Patent Application: US-94343897-A

Abstract:
fructose - 1 , 6 - diphosphate , a sugar - phosphate compound , can be useful in treating asthma , when administered as an inhalable drug , either by itself or as a component of a mixed formulation . on a cellular level , inhalable fdp appears to offer at least four beneficial effects for asthma sufferers : it reduces histamine release by activated mast cells ; it suppresses production of oxygen free radicals by polymorphonuclear cells ; it helps suppress the activation and proliferation of t - lymphocytes ; and , it helps reduce the expression of interleukin compounds by t - lymphocytes . all four effects have been measured and shown to occur in animal and / or human tests , and these effects render fdp likely to help reduce and retard the progressive worsening of asthma that occurs in many sufferers . in addition , when tested in inhalable form on humans , fdp was shown to increase bronchial flow rates . all of these effects are beneficial , and can help asthma patients treated with fdp use asthma - control drugs which impose less stress on the user than more potent , aggressive asthma - control drugs .

Description:
this invention uses fructose - 1 , 6 - diphosphate ( fdp ), a naturally occurring sugar - phosphate compound in which phosphate groups are bonded to the # 1 and # 6 carbon atoms of a fructose molecule . some scientists refer to fdp as fructose - 1 , 6 - biphosphate , or fructose - 1 , 6 - bisphosphate . the 1 , 6 - isomer of fructose diphosphate is the only isomer of interest herein . other isomers ( such as fructose - 2 , 6 - diphosphate ) exist in nature , but they are not relevant herein , and they are excluded from any references herein to fdp or fructose diphosphate . inside cells , fructose - 1 , 6 - diphosphate is created and then quickly consumed as an intermediate in the series of reactions that make up glycolysis . glycolysis is a fundamental biological process in which cells use a series of enzymatic steps to break apart glucose , a 6 - carbon sugar molecule , to generate energy . glycolysis is discussed in detail in nearly any textbook on biochemistry , physiology , or cell biology ; see , e . g ., any edition of stryer &# 39 ; s or lehninger &# 39 ; s biochemistry , guyton &# 39 ; s medical physiology , or alberts et al , molecular biology of the cell . as a short - lived intermediate that is created and then quickly consumed , fdp normally is present in cells only at relatively low concentrations . it should be noted that a large number of potential medical uses have been proposed for fdp ; for example , it has been proposed and studied as a treatment for heart attack and stroke victims , as a preservative agent for organs that are being transplanted , and as an additive for blood cells that are being frozen and stored . us patents which contain such proposals include u . s . pat . nos . 4 , 546 , 095 ( markov 1985 ), 4 , 703 , 040 ( markov 1987 ), and 4 , 757 , 052 ( markov 1988 ). scientific articles that contain similar proposals or study data include markov et al 1980 , 1986 , and 1987 , brunswick et al 1982 , marchionni et al 1985 , granot et al 1985 , farias et al 1986 and 1989 , grandi et al 1988 , zhang et al 1988 , cacioli et al 1988 , and numerous other articles . however , despite all of these published articles and patents , which stretch back at least to 1980 , a high degree of skepticism and reluctance still exists regarding fdp use to treat ischemia or hypoxia , due to a number of factors such as : ( 1 ) assumptions that fdp , a diphosphate with a strong negative charge , probably cannot enter cells in sufficient quantities to have significant effects on the intra - cellular metabolism ; ( 2 ) assumptions that fdp has a very short half - life in the blood , and will effectively disappear from the blood within a few minutes after injection or infusion ; ( 3 ) fears that administration of fdp to oxygen - starved cells would lead to substantial increases in lactic acid levels . this could be very harmful , since excess lactic acid can poison an enzyme called phosphofructokinase ( pfk ), which is the crucial rate - regulating enzyme in glycolysis ; ( 4 ) reports which state that fdp had no beneficial effects , in various studies . examples of these negative articles include eddy et al 1981 , pasque et al 1984 , tortosa et al 1993 , and angelos et al 1993 ; ( 5 ) the high level of difficulty in handling and storing , for any length of time , a compound that will readily and spontaneously hydrolyze , losing one of its phosphate groups to form fructose monophosphate , which is useless . for these and other reasons , despite all of the numerous proposals that fdp might somehow be useful in medicine , fdp simply is not being used or prescribed by any practicing physicians , for any medical conditions . the only known exceptions are ( 1 ) a few small and limited clinical trials , at research centers ; and ( 2 ) very limited use in a few countries ( such as china ) of semi - sterile preparations that cannot meet the government approval requirements for human drugs in the united states and most other industrialized nations . the foregoing is general information on fdp . it should be noted and emphasized that , to the best of the applicant &# 39 ; s knowledge and belief , no prior articles or publications have ever taught or even suggested that fdp would be a useful treatment for asthma . the discovery of that new utility is , to the best of the applicant &# 39 ; s knowledge and belief , entirely new and original . its potential for use in treating asthma was not even suspected by the inventor until recently , even though the inventor ( a cardiologist ) has been working with fdp , for other medical uses , for almost 20 years . the examples below describe several different types of tests , all of which support and help confirm the belief that fdp , when administered in inhalant form directly into the lungs of an asthma sufferer , is highly useful for treating asthma attacks . these tests are briefly summarized in the following paragraphs , and detailed protocols and results are provided in the examples , tables , and figures . example 1 describes a set of tests the release of histamine by &# 34 ; mast cells &# 34 ;, which are a certain type of white blood cells . histamine release by mast cells plays a major role in a variety of inflammatory , edematous , and anaphylactic shock reactions , and histamine has been shown to play a contributory and aggravating role in asthma attacks . microvascular leakage is also an important feature of asthma , and may be triggered by various inflammatory mediators . if excess histamine is released in or around the lungs , it can cause edema , plasma exudation into the airways , and release of kinins and complement fragments that can inhibit membrane - bonded na + -- k + atpase ; histamine has been shown to inhibit na + -- k + atpase , in gastric mucosa , to the same degree as ouabain . by contrast , fdp has been shown to help restore the activity of na + -- k + atpase in vitro , in cardiac tissue , by the same inventor herein , in tests which showed that fdp can help reduce the toxicity levels of oleander extracts . accordingly , fdp was tested to determine whether it could help reduce histamine release , under in vivo conditions . these tests used a substance known as compound 48 / 80 , which powerfully provokes the release of histamine , in test animals . in animals such as rats , if a controlled quantity of compound 48 / 80 is injected , the animal will die fairly quickly , due to various organ impairments that result from massive edema and anaphylactic shock . as described in example 1 , and as shown by the data in fig1 fdp was shown to be highly effective in protecting animals against the damaging effects of compound 48 / 80 . the system hemodynamics ( such as arterial blood pressures , and partial pressure of oxygen ( po 2 ) values ) were significantly better in the fdp - treated group than in the saline - treated control group . in rats injected with compound 48 / 80 and treated with saline , a significant increase in plasma k + was observed , indicating disruptions of the na + -- k + atpase system ; by contrast , plasma potassium levels in the fdp - treated group were much closer to normal levels , indicating effective protection of that system by the fdp . mortality rates were greatly reduced by fdp treatment ; the control group had a 100 % mortality rate , while the fdp - treated group had only 20 % mortality . all of these results , in example 1 , strongly indicate that fdp is effective in reducing histamine release in treated animals . this is a potentially very beneficial effect , in asthma sufferers . example 2 presents two different sets of tests , both of which show that fdp can help protect the functioning of a certain class of enzymes known as membrane - bound na + k + atpase enzymes , which are very important in certain aspects of cell metabolism . one set of cell culture tests used a type of toxic glycoside isolated from oleander plants , which are poisonous . these toxic glycosides can shut down the membrane - bound na + k + atpase enzyme system , and it was shown that fdp can protect that enzyme system from being shut down by those toxins . example 2 also describes in vivo tests which showed that when compound 48 / 80 was administered to intact animals , it caused a large increase in potassium ( k + ) levels in the blood plasma . this effect is caused by disruption of membrane - bound na + k + atpase enzymes , since those enzymes normally perform the function of pumping potassium ions into cells , thereby reducing the concentrations found in extracellular fluid . it was shown , in the in