to about 28:1, or from about 16:1 to about 32:1, or from about 20:1 to about 24:1, or from about 20:1 to about 28:1, or from about 20:1 to about 32:1, or from about 24:1 to about 28:1, or from about 24:1 to about 32:1, ,or from about 28: to about 32:1. The molar ratio of glutaraldehyde to the stroma-free hemoglobin will be varied depending on the length of the incubation for cross linking. Preferably, the molar ratio will be 16:1, and the incubation period may be for example about 24h at 4 C. The reaction may be stoped by addition of an excess lysine (e.g. 200:1) relative to the stroma-free hemoglobin. The mixture may be subsequently purified, dialyzed and concentrated to obtain the soluble complex.
[0067] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
File No. P1754CA00 Preparation of soluble complexes of Poly-[SFHb-SOD-CAT-CA]
MATERIALS
[0068] The Hb used is Stroma Free Hb extracted from red blood cells.
SOD, CAT and CA (EC 4.2.1.1, 2720 units/mg solid manufacturer's stated activity) were purchased from Sigma-Aldrich (Ontario, Canada). All other chemicals or reagents of analytical grade were purchased from Sigma-Aldrich.
METHOD
[0069] SOD (1050 units/mL), catalase (21,000 units/mL) and carbonic anhydrase (1070 units/mL) are added to the 20 mL solution containing stoma-free hemoglobin (7 g/dI) in 50mM sodium phosphate buffer (pH 7.4). Before the initiation of the crosslinking reaction, 1.3 M lysine is added at a molar ratio of 7:1 lysine/Hb. Nitrogen gas is used to flush the reaction vessel to prevent formation of methemoglobin. Crosslinking is started with the addition of 5%
glutaraldehyde at a molar ratio of 16:1 glutaraldehyde/Hb. Glutaraldehyde is added in four equal aliquots over a period of 15 minutes, and the reaction is allowed to crosslink for 24 hours at 4 C with constant stirring under aerobic conditions. Crosslinking is stopped by adding 2.0 M lysine at a molar ratio of 200:1 lysine/Hb. The preparation is then filtered using a sterile 0.45 pM filter, and subsequently dialyzed overnight using molecular porous dialysis membrane (MWCO: 12 000-14 000) against Ringer's lactated solution. 100 kDa microconcentrators (Amicon, Beverly, MA) are used to concentrate 500 pL aliquots of the crosslinked solution by centrifuging at 2500 g for 55 minutes at 23 C.
Molecular Weight Distribution [0070] For the analysis of molecular weight distribution of polyHb-SOD-CAT-CA a Sephacry1-300 HRTColumn is used (Vtotai=560 ml) at a flow rate of 36 File No. P1754CA00 ml/hour. This column is equilibrated with 0.1M Tris-HCI and 0.15M NaCl (pH
7.4) elution buffer. The molecular weight distribution is recorded by a 280nm UV
detector at a recording velocity of 1mm/min. Fractions collected are: (1) molecular weights higher than 450kDa, (2) 100-450kDa and (3) less than 100kDa. For animal studies, the less than 100 kDa fraction are discarded. Dialysis membrane (MWCO:
12 000-14 000) and Spectra/gel absorbent are used to concentrate the sample and the hemoglobin concentration are determined by Drabkin's method. The aliquoted samples are stored at -80 C.
Quantitative Determination of Hb Concentration [0071) The Hb concentration in the polySFHb and polySFHb-CA
preparations is colorimetrically determined by reacting the samples with Drabkin's reagent (Sigma-Aldrich), then measuring the concentration of the resulting cyanmethemoglobin solution by spectrophotometry at 540 nm.
Determination of CA Activity (0072) The hydration activity of carbon dioxide by CA is determined by an electrometric delta pH assay based on the methods of Henry (14), and of Wilbur and Anderson (15). One Wilbur- Anderson (W-A) unit of CA activity is defined as the amount of enzyme that causes the pH of a 0.02 M Tris buffer to drop from pH
8.3 to 6.3 per minute. The reagent solution consisted of test samples containing CA, and 0.02 M Tris. HCl buffer (pH 8.0) kept between 0-4 C before use.
Dissolved CO2 is prepared by bubbling CO2 through distilled water to obtain the substrate for the assay. The reaction is initiated by the addition of substrate, and the time (T) needed for the pH of the reaction mixture to drop from pH 8.3 to 6.3 is recorded. A
Fisher AccumetTM Basic pH meter (Fisher Scientific, Pittsburgh, PA) with MI-407 (P) Needle pH electrode (Microelectrodes Inc., Bedford, NH) is used to measure the change in pH caused by the hydration reaction of CO2 catalyzed File No. P1754CA00 by CA. The control (To) for the assay consists of the same mixture without the test sample. The measurements in seconds are converted into W-A units according to the following formula:
1 W-A unit = [2 x (To ¨ T)]/T.
[0073] The units are then plotted versus the Hb/CA concentration (mg/mL).
Determination of CAT Activity [0074] For catalase measurement, a UV 240nm spectrophotometer is used to measure the rate of disappearance of H202. The reaction mixture contains 2mL of 50mM phosphate buffer, pH 7.0, and 1mL of 30mM H202. The decrease of the H202 level is monitored at 240nm for 15s. The same concentrations of blood samples or phosphate buffer mixtures without H202 are applied as blank. The catalase activity is expressed in units per grams of haemoglobin.
Determination of SOD Activity [0075] The measurement for SOD activity is based on the reduction of cytochrome C by superoxide. The reagent solution consists of xantine (50 pM), cytochrome C (10pM) and CAT (500 units/ mL) in 50 mL potassium phosphate buffer, 0.1 mM EDTA, pH 7.8. The reaction system consists of test samples and reagent solution. Xanthine oxidase is added to start the reaction. 0.154 M
NaCI is used as blank. The cytochrome C reduction is recorded at 550nm by a spectrophotometer.
