Patent Application: US-201314075878-A

Abstract:
the present invention relates to mixed calcium / sodium salt of inositol tripyrophosphate , methods of preparing and methods of use . the mixed calcium / sodium salt may be a monocalcium tetrasodium salt of inositol tripyrophosphate . methods of use include administering the above salts in an effective amount to treat diseases caused by hypoxia or other conditions associated with inadequate function of the lungs or circulatory system , such as various types of cancer and alzheimer &# 39 ; s disease .

Description:
compositions that are useful in accordance with the present invention include the mixed calcium / sodium salt of inositol - tripyrophosphate ( itpp - ca / na ). the composition of the present invention may also comprise trace amounts of additional ions such as , but not limited to , strontium , barium , beryllium , magnesium , rubidium , cesium , cadmium , lead , mercury , zinc , iron , copper , nickel , cobalt , manganese , lithium , potassium , silver , and lanthanum , without departing from the scope of the present invention . itpp exhibits anti - angiogenic and anti - tumor properties , and is useful in controlling angiogenesis -, or proliferation - related events , conditions or substances . as used herein , the control of an angiogenic -, or proliferation - related event , condition , or substance refers to any qualitative or quantitative change in any type of factor , condition , activity , indicator , chemical or combination of chemicals , mrna , receptor , marker , mediator , protein , transcriptional activity or the like , that may be or is believed to be related to angiogenesis or proliferation , and that results from administering the composition of the present invention . those skilled in the art will appreciate that the invention extends to other compositions or compounds in the claims below , having the described characteristics . these characteristics can be determined for each test compound using the assays detailed below and elsewhere in the literature . other such assays include counting of cells in tissue culture plates or assessment of cell number through metabolic assays or incorporation into dna of labeled ( radiochemically , for example 3 h - thymidine , or fluorescently labeled ) or immuno - reactive ( brdu ) nucleotides . in addition , antiangiogenic activity may be evaluated through endothelial cell migration , endothelial cell tubule formation , or vessel outgrowth in ex - vivo models , such as rat aortic rings . when administered orally , itpp exhibits anti - tumor and anti - proliferative activity with little or no toxicity . itpp was tested for its ability to induce a decrease of the o 2 - affinity of hemoglobin measured as a shift of the p 50 value ( p 50 at 50 % saturation of hemoglobin ). with murine hemoglobin and whole blood , p 50 shifts to higher po 2 of up to 250 % with hemoglobin and up to 40 % with whole blood were observed . the results obtained with itpp in mice and pigs strongly suggest the possibility of its development as a therapeutic , due to its ability to enhance , in a regulated manner , oxygen delivery by red blood cells in the cases of blood flow impairment . it has been found that pigs injected intravenously with itpp - na at a rate of 1 g / kg weight had beneficial properties associated with the introduction of itpp - na into their systems ( as described in u . s . provisional patent application 60 / 585 , 804 , which is herein incorporated by reference in its entirety ); however , the introduction of itpp - na also resulted in a number of adverse side effects . these side effects included flushing , an increase in the heart rate , and a decrease in the ca 2 + plasma concentration . therefore a less toxic form of itpp that maintains a good solubility profile is needed . itpp , when administered orally , intravenously , or intraperitoneally , inhibits angiogenesis in growing tumors by enhancing po 2 in the forming tumors . this invention further provides for methods of regulation of vascular endothelial growth factor ( vegf ) in a human or animal , by administering to the human or animal an effective amount of itpp . more particularly , this invention provides for dose - dependent effects of itpp on vegf mrna and protein expressions in the llc cell line . vegf gene expression in tumor bearing c57bl / 6 mice was assayed and the effects of itpp - induced down regulation of vegf have been determined and correlated with modulation of cell proliferation . this invention resulted in the development of methods to control vegf mrna expression , protein concentration , and tumor cell proliferation . the results of these studies indicate a strong correlation between dose - dependent itpp - induced down regulation of vegf and cellular proliferation and suggests that itpp can reduce vegf mediated tumor angiogenesis , as well as the rate of tumor cell proliferation . thus , down - regulation of vegf by itpp