WO2006026735A2 - Apparatuses and methods for analyzing gas exchange properties of biological fluids - Google Patents

Abstract

The present invention relates to systems and methods for measuring oxygen saturation and for examining gas exchange of blood and other hemoglobin based oxygen carriers. The present invention includes a capillary fluid/gas exchange system and a sample mixing chamber. An oxygen electrode is used to measure hemoglobin concentration in the sample. In one embodiment, a pressure transducer and a Millipore filter are used to measure sickle cell concentration in the blood sample.

Description

APPARATUSES AND METHODS FOR ANALYZING GAS EXCHANGE PROPERTIES OF BIOLOGICAL FLUIDS

By Robert M. Winslow

TECHNICAL FIELD

[001] The present invention relates to apparatuses and methods for evaluating gas exchange properties of biological fluids, including blood and artificial blood substitutes. More particularly, it relates to an apparatus that is specifically designed to measure the oxygen carrying characteristics of hemoglobin-containing fluids.

BACKGROUND OF THE INVENTION

[002] The blood is the means for delivering oxygen and nutrients and removing waste products from the tissues. The blood is composed of plasma in which red blood cells (RBCs or erythrocytes), white blood cells (WBCs), and platelets are suspended. Red blood cells comprise approximately 99% of the cells in blood, and their principal function is the transport of oxygen to the tissues and the removal of carbon dioxide therefrom.

[003] The left ventricle of the heart pumps the blood through the arteries and the smaller arterioles of the circulatory system. The blood then enters the capillaries, where the majority of the delivery of oxygen, exchange of nutrients and extraction of cellular waste products occurs. (See, e.g., A. C. Guyton, "Human Physiology And Mechanisms Of Disease" (3rd. ed.; W. B. Saunders Co., Philadelphia, Pa.), pp. 228-229 (1982)). Thereafter, the blood travels through the venules and veins in its return to the right atrium of the heart. Though the blood that returns to the heart is oxygen-poor compared to that which is pumped from the heart, when at rest, the returning blood still contains about 75% of the original oxygen content.

[004] The reversible oxygenation function (i.e., the delivery of oxygen) of RBCs is carried out by the protein hemoglobin. In mammals, hemoglobin has a molecular weight of approximately 64,000 daltons and is composed of about 6% heme and 94% globin. In its native form, it contains two pairs of subunits (i.e., it is a tetramer), each containing a heme group and a globin polypeptide chain. In aqueous solution, hemoglobin is present in equilibrium between the tetrameric (MW 64,000) and dimeric forms (MW 32,000). Outside of the RBC, the dimers are prematurely excreted by the kidney (plasma half-life of approximately 2-4 hours). Along with hemoglobin, RBCs contain stroma (the RBC membrane), which comprises proteins, cholesterol, and phospholipids.

[005] Due to the demand for blood products in hospitals and other settings, extensive research has been directed at the development of blood substitutes. A "blood substitute" is a blood product that is capable of carrying and supplying oxygen to the tissues. Hemoglobin based oxygen carriers (HBOCs) are blood substitutes containing hemoglobins. HBOCs have a number of uses, including replacing blood lost during surgical procedures and following acute hemorrhage, and for resuscitation procedures following traumatic injury. Essentially, HBOCs can be used for any purpose in which banked blood is currently administered to patients. (See, e.g., U.S. Pat. Nos. 4,001,401 to Bonson et al, and 4,061,736 to Morris et al.)

[006] The reversible oxygenation of hemoglobin, whether in the form of blood or an HBOC, is a complex process, which is dramatically influenced by the surrounding environment. For example, if the hemoglobin is inside blood cells, the properties of these cells will affect its ability to bind and release oxygen as it travels through the blood stream. In addition, in the form of an HBOC, the oxygen carrying characteristics of hemoglobin are affected by the manipulations made to the hemoglobin in preparing the HBOC. For example, attaching polyalkylene oxide moieties to form PEG-Hb conjugates increases oxygen affinity and decreases cooperativity of the individual hemoglobin subunits.

[007] Because of the complexities of these interaction, there is a need for systems and methods to objectively evaluate the ability of biological fluids to bind and release oxygen. For example, a need exists to quickly and easily determine oxygen saturation curves for various hemoglobin based oxygen carriers. This is further complicated given the fact that such interactions are both temperature and pH dependent. What is therefore desired is a simple system which is flexible enough to perform a variety of blood/gas analyses under controllable conditions.

[008] Moreover, a need exists to measure blood cell siding such that the effectiveness of various therapies for sickle cell patients can be evaluated. US2005/031244

SUMMARY OF THE INVENTION

[0091 The present invention provides a device adapted to study blood gas exchange. The present invention can be used for many different types of analyses, including but not limited to, measuring sickle cell concentration in a blood sample, and determining an oxygen saturation curve for a hemoglobin based oxygen carrier.

[010] In preferred embodiments, the device includes: a gas exchange chamber having a fluid inlet, a fluid outlet, a gas inlet and a gas outlet; a mixing chamber having an inlet and an outlet channel, the inlet being in communication with the fluid outlet of the gas exchange chamber; a mixing system adapted to mix contents of the mixing chamber; and at least one of: an oxygen electrode adapted to measure oxygen concentration in the mixing chamber; or a pressure transducer adapted to measure pressure in the outlet channel.

[Oil] In a preferred aspect, the present invention provides a method of measuring sickle cell concentration in a blood sample, by: passing a blood sample through a mixing chamber and into an outlet channel having an exit end covered by a millipore filter; mixing the blood sample within the mixing chamber; decreasing the concentration of oxygen in the blood sample over time; measuring the concentration of oxygen in the blood sample over time; measuring the pressure of the blood sample in the outlet channel over time; and measuring sickle cell concentration in the blood sample by determining the relationship between the concentration of oxygen in the blood sample and the pressure of the blood sample in the outlet channel over time.

[012] In another preferred aspect, the present invention provides a method of determining an oxygen saturation curve for a hemoglobin based oxygen carrier, by: placing a hemoglobin based oxygen carrier into a mixing chamber; mixing the hemoglobin based oxygen carrier within the mixing chamber; changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time; while determining the concentration of oxygenated hemoglobin in the hemoglobin based oxygen carrier over time; thereby determining an oxygen saturation curve for the hemoglobin based oxygen carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[013] Fig. 1 depicts a cross sectional side view of an exemplary apparatus according to the present invention taken along line 1-1 in Fig. 2. ⁴ PCT/US2005/031244

[014] Fig. IB is a vievv similar to Fig. 1, showing additional optional features of the present invention.

[015] Fig. 2 depicts a top plan view of the apparatus of Fig. 1.

[016] Fig. 3 is an enlarged broken-away sectional view of the end portions of the gas exchange chamber of Fig. 1.

[017] Fig. 4 is a plot of fluid pressure vs. the partial pressure of oxygen of a blood sample positioned in the outlet channel against the Millipore filter of Fig. 1.

[018] Fig. 5 is a plot of an oxygen saturation curve for a hemoglobin based oxygen carrier in the device of Fig. 1.

DESCRIPTION OF THE INVENTION

[019] The present invention relates to apparatuses and methods for evaluating gas exchange properties of biological fluids, including blood and artificial blood substitutes. More particularly, it relates to an apparatus that is specifically designed to measure the oxygen carrying characteristics of oxygen-containing fluids, or "oxygen carriers". Such oxygen carriers include, inter alia, blood, hemoglobin based oxygen carriers.

[020] To facilitate understanding of the invention set forth in the disclosure that follows, a number of terms are defined below.

[021] The term "hemoglobin" refers generally to the protein contained within red blood cells that transports oxygen. Each molecule of hemoglobin has 4 subunits, 2 ά chains and 2 β 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.

[022] The term "modified hemoglobin" includes, but is not limited to, hemoglobin altered by a chemical reaction such as intra- and inter-molecular cross-linking, genetic manipulation, polymerization, and/or conjugation to other chemical groups (e.g., polyalkylene oxides, for example polyethylene glycol, or other adducts such as proteins, peptides, carbohydrates, synthetic polymers and the like). In essence, hemoglobin is "modified" if any of its structural or functional properties have been altered from its native state. As used herein, the term "hemoglobin" by itself refers both to native, unmodified, hemoglobin, as well as modified hemoglobin. 5 PCT/US2005/031244

[023] The term ''surface-modified hemoglobin" is used to refer to hemoglobin described above to which chemical groups such as dextran or polyalkylene oxide have been attached, most usually covalently. The term "surface modified oxygenated hemoglobin" refers to hemoglobin that is in the "R" state when it is surface modified.