Enzyme Activity of SFHb, polySFHb and soluble complex File No. P1754CA00 of Poly-[SFHb-SOD-CAT-CA]
[0076] Now referring to Figs. 1-3. PolySFHb-SOD-CAT-CA was formed by nanobiotechnology using glutaraldehyde as crosslink reagent. The enzymes activities of (1) SFHb, (2) PolySFHb, (3) PolySFHb+SOD+CAT+CA (SOD, CAT
and CA added in solution to PolySFHb) and (4) Poly-[SFHb-SOD-CAT-CA]
(PolySFHb crosslinked with SOD, CAT and CA) are assessed. To eliminate errors, three batches of polySFHb and Poly-[SFHb-SOD-CAT-CA] are prepared and tested separately. Crosslinking reactions significantly decreased the SOD, CAT and CA enzyme activities. Thus, PolySFHb alone did not have the same enzyme activities as RBC. The amount of SOD and CAT used in the crosslinking are much higher than those found in the RBC. This is required in order to have a therapeutic level of SOD and CAT for ischemia-reperfusion injuries. CA (1070 units/mL) also has to be added in the crosslink of PolySFHb and CA to emulate the CO2 hydration activity of SFHb in RBC. Enrichment of enzymes highly improves the activities of SOD, CAT and CA in Poly-[SFHb-SOD-CAT-CA]. An optimal Poly-[SFHb-SOD-CAT-CA] with the following addition of enzymes requires a Hb: SOD: CAT: CA ratio of 1g: 18,000:310,000:130,000U.
Animal Study [0077] Increase in tissue CO2 in hemorrhagic shock is correlated with poor recovery in patients. Thus, the effectiveness of the novel approach in lowering the elevated CO2 in hemorrhagic shock in analyzed in rats.
[0078] Study design [0079] Rats of average body weight of 300 grams. 6 groups of rats with 3 in each group. Phenobarbital Anesthesia. Cannulate femoral artery and vein on left side. The mean blood pressure (mmHg) and tissue CO2 tension (mmHg) are continuously recorded. The animals are bled 50% of their total blood volume. A
File No. P1754CA00 mean blood pressure of 30 mmHg for is maintained for 1 hour. Then each rat receives one of the following intravenous infusions.
[0080] Group 1: Saline at 3 times the volume of lost blood;
[0081] Group 2: Red blood cells (rbc 15gm/dI): reinfuse the shed loss of blood (half the total blood volume);
[0082] Group 3: PHcsc: (5 gm/di of polyhemoglobin-csc prepared by crosslinking stroma-free hemoglobin;
[0083] Group 4: PHCSC: (5 gm/di prepared by crosslinking stroma-free hemoglobin PLUS the addition of more enzymes at the concentration stated above: C = catalase; S= superoxide dismutase and C= carbonic anhydrase.
Specifically, SOD (1050 units/mL), catalase (21,000 units/mL), and carbonic anhydrase (1070 units/mL) are added to stroma-free hemoglobin (7 g/dI), then polymerized into PolySFHb-SOD-CAT-CA resulting in HB:SOD ratio of 1g: 8,000 U; Hb:CAT ratio of 1g:310,000 U after crosslinking and lib:CA ratio of 1g:130,000 U after crosslinking.
[0084] Group 5: PHcsc: (10 gm/dl prepared as in Group 3 by crosslinking stroma-free hemoglobin. Then concentrating the preparation to 10 gm/di [0085] Group 6: PHCSC: (10 gm/di prepared as in group 4 but ,then concentrating the preparation to 10gm/d1, [0086] Results [0087] Group 1: Saline at 3 times the volume of lost blood is the least effective since CO2 even increases after infusion of saline.
[0088] Group 4: 5 gm/di of the Polyhemoglobin-Catalase-Superoxidedismutase-carbonic anhydrase (PHCSC) is slightly more effective than triple the amount of red blood cells (15gm/dI).
File No. P1754CA00 [0089] Group 6: 10 gm/dl of Polyhemoglobin-Catalase-Superoxidedismutase-carbonic anhydrase (PHCSC) as shown in the lowest line is much more effective than the larger amount, 15gm/d1, of red blood cells.
[0090] Conclusion [0091] This novel approach results in a preparation that is much more effective than red blood cells in lowering the elevated tissue CO2 level.
Elevated CO2 level in hemorrhagic shock if not lowered can lead to poor recovery from shock.
[0092] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure.
Such modifications are considered as possible variants comprised in the scope of the disclosure.
= [0005] (2) Unlike RBC, polyHb can be sterilized and therefore do not have the potential of infective agents in case of epidemics (earlier examples of AIDS
and the resulting HIV contaminated donor blood can now be avoided in future epidemics of other types of infective agents), [0006] (3) Donor blood has to be stored at 4 C and is only good for about 42 days. PolyHb can be stored at room temperature for more than a year. This allows for PolyHb in plastic bags to be kept anywhere that has potential need for this product for at least 1 year and longer if kept in lower temperature.
[0007] However, polyHb only has only one of the three important functions of RBC. The three important functions of RBC are: (1) transport and supply oxygen, (2) transport and remove carbon dioxide, and (3) remove harmful oxygen radicals. At present, industrial RBC substitutes in the form of polyHb only transport oxygen, and they were not approved by the US FDA because of side effects. Furthermore, the antioxidant enzyme activities of SOD and CAT of RBC
extracts are not sufficient to prevent ischemia-reperfusion injury in severe hemorrhagic shock, stroke, myocardial infarction and other conditions. SOD and CAT have been complexed with polyHb to form polyHb-SOD-CAT (US patent File No. P1754CA00 No. 5,606,025 to D'Agnillo and Chang. However, this complex still lacks one of the important function of RBC in that it does not transport and remove carbon dioxide from blood and will therefore cause side effects. Therefore, there is a need for more complete RBC substitutes with all 3 functions of RBC to treat blood loss.
[0008] Furthermore, there is a need for more complete RBC substitutes to prevent ischemia-reperfusion injuries.