decreases tumor cell proliferation . the shifting of the p 50 value to higher o 2 - partial pressures inhibits the expression of the hypoxia gene encoding vegf in the tumors . expression of the hypoxia gene encoding vegf is necessary for angiogenesis to be stimulated in tumors . if this does not occur , angiogenesis is seriously inhibited and new vessels are not formed in tumors . the results obtained concerning vegf expression suggests that oxygen partial pressure in tumors is elevated upon administration of itpp , as this elevation is the cause of inhibition of expression of this hypoxia gene . this observation raises a very important question , namely , whether this enhancement of po 2 may act as a powerful radiosensitizer of cancer cells . oxygen is a very potent radiosensitizer and , if indeed po 2 in the tumors is enhanced by itpp , this may have major consequences in enhancing the efficacy of radiation therapy of cancer . itpp is a potential significant adjuvant in the therapy of solid tumors as inhibitor of angiogenesis on one hand , and as a radiosensitizer on the other . it is known that medial temporal oxygen metabolism is markedly affected in patients with mild - to - moderate alzheimer &# 39 ; s disease . this measure substantiated the functional impairment of the medial temporal region in alzheimer &# 39 ; s disease . it also known that mean oxygen metabolism in the medial temporal , as well as in the parietal and lateral temporal cortices is significantly lower in the patients that are shown to have alzheimer &# 39 ; s disease than in control groups without alzheimer &# 39 ; s disease ( see ishii et al ., j . nucl med . 37 ( 7 ): 1159 - 65 , july 1996 , which is herein incorporated by reference in its entirety ). thus , one potential means of treating patients shown to have alzheimer &# 39 ; s disease is to increase oxygen across the blood brain barrier . one method of doing so would be to use an allosteric effector of hemoglobin such as treatment with itpp , such as with the calcium / sodium salt of itpp . the use of itpp , such as with the calcium / sodium salt of itpp , may also help in the treatment of a variety of vascular diseases associated with various forms of dementia . because the brain relies on a network of vessels to bring it oxygen - bearing blood , if the oxygen supply to the brain fails , brain cells are likely to die and this can cause symptoms of vascular dementia . these symptoms can occur either suddenly , following a stroke , or over time through a series of small strokes . thus , one potential means of treating patients with vascular diseases associated with various forms of dementia is to increase the oxygen available to affected areas such as across the blood brain barrier . one method of doing so would be to use an allosteric effector of hemoglobin such as treatment with itpp , such as with the calcium / sodium salt of itpp . moreover , treatment of an individual with an allosteric effector of hemoglobin such as the calcium / sodium salt of itpp may have beneficial effects for both stroke victims and osteoporosis . although stroke and the bone - thinning disease osteoporosis are usually thought of as two distinct health problems , it has been found that there may be a connection between them . patients who survive strokes are significantly more likely to suffer from osteoporosis , a disease that puts them at high risk for bone fractures . often , the fractures in stroke patients occur on the side of the body that has been paralyzed from the stroke . it is known that a stroke occurs when the supply of blood and oxygen to the brain ceases or is greatly reduced . if a portion of the brain loses its supply of nutrient - rich blood and oxygen , the bodily functions controlled by that part of the brain ( vision , speech , walking , etc .) are impaired . annually , more than 500 , 000 people in the united states suffer strokes and 150 , 000 of those people die as a result thereof . one means of increasing oxygen flow to the brain is by use of an allosteric effector of hemoglobin such as treatment with the calcium / sodium salt of itpp . accordingly , a potential method of treating individuals who might potentially suffer stroke or osteoporosis is by treatment of an individual with , for example , the calcium / sodium salt of itpp . also contemplated by the present invention are implants or other devices comprised of the compounds or drugs of itpp , or prodrugs thereof , where the drug or prodrug is formulated in a biodegradable or non - biodegradable polymer for sustained release . non - biodegradable polymers release the drug in a controlled fashion through physical or mechanical processes without the polymer itself being degraded . biodegradable polymers are designed to gradually be hydrolyzed or solubilized by natural processes in the body , allowing gradual release of the admixed drug or prodrug . the drug or prodrug can be chemically linked to the polymer or can be incorporated into the polymer by admixture . both biodegradable and non - biodegradable polymers and the process by which drugs are incorporated into the polymers for controlled release are well known to those skilled in the art . examples of such polymers can be found in many references , such as brem et al ., j . neurosurg 74 : pp . 