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  • C12N5/0641—Erythrocytes

Definitions

• the major function of erythrocytes consists in the transport of molecular oxygen from the lungs to the peripheral tissues.
• the O 2 -partial pressure in the lung is about.100 mm Hg, in the capillary system is about.70 mm Hg, against which O 2 must be dissociated from the oxygenated hemoglobin.
• O 2 -partial pressure in the lung is about.100 mm Hg
• in the capillary system is about.70 mm Hg, against which O 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-O 2 adduct is not used for the O 2 transport.
• IHP like 2,3- diphosphoglycerate and other polyphosphates cannot penetrate the erythrocyte membrane. Furthermore, the depletion of DPG and ATP in stored red cells leads to a progressive increase of the oxygen affinity of hemoglobin contained therein (Balcerzak, S. et al. (1972) Adv. Exp. Med. Biol. 28, 453-447).
• the O 2 -binding isotherms are measured in the absence of CO 2 and at constant pH (pH 7.4) in order to preclude influences of these allosteric effectors on the half-saturation pressure.
• 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
• IHP is unable to pass unassisted across the erythrocyte membrane.
• IV. Enhanced Oxygen Delivery in Mammals The therapy of oxygen deficiencies requires the knowledge of parameters which characterize both the O 2 transport capacity and the O 2 release capacity of human RBCs.
• the parameters of the O 2 transport capacity i.e., Hb concentration, the number of RBCs, and hemocrit
• Hb concentration, the number of RBCs, and hemocrit are commonly used in clinical diagnosis.
• O 2 release capacity i.e., O 2 half-saturation pressure of Hb and RBCs, and the amounts of high and low oxygen affinity hemoglobins in RBCs
• Enhancement of the O 2 -release capacity of these cells brought about significant physiological effects in piglets: 1) reduced cardiac output, linearly dependent on the P 50 value of the RBCs; 2) increased arteriovenous difference; and 3) improved tissue oxygenation. Long term experiments showed that in piglets the high P 50 value of IHP- RBCs was maintained over the entire life spans of the RBCs.
• Nicolau et al. (TRANSFUSION 1995, 35, 478-486; and US Patent 5,612,207) reported the use of a large-volume, continuous-flow electroporation system for the encapsulating IHP in human RBCs. These modified RBCs possess P 50 values of approximately 50 torr, roughly twice that of unmodified human RBCs. Additionally, 85% of the RBCs survived the electroporation process, displaying hematologic indices nearly identical to those of unmodified RBCs. Nicolau' s electroporation system processes one unit of blood every ninety minutes.
• Synthetic human hemoglobin has also been produced in neonatal pigs by injection of human genes that control hemoglobin production. This product may be a less expensive product than the Somatogen synthetic hemoglobin, but it does not solve problems with oxygen affinity and breakdown of hemoglobin in the body. V. Specific Clinical Applications of Enhanced Oxygen Delivery
• compositions consisting essentially of aliphatic ammonium cations or of metal cations, such as sodium cations, and IHP derivatives comprising an internal pyrophosphate moiety.
• the present invention also relates to methods for modulating the oxygen affinity of hemoglobins comprising the use as allosteric effectors of hemoglobin of the aforementioned IHP deriviatives and the compositions comprising them.
• an aliphatic ammonium cation is substituted with one or more aliphatic groups, which can be the same or different.
• the aliphatic ammonium cation is a primary ammonium cation represented by the general formula RN'TTJ, wherein R is an aliphatic group, preferably an alkyl, preferably a lower alkyl, i.e., a C ⁇ -C 8 alkyl, and more preferably a C 3 -C ⁇ 0 cycloalkyl.
• the ammonium cation is derived from cyclic amines.
• the present invention relates to compounds, and compositions thereof, that deliver EHP into erythrocytes in vivo or ex vivo, for lowering the oxygen affinity of hemoglobin in red blood cell suspensions and whole blood. It is an object of this invention to provide methods for delivering IHP into erythrocytes in whole blood, utilizing compounds or compositions thereof that do not lose their effectiveness in the presence of whole blood.
• red blood cells or whole blood to said subject.