vivo tests , that fdp can substantially reduce that effect of compound 48 / 80 . this adds more evidence showing that fdp can help protect an important enzyme system from disruption . this is an important protective activity , since disruption of that enzyme system appears to be a significant aggravating feature in asthma attacks . examples 3 and 4 describe tests which showed that fdp can also help reduce the unwanted activation of t - type lymphocytes ( also referred to as t - cells ). t - cell activation generally refers to ( 1 ) proliferation of t - cells in an affected tissue area , and ( 2 ) release of certain intercellular mediators , called cytokines , which act as hormones that interact with other cell types . in biopsies from asthmatic patients , lymphocyte content is usually slightly increased ( compared to non - asthmatics ). a potentially more important difference is that the t - lymphocytes in asthmatics are usually more highly activated , as shown by expression of interleukin - 2 ( il - 2 ) receptors on the surfaces of the t - cells . analysis of cytokines in concentrated broncho - alveolar lavage ( bal ) obtained from asthmatic patients also reportedly demonstrate significant increases in various interleukines ( il - 5 , il - 2 , il - 1 , il - 6 , il - 8 ; see virchow et al 1995 ). those results suggest that the number of eosinophil cells and activated t - cells play an important role in asthma . it is believed that certain types of cytokines ( such as il - 5 and il - 2 ) released by t - cells react with receptors on the surfaces of other cells in the lungs , such as bronchial smooth muscle cells . these reactions trigger various cellular reactions which lead to constriction of the bronchial airways . accordingly , it is believed that treatment agents which can help reduce ( 1 ) the proliferation of t - cells in an affected region , and ( 2 ) the release of il - 2 , il - 5 , and other unwanted cytokines by t - cells in the lungs , can be a useful treatment for asthma sufferers . this is supported by test results showing that treatment with cyclosporin a can induce significant improvement in pulmonary function tests , in patients suffering from asthma ( e . g ., alexander et al 1992 ; fuguta et al 1995 ). however , cyclosporine treatment is not a promising treatment for most asthma sufferers because of its side effects , including organ toxicity and a general suppression of the immune system . accordingly , if fdp , administered either in inhalant form directly into the lungs , or in injectable form such as by intravenous infusion , can suppress t - cell activation in the bronchia and lungs without causing the unwanted complications and side effects caused by cyclosporine a , it would be greatly preferable to cyclosporine , as a treatment for asthma . the results described above indicate that it can , and that it offers a highly promising and useful agent for treating asthma . the ability of a compound such as fdp to help suppress t - cell activation is usually tested , in vitro , by stimulating t - cells with a mitogen compound known as concanavalin a ( cona ), and by testing potential treatment agents , to evaluate how well they can suppress the t - cells &# 39 ; response to the cona stimulation . when such tests were done using fdp , the results showed that fdp did indeed help to suppress both ( 1 ) the proliferation of t cells , as described in example 3 , and ( 2 ) the synthesis of cytokines by the t - cells , as described in example 4 . example 5 describes the ability of fdp to substantially reduce the severity of bronchial attacks in lab animals , which can be induced by having the animals inhale a compound called methacholine . example 6 describes the results of tests on human volunteers who do not suffer from asthma , showing that inhalation of fdp , in nebulized mist form , significantly increases bronchial and pulmonary capacity . when compared to baseline values , inhalation of nebulized fdp for 5 consecutive breaths caused a substantial increase in &# 34 ; peak expiratory flow rate &# 34 ; ( pefr ), which measures how rapidly someone can blow air out of his or her lungs , through the bronchial passageways . example 7 further indicates that in one of the test subjects who participated in the pefr tests , and who was suffering from a chronic cough , apparently due to allergic bronchitis , repeated inhalation of fdp over a period of 10 days led to a substantial improvement in the bronchitis , and to essentially complete resolution of the coughing problem . accordingly , although more testing will be required to fully evaluate these possibilities , it is believed that inhalable forms of fdp are likely to offer useful treatments , in at least some patients , for bronchitis , chronic coughing , and possibly other chronic pulmonary problems as well , such as emphysema . the main preferred