EP0804244A1

EP0804244A1 - Compositions including heme-containing proteins and methods relating thereto

Info

Publication number: EP0804244A1
Application number: EP94912841A
Authority: EP
Inventors: Dennis M. Disorbo; R. Bruce Reeves
Original assignee: BIORELEASE TECHNOLOGIES Inc
Current assignee: BIORELEASE TECHNOLOGIES Inc
Priority date: 1993-03-26
Filing date: 1994-03-23
Publication date: 1997-11-05
Also published as: AU6523194A; WO1994022482A1

Abstract

Heme-containing proteins having bound thereto nonoxygen gas ligands for in vivo and in vitro uses are provided. Cell culture preparations and pharmaceutical preparations, as well as methods for preparing the foregoing, also are provided. The preferred nonoxygen gas ligands are carbon dioxide, carbon monoxide and nitric oxide.

Description

COMPOSITIONS INCLUDING HEME-CONTAINING PROTEINS

AND METHODS RELATING THERETO

Field of the Invention

The invention relates to he e-containing proteins having bound thereto nonoxygen gas ligands for jin vitro and iji vivo uses, as well as cell culture preparations and pharmaceutical preparations relating thereto.

Background of the Invention

Cells grown under aerobic conditions can convert one molecule of glucose to carbon dioxide and water and generate 36 molecules of ATP. Under anaerobic conditions, however, one molecule of glucose is converted to lactic acid with the generation of only two molecules of ATP. Thus, oxygen depletion, or limitation, requires up to 18 times more glucose to be supplied to a cell to generate an equivalent number of ATP molecules produced under aerobic conditions. An efficient mechanism known for delivery cf oxygen to organs is through the association of oxygen with hemoglobin.

Native human hemoglobin is predominantly a tetrameric molecule with a molecular mass of 64,000 daltons. The tetramer is composed of two pairs of alpha and beta sub-units held together by non-covalent forces. Each sub-unit (16,000 daltons) is conjugated to one heme moiety, and in the tetrameric configuration, each forms a heme pocket which maintains the iron atom in the ferrous state (a gas ligand binding state.) When hemoglobin is removed from the red blood cell the ferrous atom becomes susceptible to oxidation and conversion to the ferric state. This conversion results in the formation of methemoglobin, a form of hemoglobin that does not bind oxygen or other gas ligands and has a reduced solubility in solution. Various investigators have attempted to purify, stabilize, and modify hemoglobin so that it may be used as a red blood cell substitute. Investigators have cross-linked hemoglobin inter olecularly and intramolecularly to avoid renal toxicity. In particular, when free hemoglobin is administered to a patient, it tends to dissociate into dimeric and monomeric units. These units are small enough such that they are filtered by the kidney and produce renal toxicity. By cross linking the sub-units of hemoglobin and polymerizing tetramers of hemoglobin, the dissociation of the sub-units and renal toxicity are avoided.

Cross-linkinig is not always desired because cross-linking techniques can involve multiple steps, can use potentially toxic reagents, and can adversely affect the affinity of oxygen or other gas ligands for the heme group. Nevertheless, the use of noncross-1inked hemoglobin is not generally practiced because of the toxicity associated with dimeric and monomeric units.

When culturing cells in vitro, oxygen typically is applied to the cells by bubbling oxygen into the media in which the cells are suspended. Although it has been suggested that oxygen be supplied to cells by using medium containing hemoglobin saturated with oxygen, such attempts have been largely unsuccessful. Perhaps the lack of success is caused by the problems relating to the stability of hemoglobin.

Recently, investigators have saturated hemoglobin with carbon monoxide (CO), replacing the oxygen at the heme moiety, to stabilize hemoglobin against conversion to methemoglobin. For various reasons, the carbon monoxide then would be replaced with oxygen prior to use. Carbon monoxide has been known to act as a pulmonary poison in living organisms. In addition, carbon monoxide (and nitric oxide - NO) readily displace oxygen from hemoglobin. It, therefore, would be expected that once carbon monoxide or nitric oxide has been complexed to hemoglobin, that hemoglobin should not readily act as an effective exchange agent for free oxygen in solution and should not readily increase available oxygen to cells or living tissues since hemoglobin affinity for CO and NO is substantially higher than that of oxygen. Furthermore, if the purpose of adding hemoglobin to cell culture or to blood is to enhance delivery of oxygen to cells or living tissue, then it only makes sense to preload the hemoglobin with oxygen. Thus, the clear teaching of the prior art is to replace carbon monoxide with oxygen prior to use.

The prior art also teaches that it is desirable to lower the binding affinity of hemoglobin for oxygen to promote the release of oxygen to cells or tissues. One example is the pyridoxalation of hemoglobin for the purpose of making hemoglobin a more effective oxygen delivery agent in vivo. In addition, cross-linking can improve the stability of hemoglobin.

It is known that oxygen, carbon monoxide, carbon dioxide, and nitric oxide bind to hemoglobin.

It is an object of the invention to provide methods and products for delivering nonoxygen gas ligands to cells and/or tissues.

It is an object of the invention to provide improved methods and products for culturing cells.

It is an object of the invention to provide methods for delivering nonoxygen gas ligands to cells for therapeutic purposes and for improved cell culture purposes.

These and many other objects will be understood with reference to the following description of the invention.

Summary of the Invention

The invention utilizes he e-containing proteins having bound thereto nonoxygen gas ligands for in vitro and _in vivo uses, as well as cell culture preparations and pharmaceutical preparations relating thereto.

According to one aspect of the invention, a method for delivering a nonoxygen gas ligand to a cell is provided. The

SUB environment of the cell is contacted with a nonoxygen gas ligand that is reversibl-y bound to a heme moiety of a heme-containing protein for the purpose of delivering the nonoxygen gas ligand to the cell. Preferably, the cell is contacted with a gas ligand selected from the group consisting of NO, CO, and C0₂, and preferably the heme-containing protein is selected from the group consisting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin.

The heme-containing protein may be delivered in solution or may be contained in a liposome. Likewise, the heme-containing protein may be contained in a pharmaceutically acceptable carrier or in fresh cell culture medium.