• suitable purifying' it is meant a method of washing and separating, for example by centrifugation, the red blood cell- allosteric effector or whole blood-allosteric effector suspension, and discarding the supernatant until no non-encapsulated allosteric effector can be detected.
• An exemplary method is presented in detail by Nicolau et al. in U.S. Patent No. 5,612,207, which is incorporated by reference herein.
• 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 of IHP derivatives comprising an internal pyrophosphate moiety, thereby 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 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 useful shelf-life vis-a-vis transfusions, and/or restore the oxygen carrying capacity of aged blood.
• ARDS adult respiratory distress syndrome
• Another condition which could benefit from an increase in the delivery of oxygen to the tissues is anemia.
• a significant portion of hospital patients experience anemia or a low "crit" caused by an insufficient quantity of red blood cells or hemoglobin in their blood. This leads to inadequate oxygenation of their tissues and subsequent complications.
• a physician can temporarily correct this condition by transfusing the patient with units of packed red blood cells.
• Figure 1 depicts the 31 P NMR spectrum of IHP-cholesteryloxy carbonyl hepta NN- dimethylcyclohexylammonium salt and the 31 P ⁇ MR spectrum of IHP-tripyrophosphate ("ITPP").
• Figure 3 depicts the 31 P ⁇ MR spectrum of purified IHP-cholesteryloxy carbonyl; the 31 P ⁇ MR spectrum of highly purified IHP-cholesteryloxy carbonyl; the 31 P ⁇ MR spectrum of IHP-monopyrophosphate ("EVIPP"); and the 31 P ⁇ MR spectrum of ITPP.
• Figure 5 depicts the 31 P ⁇ MR spectrum of crude IHP-benzoate; the 31 P ⁇ MR spectrum of purified IHP-benzoate; and the 31 P ⁇ MR spectrum of IHP-benzoate at pH 6.9.
• Figure 6 depicts the 31 P ⁇ MR spectrum of the crude IHP-hexanoyl derivative; the P ⁇ MR spectrum of the purified IHP-hexanoyl derivative; the P ⁇ MR spectrum of the IHP-hexanoyl derivative at pH 7.3; and the 31 P ⁇ MR spectrum of the IHP-hexanoyl derivative after heating.
• Figure 7 depicts the 31 P ⁇ MR spectra of kf56, kf53, kf31 , kf31 A, respectively.
• Figure 8 depicts 2a): mixed 31 P ⁇ MR of all compounds 5 (kf31, kf53, kf56); 2b): 2D 31 P ⁇ MR COSY experiment of compound kf53.
• Figure 9 depicts ITPP uptake in 1-octanol by cyclooctylammonium ions.
• Figure 11 depicts means of 12 P 50 -values and standard deviation are shown .
• the kfl 11 -solution was replaced by water.
• P 50 values were measured over 12 days.
• IHP was replaced by water.
• Figure 12 depicts P 50 shifts of 4 single mice (and standard deviation are shown). Mouse 1, Mouse 12, IHP control mouse, water control mouse.
• Figure 13 depicts the relation of P 50 shift [%] to erythrocytes (values are taken from Table 1). Based upon the preliminary data reported that an inverse relationship exists between the number of RBC and shift of their P 50 value. The basal value of the RBC count is restored, once ⁇ P 50 becomes 0%, 12 days after ingestion of kflll.
• Figure 15 depicts the dosis curve for 3 piglets injected via iv with 0.3, 0.5, 1*, 1.3 and 1.5g kflll per kg body weight. Means of 4 single P 50 values per blood sample and standard deviation are given. (*2 piglets injected). Detailed Description of the Invention I.
• the process of allosterically modifying hemoglobin towards a low oxygen affinity state in whole blood could be used in a wide variety of applications including in treatments for ischemia, heart disease, complications associated with angioplasty, 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, e.g., in whole blood.
• 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. In contrast, the subject 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,
• 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.
• RBCs mammalian red blood cells
• the compounds and methods of the present invention make possible the introduction or incorporation into RBCs of anionic agents, such as DNA, RNA, chemotherapeutic agents, and antibiotic agents.
• anionic agents 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 and purified RBC carrier is later injected in vivo.
• the particular anion or 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.
