WO2020077184A1 - Oxygenation media for ex-vivo preservation of organs and tissues - Google Patents
Oxygenation media for ex-vivo preservation of organs and tissues Download PDFInfo
- Publication number
- WO2020077184A1 WO2020077184A1 PCT/US2019/055797 US2019055797W WO2020077184A1 WO 2020077184 A1 WO2020077184 A1 WO 2020077184A1 US 2019055797 W US2019055797 W US 2019055797W WO 2020077184 A1 WO2020077184 A1 WO 2020077184A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hemoglobin
- oxygenation
- preparation
- weight
- constituent
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/18—Erythrocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0247—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components for perfusion, i.e. for circulating fluid through organs, blood vessels or other living parts
Definitions
- TITLE OXYGENATION MEDIA FOR EX-VIVO PRESERVATION OF ORGANS AND TISSUES
- the present disclosure generally relates to oxygenation media, and in particular, to oxygenation media and methods of making and using oxygenation media for preserving tissues and organs ex-vivo.
- an organ or tissue can be preserved in a holding container comprising a liquid medium.
- Oxygenation of the medium may be achieved by including a circulation system, i.e. a pump, configured to more or less continuously supply oxygen to the liquid medium.
- a circulation system i.e. a pump
- Systems designed to achieve organ or tissue preservation in this manner are known as machine perfusion systems.
- red blood cell viability all restrict the utility of known blood based oxygen carriers, and, in particular, the time during which organs and tissue can be preserved ex-vivo when these media are used.
- biological contaminants notably, contaminants transmitting blood borne diseases, e.g. hepatitis B and human immunodeficiency virus (HIV)
- hemoglobin and HBOCs as oxygen carriers arises from observations that certain quantities of hemoglobin, when isolated from red blood cells, are converted to a variant hemoglobin known as methemoglobin.
- methemoglobin the iron within the protein’s heme group, which serves as the molecular binding site for oxygen, is present in the ferric (Fe 3+ ) state, and not in the ferrous (Fe 2+ ), state.
- Methemoglobin is unable to bind oxygen and is therefore unable to serve as an oxygen carrier.
- methemoglobin is known to be prone to denaturation with the heme group dissociating from the protein, and unfolding of the protein. The unfolded protein can negatively affect hemoglobin preparations, including by the formation of precipitates.
- the present disclosure relates to oxygenation media for the ex-vivo preservation of organs and tissue. Accordingly, in one aspect, in accordance with the teachings herein, the present disclosure provides, in at least one embodiment, an oxygenation constituent for formulating a finished oxygenation medium, the oxygenation constituent comprising a mixture of a first and second preparation wherein:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; and wherein
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the oxygenation constituent can comprise from about 20% to about 90% by weight hemoglobin of the hemoglobin preparation.
- the oxygenation constituent can comprise from about 30% to about 80% by weight hemoglobin of the hemoglobin preparation.
- the oxygenation constituent can comprise from about 40% to about 70% by weight hemoglobin of the hemoglobin preparation.
- the oxygenation constituent can comprise from about 50% to about 60% by weight hemoglobin of the hemoglobin preparation.
- the hemoglobin preparation can comprise non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 5% to about 35% by weight hemoglobin.
- the hemoglobin preparation can comprise non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 10% to about 30% by weight hemoglobin.
- the hemoglobin preparation can comprise non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 15% to about 25% by weight hemoglobin.
- the red blood cell preparation can comprise hemoglobin in an amount of from about 5% to about 35% by weight hemoglobin.
- the red blood cell preparation can comprise hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin.
- the red blood cell preparation can comprise hemoglobin in an amount of from about 15% to about 25% by weight hemoglobin.
- the oxygenation constituent can comprise:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of from about 5% to about 15% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount from about 40% to about 60% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the oxygenation constituent can comprise:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of about 10% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of about 20% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of about 50% by weight hemoglobin of the hemoglobin preparation, and an amount equal the balance by weight hemoglobin of the red blood cell preparation.
- the hemoglobin preparation can comprise non-modified hemoglobin and is substantially free of HBOC.
- the hemoglobin preparation can comprise HBOC, and is substantially free of non-modified hemoglobin.
- the hemoglobin preparation can comprise a mixture of hemoglobin and HBOC.
- the aqueous hemoglobin diluent can be lactated Ringer's solution, or a modified form thereof.
- the aqueous red blood cell diluent can be lactated Ringer's solution, or a modified form thereof.
- a finished oxygenation medium comprising:
- the balance of the medium comprising at least one other formulary ingredient selected from a diluent or an excipient, and optionally other ingredients suitable for formulating a finished oxygenation medium.
- the finished oxygenation medium can comprise:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation;
- the finished oxygenation medium can comprise from about 5% by weight hemoglobin to about 35% by weight hemoglobin of an oxygenation constituent. [00033] In at least one embodiment, the finished oxygenation medium can comprise from about 10% by weight hemoglobin to about 30% by weight hemoglobin of an oxygenation constituent.
- the finished oxygenation medium can comprise from about 15% by weight hemoglobin to about 25% by weight hemoglobin of an oxygenation constituent, the oxygenation constituent comprising:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of from about 5% to about 15% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount from about 40% to about 60% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the finished oxygenation medium can comprise about 20% by weight hemoglobin of an oxygenation constituent, the oxygenation constituent comprising:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of about 10% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of about 20% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of about 50% by weight hemoglobin of the hemoglobin preparation, and an amount equal the balance by weight hemoglobin of the red blood cell preparation.
- the present disclosure further provides, in at least one embodiment, a method of preparing a finished oxygenation medium for ex-vivo use, the method comprising:
- hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- the finished oxygenation medium can comprise at least about 1 % by weight of hemoglobin of the oxygenation constituent. [00038] In at least one embodiment, the finished oxygenation medium can comprise from about 1 % to about 39% by weight of hemoglobin of the oxygenation constituent.
- an amount of the hemoglobin preparation can be mixed such that the oxygenation constituent comprises an amount of from about 20% to about 90% by weight hemoglobin of the hemoglobin preparation.
- an amount of the hemoglobin preparation can be mixed such that the oxygenation constituent comprises from about 30% to about 80% by weight hemoglobin of the hemoglobin preparation.