[024] The term "stroma-free hemoglobin" refers to hemoglobin from which all red blood cell membranes have been removed.

[025] The term "methemoglobin" refers to an oxidized form of hemoglobin that contains iron in the ferric state and cannot function as an oxygen carrier.

[026] The term "MaIPEG-Hb" refers to hemoglobin to which malemidyl- activated PEG has been conjugated. Such MaIPEG may be further referred to by the following formula:

Hb-(S-Y-R-CH₂-CH₂-[O-CH₂-CH₂]_n-O-CH₃)_m Formula I where Hb refers to tetrameric hemoglobin, S is a surface thiol group, Y is the succinimido covalent link between Hb and MaI-PEG, R is an alkyl, amide, carbamate or phenyl group (depending on the source of raw material and the method of chemical synthesis), [0-CH₂- CH₂],_! are the oxy ethylene units making up the backbone of the PEG polymer, where n defines the length of the polymer (e.g., MW = 5000), and 0-CH₃ is the terminal methoxy group. PHP and POE are two different PEG-modified hemoglobin.

[027] The term "plasma expander" refers to any solution that may be given to a subject to treat blood loss.

[028] The term "oxygen carrying capacity," or simply "oxygen capacity" refers to the capacity of a blood substitute^' to carry oxygen, but does not necessarily correlate with the efficiency in which it delivers oxygen. Oxygen carrying capacity is generally calculated from hemoglobin concentration, since it is known that each gram of hemoglobin binds 1.34 ml of oxygen. Thus, the hemoglobin concentration in g/dl multiplied by the factor 1.34 yields the oxygen capacity in ml/dl.

[029] Hemoglobin concentration can be measured by any known method, such as by using the β-Hemoglobin Photometer (HemoCue, Inc., Angelholm, Sweden). Similarly, oxygen capacity can be measured by the amount of oxygen released from a sample of hemoglobin or blood by using, for example, a fuel cell instrument (e.g., Lex-0₂-Con; Lexington Instruments).

[030] The term "oxygen affinity" refers to the avidity with which an oxygen carrier such as hemoglobin binds molecular oxygen. This characteristic is defined by the 2005/031244

oxygen equilibrium curve which relates the degree of saturation of hemoglobin molecules with oxygen (Y axis) with the partial pressure of oxygen (X axis). The position of this curve is denoted by the value, P50, the partial pressure of oxygen at which the oxygen carrier is half-saturated with oxygen, and is inversely related to oxygen affinity. Hence the lower the P50, the higher the oxygen affinity.

[031] The oxygen affinity of whole blood (and components of whole blood such as red blood cells and hemoglobin) can be measured by a variety of methods known in the art. (See, e.g., Winslow et al, J. Biol. Chem. 252(7):2331-37 (1977)). Oxygen affinity may also be determined using a commercially available HEMOX™ TM Analyzer (TCS Scientific Corporation, New Hope, Pennsylvania). (See, e.g., Vandegriff and Shrager in "Methods in Enzymology" (Everse et al, eds.) 232:460 (1994)).

[032] The term "oxygen-carrying component" refers broadly to a substance capable of carrying oxygen in the body's circulatory system and delivering at least a portion of that oxygen to the tissues. In preferred embodiments, the oxygen-carrying component is native or modified hemoglobin, and is also referred to herein as a "hemoglobin based oxygen carrier," or "HBOC".

[033] The term "hemodynamic parameters" refers broadly to measurements indicative of blood pressure, flow and volume status, including measurements such as blood pressure, cardiac output, right atrial pressure, and left ventricular end diastolic pressure.

[034] The term "crystalloid" refers to small molecules (usually less than 10 A) such as salts, sugars, and buffers. Unlike colloids, crystalloids do not contain any oncotically active components and equilibrate in between the circulation and interstitial spaces very quickly.

[035] The term "colloid," in contrast to "crystalloid" refers to larger molecules (usually greater than 10 A) that equilabrate across biological membranes depending on their size and charge and includes proteins such as albumin and gelatin, as well as starches such as pentastarch and hetastarch.

[036] The term "colloid osmotic pressure" refers to the pressure exerted by a colloid to equilibrate fluid balance across a membrane.

[037] The term "stable to autooxidation" refers to the ability of a HBOC to maintain a low rate of autoxidation. HBOC is considered stable at 24⁰C if the methemoglobiπ/total hemoglobin ratio does not increase more than 2% after 10 hours at 24⁰C. For example, if the rate of autoxidation is 0.2In^"1, then if the initial percentage of -,

7 PCT/US2005/031244

methemoglobin is 5%, HBOC would be considered stable at room temperature for 10 hours if this percentage did not increase above 7%.

[038] The term "methemoglobin/total hemoglobin ratio" refers to the ratio of deoxygenated hemoglobin to total hemoglobin.

[039] The term "mixture" refers to a mingling together of two or more substances without the occurrence of a reaction by which they would lose their individual properties; the term "solution" refers to a liquid mixture; the term "aqueous solution" refers to a solution that contains some water and may also contain one or more other liquid substances with water to form a multi-component solution; the term "approximately" refers to the actual value being within a range, e.g. 10%, of the indicated value.

[040] In contrast, the term "mixing" as used herein may also include simply "stirring" a single substance, such as stirring a blood sample within a mixing chamber such that blood cells do not accumulate at the bottom of the mixing chamber as the fluid sample leaves the mixing chamber.

[041] The term "polyethylene glycol" refers to liquid or solid polymers of the general chemical formula H(OCH₂CH₂)_nOH, where n is greater than or equal to 4. Any PEG formulation, substituted or unsubstituted, can be used.

[042] The term "perfusion" refers to the flow of fluid to tissues and organs through arteries and capillaries.

[043] The term "hemodynamic stability" refers to stable functioning in the mechanics of blood circulation.

[044] The term "hypotensive events" is characterized by or due to hypotension, or a lowering of blood pressure.

[045] The meaning of other terminology used herein should be easily understood by someone of reasonable skill in the art.

The Nature of Hemoglobin Oxygen Binding and Release

a. Oxygen Distribution

[046] Although the successful use of the apparatuses and methods of the present invention does not require comprehension of the underlying mechanisms of hemoglobin oxygen binding and release, basic knowledge regarding some of these mechanisms may assist in understanding some of the concepts discussed herein. [047] It has generally been assumed that the capillaries are the primary conveyors of oxygen to the tissue. However, regarding tissue at rest, current findings indicate that there is an approximately equal distribution between arteriolar and capillary oxygen release. More particularly, hemoglobin in the arterial system is believed to deliver approximately one-third of its oxygen content in the arteriolar network and one-third in the capillaries, while the remainder exits the microcirculation via the venous system.

[048] As indicated, the arteries themselves are sites of oxygen utilization. For example, the artery wall requires energy to effect regulation of blood flow through contraction against vascular resistance. Thus, the arterial wall is normally a significant site for the diffusion of oxygen out of the blood. However, oxygen carriers (e.g., HBOCs) may release too much of their oxygen content in the arterial system, which induces an autoregulatory reduction in capillary perfusion. Accordingly, the efficiency of oxygen delivery by an oxygen carrier may actually be hampered by having too much oxygen or too low an oxygen affinity.

[049] The rate of oxygen consumption by the vascular wall, which is required for both mechanical work and biochemical processes, can be determined by measuring the gradient at the vessel wall. See, e.g., Winslow, et al., in "Advances in Blood Substitutes" (1997), Birkhauser, ed., Boston, MA, pages 167-188. Present technology allows accurate oxygen partial pressure measurements in a variety of vessels. The measured gradient is directly proportional to the rate of oxygen utilization by the tissue in the region of the measurement. Such measurements show that the vessel wall has a baseline oxygen utilization that increases with increased inflammation and constriction, and is lowered by relaxation.