• IHP is preferably added to red blood cells in vitro or ex vivo, as it appears that it is toxic to animals under certain circumstances.
• IHP-treated red blood cells 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. This is because the DPG present in normal red blood cells is degraded by native enzymes, e.g., phosphatases, during storage and must be replaced by the body after transfusion. In contrast, 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.
• native enzymes e.g., phosphatases
• EHP-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, complications associated with angioplasty, 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.
• This invention is related to a method of treating a subject for any one or more of the above diseases comprising the steps of treating red blood cells or whole blood ex vivo with one or more compounds or compositions of the present invention, followed by suitably purifying said red blood cells or whole blood, and administering the thus prepared red blood cells or whole blood to said subject.
• suitably purifying it is meant a method of washing and separating the red blood cell- or whole blood-allosteric effector suspension and discarding the supernatant until no non-encapsulated allosteric effector can be detected, e.g., as devised by Nicolau et al. in U.S. Patent No. 5,612,207.
• a compound comprised of an allosteric effector can be administered directly to a subject if the compound does not have toxic effects in the subject, or at least its beneficial effects predominate over its toxicity in a subject.
• 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 P 5 o value is shifted towards higher partial pressures of O 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), RBCs or whole blood treated with the compounds of the present invention and subsequently purified 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., 112, (1987), pp. 164), decreasing the oxygen affinity of hemoglobin in circulating blood has been shown to be beneficial in the radiotherapy of tumors.
• RBCs or whole blood treated with the compounds of the present invention and subsequently purified 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.
• Administration to a patient can be achieved by intravenous or intraperitoneal injection where the dose of treated red blood cells or whole blood 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
• 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. Damaged tissues heal faster when there is better blood flow and increased oxygen tension. Therefore, the compounds, compositions and methods of this invention may be exploited to speed wound healing. Furthermore, by increasing oxygen delivery to wounded tissue, the compounds, compositions and methods of this invention may play a role in the destruction of infection causing bacteria at a wound.
• the compounds, compositions and methods of this invention may be effective in enhancing the delivery oxygen to the brain, especially before complete occlusion and reperfusion injuries occur due to free radical formation.
• 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 could be used in the treatment and prevention of ARDS by militating against lower than normal oxygen delivery to the lungs.
• cardiac bypass surgery There are several aspects of cardiac bypass surgery that make attractive the use of compounds or compositions or methods of the present invention.
• the compounds and compositions of the present invention may 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. Second,
• 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. 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.
• Red blood cells or whole blood previously treated with the compounds of the present invention and subsequently suitably purified may be used to enhance oxygen delivery in any organism, e.g., fish, that uses a hemoglobin with an allosteric binding site.
• 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.
• toxic refers to a property where the deleterious effects are greater than the beneficial effects.
• 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,
• “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 P 5 o is a measure of oxygen affinity.
• 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.
• “Skeletal 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 intrasternal injection and infusion.
• surgical refers to the treatment of diseases, injuries, and deformities by manual or operative methods. Common surgical procedures include, but are
• - 20 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.
• minimal 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).
• ED o means the dose of a drug that produces 50% of its maximum response or effect. Alternatively, the dose that produces a pre-determined response in 50% of test subjects or preparations.
• LD 5 o means the dose of a drug that is lethal in 50% of test subjects.
• therapeutic index refers to the therapeutic index of a drug defined as LD 50 /ED 5 o.
• 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
• R is selected independently for each ocurrence from the group consisting of H, cations and hydrocarbon groups.
• IHP-monopyrophosphate refers to inositol hexaphosphate where two orthopyrophosphates were condensed to one internal pyrophosphate ring.
• IHP-tripyrophosphate or “inositol tripyrophosphate” (both abbreviated as "ITPP") refers to inositol hexaphosphate with three internal pyrophosphate rings.
• DPG 2,3-diphosph-D-glyceric acid
• CPPG 2,3-cyclopyrophosphoglycerate
• 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.
• 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.
• aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
• 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 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.
• 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.
• 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.
• nitro means -NO2; the term “halogen” designates -F, -CI,
• 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:
• R9, R ⁇ Q and R' IQ each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2) m - 8, or R9 and RJQ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure;
• Rg 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 R9 or RJQ can be a carbonyl, e.g., R9, Rio and the nitrogen together do not form an imide.