It is preferred that the nonoxygen gas ligands occupy at least 25% of the heme moieties of the heme-containing protein, more preferred that the nonoxygen gas ligands occupy at least 50% of such moieties, even more preferred that the nonoxygen gas ligands occupy at least 75% of such moieties, and most preferred that the nonoxygen gas ligands saturate the heme moieties, occupying at least 90% of such moieties.

According to another aspect of the invention, a pharmaceutical preparation is provided. The pharmaceutical preparation is a mixture of a pharmaceutically acceptable carrier and a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen binding heme moieties of isolated heme containing protein. The preferred heme-containing proteins and gas ligands are as described above. Also as described above, the heme-containing protein may be contained in liposomes. The preferred concentration of the gas ligand also is as described above.

According to another aspect of the invention, a method for forming a pharmaceutical preparation is provided. The method involves mixing a pharmaceutically acceptable carrier with a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of

SUBSTITUTE isolated heme-containing protein. The preferred aspects are as described above.

According to another aspect of the invention, preparations of improved cell culture medium also are provided. Such preparations are fresh cell culture medium containing a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of isolated heme-containing protein, wherein the gas ligands occupy at least 25% of the heme-moietieε. Again, the preferred gas ligands are NO, CO, and C0₂ and the preferred heme-containing proteins are hemoglobin, native hemoglobin, myoglobin, and native myoglobin. The heme-containing protein may be free in solution or contained in liposomes contained in the medium. It is more preferred that the nonoxygen gas ligands occupy at least 50% of the heme moieties, still more preferred that the nonoxygen gas ligands occupy at least 75% of such moieties, and most preferred that the nonoxygen gas ligands saturate the moieties, occupying at least 90% of the moieties. In a particularly preferred embodiment, the fresh, cell culture medium contains isolated, stroma-free, native bovine hemoglobin that is not cross-linked and that is saturated with nonoxygen gas ligand.

The foregoing improved cell-culture media may be prepared by mixing any conventional media with the ligand-bound heme-containing proteins described above.

According, still another aspect of the invention, methods, and products for delivering biological agents are provided This aspect of the invention involves the use of heme-containing proteins as carriers for delivering biological agents to ^*he environment of cells. The heme-containing protein can have noncovalently bound to them various biological agents, including cell culture enhancers and biologically active molecules. In this manner, such treated heme-containing proteins can be used in the preparation of improved cell culture media or pharmaceutical preparations. They are particularly useful for delivering

SUBSTITUTE non-soluble agents (lipids) and for delivering precise and/or small amounts of biologically active molecules.

The heme-containing proteins, the compositions containing such proteins, and the methods utilizing such proteins can involve any one or all of the delivery of a biological agent, the delivery of a nonoxygen gas ligand, and the delivery of a stabilized oxygen-binding heme moiety. Thus, the preparations of the invention can include fresh cell culture medium or pharmaceutically acceptable carriers mixed with a heme-containing protein that has bound to it one or both of a nonoxygen gas ligand and a biological agent. The invention also provides isolated heme-containing proteins having noncovalently bound to their isolated biological agents, including lipids and cell culture enhancers.

All of the foregoing products may be used in connection with the methods of the invention. As will be readily understood, the environment of the cells can be contacted with the various products described above, including heme-containing proteins that have bound to them one or both of a nonoxygen gas ligand and a biological agent. The preferred heme-containing proteins and concentration of nonoxygen gas ligand are as described above.

Brief Description of the Drawings

Fig. 1 is a graph illustrating that the methemoglobin level remains constant throughout the period of addition of carbon monoxide to the bovine hemoglobin isolated as described in the examples below.

Fig. 2 is a graph illustrating cell growth when different doses of the material prepared according to the examples below are provided in tissue culture.

Fig. 3 is a graph illustrating that the presence of hemoglobin prepared according to the examples below results in a decrease in the rate of loss of oxygen from the medium to which the hemoglobin is applied. Detailed Description of the Invention

The invention relates, in one important aspect, to loading heme-containing proteins with nonoxygen gas ligands. As described in greater detail below, such gas-carrying proteins are useful for a variety of purposes, including both in vitro and jLn vivo. Such applications include delivering nonoxygen gas ligands to cells to stimulate metabolic activity and to provide metabolic substrates. They also include providing a stable oxygen trap that enhances cell growth and lengthens the useful life of cell-culture media. The applications also include providing blood substitutes.

As used herein, a "heme moiety" is an iron-containing prosthetic group of a protein. Such proteins are called heme-containing proteins. The heme moiety may be ferrous (II) or ferric (III) and is capable of reversibiy binding to the nonoxygen gas ligands of the invention. Preferably, the heme moiety includes a tetrapyrrole ring such as a protoporphyrin (IX) ring. It is preferred that the heme moiety be capable of reversibiy binding oxygen, although this is not necessary for all applications herein.

A "heme-containing protein" as used herein is a gas-ligand transporter that preferably is otherwise free of biological activity, such as enzymatic activity. As a result, the heme-containing proteins useful in the invention will not have biologically or medically unacceptable side effects. It will be understood, however, that the heme-containing proteins useful in the invention may have biological activity other than providing a transporter function, provided that such activity does not produce a medically unacceptable side effect or a side effect unacceptable for in vitro use.

As used herein, heme-containing proteins that are gas-ligand transporters reversibiy bind oxygen, carbon monoxide, carbon dioxide, or nitric oxide. Specifically excluded are those molecules that release gas ligands as a result of chemical modification of the molecule (e.g.

SUBSTITUTE nitroprusside) as opposed to reversibiy binding and releasing a gas ligand.

The gas-ligand transporters useful in the invention include natural transporters, such as native hemoglobin and native myoglobin, isolated from blood or produced synthetically, such as by genetic engineering. As used herein, native hemoglobin and native myoglobin are terms restricted to the foregoingN The native hemoglobins include ^α2^β2' ^α2^Υ2 ^α2^δ2' ^Alal' ^Ala2' ^Alb and A. , as well as any sub-units thereof.

