# Patent

## Title
Methods and compositions for treating conditions related to lack of blood supply, shock and neuronal injuries

## Abstract

A pharmaceutical composition comprising an amphiphilic emulsifier, a polar liquid carrier and, optionally, a lipid component. The amphiphilic emulsifier form free-moving, optionally lipid-carrying, micelles (LMs) in the polar liquid carrier. The pharmaceutical composition is free of hemoglobin and fluorocarbon and can be used for treating conditions related to lack of blood supply and to raise the blood pressure and correct hypovolemia.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 13/273,471, filed on Oct. 14, 2011, which is a continuation-in-part of U.S. patent application Ser. No. 12/314,737, filed on Dec. 16, 2008, now U.S. Pat. No. 8,036,020; which claims the priority of U.S. Provisional Patent Application Ser. No. 61/016,443, filed on Dec. 22, 2007 and U.S. Provisional Application Ser. No. 61/064,639, filed on Mar. 18, 2008; which is also a continuation-in-part of U.S. patent application Ser. No. 12/696,107, filed on Jan. 29, 2010, which claims priority of U.S. Provisional Patent Application Ser. No. 61/202,124 filed Jan. 30, 2009; which also claims priority of U.S. Provisional Patent Application Ser. No. 61/432,919, filed on Jan. 14, 2011 and U.S. Provisional Patent Application Ser. No. 61/490,816, filed on May 27, 2011. This application also claims priority of U.S. Provisional Patent Application Ser. No. 61/681,930, filed on Aug. 10, 2012. All of the aforementioned applications are incorporated by reference in their entirety.


FIELD
The technical field is medical treatment and, in particular, methods and compositions for treating conditions related to lack of blood supply, shock and neuronal injuries.
BACKGROUND
Shock is a consequence of inadequate circulation due to causes that include blood loss, vasodilation or dehydration. The loss of circulation deprives cells of oxygen and nutrients and activates a powerful inflammatory response. Such states occur after injury due to war, accident, and assault resulting in massive blood loss or due to massive infection. The current therapeutic approach is to rapidly infuse fluids in order to restore circulating volume. This rapid infusion of fluids may be combined with pharmacological agents that directly constrict blood vessels. Both the infusion of fluids and the administration of drugs that constrict blood vessels cause an increase in the blood pressure. However, the pathophysiology of shock is far more complex than that which can be remedied by simply adding volume and clamping down the blood vessels to create a higher blood pressure. Once shock has occurred and the longer it persists numerous toxic substances termed mediators are released into the blood stream. These mediators create a dangerous state that is markedly different from that which existed prior to the event that led to shock.
Current methods for treating shock are of limited efficacy. Examples of resuscitation fluids are either crystalloids that contain various salts such as Ringer's lactate that rapidly diffuses out of the bloodstream and those that contain colloids or large molecules that remain in the bloodstream a longer time such as those that contain HETASTARCHTM, a plasma volume expander derived from natural sources of starch. Numerous complications have been observed with both. HETASTARCHTM enhances bleeding, a counterproductive property after massive blood loss. Moreover, the United States Food and Drug Administration and the European Medicines Agency has issued warnings against the use of HETASTARCHTM in critically ill patients because of evidence that they increase the incidence of renal failure and mortality.
Overreliance on vasoconstrictors will raise the blood pressure but decrease tissue perfusion leading to tissue necrosis and death. Therefore, there exists a need for a low-cost therapeutic volume expander that is capable of bringing oxygen and nutrients to the tissue and reduce inflammatory responses.
SUMMARY
One aspect of the present application relates to a method for treating conditions related to lack of blood supply. The method comprises administering to a subject in need of such treatment, an effectively amount of a pharmaceutical composition comprising an amphiphilic emulsifier and a polar liquid carrier, wherein the amphiphilic emulsifier forms free-moving micelles having a lipophilic core in the polar liquid carrier and wherein the pharmaceutical composition is free of hemoglobin and fluorocarbon.
Another aspect of the present application relates to a method for raising the blood pressure in a subject with hypovolemia. The method comprises administering to said subject, an effectively amount of a pharmaceutical composition comprising an amphiphilic emulsifier and a polar liquid carrier, wherein the amphiphilic emulsifier forms free-moving micelles having a lipophilic core in the polar liquid carrier and wherein the pharmaceutical composition is free of hemoglobin and fluorocarbon.
Another aspect of the present application relates to a blood volume expander comprising an amphiphilic emulsifier, a polar liquid carrier and 1 fM-100 mM histidine or a histidine-containing peptide, wherein the amphiphilic emulsifier forms free-moving micelles having a lipophilic core in the polar liquid carrier and wherein the pharmaceutical composition is free of hemoglobin and fluorocarbon.



BRIEF DESCRIPTION OF THE DRAWINGS
The detailed description will refer to the following drawings, wherein like numerals refer to like elements, and wherein:
FIG. 1 is an illustration of a micelle containing a lipophilic core and encapsulated by an emulsifier which may be a phospholipid or another amphiphilic molecule. The polar liquid carrier surrounds the micelle. These components together create an emulsion. The polar liquid may be water. This emulsion may be formed by adding energy to the mixture by sonication, using a homogenizer or a microfluidizer.
FIG. 2 is a graph showing the linear relationship between micelle concentration and oxygen content. Oxygen content was measured using mass spectroscopy. The Y axis, designated “Carrying Capacity” is the ratio of the oxygen content of the emulsion over that of water in which both fluids were exposed to the same conditions. The X axis is the % of the emulsion comprised of micelles. The emulsion was prepared by sonication of a mixture comprised of 1-3 grams of purified soybean oil, 0.1 grams of soy lecithin and 0.24 grams of glycerol and water to bring the mixture to a volume of 10 ml. Each point is the mean of 5 samples.
FIG. 3 is a diagram showing systolic blood pressure in mice treated with different pharmaceutical compositions after severe hemorrhagic shock. LM is a commercially available model 20% soybean oil emulsion (Intralipid) alone. RL is Ringer's (L) lactate. The mean blood pressure immediately before infusion across all experiments was 4.6+/−1.2 mmHg. The systolic pressure immediately before infusion of the fluid is subtracted out. Each point is the mean of 6-7 mice. **=p<0.01, *=p<0.05 for both the systolic and diastolic pressures of FIGS. 3 and 4, using a two tailed unpaired Student's t test.
FIG. 4 is a diagram showing diastolic blood pressure in mice treated with different pharmaceutical compositions after severe hemorrhagic shock. LM is a commercially available model 20% soybean oil emulsion (Intralipid) alone. RL is Ringer's (L) lactate. The mean blood pressure immediately before infusion across all experiments was 4.6+/−1.2 mmHg. The diastolic pressure immediately before infusion of the fluid is subtracted out. Each point is the mean of 6-7 mice. P values are as with the corresponding systolic pressure in FIG. 3.
FIGS. 5 and 6 are diagrams showing the systolic and diastolic pressures after infusion as a percentage of the systolic and diastolic pre-hemorrhage blood pressure in mice treated with albumin-containing pharmaceutical composition of the present application and mice treated with shed blood after severe hemorrhagic shock. The zero time point is the first pressure after initial infusion. There was no significant difference between the pre-infusion pressures of any of the groups. Each point is the mean of 6-7 mice. VS is the combination of 5% albumin with 20% Intralipid. NSA is 5% albumin in 154 mM NaCL. RLA is 5% albumin in Ringer's lactate. VS is significantly higher (p<0.05) than shed blood at 5, 15 and 30 minutes using a two tailed, unpaired Student's t test.