SUMMARY
[0009] According to an embodiment, there is provided a soluble complex comprising hemoglobin cross-linked with an antioxidant enzyme for reduction of reactive oxygen species and an acid-base and carbon dioxide transport enzyme for CO2 and 02 transport.
[0010] The antioxidant enzyme may be chosen from super oxide dismutase (SOD), catalase (CAT), or combinations thereof.
[0011] The antioxidant enzyme may be a combination of super oxide dismutase (SOD) and catalase (CAT).
[0012] The ratio of hemoglobin to super oxide dismutase (SOD) may be from about 1 g : 4 000 U to about 1 g : 25 000 U.
[0013] The ratio of hemoglobin to super oxide dismutase (SOD) may be about 1 g : 18 000 U.
[0014] The ratio of hemoglobin to catalase (CAT) may be from about 1 g:
25,000 U to about 1 g : 310,000 U.
[0015] The ratio of hemoglobin to catalase (CAT) may be about 1 g :
310 000 U.
[0016] The acid-base and carbon dioxide transport enzyme may comprise carbonic anhydrase (CA).
File No. P1754CA00 [0017] The ratio of hemoglobin to carbonic anhydrase (CA) may be from about 1 g : 80 000 U to about 1 g : 250 000 U.
[0018] The ratio of hemoglobin to carbonic anhydrase (CA) may be about 1 g : 130 000 U.
[0019] The hemoglobin may comprise stroma free hemoglobin (SFHb) or hemoglobin (Hb).
[0020] The soluble complex may have a molecular weight of more than 100 kDa.
[0021] The soluble complex may have a molecular weight from about more than 100 kDa to more than 450 kDa.
[0022] According to another embodiment, there is provided an artificial blood substitute comprising a soluble complex according to the present invention, in a suitable pharmaceutical fluid.
[0023] According to another embodiment, there is provided a method of treating a medical condition comprising administering a therapeutically effective amount of a soluble complex according to the present invention, or an artificial blood substitute according to the present invention to a patient in need thereof.
[0024] The medical condition may be chosen from a blood loss, a condition with accumulation of carbon dioxide, an ischemia-reperfusion injury, and a diver's disease.
[0025] The ischemia-reperfusion injury may comprise a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
[0026] According to another embodiment, there is provided a method for removing carbon dioxide from a fluid comprising treating the fluid with an effective amount of a soluble complex according to the present invention, or an artificial blood substitute according to the present invention.
File No. P1754CA00 [0027] The fluid may be chosen from blood and air. The fluid may be blood in a dialysis. The fluid may be blood in an artificial lung. The fluid may be air undergoing environmental removal of CO2.
[0028] According to another embodiment, there is provided a use of a soluble complex according to the present invention for the preparation of a medicament for the treatment of a medical condition.
[0029] According to another embodiment, there is provided a use of a soluble complex according to the present invention for the treatment of a medical condition.
[0030] According to another embodiment, there is provided a use of an artificial blood substitute according to the present invention for the preparation of a medicament for the treatment of a medical condition.
[0031] According to another embodiment, there is provided a use of an artificial blood substitute according to the present invention for the treatment of a medical condition.
[0032] The condition may be chosen from a blood loss, a condition with accumulation of carbon dioxide, an ischemia-reperfusion injury, a diver's disease.
[0033] The ischemia-reperfusion injury may comprise a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
[0034] According to another embodiment, there is provided a use of a soluble complex according to the present invention or an artificial blood substitute according to the present invention for treating a fluid for removing carbon dioxide therefrom.
[0035] The fluid may be chosen from blood and air. The fluid may be blood in an artificial lung. The fluid may be blood in a dialysis.
File No. P1754CA00 [0036] According to another embodiment, there is provided a method of preparing a soluble complex comprising the steps of:
a) cross-linking a mixture comprising = a stroma-free hemoglobin or hemoglobin;
= at least one antioxidant enzyme;
= an acid-base and carbon dioxide transport enzyme, and = lysine b) purifying and concentrating said mixture to obtain said soluble complex.
[0037] The molar ratio of lysine to the stroma-free hemoglobin may be from about 7 : 1 to about 12 : 1. Preferably the molar ratio of lysine to the stroma-free hemoglobin may be 7:1.
[0038] The step a) may be performed with glutaraldehyde.
[0039] The molar ratio of glutaraldehyde to stroma-free hemoglobin may be from about 8:1 to about 32:1. Preferably, the molar ratio of glutaraldehyde to the stroma-free hemoglobin may be 16:1.
[0040] The step of cross-linking may be performed for 24h at 4 C.
[0041] The step of cross-linking may be stopped with lysine at molar ratio to the stroma-free hemoglobin of 200:1.
[0042] The step of purifying and concentrating may be performed by filtering the mixture, dialyzing the mixture, microconcentrating the mixture, or combinations thereof.
[0043] The following terms are defined below.
[0044] The term "pharmaceutically acceptable fluid" is intended to mean a preservative solution, a saline solution, an isotonic (about 0.9%) saline solution, or about a 5% albumin solution, suspension, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic File No. P1754CA00 substances suitable for delivery to a patient may be included in the compositions of the present invention. The compositions may be solutions, suspensions or any appropriate formulation suitable for administration, and are typically sterile and free of undesirable particulate matter. The compositions may be sterilized by conventional sterilization techniques.
[0045] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
[0047] Fig. 1 illustrates the carbonic anhydrase activity (CA): (1) SFHb ¨
content of red blood cells (SFHb) with CA activity; (2) PolySFHb ¨ content of red blood cells crosslinked into polySFHb with very low CA activity;(3) SFHb + SOD
+ CAT + CA ¨ enzymes added to extracts of red blood cell content without crosslinking, and (4) Poly-[Hb-SOD-CAT-CA] represent the previous components crosslinked into Poly-[Hb-SOD-CAT-CA] according to an embodiment of the present invention with resulting good CA activity. SFHb: stroma free hemoglobin;
PolySFHb: Crosslinked SFHb; SFHb+SOD+CAT+CA: add 15000 U SOD, 300,000 U CAT and 15,300 U CA to each gram of SFHb; Poly-[SFHb-SOD-CAT-CA] : crosslinked SFHb+SOD+CAT+CA.