The gas-ligand transporters useful in the invention also include various modifications of the foregoing native transporters, such as those that result from modification by intermolecular and/or intramolecular cross-linking or from complexing with other molecules. As used herein, the terms hemoglobin and myoglobin encompass not only native hemoglobin and native myoglobin, but also the various recombinant derivatives and protein, chemically modified derivatives as described in more complete detail below.

Some other examples of gas-ligand transporters that are heme-containing proteins include the following analogs and derivatives of hemoglobin: (1) 2, 3-diphosphoglycerate (DPG) or DPG analogs such as pyridoxal-5'-phoεphate-(PLP) covalently attached to hemoglobin in its deoxy state (Greenberg et al. , Surgery, Volume 86 (1979); (2) polymerized PLP-hemoglobin intermolecularly cross-linked by a nonspecific crosslinker, Bonhard et al., U.S. Pat.No. ,136,093;

(3) PLP-hemoglobin conjugated polyethelene glycol, Iwasaki et al., Artificial Organs, Volume 10, No. 5 (1986);

(4) hemoglobin conjugated with dextran, Tan et al. Pros. Nat.'l Acad. Sci. U.S.A. Vol. 73 (1976); (5) intramolecularly polymerized hemoglobin, U.S. Pat.No. 4,061,736;

(6) hemoglobin conjugated to polyalkylene glycol, Iwashida U.S. Pat.Nos.4,412,989 and 4,301,144; (7) hemoglobin conjugated to polyalkylene oxide, Iwasaki, U.S. Pat .No.4,670,417; (8) hemoglobin conjugated with inosotol phoεphate, Nicolau U.S. Pat.Noε.4,321,259 and 4,473,563;

(9) hemoglobin conjugated with an inosotol phosphate and a polysaccharide, Wang U.S. Pat.No.4,710,488; (10) hemoglobin conjugated proteins and gelatin derivates, Bonhard, U.S.

Pat.No.4,336,248; (11) intramolecularly cross-linked hemoglobin, Walder, U.S. Pat.Nos.4,598,064 and 4,600,531;

(12) hemoglobin conjugated with insulin, Ajisaka, U.S.

Pat.No.4,377,512; (13) cloned hemoglobin and mutants thereof,

Hoffman, U.S. Pat.No.5,028,588; Liebhaber, P.N.A.S. (U.S.A.)

77(1980); and Marotta, et al. J.Biol.Chem. , 252(1977) and

(14) hemoglobin cross-linked with diaspirin, U.S.

Pat.No.4,529,719; (15) BIS-diamide covalently cross linked, pyridoxal-5'-phosphate covalently modified tetrameric hemoglobin, Tye, U.S. Pat. No. 4,529,719; and

(16) α,-Ω-dialdehyde cross-linked hemoglobin, Scannon,

U.S. Pat. No. 4,473,496.

The foregoing examples are but representative of the various heme-containing proteins known to those of ordinary skill in the art, and the invention is not intended to be restricted to the foregoing list. The entire diεcloεureε of the foregoing patents and references are incorporated herein by reference.

A nonoxygen gas ligand, as used herein, is a molecule that is a gas at room temperature. Such nonoxygen gas ligands include only those ligands that have an electro-negative charge and that are capable of reversibiy binding to the heme moiety of the heme-containing proteins used in the invention. Such reversible binding haε been well characterized for oxygen, which iε εpecifically excluded from the term nonoxygen gaε ligand. The nonoxygen gas ligands include, but are not limited to: carbon monoxide (CO); nitric oxide (NO); and carbon dioxide (C0₂).

In connection with hemoglobin, which is a tetramer, the dynamics of gaε ligand binding are well known to thoεe of ordinary εkill in the art. As discusεed above, certain aspects of the invention specify that the gas ligand be bound to at least 25% of the heme moieties present. For a molecule such as hemoglobin, that would indicate that at least one heme moiety of the four heme moieties of hemoglobin has bound to it a gas ligand. It is preferred, however, that at least 50% of the heme moieties (even more preferred that at least 75% of the heme moieties) have bound to them a gas ligand. This would correspond to at least two (or three) of the four heme moieties of hemoglobin being saturated. Most preferably, the heme moieties of the heme-containing proteins are completely saturated with nonoxygen gas ligand. Using the detection equipment available to the inventors, readings of about 90% saturation were obtained with material described in the examples below. Nevertheless, it is believed that such molecules are completely saturated and that the readings indicating less than 100% saturated result from limitations relate to the detection equipment, as opposed to the ability to completely saturate the heme moieties.

The methods for saturating the heme moieties of the heme-containing proteins are well known to those of ordinary skill in the art, and typically involve bubbling the desired nonoxygen gas ligand through the solution containing the protein. The resulting proteins are loaded with nonoxygen gas ligand, and the hemoglobin is stabilized against conversion to methemoglobin.

The heme-containing proteins loaded with nonoxygen gas ligand are contacted with the environment of cells. This iε a departure from the prior art, which replaced the nonoxygen gas ligand CO with oxygen prior to uεe. The environment of the cell, when used in connection with _.in vitro procedures means the fluid or medium in which the cells are suεpended or growing. The environment of the cell, when uεed in connection with in vivo applicationε, means contacted with a living animal via topical adminiεtration, parenteral administration, systemic administration, and the like.

For cell culture, the heme-containing proteinε of the invention are mixed with freεh cell culture medium. Freεh

SUBSTITUTE SHEET cell culture medium, as used herein, means cell culture medium that has not yet been applied to cells, but rather is of the type that is stored in sterile containers for the intended use with cell culturing. Cell culture media are well known to those of ordinary skill in the art, and include commercially available products such as RPMI-1640, HAM F12, Medium 199, Eagles Minimun Essential Medium, Hybridoma Serum Free Medium, SF900 Insect Cell Medium, Excell 400, Ultra CHO, AIM-V, Keratinocyte SFM, Macrophage SFM, Endothelial SFM, LC-115, and Hrbrido a PFHM.

Cell culture medium may be prepared according to procedures well known to those of ordinary skill in the art. In the applications herein, fresh cell culture medium is admixed with a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of heme-containing protein, wherein the gas ligands occupy at least 25% of the heme moieties. Likewise, the fresh cell culture medium may be admixed with heme-containing protein having noncovalently bound to it isolated cell culture enhancers or biological agents. This will be discussed in greater detail below.