DETAILED DESCRIPTION
One aspect of the present application relates to a pharmaceutical composition for treating conditions related to lack of blood supply with a pharmaceutical composition. The pharmaceutical composition comprises an amphiphilic emulsifier and a polar liquid carrier, wherein the emulsifier forms single layer micelles with a polar outer surface and an inner hydrophobic space. In some embodiment, the pharmaceutical composition further comprises a lipophilic component that fills the inner hydrophobic space of the micelles. The pharmaceutical composition can be used to increase blood pressure and to carry oxygen to tissues in the absence of natural or modified hemoglobin. The pharmaceutical composition is free of hemoglobin, derivatives of hemoglobin, perfluorocarbon and derivatives of perfluorocarbon.
As used herein, a composition is “free of hemoglobin, derivatives of hemoglobin, perfluorocarbon and derivatives of perfluorocarbon” if the composition does not contain any hemoglobin, derivatives of hemoglobin, perfluorocarbon and derivatives of perfluorocarbon, or if the composition contains hemoglobin, derivatives of hemoglobin, perfluorocarbon and derivatives of perfluorocarbon at levels below 0.1% w/w.
FIG. 1 shows an embodiment wherein the lipid component and the amphiphilic emulsifier form lipid-containing micelles (LM) that are surrounded by the polar carrier fluid (shown as the white space around the amphiphilic molecules). The amphiphilic emulsifier molecules occupy the periphery of the lipid boundary. The lipophilic ends of the amphiphilic emulsifier molecules are directed inward toward the lipid and the polar ends of the amphiphilic emulsifier molecules are directed outward toward the polar carrier fluid. Because hydrophobic gases, such as oxygen, preferentially dissolve in the lipid core of the micelles relative to water or other aqueous environments, the pharmaceutical composition of the present application provides the ability to carry oxygen or other hydrophobic gases to body tissue. Emulsion comprised of the LM of the present application is able to reverse the hypotension characteristic of hemorrhagic shock, and absorb and release lipophilic gases such as oxygen and nitric oxide. The LMs in the pharmaceutical composition are also capable of exerting an osmotic force and absorbing mediators of tissue injury, such as prostaglandins, nitric oxide, leukotrienes, and thromboxane, and other lipophilic mediators such as platelet activating factors.
In certain embodiments, the LM have diameters in the range of 10 to 5000 nm. The colloidal properties of the LM with a diameter range from 10 to 5000 nm promotes their retention in the intravascular space. In other embodiments, the LM have diameters in the range of 100 to 500 nm. In yet other embodiments, the LM have diameters that are less than 100 nm (nanoemulsions). In shock of any etiology in which the capillary permeability is increased, nanoemulsions would more readily get into the interstitial space than larger structures. In certain embodiments, the nanoemulsion comprises a mixture of micelles of 2-300 nm in diameter. In another embodiment, the nanoemulsion comprises nano-micelles with an average diameter in the range of about 0.1-30 nm, 0.1-10 nm, 0.1-3 nm, 0.1-1 nm, 0.1-0.3 nm, 0.3-30 nm, 0.3-10 nm, 0.3-3 nm, 0.3-1 nm, 1-30 nm, 1-15 nm, 1-10 nm, 1-3 nm, 3-30 nm, 3-15 nm, 3-10 nm or 10-30 nm. At these sizes the micelle are able to get past the endothelial cell layer and enter the interstitial space. The nano-micelles may be employed in situations where the permeability of the vascular space has not increased or to promote cellular absorption of lipophilic mediators or to promote entry of molecules or cellular components that can favorably modulate intracellular mechanisms. Such modulatory effects may also be effected in the interstitial space especially in cases of increased vascular wall permeability. For example, one of the mechanisms of shock from hemorrhage or sepsis is the capillary leak. This capillary leak is caused by the death of endothelial cells and the actions of neutrophils. It is mediated by cytokines such as IL-1 and TNF as well as nitric oxide. Neutrophils adhere to damaged endothelial cells and release reactive oxygen species and cell wall damaging enzymes such as myeloperoxidase. The nano-micelles could get into the interstitium via the capillary leak and provide an anti inflammatory effect within the interstitial space.
In some embodiments, the pharmaceutical composition comprises a mixture of large micelles with diameters of 100 nm or greater, and nano-micelles with diameters of 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5 nm, 2 nm, 1 nm or less. The larger micelles would tend to stay in the intravascular space and maintain intravascular volume while the nano-micelles could get into the interstitium where they could favorably affect survival by blocking reactive oxygen species and delivering salutary molecules into cells, such as inhibitors of apoptosis (e.g., Z-VAD-FMY, an apoptosis inhibiting peptide) or protectors of mitochondrial integrity (e.g., Cyclosporin A, an inhibitor of mitochondrial inner pore opening). The nano-micelles may also be loaded with modulators of signal transduction such as diacylglycerol or cyclic GMP, or antioxidant such as Coenzyme Q10. In certain embodiments, the large micelles make up 10-40% (w/w) of the pharmaceutical composition, while the nano-micelles make up 5-30% (w/w) of the pharmaceutical composition. In other embodiments, the large micelles make up 10%-80% of the total micelles in the pharmaceutical composition, while the nano-micelles make up the remainder of the micelles of the pharmaceutical composition. In other embodiments, the large micelles are made of soybean oil and the nano-micelles are made of chia bean oil, which has a greater anti inflammatory effect than that of soybean oil.
The solubility of hydrophobic gases in the lipophilic core promotes the uptake and transport of these gases to tissues. The endogenously produced gases carbon monoxide, nitric oxide and hydrogen sulfide can also be carried in the emulsion for the modulation of the vascular tone and apoptotic processes. Oxygen may also be loaded for delivery to tissues and the enhancement of aerobic metabolism. Xenon and argon are hydrophobic gases that could provide protection of the brain in hemorrhagic shock and in other pathological states such as seizures. Delivery of these gases to the brain may also provide pain relief.
In certain embodiments, the micelles in the pharmaceutical composition of the present application are free-moving micelles that are not encapsulated in any type of particles. Further, the wall of the micelles is comprised of either a single layer or a double layer of the amphiphilic emulsifier molecules so that the micelles may easily merge with the cell membrane of the tissue that comes in contact with the pharmaceutical composition. Further, the micelles in the pharmaceutical composition of the present application are free of hemoglobin, derivatives of hemoglobin, perfluorocarbon and derivatives of perfluorocarbon.
The conditions related to lack of blood supply include, but are not limited to, hypovolemia caused by bleeding, dehydration, vomiting, severe burns, systemic inflammatory response syndrome (SIRS) and drugs such as diuretics or vasodilators. Severe hypovolemia may occur in conjunction with capillary leak (CL), which is present in different conditions such as multiorgan dysfunction (MODS), sepsis, trauma, burn, hemorrhagic shock, post-cardiopulmonary bypass, pancreatitis and systemic capillary leak syndrome, and causes morbidity and mortality among a large number of hospital patients. In one embodiment, conditions related to lack of blood supply is severe hemorrhagic shock. In other embodiments, conditions related to lack of blood supply include loss of blood in the amount of greater than 10%, 15%, 20%, 25% or 30% of total blood volume. In other embodiments, conditions related to lack of blood supply include loss of blood in the amount of greater than 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml or 1000 ml.
Amphiphilic Emulsifier
The amphiphilic emulsifier can be any amphiphiles or amphiphilic molecule that is capable of forming single layer micelles in a polar liquid carrier. Examples of amphiphilic emulsifiers include, but are not limited to, phospholipids, glycolipids, sterols, fatty acids, bile acids, saponins, amphiphilic peptides, hydrocarbon based surfactants such as sodium dodecyl sulfate (anionic), benzalkonium chloride (cationic), cocamidopropyl betaine (zwitterionic) and long chain alcohol (non-ionic).
Examples of phospholipids include natural or synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lisophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, and a hydrogenated phospholipid.
Examples of the glycolipids include glyceroglycolipids and sphingoglycolipids. Examples of glyceroglycolipids include digalactosyl diglycerides (such as digalactosyl dilauroyl glyceride, digalactosyl dimyristoyl glyceride, digalactosyl dipalmitoyl glyceride, and digalactosyl distearoyl glyceride) and galactosyl diglycerides (such as galactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride, galactosyl dipalmitoyl glyceride, and galactosyl distearoyl glyceride). Examples of sphingoglycolipids include galactosyl cerebroside, lactosyl cerebroside, and ganglioside.
Examples of the sterols include cholesterol, cholesterol hemisuccinate, 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol, ergosterol, and lanosterol.
In one embodiment, the amphiphilic emulsifier is egg phospholipids. In another embodiment, the emulsifier is soybean lecithin or alpha-phosphatidylcholine.
Amphiphilic peptide molecules typically have three regions: a hydrophobic tail, a region of beta-sheet forming amino acids, and a peptide epitope designed to allow solubility of the molecule in water.
In some embodiments, the amphiphilic emulsifier is a non-lipid amphiphilic emulsifier, such as amphiphilic peptides and hydrocarbon based surfactants.
In some embodiments, the amphiphilic emulsifier constitutes about 0.1-50% (w/v) of the pharmaceutical composition. In certain embodiments, the emulsifier constitutes about 0.1-0.5%, about 0.1-2%, about 0.1-5%, about 0.1-10%, about 0.1-15%, about 0.1-20%, about 0.1-30%, about 0.1-40%, about 0.5-2%, about 0.5-5%, about 0.5-10%, about 0.5-15%, about 0.5-20%, about 0.5-30%, about 0.5-40%, about 0.5-50%, about 2-5%, about 2-10%, about 2-15%, about 2-20%, about 2-30%, about 2-40%, about 2-50%, about 5-10%, about 5-15%, about 5-20, about 5-30%, about 5-40%, about 5-50%, about 10-15%, about 10-20%, about 10-30%, about 10-40%, about 10-50%, about 15-20%, about 15-30%, about 15-40%, about 15-50%, about 20-30%, about 20-40%, about 20-50%, about 30-40%, about 30-50% or about 40-50%, (w/v) of the pharmaceutical composition.
In certain embodiments, the emulsifier constitutes about 0.5%, about 0.75%, about 1%, about 1.2%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, about 4%, about 5%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/v) of the pharmaceutical composition.
Polar Liquid Carrier
The polar liquid carrier can be any pharmaceutically acceptable polar liquid that is capable of forming an emulsion with the lipid. The term “pharmaceutically acceptable” refers to molecular entities and compositions that are of sufficient purity and quality for use in the formulation of a composition or medicament of the present application and that, when appropriately administered to an animal or a human, do not produce an adverse, allergic or other untoward reaction. Since both human use (clinical and over-the-counter) and veterinary use are equally included within the scope of the present application, a pharmaceutically acceptable formulation would include a composition or medicament for either human or veterinary use. In one embodiment, the polar liquid carrier is water or a water based solution. In another embodiment, the polar liquid carrier is a non-aqueous polar liquid such as dimethyl sulfoxide, polyethylene glycol and polar silicone liquids.
A water-based solution generally comprises a physiologically compatible electrolyte vehicle isosmotic or near isosmotic with whole blood. The carrier can be, for example, physiological saline, a saline-glucose mixture, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's balanced saline, heparinized sodium citrate-citric acid-dextrose solution, and polymeric plasma substitutes, such as polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol and ethylene oxide-propylene glycol condensates. The pharmaceutical composition may additionally comprise other constituents such as pharmaceutically-acceptable carriers, diluents, fillers and salts, the selection of which depends on the dosage form utilized, the condition being treated, the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field and the properties of such additives.
Lipophilic Component
The lipophilic component can be any pharmaceutically acceptable lipophilic material, such as lipids. As shown in FIG. 1 the lipophilic component is carried in the hydrophobic core of a micelle. The hydrophobic core may also comprise additives that may enhance the uptake of hydrophobic gases such as hyperbranched molecules an example of which is hyperbranched polyglycerol esters. In other embodiments, the lipophilic component is trapped in the forms of structures, such as erythrocyte ghosts.
As used herein, the term “lipid” refers to a fat-soluble material that is naturally occurring, or non-naturally occurring, such a synthetic fat-soluble molecule. Examples of lipids include but are not limited to, fatty acyls, glycerolipids, phospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, polyketides, non-natural lipid(s), cationic lipid(s), amphipathic alkyl amino acid derivative, adialkyldimethylammonium, polyglycerol alkyl ethers, polyoxyethylene alkyl ethers, tri-n-octylamine, boric acid, tris(3,5-dimethyl-4-heptyl) ester, triglycerides, diglycerides and other acylglycerols, such as tetraglycerol, pentaglycerol, hexaglycerol, heptaglycerol, octoglycerol, nonaglyceol and decaglycerol, and mixtures thereof. In certain embodiments, the lipophilic component comprises soybean oil, chia bean oil or algae oil.
In one embodiment, the lipophilic component is soybean oil. In another embodiment, the lipophilic component is chia bean oil. In another embodiment, the lipophilic component is algae oil. Soybean oil is thrombogenic and procoagulant, and therefore would be preferred for the initial phase of hemorrhagic shock when clotting is desired. After bleeding is no longer an issue, oils rich in omega 3 fatty acids would be favored because of their anti-thromobogenic properties. Oils rich in omega 3 fatty acids include, but are not limited to chia oil, algae oil, pumpkin oil, flaxseed oil or fish oil.
In certain embodiments, the lipid component comprises an unsaturated fatty acid with one or more alkenyl functional groups in cis or trans configuration. A cis configuration means that adjacent hydrogen atoms or other groups are on the same side of the double bond. In a trans configuration these moieties are on different sides of the double bond. The rigidity of the double bond freezes its conformation and, in the case of the cis isomer, causes the chain to bend and restricts the conformational freedom of the fatty acid. In general, the more double bonds the chain has, the less flexibility it has. When a chain has many cis bonds, it becomes quite curved in its most accessible conformations. For example, oleic acid, with one double bond, has a “kink” in it, while linoleic acid, with two double bonds, has a more pronounced bend. Alpha-linolenic acid, with three double bonds, favors a hooked shape. The effect of this is that in restricted environments, such as when fatty acids are part of a phospholipid in a lipid bilayer, or triglycerides in lipid droplets, cis bonds limit the ability of fatty acids to be closely packed and therefore could affect the melting temperature of the membrane or of the fat. In some embodiments, the lipid component comprises up to 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% (w/w) unsaturated fatty acid(s) that have one or more alkenyl functional groups in cis configuration.
Examples of cis-unsaturated fatty acids include, but are not limited to, obtusilic acid, linderic acid, tsuzuic acid, palmito-oleic acid, oleic acid, elaidic acid, vaccenic acid, petroselinic acid, gadoleic acid, eicosenoic acid, erucic acid, cetoleic acid, nervonic acid, ximenic acid and lumepueic acid; n-3 type unsaturated fatty acids such as α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid; n-6 type unsaturated fatty acids such as linoleic acid, linoelaidic acid, γ-linolenic acid, bis-homo-γ-linolenic acid and arachidonic acid; conjugated fatty acids such as conjugated linoleic acid and α-eleostearic acid; fatty acids carrying double bonds at the 5-position thereof such as pinolenic acid, sciadonic acid, juniperic acid and columbinic acid; polyvalent unsaturated fatty acids, other than those listed above, such as hiragonic acid, moroctic acid, clupanodonic acid and nishinic acid; branched fatty acids such as isobutyric acid, isovaleric acid, iso acid and anti-iso acid; hydroxy fatty acids such as α-hydroxy acid, β-hydroxy acid, mycolic acid and polyhydroxy acid; epoxy-fatty acids; keto-fatty acids; and cyclic fatty acids. In certain embodiments, the lipophilic component also comprises amphiphilic molecules.
In some embodiments, the lipid component constitutes about 0-70% (w/v) of the pharmaceutical composition. In certain embodiments, the lipid component constitutes about 0.01-70%, 0.1-70%, 1-70%, 3-70%, 10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 0.01-60%, 0.1-60%, 1-60%, 3-60%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%, 0.01-50%, 0.1-50%, 1-50%, 3-50%, 10-50%, 20-50%, 30-50%, 40-50%, 0.01-40%, 0.1-40%, 1-40%, 3-40%, 10-40%, 20-40%, 30-40%, 0.01-30%, 0.1-30%, 1-30%, 3-30%, 10-30%, 20-30%, 0.01-20%, 0.1-20%, 1-20%, 3-20%, 10-20%, 0.01-10%, 0.1-10%, 1-10%, 3-10%, 0.01-3%, 0.1-3%, 1-3%, 0.01-1%, 0.1-1% or 0.01-0.1% (w/v) of the pharmaceutical composition. In certain embodiments, the lipid component comprises soy bean oil, chia bean oil, algea oil, or combinations thereof, and constitutes less than about 0.01%, about 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or about 70% (w/v) of the pharmaceutical composition.
Gas Carrying Capacity