• R9 and RJO 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 RJQ ⁇ S an alkyl group.
• acylamino is art-recognized and refers to a moiety that can be represented by the general formula:
• R 9 is as defined above, and R' ⁇ ⁇ 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, R ⁇ ⁇ are as defined above.
• Preferred embodiments of the amide will not include imides which may be unstable.
• 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 ⁇ ⁇ represents a hydrogen, an alkyl, an alkenyl, -(CH2) m -R or a pharmaceutically acceptable salt
• R' ⁇ ⁇ represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -R-8 > where m and Rg are as defined above.
• 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, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
• triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-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, p-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.
• R4 ⁇ is as defined above.
• sulfonyl 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, 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.
• 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. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
• 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
• 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.
• 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 marmer 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 also known as allosteric effectors 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
• the present invention relates to compositions, and methods of use thereof, consisting essentially of a nontoxic ammonia cation (preferably water-soluble), and inositol hexaphosphate (IHP, phytic acid) derivatives comprising an internal pyrophosphate ring.
• IHP is the most abundant form of phosphate in plants. DTP binds hemoglobin 1000 times more tightly than DPG and therefore triggers a decrease of the O 2 /hemoglobin affinity with a subsequent release of oxygen. Because of IHP' s superior hemoglobin binding properties over DPG, IHP represents a good pharmaceutical candidate for diseases characterized by a limited oxygen flow to organ tissues. Under normal physiological conditions, IHP bears at least 7 charges, making it very difficult for it to be transported across cell membranes. In order to answer the IHP delivery problem two approaches have been investigated: a) the
• the present invention expands upon approach b) wherein the covalently bound linker is an adjacent phosphate group or an acyl phosphate group including a cholesteryloxy carbonyl group, which under certain conditions eliminate to give an internal pyrophosphate ring.
• the nontoxic ammonium cation is represented by the general formula N ⁇ R) ⁇ wherein R is, independently for each occurrence, H or an aliphatic group, which aliphatic group is preferably an alkyl, preferably a lower (C1-C8) alkyl, and more preferably a C3-C10 cyclic alkyl.
• the ammonium cation is preferably derived from cyclic organic bases.
• the ammonium cation is NN-dimethylcyclohexylarnmonium (NN-DMCHA) for the following reasons: a) it increases the lipophilisity of IHP and makes the molecule soluble in all organic solvents, without affecting its solubility in water, and b) as an ammonium salt of a tertiary amine, it doesn't react with the acyl anhydrides or alkyl formates.
• the present invention is related to compounds, and compositions thereof, which deliver IHP into erythrocytes in vivo, in vitro, or ex vivo.
• the invention is directed to the use of the compounds or compositions thereof that are effective in delivering IHP into erythrocytes, lowering the oxygen affinity state in red blood cell suspensions and whole blood. It is an object of this invention to provide methods for delivering IHP into erythrocytes in whole blood, 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 related to a method of treating red blood cells or whole blood in vivo, in vitro, or ex vivo with one or more nontoxic compounds or compositions of the present invention, suitably purifying said red blood cells or whole blood, and administering said purified red blood cells or whole blood to a subject for any treatment where an increase in oxygen delivery by hemoglobin would be a benefit.
• the present invention is directed toward the design of water-soluble membrane compatible molecules comprising ammonium cationic moieties, e.g., lipophilic ammonium groups. These molecules form complexes with IHP derivatives comprising an internal pyrophosphate ring; such complexes are useful for the delivery of IHP into the cytoplasm of erythrocytes.
• IHP derivatives comprising an internal pyrophosphate ring
• metal cations e.g., sodium cations, may allow deliver of IHP into the cytoplasm of erythrocytes.
• C + represents independently for each occurrence an aliphatic ammonium cation, an alkali metal cation, an alkaline earth cation, or other suitable metal cation;
• a n ⁇ represents a conjugate base of inositol hexaphosphate comprising an internal pyrophosphate ring or an acyl group, wherein n equals the number of cations comprised by nC + .
• the present invention relates to a pharmaceutical composition, comprising a nontoxic compound of the present invention; and a pharmaceutically acceptable excipient.