As used herein in connection with proteins, agents, and the like, iεolated means separated from the native environment in substantially pure form. According to the procedures set forth in the examples below, isolated bovine hemoglobin means bovine hemoglobin that has been separated from red blood cell stroma and red blood cell cytoplasm. The heme-containing proteins of the invention, of course, can be further isolated and purified as described below.

The term isolated used in connection with biological agents, lipids, or cell culture enhancers means isolated separate and apart from hemoglobin and from a source other than red blood cells. Specifically excluded are those preparations of hemoglobin-associated materials, such as superoxide dismutase and carbonic anhydride that tend to co-iεolare with hemoglobin prepared for example according to

SUBSTITUTE SHEET the examples below. Thus, it should be apparent to thoεe of ordinary skill in the art that the term "isolated heme-containing protein having non-covalently attached to it isolated biological agents" specifically excludes those compositionε that may occur during the course of isolating hemoglobin from red blood cells. Iεolated alεo refers to recombinantly-derived materials.

The heme-containing proteins loaded with nonoxygen gas ligands alεo are useful n vivo in treating animal subjects. Preferably the animal subjects are mammals and, most preferably the mammals are humans, primates, horseε, cows, dogs, cats, goats, sheep, and pigs. The present invention, thus, involveε the use of pharmaceutical formulations which include the heme-containing proteinε of the invention together with one or more pharmaceutically acceptably carriers and optionally other therapeutic ingredients. The carrier(s) and other ingredientε muεt, of courεe, be pharmaceutically acceptable.

In therapeutic applicationε, the moleculeε of the invention can be formulated for a variety of modes of administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mac Pub. Co., Easton, Pa.

The particular administration route selected will depend upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally εpeaking, may be practiced uεing any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the heme-containing proteins of the invention, without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g. εubcutaneouε, intramuscular, and intravenous) routes. Other routes include intraparenchymal injection into targeted areas of an organ, such as a heart.

SUBSTITU Compositions suitable for parentaral administration conveniently comprise a sterile aqueous preparation which is preferably isotonic with the blood of the recipient. Such aqueous preparations may be formulated according to known methodε. Among the acceptable vehicles and solvents that may be employed are water, Ringer's Solution, and isotonic sodium chloride solution. For transmucoεal or tranεdermal administration, penetrants appropriate to the barrier to be permeated are uεed in the formulation. Such penetrantε are generally known in the art, and include, for example, wetting agents. In addition, detergents may be used to facilitate permeation.

When used as a therapeutic agent, the compositions of the invention are administered in therapeutically effective amounts. A therapeutically effective amount means that amount necessary to delay the onset of, inhibit the progresεion of, or halt altogether the onset or progresεion of, the particular condition being treated. Such amounts will depend, of courεe, on the particular condition being treated, the severity of the condition and individual patient parameters such as age, physical condition, size, weight, and concurrent treatment. These factors are well known to thoεe of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum doεe be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasonε or virtually any other reason.

When administering the heme-containing proteins of the invention ij vivo, isεueε such as renal toxicity, half-life of the heme-containing protein, and the presence of endotoxins should be addressed. In general, endotoxins are avoided by preparing hemoglobin using sterile techniques; residual endotoxins are then removed by establiεhed techniqueε. Procedureε for accompliεhing such preparations are well-known to those of ordinary skill in the art, and include procedures such as that described by: Eichentoph, U.S. Patent 4,526,715; Fesla et al . , Surgery, Gynecology and Obstetrics, November 1983, Volume 157, Number 5, p. 399-408; Sheffield et al . , Biotechnology and Applied Biochemistry; Volume 9, 1987, 230-238.

To avoid renal toxicity and to extend the half-life of the heme-containing protein, the heme-containing protein can be intermolecularly crosslinked and/or intra olecularly cross linked. The heme-containing protein also can be derivatized with various agents, as well described in the prior art (see the patents and references listed above) .

In addition, heme-containing proteins may be incorporated into liposomeε. In general, a lipoεome is a spherical particle of lipid substance suspended in an aqueous medium. The use of liposomes has the additional advantage of protecting the hemoglobin from interaction with the kidney. Liposomes include those with membranes consisting of, but not limited to, hydrogenated phospholipid (U.S. Patent 5,649,391), cholesterol, saturated phosphatidylcholine with an acyl chain in excess of 14 carbons and negatively charged lipid (U.S. Patent 4,911,929), and hydrogenated soy phosphatidylcholine and distearoyl phosphatidylcholine, cholesterol, dimyristoyl phoεphatidylglycerol, and alpha-tocopherol (U.S. Patent 4,776,991). The entire disclosureε of the foregoing patentε are incorporated herein by reference. In addition, other techniques for forming liposomes are well known to thoεe of ordinary εkill in the art .

The utilities of the invention are many fold. When used in vitro, the compositions of the invention are useful, for example, in stabilizing heme-containing proteins, in extending the length of the life of culture medium, in enhancing cell growth, in providing metabolic subεtrateε, and in εtimulating metabolic activity. When hemoglobin is loaded

SUBSTITUT with nonoxygen gas ligands and mixed with fresh culture medium it is stabilized both prior to application to the cells (during storage) and after application to the cells (during use). In addition, an important improvement provided by the invention is that cell culture medium containing stabilized hemoglobin will extend the useful life of the cell culture medium. This is demonstrated in the examples below, which show that nearly double the amount of cells can be produced using a fixed quantity of the medium of the invention as compared with prior art medium. Although the inventors do not wish to be bound by any particular theory of the invention, it is believed that the medium may become more useful because: (1) the hemoglobin is stabilized against methemoglobin conversion even after being introduced into the environment of the cells; (2) the heme-containing proteins of the invention act to trap oxygen in the media from escaping to the atmoεphere; and (3) the nonoxygen gaε ligandε may stimulate metobolic activity, thereby enhancing cell growth. We have demonstrated, for example, that there is lesε loss of oxygen to the atmosphere from cell culture medium that contains the heme-containing proteins loaded with carbon monoxide as compared to cell culture medium that doeε not containε heme-containing protein loaded with carbon monoxide.

