EP1223942A2 - Renforcement de l'apport en oxygene chez des mammiferes, procedes et reactifs correspondants - Google Patents

Renforcement de l'apport en oxygene chez des mammiferes, procedes et reactifs correspondants

Info

Publication number
EP1223942A2
EP1223942A2 EP00957519A EP00957519A EP1223942A2 EP 1223942 A2 EP1223942 A2 EP 1223942A2 EP 00957519 A EP00957519 A EP 00957519A EP 00957519 A EP00957519 A EP 00957519A EP 1223942 A2 EP1223942 A2 EP 1223942A2
Authority
EP
European Patent Office
Prior art keywords
compound
hemoglobin
molecule
ligand
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00957519A
Other languages
German (de)
English (en)
Inventor
Yves Claude Nicolau
Jean-Marie Lehn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GMP Companies Inc
GMP Cos Inc
Original Assignee
GMP Companies Inc
GMP Cos Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by GMP Companies Inc, GMP Cos Inc filed Critical GMP Companies Inc
Publication of EP1223942A2 publication Critical patent/EP1223942A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock

Definitions

  • Ischemia Ischemic insult i.e., the localized deficiency of oxygen to an organ or skeletal tissue
  • Ischemic insult is a common and important problem in many clinical conditions. The problem is especially acute in organ transplant operations in which a harvested organ is removed from a body, isolated from a blood source, and thereby deprived of oxygen and nutrients for an extended period of time. Ischemic insult also occurs in certain clinical conditions, such as sickle cell anemia and septic shock, which may result from hypotension or organ dysfunction. Depending on the duration of the insult, the ischemia can disturb cellular metabolism and ion gradients, and ultimately cause irreversible cellular injury and death.
  • Ischemia is also associated with various clinical conditions, such as septic shock.
  • Septic shock as a result of hypotension and organ dysfunction in response to infectious sepsis is a major cause of death.
  • the manifestations of sepsis include those related to the systemic response to infection (tachycardia, tachypnea alterations in temperature and leukocytosis) and those related to organ-system dysfunction (cardiovascular, respiratory, renal, hepatic and hematologic abnormalities).
  • the lipopolysaccharide (LPS) of gram-negative bacteria is considered to be the most important exogenous mediator of acute inflammatory response to septic shock.
  • the LPS or endotoxin released from the outer membrane of gram-negative bacteria results in the release of cytokines and other cellular mediators, including tumor necrosis factor alpha (TNF alpha), interleukin-1 (II- 1), interleukin-6 (11-6) and thromboxane A2. Extreme levels of these mediators are known to trigger many pathological events, including fever, shock, and intravascular coagulation, leading to ischemia and organ failure.
  • Sickle cell anemia is another condition associated with ischemia.
  • Sickle cell anemia is a classical phenotype of hereditary hemoglobinopathy with hemoglobin S instead of normal hemoglobin A.
  • Sickle cell anemia is associated with hypoxia because of decreased oxygen tension with hemoglobin S. This condition leads to a systemic hypoxic condition.
  • the viscosity of deoxygenated blood is related to the proportion of sickled red cells, capillary stasis and pain crisis.
  • heart attacks and stroke are the most widely recognized example of the damage resulting from ischemia.
  • Myocardial ischemia is a condition wherein there is insufficient blood supply to the myocardium (the muscles of the heart) to meet its demand for oxygen.
  • the ultimate result of persistent myocardial ischemia is necrosis or death of a portion of cardiac muscle tissue, known as a myocardial infarct, commonly known as a heart attack.
  • Insufficient blood supply to the myocardium is generally due to an obstruction or thrombus in an artery supplying blood to the myocardium.
  • Another cause can be atrial fibrillation, wherein the increased heart rate associated with atrial fibrillation increases the work, and hence the blood demand of the myocardium, while the atrial fibrillation at the same time reduces the blood supply.
  • stroke is defined as a sudden impairment of body functions caused by a disruption in the supply of blood to the brain.
  • a stroke occurs when blood supply to the brain is interrupted for any reason, including hemorrhage, low blood pressure, clogging by atherosclerotic plaque, a blood clot, or any particle.
  • Brain tissue that receives an inadequate supply of blood is said to be ischemic. Deprived of oxygen and nutrients, nerve cells and other cell types within the brain begin to fail, creating an infarct (an area of cell death, or necrosis). As the neurons fail and die, the part of the body controlled by those neurons can no longer function.
  • the devastating effects of ischemia are often permanent because brain tissue has very limited repair capabilities and lost neurons are typically not regenerated.
  • Cerebral ischemia may be incomplete (blood flow is reduced but not entirely cut off), complete (total loss of tissue perfusion), transient or permanent. If ischemia is incomplete and persists for no more than ten to fifteen minutes, neural death may not occur. More prolonged or complete ischemia results in infarction. Depending on the site and extent of the infarction, mild to severe neurological disability or death will follow.
  • the brain is protected against cerebral ischemia by compensatory mechanisms, including collateral circulation (overlapping local blood supplies), and arteriolar auto-regulation (local smooth muscle control of blood flow in the smallest arterial channels).
  • compensatory mechanisms include collateral circulation (overlapping local blood supplies), and arteriolar auto-regulation (local smooth muscle control of blood flow in the smallest arterial channels).
  • collateral circulation overlapping local blood supplies
  • arteriolar auto-regulation local smooth muscle control of blood flow in the smallest arterial channels.
  • Arteriolar auto-regulation works by shunting blood from noncritical regions to infarct zones.
  • auto-regulation may be sufficient to adjust the circulation and thereby preserve the vitality and function of brain or heart tissue.
  • ischemia may be sufficiently prolonged and compensatory mechanisms sufficiently inadequate that a catastrophic stroke or heart attack results.
  • Hemoglobin is a tetrameric protein which delivers oxygen via an allosteric mechanism. Oxygen binds to the four hemes of the hemoglobin molecule. Each heme contains porphyrin and iron in the ferrous state. The ferrous iron-oxygen bond is readily reversible. Binding of the first oxygen to a heme releases much greater energy than binding of the second oxygen molecule, binding of the third oxygen releases even less energy, and binding of the fourth oxygen releases the least energy.
  • hemoglobin In blood, hemoglobin is in equilibrium between two allosteric structures. In the “T” (for tense) state, hemoglobin is deoxygenated. In the “R” (for relaxed) state, hemoglobin is oxygenated. An oxygen equilibrium curve can be scanned to observe the affinity and degree of cooperativity (allosteric action) of hemoglobin. In the scan, the Y-axis plots the percent of hemoglobin oxygenation and the X-axis plots the partial pressure of oxygen in millimeters of mercury (mm Hg).
  • mm Hg millimeters of mercury
  • a value commonly known as the P50 is determined (i.e., this is the pressure in mm Hg when the scanned hemoglobin sample is 50% saturated with oxygen).
  • the P50 value for normal adult hemoglobin (HbA) is around 26.5 mm Hg. If a lower than normal P50 value is obtained for the hemoglobin being tested, the scanned curve is considered to be "left-shifted” and the presence of high oxygen-affinity hemoglobin is indicated. Conversely, if a higher than normal P50 value is obtained for the hemoglobin being tested, the scanned curve is considered to be "right-shifted", indicating the presence of low oxygen-affinity hemoglobin.
  • HbA Human normal adult hemoglobin
  • erythrocytes help maintain hemoglobin in its reduced, functional form.
  • the heme-iron atom is susceptible to oxidation, but may be reduced again by one of two systems within the erythrocyte, the cytochrome b5, and glutathione reduction systems.
  • Hemoglobin is able to alter its oxygen affinity, thereby increasing the efficiency of oxygen transport in the body due to its dependence on 2,3-DPG, an allosteric regulator.
  • 2,3-DPG is present within erythrocytes at a concentration that facilitates hemoglobin to release bound oxygen to tissues.