441 - 446 ( 1991 ), which is herein incorporated by reference in its entirety . these implants or devices can be implanted in the vicinity where delivery is desired , for example , at the site of a tumor . in addition to the compounds of the present invention , the pharmaceutical composition of this invention may also contain , or be co - administered ( simultaneously or sequentially ) with , one or more pharmacological agents of value in treating one or more disease conditions referred to hereinabove . a person skilled in the art will be able by reference to standard texts , such as remington &# 39 ; s pharmaceutical sciences 17 th edition , to determine how the formulations are to be made and how these may be administered . in a further aspect of the present invention there is provided use of compounds of itpp , such as itpp - ca / na or prodrugs thereof , according to the present invention for the preparation of a medicament for the prophylaxis or treatment of conditions associated with angiogenesis or accelerated cell division or inflammation . in a further aspect of the present invention there is provided a pharmaceutical composition comprising compounds of itpp , such as itpp - ca / na or prodrugs thereof , according to the present invention , together with a pharmaceutically acceptable carrier , diluent , adjuvant or excipient . the pharmaceutical composition may be used for the prophylaxis or treatment of conditions associated with angiogenesis or accelerated cell division or inflammation , for treatment of alzheimer &# 39 ; s disease , treatment of stroke and / or osteoporosis . in a still further aspect of the present invention there is provided a method of prophylaxis or treatment of a condition associated with angiogenesis or accelerated or increased amounts of cell division , hypertrophic growth , or inflammation , said method including administering to a patient in need of such prophylaxis or treatment an effective amount of compounds of itpp , such as itpp - ca / na or prodrugs thereof , according to the present invention , as described herein . it should be understood that prophylaxis or treatment of said condition includes amelioration of said condition . by “ an effective amount ” as referred to in this specification , it is meant a therapeutically or prophylactically effective amount . such amounts can be readily determined by an appropriately skilled person , taking into account the condition to be treated , the route of administration and other relevant factors . such a person will readily be able to determine a suitable dose , mode and frequency of administration . “ individual ” as referred to in this application refers to any animal that may be in need of treatment for a given condition . “ individual ” includes humans , other primates , household pets , livestock , rodents , other mammals , and any other animal ( s ) that may typically be treated by a veterinarian . the compositions described above can be provided as physiologically acceptable formulations using known techniques , and these formulations can be administered by standard routes . in general , the combinations may be administered by the topical , oral , rectal , intraperitoneal or parenteral ( e . g ., intravenous , subcutaneous or intramuscular ) route . in addition , the combinations may be incorporated into polymers allowing for sustained release , the polymers being implanted in the vicinity of where delivery is desired , for example , at the site of a tumor , or into an a cavity or blood vessel that will lead to easy delivery to the place to be treated . the dosage of the composition will depend on the condition being treated , the particular derivative used , and other clinical factors such as weight and condition of the patient and the route of administration of the compound . however , for oral administration , a recommended dosage is in the range of 0 . 1 to 5 . 0 g / kg / day . a dosage for oral administration is in the range of 0 . 5 to 2 . 0 g / kg / day or alternatively , about 0 . 5 to about 1 . 5 g / kg / day . in an alternate embodiment , a dosage for oral administration is in the range of about 0 . 80 to 1 . 0 g / kg / day or alternatively , about between 0 . 9 to 1 . 