method for using fdp to treat asthma involves inhalation of the fdp , in the form of an aerosol , so that the fdp should come in direct contact with mast cells and t - cells in the bronchial mucosa . such aerosol or nebulized formulations can use liquid carrier solutions if desired , made by methods such as dissolving fdp in a suitable aqueous carrier , such as a normal saline solution . if inhalable forms of fdp are used , dosages should generally be in the range of about 2 to about 100 mg / breath , in nebulized form , depending on the age and size of the patient , the severity of the attack , and other such factors . like most sugar compounds , fdp is highly soluble in water , and aqueous solutions containing nearly any desired concentration of fdp can be provided . preferred upper concentrations for inhalers will usually reflect an effort to balance two competing factors against each other : ( 1 ) a desire to package a substantial quantity of fdp in a small container , and ( 2 ) the need to avoid tissue irritation , which may be encountered as concentrations are increased . in general , it is anticipated that aqueous solutions containing about 5 % to about 40 % ( w / v ) fdp are suitable , and that solutions containing about 10 % to about 30 % are likely to be preferred . as described in example 6 , healthy human volunteers were tested to evaluate the effect of inhaled fdp on &# 34 ; forced expiratory flow rate &# 34 ; ( fefp ). these tests used a nebulizer connected to a bottle containing 10 % ( w / v ) fdp in water . each patient took 5 consecutive breaths from the nebulizer during these tests , and the total quantity of fdp inhaled by each patient averaged about 12 mg . one test subject , who inhaled higher dosages of fdp to evaluate possible side effects , inhaled 700 mg of the sodium salt of fdp using a 20 % w / v solution ; during a different test , the same volunteer inhaled 700 mg of sodium fdp , using a 10 % w / v solution . each such test involved a ten minute inhalation period . in both tests , no side effects were detected . it may also be possible to provide an inhalable form of fdp , for treating asthma , in a lyophilized or partially lyophilized , powdered or microcrystalline form . briefly , a method for partially lyophilizing fdp has been developed by cypros pharmaceutical corporation of carlsbad , calif . ( the assignee herein ), wherein the preparation contains a moisture content of about 10 % to about 25 %, by weight . this is a surprisingly high moisture content , in view of the fact that most other lyophilized drug preparations are dried to a point where the residual water content is about 2 % or less . this method and preparation is described in detail in u . s . pat . no . 5 , 731 , 291 , which has been examined and allowed , and which will issue in late 1997 or early 1998 . the contents of that application are hereby incorporated by reference . various methods are known in the art for generating microcrystalline powders which are loaded into inhalation devices ; for example , cakes or powders can be generated under sterile conditions , then ground into fine powders , and then loaded into inhaler cylinders , using clean - room facilities and procedures . alternately or additionally , various anti - crystallizing compounds are known , and such compounds , as well as agitator particles or devices which can pulverize any agglomerations or oversized clumps into fine powders , can be packaged into an inhaler device along with the fdp . if desired , any or all of these methods can be adapted for use in creating dried fdp powders for inhalation by asthma sufferers , as described herein . in addition , this type of lyophilized fdp powder might also be added to a powdered or other dry preparation containing one or more other drugs for treating asthma . any such dry preparation would need to be tested , to ensure that it does not irritate any tissue or cause other adverse effects . if desired , fdp can also be intravenously injected , to treat severe attacks of asthma . this method of administration can have two distinct benefits : systemic fdp can help the entire body cope with the hypoxic stresses that are caused by the patient &# 39 ; s inability to breathe in enough air , and any fdp which enters the lungs can help directly treat the various mechanisms that are aggravating the attack . if injectable fdp is used , a solution such as about 5 to about 30 % ( weight per volume , w / v ) fdp in aqueous solution ( distilled water , normal saline solution , etc .) can be administered , in any of several ways . for example , to achieve a relatively high blood concentration fairly quickly , a slow intravenous bolus can be administered over 5 to 15 minutes , in a dosage range such as about 75 to about 350 mg / kg . following this initial bolus , fdp can be administered via continuous infusion at a rate such as about 0 . 