  • Naturally-occurring hemoglobin includes any hemoglobin identical to hemoglobin naturally existing within a cell.
  • Naturally-occurring hemoglobin is predominantly wild-type hemoglobin, but also includes naturally-occurring mutant hemoglobin. Wild-type hemoglobin is hemoglobin most commonly found within natural cells. Wild-type human hemoglobin includes hemoglobin A, the normal adult human hemoglobin having two alpha - and two beta-globin chains.
  • Mutant hemoglobin has an amino-acid sequence that differs from the amino-acid sequence of wild-type hemoglobin as a result of a mutation, such as a substitution, addition or deletion of at least one amino acid.
  • Adult human mutant hemoglobin has an amino- acid sequence that differs from the amino-acid sequence of hemoglobin A.
  • Naturally-occurring mutant hemoglobin has an amino-acid sequence that has not been modified by humans.
  • the naturally-occurring hemoglobin of the present invention is not limited by the methods by which it is produced. Such methods typically include, for example, erythrocytolysis and purification, recombinant production, and protein synthesis.
  • hemoglobin specifically binds small polyanionic molecules, especially 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP), present in the mammalian red cell (Benesch and Benesch, Nature, Vol. 221, p. 618, 1969). This binding site is located at the centre of the tetrameric structure of hemoglobin (Arnone, A., Nature, Vol. 237, p. 146, 1972). The binding of these polyanionic molecules is important in regulating the oxygen-binding affinity of hemoglobin since it allosterically affects the conformation of hemoglobin leading to a decrease in oxygen affinity (Benesch and Benesch, Biochem. Biophys. Res.
  • DPG 2,3-diphosphoglycerate
  • ATP adenosine triphosphate
  • hemoglobin Conversely, the binding of oxygen allosterically reduces the affinity of hemoglobin for the polyanion. (Oxy) hemoglobin therefore binds DPG and ATP weakly. This is shown, for example, by studies of spin-labelled ATP binding to oxy- and deoxyhemoglobin as described by Ogata and McConnell (Ann. N.Y. Acad. Sc, Vol. 222, p. 56, 1973). In order to exploit the polyanion-binding specificity of hemoglobin, or indeed to perform any adjustment of its oxygen-binding affinity by chemically modifying the polyanion binding site, it has been necessary in the prior art that hemoglobin be deoxygenated.
  • hemoglobin as it exists in solutions, or mixtures exposed to air, is in its oxy state, i.e., (oxy)hemoglobin.
  • oxy i.e., (oxy)hemoglobin.
  • deoxy deoxy
  • affinity chromatography has not been used in the prior art to purify hemoglobin.
  • Hemoglobin has also been administered as a pretreatment to patients receiving chemotherapeutic agents or radiation for the treatment of tumors (U.S. Pat. No. 5,428,007; WO 92/20368; WO 92/20369), for prophylaxis or treatment of systemic hypotension or septic shock induced by internal nitric oxide production (U.S. Pat. No. 5,296,466), during the perioperative period or during surgery in a method for maintaining a steady-state hemoglobin concentration in a patient (WO 95/03068), and as part of a perioperative hemodilution procedure used prior to surgery in an autologous blood use method (U.S. Pat. Nos. 5,344,393 and 5,451,205).
  • a trauma i.e., a wound or injury
  • the trauma disturbs the patient's homeostasis.
  • the patient's body biologically reacts to the trauma to restore homeostasis. This reaction is referred to herein as a naturally occurring stress response. If the body's stress response is inadequate or if it occurs well after the trauma is suffered, the patient is more prone to develop disorders.
  • the major function of erythrocytes consists in the transport of molecular oxygen from the lungs to the peripheral tissues.
  • the 0 2 -partial pressure in the lung is about.100 mm Hg, in the capillary system is about.70 mm Hg, against which 0 2 must be dissociated from the oxygenated hemoglobin.
  • only about 25% of the oxygenated hemoglobin may be deoxygenated; about.75% is carried back to the lungs with the venous blood.
  • the major fraction of the hemoglobin-0 2 adduct is not used for the 0 2 transport.
  • ACD acid-citrate-dextrose conservation
  • clofibric acid and bezafibrate bind to the same sites in the central water cavity of deoxyhemoglobin, and that one bezafibrate molecule will span the sites occupied by two clofibric acid molecules.
  • Bezafibrate and clofibric acid act by stabilizing the deoxy structure of hemoglobin, shifting the allosteric equilibrium toward the low affinity deoxy form. Bezafibrate and clofibric acid do not bind in any specific manner to either oxy- or carbonmonoxyhemoglobin.
  • 2,3-Diphosphoglycerate (2,3-DPG) is the normal physiological ligand for the allosteric site on hemoglobin.
  • phosphorylated inositols are found in the erythrocytes of birds and reptiles. Specifically, inositol hexaphosphate (IHP), as known as phytic acid, displaces hemoglobin-bound 2,3-DPG, binding to the allosteric site with one-thousand times greater affinity.
  • IHP inositol hexaphosphate
  • IHP inositol hexaphosphate
  • phytic acid displaces hemoglobin-bound 2,3-DPG, binding to the allosteric site with one-thousand times greater affinity.
  • IHP is unable to pass unassisted across the erythrocyte membrane.
  • the therapy of oxygen deficiencies requires the knowledge of parameters which characterize both the 0 2 transport capacity and the 0 2 release capacity of human RBCs.
  • the parameters of the 0 2 transport capacity i.e., Hb concentration, the number of RBCs, and hemocrit, are commonly used in clinical diagnosis.
  • the equally important parameters of the O, release capacity i.e., 0 2 half-saturation pressure of Hb and RBCs, and the amounts of high and low oxygen affinity hemoglobins in RBCs, are not routinely determined and were not given serious consideration until pioneering work by Gerosonde and Nicolau (Blut, 1979, 39, 1 - 7).
  • Enhancement of the 0 2 -release capacity of these cells brought about significant physiological effects in piglets: 1) reduced cardiac output, linearly dependent on the P50 value of the RBCs; 2) increased arteriovenous difference; and 3) improved tissue oxygenation. Long term experiments showed that in piglets the high P50 value of IHP -RBCs was maintained over the entire life spans of the RBCs.
  • Enhanced blood oxygenation may also reduce the number of heterologous transfusions and allow use of autologous transfusions in more case.
  • the current method for treatment of anemia or replacement of blood loss is transfusion of whole human blood. It is estimated that three to four million patients receive transfusions in the U.S. each year for surgical or medical needs. In situations where there is more time it is advantageous to completely avoid the use of donor or heterologous blood and instead use autologous blood.
  • IHP-treated RBCs may release up to 2-3 times as much oxygen as untreated red cells, in many cases, a physician will need to transfuse fewer units of IHP-treaded red cells. This exposes the patient to less heterologous blood, decreases the extent of exposure to vital diseases from blood donors and minimizes immune function disturbances secondary to transfusions. The ability to infuse more efficient red blood cells is also advantageous when the patients blood volume is excessive. In more severe cases, where oxygen transport is failing, the ability to improve rapidly a patient's tissue oxygenation is life saving.
  • Synthetic human hemoglobin has also been produced in neonatal pigs by injection of human genes that control hemoglobin production. This product may be less expensive product than the Somatogen synthetic hemoglobin, but it does not solve problems with oxygen affinity and breakdown of hemoglobin in the body.
  • the present invention relates to compositions, and methods of use thereof, consisting essentially of a cationic, lipophilic, water-soluble molecule (e.g., a bis-guanidinium cholesterol), and an anionic ligand for the allosteric site of hemoglobin, e.g., inositol hexaphosphate (IHP).
  • a cationic, lipophilic, water-soluble molecule e.g., a bis-guanidinium cholesterol
  • an anionic ligand for the allosteric site of hemoglobin e.g., inositol hexaphosphate (IHP).
  • IHP inositol hexaphosphate
  • the invention is directed to the use of the compounds or compositions thereof that are effective in delivering into erythrocytes allosteric modifiers of hemoglobin, lowering the oxygen affinity state in red blood cell suspensions and whole blood. It is an object of this invention to provides methods for delivering into erythrocytes allosteric modifiers of hemoglobin in whole blood and in vivo, utilizing compounds or compositions thereof that do not lose their effectiveness in the presence of normal concentrations of the remaining components of whole blood.