[050] Whereas the vessel wall gradient is directly proportional to the rate of oxygen utilization, it is not surprisingly inversely proportional to tissue oxygenation. Vasoconstriction increases the oxygen gradient (tissue metabolism), while vasodilation lowers the gradient. Higher gradients are indicative of the fact that more oxygen is used by the vessel wall, while less oxygen is available for the tissue. The same phenomenon is believed to be present throughout the microcirculation.

b. The Relationship Between Vasoconstriction and Oxygen Affinity

[051] The rationale for developing an HBOC with high oxygen affinity is based, in part, on past studies using cell-free hemoglobins as alternatives to red blood cell transfusions. Some of the physiological effects of these solutions remain incompletely understood. Of these, perhaps the most controversial is the propensity to cause vasoconstriction, which may be manifested as hypertension in animals and man (Amberson, W., "Clinical experience with hemoglobin-saline solutions,". Science 106: 117-117 (1947)) (Keipert, P., A. Gonzales, C. Gomez, V. Macdonald, J. Hess, and R. Winslow, "Acute changes in systemic blood pressure and urine output of conscious rats following exchange transfusion with diaspirin-crosslinked hemoglobin solution," Transfusion 33: 701-708, (1993)).

[052] Human hemoglobin crosslinked between α chains with bis- dibromosalicyl-fumarate (ααHb) was developed by the U. S. Army as a model red cell substitute, but was abandoned by the Army after demonstration of severe increases in pulmonary and systemic vascular resistance (Hess, J,, V. Macdonald, A. Murray, V. Coppes, and C. Gomez, "Pulmonary and systemic hypertension after hemoglobin administratio," Blood 78: 356A (1991)). A commercial version of this product was also abandoned after a disappointing Phase III clinical trial (Winslow, R. M. "αα-Crosslinked hemoglobin: Was failure predicted by preclinical testing?" Vox sang 79: 1-20 (2000).

[053] The most commonly advanced explanation for the vasoconstriction produced by cell-free hemoglobin is that it readily binds the endothelium-derived relaxing factor, nitric oxide (NO). In fact, recombinant hemoglobins with reduced affinity for NO have been produced which appear to be less hypertensive in top-load rat experiments (Doherty, D. H., M. P. Doyle, S. R. Curry, R. J. VaIi, T. J. Fattor, J. S. Olson, and D. D. Lemon, "Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin," Nature Biotechnology 16: 672-676 (1998)) (Lemon, D. D., D. H. Doherty, S. R. Curry, A. J. Mathews, M. P. Doyle, T. J. Fattor, and J. S. Olson, "Control of the nitric oxide-scavenging activity of hemoglobin," Art Cells, Blood Subs., andlmmob. Biotech 24: 378 (1996)).

[054] However, studies suggest that NO binding may not be the only explanation for the vasoactivity of hemoglobin. It has been found that certain large hemoglobin molecules, such as those modified with polyethylene glycol (PEG), were virtually free of the hypertensive effect, even though their NO binding rates were identical to those of the severely hypertensive ααHb (Rohlfs, R. J., E. Bruner, A. Chiu, A. Gonzales, M. L. Gonzales, D. Magde, M. D. Magde, K. D. Vandegriff, and R. M. Winslow, "Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide," J Biol Chem 273: 12128-12134 (1998)). Furthermore, it was found that PEG-hemoglobin was extraordinarily effective in preventing the consequences of hemorrhage when given as an exchange transfusion prior to hemorrhage (Winslow, R. M., A. Gonzales, M. Gonzales, M. Magde, M. McCarthy, R. J. Rohlfs, and K. D. Vandegriff, "Vascular resistance and the efficacy of red cell substitutes," J Appl Physiol 85: 993-1003 (1998)).

[055] This protective effect correlated with the lack of hypertension, suggesting that vasoconstriction is responsible for the disappointing performance of many of the hemoglobin-based products studied to date. Based on these observations, a hypothesis was developed to explain vasoconstriction, as an alternative, or possibly in addition to, the effect of NO binding. Although not wishing to be bound by any particular theory, it is believed that a substantial component of hemoglobin's vasoactive effect is a reflexive response to the diffusion of hemoglobin in the cell-free space. This hypothesis was tested in an in vitro capillary system, and it was demonstrated that PEG-hemoglobin, which has a reduced diffusion constant, transferred O₂ in a manner very similar to that of native red blood cells (McCarthy, M. R., K. D. Vandegriff, and R. M. Winslow, "The role of facilitated diffusion in oxygen transport by cell-free hemoglobin: Implications for the design of hemoglobin-based oxygen carriers," Biophysical Chemistry 92: 103-117 (2001)). Oxygen affinity would be expected to play a role in its facilitated diffusion by hemoglobin in the plasma space, since the change in saturation from the hemoglobin to the vessel wall is a determinant of the diffusion gradient of the hemoglobin itself.

[056] Oxygen affinity of cell-free hemoglobin may play an additional role in the regulation of vascular tone, since the release of O₂ to vessel walls in the arterioles will trigger vasoconstriction (Lindbom, L., R. Tuma, and K. Arfors, "Influence of oxygen on perfusion capillary density and capillary red cell velocity in rabbit skeletal muscle," Microvasc Res 19: 197-208 (1980)). In the hamster skinfold, the PO₂ in such vessels is in the range of 20-40 Torr, where the normal red cell oxygen equilibrium curve is steepest (Intaglietta, M., P. Johnson, and R. Winslow, "Microvascular and tissue oxygen distribution," Cardiovasc Res 32: 632-643 (1996)). Thus from a theoretical point of view, it may be important for the P50 of cell-free hemoglobin to be lower than that of red cells (i.e., higher O2 affinity), in order to prevent release Of O₂ in arteriolar regulatory vessels. 1 1 PCT7US2005/031244

c. Oxygen Release

[057] In addition to oxygen affinity, the oxygen binding properties of the individual hemoglobin subunits, i.e. cooperativity and allosteric effects, may play a crucial role in the release of oxygen by HBOCs. It has been observed that polyalkylene oxide binding to hemoglobin results in a general "tightening" of the globin structure. This is attributed to the osmotic effects of having a hydrophilic shell surrounding the hemoglobin, and also depends on the nature and location of the linking groups used to attach the polyalkylene oxide. Most conventional wisdom is that the design of HBOCs should mimic the characteristics of native red blood cells. However, it has unexpectedly been found that perturbation of the quaternary conformation of the hemoglobin can have advantages, particularly in the context of oxygen offloading.

[058] A protein is considered to be "allosteric" if its characteristics change as a result of binding to an effector molecule, i.e. a ligand, at its allosteric site. In the case of hemoglobin, the ligand is oxygen. Each subunit of the hemoglobin tetramer is capable of binding one oxygen molecule. Each subunit also exists in one of two conformations - tense (T) or relaxed (R). In the R state, it can bind oxygen more readily than in the T state.

[059] Hemoglobin exhibits a concerted effect, or cooperativity, among individual subunits binding oxygen. The binding of oxygen to one subunit induces a conformational change that causes the remaining active sites to have an enhanced oxygen affinity. This is because binding of the first oxygen destabilizes both the intrachain and interchain ionic interactions (hydrogen bonds and salt bridges), which causes a general "loosening" of the tertiary structure. Accordingly, each sequential oxygen to be bound to the hemoglobin molecule attaches more readily than the one before, until the hemoglobin molecule has achieved the R, or "liganded" state, with four attached oxygen molecules.

[060] In the reverse, native hemoglobin exhibits a concerted effect in terms of its efficiency to release oxygen. The first molecule is more tightly attached and takes more energy to be released than the next one, and so on. Accordingly, the conventional teachings towards the design of blood substitutes that mimic the cooperativity of native hemoglobin may adversely affect its ability to release oxygen once bound.

[061] It is now believed that HBOCs with less, not more, cooperativity than native hemoglobin may be more beneficial for use in certain applications. 44

FlBOC Design

a. Hemoglobin Sources

[062] The hemoglobin may be either native (unmodified); subsequently modified by a chemical reaction such as intra- or inter-molecular cross-linking, polymerization, or the addition of chemical groups (e.g. , polyalkylene oxides, or other adducts); or it may be recombinantly engineered. Human alpha- and beta-globin genes have both been cloned and sequenced. Liebhaber, et al, P.N.A.S. 77: 7054-7058 (1980); Marotta, et al., J. Biol. Chem. 353: 5040-5053 (1977) (beta-globin cDNA). In addition, many recombinantly produced modified hemoglobins have now been produced using site-directed mutagenesis. See, e.g., Nagai, et al., P.N.A.S., 82: 7252-7255 (1985).