1 g / kg / day . the formulations in accordance with the present invention can be administered in the form of tablet , a capsule , a lozenge , a cachet , a solution , a suspension , an emulsion , a powder , an aerosol , a suppository , a spray , a pastille , an ointment , a cream , a paste , a foam , a gel , a tampon , a pessary , a granule , a bolus , a mouthwash , or a transdermal patch . the formulations include those suitable for oral , rectal , nasal , inhalation , topical ( including dermal , transdermal , buccal and sublingual ), vaginal , parenteral ( including subcutaneous , intramuscular , intravenous , intraperitoneal , intradermal , intraocular , intratracheal , and epidural ) or inhalation administration . the formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques . such techniques include the step of bringing into association the active ingredient and a pharmaceutical carrier ( s ) or excipient ( s ). in general , the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both , and then , if necessary , shaping the product . formulations contemplated as part of the present invention include nanoparticles formulations made by methods disclosed in u . s . patent application ser . no . 10 / 392 , 403 ( publication no . 2004 / 0033267 ) which is hereby incorporated by reference in its entirety . by forming nanoparticles , the compositions disclosed herein are shown to have increased bioavailability . preferably , the particles of the compounds of the present invention have an effective average particle size of less than about 2 microns , less than about 1900 nm , less than about 1800 nm , less than about 1700 nm , less than about 1600 nm , less than about 1500 nm , less than about 1400 nm , less than about 1300 nm , less than about 1200 nm , less than about 1100 nm , less than about 1000 nm , less than about 900 nm , less than about 800 nm , less than about 700 nm , less than about 600 nm , less than about 500 nm , less than about 400 nm , less than about 300 nm , less than about 250 nm , less than about 200 nm , less than about 150 nm , less than about 100 nm , less than about 75 nm , or less than about 50 nm , as measured by light - scattering methods , microscopy , or other appropriate methods well known to those of ordinary skill in the art . formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules , cachets or tablets each containing a predetermined amount of the active ingredient ; as a powder or granules ; as a solution or a suspension in an aqueous liquid or a non - aqueous liquid ; or as an oil - in - water liquid emulsion or a water - in - oil emulsion , etc . a tablet may be made by compression or molding , optionally with one or more accessory ingredients . compressed tablets may be prepared by compressing , in a suitable machine , the active ingredient in a free - flowing form such as a powder or granules , optionally mixed with a binder , lubricant , inert diluent , preservative , 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 may optionally be coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein . formulations suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis , usually sucrose and acacia or tragacanth ; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin , or sucrose and acacia ; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier . formulations suitable for topical administration to the skin may be presented as ointments , creams , gels and pastes comprising the ingredient to be administered in a pharmaceutically acceptable carrier . a preferred topical delivery system is a transdermal patch containing the ingredient to be administered . formulations for rectal administration may be presented as a suppository with a suitable base comprising , for example , cocoa butter and / or a salicylate . formulations suitable for nasal administration , wherein the carrier is a solid , include a coarse powder having a particle size , for example , in the range of 20 to 500 microns which is administered in the manner in which snuff is taken ; i . e ., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose . suitable formulations , wherein the carrier is a liquid , for administration , as for example , a nasal spray or as nasal drops , include aqueous or oily solutions of the active ingredient . formulations suitable for vaginal administration may be presented as pessaries , tampons , creams , gels , pastes , foams or spray formulations containing , in addition to the active ingredient , ingredients such as carriers as are known in the art to be appropriate . formulation suitable for inhalation may be presented as mists , dusts , powders or spray formulations containing , in addition to the active ingredient , ingredients such as carriers as are known in the art to be appropriate . formulations suitable for parenteral administration include aqueous and non - aqueous sterile injection solutions which may contain anti - oxidants , buffers , bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient ; and aqueous and non - aqueous sterile suspensions which may include suspending agents and thickening agents . the formulations may be presented in unit - dose or multi - dose containers , for example , sealed ampules and vials , and may be stored in freeze - dried ( lyophilized ) conditions requiring only the addition of a sterile liquid carrier , for example , water for injections , immediately prior to use . extemporaneous injection solutions and suspensions may be prepared from sterile powders , granules and tablets of the kinds previously described . preferred unit dosage formulations are those containing a daily dose or unit , daily sub - dose , as herein above recited , or an appropriate fraction thereof , of the administered ingredient . it should be understood that in addition to the ingredients , particularly those mentioned above , the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question , for example , those suitable for oral administration may include flavoring agents or other agents to make the formulation more palatable and more easily swallowed . for the in vitro experiments , itpp was dissolved in deionized water , ph was adjusted at ph 7 and , for incubation with whole blood , the osmolarity of the itpp solutions was adjusted with glucose to 270 - 297 mosm . mixtures of hemoglobin and itpp were measured with a hemox analyzer ( pd marketing , london ) immediately after mixing . red blood cells were incubated with itpp for 1 hour at 37 ° c . following incubation , the cells were washed 3 times with bis - tris - buffer ( ph = 7 . 0 ) and then used for p 50 measurement . in experiments conducted in vivo in which itpp was administered orally , a significant shift of the p 50 value of circulating rbcs was observed . itpp was dissolved in drinking water at a 20 g / l - concentration ( 0 . 27 mm , ph ˜ 7 . 0 .) and offered for drinking ad libitum . the following examples illustrate but do not limit the invention . thus , the examples are presented with the understanding that modifications may be made and still be within the spirit and scope of the invention . when myo - inositol tripyrophosphate - sodium salt ( itpp - na ) is mixed with cacl 2 , a mixture of itpp - na ( myo - inositol tripyrophosphate - sodium salt ) and itpp - ca ( myo - inositol tripyrophosphate - calcium salt ) is obtained . this mixture , when added to free hemoglobin or to whole blood induces a p 50 shift of 170 % and 25 %, respectively as shown in tables 2 and 3 below . please see the results in tables 2 and 3 for compound 15 . the compounds in tables 2 and 3 are as follows : 4 is the pyridinium salt of itpp , 5 is the sodium salt of itpp ( i . e ., itpp - na ), 7 is the n , n - dimethylcyclohexyl ammonium salt of itpp , 11 is the cycloheptyl ammonium salt of itpp , 12 is the cyclooctyl ammonium salt of itpp , 13 is the piperazinium salt of itpp , 14 is the tripiperazinium salt of itpp , and 15 is the calcium salt of itpp ( i . e ., itpp - ca ). in tables 2 and 3 , the effectiveness of all of the salts of itpp regarding their ability to act as allosteric effectors of hemoglobin can be seen . the sodium salt and the calcium salt of itpp appear to be the best allosteric effectors for both free hemoglobin ( table 2 ) and in whole blood ( table 3 ). however , pigs injected intravenously with itpp - na at a rate of 1 g / kg weight resulted in a number of adverse side effects . the intravenous injection of pigs with itpp - na resulted in flushing , an increase in the heart rate , and a decrease in the ca 2 + plasma concentration from 2 . 38 mmol / l to 1 . 76 mmol / l . administration of the mixture of the sodium and calcium salt of itpp , at the same dosage did not induce any of the cited effects and the ca 2 + plasma concentration stayed unchanged at 2 . 38 mmol / l . fig1 shows the ability of itpp - ca / na loaded red blood cells suppression of hif - 1 induction , vegf and angiogenesis of hypoxic endothelial cells in vitro . fig2 demonstrates the potential of itpp - ca / na as a radiation sensitizer and as an angiogenesis inhibitor in pancreatic and rectal cancers . h = human ; m = murine ; p = porcine free hb . concentration of the compound solution was 60 mm ; means of p 50 shifts in % are shown . sd = standard deviation . compounds 4 , 7 , 11 , 12 , 14 and 15 : three p 50 values each were used for the calculation of means ; compound 5 : with human blood : five values , murine blood : ten values and porcine blood ; three values were used for the calculation of the means of p 50 shifts in %. itpp , when administered orally , intravenously , or intraperitoneally , inhibits angiogenesis in growing tumors by enhancing the po 2 in the forming tumors . thirty ( 30 ) c57bl / 6 mice received 20 g / l of itpp orally until the p 50 value showed a shift of at least 20 % above the control value . thereafter , all animals received 1 × 10 6 lewis lung carcinoma ( llc ) cells , injected in the dorsal cavity . at different time points , the vegf mrna were assayed by rt - pcr in the tumors growing in both groups of mice . tumor tissue samples were ground in a ripa lysis buffer ( 1 % nonidet p - 40 detergent , 50 mm tris ph 8 . 