  • Ligands for the allosteric site of hemoglobin interact with the hemoglobin molecule and impact its ability to bind oxygen.
  • This invention is particularly concerned with the delivery into erythrocytes of ligands for the hemoglobin allosteric site, causing oxygen to be bound relatively less tightly to hemoglobin, such that oxygen is off-loaded from the hemoglobin molecule more easily.
  • the process of allosterically modifying hemoglobin towards a lower oxygen affinity state in whole blood and in vivo may be used in a wide variety of applications including treatments for ischemia, heart disease, wound healing, radiation therapy of cancer, and adult respiratory distress syndrome (ARDS). Furthermore, a decrease in the oxygen affinity of hemoglobin in whole blood will extend its shelf-life, or restore the oxygen carrying capacity of aged blood.
  • Figure 1 presents a summary of certain experiments forming inositol hexaphosphate- bisguanidinium cholesterol (IHP-BGTC) complexes.
  • Figure 2 depicts an Hb-0 2 dissociation curve in human RBCs after incubation with the IHP-BGTC system for 60 min. at room temperature
  • C a controls incubated with IHP (1 mM)- DMF (3%);
  • la RBCs + IHP (1 mM)-BGTC (0.35 mM)-DMF (3%);
  • 2a RBCs + IHP (1 mM)- BGTC (3.5 mmol)-DMF (3%);
  • 3a IHP (2 mM)-BGTC (0.35 mM)-DMF (3%)].
  • the process of allosterically modifying hemoglobin towards a low oxygen affinity state in whole blood and in vivo could be used in a wide variety of applications including in treatments for ischemia, heart disease, wound healing, radiation therapy of cancer, adult respiratory distress syndrome (ARDS), etc., in extending the shelf-life of blood or restoring the oxygen carrying capacity of out-dated blood, and as sensitizers for x-ray irradiation in cancer therapy, as well as in many other applications.
  • ARDS adult respiratory distress syndrome
  • This invention is related to the use of allosteric hemoglobin modifier compounds in red blood cell suspensions, in whole blood, and in vivo.
  • Serum albumin which is the most abundant protein in blood plasma, has been identified as inhibiting the allosteric effects of clofibric acid, bezafibrate, and L3,5/L3,4,5. The precise nature of this inhibition is not fully understood, but appears to be related to these compounds binding to the serum albumin.
  • the RSR compounds have been found to be relatively unaffected by the presence of serum albumin.
  • Ligands for the allosteric site of hemoglobin that are not adversely effected by serum albumin represent particularly good candidates for drug applications, since the performance of the drug will not be frustrated by the presence of serum albumin present in a patient's blood.
  • This invention relates to the incorporation of a wide variety of therapeutically useful substances into mammalian red blood cells (RBCs), which could not previously be accomplished without unacceptable losses of RBC contents and/or integrity. More particularly, the compounds and methods of the present invention makes possible the introduction or incorporation of anionic agents into RBCs, such as DNA, RNA, chemotherapeutic agents, and antibiotic agents. These and other water soluble substances may be used for a desired slow continuous delivery or targeted delivery when the treated RBC carrier is later injected in vivo.
  • the particular polyanion to be selected can be based on whether an allosteric effector of hemoglobin would be desirable for a particular treatment.
  • the present invention provides a novel method for increasing the oxygen-carrying capacity of erythrocytes.
  • the IHP combines with hemoglobin in a stable way, and shifts its oxygen releasing capacity.
  • Erythrocytes with IHP-hemoglobin can release more oxygen per molecule than hemoglobin alone, and thus more oxygen is available to diffuse into tissues for each unit of blood that circulates. Injected in vivo, IHP is toxic and cannot be tolerated as an ordinary drug.
  • Another advantage of IHP-treated red blood cells is that they show the Bohr effect in circulation and when stored. Normal red blood cells that have been stored do not regain their maximum oxygen carrying capacity in circulation for approximately 24 hours.
  • red blood cells treated according to the present invention retain their maximum oxygen carrying capacity during storage and therefore can deliver oxygen to the tissues in response to demand immediately after transfusion into a human or animal because there are no native enzymes in erythrocytes which degrade IHP.
  • IHP-treated RBCs may be used in the treatment of acute and chronic conditions, including, but not limited to, hospitalized patients, cardiovascular operations, chronic anemia, anemia following major surgery, coronary infarction and associated problems, chronic pulmonary disease, cardiovascular patients, autologous transfusions, as an enhancement to packed red blood cells transfusion (hemorrhage, traumatic injury, or surgery), congestive heart failure, myocardial infarction (heart attack), stroke, peripheral vascular disease, intermittent claudication, circulatory shock, hemorrhagic shock, anemia and chronic hypoxia, respiratory alkalemia, metabolic alkalosis, sickle cell anemia, reduced lung capacity caused by pneumonia, surgery, pneumonia, trauma, chest puncture, gangrene, anaerobic infections, blood vessel diseases such as diabetes, substitute or complement to treatment with hyperbaric pressure chambers, intra-operative red cell salvage, cardiac inadequacy, anoxia-secondary to chronic indication, organ transplant, carbon monoxide, nitric oxide, and cyanide poisoning.
  • the volume of IHP-treated red blood cells that is administered to the human or animal will depend upon the value of P50 for the IHP- treated RBCs. It is to be understood that the volume of IHP-treated red blood cells that is administered to the patient can vary and still be effective. IHP-treated RBCs are similar to normal red blood cells in every respect except that their P50 value is shifted towards higher partial pressures of 0 2 .
  • Erythrocytes release oxygen only in response to demand by organs and tissue. Therefore, the compounds, compositions thereof, and methods of the present invention will only restore a normal level of oxygenation to healthy tissue, avoiding the cellular damage that is associated with an over-abundance of oxygen.
  • the compounds, compositions, and methods of the present invention are capable of allosterically modifying hemoglobin to favor the low oxygen affinity "T" state (i.e., right shifting the equilibrium curve), they will be useful in treating a variety of disease states in mammals, including humans, wherein tissues suffer from low oxygen tension, such as cancer and ischemia. Furthermore, as disclosed by Hirst et al. (Radiat. Res., Vol. 112, (1987), pp. 164), decreasing the oxygen affinity of hemoglobin in circulating blood has been shown to be beneficial in the radiotherapy of tumors.
  • the compounds and compositions may be administered to patients in whom the affinity of hemoglobin for oxygen is abnormally high.
  • certain hemoglobinopathies certain respiratory distress syndromes, e.g., respiratory distress syndromes in new born infants aggravated by high fetal hemoglobin levels, and conditions in which the availability of hemoglobin/ oxygen to the tissues is decreased (e.g., in ischemic conditions such as peripheral vascular disease, coronary occlusion, cerebral vascular accidents, or tissue transplant).
  • the compounds and compositions may also be used to inhibit platelet aggregation, antithrombotic purposes, and wound healing. Topical application could be used for wound healing.
  • the compounds and compositions of the present invention can be added to whole blood or packed cells preferably at the time of storage or at the time of transfusion in order to facilitate the dissociation of oxygen from hemoglobin and improve the oxygen delivering capability of the blood.
  • the hemoglobin in the blood tends to increase its affinity for oxygen by losing 2,3-diphosphoglycerides.
  • the compounds and compositions of this invention are capable of reversing and/or preventing the functional abnormality of hemoglobin observed when whole blood or packed cells are stored.
  • the compounds and compositions may be added to whole blood or red blood cell fractions in a closed system using an appropriate reservoir in which the compound or composition is placed prior to storage or which is present in the anticoagulating solution in the blood collecting bag.