• IHP derivatives comprising an internal pyrophosphate ring were prepared by heating IHP with acyl anhydrides or acyl chlorides as depicted in Scheme 1.
• the proposed mechanism for this reaction is depicted in Scheme 2.
• the key step is presumed to be conversion of a phosphate oxygen into a leaving group by forming an acyl phosphate ester. Once the acyl phosphate ester forms, a vicinal phosphate group is well positioned to attack nucleophilically the central phosphorous atom and form the internal pyrophosphate ring.
• a 7 membered pyrophosphate ring of 1,4,5 myo-inositol triphosphate was formed as a byproduct in a sequence of reactions where acetic anhydride was involved.
• DPG was exposed to the same conditions as with IHP, i.e. heating with an excess of acyl anhydride.
• IHP octa N,N-dimethyl-cyclohexylammonium salt, 7 (kf36A), reacted with Ac 2 O (6 equiv in CH 2 C1 at rt for 4 days).
• the 31 P NMR showed a very low
• Inositol Tripyrophosphate (ITPP) ammonium salts from phytic acid and ITPP pyridinium salt
• Route A starts with compound 2, the perprotonated IHP molecule, and proceeds to the corresponding DTP compound IV.
• Compound IV bears the maximum of the counter cations they can hold.
• Compound IV was treated with DCC to give various results depending on the nature of the counter cation. For example, in the case of the N,N- dimethylcyclohexylammonium salt of compound IV, the reaction goes to almost completion. In the case of the n-hexylammonium-, cycloheptylammonium-, or cyclooctylammonium salts, 50% of the pyrophosphate product is hydrolyzed.
• the primary amine salt solutions are strongly basic (pH>10) and it is believed that the high basicity causes hydrolysis of the initially formed pyrophosphates.
• compound IV bearing 6 or less counter cations were synthesized. Their reactions with DCC gave much better results, but not the desirable pure compounds, (except in the case of the tertiary ammonium salt).
• this route was more strenuous because each pyrophosphate had to be synthesized individually from its corresponding DTP ammonium salt in 3 steps.
• the more advanced compound 4 having the pyrophosphates already formed could be used as a starting material for the construction of desired compounds III. Additionally, 4 can be synthesized in large quantities and in very clean form.
• ITPP pyridinium salt 4 (kf50A) the ⁇ a salt 5 (kf77) was prepared in the way shown in Scheme 3. All five compounds were initially examined in vitro with free hemoglobin and whole blood. VII. Solvent effects on IHP-acyloxy carbonyl formation vs. IHP-pyrophosphate formation.
• the first step in forming an internal pyrophosphate ring is acyl phosphoester formation.
• This mechanism also applies to compounds comprising an alkyloxy carbonyl group.
• Experiments were carried out to determine what conditions favored the acyl or alkyloxy carbonyl intermediate versus internal pyrophsophate ring formation.
• the effect of solvent on internal pyrophosphate ring formation was determined for CH 2 Cl 2 /l,4-dioxane, CH 3 C ⁇ , and CH 3 CN/THF. The effect of these solvents during purification was also investigated, as well as the stability of the acyl phosphoesters in water.
• Figure 1 depicts a representative example of the P-NMR spectrum.
• the spectra depict 3 bands of peaks.
• the first band covered the area 4 to - 1 ppm, that is the part of the spectrum where the orthophosphates absorb.
• the second band covered the area —4 to -9 ppm, and the third one covered the area from -9 to -14 ppm.
• reaction kf89 1.5 equiv of cholesteryl chloroformate was used for reaction kf89 1.5 equiv of cholesteryl chloroformate. According to the integration values of the 31 P-NMR spectrum, in 22 h statistically 1 cholesterol moiety was attached on DTP. The reaction was not forced to proceed for a longer period of time. Although the concentration of the reaction mixture in these dilute conditions had no effect (see reaction kf92), the increase of the reaction time was acting against the cholesterol derivatives (see reaction kfl 00). Reactions kf90, kf93, kf93p, and kfl 01 represent analogous reactions using 2 eq of cholesteryl chloroformate.