[063] The present invention is not limited by the source of the hemoglobin. For example, the hemoglobin may be derived from animals and humans. Preferred sources of hemoglobin for certain applications are humans, cows and pigs. In addition, hemoglobin may be produced by other methods, including chemical synthesis and recombinant techniques. The hemoglobin can be added to the blood product composition in free form, or it may be encapsulated in a vesicle, such as a synthetic particle, microballoon or liposome. The preferred oxygen-carrying components of the present invention should be stroma free and endotoxin free. Representative examples of oxygen-carrying components are disclosed in a number of issued United States Patents, including U.S. Pat. No. 4,857,636 to Hsia; U.S. Pat. No. 4,600,531 to Walder, U.S. Pat. No. 4,061,736 to Morris et al; U.S. Pat. No. 3,925,344 to Mazur; U.S. Pat. No. 4,529,719 to Tye; U.S. Pat. No. 4,473,496 to Scannon; 4,584,130 to Bocci et al; U.S. Pat. No. 5,250,665 to Kluger et al.; U.S. Pat. No. 5,028,588 to Hoffman et al; and U.S. Pat. No. 4,826,811 and U.S. Pat. No. 5,194,590 to Sehgal et al.

[0641 In addition to the aforementioned sources of hemoglobin, it has recently been found that horse hemoglobin has certain advantages as the oxygen carrying component in the compositions of the present invention. One advantage is that commercial quantities of horse blood are readily available from which horse hemoglobin can be purified. Another unexpected advantage is that horse hemoglobin exhibits chemical properties that may enhance its usefulness in the blood substitutes of the present invention.

[065] Previous reports have indicated that horse hemoglobin auto-oxidizes to methemoglobin faster than human hemoglobin, which would make it less desirable as a blood substitute component. See, e.g., J.G. McLean and LM. Lewis, Research in Vet. Sci., 19:259- PCWS2005/03U44

262 (1975). In order to minimize auto-oxidation, McLean and Lewis used a reducing agent, glutathione, after red blood cell lysis. However, the hemoglobin that is used to prepare the compositions of the present invention, regardless of whether the source of hemoglobin is human or horse, do not require the use of reducing agents to prevent auto-oxidation after red blood cell lysis.

[066] More recently, it has been reported that horse hemoglobin has an oxygen affinity that is different from that of human hemoglobin. See, e.g., M. Mellegrini, et al, Eur. J. Biochem., 268: 3313-3320 (2001). Such a difference would discourage the selection of horse hemoglobin to prepare blood substitutes that mimic human hemoglobin. However, when incorporated into the compositions of the present invention, no significant difference (less than 10%) in oxygen affinity between human and horse hemoglobin-containing conjugates is observed. Accordingly, contrary to these seemingly undesirable properties, in the compositions of the present invention, horse hemoglobin is equivalent if not superior to human hemoglobin.

[067] For certain applications, an HBOC will have an oxygen affinity that is greater than whole blood, and preferably twice that of whole blood, or alternatively, greater than that of stroma-free hemoglobin (SFH), when measured under the same conditions. In most instances, this means that the HBOC in the blood substitute will have a P50 less than 10, and more preferably less than 7. In the free state, SFH has a P50 of approximately 15 torr, whereas the P50 for whole blood is approximately 28 torr. It has previously been suggested that increasing oxygen affinity, and thereby lowering the P50, may enhance delivery of oxygen to tissues, although it was implied that a P50 lower than that of SFH would not be acceptable. See Winslow, et al., in "Advances in Blood Substitutes" (1997), Birkhauser, ed., Boston, MA, at page 167, and U.S. Patent No. 6,054,427. This suggestion contradicts the widely held belief that modified hemoglobins for use as blood substitutes should have lower oxygen affinities, and should have P50s that approximate that of whole blood. Hence, many researchers have used pyridoxyl phosphate to raise the P50 of SFH from 10 to approximately 20-22, since pyridoxylated hemoglobin more readily releases oxygen when compared to SFH.

[068] There are many different scientific approaches to manufacturing HBOCs with high oxygen affinity {i.e. those with P50s less than SFH). For example, studies have identified the amino acid residues that play a role in oxygen affinity, such as B-93 Cysteine, and thus site-directed mutagenesis can now be easily carried out to manipulate oxygen 14 PCT7US2005/031244

affinity to the desired level. See, e.g., U.S. Patent No. 5,661,124. Many other approaches are discussed in U.S. Patent No. 6,054,427.

b. Hemoglobin Associated Toxicity

[069] Hemoglobin is known to exhibit autooxidation when it reversibly changes from the ferrous (Fe²⁺) to the ferrie (Fe³⁺) or methemoglobin form. When this happens, molecular oxygen dissociates from the oxyhemoglobin in the form of a superoxide anion (O₂- ). This also results in destabilization of the heme-globin complex and eventual denaturation of the globin chains. Both oxygen radical formation and protein denaturation are believed to play a role in vivo toxicity of HBOCs (Vandegriff, K. D., Blood Substitutes, Physiological Basis of Efficacy, pages 105-130, Winslow et al, ed., Birkhauser, Boston, MA (1995).)

[070] With most HBOCs, there is a negative correlation between oxygen affinity and hemoglobin oxidation, i.e., the higher the oxygen affinity, the lower the rate of autooxidation. However, the effects of different hemoglobin modifications on oxygen affinity and the rate of autooxidation are not always predictable. In addition, the optimal balance between oxygen affinity and autooxidation rate is not well understood.

[071] In one embodiment, the compositions of the present invention contain PEG-Hb conjugates that exhibit very low rates of autooxidation. When measured as a rate of oxidation, this value should be as low as possible (i.e.,0.2% per hour of total hemoglobin, more preferably 0.1% per hour of total hemoglobin, at room temperature for at least 3 hours, and more preferably at least 10 hours.) Thus, exemplary HBOCs of the present invention remain stable during administration and/or storage at room temperature.

c. Hemoglobin Modifications

[072] In an exemplary embodiment, the HBOC is polyalkylene oxide (PAO) modified hemoglobin. Suitable PAOs include, inter alia, polyethylene oxide ((CH₂CH₂O)_n), polypropylene oxide ((CH(CH₃)CH₂O)_n) or a polyethylene/polypropylene oxide copolymer ((CH₂CH₂O)_n-(CH(CH₃)CH₂O)_n). Other straight, branched chain and optionally substituted synthetic polymers that would be suitable in the practice of the present invention are well known in the medical field.

[073] Most commonly, the chemical group attached to the hemoglobin is polyethylene glycol (PEG), because of its pharmaceutical acceptability and commercial 15 PCT7US2005/031244

availability. PEGs are polymers of the general chemical formula H(OCH₂CHi)_nOH, where n is generally greater than or equal to 4. PEG formulations are usually followed by a number that corresponds to their average molecular weight. For example, PEG-200 has an average molecular weight of 200 and may have a molecular weight range of 190-210. PEGs are commercially available in a number of different forms, and in many instances come preactivated and ready to conjugate to proteins.

[074] In one embodiment, surface modification of the HBOC takes place when the hemoglobin is in the oxygenated or "R" state. This is easily accomplished by allowing the hemoglobin to equilibrate with the atmosphere (or, alternatively, active oxygenation can be carried out) prior to conjugation. By performing the conjugation to oxygenated hemoglobin, the oxygen affinity of the resultant hemoglobin is enhanced. Such a step is generally regarded as being contraindicated, since many researchers describe deoxygenation prior to conjugation to diminish oxygen affinity. See, e.g., U.S. Pat. No. 5,234.903.

[075] Although in many respects the performance of PAO modified hemoglobins is independent of the linkage between the hemoglobin and the modifier (e.g. PEG)₅ it is believed that more rigid linkers such as unsaturated aliphatic or aromatic Cj to C₆ linker substituents may enhance the manufacturing and/or characteristics of the conjugates when compared to those that have more flexible and thus deformable modes of attachment.