  • Administration to a patient can be achieved by intravenous or intraperitoneal injection where the dose and the dosing regiment is varied according to individual's sensitivity and the type of disease state being treated.
  • Solid tumors are oxygen deficient masses.
  • the compounds, compositions and methods of this invention may be exploited to cause more oxygen to be delivered to tumors, increasing radical formation and thereby increasing tumor killing during radiation.
  • IHP-treated blood will only be used in conjunction with radiotherapy.
  • the compounds, compositions and methods of this invention may be exploited to cause more oxygen to be delivered at low blood flow and low temperatures, providing the ability to decrease or prevent the cellular damage, e.g., myocardial or neuronal, typically associated with these conditions.
  • the compounds, compositions and methods of this invention may be exploited to decrease the number of red blood cells required for treating hemorrhagic shock by increasing the efficiency with which they deliver oxygen.
  • the compounds, compositions and methods of this invention will be effective in enhancing the delivery oxygen to the brain, especially before complete occlusion and reperfusion injuries occur due to free radical formation. Furthermore, the compounds, compositions and methods of this invention of this invention should reduce the expansion of arterioles under both hypoxic and hypotensive conditions.
  • the compounds, compositions and methods of this invention of this invention should be capable of increasing oxygen delivery to blocked arteries and surrounding muscles and tissues, thereby relieving the distress of angina attacks.
  • Acute respiratory disease syndrome is characterized by interstitial and/or alveolar edema and hemorrhage as well as perivascular lung edema associated with the hyaline membrane, proliferation of collagen fibers, and swollen epithelium with increased pinocytosis.
  • the enhanced oxygen delivering capacity provided to RBCs by the compounds, compositions and methods of this invention can be used in the treatment and prevention of ARDS by militating against lower than normal oxygen delivery to the lungs.
  • the compounds and compositions of the present invention act as neuroprotective agents. After cardiac bypass surgery, up to 50-70% of patients show some signs of cerebral ischemia based on tests of cognitive function. Up to 5% of these patients have evidence of stroke.
  • cardioplegia is the process of stopping the heart and protecting the heart from ischemia during heart surgery. Cardioplegia is performed by perfusing the coronary vessels with solutions of potassium chloride and bathing the heart in ice water. However, blood cardioplegia is also used. This is where potassium chloride is dissolved in blood instead of salt water. During surgery the heart is deprived of oxygen and the cold temperature helps slow down metabolism.
  • the heart Periodically during this process, the heart is perfused with the cardioplegia solution to wash out metabolites and reactive species. Cooling the blood increases the oxygen affinity of its hemoglobin, thus making oxygen unloading less efficient.
  • treatment of blood cardioplegia with compounds or compositions of the present invention will counteract the effects of cold on oxygen affinity and make oxygen release to the ischemic myocardium more efficient, possibly improving cardiac function after the heart begins to beat again.
  • the patient's blood is diluted for the process of pump prime. This hemodilution is essentially acute anemia. Because the compounds and compositions of the present invention make oxygen transport more efficient, their use during hemodilution (whether in bypass surgery or other surgeries, such as orthopedic or vascular) would enhance oxygenation of the tissues in an otherwise compromised condition.
  • hemoglobin includes all naturally- and non-naturally-occurring hemoglobin.
  • hemoglobin preparation includes hemoglobin in a physiologically compatible carrier or lyophilized hemoglobin reconstituted with a physiologically compatible carrier, but does not include whole blood, red blood cells or packed red blood cells.
  • Non-naturally-occurring hemoglobin includes synthetic hemoglobin having an amino- acid sequence different from the amino-acid sequence of hemoglobin naturally existing within a cell, and chemically-modified hemoglobin.
  • Such non-naturally-occurring mutant hemoglobin is not limited by its method of preparation, but is typically produced using one or more of several techniques known in the art, including, for example, recombinant DNA technology, transgenic DNA technology, protein synthesis, and other mutation-inducing methods.
  • “Chemically-modified hemoglobin” is a natural or non-natural hemoglobin molecule which is bonded to another chemical moiety.
  • a hemoglobin molecule can be bonded to pyridoxal-5'-phosphate, or other oxygen-affinity-modifying moiety to change the oxygen-binding characteristics of the hemoglobin molecule, to crosslinking agents to form crosslinked or polymerized hemoglobin, or to conjugating agents to form conjugated hemoglobin.
  • Oxygen affinity means the strength of binding of oxygen to a hemoglobin molecule. High oxygen affinity means hemoglobin does not readily release its bound oxygen molecules. The P50 is a measure of oxygen affinity.
  • treatment is intended to encompass also prophylaxis, therapy and cure.
  • Ischemia means a temporary or prolonged lack or reduction of oxygen supply to an organ or skeletal tissue. Ischemia can be induced when an organ is transplanted, or by conditions such as septic shock and sickle cell anemia.
  • Sketal tissue means the substance of an organic body of a skeletal organism consisting of cells and intercellular material, including but not limited to epithelium, the connective tissues (including blood, bone and cartilage), muscle tissue, and nerve tissue.
  • Ischemic insult means damage to an organ or skeletal tissue caused by ischemia.
  • Subject means any living organism, including humans, and mammals.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • the term "surgery” refers to the treatment of diseases, injuries, and deformities by manual or operative methods.
  • Common surgical procedures include, but are not limited to, abdominal, aural, bench, cardiac, cineplastic, conservative, cosmetic, cytoreductive, dental, dentofacial, general, major, minor, Moh's, open heart, organ transplantation, orthopedic, plastic, psychiatric, radical, reconstructive, sonic, stereotactic, structural, thoracic, and veterinary surgery.
  • the method of the present invention is suitable for patients that are to undergo any type of surgery dealing with any portion of the body, including but not limited to those described above, as well as any type of any general, major, minor, or minimal invasive surgery.
  • Minimally invasive surgery involves puncture or incision of the skin, or insertion of an instrument or foreign material into the body.
  • minimal invasive surgery include arterial or venous catheterization, transurethral resection, endoscopy (e.g., laparoscopy, bronchoscopy, uroscopy, pharyngoscopy, cystoscopy, hysteroscopy, gastroscopy, coloscopy, colposcopy, celioscopy, sigmoidoscopy, and orthoscopy), and angioplasty (e.g., balloon angioplasty, laser angioplasty, and percutaneous transluminal angioplasty).
  • endoscopy e.g., laparoscopy, bronchoscopy, uroscopy, pharyngoscopy, cystoscopy, hysteroscopy, gastroscopy, coloscopy, colposcopy, celioscopy, sigmoidoscopy, and orthoscopy
  • angioplasty e.g.
  • ED 50 means the dose of a drug which produces 50% of its maximum response or effect. Alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations.
  • LD 50 means the dose of a drug which is lethal in 50% of test subjects.
  • therapeutic index refers to the therapeutic index of a drug defined as
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • SAR structure-activity relationship
  • bis-guanidinium-tren-cholesterol and "BGTC” refer to the structure below.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
  • electron- withdrawing group is recognized in the art, and denotes the tendency of a substituent to attract valence electrons from neighboring atoms, i.e., the substituent is electronegative with respect to neighboring atoms.
  • ⁇ Hammett sigma
  • Exemplary electron-withdrawing groups include nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the like.