- an amount of the hemoglobin preparation can be mixed such that the oxygenation constituent comprises from about 40% to about 70% by weight hemoglobin of the hemoglobin preparation.
- an amount of the hemoglobin preparation can be mixed such that the oxygenation constituent comprises from about 50% to about 60% by weight hemoglobin of the hemoglobin preparation.
- the hemoglobin preparation can comprise non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 5% to about 35% by weight hemoglobin.
- the hemoglobin preparation can comprise non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 10% to about 30% by weight hemoglobin.
- the hemoglobin preparation can comprise non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 15% to about 25% by weight hemoglobin.
- the red blood cell preparation can comprise hemoglobin in an amount of from about 5% to about 35% by weight hemoglobin.
- the red blood cell preparation can comprise hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin.
- the red blood cell preparation can comprise hemoglobin in an amount of from about 15% to about 25% by weight hemoglobin.
- the oxygenation constituent can comprise:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of from about 5% to about 15% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount from about 40% to about 60% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation;
- the finished oxygenation medium can comprise from about 15% by weight hemoglobin to about 25% by weight hemoglobin of the oxygenation constituent.
- the present disclosure further provides, in at least one embodiment, a use of an oxygenation constituent to formulate a finished oxygenation medium, the oxygenation constituent comprising a mixture of a first and second preparation wherein:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; and wherein
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the present disclosure further provides, in at least one embodiment, a use of a finished oxygenation medium for the ex- vivo preservation of a tissue or an organ, the finished oxygenation medium comprising:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation;
- the ex-vivo preservation can be static ex- vivo preservation.
- the ex-vivo preservation can be dynamic ex-vivo preservation.
- the ex-vivo preservation can be dynamic ex-vivo preservation involving the use of a machine perfusion system configured to comprise a filtering device to filter the finished oxygenation medium.
- the ex-vivo preservation can be dynamic ex-vivo preservation involving the use of a machine perfusion system configured to comprise a continuous or semi continuous supply assembly for the finished oxygenation medium, or an excipient or diluent, and a filtering device to continuously filter the finished oxygenation medium.
- a machine perfusion system configured to comprise a continuous or semi continuous supply assembly for the finished oxygenation medium, or an excipient or diluent, and a filtering device to continuously filter the finished oxygenation medium.
- the filtering device can be a tangential flow based filtering device.
- the organ can be an organ for transplantation to a transplant recipient.
- the organ can be a lung, a heart, a liver or a kidney.
- the organ can be a human organ.
- the tissue can be for grafting to a graft recipient.
- the tissue can be for tissue engineering.
- an organ preservation system comprising a circuitous perfusion fluid system operable to perfuse a perfusion fluid through an organ, the preservation system comprising a fluid reservoir to support an organ, the fluid reservoir in fluid connection with a continuous or semi-continuous supply assembly for supplying a perfusion fluid or an excipient to the fluid reservoir, the fluid reservoir further in fluid connection with a separation device permitting the filtration of the perfusion fluid upon removal of the perfusion fluid from the fluid reservoir to separate and remove spent constituents from the perfusion fluid, and a pump for circulating the perfusion fluid through the circuitous fluid system.
- the organ preservation system can be used for perfusion with a hemoglobin based perfusion fluid.
- the organ preservation system can be used for perfusion with a red blood cell based perfusion fluid.
- the organ preservation system can be used for perfusion with a finished oxygenation medium of the present disclosure.
- any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub-ranges are individually and specifically disclosed (e.g. a range of 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5).
- other terms of degree such as “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.
- red blood cell or “erythrocyte”, as may be used interchangeably herein, mean a non-nucleated cell containing hemoglobin that circulates in blood, generally responsible for the red color of blood.
- ex-vivo is to denote outside a human or animal body such that an organ, tissue or cells are wholly or partially separated from the human or animal’s normal blood circulation.
- Formulating the oxygenation constituent to form a finished oxygenation medium it is meant that the oxygenation constituent is contacted with at least one other ingredient, including, but not limited to, a diluent or excipient and mixed, homogenized or prepared until a finished oxygenation medium is formed.
- the term“finished oxygenation medium”, as used herein, refers to a fully formulated oxygenation medium comprising an oxygenation constituent suitable for ex-vivo use.
- the terms“HBOC”, or“hemoglobin based oxygen carrier”, as used herein, refer to an oxygen carrying hemoglobin derivative wherein the hemoglobin molecule has been modified, for example by chemically cross-linking of hemoglobin molecules, polymerization, or by conjugation of hemoglobin to other molecules, for example, conjugation to polyethylene glycol (PEG) or dextran.
- PEG polyethylene glycol
- hemoglobin and “hemoglobin molecule”, as used interchangeably herein, refer to the protein contained within erythrocytes that transports oxygen in living organisms. Each molecule of hemoglobin has 4 subunits, 2 a-chains and 2 b-chains, which are arranged in a tetrameric structure. Each subunit also contains one heme group, which is the iron-containing center that binds oxygen. Thus, each hemoglobin molecule can bind 4 oxygen molecules.
- the term by itself refers to native hemoglobin, including naturally occurring variants thereof, and further includes hemoglobin obtainable from any invertebrate or vertebrate animal, or a human, including, without limitation, e.g. human hemoglobin, bovine hemoglobin, ovine hemoglobin, and porcine hemoglobin.
- substantially pure describes an entity, e.g. a cell or chemical or biochemical compound, such as hemoglobin, which has been separated from constituents that naturally accompany it.
- an entity is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the entity of interest.
- the term further refers to the separation from of non- erythrocyte blood cells, e.g.
- leukocytes and thrombocytes including a preparation comprising no more than 5%, 4%, 3%, 2% or 1 % of non-erythrocyte blood cells. Purity can be measured by any appropriate method, e.g., in the case of proteins, by chromatography, gel electrophoresis or HPLC analysis, and in the case of red blood cells, flow cytometry.
- preparations comprising non-modified hemoglobin, HBOC, or a mixture thereof when mixed with certain quantities of red blood cells can be used as oxygenation constituents.
- hemoglobin preparations comprising from about 10% to about 99% by weight hemoglobin of the oxygenation constituents, can be mixed with red blood cell preparations comprising the balance by weight hemoglobin of the oxygenation constituents.