[076] The number of PEGs to be added to the hemoglobin molecule may vary, depending on the size of the PEG. However, the molecular size of the resultant modified hemoglobin should be sufficiently large to avoid being cleared by the kidneys to achieve the desired half-life. Blumenstein, et al., determined that this size is achieved above 84,000 molecular weight. (Blumenstein, et al., in "Blood Substitutes and Plasma Expanders," Alan R. Liss, editors, New York, New York, pages 205-212 (1978).) Therein, the authors conjugated hemoglobin to dextran of varying molecular weight. They reported that a conjugate of hemoglobin (with a molecular weight of 64,000) and dextran (having a molecular weight of 20,000) "was cleared slowly from the circulation and negligibly through the kidneys," but increasing the molecular weight above 84,000 did not alter the clearance curves. Accordingly, as determined by Blumenstein, et ah, it is preferable that the HBOC have a molecular weight of at least 84,000. [077] In one embodiment of the present invention, the HBOC is a "MaIPEG,¹' which stands for hemoglobin to which malemidyl-activated PEG has been conjugated. Such MaIPEG may be further referred to by the following formula:

Hb-(S-Y-R-CH₂-CH₂-[O-CH₂-CH₂]_n-O-CH₃)_m Formula I where Hb refers to tetrameric hemoglobin, S is a surface thiol group, Y is the succinimido covalent link between Hb and MaI-PEG, R is an alkyl, amide, carbamate or phenyl group (depending on the source of raw material and the method of chemical synthesis), [OCH₂- CH₂]_n are the oxyethylene units making up the backbone of the PEG polymer, where n defines the length of the polymer (e.g., MW = 5000), and 0-CH₃ is the terminal methoxy group.

d. HBOC Formulations

[078] HBOC-based blood substitutes are formulated by mixing the HBOC and other optional excipients with a suitable diluent. Although the concentration of the HBOC in the diluent may vary according to the application, and in particular based on the expected post-administration dilution, in preferred embodiments, because of the other features of the compositions of the present invention that provide for enhanced oxygen delivery and therapeutic effects, it is usually unnecessary for the concentration to be above 6 g/dl, and is more preferably between 0.1 to 4 g/dl.

[079] Suitable (i.e. pharmaceutically acceptable for intravenous injection) diluents include, intra alia, solutions of proteins, glycoproteins, polysaccharides, and other colloids or other non-oxygen carrying components. The HBOC formulation usually has a viscosity of at least 2.5 cP. In some preferred embodiments, the viscosity is between 2.5 and 4 cP or higher.

Other Oxygen Carriers

[080] In addition to being useful for studying the oxygen carrying characteristics of HBOCs, the apparatuses and methods of the present invention are useful in evaluating suspensions of blood cells, as well as whole blood. In particular, the present invention is easily adapted to study the oxygen carrying characteristics of blood cells and whole blood from patients suffering from sickle cell disease and other hemoglobinopathies. .._

17 PCT/US2005/031244

Oxygen Measurements

[081] Analyzing the oxygen cany ing capacity of blood and other oxygen carriers provides information about the overall cardiopulmonary system. Accordingly, the present invention presents apparatuses and methods to analyze the oxygen carrying characteristics of oxygen carriers, and in particular hemoglobin based oxygen carriers (HBOCs).

a. Measuring hemoglobin oxygen saturation

[082] Measuring hemoglobin oxygen saturation can be accomplished using a blood gas machine, a co-oximeter, a pulse-oximeter or an intravascular fiber optic oximeter. Arterial hemoglobin oxygen saturation is usually measured via arterial blood gas analysis or by co-oximetry. Using a co-oximeter, oxyhemoglobin can be distinguished from deoxyhemoglobin on the basis of different light refraction properties.

[083] An alternative way of measuring hemoglobin oxygen saturation is by directly measuring the partial pressure of oxygen and then plotting the PO2 on an oxygen dissociation curve. Another way is by using a pulse oximeter which measures the transdermal absorption of light by hemoglobin flowing through the microcapillary system.

b. The alveolar-arterial oxygen gradient

[084] PaO₂ is the partial pressure of arterial oxygen. Oxygen molecules free in plasma (not bound to hemoglobin) are detectable by an oxygen electrode that detects randomly moving dissolved oxygen molecules in plasma. PaO₂ is unrelated to the characteristics of hemoglobin, since it is a measure of free oxygen. Oxygen molecules that are inhaled transit through the alveolar-capillary membrane and enter the plasma. Most enter red blood cells and become bound to hemoglobin. The more the dissolved oxygen, the higher the concentration of bound oxygen.

[085] The alveolar oxygen saturation is calculated, and given by PaO₂. PaO₂ is always higher than PaO₂ because of the circulatory architecture that leads to a mixing of venous (oxygen depleted) blood with the pulmonary circulatory system. The difference defines the alveolar-arterial oxygen gradient. Knowledge about the gradient tells you if the lungs are taking up oxygen efficiently. If the gradient is high, they are not. 18 PCT7US2005/031244

[086] SaO2 is the percent of total hemoglobin oxygen, binding sites that are actually occupied by bound oxygen. The oxygen content of blood in ml O₂/dl is given by: [0871 CaO₂ = SaO₂ X Hb (g/dl) X 1.34 ml 0₂/gm Hb

c. Sickle Cell Disease and Oxygen Delivery

[088] Sickle cell disease is a genetic disorder characterized by a single amino acid substitution in one of the hemoglobin molecule subunits. More specifically, it is caused by the substitution of the amino acid valine for glutamic acid at the sixth residue of the beta- globin chain. This abnormal form of hemoglobin is referred to as "HbS". When HbS is deoxygenated, it forms fibers within the red blood cells, which causes them to sickle and become rigid. This in turn affects their ability to flow through the microcapillary system, which causes blockages that lead to tissue hypoxia, since no oxygen can be delivered to the tissues downstream from the blockage. In severe cases, sickle cell disease results in acute pain.

[089] As indicated, the formation of HbS fibers is initiated when HbS is deoxygenated and thus becomes less soluble than its oxygenated counterpart. In particular, under low oxygen conditions, a small aggregate of deoxygenated HbS molecules form a critical nucleus that initiates fiber formation. Although this condition is reversible by oxygenation, repeated sickling and unsickling eventually damages the red blood cell membrane.

[090] In the case of normal hemoglobin, all of the different methods for measuring oxygen saturation have very good correlation. However when the population of sickle cells in a blood sample is significant, the measurements can be skewed. For example, pulse oximetry underestimates oxygenation of sickled blood, whereas the calculated method greatly overestimates hemoglobin oxygen saturation.

Gas Exchange Measuring System ("GEMS"^{^})

[091] The apparatuses and methods of the present invention can be used in determining oxygen saturation curves for various hemoglobin based oxygen carriers. More generally, the present invention can be used to study blood gas exchange. In one particular aspect, the apparatuses and methods of the present invention can be used to measure the degree of sickling of cells in a blood sample. , _r

19 PCT7US2005/031244

Representative Uses of the Present Invention:

[092] As described herein, the apparatuses of the present invention can be used to:

Evaluate the sickling characteristics of a patient's blood.

Evaluate the oxygen carrying capacity of a hemoglobin based oxygen carrier.

Measure the oxygen saturation curves of oxygen carriers.

Monitor the effects of therapies on oxygen carrying characteristics.

Basic Apparatus Design

[093] Figs. 1 and 2 show a preferred embodiment of the present gas exchange measuring system (GEMS) device 5. As will be explained, GEMS device 5 is useful in evaluating the oxygen carrying characteristics of any hemoglobin based oxygen carrier. In addition, GEMS device 5 is useful in evaluating different test conditions to enhance oxygen carrying characteristics of various oxygen carriers.

[094] GEMS device 5 includes a gas exchange member 10 and a mixing and measuring member 20. As seen in Figs. 1, 2 and 3, gas exchange member 10 has a fluid inlet 11 and a fluid outlet 13. Gas exchange member 10 also has gas inlet 14 and a gas outlet 16.

[095] As is seen in more detail in Fig. 3, gas exchange member 10 includes a plurality of gas exchange capillaries 12. Gas exchange capillaries 12 are small structures that are permeable to gas but are impermeable to fluid. In preferred embodiments, gas exchange capillaries 12 maybe made of small tubes of polydimethylsiloxane. An example of a suitable gas exchange capillary system can be seen in US Patent 6,269,679 (See Fig. 9 in particular). Capillaries 12 may be received into glass tubes 15 which are together received into a silicone sealing body 17.