• Figure 2 depicts (a) the 31 P NMR spectrum of the compound from kf96 in CDC1 3 , and (b) the same purified compound in D 2 O.
• DTP alkyloxy carbonyl derivatives are quite unstable in water. They hydrolyse immediately either to pyrophosphate through an intramolecular attack from a neighboring phosphate, or to orthophosphate through an intermolecular attack from water. (The latter transformation was hypothesized due to the loss of the 5:1 ratio of the integration between orthophosphates and pyrophosphates in the spectra). This observation also explained, why prolonged storage in air was changing the composition of the highly hygroscopic cholesteryloxycarbonyl compounds like kf49 in Table 1. VIII. Synthesis of IHP monopyrophosphate (IMPP) Further experimentation led to control over the number of internal pyrophosphate rings formed.
• IHP monopyrophosphate IHP monopyrophosphate
• CH 2 C1 2 was avoided for reasons explained before. Instead, we examined the reactions of IHP with 1-3 equivalents of Ac 2 O, Bz 2 O and hexanoic anhydride in either CH 3 CN or a mixture of CH 3 CN/THF.
• the CH 3 CN/THF solvent system was superior because it gave more loading of acyls on DTP in the same amount of time. No pyrophosphates formed in all cases using these solvents.
• the 31 P-NMR of kfl 05 in CDC1 3 is shown in Figure 4(a). The abso ⁇ tion of the phosphorous peaks at -6 ppm indicates a benzoate substitution, since there was no phosphorous observed in the area of -8 to -14 ppm.
• ITPP ability of ITPP to react further with acylating or other agents was investigated through three preliminary experiments.
• ITPP NN-dimethylcyclohexyl ammonium salt was heated extensively with acyl anhydrides and no change was observed.
• the second experiment attempted to react ITPP pyridinium salt 9 with acyl anhydride in the presence of pyridine and DMAP, but solubility ofthe starting material in CH 2 C1 2 proved problematic.
• a reaction with triphosgene which was expected to exchange an OH with CI
• subsequent reaction with cycloheptyl amine gave, after extraction ofthe reaction mixture with H 2 O and CH 2 C1 2 separately, phosphorous containing compounds in both phases (TLC, ⁇ MR).
• TLC, ⁇ MR phosphorous containing compounds
• ITPP was dissolved in a buffer solution of a pH 4.6 and heated in the presence of baker's yeast for 12 h at 45 °C. Non selective hydrolysis of approximately 25% occurred.
• Partition coefficients relate to the distribution of a solute between two immiscible liquid phases and are defined as the ratios of concentrations (or molar fraction) ofthe distributed solute. These data have been used to predict and rationalize numerous drug properties such as quantitative structure/activity relationship, lipophilicity, and pharmacokinetic characteristics.
• 1-Octanol has been found to properly mimic biological membranes, and it has been estimated that 1-octanol/water (KQ W ) partition coefficients of more than 18000 substances are now available in the literature.
• mice Twelve C57BL/6 mice drank kflll over 4 days (about 25ml/24 hrs). Three control mice drank either pure water, or a solution of IHP (inositol hexaphosphate) at the same concentration and pH as kflll (4 mice). The amount of drunken fluid was the same when offering pure water, DTP- water or kflll- water, indicating that kflll-, or IHP-solution was not rejected by the mice. Blood was collected from the tail vein ofthe 19 C57BL/6 mice on day 0 (before treatment started), 1, 2, 4, 6, 7, 8, 10, 11 and 12, in order to measure P 5 o values. The following remarks can be made:
• mice Blood from mice, having ingested kflll or DTP in water (for 4 days) or water only was collected on day 0, 7 and 11, in order to assess any differences in the blood count (and the amount of erythropoietin in the sera) of treated and control mice.
• Two major observations were made: 1.) The number of RBC in mice having ingested kflll was reduced significantly, and 2.) There were no major differences in the number of white blood cells (e.g. granulocytes, macrophages ect.) in blood from mice in different groups. Table 1 shows the RBC counts for mice with shifted ODC as compared to controls. Erythropoietin assays in all mice sera will be reported soon.
• mice having received kflll, and 2 mice having received water only and 1 mouse having received IHP/water are shown.
