EP0804244A1 - Häm-proteine enthaltende zusammensetzungen und dazu gehörende methoden - Google Patents

Häm-proteine enthaltende zusammensetzungen und dazu gehörende methoden

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Publication number
EP0804244A1
EP0804244A1 EP94912841A EP94912841A EP0804244A1 EP 0804244 A1 EP0804244 A1 EP 0804244A1 EP 94912841 A EP94912841 A EP 94912841A EP 94912841 A EP94912841 A EP 94912841A EP 0804244 A1 EP0804244 A1 EP 0804244A1
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EP
European Patent Office
Prior art keywords
heme
containing protein
gas
hemoglobin
moieties
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94912841A
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English (en)
French (fr)
Inventor
Dennis M. Disorbo
R. Bruce Reeves
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BIORELEASE TECHNOLOGIES Inc
Original Assignee
BIORELEASE TECHNOLOGIES Inc
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Publication date
Application filed by BIORELEASE TECHNOLOGIES Inc filed Critical BIORELEASE TECHNOLOGIES Inc
Publication of EP0804244A1 publication Critical patent/EP0804244A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6445—Haemoglobin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin

Definitions

  • 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.
  • 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.
  • 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.
  • free hemoglobin 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.
  • 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.
  • oxygen 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.
  • 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.
  • cross-linking can improve the stability of hemoglobin.
  • 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.
  • a method for delivering a nonoxygen gas ligand to a cell is provided.
  • 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.
  • the cell is contacted with a gas ligand selected from the group consisting of NO, CO, and C0 2 , 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.
  • 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.
  • 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.
  • the heme-containing protein may be contained in liposomes.
  • the preferred concentration of the gas ligand also is as described above.
  • a method for forming a pharmaceutical preparation 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
  • preparations of improved cell culture medium 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-moietie ⁇ .
  • the preferred gas ligands are NO, CO, and C0 2 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.
  • 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.
  • 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.
  • 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.
  • cell culture enhancers include cell culture enhancers and biologically active molecules.
  • treated heme-containing proteins can be used in the preparation of improved cell culture media or pharmaceutical preparations. They are particularly useful for delivering
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention relates, in one important aspect, to loading heme-containing proteins with nonoxygen gas ligands.
  • 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.
  • 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.
  • 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.
  • biological activity such as enzymatic activity.
  • 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.
  • 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.
  • 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.
  • native hemoglobin and native myoglobin are terms restricted to the foregoingN
  • the native hemoglobins include ⁇ 2 ⁇ 2' ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2' A lal' A la2' A lb and A. , as well as any sub-units thereof.
  • 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.
  • 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.
  • 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'-pho ⁇ phate-(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;
  • DPG 2, 3-diphosphoglycerate
  • PPG analogs such as pyridoxal-5'-pho ⁇ phate-(PLP) covalently attached to hemoglobin in its deoxy state
  • 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 pho ⁇ phate, Nicolau U.S. Pat.No ⁇ .4,321,259 and 4,473,563;
  • 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;
  • a nonoxygen gas ligand 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 ha ⁇ been well characterized for oxygen, which i ⁇ ⁇ pecifically excluded from the term nonoxygen ga ⁇ ligand.
  • the nonoxygen gas ligands include, but are not limited to: carbon monoxide (CO); nitric oxide (NO); and carbon dioxide (C0 2 ).
  • hemoglobin which is a tetramer
  • the dynamics of ga ⁇ ligand binding are well known to tho ⁇ e of ordinary ⁇ kill in the art.
  • certain aspects of the invention specify that the gas ligand be bound to at least 25% of the heme moieties present.