1 to about 1 . 0 mg / kg per minute , until the asthmatic crisis is resolved . alternatively , a slow iv bolus can be given every few hours such as every 6 hours , at dosages such as suggested by the above . it should also be noted that fdp , in either inhalable or injectable form , can be mixed , or otherwise administered in conjunction with , various other drugs that are useful for treating asthma . as one example , a liquid or crystalline preparation of fdp can be packaged in the same inhalant cylinder that contains a second asthma - treating drug ; alternately , an asthma sufferer who is struggling with an attack may alternate fdp inhalation with inhalation of a second drug . in general , oral administration of fdp is not likely to be effective , since the fdp will be substantially degraded by stomach acidity and other factors before it can enter the bloodstream . this invention also discloses articles of manufacture comprising inhaler cylinders or other inhalation devices which have been loaded with fdp in a form and in a dosage or concentration that is ready for inhalation by an asthma sufferer . preferably , such devices should include small inhaler cylinders that are small enough to fit into a conventional pocket or purse , so they can be carried anywhere by asthma sufferers with little or no inconvenience . such devices might hold up , for example , about 50 to 100 grams ( about 2 to 4 ounces ) of material . in addition , such inhalation devices should also include larger cylinders , which preferably can be refilled when empty , comparable to the oxygen - containing cylinders that are often carried by emphysema sufferers , for use in homes , clinics , and other such locations . such cylinders are conventionally made of aluminum , molded plastic or other suitable materials , and are manufactured with orifices that are suitable for accommodating inhaler nozzles which deliver a pre - determined dosage of material each time the nozzle is depressed or otherwise manipulated . personal - use inhalers are usually sold with such nozzles already attached . as noted above , the release of histamine by mast cells plays a significant role in aggravating asthma attacks . fdp was evaluated to determine whether it could help suppress histamine release by mast cells , in tests that use a compound called &# 34 ; 48 / 80 &# 34 ;, which is known to trigger the release of histamine by mast cells . this compound , if injected into an animal at an appropriate dosage , will trigger sufficient histamine release to send the animal into anaphylactic shock , which poses a lethal challenge . useful physiological measurements can be made during the test period , and if mortality is studied as an endpoint , an animal is deemed to survive if it lives for 48 hours after injection of the 48 / 80 compound . anesthetized male sprague - dawley rats ( n = 20 ) weighing 448 ± 7 . 4 gm were randomly assigned into 2 groups . animals in the fdp - treated group were injected intraperitoneally ( ip ) with a 10 % ( w / v ) solution of fdp in water , at a dosage of 2 . 5 gm / kg ( i . e ., 2 . 5 grams of fdp per kilogram of body weight ). animals in the control group ( n = 10 ) were injected with comparable volumes of normal saline , at 25 ml / kg . thirty minutes later , all rats were injected ip with compound 48 / 80 , at 5 mg / kg . during the test period , the arterial pressure in the fdp group was significantly higher than in the saline group ( p & lt ; 0 . 005 ) ( fig1 ). plasma potassium in the fdp group was lower ( p & lt ; 0 . 01 ) ( fig2 ). the average arterial partial pressure of oxygen ( po 2 ) in the fdp group was 121 ± 6 . 1 mm hg and the saline group 98 ± 9 . 2 mm hg . mortality in the saline ( control ) group was 100 %; by contrast , mortality in the fdp - treated group was only 20 % ( p & lt ; 0 . 001 ). the mean survival time for the rats treated with saline only ( controls ) was 32 . 7 ± 6 min after injection with the 48 / 80 compound ( time range : 20 to 70 min ). only two rats in the fdp group died ( at 10 and 50 min ). in summary , death as a result of compound 48 / 80 - induced shock appears to be due to hypotension , bronchospasm and negative inotropic effect caused by histamine . it is concluded that fdp is an effective mast cell inhibitor in vivo , and is effective in counteracting the mast cell histamine release induced by injection of compound 48 / 80 . various processes involved in asthma attacks can disrupt the proper functioning of membrane - bonded na + -- k + atpase enzymes , which are important in various aspects of cellular metabolism , including the ability of cells to sustain the proper concentration gradients of sodium and potassium ions across the cell membranes . in a research project which was not initially related to asthma , the applicant and his coworkers investigated the potential