- the novel oxygenation constituents can be used to prepare finished oxygenation media.
- the finished oxygenation media are suitable to provide tissue or organ oxygenation and thereby mediate ex-vivo preservation of organs and tissues.
- the finished oxygenation media of the present disclosure permits for prolonged oxygenation and preservation of organs or tissues. Furthermore, the oxygenation constituents and the finished oxygenation media of the present disclosure exhibit limited red blood cell lysis, and limited coagulation.
- the finished oxygenation media of the present disclosure permit the ex-vivo preservation of organs and tissues for extended periods of time, for example, for at least about 6 hours, under static conditions, or for a period of at least 12 hours using a machine perfusion system.
- a yet further significant benefit of the finished oxygenation media of the present disclosure is that they can be used within a wide temperature range.
- the finished oxygenation media it is, for example, possible to use the finished oxygenation media at a temperature as low as about 2 °C, and as high as about 40 °C.
- the oxygenation constituents and finished oxygenation media can be safely and economically manufactured and methods for manufacture are also included herein.
- the disclosure provides novel oxygenation constituents which can be used to prepare finished oxygenation media for ex-vivo use, for example, during surgical procedures, in order to preserve tissues and organs for transplantation, and for other uses.
- an oxygenation constituent for formulating a finished oxygenation medium comprising a mixture of a first and second preparation wherein:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; and wherein
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the oxygenation constituents can be prepared by initially providing a hemoglobin preparation or HBOC preparation or a mixture thereof, and a red blood cell preparation.
- red blood cells, hemoglobin and HBOC preparations can be manufactured using whole blood as a source.
- Suitable blood that may be used in accordance herewith includes invertebrate or vertebrate animal or human blood, including, without limitation, mammalian and avian blood, including without limitation, human blood, bovine blood, porcine blood, equine blood and ovine blood.
- Blood solutions may be collected from live or dead organisms and may be collected using any techniques and devices known to the art, including, for example, the methodologies described in U.S. Patents 5,084,558 and 5,296,465.
- the blood may be fresh or from an older sample, for example expired blood from a blood bank institute.
- the blood may have been stored and/or frozen.
- the blood solution is screened for the presence of blood borne pathogens, for example where human blood is used, HIV and hepatitis B.
- blood free of blood borne pathogens is selected for use in accordance with the methods of the present disclosure.
- an anti-coagulant is added to the blood to prevent blood clotting.
- Anti-coagulants include heparin, hirudin, sodium citrate, ethylenediaminetetraacetic acid.
- the anticoagulant may be provided as an aqueous solution or in particulate form.
- red blood cell separation technique known to the art may be used. This includes the use of centrifugation and/or straining and filtering techniques to remove large blood aggregates (e.g. 50 pm and larger) and debris. Thus, one or more propylene 800 pm to 50 pm filters may be used. It is noted in this regard that red blood cells are about 5 - 10 pm in size.
- the red blood cells can be further isolated from blood by diafiltration using an isotonic solution, having a pH and osmolarity which preserves the integrity of the erythrocyte cellular membrane, for example, a sodium citrate (about 6.0 g/l) and sodium chloride (about 8.0 g/l) solution having an osmolarity of 285 - 315 mOsm.
- Acceptable diafiltration filters that can be used in accordance herewith include microporous membranes which substantially separate erythrocytes from smaller components for example, a modified polyethersulfone hollow fiber tangential flow filtration membrane obtainable from Spectrum labs.
- the isotonic solution can be added in batches or continuously, typically approximately at the same rate at which filtrate is lost.
- components of the blood solution smaller than red blood cells generally the plasma portion of the blood, including extracellular blood proteins, e.g. antibodies and serum albumins, are separated as filtrate from the red blood cells, which are retained and continuously or batch-wise added to the isotonic solution.
- Volumes of isotonic solution used may vary and may for example be at least 2x, 3x, 4x, 5x, 6x or 7x the volume of blood solution.
- sufficient volumes of isotonic solution are used to remove at least from about 90% to 95% (mole/mole) of blood plasma proteins and obtain a substantially pure red blood cell preparation, for example a red blood cell preparation that is at least about 90%, at least about 95%, or at least about 99% pure.
- the techniques used to isolate red blood cells from blood and to obtain a substantially pure red blood cell preparation may be as desired, and include any techniques known to the art, including, for example, the methods for isolating erythrocytes are described in U.S. Patent 5,955,581.
- the red blood cell preparation can be mixed with a physiologically compatible aqueous red blood cell diluent, i.e. an aqueous diluent in which the red blood cells remain substantially intact, and in which the red blood cells substantially retain their physiological oxygen carrying capabilities.
- a physiologically compatible aqueous red blood cell diluent i.e. an aqueous diluent in which the red blood cells remain substantially intact, and in which the red blood cells substantially retain their physiological oxygen carrying capabilities.
- the red blood cell preparation can be diluted using a saline solution, or a lactated Ringer’s solution, or a modified lactated Ringer’s solution.
- the amount of aqueous red blood cell diluent can vary, and can, for example, be selected so that the fully diluted red blood cell preparation comprises hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; or from about 5% to about 35% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; or from about 10% to about 30% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; or from about 15% to about 25% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; or from about 20% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent.
- isolated red blood cells may be used as a source whence hemoglobin can be extracted to obtain an isolated hemoglobin preparation.
- isolated red blood cells can be lysed. Any technique for lysing erythrocytes known to the art may be used, including any mechanical lysis technique or chemical lysis technique, provided however that such technique does not substantially negatively affect the ability of hemoglobin to transport and release oxygen.
- the isolated red blood cells can be lysed by subjecting the red blood cells to a hypotonic shock to obtain a red blood cell lysate.
- Suitable hypotonic solutions that may be used in accordance herewith include, for example, a phosphate buffer 3.75 mM, pH 7.2 or water which may be mixed with the red blood cells, and the mixture may be incubated on ice, for example for 1 hour to obtain a lysed red blood cell preparation.
- a hemoglobin fraction can be obtained from the red blood cell lysate using a variety of suitable protein purification techniques, including chromatographic separation techniques which include absorption based methods, involving initial retention of hemoglobin on solid chromatographic media, and thereafter solvent based elution, or flow-through based techniques, involving retention of impurities and flow through of hemoglobin.