0 , 137 mm nacl , 10 % glycerol ) supplemented with protease inhibitor cocktail ( roche , reinach , switzerland ). after centrifugation for 10 minutes at 4 ° c . and 12 , 000 g , protein concentrations of tissue extracts were determined according to the bradford method . detergent soluble protein samples ( 10 mg ) were separated by size on a sds - page in 10 % acrylamide gels and transferred to nitrocellulose membrane ( protran ba 85 , schleicher and schuell , dassel , germany ). membranes were blocked for 3 hours at room temperature in 10 % skim milk in tris buffer saline containing 0 . 1 % tween , before an overnight incubation at 4 ° c . with rabbit polyclonal antibodies recognizing human , mouse and rat vascular endothelial growth factor ( vegf a - 20 , sc - 152 , santa cruz biotechnology , santa cruz , calif .) at a dilution of 1 : 200 . membranes were then probed for primary antibody with anti - rabbit ( 1 : 16 , 000 ) peroxidase conjugates ( sigma - aldrich , l &# 39 ; isle d &# 39 ; abeau chesnes , france ) for 60 minutes at room temperature . the resulting complexes were visualized by enhanced chemiluminescence autoradiography ( amersham pharma biotech , orsay , france ). there was a difference in the level of mrna of the vegf gene in both groups . fig3 shows an agarose gel indicating the vegf mrna concentrations in tumors from control and itpp drinking animals . the rt - pcr agarose gel assay of vegf mrnas from tumor tissue taken from 2 mice each on day 15 after inoculation of llc cells ( track 1 : controls , track 2 : itpp treated animals ) and day 30 after inoculation ( track 3 : control animals , track 4 : itpp treated animals ). fig4 shows the western blot assay of the expressed vegf in tumors of control and itpp - treated llc tumor - bearing animals . quantification of the gel assays indicated a reduction by a factor of 10 , 000 of the amount of vegf mrnas detected in the tumors of animals having received itpp , at day 9 and then , while differences remain between treated and untreated animals , they tend to decrease . this indicates that itpp taken up by circulating red blood cells significantly increases tumor po 2 . 1 . myo - inositol hexakisphosphate dodecasodium salt ( product number : p0109 , sigma ). 2 . dicyclohexylcarbodiimide ( product number : d80002 , aldrich ). 3 . triethylamine ( product number : 15791 , acros ) 4 . dowex 50wx8 hydrogen form ( product number : 217506 , aldrich ). 5 . ca ( oh ) 2 ( product number : 239232 , aldrich ). 6 . naoh ( product number : 1040017 , sds ). 7 . acetonitrile ( product number : 34851 , aldrich ). 8 . deionized water . the following synthesis scheme is shown in fig5 . dowex 50wx8 - 200 ion exchange resin ( 800 g ) was washed with water until the elute was colorless . myo - inositol hexakisphosphate dodecasodium salt ( note - 1 ) ( 100 g , 0 . 108 mol , 1 . 0 eq ) was added portionwise ( 10 g / portion in about 45 minutes ) to 500 ml of water . each portion was dissolved with stirring at room temperature ( 23 ° c .) before the next portion was added . this solution was then passed through the column containing the above washed dowex 50wx8 - 200 ion exchange resin and eluted with water ( 4 × 200 ml ) to obtain the free phytic acid ( note - 2 ). to the combined acidic fractions , triethylamine ( 400 ml , 2 . 87 mol , 26 . 5 eq , about twice the theoretical quantity ) was added over 1 to 2 minutes at room temperature ( 23 ° c .) and the mixture was stirred vigorously for 15 minutes ( note - 3 ). then the solvent was evaporated on a rotary evaporator ( 60 ° c ., 68 - 22 mbar ) ( note - 4 ) and the residue was dried under high vacuum for 1 hr at room temperature ( 23 ° c .) to give the hexatriethylammonium myo - inositol hexakisphosphate ( note - 5 ). to this hexatriethylammonium myo - inositol hexakisphosphate dissolved in water ( 800 ml ), dicyclohexylcarbodiimide ( 142 g , 0 . 68 mol , 6 . 3 eq ) dissolved in acetonitrile ( 1600 ml ) was added at once and the mixture was refluxed for 12 h ( note - 6 ). one more equivalent of dicyclohexylcarbodiimide ( 22 g , 0 . 108 mol , 1 . 0 eq ) dissolved in acetonitrile ( 40 ml ) was added and refluxed for further 6 h ( note - 7 ). the mixture was cooled to room temperature ( 23 ° c .) and the dicyclohexylurea formed was filtered through a sintered funnel ( note - 8 ) and washed with water ( 3 × 200 ml ). the filtrate was evaporated on a rotary evaporator ( 60 ° c ., 68 - 22 mbar ) and dried under high vacuum at room temperature ( 23 ° c .) ( note - 9 ). the resulting sticky syrupy residue was redissolved in 400 ml of water to remove all dicyclohexylurea that had remained dissolved in acetonitrile , filtered through a sintered funnel ( note - 8 ), and washed with water ( 2 × 100 ml ). the filtrate was evaporated on a rotary evaporator ( 60 ° c ., 68 - 22 mbar ) and dried under high vacuum at room temperature ( 23 ° c .). the resulting residue was redissolved in 200 ml of water to remove any further dicyclohexylurea that had remained dissolved in solution , filtered through a sintered funnel ( note - 8 ), and washed with water ( 2 × 100 ml ). the filtrate was evaporated on a rotary evaporator ( 60 ° c ., 68 - 22 mbar ) and dried under high vacuum at room temperature ( 23 ° c .) affording hexatriethylammonium myo - inositol 1 , 6 : 2 , 3 : 4 , 5 trispyrophosphate ( note - 10 ). this hexatriethylammonium myo - inositol 1 , 6 : 2 , 3 : 4 , 5 trispyrophosphate salt was dissolved in 400 ml of water , passed through a column ( note - 11 ) containing prewashed dowex 50wx8 - 200 ( 400 g ) ion exchange resin and eluted with water ( 4 × 100 ml ) ( note - 12 ). to the combined acidic fractions was immediately added solid ca ( oh ) 2 ( 5 . 