use fdp as an antidote against oleander poisoning . oleanders are a well - known flowering shrub , widely used as a landscaping plant . despite their widespread use , they are highly poisonous . a number of deaths have been attributed to it , usually involving the burning of oleander leaves and stems in cooking fires , or the use of oleander stems as spits on which meat was suspended while being roasted or barbecued . the poisonous toxins in oleander plants involve certain types of glycosides which can severely disrupt various gastrointestinal and cardiac functions . one of their main modes of activity is disruption of membrane - bonded na + -- k + atpase enzymes . accordingly , oleander toxins offer a model for testing the ability of candidate drugs to help protect na + -- k + atpase enzymes against attack and disruption by various toxins , and by other compounds which are involved in asthma attacks , such as histamine , certain types of kinins , and certain types of enzyme - disrupting complement fragments . oleander toxins were prepared by air - drying leaves from red - flowering nerium oleander plants for 4 days , pulverizing the leaves to a fine powder in a blender , and extracting the leaves in a 95 % ethyl alcohol solution for 10 days . after extraction , the tincture was filtered , and part of the alcohol solvent was removed by flash evaporation , to a volume where 1 gram of crude drug was equal to 1 ml of alcohol solution . the extract was stored as a stock solution at 0 ° c ., and periodically analyzed for alkaloid content by high pressure liquid chromatography , using oleandrin as an external standard . to carry out the in vitro tests described below , 1 to 2 μl aliquots of the stock solution were used , containing 0 . 1 top 2 mg of the toxins . ethanol , at the concentrations used , was also tested , and was shown to have no effect on membrane atpase activity . to prepare cardiac membrane fractions with membrane - bonded na + -- k + atpase enzymes , adult sprague - dawley rats , 250 ± 25 g body weight , were anesthetized with ketamine hcl ( 50 mg / kg body weight ) by intraperitoneal injection . whole hearts were dissected , washed in saline , quickly frozen in liquid nitrogen , and stored at - 80 ° c . until use . when ready for use , intact isolated rat hearts were transferred to ice - cold homogenizing medium ( 50 mm na 2 hpo 4 , 10 mm na 2 edta , and 25 mm naf , ph 7 . 4 ). the ventricular sections of the hearts were dissected , and blood was removed by squeezing the ventricles on absorbing paper . the membrane vesicles were prepared as described by jones et al 1979 . briefly , the minced tissue from each ventricle was homogenized in 10 ml of homogenizing medium using polytron at a power setting of 7 with 3 bursts of 15 sec each . an additional 5 ml of medium was added and the homogenate was sedimented twice for 20 min at 14 , 000 g . the supernatant from the second spin was then centrifuged at 45 , 000 g for 30 min . the resulting pellet was suspended in 10 ml of homogenizing medium containing 0 . 6 ml nacl ( ph 7 . 0 ). this suspension was centrifuged again at 45 , 000 g for 30 min . the pellet obtained after this centrifugation , consisting of crude membrane vesicles , was suspended in a storage buffer ( 30 mm histidine , 0 . 25 m sucrose , 10 mm edta , and 10 mm naf , ph 7 . 5 ) and stored at - 80 ° c . until use . protein content was determined using a biorad protein assay kit , with gamma globulin as a standard . the na + -- k + atpase activity levels for these sarcolemmal membrane preparations was determined calorimetrically by measuring how much inorganic phosphate ( pi ) was liberated when the atpase enzyme hydrolyzed atp to release phosphate groups . a 1 ml reaction mixture was used , and contained ( in final concentration ) 5 mm atp , 5 mm magnesium chloride ( mgcl 2 ), 100 mm nacl , 20 mm kcl , 135 mm imidazole / hcl buffer ( ph 7 . 5 ), and 50 g of enzyme protein . the total cationic ligand - stimulated atpase activity was measured with na + , k + and mg ++ present in the reaction mixture . mg ++ atpase activity was measured in the presence of 1 mm ouabain , a specific inhibitor of na + -- k + - atpase . thus , delineation of the ( na + -- k + )- activated component of total atpase was obtained by determining the difference between total atpase activity , and mg ++ atpase activity . the incubation was carried out at 37 ° c . for 30 min and the reaction was stopped with trichloroacetic acid at a final concentration of 5 %. samples then were assayed for inorganic phosphate . enzyme activity was expressed as micromoles of inorganic phosphate released from the atp substrate , per milligram of enzyme protein , per hour . the effects of oleander extract , in the absence of fdp ( controls ) or after pretreatment with 500 μm fdp , were assessed by preincubating the na + -- k + atpase enzyme preparations with various concentrations of oleander toxins for 5 minutes , then adding atp to supply the necessary substrate for enzymatic hydrolysis which released phosphate groups . the results , shown in table 1 , clearly show that fdp reduced disruption of the na + -- k + atpase enzyme system by the glycosidic toxins in oleander plants . all fdp treatment results are statistically significant at levels of more than 99 %. table 1______________________________________protection of na . sup .