- chromatographic separation techniques include absorption based methods, involving initial retention of hemoglobin on solid chromatographic media, and thereafter solvent based elution, or flow-through based techniques, involving retention of impurities and flow through of hemoglobin.
- Other chromatography based techniques include affinity chromatography and high performance liquid based chromatography (HPLC). Chromatographic techniques to isolate hemoglobin and obtain an isolated hemoglobin preparation that can be used further include, for example, those described in U.S. Patent 5,691 ,453, Andrade et al., Int. J. Biol.
- the red blood cell lysates may be subjected non-chromatography based purification techniques, including, for example, aqueous phase extraction, membrane based micro- and ultrafiltration techniques and tangential flow based techniques.
- Ultrafiltration based techniques that can be used include those described by Feins et al. 2005, J. Membr. Sci 248: 137-148.
- Tangential flow based techniques that can be used include those described for example by Palmer et al., 2009, in Biotechnol.
- Two-phase aqueous extraction techniques that can be used to obtain hemoglobin include those described in U.S. Patent 5,407,579.
- substantially pure hemoglobin preparations can be obtained, including, for example, preparations which are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% pure.
- hemoglobin obtained using the foregoing techniques, or combinations or modifications thereof, or other suitable techniques is non- modified hemoglobin, i.e. hemoglobin which has not been chemically modified.
- the hemoglobin obtained in accordance with the foregoing techniques, combinations or modifications thereof can be used to prepare hemoglobin preparations comprising non-modified hemoglobin in accordance herewith.
- non-modified hemoglobin can be mixed with a physiologically compatible aqueous hemoglobin diluent.
- hemoglobin can be diluted using a saline solution, or a lactated Ringer’s solution, or a modified lactated Ringer’s solution.
- the amount of aqueous hemoglobin diluent can vary, and can, for example, be selected so that the fully diluted hemoglobin preparation comprises non-modified hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 5% to about 35% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 5% to about 35% by weight hemoglobin; or in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 15% to about 25% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent.
- the foregoing hemoglobin preparations are substantially free of modified hemoglobin.
- a hemoglobin formulating agent can be included to prepare the hemoglobin preparation, for example, a formulating agent to encapsulate isolated hemoglobin in liposomes, and form liposome encapsulated hemoglobin (LEH), see for example: U.S. Patent Application No. 2014/0212477.
- the hemoglobin obtained from red blood cells can also be used to prepare HBOC preparations, including polymerized HBOC preparations, for example, by cross-linking of hemoglobin molecules, or by chemically modifying hemoglobin molecules. Examples of polymerized hemoglobin molecules are described in U.S. Patent 5,955,581 ; U.S. Patent 5,895,810; U.S.
- chemically modified hemoglobin molecules include polysaccharide conjugated hemoglobin (e.g . PCT Patent Application PCT/CA99/00260), polyalkylene oxide-conjugated hemoglobin (e.g. U.S. Patent 5,650,388) and polyethylene glycol (PEG) conjugated hemoglobin (e.g. U.S. Patent 5,750,725, U.S. Patent 7,144,989 and U.S. Patent 8,609,815).
- the HBOC preparation obtained in accordance with the foregoing techniques, combinations or modifications thereof can be used to prepare hemoglobin preparations comprising HBOC in accordance herewith.
- the HBOC preparation can be mixed with a physiologically compatible aqueous hemoglobin diluent.
- the HBOC preparation can be diluted using a saline solution, or a lactated Ringer’s solution, or a modified lactated Ringer’s solution.
- the amount of aqueous hemoglobin diluent can vary, and can, for example, be selected so that the fully diluted hemoglobin preparation comprises HBOC in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 5% to about 35% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 5% to about 35% by weight hemoglobin; or in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 15% to about 25% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent.
- the foregoing HBOC preparations can be substantially free of non-modified hemoglobin.
- the HBOC preparations can contain less 10% (w/w), less than 5% (w/w), or less then 1 % (w/w) of non-modified hemoglobin.
- a mixture of non-modified hemoglobin and HBOC can be used to prepare a hemoglobin preparation.
- a mixture can be prepared by obtaining a hemoglobin preparation comprising non-modified hemoglobin and an HBOC preparation, using techniques or combinations or modifications thereof as is hereinbefore described, and contacting and mixing the two preparations in order to obtain a substantially homogenous mixture of non- modified hemoglobin and HBOC.
- the relative amounts of HBOC preparation and non-modified hemoglobin preparation within the mixture may be varied, and selected as desired.
- the preparations comprising a mixture of non-modified hemoglobin and HBOC can be selected so that the fully diluted hemoglobin preparation comprises HBOC and non-modified hemoglobin together in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 5% to about 35% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 5% to about 35% by weight hemoglobin; or in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent; or in an amount of from about 15% to about 25% by weight hemoglobin, and the balance of the hemoglobin preparation comprising the aqueous hemoglobin diluent.
- Red blood cell preparations and hemoglobin preparations can also be obtained from a commercial supplier.
- Commercial hemoglobin suppliers include Thomas Scientific and Sigma Aldrich.
- Commercial red blood cell suppliers include blood banks such as the Red Cross.
- a preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof, and a red blood cell preparation
- the two preparations can be contacted and mixed until a substantially homogenous mixture is obtained.
- the amounts of hemoglobin and red blood cell preparation can be varied.
- the oxygenation constituent can be prepared to constitute an amount of the hemoglobin preparation so that the hemoglobin preparation comprises from about 10% to about 99% by weight hemoglobin of the oxygenation constituent, and an amount of the red blood cell preparation so that the balance by weight hemoglobin to 100% (i.e. from about 1 % to about 90%) of the oxygenation constituent constitutes the red blood cell preparation.
- the oxygenation constituent can be prepared to constitute an amount of the hemoglobin preparation so that the hemoglobin preparation comprises from about 20% to about 90% by weight hemoglobin of the oxygenation constituent, and an amount of the red blood cell preparation so that the balance by weight hemoglobin to 100% (i.e. from about 10% to about 80%) of the oxygenation constituent constitutes the red blood cell preparation.