The lipophilic portion of the micelles (i.e., the hydrophobic interior of the micelles), as well as the lipid component of the pharmaceutical composition of the present application, provides the ability for the pharmaceutical composition to carry a larger amount of lipophilic gases than of a purely aqueous solution. In some embodiments, the lipophilic gases are diffused into the hydrophobic interior of the micelles. In certain embodiments, the interior of the micelles are filled with the lipid component or other hydrophobic liquid.
In one embodiment, the lipophilic gas is oxygen. Oxygen is 4.41 times more soluble in lipid than in water (Battion et al., J. Amer. Oil Chem. Soc. 1968, 45:830-833). Accordingly, a composition with a higher lipid content would be able to carry more oxygen than a composition with a lesser lipid content. In one embodiment, the composition of the present application has a lipid content of about 0-70% (w/v). In other embodiments, the composition of the present application has a lipid content of about 1-70% (w/v), 1-60% (w/v), about 1-50% (w/v), about 1-40% (w/v), about 1-30% (w/v), about 1-20% (w/v), about 1-10% (w/v), about 1-6% (w/v) or about 1-3% (w/v). In yet another embodiment, the composition of the present application has a lipid content of about 21.8% (w/v). In certain embodiments, the composition of the present application is prepared by mixing the emulsifier and the polar liquid component, or by mixing the lipid component, the emulsifier and the polar liquid component, in the presence of regular air. In other embodiment, the composition of the present application is further oxygenated by bubbling regular air, pure oxygen or an oxygen containing gas mixture through the composition for a desired period of time. Since bubbles are undesirable in the circulation due to the possibility of air embolization a bubble trap would have to be added to remove bubbles leaving only the gas that has been solubilized in the core of the micelle, in the polar carrier or attached to proteins or other additives. The gas may also be loaded onto the micelles by equilibration of the micelles with an atmosphere enriched with the gas combined with gentle movement of the composition of the present application in a mixture chamber in order to avoid the creation of bubbles. Loading may also be done under pressures greater than 1 atmosphere followed by release of the pressure to allow the release of excess gas.
In another embodiment, the lipophilic gas is xenon (Xe) or argon (Ar). In another embodiment, the lipophilic gas is nitric oxide (NO). In another embodiment, the lipophilic gas is hydrogen sulfide (H2S). In yet another embodiment, the lipophilic gas is carbon monoxide (CO).
In some embodiments, the pharmaceutical composition contains micelles loaded with a gas mixture. In certain embodiments, the gas mixture is a mixture of two or more gases selected from the group consisting of oxygen, hydrogen sulfide, carbon monoxide, xenon, argon and nitric oxide). In other embodiments, the pharmaceutical composition contains a mixture of micelles loaded with various gases. In some embodiments, the mixture of micelles contains two or more different types of micelles selected from the group consisting of O2-loaded micelles, H2S-loaded micelles, NO-loaded micelles, CO-loaded micelles, Xe-loaded micelles, Ar-loaded micelles and micelles loaded with the gas mixtures described above. In one embodiment, the mixture of micelles contain 50% NO-loaded micelles and 50% O2-loaded micelles.
Buffering Agent
The pharmaceutical composition of the present application may further comprise a biological buffer to maintain the pH of the fluid at the physiological range of pH7-8. Examples of biological buffers include, but are not limited to, N-2-Hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), 2-([2-Hydroxy-1,1-bis(hydroxymethypethyl]amino)glyci ethanesulfonic acid (TES), 3-[N-tris(Hydroxy-methyl)methylamino]-2-hydroxyethyl]-1-piperazinep ropanesulfonic acid (EPPS), Tris[hydrolymethyl]-aminoethane (THAM), and Tris[Hydroxylmethyl]methyl aminomethane (TRIS).
In some embodiments, the buffering agent is histidine, substituted histidine such as glycylhistidine, imidazole, imidazole derivatives retaining the amphoteric site of the imidazole ring, glycylglycine, carnosine, oligopeptides containing histidine such as gly-his-gly, or mixtures thereof. In some embodiments, the buffering agent is used in a concentration range of about 0.1 mM to about 300 mM. In other embodiment, the buffering agent is used in a concentration range of about 0.1-100 mM, about 0.1-30 mM, about 0.1-10 mM, about 0.1-3M, about 0.1-1 mM, about 0.1-0.3 mM, about 0.3-300 mM, about 0.3-100 mM, about 0.3-30 mM, about 0.3-10 mM, about 0.3-3 mM, about 0.3-1 mM, about 1-300 mM, about 1-100 mM, about 1-30 mM, about 1-10 mM, about 1-3 mM, about 3-300 mM, about 3-100 mM, about 3-30 mM, about 3-10 mM, 10-300 mM, about 10-100 mM, about 10-30 mM, about 30-300 mM, about 30-100 mM or about 100-300 mM, In other embodiments, the buffering agent is used in a concentration range of about 5-20 mM. In other embodiments, the buffering agent is used in a concentration range of about 1-10 mM. In other embodiments, the buffering agent is used in a concentration of about 10 mM.
In some embodiment, the buffering agent is histidine, which is also capable of reducing reactive oxygen species and inhibiting cell shrinkage. (see e.g., Simpkins et al., J Trauma. 2007, 63:565-572). In some embodiments, the composition of the present application comprises a buffer agent at a concentration range of 1 uM to 300 mM, 10 uM to 300 mM, 100 uM to 300 mM, 1 mM to 300 mM, 10 mM to 300 mM, 30 mM to 300 mM, 100 mM to 300 mM, 1 uM to 100 mM, 10 uM to 100 mM, 100 uM to 100 mM, 1 mM to 100 mM, 10 mM to 100 mM, 30 mM to 100 mM, 1 uM to 30 mM, 10 uM to 30 mM, 100 uM to 30 mM, 1 mM to 30 mM, 10 mM to 30 mM, 1 uM to 10 mM, 10 uM to 10 mM, 100 uM to 10 mM, 1 mM to 10 mM, 3 mM to 10 mM, 1 uM to 3 mM, 10 uM to 3 mM, 100 uM to 3 mM, 1 mM to 3 mM, 1 uM to 1 mM, 10 uM to 1 mM, 100 uM to 1 mM, 300 uM to 1 mM, 1 uM to 300 uM, 10 uM to 300 uM, 1 uM to 100 uM, 10 uM to 100 uM, 10 uM to 30 uM or 100 uM to 300 uM. In some embodiments, the buffering agent is selected from the group consisting of histidine, imidazole, glycylglycine, carnosine, and histidine-containing peptides such as glycylhistidine, his-gly-gly and his-gly-gly-his. As used herein, the term “histidine-containing peptides” refers to peptides of 2-50 amino acid residues, preferably 2-15 amino acid residues, with a histidine content of 10% or higher (e.g., a 50 amino acid histidine-containing peptide would contain at least 5 histidine residues). In some embodiments, the histidine-containing peptides have a histidine content of 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, or 70% or higher.
In one embodiment, a buffering agent, such as histidine, glycylglycine or a histidine-containing peptide, is used at a concentration of about 0.5-20 mM. In another embodiment, a buffering agent, such as histidine, glycylglycine, histidine-containing peptide, is used at a concentration of about 1-10 mM. In other embodiment, a buffering agent, such as histidine, glycylglycine, or histine-containing peptide, is used at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM.
The histidine, glycylglycine or a histine-containing peptide may also be used as an anti-apoptotic agent. Under such circumstances, the histidine, glycylglycine or a histidine-containing peptide may be used in a concentration range of 1 fM to 300 mM.
In yet other embodiments, the pharmaceutical composition of the present application comprises two or more buffering agents, each is used within the concentration range of 1 uM to 300 mM, 10 uM to 300 mM, 100 uM to 300 mM, 1 mM to 300 mM, 10 mM to 300 mM, 30 mM to 300 mM, 100 mM to 300 mM, 1 uM to 100 mM, 10 uM to 100 mM, 100 uM to 100 mM, 1 mM to 100 mM, 10 mM to 100 mM, 30 mM to 100 mM, 1 uM to 30 mM, 10 uM to 30 mM, 100 uM to 30 mM, 1 mM to 30 mM, 10 mM to 30 mM, 1 uM to 10 mM, 10 uM to 10 mM, 100 uM to 10 mM, 1 mM to 10 mM, 3 mM to 10 mM, 1 uM to 3 mM, 10 uM to 3 mM, 100 uM to 3 mM, 1 mM to 3 mM, 1 uM to 1 mM, 10 uM to 1 mM, 100 uM to 1 mM, 300 uM to 1 mM, 1 uM to 300 uM, 10 uM to 300 uM, 1 uM to 100 uM, 10 uM to 100 uM, 10 uM to 30 uM or 100 uM to 300 uM. In other embodiments, the two or more buffering agent is used in a concentration range of about 1 mM to about 10 mM, about 10 mM to about 30 mM or about 30 mM to about 100 mM. In one embodiment, the pharmaceutical composition of the present application comprises histidine in the range of 1-10 mM and carnosine in the range of 1-10 mM.
In another embodiment, the pharmaceutical composition of the present application uses normal biological components to maintain in vivo biological pH. Briefly, some biological compounds, such as lactate, are capable of being metabolized in vivo and act with other biological components to maintain a biologically appropriate pH in an animal. The biological components are effective in maintaining a biologically appropriate pH even at hypothermic temperatures and at essentially bloodless conditions. Examples of the normal biological components include, but are not limited to carboxylic acids, salt and ester thereof. Carboxylic acids have the general structural formula of RCOOX, where R is an alkyl, alkenyl, or aryl, branched or straight chained, containing 1 to 30 carbons which carbons may be substituted, and X is hydrogen or sodium or other biologically compatible ion substituent which can attach at the oxygen position, or is a short straight or branched chain alkyl containing 1-4 carbons, e.g.,—CH3, —CH2CH3. Examples of carboxylic acids and carboxylic acid salts include, but are not limited to, lactate and sodium lactate, citrate and sodium citrate, gluconate and sodium gluconate, pyruvate and sodium pyruvate, succinate and sodium succinate, and acetate and sodium acetate. Amino acids at concentrations of 100 mM or more can also act as oncotic agents.
Plasma Component
The pharmaceutical composition of the present application may further comprise a plasma component. In one embodiment, the plasma is an animal plasma. In another embodiment, the plasma is human plasma. Although not wishing to be bound by any particular scientific theory, it is believed that the administration of a large amount of the pharmaceutical composition of the present application as a volume expander may dilute the concentration of coagulation factors to an undesirable level. Accordingly, using plasma as the diluent for the oxygen carrying component avoids this problem. Plasma can be collected by any means known in the art, provided that red cells, white cells and platelets are essentially removed. Preferably, it is obtained using an automated plasmaphoresis apparatus. Plasmaphoresis apparatuses are commercially available and include, for example, apparatuses that separate plasma from the blood by ultrafiltration or by centrifugation. An ultrafiltration-based plasmaphoresis apparatus such as manufactured by Auto C, A200 (Baxter International Inc., Deerfield, Ill.) is suitable because it effectively removes red cells, white cells and platelets while preserving coagulation factors.
Plasma may be collected with an anticoagulant, many of which are well known in the art. Preferred anti-coagulants are those that chelate calcium such as citrate. In one embodiment, sodium citrate is used as an anticoagulant at a final concentration of 0.2-0.5%, preferably 0.3-0.4%, and most preferably at 0.38%. The plasma may be fresh, frozen, pooled and/or sterilized. While plasma from exogenous sources may be preferred, it is also within the present application to use autologous plasma that is collected from the subject prior to formulation and administration of the pharmaceutical composition.
In addition to plasma from natural sources, synthetic plasma may also be used. The term “synthetic plasma,” as used herein, refers to any aqueous solution that comprises at least one plasma protein. Proteins resembling plasma protein may also be used.
Oncotic Agent
In one embodiment, the pharmaceutical composition of the present application further contains an oncotic agent in addition to the micelles. The oncotic agent is comprised of molecules whose size is sufficient to prevent their loss from the circulation by traversing the fenestrations of the capillary bed into the interstitial spaces of the tissues of the body. Examples of oncotic agents include, but are not limited to, dextran (e.g., a low-molecular-weight dextran), dextran derivatives (e.g., carboxymethyl dextran, carboxydextran, cationic dextran, and dextran sulfate), hydroxyethyl starch, hydroxypropyl starch, branched, unsubstituted or substituted starch, gelatin (e.g., modified gelatin), albumin (e.g., human plasma, human serum albumin, heated human plasma protein, and recombinant human serum albumin), PEG, polyvinyl pyrrolidone, carboxymethylcellulose, acacia gum, glucose, a dextrose (e.g., glucose monohydrate), oligosaccharides (e.g., oligosaccharide), a polysaccharide degradation product, an amino acid, and a protein degradation product. Among those, particularly preferable are low-molecular-weight dextran, hydroxyethyl starch, modified gelatin, and recombinant albumin.
Because of its antioxidant effects, albumin may also be used to minimize reactive oxygen species interaction with the components of the micelle and may also stabilize the micelle structure. In one embodiment, the oncotic agent is about 2%, 5%, 7% or 10% (w/v) albumin. In another embodiment, the oncotic agent is a polysaccharide, such as Dextran, in a molecular weight range of 30,000 to 50,000 daltons (D). In yet another embodiment, the oncotic agent is a polysaccharide, such as Dextran, in a molecular weight range of 50,000 to 70,000 D. High molecular weight dextran solutions are more effective in preventing tissue swelling due to their lower rates of leakage from capillaries.
In one embodiment, the concentration of the polysaccharide is sufficient to achieve (when taken together with chloride salts of sodium, calcium and magnesium, organic ion from the organic salt of sodium and hexose sugar discussed above) colloid osmotic pressure approximating that of normal human serum, about 28 mm Hg.
In another embodiment the oncotic agent is glycerol or mannitol in an amount of 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or up to 30% (w/v) of the pharmaceutical composition. In other embodiments, pharmaceutical composition comprises glycerol or mannitol in an amount of 1-2%, 1-3%, 1-4%, 1-5%, 2-3%, 2-4%, 2-5%, 3-4%, 3-5% or 4-5% (w/v).
Crystalloid Agent
The pharmaceutical composition of the present application may also comprise a crystalloid agent. The crystalloid agent can be any crystalloid which, in the form of the pharmaceutical composition, is preferably capable of achieving an osmolarity greater than 800 mOsm/l, i.e. it makes the pharmaceutical composition “hypertonic”. Examples of suitable crystalloids and their concentrations in the pharmaceutical composition include, but are not limited to, 3% w/v NaCl, 7% NaCl, 7.5% NaCl, and 7.5% NaCl in 6% w/v dextran. In one embodiment, the pharmaceutical composition has an osmolarity of between 800 and 2400 mOsm/1. In other embodiment, the pharmaceutical composition has an osmolarity of 800-1200 mOsm/l, 1200-1600 mOsm/l, 1600-2000 mOsm/l, or 2000-2400 mOsm/l.
When the pharmaceutical composition further comprises a crystalloid and is hypertonic, the pharmaceutical composition may provide improved functionality for rapid recovery of hemodynamic parameters over other compositions, which include a colloid component. Small volume highly hypertonic crystalloid infusion (e.g., 1-10 ml/kg) provides significant benefits in the rapid and sustained recovery of acceptable hemo dynamic parameters in controlled hemorrhage. In another embodiment, the lipid emulsion used is Intralipid®. In another embodiment, the lipid emulsion used is 20% Intralipid®. In one embodiment, the lipid comprises anti-inflammatory lipids such as omega-3 fatty acids. Hypertonicity may also be achieved by adding glycerol.
Anti-Inflammatory and Immunomodulatory Agent
In one embodiment, the pharmaceutical composition of the present application further includes an anti-inflammatory or immunomodulatory agent. Examples of the anti-inflammatory agent shown to inhibit reactive oxygen species including, but are not limited to, histidine, albumin, (+) naloxone, prostaglandin D2, molecules of the phenylalkylamine class. Other anti-inflammatory compounds and immunomodulatory drug include interferon; interferon derivatives comprising betaseron, β-interferon; prostane derivatives comprising iloprost, cicaprost; glucocorticoids comprising cortisol, prednisolone, methyl-prednisolone, dexamethasone; immunsuppressives comprising cyclosporine A, methoxsalene, sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitors comprising zileutone, MK-886, WY-50295, SC-45662, SC-41661A, BI-L-357; leukotriene antagonists; peptide derivatives comprising ACTH and analogs thereof; soluble TNF-receptors; anti-TNF-antibodies; soluble receptors of interleukins or other cytokines; antibodies against receptors of interleukins or other cytokines, T-cell-proteins; and calcipotriols and analogues thereof taken either alone or in combination.
Electrolytes
In one embodiment, the pharmaceutical composition of the present application includes one or more electrolytes. The electrolyte to be used in the present application typically includes various electrolytes to be used for medicinal purposes. Examples of the electrolyte include sodium salts (e.g., sodium chloride, sodium hydrogen carbonate, sodium citrate, sodium lactate, sodium sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate, sodium glycerophosphate, sodium carbonate, an amino acid sodium salt, sodium propionate, sodium β-hydroxybutyrate, and sodium gluconate), potassium salts (e.g., potassium chloride, potassium acetate, potassium gluconate, potassium hydrogen carbonate, potassium glycerophosphate, potassium sulfate, potassium lactate, potassium iodide, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium citrate, an amino acid potassium salt, potassium propionate, and potassium β-hydroxybutyrate), calcium salts (e.g., calcium chloride, calcium gluconate, calcium lactate, calcium glycerophosphate, calcium pantothenate, and calcium acetate), magnesium salts (e.g., magnesium chloride, magnesium sulfate, magnesium glycerophosphate, magnesium acetate, magnesium lactate, and an amino acid magnesium salt), ammonium salts (e.g., ammonium chloride), zinc salts (e.g., zinc sulfate, zinc chloride, zinc gluconate, zinc lactate, and zinc acetate), iron salts (e.g., iron sulfate, iron chloride, and iron gluconate), copper salts (e.g., copper sulfate), and manganese salts (for example, manganese sulfate). Among those, particularly preferable are sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lactate, sodium acetate, sodium citrate, potassium acetate, potassium glycerophosphate, calcium gluconate, calcium chloride, magnesium sulfate, and zinc sulfate.
