Patent Application: US-201113212612-A

Abstract:
inositol - tripyrophosphate is an allosteric effector of hemoglobin due to its ability to cross the plasma membrane of red blood cells and deliver oxygen to solid tumors , by lowering the oxygen affinity of the hemoglobin of red blood cells . the present invention is directed to the use of inositol - tripyrophosphate to reduce hemoglobin &# 39 ; s affinity for oxygen in circulating red blood cells . the present invention is further directed to the use of inositol - tripyrophosphate to inhibit angiogenesis and enhance radiation sensitivity of hypoxic tumors . the present invention is further directed to the use of inositol - tripyrophosphate to enhance po 2 in hypoxic tumors .

Description:
compositions that are useful in accordance with the present invention include inositol - tripyrophosphate ( itpp ) ( fig1 ) and salts thereof like na , ca , nh 4 salts of itpp , which exhibits anti - angiogenic and anti - tumor properties , and are 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 . 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 not 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 radiosensitizer on the other . 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 ). 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 17th 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 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 or prodrugs thereof according to the present invention , together with a pharmaceutically acceptable carrier , diluent or excipient . the pharmaceutical composition may be used for the prophylaxis or treatment of conditions associated with angiogenesis or accelerated cell division or inflammation . 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 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 ” 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 . 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 . 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 preferred dosage for oral administration is in the range of 0 . 5 to 2 . 0 g / kg / day . an especially preferred dosage for oral administration is in the range of 0 . 80 to 1 . 0 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 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 pharmaceutical 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 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 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 . 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 . 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 (= 27 mm , ph ˜ 7 . 0 .) and offered for drinking ad libitum . the following examples illustrate but not limit the invention . thus , the examples are presented with the understanding that modifications may be made and still be within the spirit of the invention . induced right shift of the o 2 - hemoglobin dissociation curve ( odc ) in mice ( orally administered ) twelve ( 12 ) c57bl / 6 mice were fed the itpp - solution ( 20 g / l - concentration = 27 mm , ph ˜ 7 . 0 ) for 4 days ( up to 25 ml per 24 hrs ). three ( 3 ) control mice drank pure water , and four ( 4 ) control mice a solution of myo - inositol hexaphosphate ( ihp ) ( same concentration and ph as itpp ). blood was collected from all mice on day 0 ( before treatment started ), and on days 1 , 2 , 4 , 6 , 7 , 8 , 10 , 11 and 12 ( after treatment had started ), in order to measure p 50 values . itpp was neither rejected by the mice , nor harmful to the animals . oral application of itpp caused significant right shifts of p 50 ( up to 31 %) in mice . itpp , when orally administered at a concentration of 27 mm , causes a right shift of the p 50 value in murine circulating red blood cells ( fig2 ). there is a time lag of approximately 48 hrs before the maximum shift is attained . maximal p 50 shifts are reached between day 2 and day 4 , after beginning oral administration of itpp . after 12 days , p 50 values are back to control values , when ingestion is stopped on day 4 . there is a significant effect of itpp ingestion on the number of red blood cells . although not wishing to be bound by theory , it is believed that effect of itpp ingestion on the number of red blood cells ( down - regulation of erythropoiesis ) is due to the increased p 50 . hemolysis can be ruled out , as lysis of the red blood cells never occurred in vitro . the level of ions , such as sodium and potassium and calcium were normal after oral application of itpp in mice ( fig3 ). fig3 contains the mean values and sd for the serum concentration of sodium , potassium and calcium obtained on day 0 , 7 and 11 after start drinking itpp ( 4 mice ), ihp ( 3 mice ) or water ( 3 mice ). blood counts were measured from all mice , on day 0 , 7 and 11 . the number of red blood cells in mice having ingested itpp was reduced . there were no significant differences in the number of white blood cells ( e . g . granulocytes , macrophages etc .) in blood from mice in different groups . fig4 shows the rbc counts for mice with shifted odc as compared to controls . fig4 further shows the relation of p 50 shift [%] to number of erythrocytes / mm 3 in mice having received itpp . it appears , based upon preliminary data , that an inverse relationship exists between the number of red blood cells and shift of their p50 value . the basal value of the red blood cell count is restored , once δp 50 becomes 0 %, 12 days after ingestion of itpp . when itpp ( ph 7 , 200 μl ) was injected intraperitoneally in mice , the p 50 values of circulating red blood cells were shifted up to 23 %. fig5 demonstrates that itpp was well tolerated by mice , up to a concentration of 150 mm . the level of ions , such as sodium , potassium and calcium were normal after ip injection . six ( 6 ) mice were each injected intraperitoneally with 45 - 150 mm (= 0 . 17 - 0 . 88 g / kg body weight ) of itpp . means of % shift and standard deviation are shown in fig5 . the concentration dependence of the p 50 shifts induced by itpp is an additional indication that this compound crosses the plasma membrane of the red blood cells . itpp was also injected intravenously ( iv ) in piglets . a right shift of p 50 was observed , when the compound was injected at a 1 g / kg body weight dose . in order to check possible side effects of itpp the level of calcium in the serum of the injected piglet was determined . a strong drop in the ca 2 + concentration in the animal &# 39 ; s blood immediately after infusion indicated the possibility that itpp , with 3 dissociated phosphate groups binds ca 2 + , reducing thus its availability as free ion in the blood . one day after infusion the concentration of ca 2 + in the piglets &# 39 ; blood was restored to the normal value . the results are shown in table 1 . based upon this observation , a cacl 2 ( equimolar to itpp ) solution was injected with the itpp solution , so that the dissociated phosphate groups of itpp were saturated . none of the side effects observed previously occurred . the level of calcium remained constant and the p 50 shift was again approximately 20 % of the basal value . the level of the other ions sodium and potassium was unchanged after iv injection of itpp in piglets . effect of in vivo lowering of hemoglobin &# 39 ; s affinity for o 2 by itpp on intratumoral po 2 , angiogenesis and expression of vegf mrna itpp , when administered orally , intravenously , or intraperitoneally , inhibits angiogenesis in growing tumors by enhancing 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 . all animals received then 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 ( 10 minutes , 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 size separated by 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 . fig6 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 ). fig7 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 . 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 . the claims are meant to cover the claimed components and steps in any sequence that is effective to meet the objectives there intended , unless the context specifically indicates to the contrary . 1 . fylaktakidou , k ., lehn , j .- m ., greferath , r ., and nicolau , c . 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