[096] The sample fluid to be analyzed enters fluid inlet 11 , simultaneously passing along through the individual gas exchange capillaries 12, and then exits from fluid outlet 13. The gas passing from gas inlet 14 to gas outlet 16 passes around each of capillaries 12. Therefore, as the sample fluid passes along, through gas exchange capillaries 12 it equilibriates with the gas simultaneously passing through gas exchange member 10. For example, if oxygen is passed through gas exchange member 10 (i.e. from gas inlet 14 to gas outlet 16) the fluid sample passing through capillaries 12 will tend to become oxygenated. Conversely, if nitrogen is passed through gas exchange member 10 (i.e. from gas inlet 14 to 20 PCT7US2005/031244

gas outlet 16) the fluid sample passing through capillaries 12 will tend to become de- oxygenated.

[097] Therefore, by varying the oxygen concentration passing through gas exchange member 10, the oxygen concentration in the fluid sample (in capillaries 12) can be varied. When the fluid sample assign through gas exchange member 10 is blood or other hemoglobin based oxygen carrier, it is therefore possible to selectively fully oxygenate or de- oxygenate the blood / hemoglobin based oxygen carrier.

[098] As a result, when the oxygen concentration of the gas passing through gas exchange member 10 is varied over time, the oxygen concentration in the sample fluid exiting fluid outlet 13 correspondingly varies over time. For example, by replacing oxygen in gas exchange member 10 with nitrogen over time, the sample fluid exiting fluid outlet 13 becomes progressively de-oxygenated over time. Conversely, by replacing nitrogen in gas exchange member 10 with oxygen over time, the sample fluid exiting fluid outlet 13 becomes progressively oxygenated over time. In this embodiment, the sample can be cycled back through the oxygenator. As a result, gas exchange member 10 can be used in providing a sample fluid having oxygenation characteristics that very over time. As will be shown, this is especially useful both in determining oxygen saturation curves and when measuring sickle cell formation.

[099] In optional embodiments, delivery of the fluid sample into the fluid inlet 11 of gas exchange member 10 can optionally be accomplished by a syringe or via a pumping device (not shown). A luer lock may be provided at fluid inlet 11 to facilitate connection of such a syringe/ luer lock. In various embodiments, the fluid sample may be delivered into fluid inlet 11 by manual pressure, or be pumped at a constant flow rate.

[0100] The sample fluid exiting fluid outlet 13 of gas exchange member 10 enters directly into inlet 21 of mixing and measuring member 20, thereby passing into mixing chamber 22. The mixing and measuring member 20 can be made of any suitable material. For convenience, it is constructed of Lucite material or another clear plastic material.

[0101] Mixing chamber 22 includes a mixing (i.e.: "mixing" or simple "stirring") system that ensures that cells or other components do not settle in the bottom of mixing chamber 22. Any suitable system that provides mixing or stirring of the contents of chamber 22 can be used. In one exemplary embodiment, a small magnetic stir bar 25 is used. Stir bar 25 is rotated by a remote rotating magnet mixing apparatus 26. A particular advantage of using a magnetic stir bar 25 is that the rotating magnet mixing apparatus 26 may be placed 21 PCT/US2005/031244

outside of mixing chamber 22. There is no physical contact between stir bar 25 and rotating magnet mixing apparatus 26. This substantially reduces the potential for introducing contamination into mixing chamber 22.

[0102] Various measurements and analyses of the fluid sample in mixing chamber 22 can be conducted within mixing and measuring member 20. In preferred embodiments, member 20 includes at least one of: an oxygen electrode 23, or a pressure transducer 24.

[0103] Oxygen electrode 23 may be positioned in port 27, as shown, to measure the oxygen concentration of the fluid sample in mixing chamber 22. (In accordance with the present invention, however, oxygen electrode 23 may instead be located with outlet channel 28 or even at inlet 21 of member 20.)

[0104] Pressure transducer 24 may be located within port 29, as shown, to measure the fluid pressure within outlet channel 28. In accordance with the present invention, pressure transducer 24 may also be located within mixing chamber 22. (However, locating pressure transducer 24 within outlet channel 28 may be preferred so as to minimize the effects of pressure changes in the fluid induced by rotating stir bar 25.)

[0105] The fluid sample to be analyzed in member 20 enters by way of inlet 21 , passes into mixing chamber 22 where it is stirred (or optionally mixed with another substance, as will be explained). Thereafter, the fluid sample exits member 20 by way of outlet channel 28.

[0106] In one preferred embodiment of the invention, mixing chamber 22 has a narrowed upper portion 29 (e.g.: an inverted funnel portion, as shown) connecting outlet channel 28 to mixing chamber 22. The narrowed upper portion 29 may be useful in permitting any bubbles in the fluid sample to exit from mixing chamber 22 (through outlet channel 28) prior to the start of any fluid sample analysis.

[0107] In preferred embodiments of the invention, outlet channel 28 extends vertically away from mixing chamber 22, as shown. However, the present invention is not so limited. For example fluid samples instead may exit from mixing chamber 22 through other outlet channels, or other channel orientations. However, an advantage of having outlet channel 28 extend vertically out of the top of mixing chamber 22 is that it also facilitates the removal of bubbles from mixing chamber 22 prior to the start of any fluid sample analysis.

[0108] In preferred embodiments, member 20 may be fabricated from a block of Lucite™, but is not so limited. As illustrated, member 20 may be fabricated from a top block 2005/031244

of material 20A and a bottom block of material 2OB, held together by screws 30. It is to be understood that such design is merely exemplary, and is not limiting.

[0109] In optional embodiments, member 20 may further include an optional Millipore filter 40 disposed across the top end of outlet channel 28. As will be explained, Millipore filter 40 is useful in determining the degree of sickling / profiles in the fluid sample. In various optional embodiments, outlet channel 28 is at least 1 cm long. As will also be explained, having output channel 28 be of a sufficient length is also useful in determining sickle cell concentrations / profiles in the fluid sample.

[0110] In optional embodiments, as shown in Fig. IB, member 20 may further include an optical probe 50 configured to measure color changes in the fluid sample in mixing chamber 22. Such an optical probe may be especially useful in measuring oxygen concentration in a blood (or other hemoglobin based oxygen carrier) sample since the concentration of oxygenated hemoglobin in the sample changes the color of the sample significantly.

[0111] In optional embodiments, member 20 may also include a carbon dioxide electrode 52 to measure the pH of the fluid sample in mixing chamber 22.

[0112] In further optional embodiments, device 20 may also include a separate fluid access channel 32 into mixing chamber 22. Fluid access channel 32 permits fluid to be added into mixing chamber 22 by a path other than from inlet 21. As will be explained, fluid access channel 32 can be used to add a reaction substance into mixing channel 22 after the sample fluid has been passed through. In such embodiments, the fluid sample entering mixing chamber 22 through inlet 21 and fluid access channel 32 both exit mixing chamber 22 through outlet channel 28.

[0113] In further optional embodiments, the volume of mixing chamber 22 may be adjustable. For example, mixing device 26 may be located on a movable piston 54 that forms the bottom of mixing chamber 22.

[0114] Having set forth the preferred embodiments of the present invention, various methods of using the present invention will now be set forth.

Sickle Cell Analysis:

[0115] In one preferred aspect, the present invention is used to perform an analysis of sickling of blood cells in a blood sample. Fig. 4 illustrates the fluid pressure vs. 23 PCT/US2005/031244

the partial pressure of oxygen in the blood sample when the analysis has been performed according to the preferred method.

[0116] The fluid pressure in Fig. 4 is measured by pressure transducer 24 in outlet channel 28; and the partial pressure of oxygen is measured by oxygen electrode 23 in the mixing chamber. Millipore filter 40 is positioned on top of outlet channel 28 as shown in

Fig. 1.

[0117] The viscosity of blood increases when blood cells sickle. This property of blood is used in measuring the extent of sickling (and in evaluating therapies for sickle cell patients) as follows.

[0118] A blood sample is passed through gas exchange member 10 concurrently with (pure) oxygen being passed through gas exchange member 10. As a result, the blood sample equilibriates with the oxygen, thus becoming fully oxygenated when entering mixing chamber 22. Oxygen electrode 23 measures oxygen concentration by measuring the partial pressure of oxygen in the (fully) oxygenated blood sample while pressure transducer 24 measures the fluid pressure in outlet channel 28 (at a position adjacent to Millipore filter 40). The partial pressure of oxygen and the fluid pressure in the (fully) oxygenated blood sample are shown as point Pl in Fig. 4.