  • Exemplary electron-donating groups include amino, methoxy, and the like.
  • alkyl refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1 -C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
  • a halogen such as a carboxy
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
  • Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.
  • alkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • aryl as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, - CN, or the like.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • ortho, meta and para apply to 1,2-, 1,3- and 1 ,4-disubstituted benzenes, respectively.
  • the names 1 ,2-dimethylbenzene and ort ⁇ o-dimethylbenzene are synonymous.
  • heterocyclyl or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles.
  • Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofiiran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,
  • polycyclyl or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl
  • carrier refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
  • nitro means -NO2; the term “halogen” designates -F, -Cl, -Br or -I; the term “sulfhydryl” means -SH; the term “hydroxyl” means -OH; and the term “sulfonyl” means -S02--
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: p I l0
  • R9, Ri o and R' J Q each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2) m -R8, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure;
  • R3 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and
  • m is zero or an integer in the range of 1 to 8.
  • only one of Rq or RI Q can be a carbonyl, e.g., R9, RJQ and the nitrogen together do not form an imide.
  • R9 and RI Q each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2) m -R8-
  • alkylamine as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and RJ Q is an alkyl group.
  • acylamino is art-recognized and refers to a moiety that can be represented by the general formula:
  • R is as defined above, and R' ⁇ i represents a hydrogen, an alkyl, an alkenyl or -(CH2) m - Rg, where m and Rg are as defined above.
  • amino is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
  • R9, R10 are as defined above.
  • Preferred embodiments of the amide will not include imides which may be unstable.
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S- alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2) m -Rg, wherein m and Rg are defined above.
  • alkylthio groups include methylthio, ethyl thio, and the like.
  • carbonyl is art recognized and includes such moieties as can be represented by the general formula:
  • X is a bond or represents an oxygen or a sulfur
  • R j 1 represents a hydrogen, an alkyl, an alkenyl, -(CH2) m -Rg or a pharmaceutically acceptable salt
  • R' ⁇ 1 represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -Rg, where m and Rg are as defined above.
  • X is an oxygen and Ri 1 or R' ⁇ 1 is not hydrogen
  • the formula represents an "ester”.
  • Ri 1 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Ri 1 is a hydrogen, the formula represents a "carboxylic acid".
  • X is an oxygen, and
  • R'l 1 is hydrogen, the formula represents a "formate".
  • the formula represents a "thiolcarbonyl” group.
  • X is a sulfur and Ri 1 or R'j 1 is not hydrogen, the formula represents a "thiolester.”
  • X is a sulfur and Ri 1 is hydrogen, the formula represents a "thiolcarboxylic acid.”
  • X is a sulfur and Ri ]' is hydrogen, the formula represents a "thiolformate.”
  • X is a bond, and Ri i is not hydrogen, the above formula represents a "ketone" group.
  • Ri 1 is hydrogen
  • the above formula represents an "aldehyde” group.
  • alkoxyl or "alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O- (CH2) m -Rg, where m and Rg are described above.
  • R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
  • triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, -toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
  • triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, />-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
  • Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, jc-toluenesulfonyl and methanesulfonyl, respectively.
  • a more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
  • R9 and R'j 1 are as defined above.
  • R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.
  • sulfoxido refers to a moiety that can be represented by the general formula:
  • R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.
  • a "phosphoryl” can in general be represented by the formula: Qi _ " _
  • Q represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N.
  • Q ⁇ is an S
  • the phosphoryl moiety is a "phosphorothioate”.
  • Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
  • each expression e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 nd ed.; Wiley: New York, 1991).
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof, wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound.
  • the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
  • hydrocarbon is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom.
  • permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted.
  • Vigneron et al. ⁇ Proc. Natl. Acad. Sci. USA 1996, 93, 9682-86
  • BGSC bis-guanidinium-spermidine-cholesterol
  • BGTC bis- guanidinium-tren-cholesterol
  • Lehn et al. have filed a PCT patent application (WO 97/31935) directed toward novel amidinium-bearing cholesterol derivatives and pharmaceutical compositions thereof, which are particularly useful in gene therapy for transferring therapeutic genes into cells.
  • the guanidinium moiety has been shown to be well-suited to complexation of carboxylates and phosphates, due to its high pKa value (relative to that of the ammonium moiety), and its ability to form two well-organized, strong zwitterionic hydrogen bonds with those functional groups.
  • Dietrich et al. J. Chem. Soc, Chem. Commun. 1978, 934 have disclosed a general method for the introduction of guanidinium groups into macrocyclic molecules.
  • Various cationic lipids have been shown to induce efficient transfection of a large variety of eukaryotic cells. Most of them have been formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidyl ethanolamine (DOPE).
  • DOPE dioleoylphosphatidyl ethanolamine
  • some lipids e.g., lipopolyamines
  • Bezafibrate another antilipidemic drug, was later found to be much more effective in lowering the oxygen affinity of hemoglobin solutions and suspensions of fresh, intact red cells (Perutz et al., Lancet, 881, Oct. 15, 1983). Subsequently, X-ray crystallographic studies have demonstrated that clofibric acid and bezafibrate bind to the same sites in the central water cavity of deoxyhemoglobin, and that one bezafibrate molecule will span the sites occupied by two clofibric acid molecules. Bezafibrate and clofibric acid act by stabilizing the deoxy structure of hemoglobin, shifting the allosteric equilibrium toward the low affinity deoxy form. Bezafibrate and clofibric acid do not bind in any specific manner to either oxy- or carbonmonoxyhemoglobin.
  • certain allosteric hemoglobin modifier compounds are hydrophobic molecules that can be bound to the body's neutral fat deposits and lipophilic receptors sites, thus lowering their potency due to a decreased concentration in RBCs.
  • Administration of a hydrophobic compound, such as a mixture of anesthetic molecules will saturate the body's neutral fat deposits and lipophilic receptor sites, and thereby increase the concentration of this type of allosteric modifiers in RBCs, where higher concentrations of effector will increase its ability to interact with hemoglobin, causing delivery of more oxygen.
  • Ligands for the allosteric site of hemoglobin include 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), LR16 and L35 (two recently synthesized derivatives of Bzf), and pyridoxal phosphate.
  • DPG 2,3-diphosphoglycerate
  • IHP inositol hexakisphosphate
  • Bzf bezafibrate
  • LR16 and L35 two recently synthesized derivatives of Bzf
  • pyridoxal phosphate pyridoxal phosphate.
  • hemoglobin's affinity for oxygen can be modulated through electrostatic interactions with chloride and/or organophosphate anions present in RBCs.
  • compositions, and methods of use thereof consisting essentially of a cationic, lipophilic, water-soluble molecule (e.g., a bis-guanidinium cholesterol), and an anionic ligand for the allosteric site of hemoglobin, e.g., inositol hexaphosphate (IHP).
  • a cationic, lipophilic, water-soluble molecule e.g., a bis-guanidinium cholesterol
  • anionic ligand for the allosteric site of hemoglobin e.g., inositol hexaphosphate (IHP).