- the oxygenation constituent can be prepared to constitute an amount of the hemoglobin preparation so that the hemoglobin preparation comprises from about 30% to about 80% by weight hemoglobin of the oxygenation constituent, and an amount of the red blood cell preparation so that the balance by weight hemoglobin to 100% (i.e. from about 20% to about 70%) of the oxygenation constituent constitutes the red blood cell preparation.
- the oxygenation constituent can be prepared to constitute an amount of the hemoglobin preparation so that the hemoglobin preparation comprises from about 40% to about 70% by weight hemoglobin of the oxygenation constituent, and an amount of the red blood cell preparation so that the balance by weight hemoglobin to 100% (i.e. from about 30% to about 60%) of the oxygenation constituent constitutes the red blood cell preparation.
- the oxygenation constituent can be prepared to constitute an amount of the hemoglobin preparation so that the hemoglobin preparation comprises from about 50% to about 60% by weight hemoglobin of the oxygenation constituent, and an amount of the red blood cell preparation so that the balance by weight hemoglobin to 100% (i.e. from about 40% to about 50%) of the oxygenation constituent constitutes the red blood cell preparation.
- the present disclosure includes an oxygenation constituent for formulating a finished oxygenation medium, the oxygenation constituent comprising a mixture of a first and second preparation wherein:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of from about 5% to about 15% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; and wherein
- the mixture is formed such that the oxygenation constituent comprises an amount from about 40% to about 60% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the oxygenation constituent can comprise an oxygenation constituent for formulating a finished oxygenation medium, the oxygenation constituent comprising a mixture of a first and second preparation wherein:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of about 10% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of about 20% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of about 50% by weight hemoglobin of the hemoglobin preparation, and an amount equal the balance by weight hemoglobin of the red blood cell preparation.
- the thus obtained mixture can be used as an oxygenation constituent to formulate a finished oxygenation medium.
- a non-modified hemoglobin preparation or, as the case may be, an HBOC preparation or a preparation containing a mixture of non- modified hemoglobin and HBOC, is provided, and separately a red blood cell preparation is provided.
- the preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof and the red blood cell preparation are mixed to obtain an oxygenation constituent.
- the oxygenation constituent can be contacted with at least one other ingredient suitable for use in finished oxygenation, notably a diluent or excipient.
- a finished oxygenation medium comprising:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation;
- the present disclosure further provides, in at least one embodiment, a use of a use of an oxygenation constituent to formulate a finished oxygenation medium, the oxygenation constituent comprising a mixture of a first and second preparation wherein:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent; and wherein
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the oxygenation constituent and diluent or excipient are mixed, homogenized or prepared, preferably until a homogenous mixture of the diluent, or excipient and oxygenation constituent is formed, wherein such mixture is suitable for use as an oxygenation medium.
- the diluent or excipient may be any suitable diluent or excipient, and the diluent or excipient may be provided in any form, including, for example, as a solution, suspension, gel, or liquid. Diluents that can be included include buffers, such as isotonic saline, 2-(N- morpholino)ethanesulfonic acid (MES), and excipients that can be used include lactated Ringer’s Solution.
- MES 2-(N- morpholin
- a plurality of other ingredients can be provided, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ingredients, in addition to the oxygenation constituent and diluent or excipient, to prepare the finished oxygenation medium.
- such ingredients may be mixed sequentially or simultaneously.
- the additional ingredients can include compounds normally found in blood, for example, ions normally found in blood, including calcium ions; chloride ions; sodium ions; magnesium ions; phosphate ions; or mixtures thereof, each of which can be provided in a variety of chemical forms, including, for example sodium chloride, and a variety of formulations, for example in the form of a saline solution.
- ions normally found in blood including calcium ions; chloride ions; sodium ions; magnesium ions; phosphate ions; or mixtures thereof, each of which can be provided in a variety of chemical forms, including, for example sodium chloride, and a variety of formulations, for example in the form of a saline solution.
- buffering agents for example 2- (N-morpholino)ethanesulfonic acid (MES); redox agents, for example sulfhydryls, such as glutathione, ascorbic acid (ascorbate), n-acetyl cysteine, nicotinamide adenine dinucleotide phosphate (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH); colloids, such as hydroxyethyl starch or albumin; sugars, including monosaccharides or disaccharides, such as glucose or lactobionic acid; amino acids; agents to limit the presence of oxygen radicals, including superoxide dismutase; agents to limit the formation of methemoglobin, methemoglobin reductase, for example; antibiotics; or mixtures thereof.
- MES 2- (N-morpholino)ethanesulfonic acid
- redox agents for example sulfhydryls, such
- pharmacological compounds capable of ameliorating a disease state or medical condition can be included.
- the quantities of the other ingredients, including the excipient or diluent can vary, however typically the oxygenation constituent constitutes at least 1 % by weight of hemoglobin of the finished oxygenation medium.
- the finished oxygenation medium can comprise from about 1 % by weight hemoglobin to about 39% by weight hemoglobin of the oxygenation constituent.
- the finished oxygenation medium can comprise from about 5% by weight hemoglobin to about 35% by weight hemoglobin of the oxygenation constituent.
- the finished oxygenation medium can comprise from about 10% by weight hemoglobin to about 30% by weight hemoglobin of the oxygenation constituent.
- the finished oxygenation medium can comprise from about 15% by weight hemoglobin to about 25% by weight hemoglobin of an oxygenation constituent, the oxygenation constituent comprising:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of from about 5% to about 15% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of from about 10% to about 30% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount from about 40% to about 60% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation.
- the finished oxygenation medium can comprise about 20% by weight hemoglobin of an oxygenation constituent, the oxygenation constituent comprising:
- a hemoglobin preparation comprising non-modified hemoglobin, HBOC or a mixture thereof in an amount of about 10% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- a red blood cell preparation comprising hemoglobin in an amount of about 20% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of about 50% by weight hemoglobin of the hemoglobin preparation, and an amount equal the balance by weight hemoglobin of the red blood cell preparation.
- the present disclosure includes, in a further embodiment, a method of preparing a finished oxygenation medium for ex-vivo use, the method comprising:
- hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent;
- the finished oxygenation media may be stored for shorter or longer periods of time, for example, from 1 -2 days up to 1 year or more.