[0119] Next, the concentration of oxygen in the blood sample (in mixing chamber 22) is decreased over time. This can be done by passing the blood sample through gas exchange member 10 while simultaneously passing a non-oxygen gas through gas exchange member 10. Such a non-oxygen gas may include an inert gas such as nitrogen, but is not so limited.

[0120] As a result, the concentration of oxygen in the blood sample (passing through gas exchange member 10 and into mixing chamber 22) can be decreased (gradually) over time by simply replacing oxygen passing through the gas exchange chamber with a non- oxygen gas overtime, i.e. decreasing the concentration of oxygen passing through gas exchange member 10 over time.

[0121] As the concentration of oxygen in the blood sample (gradually) decreases over time, the cells in the blood will eventually start to sickle. Such sickling will cause the blood sample to become more viscous. During operation, the blood sample (leaving mixing chamber 22 through outlet channel 28) passes continuously through Millipore filter 40. As sickling increases, it becomes increasingly more difficult for the blood sample to pass through Millipore filter 40. As a result, the pressure in the blood sample (in both mixing 24 PCT7US2005/031244

chamber 22 and outlet channel 28) will increase. Therefore, when fluid is passed through device 20 at a constant rate, the pressure measured in the outlet channel 28 (e.g. near Millipore filter 40) will increase over time. Rotating magnetic stir bar 25 stirs the blood sample, thus ensuring that the blood cells do not settle to the bottom of mixing chamber 22, but instead reach Millipore filter 40.

[01221 As can be seen in Fig. 4, as the oxygen concentration in the blood sample is decreased over time, the pressure in the blood sample will increase (moving from point Pl to P2). Thus, the relationship of blood cell sickling vs. partial pressure of oxygen in the blood sample can be determined.

[0123] The present invention can also be used to measure the effectiveness of sickle cell therapies, as follows. The relationship of blood cell sickling vs. partial pressure of oxygen can be measured in a first blood sample (line 100 between points Pl and P2, as explained above). Next, a therapeutic agent (to reduce blood cell sickling) can be added to the blood sample, with the above test repeated. If successful, the relationship of blood cell sickling vs. partial pressure of oxygen will appear as line 102 (between the same starting point Pl and a new ending point P3). As can be seen, although the partial pressure of oxygen is the same at points P2 and P3, the fluid pressure at point P3 is lower than that at point P2. This indicates the effectiveness of the therapeutic agent since there is less sickling at the same partial pressure of oxygen. As a result, the present invention can be used to evaluate the effectiveness of different therapeutic agents in preventing or reducing blood cell sickling.

[0124] In addition, the present invention can be used to evaluate differences in blood cell sickling for the same patient, measured at different periods of time. Moreover, the present invention can be used to evaluate differences in blood cell sickling among different patients.

[0125] As explained above, the concentration (partial pressure) of oxygen can be measured by oxygen electrode 23. It is to be understood, however, that the present invention is not so limited. For example, in an alternate embodiment of the invention, the concentration of oxygen in the blood sample can also be measured with optical probe 50 configured to measure color changes in the blood sample. _c

25 PCT/US2005/031244

Oxygen Saturation Curve, Anal^ysis:

[0126] In various preferred aspects, the present invention is used to determine an oxygen saturation curve for a hemoglobin based oxygen carrier. Fig. 5 is a plot of an oxygen saturation curve for a hemoglobin based oxygen carrier when the analysis has been performed according to the preferred method. Specifically, Fig. 5 illustrates the relationship between the concentration of oxygenated hemoglobin and the partial pressure of oxygen in the hemoglobin based oxygen.

[0127] In accordance with the present invention, an oxygen saturation curve for a hemoglobin based oxygen carrier is determined by: placing a hemoglobin based oxygen carrier into mixing chamber 22; mixing (including simply "stirring") the hemoglobin based oxygen carrier within mixing chamber 22; changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time; while determining the concentration of oxygenated hemoglobin in the hemoglobin based oxygen carrier over time; thereby determining an oxygen saturation curve for the hemoglobin based oxygen carrier.

[0128] Similar to the sickle cell method described above, the saturation curve of Fig. 5 is determined by: measuring the oxygen concentration in the fluid (i.e.: the hemoglobin based oxygen carrier) sample with oxygen electrode 23, while varying the partial pressure of oxygen in the fluid (i.e.: the hemoglobin based oxygen carrier).

[0129] In one preferred aspect of the method, the hemoglobin based oxygen carrier is de-oxygenated prior to placing it into mixing chamber 22. As was described above, this can be done by passing the hemoglobin based oxygen carrier through gas exchange member 10, thereby equilibriating the hemoglobin based oxygen carrier with a de- oxygenating gas passing through gas exchange member 10.

[0130] In various aspects of the invention, the saturation curve of Fig. 5 can be determined by either: (a) shutting off flow from gas exchange member 10 into mixing chamber 22 prior to changing (and measuring) the partial pressure of oxygen in the hemoglobin based oxygen carrier over time, or (b) permitting continuous flow from gas exchange member 10 into mixing chamber 22, while changing (and measuring) the partial pressure of oxygen in the hemoglobin based oxygen carrier over time.

[0131] In the first approach, flow into mixing chamber 22 is shut off prior to changing (and measuring) the partial pressure of oxygen. The partial pressure of oxygen in the hemoglobin based oxygen carrier in mixing chamber 22 is then varied by introducing a substance into mixing chamber 22 through fluid access channel 32. For example, the hemoglobin based oxygen carrier may have been de-oxygenated (in gas exchange member 10) prior to it being placed into mixing chamber 22. Then, the flow from gas exchange member 10 into mixing chamber 22 is shut off. An oxygenating substance is then introduced into mixing chamber 22 through fluid access channel 32. Such oxygenating substance may include, but is not limited to, H₂O₂ or oxygenated hemoglobin.

[0132] In an alternate aspect of the above method, the hemoglobin based oxygen carrier may have been oxygenated prior to being placed it into mixing chamber 22. Then, a de-oxygenating substance may be introduced (through fluid access channel 32) into mixing chamber 22. Such a de-oxygenating substance may be an oxygen-consuming enzyme, but is not so limited. In this embodiment, it may be desired to have an optical channel to measure hemoglobin saturation. However, if the rate of oxygen consumption by the enzyme is known, then the total oxygen can be calculated as a function of time. The PO₂ measured with an electrode can be converted to dissolved oxygen concentration. Then, the difference between total oxygen and dissolved oxygen is bound oxygen. Bound oxygen divided by total oxygen capacity is the same as saturation.

[0133] In one approach (in which flow from gas exchange member 10 into mixing chamber 22 is shut off), the concentration of oxygenated hemoglobin is determined empirically.

[0134] In a second approach, there is continuous flow between gas exchange member 10 and mixing chamber 22 while varying (and measuring) the partial pressure of oxygen in the hemoglobin based oxygen carrier over time. For example, the partial pressure of oxygen in the hemoglobin based oxygen carrier may be varied over time by: passing the hemoglobin based oxygen carrier through gas exchange member 10 while changing the oxygen concentration of the gas over time. The concentration of oxygenated hemoglobin in mixing chamber 22 can be measured by either of: oxygen electrode 23 in mixing chamber 22, or by optical probe 50 measuring oxygen concentration in mixing chamber 22. Actually, the oxygen electrode does not measure dissolved oxygen directly. Dissolved oxygen is related to PO₂ by the solubility coefficient, i.e. O₂ - alpha X PO₂, where alpha is the solubility coefficient unique to any particular gas.

[0135] As was described above, rotating magnetic stir bar 25 within mixing chamber 22 ensures that cells and other components do not settle in the bottom of mixing chamber 22. ^ PCWS2005/03U44

[0136] In various aspects of the invention, the hemoglobin based oxygen carrier in the mixing chamber may be blood or native hemoglobin. Optionally, such hemoglobin may be surface modified with PEG or any other modified form of hemoglobin.

[0137] Oxygen saturation curves are known to be very temperature dependent. Therefore, in accordance with both of the above aspects of the method of determining saturation curves, the temperature at which the device operates is preferably closely controlled.