  • the present invention is related to compounds and compositions thereof which deliver into erythrocytes allosteric modifiers of hemoglobin in vivo. Additionally, the invention is directed to the use of the compounds or compositions thereof that are effective in delivering into erythrocytes allosteric modifiers of hemoglobin, lowering the oxygen affinity state in red blood cell suspensions and whole blood. It is an object of this invention to provides methods for delivering into erythrocytes allosteric modifiers of hemoglobin in whole blood and in vivo, utilizing compounds or compositions thereof that do not lose their effectiveness in the presence of normal concentrations of the remaining components of whole blood.
  • the present invention is directed toward the design of water-soluble membrane compatible molecules bearing novel cationic moieties, e.g., guanidinium-bearing sterol derivatives. These molecules form complexes with anionic molecules, e.g., ligands for the allosteric site of hemoglobin; such complexes are useful for the deliver of said anionic molecules into the cytoplasm of mammalian cells, e.g., erythrocytes.
  • novel cationic moieties e.g., guanidinium-bearing sterol derivatives.
  • the compounds and compositions thereof of the present invention may be used to prepare cationic liposomes, and should also be effective directly as aqueous (micellar) solutions, thereby avoiding the need to prepare cationic liposomes and the difficulties associated with them.
  • the cationic membrane compatible molecules are designed to form complexes with anionic molecules, e.g., ligands for the allosteric site of hemoglobin. These complexes will react with mammalian cells in vitro and/or in vivo to deliver their anionic component into the cytoplasm of the cells.
  • the guanidinium group of the cationic component of the compounds of the present invention is particularly well suited for interaction with the phosphate residues of IHP and congeners thereof because a pair of hydrogen bonds can be established between the two moieties.
  • We report here the use of bisguanidinium cholesterol lipids for the efficient delivery into mammalian erythrocytes of phosphate-containing ligands for the allosteric site of hemoglobin.
  • Our data demonstrate the usefulness, convenience, and versatility of cationic cholesterol derivatives with guanidinium polar head groups for delivery of small anionic molecules into the cytoplasm of mammalian cells.
  • the compounds of the present invention are represented by generalized structure 1 :
  • C+ represents a lipophilic water-soluble molecule bearing at least one positive charge
  • A- represents a ligand for a mammalian cellular receptor, wherein said ligand bears at least one negative charge.
  • the compounds of the present invention are represented by generalized structure 1, and the attendant definitions, wherein A- is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention are represented by generalized structure 1, and the attendant definitions, wherein C+ comprises at least one cationic functional group selected from the group consisting of guanidinium, imidazolium, 1 ,2- diammoniumethylene, 1,8-diammoniumnaphthyl, and 2,2'-bipyridinium.
  • the compounds of the present invention are represented by generalized structure 1, and the attendant definitions, wherein C+ comprises at least one guanidinium moiety.
  • the compounds of the present invention are represented by generalized structure 1, and the attendant definitions, wherein C+ comprises two guanidinium moieties.
  • the compounds of the present invention are represented by generalized structure 1, and the attendant definitions, wherein C+ comprises at least one cationic functional group selected from the group consisting of guanidinium, imidazolium, 1 ,2- diammoniumethylene, 1,8-diammoniumnaphthyl, and 2,2'-bipyridinium; and A- is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention are represented by generalized structure 1 , and the attendant definitions, wherein C+ comprises at least one guanidinium moiety; and A- is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention are represented by generalized structure 1, and the attendant definitions, wherein C+ comprises two guanidinium moieties; and A- is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and a phosphorylated inositol.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and IHP.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of a lipophilic water-soluble molecule, wherein said lipophilic water-soluble molecule comprises at least one guanidine or guanidinium moiety; and a phosphorylated inositol, wherein said phosphorylated inositol is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and a phosphorylated inositol.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and IHP.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of a sterol, wherein said sterol comprises at least one guanidine or guanidinium moiety; and a phosphorylated inositol, wherein said phosphorylated inositol is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and a phosphorylated inositol.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and IHP.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of cholesterol, wherein said cholesterol comprises at least one guanidine or guanidinium moiety; and a phosphorylated inositol, wherein said phosphorylated inositol is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and a phosphorylated inositol.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and IHP.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of BGSC or BGTC; and a phosphorylated inositol, wherein said phosphorylated inositol is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of BGTC; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety.
  • the compounds of the present invention consist essentially of BGTC; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety.
  • the compounds of the present invention consist essentially of BGTC; and a phosphorylated inositol.
  • the compounds of the present invention consist essentially of BGTC; and IHP.
  • the compounds of the present invention consist essentially of BGTC; and a second molecule comprising at least one carboxylic acid, phosphoric acid, phosphonic acid, sulfuric acid, or sulfonic acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of BGTC; and a second molecule comprising at least one carboxylic acid or phosphoric acid moiety, wherein said second molecule is a ligand for the allosteric site of hemoglobin.
  • the compounds of the present invention consist essentially of
  • BGTC BGTC
  • phosphorylated inositol wherein said phosphorylated inositol is a ligand for the allosteric site of hemoglobin.
  • a compound of the present invention is a component of a pharmaceutical composition.
  • a compound of the present invention is formulated in a liposome. In certain embodiments, a compound of the present invention is formulated for intravenous administration.
  • the method of the present invention comprises the step of administering to a subject a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject experiencing ischemia a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject experiencing ischemia a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject experiencing cardiac arrhythmia a compound or composition of the present invention. In certain embodiments, the method of the present invention comprises the step of administering to a subject experiencing cardiac arrhythmia a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject experiencing a heart attack a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject experiencing a heart attack a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject experiencing a stroke a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject experiencing a stroke a compound or composition of the present invention, wherein said administration is intravenous. In certain embodiments, the method of the present invention comprises the step of administering to a subject experiencing hypoxia a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject experiencing hypoxia a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject afflicted with sickle cell anemia a compound or composition of the present invention. In certain embodiments, the method of the present invention comprises the step of administering to a subject afflicted with sickle cell anemia a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject suffering from hypotension a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject suffering from hypotension a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of administering to a subject suffering from arteriosclerosis a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject suffering from arteriosclerosis a compound or composition of the present invention, wherein said administration is intravenous. In certain embodiments, the method of the present invention comprises the step of administering to a subject suffering from altitude sickness a compound or composition of the present invention.