[0048] Fig. 2 illustrates the superoxide dismutase (SOD): SFHb ¨ content of red blood cells (SFHb) with very low SOD activity; PolySFHb ¨ content of red File No. P1754CA00 blood cells crosslinked into polySFHb with very low SOD activity; SFHb + SOD +
CAT + CA ¨ enzymes added to extracts of red blood cell content without crosslinking, and Poly-[SFHb-SOD-CAT-CA] represent the previous components crosslinked into Poly-[SFHb-SOD-CAT-CA] according to an embodiment of the present invention with resulting greatly enhanced SOD activity. SFHb: stroma free hemoglobin; PolySFHb: Crosslinked SFHb; SFHb+SOD+CAT+CA : Add 1500 U SOD, 300,000 U CAT and 15,300 U CA to each gram of SFHb; Poly-[SFHb-SOD-CAT-CA]: crosslinked SFHb+SOD+CAT+CA.
[0049] Fig. 3 illustrates the catalase activity (CAT): SFHb ¨ content of red blood cells (SFHb) with very low SOD activity; PolySFHb ¨ content of red blood cells crosslinked into polySFHb with very low SOD activity; SFHb + SOD + CAT
+ CA ¨ enzymes added to extracts of red blood cell content without crosslinking, and Poly-[SFHb-SOD-CAT-CA] represent the previous components crosslinked into Poly-[SFHb-SOD-CAT-CA] according to an embodiment of the present invention with resulting markedly enhanced CAT activity. SFHb: stroma free hemoglobin; PolySFHb: Crosslinked SFHb; SFHb+SOD+CAT+CA : Add 1500 U
SOD, 300,000 U CAT and 15,300 U CA to each gram of SFHb; Poly-[SFHb-SOD-CAT-CA] : crosslinked SFHb+SOD+CAT+CA.
[0050] Fig. 4 illustrates an in vivo trial of the soluble complex according to an embodiment of the present invention. RBC: whole blood, PHcsc PolyHb with low enzyme, PHCSC: PolyHb with high enzyme = PolyHbCAT-SOD-CA.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] In embodiments there is disclosed a soluble complex formed by crosslinking hemoglobin with antioxidant enzyme and acid-base and carbon dioxide enzyme. Extracts from red blood cell contains hemoglobin contain very low antioxidant enzymes activity (e.g. SOD and CAT) and an acid-base and carbon dioxide transport activity (CA). However, when direct cross-linking of these enzymes is performed, it significantly decreases the SOD, CAT and CA
enzyme activity to levels much below the optimal levels. Furthermore, the File No. P1754CA00 antioxidant enzyme (e.g. of SOD and CAT) present in red blood cell extracts are insufficient to prevent ischemia-reperfusion injuries that occur in severe hemorrhagic shock, stroke, myocardial infarction and other conditions.
Furthermore, after crosslinking, the CA level is no longer sufficient for acid-base and carbon dioxide transport functions, especially in severe ischemia.
[0052] Therefore, according to an embodiment of the present invention, the soluble complex (e.g. Poly-[SFHb-SOD-CAT-CA]) of the present invention comprises the three functions of RBC: It is an oxygen carrier (PolySFHb) with antioxidant activity (SOD, CAT) and also the ability to facilitate the transport and acid-base functions (CA activity). It also comprises enhanced SOD, CAT and CA
activities to enhance the function of this novel red blood cell substitute.
The SOD
and CAT levels in this Poly-[SFHb-SOD-CAT-CA] cross-linked polyhemoglobin are much higher than those in RBC. Therefore, it is believed that it may also be used as a therapeutic agent for the treatment or prevention of ischemia-reperfusion injuries and to facilitate the removal of accumulated carbon dioxide in the case of severe hemorrhagic shock and other conditions.
[0053] According to an embodiment of the present invention, the hemoglobin component of the soluble complex acts as an oxygen carrier. The hemoglobin component of the soluble complex may be prepared from any suitable hemoglobin extracted from red blood cells, from human or animals (bovine, Porcine etc) or recombinant Hb or other types of organisms and even synthetic hemoglobin.
[0054] The soluble complex of the present invention also includes a therapeutically effective amount of an antioxidant enzyme. This antioxidant enzyme may be any suitable antioxidant enzymes. The antioxidant enzyme may be either super oxide dismutase (SOD), catalase (CAT), or combinations thereof.
Preferably, the antioxidant enzyme is a combination of super oxide dismutase (SOD) and catalase (CAT). These enzymes may be from sources exogenous from the hemoglobin. The SOD enzyme may be present in a ratio with the File No. P1754CA00 hemoglobin component corresponding to from about 1g of hemoglobin to 4 000 U to 25 000 U of SOD. Preferably, the optimal ratio of the-hemoglobin to the super oxide dismutase (SOD) is about 1 g :18 000 U. The CAT enzyme may be present in a ratio with the hemoglobin corresponding from about 1g of hemoglobin to 25 000 U to 310,000 U of CAT. Preferably, the ratio of hemoglobin preparation to said catalase (CAT) is about 1 g : 310 000 U.
[0055] The soluble complex of the present invention also includes a therapeutically effective amount of an acid-base and carbon dioxide transport enzyme. Preferably, the acid-base and carbon dioxide transport enzyme comprises carbonic anhydrase (CA), but synthetic enzymes (e.g. synthetic enzyme bearing the active sites necessary to provide CA activity) with the same activity may also be used. The CA enzyme may be from an exogenous source.
The CA enzyme may be present in a ratio with the hemoglobin component corresponding from about 1g of hemoglobin to 80 000 U to 250 000 U of CA.
Preferably, the ratio of hemoglobin to said carbonic anhydrase (CA) is about 1 g:
130 000 U.