The invention also contemplates loading the heme-containing protein with a gas ligand that can act as a substrate or starter material for cell culture, in addition to enhancing the buffering capabilities of the cell culture medium. It is known that bicarbonate is used both as a buffer and nutrient by living cells. I_n vitro, bicarbonate can be supplied to cell cultures by adding sodium bicarbonate to the liquid medium or by gasing the cultures with carbon dioxide, a process which readily forms carbonic acid and then bicarbonate ions. The culturing is usually performed in an 'open system' that allows the exchange of gases between the atmosphere and the liquid medium. However, when there is a need to grow cells in a closed environment, a mechanism is needed to supply a source of carbon dioxide to cells in a way other than via the atmosphere. Closed environment cell culturing is becoming popular because of the HIV virus and other infectious agents. In addition, closed environments are useful in experimental situations when ready supplies of carbon dioxide are not available, such as aboard spacecraft.

The present invention provides a mechanism for loading a heme-containing protein with carbon dioxide and delivering the bound carbon dioxide directly to the culture medium. Since the carbon dioxide is reversibiy bound, the concentration of carbon dioxide provided to the cell culture can be controlled by the amount of hemoglobin dissolved in the medium. Thus, heme-containing proteins loaded with carbon dioxide not only serve to trap oxygen, but also act to provide substrates for cell growth and act to provide a buffering agent to the medium. Provision of cell substrates is particularly important in situations where the medium is serum-free.

The compositions of the invention further have a variety of uses in vivo. First, they provide a more stable heme-containing protein for trapping oxygen already in the blood stream. When ligands such as CO and NO are used, they provide the generalized effect of stimulating metabolic activity. This can be particularly useful in trauma situations when blood substitutes generally are appropriate.

The compositions of the invention further are useful for delivering nonoxygen gas ligands to cells in vivo. Nitric oxide is a vasodilator, a smooth muscle relaxant, a platelet inhibitor, an anti-microbial agent, a modifier of cell adhesion, a modifier of neurotransmiεεion, an enhancer of penile erection, an enzyme regulator, an immune regulator, and a cytotoxic modulator. Aε a result, it has numerous clinical applications.

NO can be useful in, and not limited to, the treatment of systemic hypertension including malignant hypertension, transient ischemic attacks, cerebral or myocardial ischemia, coronary insufficiency, -intestinal or renal ischemia, peripheral occlusive diseaseε, congeεtive heart failure, and angina pectoriε. It alεo can be used to enhance transdermal absorption of pharmaceutical agentε due to itε vaεodilatory capabilitieε. Thus, the compositions of the invention may be combined with drugs or agents that are delivered transdermally.

Nitric oxide also can be useful in the treatment of biliary colic, esophageal or intestinal spaεm, ureteral spasm, and uterine spasm. It further can be useful in the prevention and/or treatment of artherosclerotic plaques and the prevention of coronary artery occlusion, as well as subsequent prevention of myocardial ischemia and/or infarction. It also may be useful in the adjunctive therapy of bacteria which normally infect humans.

NO also is implicated in the treatment of learning disorderε, the treatment of Alzheimer'ε Disease, and enhancement of memory. It further is implicated as adjunctive treatment for both nonsolid and solid tumors, treatment of autoimmune diseases such as rheumatoid arthritis, lupus, erythematosus, diabeteε mellituε and thyroiditiε, and prevention of bone marrow, renal, hepatic, and cardiac transplant rejection.

Carbon monoxide recently has been implicated for the same indications as discussed above in connection with nitric oxide.

Heme-containing proteins are purified, endotoxin is removed, and a fluid is added to produce a "blood substitute" that is electrolytically, oncotically, and oεmotically compatible with blood to provide the benefits of volume expansion and oxygen transport. The heme-containing protein incorporated into the blood substitute is stabilized intra- and/or intermolecularly by methods such as those described above. Heretofore dextran and albumin have been utilized as volume expanderε; they have limited benefit, if any, to enhance oxygen transport and eventual delivery of oxygen to

TUTE SHEET tiεεue. Blood substitution is indicated in situations of acute blood loss, such as trauma with hemorrhage (surgical, accident-related, war related, etc.) or hemorrhage related to medical disorders, e.g. peptic ulcer disease, variceal bleeds, diverticular bleeding. Blood substitutes could be used for full replacement until bleeding is stanched or conjunction with dextran and/or albumin. Blood substitutes can be used in instances of severe anemia such as the anemia secondary to end-stage renal diseaεe or εickle-cell anemia. Blood substitutes can be administered intravenously.

Still another important aspect of the invention involves the use of heme-containing proteins to deliver biological agents. Biological agents may be difficult to administer to a cell because they are lipids (i.e., nonsoluble) or because they are needed in such small or precise amounts that it is necessary to associate them with another carrier molecule to avoid the loss of the agent during the preparation process or culturing process as a result of the agent, for example, adhering to glass or plastic. Albumin has been frequently used in the prior art as such a carrier of biological agents. According to the invention, heme-containing proteins can be used as such carriers. This is particularly important in cell culture applications when serum-free medium is required. When using serum-free medium cells often do not have the necessary factors for the desired growth or activity (e.g. gene regulation, enhanced cell division, etc.). The media then must be supplemented with the desired cell culture enhancers. Such enhancers may need to be noncovalently bound to the carriers to get them into solution or to deliver them in accurate amounts. Thus, the invention employs heme-containing proteinε for this purpose and avoids the need to use recombinantly derived and/or isolated albumin for the same purpose. The invention further contemplates using heme-containing proteins for multiple purposes, including ligand-related purposes, as described above, in addition to biological agent delivery. The preferred biological agents