  • the method of the present invention comprises the step of administering to a subject suffering from altitude sickness a compound or composition of the present invention, wherein said administration is intravenous.
  • the method of the present invention comprises the step of adding to mammalian blood a compound or composition of the present invention.
  • the method of the present invention comprises the step of adding to plasma comprising mammalian erythrocytes a compound or composition of the present invention.
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam.
  • terapéuticaally-effective amount means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • certain embodiments of the present compounds contain a cationic or basic functional group, such as an ammonium ion, or amino or alkylamino group, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids, e.g., a ligand for the allosteric site of hemoglobin such as inositol hexaphosphate (IHP).
  • a ligand for the allosteric site of hemoglobin such as inositol hexaphosphate (IHP).
  • IHP inositol hexaphosphate
  • the chloride salt of a guanidinylated sterol may be combined with the potassium salt of IHP to give potassium chloride and a salt of the present invention comprising the guanidinylated sterol and IHP.
  • representative anions include bromide, chloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate; and representative cations include sodium, potassium, lithium, cesium, magnesium, calcium, and barium.
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases.
  • pharmaceutically-acceptable salts in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • Formulations of the present invention include, but are not limited to, those suitable for parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable earners, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl py ⁇ olidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonimtating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonimtating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically- acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, or phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, or phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars,
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug, e.g., from a subcutaneous or intramuscular injection.
  • This goal may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
  • the rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers, such as polylactide-polyglycolide.
  • the monomers of the biodegradable polymer comprise the functionalized sterol of the salts of the present invention, so that as the polymer biodegrades the salt of the present invention is released over a desired period of time.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Intravenous administrations are preferred.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, mtracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • composition While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the subject compounds, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam.
  • the compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.
  • Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • Administration of the Compounds of the Present Invention thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • the above drug delivery systems tend to be systemic. In other words, administration of the drug is delivered throughout the body by the blood stream.
  • Several devices have been developed for the purpose of accessing specific body lumens or passageways (i.e., blood vessels, gastrointestinal tract, urinary tract) and delivering therapeutic agents transmurally to specific subregions of tissue.
  • a double-balloon catheter has been used to administer agents to the area confined by the balloons.
  • a disadvantage of this system is that drugs may be lost through communicating vessels between the balloons.
  • a perforated balloon has been developed to deliver agents directly into the vessel wall.
  • a major disadvantage with both of these systems in certain desired applications is that the drug is delivered radially in all directions.
  • DMSO dimethyl methacrylate
  • Other fluid diffusion enhancement compositions include propylene glycol, azone and ionic or non-ionic surfactants.
  • the parenteral administration of medical liquids is an established clinical practice.
  • the liquids are administered particularly intravenously, and the practice is used extensively as an integral part of the daily treatment of medical and surgical patients.
  • the liquids commonly administered include blood and blood substitutes, dextrose solution, electrolyte solution, and saline.
  • the liquids are administered from an intravenous delivery system having a container suspended above the patient, with the liquid flowing through a catheter hypodermic needle set to the patient.
  • a beneficial agent is often administered intravenously by (1) temporarily removing the intravenous system and halting the flow of liquid, and then intravenously administering the agent to the patient followed by reinserting the intravenous system into the patient; (2) the agent is added to the liquid in the container and then carried by the flow of the liquid to the patient; (3) agent is added to a liquid in a separate container called a partial fill that is connected to the primary intravenous line through which line the agent is carried by the flow of liquid to the patient; (4) an agent is contained in a piggyback vial into which is introduced an intravenous fluid, with the vital subsequently connected to the primary line through which the drug is administered to the patient; or (5) the agent is administered by a pump that exerts a force on a liquid containing agent for intravenously adminster
  • Such devices have comprised pumps driven by a power source, and which controls the delivery of drug to the body. Some of these pumps may be mounted externally to the body and are connected to a catheter introduced to the body of the patient. Other devices have comprised pump which is mounted subcutaneously to the body of the patient and which delivers a drug to the body at a desired location.
  • a manually operated pump which may be mounted externally to the body or subcutaneously in the body of the patient whereby the pump can be activated by the patient for the delivery of the drug as need arises for that drug.
  • Examples of such devices are disclosed in U.S. Pat. Nos. 4,588,394, 4,681,560 and, 5,085,644, and comprise devices whereby a pumping chamber is connected via catheter directly into the body and derives its source of drug from a holding reservoir.
  • the RBCs were washed twice, resuspended gently, and then centrifuged again as outlined above.
  • the cells were then similarly washed once in the following IHP solution: 35 mM inositol hexaphosphoric acid, dodecasodium salt (P-8810, Sigma, St. Louis, MO), 33 mM
  • a blood-gas monitor Hemox-Analyzer, TCS Medical Products Co., Huntington Valley, PA was used to measure IHP intemalization in the RBCs on the basis of the P50 shift of the Hb- 0 2 dissociation curve.
  • the form of the curve and the Hill coefficients (control: 2.05; RBC-IHP: 1.54-1.58) suggest that the great majority of the incubated RBCs have internalized IHP.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne des composés, des compositions réalisées avec ces derniers et des procédés qui permettent d'apporter une grande diversité de molécules anioniques au cytoplasme de cellules de mammifères. Dans certaines formes de réalisation, la présente invention concerne des composés, des compositions préparées avec ces derniers et des procédés qui permettent de renforcer l'aptitude des érythrocytes à apporter de l'oxygène au cytoplasme des globules sanguins, au moyen de l'envoi d'un ligand destiné au site allostérique de l'hémoglobine.
EP00957519A 1999-08-25 2000-08-17 Renforcement de l'apport en oxygene chez des mammiferes, procedes et reactifs correspondants Withdrawn EP1223942A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US15057499P 1999-08-25 1999-08-25
US150574P 1999-08-25
PCT/US2000/022583 WO2001013933A2 (fr) 1999-08-25 2000-08-17 Renforcement de l'apport en oxygene chez des mammiferes, procedes et reactifs correspondants