Concentration of calcium, sodium, magnesium and potassium ion is typically within the range of normal physiological concentrations of said ions in plasma. In general, the desired concentration of these ions is obtained from the dissolved chloride salts of calcium, sodium and magnesium. The sodium ions may also come from a dissolved organic salt of sodium that is also in solution.
In one embodiment, the sodium ion concentration is in a range from 70 mM to about 160 mM. In another embodiment, the sodium ion concentration is in a range of about 90 to 130 mM. In another embodiment, the sodium ion concentration is about 102 mM.
In one embodiment, the concentration of calcium ion is in a range of about 0.5 mM to 4.0 mM. In another embodiment, the concentration of calcium ion is in a range of about 2.0 mM to 2.5 mM. In another embodiment, the pharmaceutical composition of the present application does not contain calcium ion.
In one embodiment, the concentration of magnesium ion is in a range of 0 to 10 mM. In another embodiment, the concentration of magnesium ion is in a range of about 0.3 mM to 0.45 mM. It is best not to include excessive amounts of magnesium ion in the pharmaceutical composition of the invention because high magnesium ion concentrations negatively affect the strength of cardiac contractile activity. In one embodiment, pharmaceutical composition of the present application contains subphysiological amounts of magnesium ion. In another embodiment, the pharmaceutical composition of the present application does not contain magnesium ion.
In one embodiment, the concentration of potassium ion is in a subphysiological range of between 0-5 mEq/l K+ (0-5 mM), preferably 2-3 mEq/1 K+ (2-3 mM). Thus, the pharmaceutical composition allows for dilution of the potassium ion concentration in stored transfused blood. As a result, high concentrations of potassium ion and potential cardiac arrhythmias and cardiac insufficiency caused thereby can be more easily controlled. The pharmaceutical composition containing a subphysiological amount of potassium is also useful for purposes of blood substitution and low temperature maintenance of a subject. In some embodiments, the pharmaceutical composition of the present application contain potassium ion at a concentration of about 4 mM.
In one embodiment, the concentration of chloride ion is in the range of 70 mM to 160 mM. In another embodiment, the concentration of chloride ion is in the range of 110 mM to 125 mM.
Other sources of ions include sodium salts (e.g., sodium hydrogen carbonate, sodium citrate, sodium lactate, sodium sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate, sodium glycerophosphate, sodium carbonate, an amino acid sodium salt, sodium propionate, sodium β-hydroxybutyrate, and sodium gluconate), potassium salts (e.g., potassium acetate, potassium gluconate, potassium hydrogen carbonate, potassium glycerophosphate, potassium sulfate, potassium lactate, potassium iodide, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium citrate, an amino acid potassium salt, potassium propionate, and potassium β-hydroxybutyrate), calcium salts (e.g., calcium gluconate, calcium lactate, calcium glycerophosphate, calcium pantothenate, and calcium acetate), magnesium salts (e.g., magnesium sulfate, magnesium glycerophosphate, magnesium acetate, magnesium lactate, and an amino acid magnesium salt), ammonium salts, zinc salts (e.g., zinc sulfate, zinc chloride, zinc gluconate, zinc lactate, and zinc acetate), iron salts (e.g., iron sulfate, iron chloride, and iron gluconate), copper salts (e.g., copper sulfate), and manganese salts (for example, manganese sulfate). Among those, particularly preferable are sodium chloride, potassium chloride, magnesium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lactate, sodium acetate, sodium citrate, potassium acetate, potassium glycerophosphate, calcium gluconate, calcium chloride, magnesium sulfate, choline chloride and zinc sulfate.
Carbohydrates and Amino Acids
The pharmaceutical composition may contain a carbohydrate or a mixture of carbohydrates. Suitable carbohydrates include, but are not limited to, simple hexose (e.g., glucose, fructose and galactose), mannitol, sorbitol or others known to the art. In one embodiment, the pharmaceutical composition includes physiological levels of a hexose. “Physiological levels of a hexose” includes a hexose concentration of between 2 mM to 50 mM. In one embodiment, the pharmaceutical composition contains 5 mM glucose. At times, it is desirable to increase the concentration of hexose in order to provide nutrition to cells. Thus the range of hexose may be expanded up to about 50 mM if necessary to provide minimal calories for nutrition. Micelles may also be used as a nutritional source.
Other suitable carbohydrates include various saccharides to be used for medicinal purposes. Examples of the saccharides include xylitol, dextrin, glycerin, sucrose, trehalose, glycerol, maltose, lactose, and erythritol.
The pharmaceutical composition may contain an amino acid or a mixture of amino acids. Suitable amino acids include, but are not limited to, alanine, arginine, aspartate, asparagine, cysteine, glutamate, glutamine, glycine, histidine, isoleuc leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, threonine, tryptophan, valine and 2-aminopentaenoic acid. In one embodiment, the amino acid is selected from the group consisting of histidine, tyrosine, phenylalanine and cysteine. In another embodiment, the pharmaceutical composition comprises an amino acids known to prevent apoptosis. Examples of such amino acids include glutamine, glycine, proline and 2-aminopentaenoic acid.
The amino acid may be used in the concentration range of 0.1 fM-200 mM, 0.1 fM-20 mM, 0.1 fM-1 mM, 0.1 fM-100 μM, 0.1 fM-10 μM, 0.1 fM-1 μM, 0.1 fM-100 pM, 0.1 fM-10 pM, 0.1 fM-1 pM, 0.1 fM-100 fM, 0.1 fM-10 fM, 0.1 fM-1 fM, 1 fM-200 mM, 1 fM-20 mM, 1 fM-1 mM, 1 fM-100 μM, 1 fM-10 μM, 1 fM-1 μM, 1 fM-100 pM, 1 fM-10 pM, 1 fM-1 pM, 1 fM-100 fM, 1 fM-10 fM, 10 fM-200 mM, 10 fM-20 mM, 10 fM-1 mM, 10 fM-100 μM, 10 fM-10 μM, 10 fM-1 μM, 10 fM-100 pM, 10 fM-10 pM, 10 fM-1 pM, 10 fM-100 fM, 100 fM-200 mM, 100 fM-20 mM, 100 fM-1 mM, 100 fM-100 μM, 100 fM-10 μM, 100 fM-1 μM, 100 fM-100 pM, 100 fM-10 pM, 100 fM-1 pM, 1 pM-200 mM, 1 pM-20 mM, 1 pM-1 mM, 1 pM-100 μM, 1 pM-10 μM, 1 pM-1 μM, 1 pM-100 pM, 1 pM-10 pM, 10 pM-200 mM, 10 pM-20 mM, 10 pM-1 mM, 10 pM-100 μM, 10 pM-10 μM, 10 pM-1 μM, 10 pM-100 pM, 100 pM-200 mM, 100 pM-20 mM, 100 pM-1 mM, 100 pM-100 μM, 100 pM-10 μM, 100 pM-1 μM, 1-200 mM, 1 μM-20 mM, 1 μM-1 mM, 1-100 μM, 1 μM-10 μM, 10 μM-200 mM, 10 μM-20 mM, 10 μM-1 mM, 10 μM-100 μM, 100 μM-200 mM, 100 μM-20 mM, 100 μM-1 mM, 1 mM-200 mM, 1 mM-20 mM, 1 mM-10 mM, 1 mM-5 mM or 5 mM-20 mM.
In one embodiment, the amino acid is used at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM.
Coagulation Enhancers
Aggressive high volume resuscitation, without controlling the bleeding, can exacerbate the hemorrhage by disrupting the early formed soft thrombi, and by diluting coagulation factors. In certain embodiments, the pharmaceutical composition may further comprise one or more coagulation enhancers. Examples of coagulation factors include, but are not limited to, factor VII, thrombin, platelets and tranexemic acid. These factors may be from natural or non-natural sources. In certain embodiments, factor 7 is added to the pharmaceutical composition at a concentration of 70-150 IU/kg body weight, prothrombin complex is added to the pharmaceutical composition at a concentration of 15-40 IU/kg body weight, and fibrinogen is added to the pharmaceutical composition at a concentration of 50-90 mg/kg body weight. Naturally-derived or synthetic platelets or platelet substitutes may also be added.
Antioxidants
In certain embodiments, the pharmaceutical composition may further comprise one or more antioxidants. Examples of antioxidants include, but are not limited to, sodium hydrogen sulfite, sodium sulfite, sodium pyrosulfite (e.g., sodium metabisulfite), rongalite (CH2OHSO2Na), ascorbic acid, sodium ascorbate, erythorbic acid, sodium erythorbate, cysteine, cysteine hydrochloride, homocysteine, glutathione, thioglycerol, α-thioglycerin, sodium edetate, citric acid, isopropyl citrate, potassium dichloroisocyanurate, sodium thioglycolate, sodium pyrosulfite 1,3-butylene glycol, disodium calcium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, an amino acid sulfite (e.g, L-lysine sulfite), butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), propyl gallate, ascorbyl palmitate, vitamin E and derivatives thereof (e.g., dl-α-tocopherol, tocopherol acetate, natural vitamin E, d-δ-tocopherol, mixed tocopherol, and trolox), guaiac, nordihydroguaiaretic acid (NDGA), L-ascorbate stearate esters, soybean lecithin, palmitic acid ascorbic acid, benzotriazol, and pentaerythrityl-tetrakis[3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate]2-mercaptobenzimidazole. Among those, preferable are sodium hydrogen sulfite, sodium sulfite, ascorbic acid, homocysteine, dl-α-tocopherol, tocopherol acetate, glutathione, and trolox.
Other Components
In addition to the components discussed above, the pharmaceutical composition may further comprise other additives that include, but are not limited to, antibiotics, such as penicillin, cloxacillin, dicloxacillin, cephalosporin, erythromycin, amoxicillin-clavulanate, ampicillin, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, aminoglycoside (e.g., tobramycin and gentamicin), streptomycin, sulfa drugs, kanamycin, neomycin, land monobactams; anti-viral agents, such as amantadine hydrochloride, rimantadin, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine, valacyclovir, valgancyclovir, pencyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine; anti-fungal agents such as terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid, voriconazole, caspofungin, and selenium sulfide; vitamins, amino acids, vessel expanders such as alcohols and polyalcohols, surfactants, antibodies against harmful cytokines such as tumor necrosis factor (TNF) or interleukins, and mediators of vascular potency and immunomoduators, such as prostaglandins, leukotrienes, pro-opiomelanocortin fragments and platelet activating factors.
In certain embodiments, the pharmaceutical composition may further contain beneficial anions such as lactate or glutamate. Hypertonic lactate containing compositions have been found to be effective in reducing brain edema in patients with acute hemodynamic distress. In one embodiment, the pharmaceutical composition contains 250 to 2400 mM of lactic acid or lactate. In other embodiments, the pharmaceutical composition contains 250 to 500 mM, 500 to 1000 mM, 1000 to 1500 mM, 1500 to 2000 mM or 2000 to 2400 mM of lactic acid or lactate.
In certain other embodiments, the pharmaceutical composition may contain substituted cations. For example, the pharmaceutical composition may contain choline to substitute sodium ions.
In some other embodiments, the pharmaceutical composition further comprises a potassium channel blocker, which is capable of inhibiting programmed cell death by preventing potassium efflux.
In certain embodiments, the pharmaceutical composition further contains anti-cancer drugs and/or intracellular signal molecules, such as cAMP and diacylglycerol. In other embodiments, the pharmaceutical composition further contain one or more organelles or organelle components such as endoplasmic reticulum, ribosomes, and mitochondria in whole or in part.
In other embodiments, the pharmaceutical composition may be combined with red blood cells, modified red blood cells or other cellular components of blood.
In yet other embodiments, the pharmaceutical composition further comprises proopiomelanocortin fragments, such as beta endorphin, melonocyte stimulating hormone enkephalins or opiates to modify the immune response and to provide analgesia. Beta endorphin may also be used at a final concentration of 0.01-100 nm. In some embodiments, the pharmaceutical composition comprises beta endorphin in the concentration range of 0.1-10 nm. In other embodiments, the pharmaceutical composition comprises beta endorphin at a concentration of about 0.2, 0.5, 1, 2 or 5 nm, to modulate neutrophilic function in the septic state.
In yet other embodiments, the pharmaceutical composition further comprises one or more neurotrophic agents for treatment of psychiatric disease or prevention of psychiatric disease. Neurotrophic agents that are important for survival, growth, or differentiation of discrete neuronal populations. Examples of neurotrophic agents include, but are not limited to, factors in the neurotrophin family, such nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrohin-3 (NT-3) and neurotrohin-4 (NT-4); factors in the CNTF family, such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6); and factors in the GDNF family, such as glial cell line derived neurotrophic factor (GDNF), neurturin (NTN), artemin (ART) and persephin (PSP).
The pharmaceutical composition of the present application possesses the ability to absorb toxic chemical molecules/biomolecules produced as the result of trauma or hemorrhagic shock. For example, lymph factors produced in gut and thoracic duct lymph nodes may result in acute lung injury and red blood cell deformability after trauma/hemorrhagic shock. Other toxic chemical molecules/biomolecules include, but are not limited to, leukotrienes, prostaglandins, nitric oxide, endotoxin and tumor necrosis factor (TNF). The lipid emulsion in the pharmaceutical composition allows effective absorption of lipophilic chemical molecules/biomolecules. In certain embodiment, the pharmaceutical composition further contains antagonists to toxic chemical molecules/biomolecules, such as antibodies to endotoxins.
In some embodiments, the pharmaceutical composition comprises 10-50% (w/v) soybean oil, chia bean oil or algae oil, 1-6% (w/v) soybean lecithin and 1-5% (w/v) glycerol or mannitol. In other embodiments, the above pharmaceutical composition further comprises a buffering agent at a concentration of 1-100 mM. In some embodiments, the buffering agent comprises 1-10 mM histidine, 1-10 mM glycylglycine, 1-10 mM carnosine, 1-10 mM glycylhistidine, 1-10 mM gly-his-gly, 1-10 mM other polyhistidine, polyglycine or his/gly-containing peptides of 3-10 amino acids or combinations thereof. In other embodiments, the above pharmaceutical composition further comprises lysine at a concentration of 1-10 mM. In other embodiments, the above pharmaceutical composition further comprises lysine at a concentration of about 4 mM. In other embodiments, the above pharmaceutical composition further comprises albumin at a concentration of 1-10% (w/v). In other embodiments, the above pharmaceutical composition further comprises albumin at a concentration of about 5% (w/v).
In others embodiment, the pharmaceutical composition comprises 10-40% (w/v) soybean oil, chia bean oil or algae oil, 1-5% (w/v) phospholipid or soybean lecithin or phospholipid, 1-4% (w/v) glycerol or mannitol, about 102 mM NaCl, about 28 mM Na(L) lactate, and about 4 mM KCl. In some embodiments, the pharmaceutical composition further comprises a buffering agent selected from the group consisting of 1-10 mM histidine, 1-10 mM glycylglycine, 1-10 mM carnosine, 1-10 mM glycylhistidine, 1-10 mM gly-his-gly or combinations thereof. In some embodiments, the pharmaceutical composition does not comprise albumin.
In another embodiment, the pharmaceutical composition comprises 35% (w/v) soybean oil, 3.94% (w/v) alpha phosphatidylcholine, 2.25% (w/v) glycerol, about 102 mM NaCl, about 28 mM Na(L) lactate, about 4 mM KCl, about 1 mM histidine, about 4 mM lysine and no albumin. In another embodiment, the pharmaceutical composition comprises 35% (w/v) algae oil, 3.94% (w/v) alpha phosphatidylcholine, 2.25% (w/v) glycerol, about 102 mM NaCl, about 28 mM Na(L) lactate, about 4 mM KCl, about 1 mM histidine, about 1 mM arginine and no albumin.
In another embodiment, the pharmaceutical composition comprises 10-30% (w/v) soybean oil, chia bean oil or algae oil, 1-2% (w/v) phospholipid or soybean lecithin, 2-3% (w/v) glycerol or mannitol, about 102 mM NaCl, about 28 mM Na(L) lactate, about 4 mM KCl and about 1 mM histidine, tyrosine, phenylalanine or cysteine. In some embodiments, the pharmaceutical composition further comprises a buffering agent selected from the group consisting of 1-10 mM histidine, 1-10 mM glycylglycine, 1-10 mM carnosine, 1-10 mM glycylhistidine, 1-10 mM gly-his-gly or combinations thereof.
In another embodiment, the pharmaceutical composition comprises 10-30% (w/v) soybean oil, chia bean oil or algea oil, 1-2% (w/v) phospholipid or soybean lecithin, 2-3% (w/v) glycerol or mannitol, about 102 mM NaCl, about 28 mM Na(L) lactate, about 4 mM KCl and about 1 mM histidine, tyrosine, phenylalanine or cysteine.
In another embodiment, the pharmaceutical composition comprises 20-35% (w/v) soybean oil, 1.2-2.1% (w/v) egg phospholipid, 2.25% glycerin (w/v), about 102 mM NaCl, about 28 mM Na(L) lactate, about 4 mM KCl, about 1 mM histidine, about 4 mM lysine and no albumin.
In some embodiments, the above-described pharmaceutical composition is prepared by preparing a micelle emulsion that contains the oil, emulsifier and glycerol/mannitol. The other components are added prior to use in order to prevent destabilization of the micelles. In other embodiments, the above-described pharmaceutical composition is prepared by preparing a micelle component that contains the oil, emulsifier, glycerol/mannitol and all other ingredients except K+, which is added just prior to use (e.g., within 24 hour of use). In some embodiments, the above-described pharmaceutical composition is used as a blood volume expander.
The pharmaceutical composition is prepared under conditions that form micelles with an average diameter of 5 μm or smaller. In one embodiment, the micelles in the pharmaceutical composition have an average diameter of 1 μm or smaller. In another embodiment, the micelles in the pharmaceutical composition have an average diameter of 300 nm or smaller. In another embodiment, the micelles in the pharmaceutical composition have an average diameter of 100 nm or smaller. In another embodiment, the micelles in the pharmaceutical composition have an average diameter of 30 nm or smaller. In yet another embodiment, the micelles in the pharmaceutical composition have an average diameter of 10 nm or smaller. In some embodiments, the micelles have an average diameter in the range of 3-300 nm, 3-30 nm, 30-100 nm or 100-300 nm. In other embodiments, the micelles have an average diameter of about 10 nm, 30 nm, 50 nm, 80 nm, 100 nm, or 300 nm.