SUBSTITUTE SHEET are cell culture enhancers. Cell culture enhancers mean any molecule that is useful in connection with positively influencing cell culture. Such molecules are well known to those of ordinary skill in the art and include a wide array of agents that are used for a variety of cell culturing purposes. They typically enhance cell viability, growth or metabolism. Cell culture enhancers are commercially available as constituents of cell media and may be purchased separately; enhancers may be added alone to the media or be added in association with another carrier molecule such aε albumin. Examples of lipid molecules that can be noncovalently bound to hemoglobin and thus solubilized in medium for cell culture include sterols, fatty acids, glycerols, prostaglandins, leucotrienes, triacylglycerolε (triglycerideε) , and sphingolipids. Particular agents include glucocorticoids such as desoxycorticoεterone, 11-deεoxycortisol, cortisol, corticosterone, aldosterone, 18-hydroxycorticosterone, and the synthetic glucocorticoids such as triamcinolone acetonide, dexamethasone and prednisolone; androgens such as testosterone, dihydrotestosterone, androsterone and the εynthetic analogs stanozolol, danazol, testosterone cypionate, and nandrolone decanoate; estrogens such as estradiol, estrone, estriol and the synthetic estrogens quinestrol and estridiol cypionate; progestins such as progeterone, 17-hydroxyprogesterone and the synthetic progestins medroxyprogesterone acetate and megestrol acetate; nonsteroidal compounds such as tamoxifen, clomiphene and diethylstilbeεtrol; fatty acids including naturally occurring fatty acids and in particular palmitic acid, stearic acid, linoleic acid, oleic acid and arachidonic acid; and phosphacylglycerols such as phosphatidylcholineε, phosphatidylserines, phoεphatidylethanolamineε, and phosphatidylinosotol.

In addition to solubilizing nonaqueouε materialε, heme-containing proteins may be used to bind trace metals εuch aε zinc, nickel, copper, and εelenium. They also may be

UTE SHEET used as a carrier of growth factors such as epidermal growth factor, growth hormone, insulin and fibroblast growth factor. When used in conjunction with trace amounts of itogenic protein, the heme-containing proteins will prevent nonspecific binding of the mitogens thus effectively maintaining biologically active concentrationε of the mitogenε.

The biological agent delivering propertieε of heme-containing proteinε also may be used for in vivo applications. Agents useful for in vivo applications are those that are appropriate for the condition being treated. Moεt preferred are thoεe agents useful in connection with conditions that also require the gas ligand related function of the heme-containing proteins described herein. In addition to the agents described above in connection with in vitro applications, the heme-containing proteins can be used to assist in the delivery of most enzymes and drugs, as will be well recognized by those of ordinary skill in the art.

The present invention also provides for experimental model systems for studying the ability of the heme-containing proteins of the invention to deliver biological agents and enhancers. In these model systems, the biological agents and/or enhancers are noncovalently bound to the heme-containing proteins (which proteins may or may not be loaded with nonoxygen gas ligand) . These molecules then are provided to cells via tissue culture medium or to animals in vivo, and the effects are compared to controls which use the same heme-containing protein, but not coated with a biological agent and/or cell culture enhancer. In this manner, methods for discovering useful cell culture medium additives and therapeutics are provided.

Example 1 Sterile Collection of Blood: Bovine blood (living donor or siaughterhouεe) waε collected using sterile procedures and equipment known to those skilled in the art. All containers, equipment, and tubing were chemically sanitized or autoclaved before use. Deionized water was used throughout the entire process. Slaughterhouse blood was collected in depyrogenated buckets containing anticoagulant (48.8 mM sodium nitrate • 2 H_0, 139 M sodium chloride, 1.38 mM citric acid anhydrous) . Donor blood was collected in plastic bags containing anticoagulant to blood in a 1:4 ratio.

Red Blood Cell Separation: Slaughterhouse blood was transferred to 1.0 liter depyrogenated containers and placed in a Sorval RC-3 refrigerated centrifuge (4°C). When donor blood was used, the blood was collected in donor blood bagε, and these bags were placed directly in the centrifuge. The blood was centrifuged at 3000 RPM for 30 minutes. Immediately after centrifugation the plasma and buffy coat was removed aseptically.

Cell Washing: The packed red blood cells were poured through sanitized cheese cloth into a 5-gallon depyrogenated collection tank. The cheese cloth was rinsed with dialysate solution (10 mM sodium phosphate, 200 mM sodium chloride, pH 6.7), and then the red blood cell solution was transferred into a 100-liter processing tank adjusting the volume to 25-30 liters using dialysate solution. The red blood cell solution was diafiltered (croεs flow filtration technique) by circulating through a Millipore proεtack (16 εquare feet of membrane, 0.65 μ size) using a Watεon-Marlow pump (Model 710R) . For 20-26 liters of blood, 180 liters of dialysate buffer are required. Dialysate buffer was pumped into processing tank at an equivalent rate to permeate flow rate in order to maintain reservoir volume (initial starting volume). After washing the red blood cells, cells were concentrated to 18-25 grams of hemoglobin/100 ml. (Hemoglobins concentrations were measured using a CO-Oximeter) .

Isolation of Hemoglobin: To the concentrated red blood cell solution 1.5 volumes of lysing εolution (lOmM εodium phoεphate, pH 6.7) waε added per volume of concentrated solution. Osmolarity of solution waε 130-160 m Osm. The solution was circulated through a system using a prefilter bypass for 5 minutes. The bypass consisted of a prefilter contained in a stainless steel sanitary holder capable of removing stroma without causing excesεive back preεsure on the syεtem. Upon completion of prefiltration, lysed εolution waε circulated through a Millipore proεtack. The permeate valve was opened and filtrate was collected (hemoglobin stock solution being approximately 100 g/liter) into a sterile container. To the filtrate was added magnesium chloride (1.0 mM) , sodium chloride (25.0 mM) , lactose (5.5 mM) , and pH of the solution was adjusted to 7.5. Ingredients were mixed for 15 minutes. The final osmolarity was 240-270 m Osm.

Stabilization of Hemoglobin: Hemoglobin was εaturated by bubbling carbon monoxide through the εolution. The percentage of carbon monoxide bound to hemoglobin waε monitored using a CO-Oximeter. Ninety percent saturation was achieved.

Example 2 Carbon Monoxide Stabilized Hemoglobin:

Hemoglobin was isolated as described above. Carbon monoxide was bubbled into a stirred hemoglobin solution. The percentage of carbon monoxide bound to hemoglobin was measured using a CO-Oximeter. At each of the indicated time points, methemoglobin levels were recorded as well. Referring to Fig. l, 50% saturation with carbon monoxide was reached after approximately 8 minutes and 90% saturation with CO was reached after approximately 25 minutes. The methemoglobin levels remained constant throughout the 25-minute period.