  • a gas ligand 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.
  • the heme moieties of hemoglobin are completely saturated with nonoxygen gas ligand.
  • 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.
  • the environment of the cell when used in connection with . in vitro procedures means the fluid or medium in which the cells are su ⁇ pended or growing.
  • the environment of the cell when u ⁇ ed in connection with in vivo application ⁇ , means contacted with a living animal via topical admini ⁇ tration, parenteral administration, systemic administration, and the like.
  • the heme-containing protein ⁇ of the invention are mixed with fre ⁇ h cell culture medium.
  • SUBSTITUTE SHEET cell culture medium 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.
  • 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.
  • 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.
  • i ⁇ olated means separated from the native environment in substantially pure form.
  • 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 can be further isolated and purified as described below.
  • 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-i ⁇ olare with hemoglobin prepared for example according to
  • I ⁇ olated heme-containing protein having non-covalently attached to it isolated biological agents specifically excludes those composition ⁇ that may occur during the course of isolating hemoglobin from red blood cells.
  • I ⁇ olated al ⁇ o refers to recombinantly-derived materials.
  • the heme-containing proteins loaded with nonoxygen gas ligands al ⁇ o are useful n vivo in treating animal subjects.
  • the animal subjects are mammals and, most preferably the mammals are humans, primates, horse ⁇ , cows, dogs, cats, goats, sheep, and pigs.
  • the present invention thus, involve ⁇ the use of pharmaceutical formulations which include the heme-containing protein ⁇ of the invention together with one or more pharmaceutically acceptably carriers and optionally other therapeutic ingredients.
  • the carrier(s) and other ingredient ⁇ mu ⁇ t, of cour ⁇ e be pharmaceutically acceptable.
  • the molecule ⁇ 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 may be practiced u ⁇ ing 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.
  • modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g. ⁇ ubcutaneou ⁇ , 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.
  • aqueous preparations may be formulated according to known method ⁇ .
  • acceptable vehicles and solvents that may be employed are water, Ringer's Solution, and isotonic sodium chloride solution.
  • penetrants appropriate to the barrier to be permeated are u ⁇ ed in the formulation.
  • penetrant ⁇ are generally known in the art, and include, for example, wetting agents.
  • detergents may be used to facilitate permeation.
  • 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 maximal ⁇ ion of, or halt altogether the onset or maximal ⁇ ion of, the particular condition being treated. Such amounts will depend, of cour ⁇ e, 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 tho ⁇ e of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum do ⁇ e 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 reason ⁇ or virtually any other reason.
  • 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) .
  • heme-containing proteins may be incorporated into liposome ⁇ .
  • a lipo ⁇ ome 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 hydrogenated soy phosphatidylcholine and distearoyl phosphatidylcholine, cholesterol, dimyristoyl pho ⁇ phatidylglycerol, and alpha-tocopherol
  • U.S. Patent 4,776,991 The entire disclosure ⁇ of the foregoing patent ⁇ are incorporated herein by reference.
  • other techniques for forming liposomes are well known to tho ⁇ e of ordinary ⁇ kill in the art .
  • 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 sub ⁇ trate ⁇ , and in ⁇ timulating metabolic activity.
  • hemoglobin 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).
  • 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.
  • 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 atmo ⁇ phere; and (3) the nonoxygen ga ⁇ ligand ⁇ may stimulate metobolic activity, thereby enhancing cell growth.
  • 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 atmo ⁇ phere; and (3) the nonoxygen ga ⁇ ligand ⁇ may stimulate metobolic activity, thereby enhancing cell growth.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the invention further have a variety of uses in vivo.
  • they provide a more stable heme-containing protein for trapping oxygen already in the blood stream.
  • 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.
  • 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 neurotransmi ⁇ ion, an enhancer of penile erection, an enzyme regulator, an immune regulator, and a cytotoxic modulator.