56 g , 0 . 075 mol , 1 . 0 eq ) followed by addition of a naoh solution [( 12 . 0 g , 0 . 300 mol , 4 . 0 eq ) in 25 ml of water )] at room temperature ( 23 ° c .) ( note - 13 ). then the ph of the reaction mixture was carefully adjusted to around 6 . 99 with a solution of 1 : 4 ca ( oh ) 2 : naoh ( 1 . 5 g of ca ( oh ) 2 and 3 . 23 g of naoh in 1500 ml of water , ˜ 1385 ml brought the ph to ˜ 6 . 99 ) ( note - 14 ). finally , the solvent was evaporated on a rotary evaporator ( 60 ° c ., 68 - 22 mbar ) and dried under high vacuum at room temperature ( 23 ° c .) to yield the monocalcium tetrasodium myo - inositol 1 , 6 : 2 , 3 : 4 , 5 trispyrophosphate , itpp ca4na ( 77 . 2 g , 97 %) as a white solid . the compound obtained has been characterized by proton and phosphorous - 31 nmr spectroscopy , mass spectroscopy , elemental analysis , cation determination by atomic absorption and water content . it contains less than 2 % other phosphorous compounds . elemental analysis ( icp atomic absorption ): p 20 %; ca 4 . 2 %; na 10 . 3 % ( calc . : p 25 . 4 %; ca 5 . 5 %; na 12 . 6 %). water content : about 18 - 23 % depending on drying conditions . note - 1 . purity checked in - house by 1 h and 31 p nmr (& gt ; 98 %), as well as hplc , elemental analysis and atomic absorption for cation determination . note - 2 . collect the elutes which are acidic ( ph paper ). when all phytic acid is eluted , the elute becomes neutral . note - 4 . lower temperature can be used if evaporation can be achieved with the equipment available . note - 5 . purity and characterization was checked by 1 h and 31 p nmr . note - 6 . reflux temperature was about 80 ° c . heated with a mantle . note - 7 . after 12 h , more than 98 % product has been formed . addition of more dicyclohexylcarbodiimide led to & gt ; 99 % product formation . note - 8 . the porosity of the sintered funnel used was 4 and this will effectively filter off the dicyclohexylurea byproduct . note - 9 . thorough drying is necessary in order to be able to remove all the remaining dicyclohexylurea byproduct . note - 10 . characterized by 1 h and 31 p nmr , purity & gt ; 99 %. the column frit porosity was 1 . the diameter of the column was 8 cm and the length of the dowex bed was 9 . 5 cm . the solution was eluted first in 15 minutes without any pressure and then the washings under some pressure were eluted within 5 minutes . the column frit porosity was 2 . the diameter of the column was 6 cm and the length of the dowex bed was 16 . 5 cm . the solution was eluted first in 45 minutes without any pressure and then the washings under some pressure were eluted within 5 - 10 minutes . a ) a column frit porosity of 1 . b ) a dowex column with a height / diameter ratio of 1 . 5 - 2 . 0 , so that the elution time be less than 30 minutes . if the elution is too slow then flush with some pressure . note - 12 . caution : as the myo - inositol 1 , 6 : 2 , 3 : 4 , 5 trispyrophosphate free acid may hydrolyse after standing for a long time at low ph (& lt ; 1 . 0 ), the ph should be adjusted quickly to about 3 - 4 in order to avoid any such hydrolysis . check then for absence of triethylamine signals in 1 h nmr ( 4 - 1 ppm ) and for absence of phosphorous signals around 2 - 4 ppm . in the unlikely case that the 1 h nmr shows the presence of triethylamine , the whole solution has to be passed again over a fresh dowex - h + in order to remove it . it is very important that there be no triethylamine left , as it would remain in the final material . note - 13 . it is very important to add first solid ca ( oh ) 2 and make sure that it is completely dissolved . after addition of the naoh solution , the ph of the reaction mixture was ˜ 1 . 6 . the total amount of ca ( oh ) 2 : naoh required to neutralize the reaction mixture to ph ˜ 6 . 9 was ˜ 6 . 9 : 14 . 9 g , respectively . in order to minimize the amount of 1 : 4 ca ( oh ) 2 : naoh solution required at the end ( and reduce the final volume ), add initially 6 . 3 g of solid ca ( oh ) 2 followed by 13 . 6 g of naoh ( in 25 ml of water ). thereafter , the amount of 1 : 4 ca ( oh ) 2 : naoh solution required to bring the ph to 6 . 9 - 7 . 