We also tested the stability of hemoglobin saturated with carbon monoxide for seventy-three days stored at 4°C. There was virtually no conversion to methemoglobin over this period of time. Example 3

In Vitro Biological Efficacy:

Insect cells (SF9) were seeded at a density of 3 x 10 cells/ml into a 500 ml spinner flask containing 300 ml of

SF900 insect cell medium (GIBCO) . At time zero (seeding), the medium was then supplemented with hemoglobin εaturated with carbon monoxide, the hemoglobin added to final concentrations of lg/L, 3g/L, and 6g/L. A stock solution having a concentration of approximately 79g/L was used as the starting material and diluted to the various final concentrations. The stock solution was prepared as described above in Example 1. The control medium did not receive any supplementation. Incubation of the insect cells were performed at 28°C. Cell counts were taken approximately every 24 hours. The data, appearing in Fig. 2, are plotted aε viable cells per ml. As can be seen from Fig. 2, the cells grown in control media exhibited the least amount of growth and the cells receiving hemoglobin-containing carbon monoxide grew faster and in a dose dependent fashion. For example, at about 100 hours, the concentration of cellε in g the control medium waε about 2 x 10 whereaε the concentration of cellε in media containing hemoglobin at 6g/L was approximately 4 x 10 . Likewise, at about 130 hourε, the differentials were 4 x 10 (control) vs. 8.25 x 10

(6g/L).

Example 4

Oxygen 'Trapping' by Carbon Monoxide Bound Hemoglobin: ₂ Two T-25cm flaskε containing 5 ml of hybridoma εerum

₇ free medium (HSFM) and 1 x 10 AE-l cells/ml were incubated in air at 25°C for three hourε to equilibrate the εyεtem. The flaskε were deεignated aε control (not εupplemented with carbon monoxide-bound hemoglobin) and treated (εupplemented) with carbon monoxide bound hemoglobin to a final concentration of lg/L from a εtock concentration of 74g/L. After the incubation period, the flaεks were agitated to

ET increase the diεεolved oxygen levels to 21% (time zero) . The disεolved oxygen concentration was then measured over time using a microoxygen probe. The results are shown in Fig. 3. As can be seen, the treated medium contained more oxygen than the untreated medium over the course of time.

The foregoing results were surpriεing. It waε not expected that hemoglobin complexed with carbon monoxide would act as an oxygen trap because it was believed from the teachings of the prior art that the carbon monoxide would continue to occupy the binding sites on the hemoglobin because of the higher affinity of carbon monoxide for such binding sites. Furthermore, because of the known toxicity of carbon monoxide, the results further are surprising. Thus the higher levels of disεolved oxygen and enhanced cell growth were unexpected.

It further is unexpected that the foregoing results were achieved using noncrosslinked, nonpyridoxilated hemoglobin. The art has taught that better resultε are achieved when the affinity for oxygen is lowered by crosεlinking and pyridoxilating the hemoglobin. Thus, the invention in a preferred aspect involves the use of noncroεεlinked, nonpyridoxilated native hemoglobin containing the nonoxygen gas ligand. This has advantages in many applications including the reduction in the steps necessary to prepare the product and consequent reduction in use of toxic crosεlinking materials and in cost.

Those skilled in the art will be able to ascertain with no more than routine experimentation numerous equivalentε to the εpecific embodiments of the invention described herein. Such equivalentε are conεidered to be within the scope of the invention and are intended to be embraced by the following claims in which we claim:

Claims

1. A method for delivering a nonoxygen gas ligand to a cell, comprising contacting the environment of the cell with a nonoxygen gas ligand that is reversibiy bound to a heme moiety of a heme-containing protein for the purpose of delivering the nonoxygen gas ligand to the cell.

2. A method as claimed in claim 1 wherein the environment of the cell is contacted with a gas ligand selected from the group consisting of NO, CO, and CO,; and wherein the heme-containing protein is selected from the group consisting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin.

3. A method as claimed in claim 1 wherein the environment of the cell is contacted with a gas ligand bound to bovine hemoglobin.

4. A method as claimed in claim 1 wherein the gas ligand bound to the heme-containing protein is contained in a liposome.

5. A method as claimed in claim 2 wherein the gas ligand bound to the heme-containing protein is contained in a liposome.

6. A method aε claimed in claim 1 wherein the gas ligand bound to the heme-containing protein is contained in a pharmaceutically acceptable carrier.

7. A method as claimed in claim 1 wherein the gas ligand bound to the heme-containing protein is contained in fresh cell culture medium.

8. A method as claimed in claim 1 wherein the heme-containing protein has a plurality of heme moietieε and the gaε ligand occupieε at leaεt 25% of the moietieε.

9. A method as claimed in claim 8 wherein the gas ligand occupies at least 50% of the moieties.

10. A method as claimed in claim 8 wherein the gas ligand occupies at least 75% of the moieties.

11. A method aε claimed in claim 8 wherein the gaε ligand occupies at least 90% of the moieties.

12. A pharmaceutical preparation comprising a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of isolated heme-containing protein, wherein the gas ligands occupy at least 25% of the heme moieties.

13. A pharmaceutical preparation as claimed in claim 12 wherein the gas ligands are selected from the group consisting of NO, CO, and C0₂; and wherein the heme-containing protein is selected from the group consiεting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin.

14. A pharmaceutical preparation as claimed in claim 12 wherein the gas ligand bound to the heme-containing protein are contained in liposomes.

15. A pharmaceutical preparation as claimed in claim 12 wherein the heme-containing protein haε non-covalently attached to it a plurality of iεolated biologically active moleculeε.

16. A pharmaceutical preparation aε claimed in claim 12 wherein the heme-containing protein has non-covalently attached to it a plurality of isolated lipid moleculeε.