[0056] The components of the soluble complex are chemically bonded because hemoglobin and the enzymes, when in the free form, are rapidly removed after infusion and they may also cause harmful effects. Especially sensitivity, allergic and immunological types of reactions. Thus, according to one embodiment of the present invention, the soluble complex may be Poly-[SFHb-SOD-CAT-CA], where the SFHb, SOD, CAT and CA are cross-linked to each other.
[0057] The SOD and CAT activity of the present invention is much higher than that normally present in the RBC and can therefore enhance the antioxidant activity. This enhanced ability to prevent the harmful effects of oxygen radicals is important in ischemia-reperfusion encountered in severe hemorrhagic shock, stroke, myocardial infarction. The CA activity is important because in any File No. P1754CA00 hemorrhagic shock or ischemic condition, there is also accumulation of carbon dioxide that also needs to be removed.
[0058] According to another embodiment, the soluble complex of Poly-[SFHb-SOD-CAT-CA] may have a molecular weight of at least 100 kDa.
Preferably, the soluble complex of Poly-[SFHb-SOD-CAT-CA] has a molecular weight from about 100 kDa to more than 450 kDa, and most preferably, the soluble complex of Poly-[SFHb-SOD-CAT-CA] has a molecular weight of more than 450 kDa. Table I below, shows the molecular distribution of Poly-[SFHb-SOD-CAT-CA] and enzyme activities. Poly-[SFHb-SOD-CAT-CA] shows three molecular components: (1) low (<100 kDa), (3) intermediate (100-450 kDa), and (3) high molecular weight (>450kDa). The sample contains about 86%
components with molecular weight higher than 100 kDa. The low molecular weight fraction (<100 kDa) is discarded. Most of the Hb, SOD, CAT and CA
activity (70%, 82%, 90%, and 84% respectively) are in the Poly-[SFHb-SOD-CAT-CA] fraction with a molecular weight of greater than 450 kDa. The fraction with molecular weight between 100 kDa and 450 kDa contains Hb, SOD, CAT
and CA activities of 16%, 11%, 9%, and 13% respectively. In the fraction with molecular weight of less than 100 kDa the activities of SOD, CAT and CA
activities are even lower (14%, 5%, 0.5%, and 0.05%) ¨ this fraction contains the uncrosslinked Hb and enzymes and is discarded.
Table I. Molecular Distribution of PolySFHb-SOD-CAT-CA and enzyme activities Molecular MW Component SOD Activity CAT Activity CA Activity Weight (%) (%) (%) (%) >450 kDa 70 82 + 3 90 1 84 2 100-450 kDa 16 11 1 9 1 13 2 <100kDa 14 5 1 0.5 10. 05 3 1 File No. P1754CA00 [0059] According to another embodiment of the present invention, there are disclosed methods of treating medical conditions by administering a therapeutically effective amount of a soluble complex according to the present invention.
[0060] According to some embodiment the medical condition may be blood loss, conditions with accumulation of carbon dioxide, ischemia-reperfusion injuries, and diver's diseases.
[0061] The ischemia-reperfusion injury may comprise a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
[0062] According to another embodiment, there are disclosed methods of removing carbon dioxide from a fluid with an effective amount of a soluble complex according to the present invention, or with an artificial blood substitute according to the present invention.
[0063] The fluid may be for example blood and air, but could be any fluid from which carbon dioxide may be advantageously be removed. For example, the fluid may be blood in a dialysis or in artificial lung. According to another embodiment, the fluid may be air undergoing environmental removal of 002.
[0064] According to another embodiment of the present invention, there is disclosed a method of preparing a soluble complex according to the present invention. The method comprises the steps of cross-linking a mixture which comprises stroma-free hemoglobin, antioxidant enzymes, an acid-base and carbon dioxide transport enzyme, and lysine.
[0065] The molar ratio lysine to the stroma-free hemoglobin may be from about 7: 1 to about 12 : 1, or from about 8:1 to about 12:1, or from about 9:1 to about 12:1, or from about 10:1 to about 12:1 or from about 11:1 to about 12:1, or from about 7:1 to about 8:1, or from about 7:1 to about 9:1, or from about 7:1 to about 10:1, or from about 7:1 to about 11:1, or from about 8:1 to about 9:1, or from about 8:1 to about 10:1, or from about 8:1 to about 11:1, or from about 8:1 File No. P1754CA00 to about 12:1, or from about 9:1 to about 10:1, or from about 9:1 to about 11:1, or from about 9:1 to about 12:1, or from about 10:1 to about 11:1, or from about 11:
to about 12:1. Preferably, the molar ratio of lysine to the stroma-free hemoglobin is 7:1.
[0066] According to an embodiment, the cross-linking is performed with glutaraldehyde. The molar ratio of glutaraldehyde to the stroma-free hemoglobin may be from about 8:1 to about 32:1, or from about 8:1 to about 12:1, or from about 8:1 to about 16:1, or from about 8:1 to about 20:1, or from about 8:1 to about 24:1, or from about 8:1 to about 28:1, or from about 12:1 to about 16:1, or from about 12:1 to about 20:1, or from about 12:1 to about 24:1, or from about 12:1 to about 28:1, or from about 12:1 to about 32:1, or from about 16:1 to about 20:1, or from about 16:1 to about 24:1, or from about 16:1 to about 28:1, or from about 16:1 to about 32:1, or from about 20:1 to about 24:1, or from about 20:1 to about 28:1, or from about 20:1 to about 32:1, or from about 24:1 to about 28:1, or from about 24:1 to about 32:1, ,or from about 28: to about 32:1. The molar ratio of glutaraldehyde to the stroma-free hemoglobin will be varied depending on the length of the incubation for cross linking. Preferably, the molar ratio will be 16:1, and the incubation period may be for example about 24h at 4 C. The reaction may be stoped by addition of an excess lysine (e.g. 200:1) relative to the stroma-free hemoglobin. The mixture may be subsequently purified, dialyzed and concentrated to obtain the soluble complex.
[0067] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
File No. P1754CA00 Preparation of soluble complexes of Poly-[SFHb-SOD-CAT-CA]
MATERIALS
[0068] The Hb used is Stroma Free Hb extracted from red blood cells.