- Finished oxygenation media are preferably stored in sterile, sealed containers, for example, sealed glass containers, stainless steel containers or storage bags, having a low oxygen environment.
- Storage containers are further preferably impermeable to the transfer of water in order to prevent evaporation of water and concentration of the media.
- storage containers may be blanketed with, for example, a nitrogen atmosphere prior to sealing.
- the finished oxygenation medium in order to prevent auto- oxidation, i.e. the formation of methemoglobin, the finished oxygenation medium can be treated with carbon monoxide.
- the containers can be refrigerated (0 °C to 4 °C) for storage, or the media may be frozen and stored in a freezer, for example from -20 °C to -80 °C.
- the present disclosure provides, in one embodiment,
- a finished oxygenation medium for the ex-vivo preservation of a tissue or an organ comprising:
- the first preparation is a hemoglobin preparation comprising non-modified hemoglobin, HBOC, or a mixture thereof in an amount of from about 1 % to about 40% by weight hemoglobin, the balance of the hemoglobin preparation comprising an aqueous hemoglobin diluent; and
- the second preparation is a red blood cell preparation comprising hemoglobin in an amount of from about 1 % to about 40% by weight hemoglobin, and the balance of the red blood cell preparation comprising an aqueous red blood cell diluent;
- the mixture is formed such that the oxygenation constituent comprises an amount of from about 10% to about 99% by weight hemoglobin of the hemoglobin preparation, and an amount equal to the balance by weight hemoglobin of the red blood cell preparation;
- the finished oxygenation medium of the present disclosure can be used to maintain the oxygen content of an organ or tissue ex- vivo in order to preserve an organ, for example, when organs are stored for later transplantation to a patient, or for reimplantation in a patient, or when organs require transportation.
- Organ transplants may be autotransplants (i.e. transplantation of an organ from one part of the body to another part of the body of the same subject), allotransplants (i.e. transplantation of an organ from one subject to another subject), or xenotransplants (i.e. transplantation of an organ from a subject of one species to a subject of another species).
- organs that may be used in this regard include, without limitation liver, kidney, heart, lung, intestine and pancreas.
- the organ can be in any stage of development or life, or of any age, or of any origin including a human organ, or an organ obtainable from another vertebrate.
- the finished oxygenation medium of the present disclosure can be used to maintain the oxygen content of a tissue ex-vivo in order to preserve a tissue, or for tissue engineering.
- tissue can be preserved, for example, when tissue is stored for later transplantation or grafting to a patient, or for reimplantation in a patient, or when tissue requires transportation, or when tissue is used for experimental ex vivo purposes, for example, the study of cancer tumors and treatment thereof.
- Tissue grafts may be autografts (i.e. transplantation of tissue from one part of the body to another part of the body of the same subject), allografts (i.e. transplantation of tissue from one subject to another subject), or xenografts (i.e. transplantation of tissue from a subject of one species to a subject of another species).
- tissue transplants include composite tissue allotransplants, e.g. limbs, face.
- the finished oxygenation media of the present disclosure can be used to maintain organs for research and development purposes, for example, for the discovery of biomarkers, for tissue engineering or biofabrication, of for use of organ tissue as a bioreactor.
- the organs or tissue can be preserved using a preservation system permitting preservation in static mode.
- the organs or tissue can be preserved using a preservation system permitting preservation in dynamic mode.
- static mode the organs are bathed in a solution comprising the oxygenation medium of the present disclosure.
- dynamic mode the organ is perfused using the finished oxygenation medium of the present disclosure and one or more mechanical devices including, for example, a pump system, or devices for regulating temperature, i.e. a machine perfusion system.
- Various preservation systems are known to the art and include, by way of example, the systems described in PCT Patent Applications: PCT/US2013/049573 and PCT/US2016/051122, and US Patent Applications: U.S. 2008/0038811 and U.S. 2010/0304352.
- the preservation system can be configured to include a separation device.
- separation devices allow for removal of certain constituents from the finished oxygenation medium, including non-modified hemoglobin, HBOCs or red blood cells, or any of the additional ingredients, and replacement with fresh constituents.
- a preservation system can be configured to include a continuous or semi- continuous supply assembly for a finished oxygenation medium or an excipient, a separation device, and a pump, and further optionally an oxygenation cartridge.
- the preservation system can be operated to supply oxygenation medium or excipient on a continuous or semi-continuous basis, and filter the finished oxygenation medium or excipient, and replace it with fresh oxygenation medium or excipient from the supply assembly, thus allowing for continuous or semi- continuous refreshment of the excipient or finished oxygenation medium.
- Separation devices that may be used include filters which may be operated in line on a substantially continuous basis, using, for example, a tangential flow system, permitting continuous refreshment of the oxygenation medium or excipient, or filters which may be installed for use on a periodic basis.
- filters and pore size of the filters may be selected in order to facilitate the selective removal of certain constituents of the finished oxygenation medium.
- the present disclosure further includes in one embodiment, an organ preservation system comprising a circuitous perfusion fluid system operable to perfuse a perfusion fluid through an organ, the preservation system comprising a fluid reservoir to support an organ, the fluid reservoir in fluid connection with a continuous or semi-continuous supply assembly for supplying a perfusion fluid or an excipient to the fluid reservoir, the fluid reservoir further in fluid connection with a separation device permitting the filtration of the perfusion fluid upon removal of the perfusion fluid from the fluid reservoir, to separate and remove spent constituents from the perfusion fluid, and a pump for circulating the perfusion fluid through the circuitous fluid system.
- the system can further optionally contain an oxygenation cartridge.
- the organ preservation can be used in conjunction with the finished oxygenation media of the present disclosure, or in conjunction with any other perfusion fluid, for example, other hemoglobin based perfusion fluid or other red blood cell based perfusion fluids.
- an organ or tissue can be statically preserved for at least about 6 hours, at least about 8 hours, at least about 10 hours, or at least about 12 hours using a finished oxygenation medium of the present disclosure; or for example, an organ or tissue can be dynamically preserved for at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 96 hours, or at least about 1 week, about 2 weeks, or about 4 weeks using a finished oxygenation medium of the present disclosure.