17. A pharmaceutical preparation aε claimed in claim 12 wherein the gaε ligand occupies at least 50% of the moietieε.

18. A pharmaceutical preparation aε claimed in claim 12 wherein the gas ligand occupies at least 75% of the moieties.

19. A pharmaceutical preparation as claimed in claim 12 wherein the gas ligand occupies at least 90% of the moieties.

20. A method for forming a pharmaceutical preparation comprising mixing with a pharmaceutically acceptable carrier a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of isolated heme-containing protein, wherein the gaε ligands occupy at least 25% of the heme moieties.

TTUTE SHEET

21. A method as claimed in claim 20 wherein the gaε ligands are selected from the group consisting of NO, CO, and C0₂; and wherein the heme-containing protein is selected from the group consiεting of hemoglobin, native hemoglobin, myoglobin and native myoglobin.

22. A method aε claimed in claim 20 wherein the gas ligands bound to the heme-containing protein are contained in liposomes.

23. A method as claimed in claim 20 wherein the heme-containing protein has non-covalently attached to it a plurality of isolated biologically active molecules.

24. A method as claimed in claim 20 wherein the heme-containing protein has non-covalently attached to it a plurality of isolated lipid molecules.

25. A method as claimed in claim 20 wherein the gas ligand occupies at least 50% of the moieties.

26. A method as claimed in claim 20 wherein the gas ligand occupies at least 75% of the moieties.

27. A method as claimed in claim 20 wherein the gaε ligand occupies at least 90% of the moieties.

28. A preparation comprising fresh cell culture medium containing a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of isolated heme-containing protein, wherein the gas ligands occupy at least 25% of the heme moieties.

29. A preparation as claimed in claim 28, wherein the gas ligands are selected from the group consisting of NO, CO, and C0₂; and wherein the heme-containing protein is selected from the group consisting of hemoglobin, narive hemoglobin, myoglobin, and native myoglobin.

30. A method as claimed in claim 28 wherein the gas ligand bound to the heme-containing protein are contained in liposomes.

31. A method as claimed in claim 28 wherein the heme-containing protein has non-covalently attached to it a plurality of isolated biologically active molecules.

SUBSTITUTE

32. A preparation as claimed in claim 28 further comprising an isolated cell culture enhancer bound to the heme-containing protein.

33. A method as claimed in claim 28 wherein the heme-containing protein has noncovalently attached to it a plurality of isolated lipid molecules.

34. A method as claimed in claim 28 wherein the gas ligand occupies at least 50% of the moieties.

35. A method as claimed in claim 28 wherein the gas ligand occupies at least 75% of the moieties.

36. A method as claimed in claim 28 wherein the gas ligand occupies at least 90% of the moietieε,

37. A method for forming improved freεh cell culture media, comprising mixing with fresh cell culture medium a plurality of nonoxygen gas ligands that are reversibiy bound to a plurality of oxygen-binding heme moieties of isolated heme-containing protein, wherein the gaε ligands occupy at least 25% of the heme-moieties.

38. A method aε claimed in claim 37 wherein the gas ligands are selected from the group consisting of NO, CO, and C0₂; and wherein the heme-containing protein is selected from the group consiεting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin.

39. A method as claimed in claim 37 wherein the gas ligand bound to the heme-containing protein are contained in liposomeε.

40. A method as claimed in claim 37 further comprising binding non-covalently a cell culture enhancer to the heme-containing protein.

41. A method as claimed in claim 37 further comprising an isolated biologically active molecule to the heme-containing protein.

42. A method as claimed in claim 37 further comprising binding noncovalently an isolated lipid to the heme-containing protein.

43. A method as claimed in claim 37 wherein the gas ligand occupies at least 50% of the moieties.

44. A method as claimed in claim 37 wherein the gaε ligand occupies at least 75% of the moieties.

45. A method as claimed in claim 37 wherein the gas ligand occurpies at least 90% of the moieties.

46. A preparation comprising fresh cell culture medium containing isolated heme-containing protein having non-covalently bound to it isolated cell culture enhancers.

47. A preparation as claimed in claim 46 wherein the isolated heme-containing protein is selected from the group consisting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin.

48. A preparation as claimed in claim 46 wherein the cell culture enhancer is a biologically active molecule.

49. A preparation as claimed in claim 46 wherein the cell culture enhancer is a lipid.

50. A method for preparing improved fresh cell culture comprising mixing with fresh cell culture medium a heme-containing protein that has noncovalently attached to it cell culture enhancers.

51. A method as claimed in claim 50 wherein the fresh cell culture medium is mixed with a heme-containing protein selected from the group consisting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin.

52. Isolated heme-containing protein having noncovalently bound to it isolated biological agents.

53. Isolated heme-containing protein having noncovalently bound to it isolated lipid.

54. Isolated heme-containing protein having noncovalently bound to it isolated cell culture enhancers.

55. A method for delivering a biological agent to a cell comprising noncovalently binding the agent to a heme-containing protein and then delivering the heme-containing protein to the environment of the cell.

56. A method as claimed in claim 55 wherein isolated lipid is noncovalently bound to protein.

57. A method as claimed in claim 55 wherein isolated cell culture enhancer is noncovalently bound to the protein.

58. A method for culturing cells, comprising contacting the environment of the cells with a protein containing oxygen-binding heme moieties, wherein at least 25% of the heme-moieties are occupied with a nonoxygen gaε ligand.

59. A method as claimed in claim 58 wherein the environment of the cells is contacted with a protein selected from the group consisting of hemoglobin, native hemoglobin, myoglobin, and native myoglobin; and wherein the gas ligand iε εelected from the group consisting of: NO, CO, and C0₂-

60. A method as claimed in claim 58 wherein at least 50% of the heme-moieties are occupied with the nonoxygen gas ligand.

61. A method as claimed in claim 58 wherein at least 75% of the heme-moieties are occupied with the nonoxygen gas ligand.

62. A method aε claimed in claim 58 wherein at leaεt 90% of the heme-moietieε are occupied with the nonoxygen gas ligand.

63. A method as claimed in claim 58 wherein the gas ligands is NO.

64. A method as claimed in claim 58 wherein the gas ligand is C0₂.

65. A method as claimed in claim 58 wherein a cell culture enhancer is noncovalently bound to the heme-containing protein prior to contacting the environment of the cells with the protein.

66. A method as claimed in claim 65 wherein the biological agent is an isolated lipid.