SOD, CAT and CA (EC 4.2.1.1, 2720 units/mg solid manufacturer's stated activity) were purchased from Sigma-Aldrich (Ontario, Canada). All other chemicals or reagents of analytical grade were purchased from Sigma-Aldrich.
METHOD
[0069] SOD (1050 units/mL), catalase (21,000 units/mL) and carbonic anhydrase (1070 units/mL) are added to the 20 mL solution containing stoma-free hemoglobin (7 g/dI) in 50mM sodium phosphate buffer (pH 7.4). Before the initiation of the crosslinking reaction, 1.3 M lysine is added at a molar ratio of 7:1 lysine/Hb. Nitrogen gas is used to flush the reaction vessel to prevent formation of methemoglobin. Crosslinking is started with the addition of 5%
glutaraldehyde at a molar ratio of 16:1 glutaraldehyde/Hb. Glutaraldehyde is added in four equal aliquots over a period of 15 minutes, and the reaction is allowed to crosslink for 24 hours at 4 C with constant stirring under aerobic conditions. Crosslinking is stopped by adding 2.0 M lysine at a molar ratio of 200:1 lysine/Hb. The preparation is then filtered using a sterile 0.45 pM filter, and subsequently dialyzed overnight using molecular porous dialysis membrane (MWCO: 12 000-14 000) against Ringer's lactated solution. 100 kDa microconcentrators (Amicon, Beverly, MA) are used to concentrate 500 pL aliquots of the crosslinked solution by centrifuging at 2500 g for 55 minutes at 23 C.
Molecular Weight Distribution [0070] For the analysis of molecular weight distribution of polyHb-SOD-CAT-CA a Sephacry1-300 HRTColumn is used (Vtotai=560 ml) at a flow rate of 36 File No. P1754CA00 ml/hour. This column is equilibrated with 0.1M Tris-HCI and 0.15M NaCl (pH
7.4) elution buffer. The molecular weight distribution is recorded by a 280nm UV
detector at a recording velocity of 1mm/min. Fractions collected are: (1) molecular weights higher than 450kDa, (2) 100-450kDa and (3) less than 100kDa. For animal studies, the less than 100 kDa fraction are discarded. Dialysis membrane (MWCO:
12 000-14 000) and Spectra/gel absorbent are used to concentrate the sample and the hemoglobin concentration are determined by Drabkin's method. The aliquoted samples are stored at -80 C.
Quantitative Determination of Hb Concentration [0071) The Hb concentration in the polySFHb and polySFHb-CA
preparations is colorimetrically determined by reacting the samples with Drabkin's reagent (Sigma-Aldrich), then measuring the concentration of the resulting cyanmethemoglobin solution by spectrophotometry at 540 nm.
Determination of CA Activity (0072) The hydration activity of carbon dioxide by CA is determined by an electrometric delta pH assay based on the methods of Henry (14), and of Wilbur and Anderson (15). One Wilbur- Anderson (W-A) unit of CA activity is defined as the amount of enzyme that causes the pH of a 0.02 M Tris buffer to drop from pH
8.3 to 6.3 per minute. The reagent solution consisted of test samples containing CA, and 0.02 M Tris. HCl buffer (pH 8.0) kept between 0-4 C before use.
Dissolved CO2 is prepared by bubbling CO2 through distilled water to obtain the substrate for the assay. The reaction is initiated by the addition of substrate, and the time (T) needed for the pH of the reaction mixture to drop from pH 8.3 to 6.3 is recorded. A
Fisher AccumetTM Basic pH meter (Fisher Scientific, Pittsburgh, PA) with MI-407 (P) Needle pH electrode (Microelectrodes Inc., Bedford, NH) is used to measure the change in pH caused by the hydration reaction of CO2 catalyzed File No. P1754CA00 by CA. The control (To) for the assay consists of the same mixture without the test sample. The measurements in seconds are converted into W-A units according to the following formula:
1 W-A unit = [2 x (To ¨ T)]/T.
[0073] The units are then plotted versus the Hb/CA concentration (mg/mL).
Determination of CAT Activity [0074] For catalase measurement, a UV 240nm spectrophotometer is used to measure the rate of disappearance of H202. The reaction mixture contains 2mL of 50mM phosphate buffer, pH 7.0, and 1mL of 30mM H202. The decrease of the H202 level is monitored at 240nm for 15s. The same concentrations of blood samples or phosphate buffer mixtures without H202 are applied as blank. The catalase activity is expressed in units per grams of haemoglobin.
Determination of SOD Activity [0075] The measurement for SOD activity is based on the reduction of cytochrome C by superoxide. The reagent solution consists of xantine (50 pM), cytochrome C (10pM) and CAT (500 units/ mL) in 50 mL potassium phosphate buffer, 0.1 mM EDTA, pH 7.8. The reaction system consists of test samples and reagent solution. Xanthine oxidase is added to start the reaction. 0.154 M
NaCI is used as blank. The cytochrome C reduction is recorded at 550nm by a spectrophotometer.
Enzyme Activity of SFHb, polySFHb and soluble complex File No. P1754CA00 of Poly-[SFHb-SOD-CAT-CA]
[0076] Now referring to Figs. 1-3. PolySFHb-SOD-CAT-CA was formed by nanobiotechnology using glutaraldehyde as crosslink reagent. The enzymes activities of (1) SFHb, (2) PolySFHb, (3) PolySFHb+SOD+CAT+CA (SOD, CAT
and CA added in solution to PolySFHb) and (4) Poly-[SFHb-SOD-CAT-CA]
(PolySFHb crosslinked with SOD, CAT and CA) are assessed. To eliminate errors, three batches of polySFHb and Poly-[SFHb-SOD-CAT-CA] are prepared and tested separately. Crosslinking reactions significantly decreased the SOD, CAT and CA enzyme activities. Thus, PolySFHb alone did not have the same enzyme activities as RBC. The amount of SOD and CAT used in the crosslinking are much higher than those found in the RBC. This is required in order to have a therapeutic level of SOD and CAT for ischemia-reperfusion injuries. CA (1070 units/mL) also has to be added in the crosslink of PolySFHb and CA to emulate the CO2 hydration activity of SFHb in RBC. Enrichment of enzymes highly improves the activities of SOD, CAT and CA in Poly-[SFHb-SOD-CAT-CA]. An optimal Poly-[SFHb-SOD-CAT-CA] with the following addition of enzymes requires a Hb: SOD: CAT: CA ratio of 1g: 18,000:310,000:130,000U.