• the amount of blood from the other mice were not sufficient to determine the blood count. (On day 0 the RBC count in the mice was 8.9-11.8 xlO 6 cells /mm 3 ).
• kflll when orally administered at a concentration of 27 mM, causes a significant right shift ofthe P 50 value in murine circulating RBC. There is a time lag of about 48 hrs before the maximum shift is attained, contrarily to the observations made after ip inoculation of kflll, where the P 50 shifts appears 2 hrs after inoculation. 2.) Maximal P 0 shifts are reached between day 2 and day 4 after beginning oral administration of kfl 11. 3.) After 12 days P 50 values are back to control values (taken on day 0), when ingestion is stopped on day 4.
• white blood cells e.g. granulocytes, macrophages, platelets ect.
• kflll was not harmful to the piglet, when applied intravenously slowly (at least 10 min for a vol. of solution of 100 ml)) at a concentration lg/kg* body weight.
• the piglets were thirsty after the treatment.
• Higher amounts of kfl 11 inj ected via iv, killed the animals.
• the Na ITPP salt kflll was the molecule with the best profile, showing no toxicity even in a concentration of 160 mM.
• the activity ofthe ITPP molecules was predicted from the discovery ofthe fate ofthe cholesteryloxy carbonyl derivative in aqueous solutions, Route 3.
• Route 1 The products of Route 1 were passing through an ion exchange Dowex 50Wx8 Na + form column, and the elute was concentrated in vacuum to give hexasodium Salt ofMyo- inositol 1, 6:2,3:4, 5 -Tripyrophosphate.
• Crystalline sodium phytate C (4 g) was dissolved with sonication in water (20 ml) and converted to the free acid by passage through a column of Dowex 50x8-200 ion- exchange resign. The column eluate was adjusted to pH 8 with pyridine and evaporated to dryness. The residue was dissolved in water (30 ml) and pyridine (130 ml) containing NN- dicyclohexylcarbodiimide (8 g) was added. The reaction mixture was heated to 65 °C for 18 h and evaporated to dryness.
• the compound was then dissolved in water (30 ml) and passed through a column Dowex 50Wx8 Na + form.
• the column eluate was concentrated to dryness to give Hexasodium Salt of Myo-inositol 1,6: 2, 3: 4, 5 -Tripyrophosphate (2.25 g, 97%) and used for biological experiments in 98.5% purity without any further purification.
• the impurity is unreacted starting material (or tripyrophosphate hydrolyzed back to starting material).
• IHP cholesteryoxy carbonyl hepta N.N-Dimethyl cyclohexyl ammonium salt initial conditions: material contaminated with monopyrophosphates, kfl 6, kf38, kf42, kf96.
• Crystalline sodium phytate C (2 g) was dissolved with sonication in water (10 ml) and converted to the free acid by passage through a column of Dowex 50x8-200 ion- exchange resign. The column eluate was adjusted to pH 8 with pyridine and evaporated to dryness. The residue was dissolved in water (14 ml) and pyridine (56 ml) containing NN- dicyclohexylcarbodiimide (438 mg, 1 equiv) was added. The reaction mixture was heated to
• the effectors kf96 and kflll were tested for P 50 shifts in whole blood of three species: human, mouse and pig.
• pH's for the compound-solutions were adjusted to -7.0, osmolarities for both solutions were determined (325-373 mOsM) prior to effectors, and whole blood volumes at 1:1 ratios were incubated. Following incubation, blood cells were washed 3 times with Bis-Tris-buffer (no lysis of RBCs was observed).
• Table 5 A summary of P 50 values for whole blood induced by the effectors is presented in Table 5.
• the concentration ofthe electrolytes sodium, potassium and calcium will be determined after injection, in order to investigate possible side effects.
• the new compound kfl37 showed a right shift of 120% with free porcine hemoglobin and su ⁇ risingly a very strong right shift with porcine whole blood (of 53% and more) under approximately iso osmolar conditions (288mOsM).
• Inositol pyrophosphates represent a new class of allosteric effectors of hemoglobin. Their highly charged nature allows them to pass through erythrocyte membranes and bind to hemoglobin, resulting in a signifficant right P 50 value. The progressive decrease ofthe

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