In another embodiment the hydrophobic component is carried within erythrocyte ghosts. Other oils such as oil from chia beans, pumpkin seeds or other sources may be used to take advantage of their ability to modulate negatively or positively the processes of coagulation, proliferation, immune response or nutrition as needed by the disease process being addressed. In certain embodiments, the above described pharmaceutical composition further comprise about 2-40% (w/v), about 2-20% (w/v), about 4-10% (w/v), or about 5% (w/v) albumin or albumin polymers or albumin polymers conjugated with amino acids or peptides, which are added to the pharmaceutical composition after the formation of micelles.
In certain embodiments the pharmaceutical composition of the present application comprises a lipid component selected from the group consisting of soybean oil, chia bean oil and algae oil, an emulsifier selected from the group consisting of phospholipids and α-phosphatidylcholine, and an amino acid or n-acetyl amino acid at a final concentration of 0.2-20 mM, 0.5-10 mM, 0.5-5 mM or 0.5-2 mM. In certain embodiments, the pharmaceutical composition has an final amino acid concentration of 0.1, 0.2, 0.5, 1, 2.5, 5, 7.5, or 10 mM. The emulsifier:the lipid component ratio (w/w) is about 1:400 to about 1:5. In some embodiments, the emulsifier:the lipid component ratio (w/w) is about 1:50 to about 1:10. In other embodiments, the emulsifier:the lipid component ratio (w/w) is about 1:30 to about 1:10. In other embodiments, the emulsifier:the lipid component ratio (w/w) is about 1:20 to about 1:15. In one embodiment, the emulsifier:the lipid component ratio (w/w) is about 1:16.7.
The pharmaceutical composition is free of Ca++, and Mg++ and Al+++, as well as hemoglobin, derivatives of hemoglobin, perfluorocarbon and derivatives of perfluorocarbon. In certain embodiments, Ca++ is added to the pharmaceutical composition just prior to use (e.g., within 24 hours prior to use). Because Al+++ is toxic to bone, brain, hematopoieisis, heme synthesis, globulin synthesis, iron absorption and metabolism, and fetal development, all oils and other components must have the minimum amount of Al+++ possible. In certain embodiments, the pharmaceutical composition contains Al+++ at a concentration of less than 25 mg/l, 20 mg/l, 10 mg/l or 5 mg/l. In other embodiments, the pharmaceutical composition is free of Al+++, i.e., undetectable by conventional methods.
In one embodiment, the pharmaceutical composition comprises soybean oil, egg yolk phospholipids and an amino acid, beta-endorphin or other modulator that acts at femtomolar concentrations or higher at a final concentration of 0.1 femtomolar (fM) to 10 mM.
Preparation of the Pharmaceutical Composition
The pharmaceutical composition may be prepared by mixing the amphiphilic emulsifier, the aqueous carrier, and optionally the lipid component and any other components, to form an emulsion. In some embodiments, no lipid or a very small amount lipid (e.g., less than 5%, 2%, 1%, 0.5%, 0.2% or 0.1% (w/v)) is present in the pharmaceutical composition. Commonly used mixing methods include, but are not limited to, stirring, shaking, homogenization, vibration, microfluidization and sonication. In one embodiment, the pharmaceutical composition is formed by mixing a pre-formed lipid emulsion, such as Intralipid® or Liposyn® with the aqueous carrier and other components. In addition, the pharmaceutical composition can be carried in erythrocyte ghosts. Specifically, the emulsion should be prepared in manners that allow the lipophilic gases dissolving into the lipophilic portion of the emulsion but not forming microbubbles which may increase the risk of gas embolization.
In certain embodiments, albumin or albumin polymers or albumin polymers conjugated with amino acids or peptides is added to the pharmaceutical composition in an amount of 2-40% (w/v), about 2-20% (w/v), about 2-10% (w/v), about 3-8%, about 4-6% or about 5% (w/v). The albumin or albumin polymers or albumin polymers conjugated with amino acids or peptides is added to the pharmaceutical composition after the formation of micelles. In one embodiment, the lipid component, the emulsifier, the aqueous carrier and any other non-albumin components are mixed to form an emulsion. Albumin, albumin polymers or albumin polymers conjugated with amino acids or peptides is then dissolved in the emulsion at the desired concentration.
In one embodiment, the pharmaceutical composition is prepared according to the following recipes:
Recipe 1
Part A
soybean oil 20%=20 grams
glycerol 2.25%=2.25 grams
egg yolk phospholipids 1.2%=1.2 grams
add water to final volume of 100 ml
stir or sonicate to produce micelles with average diameter of 300-400 nm
Part B
NaCl 102 mM=0.6 grams
Na (L) lactate 28 mM=0.31 grams
KCl 4 mM=0.03 grams
CaCl2 1.5 mM=0.06 grams
Human serum albumin 5%=5 grams
Add sodium hydroxide until pH=8.0
Part A may be used alone, or mixed with Part B within 24 hours of use.
Recipe 2
Part A
Soybean oil 20%=20 grams
Glycerol 2.25%=2.25 grams
Egg yolk phospholipids 1.2%=1.2 grams add water
Add water to final volume of 100 ml
stir or sonicate to produce micelles with average diameter of 300-400 nm
Part B
NaCl 102 mM=0.6 grams
Na (L) lactate 28 mM=0.31 grams
KCl 4 mM=0.03 grams
CaCl2 1.5 mM=0.06 grams
histidine or cysteine 1 mM=15.5 mg histidine or 12.1 mg cycteine
β-endorphin 1 nM=346.5 ng
add sodium hydroxide until pH=8.0
Part A may be used alone, or mixed with Part B within 24 hours of use.
In some embodiments, Part A or the mixture of Part A and Part B, is loaded with oxygen, nitric oxide, carbon monoxide, xenon, argon, hydrogen sulfide other hydrophobic gases or mixtures of these gases prior to use. These gases may be used in shock to deliver oxygen for aerobic metabolism after the initial bolus, provide an initial carbon monoxide bolus to protect against reperfusion injury, to open vessels in vascular diseases or states involving vascular constriction or obstruction, xenon or argon to protect against the effects of traumatic brain injury or seizures, or hydrogen sulfide to promote long-term tissue preservation. Nitric oxide loaded micelles may also be used as an anti-hypertensive medication. Either Part A, Part B, or the mixture of Part A and Part B can be sterilized by autoclaving.
In some embodiments, the soybean oil, which enhances clotting, is replaced or partially replaced with chia bean oil which is anti-inflammatory and reduces clotting. In one embodiment, a pharmaceutical composition with soybean oil is used in initial phase of the infusion in which bleeding is occurring. A pharmaceutical composition with chia bean oil is used for later stages of the infusion when bleeding is no longer an issue an issue.
In some other embodiments, the glycerol in Part A is replaced with mannitol. In other embodiments, the egg phospholipids is replaced with α-phosphatidylcholine to eliminate source of protein contamination and anaphylaxis due to contamination of egg phospholipid with egg protein. In yet other embodiments, the amino acids in Part B of Recipe 2 is replaced with N-acetyl amino acids.
In one embodiment, the pharmaceutical composition is a non-oxygenated pharmaceutical composition. As used herein, the term “non-oxygenated pharmaceutical composition” refers to a formulation that is prepared in atmospheric air and is not loaded with oxygen by any oxygenation device or method.
In certain embodiments, the pharmaceutical composition may be loaded with a lipophilic gas prior to clinical application. Examples of such gases include, but are not limited to, oxygen, xenon, argon, nitric oxide, carbon monoxide, hydrogen sulfide. As used herein, “a pharmaceutical composition loaded with a lipophilic gas” refers to a pharmaceutical composition that has been subjected to a process to increase the content of such lipophilic gas in the pharmaceutical composition. A pharmaceutical composition may be loaded with a lipophilic gas by bubbling the lipophilic gas through the pharmaceutical composition for a desired period of time, or by agitating the pharmaceutical composition in the presence of the lipophilic gas under pressure.
In one embodiment, the pharmaceutical composition is oxygenated by bubbling pure oxygen or a gas with an oxygen content in the range of 21% to 100% (v/v), preferably 40% to 100% (v/v), more preferably 60% to 100% (v/v), and most preferably 80% to 100% (v/v), through the mixture for a period of 30 seconds or longer, preferably 1-15 minutes, more preferably 1-5 minutes. Oxygen may also be added under pressure followed by a reduction of the pressure to one atmosphere. In one embodiment, the pharmaceutical composition is oxygenated immediately prior to application. The pharmaceutical composition may be oxygenated using portable oxygen tanks or portable oxygen concentrators, such the Evergo Portable Pulse Dose Oxygen concentrator produced by Philips Healthcare at Andover, Mass.
Another method could be allowing the emulsion to equilibrate with an atmosphere filled with the gas that is to be added. In most cases a bubble trap would be necessary to remove bubbles that could become gas emboli. The equilibration time for a pharmaceutical composition of a particular composition may be determined experimentally.
In one embodiment, the pharmaceutical composition comprises an oxygenated lipid emulsion. As used herein, the term “oxygenated lipid emulsion” or “oxygenated pharmaceutical composition” refers to a specific type of gassed lipid emulsion or gassed fluid which has been forced to absorb oxygen such that the total concentration of oxygen contained therein is greater than that present in the same liquid at atmospheric equilibrium conditions.
Kits
Another aspect of the present application relates to a resuscitation kit. In one embodiment, the resuscitation kit comprises an oxygenated pharmaceutical composition and at least one additive. Examples of additives include, but are not limited to, oncotic agent, crystalloid agent, vessel expander, cardioplegic, or cardiotonic agent scavengers of free radicals or mediators, cell signaling modulators, and receptor agonists or antagonists. In another experiment, the kit further contains an intravenous infusion (IV) set. In another embodiment, the oxygenated pharmaceutical composition is contained in one or more preloaded syringes for emergency application.
In another embodiment, the kit contains a pharmaceutical composition and a portable oxygen container that can be used to re-oxygenate the pharmaceutical composition immediately prior to application. The oxygen container may contain pure oxygen, or a gas mixture of oxygen with hydrogen sulfide and/or carbon monoxide and/or nitric oxide. In another embodiment, the kit contains a pharmaceutical composition, and an air pump for oxygenating the pharmaceutical composition with ambient air immediately prior to application.
In another embodiment, the kit contains pharmaceutical composition and a portable oxygen concentrator for oxygenating the pharmaceutical composition with oxygen filtered from ambient air immediately prior to application. In one embodiment, the portable oxygen concentrator is an Evergo Portable Pulse Dose Oxygen concentrator produced by Philips Healthcare at Andover, Mass.
In another embodiment, the kit contains a pharmaceutical composition and an oxygen producing canister that is capable of producing oxygen through a chemical reaction. Chemicals that may be used for the production of oxygen include, but are not limited to, sodium chlorate, sodium peroxide and potassium superoxide.
In another embodiment, the kit further contains a bubble removing device, such as a bubble trap.
In another embodiment, the kit contains syringes that are prefilled with the pharmaceutical composition of the present application and are ready to be pushed into the blood stream of shock patients. In certain embodiment, the syringes have volumes of 60-500 cc. In other embodiments, the syringes also come with delivery tubing. The pharmaceutical composition may also be preloaded with oxygen, some other gas or other beneficial substances. In another embodiment, the kit contains cartridges that are prefilled with the pharmaceutical composition of the present application. The cartridges can be snapped into an apparatus that would infuse the pharmaceutical composition into a patient at a desired rate.
Treatment Methods
Another aspect of the present application relates to a method for treating conditions related to lack of blood supply with a lipid-based pharmaceutical composition. Conditions related to a lack of blood supply include, but are not limited to, hypovolemia, ischemia, hemodilution, trauma, septic shock, cancer, anemia, cardioplegia, hypoxia and organ perfusion. The term “hypovolemia,” as used herein, refers to an abnormally decreased volume of circulating fluid (blood or plasma) in the body. This condition may result from “hemorrhage,” or the escape of blood from the vessels. The term “ischemia,” as used herein, refers to a deficiency of blood in a part of the body, usually caused by a functional constriction or actual obstruction of a blood vessel. Conditions related to lack of blood supply also include situations in which the intravascular volume may be normal but blood vessels are dilated. Examples are septic shock in which mediators such as endothelium-derived relaxing factors cause blood vessel tone and responsiveness to catecholamines to decrease. Examples of an endothelium-derived relaxing factor is nitric oxide. Prostaglandin E is another molecule that dilates blood vessels.
The pharmaceutical composition may be administered intravenously, intra-arterially or intra cardiac to a subject in need of such treatment. Administration of the pharmaceutical composition can occur for a period of seconds, hours, days or weeks depending on the purpose of the pharmaceutical composition usage, the ability to control blood loss or the ability to restore spontaneous cardiac contraction. For example, when used as a blood volume expander and an oxygen carrier for the treatment of severe hemorrhage shock, the usual time course of administration is as rapidly as possible, which may range from about 1 ml/kg/hour to about 150 ml/kg/min, or from 10 ml/kg/hour to about 10 ml/kg/min, or from about 10 ml/kg/min to about 150 ml/kg/min. For the treatment of severe hemorrhagic shock, the pharmaceutical composition is given in an amount and at an infusion rate that is sufficient to raise the blood pressure in the patient. In certain embodiments, the pharmaceutical composition is given in an amount of 500-4000 ml, 500-2000 ml, 500-1000 ml, 1000-4000 ml and 1000-2000 ml. In certain embodiments, the pharmaceutical composition is given in an amount that equals to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%, 85% or 90% of the normal blood volume of a patient in a period of 30-300 seconds. In certain embodiments, the pharmaceutical composition is given at a rate of 200-4000 ml/min. In other embodiments, the pharmaceutical composition is given at a rate of about 1000-4000 ml/min. In other embodiments, the pharmaceutical composition is given at a rate of about 200-2000 ml/min. In other embodiments, the pharmaceutical composition is given at a rate of about 100-1000 ml/min. In yet other embodiments, the pharmaceutical composition is given at a rate of about 500-700 ml/min. In some embodiments, the pharmaceutical composition is given without oxygenation. In other embodiments, the pharmaceutical composition is an oxygenated pharmaceutical composition. The infusion rate may vary from a low of 5 ml/min to 120 liters/hour. There is no upper limit of volume or rate due to the fact that blood loss can be massive and the infusion of the pharmaceutical composition would be given at a rate and volume necessary to provide sufficient perfusion of tissues and tissue viability.
Another aspect of the present application relates to a method for increasing blood pressure in a subject in need of such treatment. In certain embodiment, the blood pressure refers to systolic pressure. The method comprises administering into the patient an effective amount of the pharmaceutical composition of the present application. In one embodiment, the subject is a mammal who lost at least 15% of its blood volume. In another embodiment, the subject is a mammal who lost 15% to 30% of its blood volume. In another embodiment, the subject is a mammal who lost at least 30% of its blood volume. In another embodiment, the subject is a mammal who lost 30% to 40% of its blood volume. In another embodiment, the subject is a mammal who lost at least 40% of its blood volume. In another embodiment, the subject is in hemorrhagic shock. In yet another embodiment, the subject is in severe hemorrhagic shock. As used herein, the term “hemorrhagic shock” refers to a shock status induced by the loss of at least 15% of the blood volume. As used herein, the term “severe hemorrhagic shock” refers to a shock status induced by the loss of at least 30% of the blood volume.
In one embodiment, the “effective amount” of the pharmaceutical composition needed to increase the blood pressure in a subject with hemorrhagic shock or severe hemorrhagic shock is 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200% or 300% of the lost blood volume. Typically, the pharmaceutical composition is infused initially at a volume that equals to at least 40%, 50%, 60%, 70% 80%, 90% or 100% of the lost blood volume to raise the blood pressure. A larger amount (e.g., 150% to 300% or even more) is given on an as needed basis.
In another embodiment, the “effective amount” of the pharmaceutical composition needed to increase the blood pressure in a subject with hemorrhagic shock or severe hemorrhagic shock is the amount needed to restore the systolic pressure of the subject to 70 mmHg, 80 mmHg, 90 mmHg, or higher.
Other hemodynamic parameters, such as perfusion of brain, kidneys, heart, muscle, spleen or other tissues, cardiac output, stroke volume index, mitochondrial oxidative phosphorylation followed by near infrared spectroscopy or other means, blood lactate or membrane polarization, may also be used to determine the “effective amount” of the pharmaceutical composition needed to increase the blood pressure in a subject.