- the finished oxygenation medium may be used at various temperatures ranging from as low as about 2 °C, up to as high as about 40 °C to ex-vivo preserve organs or tissue. Other temperatures ranges at which the finished oxygenation medium may be used to ex-vivo preserve organs or tissue include temperatures from about 5 °C to about 35 °C, from about 10 °C to about 30 °C, and from about 15 °C to about 25 °C.
- the finished oxygenation medium may further be used at about physiological temperature to ex-vivo preserve organs or tissue, i.e. at 37 °C for human tissue and organs, or at room temperature, i.e. from about 18 °C to about 23 °C.
- the temperature may be varied in different circumstances, for example, depending on the specific organ or tissue that is being preserved, or the duration of the preservation time. Furthermore the temperature may be adjusted or optimized, for example by evaluation ex-vivo preservation at different temperatures or using different temperature regimens for different periods of time, and selected an optimized temperature.
- the therapeutic amounts of the oxygenation medium used may vary.
- the term "therapeutically effective amount,” for the purposes of the present disclosure, refers to the amount of oxygenation medium which is effective to achieve its intended purpose, i.e. the sufficient ex-vivo oxygenation of an organ or tissue to survive for clinical use, or for clinical or medical research or development purposes. While individual situations may vary, determination of optimal ranges for effective amounts of a finished oxygenation medium to be used is within the skill of one in the art.
- dosages required to provide therapeutically effective amounts of the oxygenation medium or the oxygenation constituent will vary, depending on the tissue, organ, duration of preservation, health or physical condition of the tissue or organ, as can be determined by one of ordinary skill in the art using conventional considerations, ( e.g . by means of an appropriate, conventional pharmacological or veterinary protocol).
- an oxygenation constituent and finished oxygenation medium can be prepared according to the methods of the present disclosure, which allows for prolonged periods of preservation of organs or tissues.
- the finished oxygen media can be applied in many ex-vivo clinical and clinical development processes.
- Example 1 Preparation of an oxygenation constituent for ex-vivo use.
- a low purity erythrocyte protein fraction consisting primarily of hemoglobin isolated by filtration was polymerized using giutaraldehyde and then reduced with cyanoborohydride and was put into a suitable buffer (iactated Ringers with n-acetyl cysteine) such that the giutaraldehyde and cyanoborohidride were removed by filtration to thereby obtain an HBOC solution that was 11 g/dL (11 %) by weight hemoglobin.
- the solution can either be end point sterilized with filtration or by irradiation.
- Packed red blood cells (pRBC) can be obtained commercially from one of numerous institutions dedicated to blood collection storage, and distribution, for example the Red Cross.
- An oxygenation constituent can be prepared by mixing, the HBOC solution and the pRBC in a 1 to 1. The oxygenation constituent can thereafter, as desired, immediately be used to prepare a finished oxygenation medium, for example as described in Example 2.
- a finished oxygenation medium can be prepared using the oxygenation constituent as described in Example 1 , and mixing the oxygenation constituent with a pre-packaged sterile isosmotic diluent containing saline, glucose, and an antibiotic.
- the oxygenation constituent and diluent can be mixed at room temperature prior to a final hemoglobin concentration of 10 g/dL (10%) by weight hemoglobin to obtain a finished oxygenation medium.
- the temperature of the finished oxygenation medium may be adjusted prior to ex-vivo use.
- An ex-vivo use of a finished oxygenation medium can include a freshly isolated human or animal organ, for example a kidney, canulated for fluid flow though the vasculature, suspended in a temperature controlled tank in the finished oxygenation medium as prepared in accordance with Examples 1 and 2 under best practices asceptic conditions.
- the finished oxygenation medium can be circulated through the organ and holding tank at appropriate flow and pressure settings to avoid damage to the organ.
- the medium can be kept oxygenated by use of an oxygenation cartridge in the circulation loop. If metabolism is desired the temperature can be controlled to physiologic norms. If just prolonged storage is desired, the temperature can be reduced.
- Glucose and metabolite levels can be monitored by use of existing analyzers and if levels are not appropriate, a diluent exchange can be initiated by diafiltration.
- the organ can be maintained until required for its intended use, for example transplantation, or until its condition is deemed to have deteriorated too far for its intended use.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Diabetes (AREA)
- Gastroenterology & Hepatology (AREA)
- Physiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA3115973A CA3115973A1 (en) | 2018-10-12 | 2019-10-11 | Oxygenation media for ex-vivo preservation of organs and tissues |
AU2019357737A AU2019357737A1 (en) | 2018-10-12 | 2019-10-11 | Oxygenation media for ex-vivo preservation of organs and tissues |
JP2021545259A JP2022514800A (en) | 2018-10-12 | 2019-10-11 | Oxygenized medium for exvivo preservation of organs and tissues |
US17/284,668 US20210352887A1 (en) | 2018-10-12 | 2019-10-11 | Oxygenation media for ex-vivo preservation of organs and tissues |
EP19870597.2A EP3863649A4 (en) | 2018-10-12 | 2019-10-11 | Oxygenation media for ex-vivo preservation of organs and tissues |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862745020P | 2018-10-12 | 2018-10-12 | |
US62/745,020 | 2018-10-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020077184A1 true WO2020077184A1 (en) | 2020-04-16 |
Family
ID=70163925
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2019/055797 WO2020077184A1 (en) | 2018-10-12 | 2019-10-11 | Oxygenation media for ex-vivo preservation of organs and tissues |
Country Status (6)
Country | Link |
---|---|
US (1) | US20210352887A1 (en) |
EP (1) | EP3863649A4 (en) |
JP (1) | JP2022514800A (en) |
AU (1) | AU2019357737A1 (en) |