Animal Study [0077] Increase in tissue CO2 in hemorrhagic shock is correlated with poor recovery in patients. Thus, the effectiveness of the novel approach in lowering the elevated CO2 in hemorrhagic shock in analyzed in rats.
[0078] Study design [0079] Rats of average body weight of 300 grams. 6 groups of rats with 3 in each group. Phenobarbital Anesthesia. Cannulate femoral artery and vein on left side. The mean blood pressure (mmHg) and tissue CO2 tension (mmHg) are continuously recorded. The animals are bled 50% of their total blood volume. A
File No. P1754CA00 mean blood pressure of 30 mmHg for is maintained for 1 hour. Then each rat receives one of the following intravenous infusions.
[0080] Group 1: Saline at 3 times the volume of lost blood;
[0081] Group 2: Red blood cells (rbc 15gm/dI): reinfuse the shed loss of blood (half the total blood volume);
[0082] Group 3: PHcsc: (5 gm/di of polyhemoglobin-csc prepared by crosslinking stroma-free hemoglobin;
[0083] Group 4: PHCSC: (5 gm/di prepared by crosslinking stroma-free hemoglobin PLUS the addition of more enzymes at the concentration stated above: C = catalase; S= superoxide dismutase and C= carbonic anhydrase.
Specifically, SOD (1050 units/mL), catalase (21,000 units/mL), and carbonic anhydrase (1070 units/mL) are added to stroma-free hemoglobin (7 g/dI), then polymerized into PolySFHb-SOD-CAT-CA resulting in HB:SOD ratio of 1g: 8,000 U; Hb:CAT ratio of 1g:310,000 U after crosslinking and lib:CA ratio of 1g:130,000 U after crosslinking.
[0084] Group 5: PHcsc: (10 gm/dl prepared as in Group 3 by crosslinking stroma-free hemoglobin. Then concentrating the preparation to 10 gm/di [0085] Group 6: PHCSC: (10 gm/di prepared as in group 4 but ,then concentrating the preparation to 10gm/d1, [0086] Results [0087] Group 1: Saline at 3 times the volume of lost blood is the least effective since CO2 even increases after infusion of saline.
[0088] Group 4: 5 gm/di of the Polyhemoglobin-Catalase-Superoxidedismutase-carbonic anhydrase (PHCSC) is slightly more effective than triple the amount of red blood cells (15gm/dI).
File No. P1754CA00 [0089] Group 6: 10 gm/dl of Polyhemoglobin-Catalase-Superoxidedismutase-carbonic anhydrase (PHCSC) as shown in the lowest line is much more effective than the larger amount, 15gm/d1, of red blood cells.
[0090] Conclusion [0091] This novel approach results in a preparation that is much more effective than red blood cells in lowering the elevated tissue CO2 level.
Elevated CO2 level in hemorrhagic shock if not lowered can lead to poor recovery from shock.
[0092] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure.
Such modifications are considered as possible variants comprised in the scope of the disclosure.
Claims (30)
1. A soluble complex comprising ( i ) hemoglobin for 02 transport, (ii) antioxidant enzymes for reduction of reactive oxygen species, and (ii) a carbonic anhydrase enzyme for blood acid-base regulation and blood CO2 transport, wherein the hemoglobin is cross-linked to (ii) the antioxidant enzymes for reduction of reactive oxygen species and (iii) the carbonic anhydrase enzyme, wherein the soluble complex removes CO2 from tissue and blood.
2. The soluble complex according to claim 1, wherein said antioxidant enzymes are chosen from the group consisting of super oxide dismutase (SOD), catalase (CAT), and combinations thereof.
3. The soluble complex according to claim 2, wherein said antioxidant enzymes are a combination of super oxide dismutase (SOD) and catalase (CAT).
4. The soluble complex according to any one of claims 2 - 3, wherein the ratio of hemoglobin to said super oxide dismutase (SOD) is from about 1 g :
4,000 U to about 1 g : 25,000 U.
4,000 U to about 1 g : 25,000 U.
5. The soluble complex according to any one of claims 2 - 3, wherein the ratio of hemoglobin to said super oxide dismutase (SOD) is about 1 g : 18,000 U.
6. The soluble complex according to any one of claims 2 - 3, wherein the ratio of hemoglobin to said catalase (CAT) is from about 1 g : 25,000 U to about 1 g : 310,000 U.
Date Recue/Date Received 2021-05-26
Date Recue/Date Received 2021-05-26
7. The soluble complex according to any one of claims 2 - 3, wherein the ratio of hemoglobin to said catalase (CAT) is about 1 g : 310,000 U.
8. The soluble complex according to claim 7, wherein the ratio of hemoglobin to said carbonic anhydrase (CA) is from about 1 g : 80,000 U
to about lg : 250,000 U.
to about lg : 250,000 U.
9. The soluble complex according to claim 7, wherein the ratio of hemoglobin to said carbonic anhydrase (CA) is about 1 g : 130,000 U.
10. The soluble complex according to any one of claims 1 - 9, wherein said hemoglobin comprises stoma free hemoglobin (SFHb).
11. The soluble complex according to any one of claims 1 - 10, wherein said soluble complex has a molecular weight of more than 100 kDa.
12. An artificial blood substitute comprising a soluble complex according to any one of claims 1 - 11 in a pharmaceutically acceptable carrier.
13. The use of a soluble complex according to any one of claims 1 - 11 for the preparation of a medicament for the treatment of a blood loss, a hemorrhagic shock, an ischemic condition, o r an ischemia-reperfusion injury.