[0138] Oxygen saturation curves are also known to be very pH dependent. Therefore, in accordance with both of the above aspects of the method of determining saturation curves, the pH within mixing chamber 22 is preferably both measured and controlled. For example, carbon dioxide electrode 52 may be used to directly measure the pH in mixing chamber 22. In addition, acids or bases may be selectively added to mixing chamber 22 to maintain a constant pH in the mixing chamber. Optionally, such acids or bases may be added through fluid access channel 32.

[0139] The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred . embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

Claims

ClaimsWhat is claimed is:

1. A gas exchange measuring device adapted to study gas exchange in a fluid, comprising: a gas exchange member having a fluid inlet, a fluid outlet, a gas inlet and a gas outlet; a mixing chamber having an inlet and an outlet channel, the inlet being in communication with the fluid outlet of the gas exchange member; a mixing system adapted to mix contents of the mixing chamber; and at least one of: an oxygen electrode adapted to measure oxygen concentration in the mixing chamber; or a pressure transducer adapted to measure pressure in the outlet channel.

2. The device of claim 1 further comprising an optical channel disposed within the gas exchange chamber.

3. The device of claim 1, wherein the gas exchange chamber comprises a plurality of gas exchange capillaries configured to pass fluid therethrough, with one end of each capillary in communication with the fluid inlet of the gas exchange chamber and the other end of capillary in communication with the fluid outlet of the gas exchange chamber.

4. The device of claim 2, wherein the gas exchange capillaries are positioned within the gas exchange chamber such that gas passes therearound as the gas moves into the gas inlet and out of the gas outlet of the gas exchange chamber.

5. The device of claim 2, wherein the each of the capillaries is made of tubes of polydimethylsiloxane.

6. The device of claim 1, wherein the mixing system comprises: a magnetic element disposed within the mixing chamber; and 29 PCT7US2005/031244

a rotating magnet configured to cause the magnetic element to rotate within the mixing chamber, the rotating magnet being disposed outside of the mixing chamber, and not being in physical contact with the magnetic element.

7. The device of claim 1 , wherein the mixing chamber has a narrowed upper portion, and wherein the outlet channel of the mixing chamber extends upwardly from the narrowed upper portion of the mixing chamber.

8. The device of claim 1 , wherein the mixing chamber is disposed in a clear plastic housing.

9. The device of claim 1, further comprising a millipore filter covering an end of the outlet channel of the mixing chamber.

10. The device of claim 1, further comprising an optical probe configured to measure optical changes in a fluid sample in the mixing chamber.

11. The device of claim 1 , wherein the length of the outlet channel is at least lcm.

12. The device of claim I₃ wherein the volume of the mixing chamber is adjustable.

13. The device of claiml , further comprising a fluid access channel into the mixing chamber.

14. The device of claim 1 , further comprising a carbon dioxide electrode adapted to measure pH in the mixing chamber.

15. A method of monitoring sickle cells in a blood sample, comprising: passing a sample containing blood cells through a mixing chamber and into an outlet channel having an exit end covered by a millipore filter; mixing the sample within the mixing chamber; decreasing the concentration of oxygen in the sample over time; 30 PCT7US2005/031244

measuring the concentration of oxygen in the sample over time; measuring the pressure of the sample over time; and monitoring sickle cells in the sample by determining the relationship between the concentration of oxygen in the sample and the pressure of the sample over time.

16. The method of claim 15, wherein decreasing the concentration of oxygen in the sample over time comprises: passing the sample through a gas exchange chamber configured to equilibriate the sample with a gas passing through the gas exchange chamber; while passing a non-oxygen gas through the gas exchange chamber, thereby de-oxygenating the sample passing through the gas exchange chamber.

17. The method of claim 16, wherein the non-oxygen gas is an inert gas.

18. The method of claim 17, wherein the inert gas is nitrogen.

19. The method of claim 16, wherein passing a non-oxygen gas through the gas exchange chamber comprises: replacing oxygen passing through the gas exchange chamber with a non-oxygen gas over time.

20. The method of claim 15, wherein mixing the sample in the mixing chamber comprises: rotating a magnetic element within the mixing chamber.

21. The method of claim 15 , wherein measuring the concentration of oxygen in the sample over time comprises: measuring the concentration of oxygen in the mixing chamber with an oxygen electrode.

22. The method of claim 15 , wherein measuring the concentration of oxygen in the sample over time comprises: 31 PCT7US2005/031244

measuring the concentration of oxygen in the mixing chamber with an optical probe configured to measure optical changes in the sample.

23. The method of claim 15, wherein measuring the pressure of the sample in the outlet channel over time comprises: measuring the pressure of the sample with a pressure transducer in the outlet channel.

24. A method of determining an oxygen saturation curve for a hemoglobin based oxygen carrier, comprising: placing a hemoglobin based oxygen carrier into a mixing chamber; mixing the hemoglobin based oxygen carrier within the mixing chamber; changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time; while determining the concentration of oxygenated hemoglobin in the hemoglobin based oxygen carrier over time; thereby determining an oxygen saturation curve for the hemoglobin based oxygen carrier.

25. The method of claim 24, further comprising: de-oxygenating the hemoglobin based oxygen carrier prior to placing it into the mixing chamber.

26. The method of claim 25, wherein de-oxygenating the hemoglobin based oxygen carrier comprises: passing the hemoglobin based oxygen carrier through a gas exchange chamber configured to equilibriate the hemoglobin based oxygen carrier with a non-oxygen gas passing through the gas exchange chamber.

27. The method of claim 24, wherein flow of the hemoglobin based oxygen carrier from the gas exchange chamber to the mixing chamber is shut off prior to: changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time, and 32 PCT7US2005/031244

determining the concentration of oxygenated hemoglobin in the hemoglobin based oxygen carrier over time.

28. The method of claim 24, wherein changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time is accomplished by: introducing a substance into the mixing chamber, the substance changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time.

29. The method of claim 28, wherein the hemoglobin based oxygen carrier has been de-oxygenated prior to being placed into the mixing chamber, and wherein the substance is an oxygenating substance.

30. The method of claim 29, wherein the oxygenating substance is H₂O₂.

31. The method of claim 29, wherein the oxygenating substance is oxygenated hemoglobin.

32. The method of claim 31 , wherein the oxygenating substance is introduced into the mixing chamber through a channel different from that which the hemoglobin based oxygen carrier is introduced into the mixing chamber.

33. The method of claim 24, wherein the concentration of oxygenated hemoglobin is determined empirically.

34. The method of claim 24, wherein the hemoglobin based oxygen carrier flows continuously from the gas exchange chamber to the mixing chamber while: changing the partial pressure of oxygen in the hemoglobin based oxygen carrier over time, and while determining the concentration of oxygenated hemoglobin in the hemoglobin based oxygen carrier over time.

35. The method of claim 24, wherein the partial pressure of oxygen in the hemoglobin based oxygen carrier is changed over time by: P C T / U S O 5 / 3 15 «Ψ4.. ³³ PCT/US²⁰⁰⁵/0³¹²⁴⁴

passing the hemoglobin based oxygen carrier through, a gas exchange chamber configured to equilibriate the hemoglobin based oxygen carrier with a gas passing through the gas exchange chamber; while changing the oxygen concentration of the gas over time.

36. The method of claim 24, wherein determining the concentration of oxygenated hemoglobin comprises: measuring the concentration of oxygenated hemoglobin by an oxygen electrode in the mixing chamber.

37. The method of claim 24, wherein determining the concentration of oxygenated hemoglobin comprises: measuring the concentration of oxygenated hemoglobin with an optical probe in the mixing chamber.

38. The method of claim 24, wherein mixing the hemoglobin based oxygen carrier in the mixing chamber comprises: rotating a magnetic element within the mixing chamber.

39. The method of claim 24, wherein the hemoglobin based oxygen carrier is native hemoglobin.

40. The method of claim 24, wherein the hemoglobin based oxygen carrier is hemoglobin that has been surface modified with polyethylene glycol.

41. The method of claim 24, further comprising: measuring the pH in the mixing chamber.

42. The method of claim 41 , further comprising: adding acids or bases to maintain a constant pH in the mixing chamber.

43. The method of claim 25, wherein the hemoglobin based oxygen carrier is deoxygenated by an oxygen-consuming enzyme.