  • 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 disease ⁇ , conge ⁇ tive heart failure, and angina pectori ⁇ . It al ⁇ o can be used to enhance transdermal absorption of pharmaceutical agent ⁇ due to it ⁇ va ⁇ odilatory capabilitiesitie ⁇ . 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 spa ⁇ m, 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 disorder ⁇ , the treatment of Alzheimer' ⁇ 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, diabete ⁇ mellitu ⁇ and thyroiditi ⁇ , and prevention of bone marrow, renal, hepatic, and cardiac transplant rejection.
  • autoimmune diseases such as rheumatoid arthritis, lupus, erythematosus, diabete ⁇ mellitu ⁇ and thyroiditi ⁇
  • prevention of bone marrow, renal, hepatic, and cardiac transplant rejection 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 o ⁇ motically 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.
  • dextran and albumin have been utilized as volume expander ⁇ ; they have limited benefit, if any, to enhance oxygen transport and eventual delivery of oxygen to
  • TUTE SHEET ti ⁇ ue 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 disea ⁇ e or ⁇ ickle-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.
  • 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.
  • the invention employs heme-containing protein ⁇ 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 include heme-containing proteins, including ligand-related purposes, as described above, in addition to biological agent delivery.
  • 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 a ⁇ albumin.
  • Particular agents include glucocorticoids such as desoxycortico ⁇ terone, 11-de ⁇ oxycortisol, cortisol, corticosterone, aldosterone, 18-hydroxycorticosterone, and the synthetic glucocorticoids such as triamcinolone acetonide, dexamethasone and prednisolone; androgens such as testosterone, dihydrotestosterone, androsterone and the ⁇ ynthetic 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 dieth
  • heme-containing proteins may be used to bind trace metals ⁇ uch a ⁇ zinc, nickel, copper, and ⁇ elenium. 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.
  • growth factors such as epidermal growth factor, growth hormone, insulin and fibroblast growth factor.
  • the heme-containing proteins 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 concentration ⁇ of the mitogen ⁇ .
  • the biological agent delivering propertie ⁇ of heme-containing protein ⁇ also may be used for in vivo applications.
  • Agents useful for in vivo applications are those that are appropriate for the condition being treated.
  • Mo ⁇ t preferred are tho ⁇ e agents useful in connection with conditions that also require the gas ligand related function of the heme-containing proteins described herein.
  • 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.
  • 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 siaughterhou ⁇ e) wa ⁇ 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.
  • Bovine blood living donor or siaughterhou ⁇ e wa ⁇ 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
  • 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 bag ⁇ , 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.
  • 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 (cro ⁇ s flow filtration technique) by circulating through a Millipore pro ⁇ tack (16 ⁇ quare feet of membrane, 0.65 ⁇ size) using a Wat ⁇ on-Marlow pump (Model 710R) .
  • 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) .
  • 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.
  • Hemoglobin was ⁇ aturated by bubbling carbon monoxide through the ⁇ olution. The percentage of carbon monoxide bound to hemoglobin wa ⁇ monitored using a CO-Oximeter. Ninety percent saturation was achieved.
  • 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.
  • 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) .
  • the medium was then supplemented with hemoglobin ⁇ aturated 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 a ⁇ viable cells per ml. As can be seen from Fig.
  • 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.
  • concentration of cell ⁇ in g the control medium wa ⁇ about 2 x 10 wherea ⁇ the concentration of cell ⁇ in media containing hemoglobin at 6g/L was approximately 4 x 10 .
  • the differentials were 4 x 10 (control) vs. 8.25 x 10
  • ET increase the di ⁇ olved oxygen levels to 21% (time zero) .
  • the dis ⁇ olved 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 achieved using noncrosslinked, nonpyridoxilated hemoglobin.
  • the art has taught that better result ⁇ are achieved when the affinity for oxygen is lowered by cros ⁇ linking and pyridoxilating the hemoglobin.
  • the invention in a preferred aspect involves the use of noncro ⁇ linked, 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 cros ⁇ linking materials and in cost.

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US6911427B1 (en) 1995-09-15 2005-06-28 Duke University No-modified hemoglobins and uses therefore
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JP2000502058A (ja) * 1995-11-30 2000-02-22 ソマトジェン，インコーポレイテッド ヘモグロビン架橋の際の機能性制御のための方法
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