CA (1) | CA3115973A1 (en) |
WO (1) | WO2020077184A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111537315A (en) * | 2020-07-13 | 2020-08-14 | 天津德祥生物技术有限公司 | Erythrocyte membrane separation liquid and erythrocyte membrane separation method |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023163750A1 (en) * | 2022-02-28 | 2023-08-31 | Ohio State Innovation Foundation | Polymerized hemoglobin size fractionated via tangential flow filtration with low auto oxidation rates |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4532130A (en) * | 1981-07-06 | 1985-07-30 | Rush-Presbyterian-St. Luke's Medical Center | Preparation of synthetic frythrocytes |
US6284481B1 (en) * | 1999-02-02 | 2001-09-04 | Astrazeneca Ab | Assay |
US6811778B2 (en) * | 2001-09-10 | 2004-11-02 | Biopure Corporation | Method for improving oxygen transport by stored red blood cells |
US8420381B2 (en) * | 1998-09-29 | 2013-04-16 | Lifeline Scientific, Inc. | Apparatus and method for maintaining and/or restoring viability of organs |
US20150031599A1 (en) * | 2009-06-09 | 2015-01-29 | Prolong Pharmaceuticals, LLC | Hemoglobin compositions |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2689023B2 (en) * | 1990-08-17 | 1997-12-10 | バクスター、インターナショナル、インコーポレイテッド | Composition based on hemoglobin oligomer and method for producing the same |
CA2266174A1 (en) * | 1999-03-18 | 2000-09-18 | Hemosol Inc. | Hemoglobin-antioxidant conjugates |
JP2009524436A (en) * | 2006-01-24 | 2009-07-02 | ノースフィールド ラボラトリーズ、インコーポレイテッド | Polymerized hemoglobin medium and its use in isolation and transplantation of islet cells |
SI2440239T1 (en) * | 2009-06-09 | 2018-01-31 | Prolong Pharmaceuticals, LLC | Hemoglobin compositions |
US20120196270A1 (en) * | 2011-02-02 | 2012-08-02 | Sangart, Inc. | Methods for preserving an organ for transplantation using a hemoglobin-carbon monoxide complex |
WO2018053634A1 (en) * | 2016-09-21 | 2018-03-29 | The Governing Council Of The University Of Toronto | Hemoglobin based oxygen carrier and method of preparation |
-
2019
- 2019-10-11 JP JP2021545259A patent/JP2022514800A/en active Pending
- 2019-10-11 US US17/284,668 patent/US20210352887A1/en active Pending
- 2019-10-11 AU AU2019357737A patent/AU2019357737A1/en active Pending
- 2019-10-11 EP EP19870597.2A patent/EP3863649A4/en active Pending
- 2019-10-11 WO PCT/US2019/055797 patent/WO2020077184A1/en unknown
- 2019-10-11 CA CA3115973A patent/CA3115973A1/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4532130A (en) * | 1981-07-06 | 1985-07-30 | Rush-Presbyterian-St. Luke's Medical Center | Preparation of synthetic frythrocytes |
US8420381B2 (en) * | 1998-09-29 | 2013-04-16 | Lifeline Scientific, Inc. | Apparatus and method for maintaining and/or restoring viability of organs |
US6284481B1 (en) * | 1999-02-02 | 2001-09-04 | Astrazeneca Ab | Assay |
US6811778B2 (en) * | 2001-09-10 | 2004-11-02 | Biopure Corporation | Method for improving oxygen transport by stored red blood cells |
US20150031599A1 (en) * | 2009-06-09 | 2015-01-29 | Prolong Pharmaceuticals, LLC | Hemoglobin compositions |
Non-Patent Citations (2)
Title |
---|
SAKAI, H ET AL.: "Encapsulation of Concentrated Hemoglobin Solution in Phospholipid Vesicles Retards the Reaction with NO , but Not CO, by Intracellular Diffusion Barrier", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, no. 3, 18 January 2008 (2008-01-18), pages 1508 - 1517, XP055704204 * |
See also references of EP3863649A4 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111537315A (en) * | 2020-07-13 | 2020-08-14 | 天津德祥生物技术有限公司 | Erythrocyte membrane separation liquid and erythrocyte membrane separation method |
Also Published As
Publication number | Publication date |
---|---|
EP3863649A4 (en) | 2022-08-10 |
AU2019357737A1 (en) | 2021-05-20 |
EP3863649A1 (en) | 2021-08-18 |
US20210352887A1 (en) | 2021-11-18 |
CA3115973A1 (en) | 2020-04-16 |
JP2022514800A (en) | 2022-02-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5045446A (en) | Lyophilization of cells | |
US8512941B2 (en) | Biological material and methods and solutions for preservation thereof | |
AU693339B2 (en) | Cryopreservation solution | |
US4473552A (en) | Anaerobic method for preserving whole blood, tissue and components containing living mammalian cells | |
US7358039B2 (en) | Fixed-dried red blood cells | |
RU2396748C2 (en) | Medium for storage of cells | |
US20100105133A1 (en) | Somatic Cells for Use in Cell Therapy | |
WO2014059316A1 (en) | Compositions and methods for organ preservation | |
AU2019357737A1 (en) | Oxygenation media for ex-vivo preservation of organs and tissues | |
AU6326198A (en) | Organ preservation solution | |
CN112753695A (en) | Immune cell cryopreservation method | |
CN115802889A (en) | Low temperature storage of biological samples | |
EP0061277B1 (en) | Anaerobic method for preserving whole blood, tissue and components containing living mammalian cells | |
CN111357737B (en) | Normal-temperature mechanical organ perfusate and application thereof | |
Rowe | Cryopreservation of red blood cells | |
CA2122140A1 (en) | Method for freezing engrafting cells | |
Zhou et al. | Effects of glycerol pretreatment on recovery and antioxidant enzyme activities of lyophilized red blood cells | |
Xashimova et al. | Modern blood substitutes with detoxification properties | |
Pristoupil et al. | Trends in exploitation of packed red blood cells | |
JP2020527608A (en) | Blood substitute containing hemoglobin and preparation method | |
Wolkers et al. | 77. Effect of gamma sterilization on proteins in AlloDermTM regenerative tissue matrix | |
JPH04504841A (en) | Synthetic plasma-free transfusionable storage medium for red blood cells and platelets | |
Tajalli et al. | Cell Biology | |
IE53734B1 (en) | Anaerobic method for preserving whole blood, tissue and components containing living mammalian cells | |
NZ201599A (en) | Preserving biological substances containing mammalian cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19870597 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2021545259 Country of ref document: JP Kind code of ref document: A Ref document number: 3115973 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2019870597 Country of ref document: EP Effective date: 20210512 |
|
ENP | Entry into the national phase |
Ref document number: 2019357737 Country of ref document: AU Date of ref document: 20191011 Kind code of ref document: A |