While the pharmaceutical composition of the present application is being administered to and circulated through the subject, various agents such as cardioplegic or cardiotonic agents may be administered either directly into the subject's circulatory system, administered directly to the subject's myocardium, or added to the pharmaceutical composition the present application. These components are added to achieve desired physiological effects such as maintaining regular cardiac contractile activity, stopping cardiac fibrillation or completely inhibiting contractile activity of the myocardium or heart muscle.
Cardioplegic agents are materials that cause myocardial contraction to cease and include anesthetics such as lidocaine, procaine and novocaine and monovalent cations such as potassium ion in concentrations sufficient to achieve myocardial contractile inhibition. Concentrations of potassium ion sufficient to achieve this effect are generally in excess of 15 mM.
During revival of a subject, the subject may be re-infused with a mixture of the pharmaceutical composition described along with blood retained from the subject or obtained from blood donors. Whole blood is infused until the subject achieves an acceptable hematocrit, generally equal to or exceeding hematocrits of about 30% and the goals of resuscitation such as a normal lactate or mixed venous oxygen saturation of between 60% and 76% have been achieved. When these goals have been achieved, rapid infusion is discontinued and the subject is revived after closure of surgical wounds using conventional procedures. In certain embodiments, the pharmaceutical composition of the present application is used to treat shock, including but are not limited to, neurogenic shock, cardiogenic shock, adrenal insufficiency shock and septic shock.
Another aspect of this invention is ability to increase blood pressure by reversible absorption of nitric oxide. Hemoglobin and nitric oxide synthase inhibitors are very effective at removing nitric oxide. However they do not readily lead to the release of nitric oxide. Such potent and irreversible uptake of nitric oxide can lead to lethal vasoconstriction even as the blood pressure increases. In contrast, the pharmaceutical composition takes up nitric oxide because of the greater solubility of nitric oxide in lipids. This mechanism is not as potent as the aforementioned methods and it is highly reversible. This mechanism method provides a variable reservoir of nitric oxide that may take up or release nitric oxide in a way that varies with local concentrations of nitric oxide.
Another aspect of the present application relates to a method of using the pharmaceutical composition described above to oxygenate patients whose lungs are severely damaged and unable to absorb oxygen even with special modes of ventilation. The oxygen loaded pharmaceutical composition may deliver oxygen to tissues via circulation and allows the lung to recover from the damage. In this regard, the pharmaceutical composition may be used to replace extracorporeal membrane oxygenation (ECMO).
Another aspect of the present application relates to a method of using the pharmaceutical composition in exchange transfusion and whole circulation perfusion to wash out blood containing harmful materials such as infectious agents, cancerous agents, and toxic agents. In both cases, the pharmaceutical composition may comprise an emulsion consisting of 20% or more lipid micelles and isotonic saline with or without albumin. The pharmaceutical composition may also be used to absorb toxic chemical molecules/biomolecules produced as the result of trauma, hemorrhagic shock or other form of shocks. Oxygen or other gases may or may not be added.
Treatment of Seizure or Neuronal Injuries with Xenon/Argon-Carrying Pharmaceutical Composition
Another aspect of the present application relates to a method for treating seizure or neuronal injury using xenon or argon carried by the pharmaceutical composition of the present application. Xenon is colorless, heavy, odorless noble gas. Xenon has been used as a general anaesthetic. Two physiological mechanisms for xenon anesthesia have been proposed. The first one involves the inhibition of the calcium ATPase pump—the mechanism cells use to remove calcium (Ca2+)—in the cell membrane of synapses (see e.g., Franks, J J et al. Anesthesiology 82:108-117). The second mechanism involves the non-specific interactions between the anesthetic and the lipid membrane (see, e.g., Heimburg, T and Jackson A D, 2007, Biophysical Journal 92:3159-65. Xenon has a minimum alveolar concentration (MAC) of 71%, making it 50% more potent than N2O as an anesthetic. Thus it can be used in concentrations with oxygen that have a lower risk of hypoxia.
Xenon is also an antagonist of N-methyl-d-aspartate receptors (NMDA receptors) and can be used to treat brain injuries. The NMDA receptors exacerbate the damage from oxygen deprivation and xenon performs better as a neuroprotectant than either ketamine or nitrous oxide, which have undesired side-effects (Ma, D. et al., 2002, British Journal of Anaesthesia 89:739-746).
Without being bound by any theory, it is believed that glutamate excitotoxicity is involved in the development of injury in conditions such as traumatic brain injury and ischemia/stroke and that xenon neuroprotection is mediated by competitive inhibition of the NMDA receptor at its glycine-binding site.
Argon (Ar) is another noble gas that has shown neuroprotective properties in in vitro models of cerebral ischemia and traumatic brain injury (Loetscher et al., Crit Care 2009, 13:R206).
In one embodiment, a xenon- or argon-carrying pharmaceutical composition is used to treat seizure, neuronal injuries and/or brain ischemia in a subject. The treatment comprises: infusing a sufficient amount of a xenon- or argon-carrying pharmaceutical composition into said subject for a desired period of time. The xenon- or argon-carrying pharmaceutical composition may be produced by mixing degased lipid component and degased polar liquid in a container filled with xenon, or by bubbling xenon through a degassed pharmaceutical composition. As noted earlier, the gas carrying capacity of the pharmaceutical composition is proportional to its lipid content. Accordingly, pharmaceutical composition with high lipid content (e.g., 30-50% v/v) is used when a high Xenon dose is needed. In another embodiment, the pharmaceutical composition is saturated with a mixture of xenon and oxygen with the percentage loaded with xenon or argon from 10 to 80% (v/v). In one embodiment, the pharmaceutical composition is saturated with a mixture of xenon/argon and oxygen at a xenon/argon:oxygen ratio of 50:50, 60:40, 70:30, 75:25, or 80:20 (v/v). In yet another embodiment, the pharmaceutical composition is a mixture of a first pharmaceutical composition saturated with xenon or argon and a second pharmaceutical composition saturated with oxygen. In yet another embodiment, xenon or argon is used in conjunction with another volatile anesthetic, such as sevoflurane.
The treatable neuronal injuries involving NMDA receptors, include but are not limited to, ischemic stroke, intracranial bleeding due to trauma, or anoxic brain injury caused by blood loss or other forms of shock or seizure disorder, and encephalitis.
In one embodiment, neuronal injuries are injuries mediated by methyl-D-aspartate (NMDA)-type glutamate receptors. In another embodiment, the neuronal injuries are injuries caused by oxygen deprivation in the brain.
In certain embodiments the xenon, argon, Xe/O2 or Ar/O2-carrying pharmaceutical composition is infused at a rate of 20 to 100,000 ml/hour, 500 to 100,000 ml/hour, or 5000 to 60,000 ml/hour. The infusion may last for a period of several minutes to several weeks, depending on the condition and specific needs of the patient. In certain embodiments, the xenon, argon, Xe/O2 or Ar/O2-carrying pharmaceutical composition is infused for a period of 3 to 600 minutes, 5-300 minutes, 10-120 minutes, 10 to 24 hours, or 1 to 7 days.
In another embodiment, the xenon-carrying pharmaceutical composition also carries a desired amount of carbon monoxide to prevent the development of pathologic conditions such as ischemia reperfusion injury.
In another embodiment, the xenon-carrying pharmaceutical composition also carries a desired amount of hydrogen sulfide to regulate brain perfusion and promote organ survival.
In another embodiment, the xenon-carrying pharmaceutical composition is used in combination with hypothemia (32° C.-34° C.) for treating neuronal injuries. Xenon may similarly be used in cardiac disease given its utility in treating heart failure (Baumert J H et al., Acta Anaesthesiol Scand. 2005 July; 49(6):743-9)
In one embodiment, the xenon/argon treatment is given within 72 hours of neuronal injury or brain ischemia.
In another embodiment, the xenon/argon treatment is given within 24 hours of neuronal injury or brain ischemia.
In another embodiment, the xenon/argon treatment is given within 8 hours of neuronal injury or brain ischemia.
In another embodiment, the xenon/argon treatment is given within 4 hours of neuronal injury or brain ischemia.
In another embodiment, the xenon/argon treatment is given within 2 hours of neuronal injury or brain ischemia.
Treatment of the Hypotension of Septic Shock with the Pharmaceutical Composition of the Present Application
Another aspect of the present application relates to a method for treating septic shock in a subject with pharmaceutical composition to absorb endogenously produced nitric oxide. Septic shock, or sepsis, is a medical condition in which acute inflammation, low blood pressure, and blood clotting cause a dangerous decrease in the delivery of blood to the organs. Because of the failure of oxygen delivery to the microcirculation and the inability to utilize oxygen, once delivered to tissues the patient's organs start to fail, one after the other. Currently, only supportive treatment is available. Nitric oxide plays a prominent role in the pathophysiology of both hemorrhagic and septic shock. Excess nitric oxide release from endothelial cells cause relaxation of arterial smooth muscle cells, loss of vascular tone and hypotension resulting in the inability to perfuse the microcirculation. In addition, nitric oxide can interact with reactive oxygen species and form toxic products such as peroxynitrite. In such cases it would beneficial to remove excess nitric oxide. Therefore, in one embodiment, the method comprises administering into said subject, an effective amount of the pharmaceutical composition for a desired period of time to absorb or remove endogenously released nitric oxide from the circulation system. The nitric oxide-absorbing pharmaceutical composition can be administered intravenously or intra-arterially at a rate of about 20 ml/hour to about 15,000 ml/min, preferably at a rate of 4000 ml/minute.
Treatment of Vasoconstriction and/or Hypertension with Nitric Oxide-Carrying Pharmaceutical Composition of the Present Application
Some diseases such as Raynaud's disease occur because of increased vascular resistance. Other examples are Prinzmetal's angina, cerebral vasospasm and atherosclerosis. In this case loading the pharmaceutical composition with nitric oxide would relieve the vasoconstriction. Also in sickle cell crisis a combination of micelles loaded with nitric oxide and oxygen would be useful with the nitric oxide loaded micelles opening constricted small vessels while oxygen loaded cells delivered oxygen to ischemic tissues. Oxygen free radicals would be scavenged by the antioxidant moieties within the emulsion. In all embodiments the nitric-oxide carrying pharmaceutical composition also carries a desired amount oxygen or other gases depending upon the physiological need. In another embodiment, the nitric-oxide carrying pharmaceutical composition also carries a desired amount of carbon monoxide to prevent the development of pathologic conditions such as ischemia reperfusion injury.
In another embodiment, the nitric-oxide carrying pharmaceutical composition also carries a desired amount of hydrogen sulfide to promote perfusion of the brain and to modulate apoptosis and induce suspended animation.
Preservation of Organs
Another aspect of the present application relates to a method of preserving the biological integrity of organs of a mammalian donor organism using the pharmaceutical composition described. In one embodiment, the subject organ is chilled and the pharmaceutical composition is perfused into the subject organ using a pumped circulating device such as a centrifugal pump, roller pump, peristaltic pump or other known and available circulatory pump. The circulating device is connected to the subject organ via cannulae inserted surgically into appropriate veins and arteries. When the pharmaceutical composition is administered to a chilled subject organ, it is generally administered via an arterial cannula and removed from the subject via a venous cannula and discarded or stored. In another embodiment, the pharmaceutical composition is perfused at a temperature in the room temperature range (15° C. to 25° C.). In another embodiment, the pharmaceutical composition is perfused at a temperature in the sub-body temperature range (25° C. to 34° C.). In another embodiment, the pharmaceutical composition is perfused at a temperature in the body temperature range (34° C. to 39° C.).
When used for organ perfusion during an organ transplantation, the pharmaceutical composition may be administered over a period of hours. In certain embodiments, the pharmaceutical composition comprises chia bean oil for its immunosuppressive effect.
Diagnosis
The pharmaceutical composition of the present application may also be used as a diagnostic tool. Because of its hydrophobic properties, the pharmaceutical composition is capable of absorbing lipophilic moieties that are produced in various disease states. Analysis of the pharmaceutical composition after it has circulated through the blood stream can reveal the presence of diseases that release such moieties into the bloodstream. An example is the production of increased carbon monoxide and nitric oxide in septic shock or other diseases that involve the induction of hemeoxygenase that leads to increased production endogenous carbon monoxide.
EXAMPLES
The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to carry out the method of the present application and is not intended to limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
Methods and Materials
Lipid Emulsions Prepared by Sonication:
Lipid emulsion of various concentrations was prepared by mixing 1-3 grams of soybean oil, 0.12 grams of soy lecithin, and 0.24 grams of glycerol and water to make the volume up to 10 ml. This mixture was put into a 15 ml vial and emulsified in an ice bath using an ultrasound cell disruptor.
Lipid Emulsions Prepared by Homogenization and Microfluidization:
An emulsion consisting of soybean oil (SIGMA, St. Louis) was prepared. The composition was composed of between 20%-50% w/v soybean oil, de-ionized water, varying amounts of an emulsifying agent (either phosphotidylcholine (Avanti Polar Lipids, Alabaster, Ala.) or egg yolk phospholipids (Sigma-Aldrich, St. Louis, Mo.), and with or without 5% human serum albumin (Sigma-Aldrich, St. Louis, Mo.). The albumin was added after the formation of the micelles. Some samples also contained 154 mM NaCl. The mixture was homogenized for five minutes with a Silverson® high-shear rotor/stator homogenizer. A M-110T® microfluidizer (Microfluidics, Newton, Mass.) was used for final homogenization of the coarse emulsion prepared by the Silverson homogenizer.
Commercially Obtained Lipid Emulsions:
20% Intralipid® (marketed and sold by Baxter International Inc., Deerfield, Ill.) was used as a model lipid emulsion. It is composed of 20% soy bean oil, 1.2% egg yolk phospholipids 2.25% glycerin, water and sodium hydroxide to adjust the pH to 8. Another model lipid emulsion obtained commercially is Liposyn® III 20% sold by Hospira in Lake Forest, Ill. This emulsion consists of 20% soybean oil, 1.2% egg phospholipids, 2.5% glycerin, water and NaOH to adjust the pH to 8.3.
Determination of Oxygen Content of Emulsions Using Mass Spectroscopy:
The technique developed in Dr. Juan Rodriguez' laboratory was employed (Cornelius J, Tran T, Turner N, Piazza A, Mills L, Slack R, Hauser S, Alexander J S, Grisham M B, Feelisch M, Rodriguez J. “Isotope tracing enhancement of chemiluminescence assays for nitric oxide research” Biol Chem. February; 390(2):181-9 (2009)). Some samples (1 ml each) were left open to air in 2.0 ml tubes for 30 minutes prior to dissolved gas analysis. In other experiments the emulsion was placed into a 15 ml tube. The headspace was filled with the gas under study and the gas was allowed to enter the emulsion by equilibration over 60 minutes with the tube on its side to increase the gas emulsion interaction. 50 μL drawn from each of these fluids were injected into a Sievers purge vessel at 37° C. containing 36 ml of a mildly acidic solution consisting of 32 ml of 1M HCL and 4 ml of 0.5M ascorbic acid. The solution was continuously purged with high purity helium to transport any oxygen released from the samples to a mass spectrometer (HP 5975) for direct gas analysis. Signals generated at m/z=32 upon injection of RL and lipid emulsion samples were integrated using Peakfit and compared to those obtained with distilled water.
Animals and Animal Procedures:
Male and female mice weighing 27-47 grams were utilized. The strains were either CD-1 or NFR2. All comparisons utilized the same strain. Mice were anesthetized using ketamine/xylazine anesthesia administered subcutaneously. In order to prevent the skewing of data due to the cardiodepressant effects of the anesthetic agent, the experiment was aborted and the mouse euthanized in the rare instance when more anesthetic was required than the calculated dose. Once it was clear that the mouse was well-anesthetized, the carotid artery was cannulated. As much blood as possible was removed in three minutes. This resulted in the loss of 55% of blood volume and 100% lethality without any infusion. Immediately after blood removal infusions were administered over two minutes.
Pharmaceutical composition, resuscitation fluid (Ringer's lactate) or shed blood was administered at a volume equal to the amount of blood that had been removed. Blood pressure was measured at the carotid artery using a BP-2 monitor made by Columbus Instruments (Columbus, Ohio). This monitor measures the blood pressure as a voltage. A standard curve was prepared. Measured voltages were converted to blood pressure (BP) using the following formula:

BP=[Voltage−0.1006]/0.0107

No warming measures were applied to the mice. No measures were taken to support respiration.
Rat Model of Hemorrhagic Shock
Experiments using rats were performed with Sprague-Dawley rats weighing between 200 and 300 g. These rats were were purchased with the carotid artery and ipsilateral jugular vein pre-cannulated. The rats were given ketamine/xylazine anesthesia subcutaneously. After the effectiveness of the anesthetic was assured the carotid cannula was connected with a blood pressure monitor. The rats were bled to a mean blood pressure of 40 mmHg and maintained at that level for 45 minutes by the removal or infusion of shed blood as needed. At the end of the 45 minute period of shock the test pharmaceutical composition was infused in a volume equal to the amount of blood removed. The blood pressure was recorded for 45 minutes after resuscitation after which surviving rats were euthanized by exsanguination.
Statistical Analysis:
Data were analyzed using the two-tailed, Student's unpaired t test.
Example 2
Oxygen and Nitric Oxide Content of the Pharmaceutical Composition of the Present Application
Intralipid® 20% I.V. Fat Emulsion (marketed and sold by Baxter International Inc., Deerfield, Ill.) was used as a sample pharmaceutical composition (LM). The composition of Intralipid® is 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, water and sodium hydroxide to adjust the pH to 8. Oxygen content in the samples was measured using mass spectrometry. As shown in Table I, the oxygen content of LM was nearly twice that of Ringer's lactate (RL), a standard resuscitation fluid infused when a large amount of blood is lost. The oxygen content of water was assigned the value of 1. The oxygen content of RL was slightly less than that of water. As shown in Table 2, the oxygen content of the LM was increased five-fold by bubbling oxygen through it for approximately 1 minute. After oxygen loading, the oxygen content of LM compared favorably to that of blood with the minimum acceptable hemoglobin level (i.e., 7.0 g/dl).







TABLE 1







Oxygen content of Ringer's lactate and Intralipid ® 20%.


Each mean is the result of 3 samples.










Ringer's lactate
Intralipid ® 20%















Oxygen Content*
0.91 ± 0.11*
1.78 ± 0.09*







*the oxygen content is expressed as the amount relative to the oxygen content in water.













TABLE 2





Oxygen solubility in various liquids at 1 atm


Oxygen Content at 25° C. and Sea level Pressure



















Blood (hemoglobin of 7.0)
72.8
mg/L



Water
8.3
mg/L+



LM (20%)
15.1
mg/L



LM (20% after oxygen perfusion)
75.5
mg/L







+Obtained from “The Engineering Toolbox” Oxygen Solubility in Fresh and Sea water at http://www.engineeringtoolbox.com/oxygen-solubility-water-d_841.html.