Publications (1)

Publication Number Publication Date
EP1223942A2 true EP1223942A2 (fr) 2002-07-24

Family

ID=22535137

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00957519A Withdrawn EP1223942A2 (fr) 1999-08-25 2000-08-17 Renforcement de l'apport en oxygene chez des mammiferes, procedes et reactifs correspondants

Country Status (4)

Country Link
EP (1) EP1223942A2 (fr)
JP (1) JP2003507430A (fr)
AU (1) AU6912500A (fr)
WO (1) WO2001013933A2 (fr)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6610702B2 (en) 2000-08-01 2003-08-26 Gmp Oxycell, Inc. Ammonium salts of inositol hexaphosphate, and uses thereof
CA2377442A1 (fr) * 2002-03-20 2003-09-20 Thomas Ichim Augmentation de la teneur en oxygene dans des tumeurs par l'administration de cellules agressees
US7084115B2 (en) 2002-04-29 2006-08-01 Oxyplus, Inc. Inositol pyrophosphates, and methods of use thereof
EP2540696B1 (fr) 2005-02-24 2020-01-01 Diffusion Pharmaceuticals LLC Transcaroténoïdes, formulation les comprenant et leurs utilisations
US20060264413A1 (en) * 2005-04-18 2006-11-23 Sri International Method and composition for inhibiting cell proliferation and angiogenesis
WO2007123126A1 (fr) * 2006-04-17 2007-11-01 Juridical Foundation Osaka Industrial Promotion Organization Agent PERMETTANT D'AMÉLIORER la TOLÉRANCE DIMINUÉE À l'exercice dans l'insuffisance cardiaque chronique
FR2944106B1 (fr) * 2009-04-03 2012-09-28 Erytech Pharma Methode de dosage de l'inositol hexaphosphate (ihp).
EP2445339B1 (fr) 2009-06-22 2019-08-07 Diffusion Pharmaceuticals LLC Composé améliorant la diffusion et son utilisation avec un thrombolytique
AU2011262361A1 (en) 2010-06-02 2013-01-10 Diffusion Pharmaceuticals Llc Oral formulations of bipolar trans carotenoids
WO2014039660A1 (fr) * 2012-09-06 2014-03-13 Biomet Biologics, Llc Procédés de production de globules rouges rajeunis
US9066909B2 (en) 2012-09-06 2015-06-30 Biomet Biologics, Llc Methods for producing and using rejuvenated red blood cells
US10253295B2 (en) 2012-09-06 2019-04-09 Biomet Manufacturing, Llc Methods for producing rejuvenated red blood cells
US9103842B2 (en) 2013-01-31 2015-08-11 Biomet Biologics, Llc Methods for rejuvenating red blood cells
US9102918B2 (en) 2013-01-31 2015-08-11 Biomet Biologics, Llc Methods for rejuvenating red blood cells
US9011408B2 (en) 2013-01-31 2015-04-21 Biomet Biologics, Llc Functionally-closed, sterile blood processing solution system and method
CA3018550A1 (fr) 2016-03-24 2017-09-28 Diffusion Pharmaceuticals Llc Utilisation de carotenoides trans bipolaires avec une chimiotherapie et une radiotherapie en vue du traitement d'un cancer
JP2018135329A (ja) * 2018-02-19 2018-08-30 ディフュージョン・ファーマシューティカルズ・エルエルシー 双極性トランスカロテノイド塩及びそれらの使用
CA3113377A1 (fr) 2018-10-11 2020-04-16 Sanifit Therapeutics S.A. Phosphates d'inositol pour le traitement de la calcification ectopique
MX2021008966A (es) * 2019-01-30 2021-11-04 Sanifit Therapeutics S A Compuestos de fosfato de inositol para su uso en el aumento de la perfusion tisular.

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2600894B1 (fr) * 1986-07-02 1989-01-13 Centre Nat Rech Scient Conjugues macromoleculaires d'hemoglobine, leur procede de preparation et leurs applications
FR2630329B1 (fr) * 1988-04-20 1991-07-05 Merieux Inst Conjugues macromoleculaires d'hemoglobine, leur procede de preparation et leurs applications
US5114768A (en) * 1988-06-02 1992-05-19 Hewlett-Packard Company Surfaces with reduced protein interactions
US4921997A (en) * 1988-06-15 1990-05-01 Montefiore Medical Center Method of synthesis and novel compounds for pharmaceutical uses
US5677330A (en) * 1990-02-12 1997-10-14 The Center For Innovative Technology Medical uses of allosteric hemoglobin modifier compounds in patient care
US5731454A (en) * 1990-02-12 1998-03-24 Virginia Commonwealth University Allosteric modifiers of hemoglobin useful for decreasing oxygen affinity and preserving oxygen carrying capability of stored blood
US5432191A (en) * 1990-02-12 1995-07-11 The Center For Innovative Technology Allosteric hemoglobin modifiers to decrease oxygen affinity in blood
US5248785A (en) * 1990-02-12 1993-09-28 Virginia Commonwealth University Using allosteric hemoglobin modifiers to decrease oxygen affinity in blood
US5872282A (en) * 1990-12-07 1999-02-16 Virginia Commonwealth University Allosteric modifiers of hemoglobin
JPH04300838A (ja) * 1991-03-28 1992-10-23 Terumo Corp 人工赤血球および人工赤血球懸濁液
JPH08511680A (ja) * 1993-03-23 1996-12-10 シービーアール・ラボラトリーズ・インコーポレーテツド 細胞内への生物学的活性物質の封入のための方法および装置
US5599974A (en) * 1994-09-19 1997-02-04 Center For Innovative Technology Aldehydic agents for allosteric modification of hemoglobin
WO1997042819A1 (fr) * 1996-05-13 1997-11-20 Alberto Haces Lipides cationiques destines a la transfection de molecules neutres ou chargees negativement a l'interieur de cellules vivantes
US5877220A (en) * 1997-03-06 1999-03-02 Genta, Incorporated Amide-based oligomeric cationic lipids
US6034135A (en) * 1997-03-06 2000-03-07 Promega Biosciences, Inc. Dimeric cationic lipids

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0113933A2 *

Also Published As

Publication number Publication date
WO2001013933A9 (fr) 2002-09-12
AU6912500A (en) 2001-03-19
WO2001013933A2 (fr) 2001-03-01
JP2003507430A (ja) 2003-02-25
WO2001013933A3 (fr) 2001-07-19

Similar Documents

Publication Publication Date Title
US9078908B2 (en) Inositol pyrophosphates, and methods of use thereof
US20020142995A1 (en) Ammonium salts of hemoglobin allosteric effectors, and uses thereof
US6610702B2 (en) Ammonium salts of inositol hexaphosphate, and uses thereof
EP1223942A2 (fr) Renforcement de l'apport en oxygene chez des mammiferes, procedes et reactifs correspondants
JP3188897B2 (ja) 細胞増殖の抑制及びｎｋ細胞活性の増強
JP5690142B2 (ja) 蘇生液
CN111406737B (zh) 用于器官持续维持的成分、装置和方法
US6506549B1 (en) Plasma expanders and blood substitutes
JP2024175025A (ja) 組織の灌流の増加における使用のためのイノシトールリン酸化合物
JP2005530786A (ja) 心損傷の処置のためのピロロキノリンキノンおよびその使用方法
US20160120887A9 (en) Tumor eradication by inositol-tripyrophosphate
US20040072801A1 (en) Sterols bearing pendant allosteric effectors of hemoglobin, and uses thereof
EP0199117A2 (fr) Application thérapeutique du phosphocréatine
EP0928191B1 (fr) Compositions pharmaceutiques renfermant des derives de s-alkylisothiouronium
US20110294732A1 (en) Erythropoletin complementation or replacement

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020315

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20050401