0 will be significantly reduced . note - 14 . the 1 : 4 ca ( oh ) 2 : naoh solution should be freshly prepared and well closed ; otherwise , co 2 from the atmosphere will be absorbed and insoluble materials will be formed . adjust the ph of the solution close to 7 and do not go beyond 7 . itpp - ca4na acts as a powerful effector of hemoglobin shifting the oxygen fixation curves to the right , with respect to the natural effector bisphosphoglycerate . the p50 values increase with concentration as show in fig6 and 7 . in fig6 , hemoglobin is incubated ( at concentrations up to 100 mm final ) with itpp ca4na , for 1 hour at 37 ° c . and measured by tcs - hemox analyzer for p50 shifts . in fig7 , whole human blood was incubated ( at concentrations up to 120 mm final ) with itpp ca4na , for 1 hour 37 ° c . and measured by tcs - hemox analyzer for p50 shifts . final formulation will contain sodium salt of inositol tripyrophosphate ( itpp - na ) at a concentration of 105 mg / ml and calcium chloride dihydrate at a concentration of 15 . 7 mg / ml , corresponding to a ca : na molar ratio of 0 . 75 to yield a monocalcium tetrasodium inositol tripryrophosphate solution . the monocalcium tetrasodium ittp solution will be prepared by adding a measured volume of 37 . 5 % stock solution of itpp - na to a measured volume of sterile water for injection , followed by slow addition of a measured volume of 10 % cacl 2 . 2h 2 o solution , with stirring . while , the present example utilizes cacl 2 as the source of calcium , one of ordinary skill in the art would readily recognize that other sources of calcium may be used . examples of suitable sources of calcium for use in the present invention include , but are not limited to , calcium carbonate , calcium acetate , calcium oxide , calcium arsenate , calcium carbide , calcium citrate , calcium gluconate , calcium hypochlorite , calcium cyclamate , calcium permanganate , calcium phosphate , calcium stearate , calcium sulfate , calcium tartrate , calcium glubionate , calcium gluceptate , calcium pyrophosphate , and calcium tetrafluoroborate . for every 100 ml of stock solution required , dissolve 37 . 5 grams of itpp - na ( on a pure weight basis , corrected for water content and impurities ) in 75 ml of sterile water for injection . adjust the ph to 7 . 5 using 1n naoh . bring final volume to 100 with additional sterile water for injection . for every 100 ml of stock solution required , dissolve 10 grams of cacl 2 . 2h 2 o in sterile water for injection and bring to a final total volume of 100 ml . for every 100 ml of monocalcium tetrasodium itpp solution required , add 56 . 2 ml of sterile water for injection to an appropriate mixing vessel . add 28 . 1 ml of 37 . 5 % itpp - na stock solution with constant mixing . follow with the slow addition of 15 . 7 ml of the 10 % cacl 2 stock solution , with constant mixing . store monocalcium tetrasodium itpp solution at approximately 5 ° c . until use , and use the test article solution within 24 to 72 hours of preparation . ittp - na sterile solution for injection will be supplied in single use , 10 ml vials containing 375 mg / ml of ittp - na ( on a pure weight basis ). 10 % cacl 2 injection , usp , and sterile water for injection will be purchased from existing manufacturers . for every 100 ml of dosing solution required , 56 . 2 ml of sterile water for injection will be added to an infusion bag . this will be followed by addition of 28 . 1 ml of itpp - na sterile solution for injection with constant mixing . then 15 . 7 ml of the 10 % cacl 2 injection , usp , will be added with constant mixing . the dosing solution will be administered through an in - line 0 . 2 micron filter . if not to be used immediately , the final dosing solution will be stored at approximately 5 ° c . until use . it should be used within 24 to 72 hours of preparation having described the invention with reference to particular compositions , method for detection , and source of activity , and proposals of effectiveness , and the like , it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms , and that modifications can be made without departing from the scope or spirit of the invention , as defined by the appended claims . it is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims . it should be understood that any of the above described one or more elements from any embodiment can be combined with any one or more element in any other embodiment . moreover , when a range is mentioned , it should be understood that it is contemplated that any real number that falls within the range is a contemplated end point . for example , if a range of 0 . 9 and 1 . 1 g / kg is given , it is contemplated that any real number value that falls within that range ( for example , 0 . 954 to 1 . 052 g / kg ) is contemplated as a subgenus range of the invention , even if those values are not explicitly mentioned . all references referred to herein are incorporated by reference in their entireties . finally , the above description is not to be construed to limit the invention but the invention should rather be defined by the below claims . 1 . fylaktakidou , k ., lehn , j .- m ., greferath , r ., and nicolau , c . 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