14. The use of a soluble complex according to any one of claims 1 - 11 for the treatment of a blood loss, a hemorrhagic shock, an ischemic condition, or an ischemia-reperfusion injury.
15. The use of an artificial blood substitute according to claim 12 for the preparation of a medicament for the treatment of a blood loss, a Date Recue/Date Received 2021-05-26 hemorrhagic shock, an ischemic condition, o r an ischemia-reperfusion injury.
16. The use of an artificial blood substitute according to claim 12 for the treatment of a blood loss, a hemorrhagic shock, an ischemic condition, o r an ischemia-reperfusion injury.
17. The use according to claim 16, wherein said ischemia-reperfusion injury comprises a severe hemorrhagic shock, a stroke, a myocardial infarction, or combinations thereof.
18. The use of a soluble complex according to any one of claims 1 - 11 or an artificial blood substitute according to claim 12 for removing carbon dioxide from a body fluid selected from the group consisting of air, plasma, interstitial fluid, intracellular fluid, blood and dialysate fluid.
19. The use according to claim 18, wherein said fluid is chosen from blood and air.
20. The use according to claim 18, wherein said fluid is blood in an artificial lung.
21. The use according to claim 18, wherein said fluid is blood in a dialysis.
22. A method of preparing a soluble complex for removing CO2 from tissue and blood comprising the steps of:
a) cross-linking a mixture comprising:
= a stroma-free hemoglobin or hemoglobin;
= at least one antioxidant enzyme;
= a carbonic anhydrase enzyme for blood acid-base regulation and blood CO2 transport; and Date Recue/Date Received 2021-05-26 = lysine, and b) purifying and concentrating the cross-linked mixture to obtain said soluble com plex.
a) cross-linking a mixture comprising:
= a stroma-free hemoglobin or hemoglobin;
= at least one antioxidant enzyme;
= a carbonic anhydrase enzyme for blood acid-base regulation and blood CO2 transport; and Date Recue/Date Received 2021-05-26 = lysine, and b) purifying and concentrating the cross-linked mixture to obtain said soluble com plex.
23. The method according to claim 22, wherein a molar ratio of said lysine to said stroma-free hemoglobin is from about 7 : Ito about 12 : 1.
24. The method according to claim 22, wherein a molar ratio of said lysine to said stroma-free hemoglobin is 7:1.
25. The method according to any one of claims 22 ¨ 24, wherein glutaraldehyde is used to cross-link the stroma-free hemoglobin or hemoglobin, the at least one antioxidant enzyme, the enzyme for blood acid-base regulation and blood CO2 transport, and the lysine in step a).
26. The method according to claim 25, wherein a molar ratio of said glutaraldehyde to said stroma-free hemoglobin is from about 8: Ito about 32:
1.
1.
27. The method according to claim 25, wherein a molar ratio of said glutaraldehyde to said stroma-free hemoglobin is 16:1.
28. The method according to any one of claims 22 - 27, wherein said step of cross-linking is performed for 24h at 4 C.
29. The method according to any one of claims 22 - 28, wherein said step of cross-linking is stopped by adding an excess of lysine at molar ratio to said stroma-free hemoglobin of 200:1.
30. The method according to any one of claims 22 - 29, wherein said step of purifying and concentrating is performed by filtering the cross-linked mixture, dialyzing the cross-linked mixture, microconcentrating the cross-linked mixture, or combinations thereof.
Date Recue/Date Received 2021-05-26
Date Recue/Date Received 2021-05-26
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161490304P | 2011-05-26 | 2011-05-26 | |
| US61490304 | 2011-05-26 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2778010A1 CA2778010A1 (en) | 2012-11-26 |
| CA2778010C true CA2778010C (en) | 2022-07-05 |
Family
ID=47262863
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2778010A Active CA2778010C (en) | 2011-05-26 | 2012-05-23 | A novel blood substitute with complete red blood cell functions |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN102861323A (en) |
| CA (1) | CA2778010C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112618705A (en) * | 2020-12-30 | 2021-04-09 | 云锦华彰(北京)生物科技有限公司 | Human-derived hemoglobin oxygen carrier and preparation method and application thereof |
| CN112546206A (en) * | 2020-12-30 | 2021-03-26 | 云锦华彰(北京)生物科技有限公司 | Preparation method of human hemoglobin oxygen carrier, product and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101357940B (en) * | 2008-09-18 | 2012-01-04 | 上海市血液中心 | Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof |
-
2012
- 2012-05-23 CA CA2778010A patent/CA2778010C/en active Active
- 2012-05-28 CN CN2012101796705A patent/CN102861323A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CA2778010A1 (en) | 2012-11-26 |
| CN102861323A (en) | 2013-01-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9498537B2 (en) | Method of enhancing efficacy of blood transfusions | |
| US5439882A (en) | Blood substitute | |
| US11116868B2 (en) | Wound spray | |
| JP6083674B2 (en) | Hemoglobin-albumin complex, artificial plasma bulking agent and artificial oxygen carrier containing the complex | |
| CA2778010C (en) | A novel blood substitute with complete red blood cell functions | |
| WO1991009615A1 (en) | Polyhemoglobin stabilized by purine derivatives and glutathione | |
| Scatena et al. | O-raffinose-polymerised haemoglobin. A biochemical and pharmacological profile of an oxygen carrier | |
| WO2004089404A1 (en) | Hemoglobin conjugate and the preparation method and its use | |
| EP0586381B1 (en) | Improved blood substitute | |
| US20140371149A1 (en) | Scarring reducing wound treatment | |
| US10960055B2 (en) | Scarring reducing wound treatment | |
| JP2001502351A (en) | Therapeutic use of hemoglobin to treat head injury | |
| BRAIN | Science of Physiology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request |
Effective date: 20170511 |
|
| EEER | Examination request |
Effective date: 20170511 |
|
| EEER | Examination request |
Effective date: 20170511 |
|
| EEER | Examination request |
Effective date: 20170511 |