These data show that LM may be loaded with sufficient oxygen to provide for aerobic metabolism. This point is further underscored by the fact that only 30% of the oxygen bound to hemoglobin is used under normal circumstances. In extreme stress such as severe hemorrhagic shock 60% might be offloaded from hemoglobin. In contrast all of the oxygen dissolved in LM should be available given the fact that oxygen is released 3 times faster from LM than from hemoglobin. The published release time for hemoglobin is 17.5 msec while the off time measured when we purged oxygen from LM was less than 5 msec.
FIG. 2 shows the linear dependence of emulsion oxygen content on the concentration of lipid micelles. Micelles were prepared by sonicating a mixture of 1-3 grams of soybean oil, 0.12 gram of soybean lecithin and 0.12 gram of glycerol and water to make a volume of 10 ml. The mixture was put into a 15 ml tube and emulsified in an ice bath using an ultrasound disruptor. Each point is the mean of 2-3 experiments. The Y axis is the oxygen content of the emulsion relative to water. The X axis is the soybean oil concentration of the emulsion.
Example 3
Nitric Oxide Uptake of the Pharmaceutical Composition of the Present Application
In another set of experiments we measured the uptake of nitric oxide by a commercially available 20% soybean oil emulsion, Liposyn III 20%. This emulsion contains 20% soybean oil, 1.2% egg phospholipids and 2.5% glycerin in water. Using NaOH pH was adjusted to 8.3. Oxygen was added by equilibration in which 200 μl of the emulsion was placed ***into a 15 ml vial. 100 ppm nitric oxide in helium was used to purge the headspace for two minutes taking care not to bubble the fluid. For sixty minutes the sample was allowed to reach gas-liquid equilibrium with the tube placed on its side to maximize gas-emulsion contact. The ***nitric oxide content was measured in ten samples that were equilibrated with nitric oxide and ten samples that were equilibrated with room air. The mean values and standard errors were 3.19×10−3+/−0.19×10−3 and 2.12×10−3+/−0.17×10−3 for 20% Liposyn and water, respectively, showing that the solubility of nitric oxide in the emulsion was 50% greater than that of water.
Example 4
The Effect of the Pharmaceutical Composition of the Present Application in Restoring Arterial Pressure in Mice with Severe Hemorrhagic Shock
The effect of the pharmaceutical composition (LM) in Example 2 on blood pressure was determined in mice. Mice were anesthetized and a cannula was placed into the carotid artery. All the blood that could be removed was removed via the carotid artery. After the blood was removed a volume of either RL or LM was given equal to the amount of blood removed. 6 mice were in the LM group and 6 mice were in the RL group. The observation period was one hour. Two of the mice given RL died within ten minutes. All mice given LM lived through the entire hour observation period and until euthanized at 1-4 hours. Animals were euthanized whenever they began to awaken from the anesthesia or at the end of the observation period to prevent suffering.
FIGS. 3 and 4 show the difference between the systolic blood pressure (FIG. 3) and diastolic blood pressure (FIG. 4) after hemorrhage and after infusion of RL or LM at time=0, 30 and 60 minutes. The Y axis represents the blood pressure attained after infusion minus the blood pressure after hemorrhage in mm of Hg. The X axis shows the specific time after the infusion. All data were analyzed for statistical significance using an unpaired two tailed t test. These graphs show that LM raised the blood pressure higher than RL (p<0.01).
In another experiment, a pharmaceutical composition containing Intralipid® 20% and 5% (w/v) albumin was prepared by dissolving albumin (Sigma Aldrich, 99% pure, fatty acid free, essentially globulin free, catalog number A3782-5G) in Intralipid® 20% to a final concentration of 50 mg/ml. The new pharmaceutical composition with albumin (VS) was tested using the experimental procedure described above. Albumin dissolved in normal saline (NSA) and Ringer's lactate (RLA) at 50 mg/ml, as well as the shed blood (i.e., the blood that had been removed from the mice), were used as controls. In FIG. 5, the Y axis shows the mean systolic blood pressure (expressed as percentage of mean pre-hemorrhage blood pressure) achieved by infusion of the various fluids. The X axis shows specific times after the infusion. The data show that VS is superior even to shed blood in maintaining blood pressure. Similar results were also obtained for the diastolic blood pressure (FIG. 6). For each time point, an average of 6-7 mice is plotted. Differences between shed blood and VS was statistically significant (P<0.05) at 5, 15 and 30 minutes.
Data collected from the experiments described above were used to compare the effects of infusion of the emulsion without albumin to 5% human albumin in normal saline. In this data analysis, 6 mice that were resuscitated with emulsion without albumin and 5% albumin in normal saline were compared. Mean initial post infusion pressure was divided by the mean prehemorrhage pressure. There was no significance difference between the emulsion or the 5% albumin/saline. However the blood pressures of the mice that received the emulsion stayed up longer than those given 5% albumin in saline. Mean BP at one hour after the emulsion divided by the mean blood pressure before hemorrhage=0.7208+/−0.041 and the same quantity after 5% albumin in normal saline is 0.534+/−0.033 n=6 in each group p=0.0056 by two tailed Student's t test.
Table 2 shows the survival time of mice receiving soybean oil emulsion or Ringer's lactate.







TABLE 2







Survival: Soybean Oil Emulsion vs Ringer's Lactate (minutes)


(Each row shows survival time of two mice, one for each Infusate)










Emulsion
Ringer's lactate














270*
4.2



180*
9.4



270*
144.2



90
100.68



280*
24.67




111.11



218.00 +/− 36.80
65.71 +/− 24.55 [mean +/− SE]







*Still alive at the indicated time points. Euthanized because the mice continued to survive beyond the termination time of the experiment.



p = 0.0062 two tailed Student's t test






Example 5
The Effect of the Pharmaceutical Composition of the Present Application in Restoring Arterial Pressure in Rats with Severe Hemorrhagic Shock
In another experiment anesthetized rats were bled to a mean pressure of 40 that was maintained for 45 minutes using shed blood. At the end of this period a test fluid was given within 2 minutes. The test fluids were the (1) emulsion containing 20% Intralipid mixed with 5% albumin NaCl 102 mM, Na(L) lactate 28 mM, KCl 4 mM, (2) Ringer's lactate, (3) Hextend and (4) shed blood. The results are shown in Table 4.







TABLE 4







Effect of the pharmaceutical composition in restoring


arterial pressure in rats with severe hemorrhagic shock














Ringer's




Emulsion
Hextend
Lactate
Blood















Number of rats
6
2
2
2


Avg survival time (min)
35.8 +/− 6.7
10.74
18.25
35.75


Avg mean arterial
64.2 +/− 2.4
38.3
34.2
64.7


pressure









Hematoxylyn and eosin histopathological examination of the lung, liver, kidneys, spleen heart and intestine was carried out. No harmful effect of the emulsion was found.
These experimental results are consistent with the fact that the lipid micelles in the pharmaceutical composition are capable of exerting an osmotic force and absorbing mediators of vascular patency, such as prostaglandins, nitric oxide, leukotrienes, and hromboxane.
Example 6
The Effect of Amino Acid-Containing Pharmaceutical Composition in Restoring Arterial Pressure in Mice with Severe Hemorrhagic Shock
In another experiment, hemorrhagic shock was induced in mice as described in ***Example 4. The mice were then resuscitated with a control emulsion containing 20% soybean oil emulsion (Intralipid), alone, or a test emulsion containing the control emulsion and 1 mM histidine or tyrosine or phenylalanine or cysteine. Table 4 shows the effect of the pharmaceutical composition in restoring arterial pressure in mice with hemorrhagic shock.







TABLE 4







Effect of the pharmaceutical composition in restoring


arterial pressure in mice with hemorrhagic shock.












#


Post infusion -



mice
Pre bleed
Shock
pressure in shock















IL alone
4
105.0 +/− 3.6 
22.8 +/− 2.8
32.3 +/− 6.8




79.0 +/− 2.3
14.5 +/− 2.4
 8.3 +/− 3.1


IL
2
112.0 +/− 13.0
16.0 +/− 0 
63.0 +/− 4.0


+

83.0 +/− 7.0
10.5 +/− 0.5
43.0 +/− 0 


Histidine


IL
2
 134 +/− 18.0
24.0 +/− 1.0
68.0 +/− 3.0


+

83.0 +/− 16 
15.0 +/− 1.0
56.5 +/− 2.5


Tyrosine


IL
2
106.5 +/− 21.5
19.5 +/− 2.5
55 5 +/− 2 5


+

 70.5 +/− 14.5
13.5 +/− 0.5
42.0 +/− 6.0


Phenylalanine


IL
2
156.5 +/− 37.5
25.0 +/− 0 
58.5 +/− 3.5


+

 99.0 +/− 14.0
16.0 +/− 3.0
45.0 +/− 5.0


cysteine









Example 7
The Effect of Oxygen Loaded Lipid Colloids in Hemorrhagic Shock
The above examples demonstrated that a soybean oil emulsion was superior to Ringers lactate in raising and maintaining the blood pressure in the 100% lethal model of hemorrhagic shock. No difference in the increase in blood pressure occurred by increasing the amino acid concentration to 10 mM. All animals survived the one hour observation period except one mouse that received 1 mm cysteine and both mice that received 10 mM cysteine. Because of the oxygen oil/water partition coefficient of 4.4, soybean oil emulsion could be loaded with clinically useful hydrophobic gases such as oxygen or nitric oxide. The uptake of NO and O2 by a commercial 20% soybean oil emulsion, Liposyn, was measured using mass spectrometry. It was found that the NO content of the emulsion was 3.19×10−3+/−0.19 moles/L and that of water was 2.12×10−3+/−0.17 moles/L. The oxygen content was linearly related to soybean oil concentration of emulsions prepared in the laboratory. Off-loading of oxygen and nitric oxide from the emulsion was rapid being nearly as fast as offloading of these gases from water.
In the experiment described below, the effect of oxygenated 20% soybean emulsion (Intralipid) and Ringer's lactate in mice with hemorrhagic shock after clinical death was compared. To approximate field conditions, no warming or respiratory support was provided. Next, 55% of blood volume was removed within 2 minutes. Without intervention 100% of the animals died. The time of clinical death was defined as the cessation of respiration. The interval+/−SEM between hemorrhage and clinical death was 3.06+/−0.456 minutes. In 5 mice a volume of Ringer's lactate equal to the volume of blood removed was administered via the internal jugular vein over 2 minutes immediately after the loss of chest wall movement. Chest wall movement was not restored in any of these animals. In 6 mice a volume of Ringer's lactate equal to the volume of blood removed was administered via the carotid artery over 2 minutes. In this case respiration was restored as shown by vigorous chest wall movement in 6 of 6 mice. Respiration continued for a variable time from 3 to 105 minutes. P=0.008 by Mann Whitney U for arterial vs. venous infusion. In the next set of experiments, infusing Ringers' lactate was delayed for 3 minutes. In two mice there was no response. In two additional mice there was no response even though the delay was reduced to 1 minute. In the next set of experiments, oxygen was bubbled into a 20% soybean oil emulsion (Intralipid) for 1 minute. A volume of emulsion equal to the volume of blood that had been removed was infused. This oxygenated emulsion was infused 5 minutes after the loss of respiration. In 6 of 6 mice breathing was restored after this single initial infusion alone. Breathing lasted from 77 to 114 minutes in 5 mice and only 1.5 minutes in one mouse.
These data show that (1) uptake of lipophilic gases such as NO and O2 is enhanced by the presence of micelles, (2) oxygenated 20% soybean oil emulsion may be used for resuscitation after clinical death, and (3) due to its low affinity for NO compared to hemoglobin, lipid micelles may act as a reservoir in which NO is released where its concentration is low and absorbed where it is present in excess. The arterial route is superior to the venous route in severe hemorrhagic shock. These data implicate that lipid colloids can deliver lipophilic gases that are therapeutically useful such as oxygen, NO, xenon, hydrogen sulphide and carbon monoxide.
The terms and descriptions used herein are set forth by way of illustration only and are not meant as limitations. Those skilled in the art will recognize that many variations are possible within the spirit and scope of the invention as defined in the following claims, and their equivalents, in which all terms are to be understood in their broadest possible sense unless otherwise indicated.

## Claims

1. A method for treating conditions related to lack of blood supply, comprising:
administering to a subject in need of such treatment, an effectively amount of a pharmaceutical composition comprising:
an amphiphilic emulsifier; and
a polar liquid carrier,
wherein said amphiphilic emulsifier forms free-moving micelles having a lipophilic core in said polar liquid carrier and wherein said pharmaceutical composition is free of hemoglobin and fluorocarbon.

2. The method of claim 1, wherein said pharmaceutical composition further comprises a lipid component and wherein said lipid component is present in said lipophilic core of said micelles.

3. The method of claim 2, wherein said lipid component comprises soybean oil or an oil rich in omega 3 fatty acid.

4. The method of claim 3, wherein said soybean oil or oil rich in omega 3 fatty acid is present in an amount of 10-70% (w/v) of said pharmaceutical composition.

5. The method of claim 3, wherein said oil rich in omega 3 fatty acid is selected from the group consisting of chia bean oil, algae oil, pumpkin oil, flaxseed oil and fish oil.

6. The method of claim 1, wherein said micelles have an average diameter in the range of 3-300 nm.

7. The method of claim 1, wherein said pharmaceutical composition further comprises histidine or a histidine-containing peptide at a final concentration of 1 fM-100 mM.

8. The method of claim 1, wherein said amphiphilic emulsifier comprises a phospholipid in an amount of 0.5-50% (w/v) of said pharmaceutical composition.

9. The method of claim 1, wherein said pharmaceutical composition further comprises glycerol or mannitol in an amount of 2-15% (w/v) of said pharmaceutical composition and soybean oil in an amount of 20-35% (w/v) of said pharmaceutical composition, and wherein said micelles have an average diameter in the range of 3-300 nm.

10. The method of claim 1, wherein said pharmaceutical composition comprises sodium ions at a concentration in the range of 70 mM to 160 mM.

11. The method of claim 10, wherein said pharmaceutical composition comprises sodium chloride and sodium lactate.

12. A method for raising the blood pressure in a subject with hypovolemia, comprising:
administering to said subject, an effectively amount of a pharmaceutical composition comprising:
an amphiphilic emulsifier; and
a polar liquid carrier,
wherein said amphiphilic emulsifier forms free-moving micelles having a lipophilic core in said polar liquid carrier and wherein said pharmaceutical composition is free of hemoglobin and fluorocarbon.

13. The method of claim 12, wherein said pharmaceutical composition further comprises a lipid component and wherein said lipid component is present in said lipophilic core of said micelles.

14. The method of claim 13, wherein said lipid component comprises soybean oil or an oil rich in omega 3 fatty acid.

15. The method of claim 14, wherein said soybean oil or an oil rich in omega 3 fatty acid is present in an amount of 10-70% (w/v) of said pharmaceutical composition.

16. The method of claim 14, wherein said oil rich in omega 3 fatty acid is selected from the group consisting of chia bean oil, algae oil, pumpkin oil, flaxseed oil and fish oil.

17. The method of claim 12, wherein said micelles have an average diameter in the range of 3-300 nm.

18. The method of claim 12, wherein said pharmaceutical composition further comprises histidine or a histidine-containing peptide at a final concentration of 1 fM-100 mM.

19. The method of claim 12, wherein said pharmaceutical composition comprises histidine amphiphilic emulsifier comprises a phospholipid in an amount of 0.5-50% (w/v) of said pharmaceutical composition.

20. The method of claim 12, wherein said pharmaceutical composition further comprises glycerol or mannitol in an amount of 2-15% (w/v) of said pharmaceutical composition and soybean oil in an amount of 10-70%(w/v) of said pharmaceutical composition, and wherein said micelles have an average diameter in the range of 3-300 nm.

21. The method of claim 12, wherein said pharmaceutical composition comprises sodium ions at a concentration in the range of 70 mM to 160 mM.

22. The method of claim 21, wherein said pharmaceutical composition comprises sodium chloride and sodium lactate.