+ -- k . sup .+ atpase enzymes by fdpoleander extract no fdp with fdp______________________________________control ( 0 mg ) 21 . 58 ± 4 . 40 -- 0 . 1 mg 18 . 04 ± 1 . 12 -- 0 . 5 mg 14 . 48 ± 0 . 52 -- 1 . 0 mg 9 . 35 ± 0 . 55 19 . 32 ± 0 . 622 . 0 mg 4 . 85 ± 0 . 57 15 . 75 ± 0 . 39______________________________________ fdp does not react with glycosidic toxins from oleanders . instead , it acts by providing a useful energy substrate for cellular components . accordingly , the oleander toxin test offers a valid model which indicates that fdp can help protect membrane - bound na + -- k + atpase enzymes against at least some types of disruption . to further evaluate the effects of fdp on membrane - bound na + -- k + atpase enzymes , a set of in vivo tests were also carried out , using rats . in rats injected with compound 48 / 80 ( described in example 1 ) and not protected by fdp , potassium ion ( k + ) levels in blood plasma substantially increased . this offers strong evidence that the na + -- k + atpase system is being disrupted , since that enzyme normally pump k + ions from extracellular fluids , into cells . by contrast , in rats treated with fdp , k + levels in blood plasma remained within normal values ( p & lt ; 0 . 01 ), despite identical treatment with compound 48 / 80 . accordingly , both sets of observations ( the oleander toxin test results , and the compound 48 / 80 test results ), in combination , offer good evidence that fdp can help protect na + -- k + atpase system against disruption and inactivation . since disruption of that enzyme system is an aggravating factor in asthma attacks , these results provide evidence of a specific cellular mechanism by which fdp can help treat and reduce the severity of asthma attacks . it has long been known that in patients with allergic asthma , exposure to relevant allergen ( s ) induces degranulation of mast cells in the bronchi ; this mast cell degranulation leads to histamine release by the mast cells , and to subsequent bronchial constriction . in recent years , biopsies on fluids or tissues obtained from the bronchial airways of asthma sufferers also show infiltration by eosinophils and mononuclear lymphocyte cells . in such biopsies from asthmatic patients , lymphocyte numbers are slightly increased . more importantly , when compared to control subjects , t - lymphocytes in asthmatic patients are much more highly activated , as shown by substantially higher numbers of certain types of interleukin receptor proteins ( especially interleukin - 2 , abbreviated as il - 2 ) on the surfaces of the activated lymphocytes . in addition , analysis of cytokine concentrations in the fluids obtained by bronchio alveolar lavage ( bal ) of asthmatic patients have demonstrated substantial increases of certain interleukines , including il - 1 , il - 2 , il - 5 , il - 6 , and il - 8 . this has led researchers to conclude that the number of activated t - cells in bronchial tissues play an important role in allergic asthma ( see , e . g ., virchow et al 1995 ). accordingly , fdp was tested to determine whether it could help suppress the activation of such t - type lymphocytes , i . e ., to determine whether it could help reduce the number of interleukin receptors expressed by the cells . these tests used a mitogen compound called concanavalin a ( con a ), which is known to activate t - cells . for comparative purposes , fdp &# 39 ; s ability to reduce t - cell activation in these tests was compared against cyclosporine , a powerful immunosuppressant drug which selectively suppresses t - cell activation . lymphocyte cells were isolated either from human venous blood or rat spleen . the cells were suspended in dulbecco &# 39 ; s media . a manual cell count was done using 0 . 1 ml of trypan blue and 0 . 1 ml of the cell media mixture . the cell count was adjusted to 1 × 10 6 cells / ml and viability ranged between 92 to 98 %. tissue culture multi - well plates were used for incubation and in each well 0 . 1 ml of cell suspension was placed . to the fdp treatment wells ( in triplicate ) were added 0 . 1 ml of fdp from 10 % stock solution , in serial dilutions which included 1 : 10 , 1 : 100 , 1 : 250 , 1 : 500 ; 1 : 1000 , and 1 : 10000 ). cyclosporine treatment wells received 0 . 1 ml cyclosporine a , at various concentrations including 500 ng / ml , 50 ng / ml ; 5 ng / ml and 0 . 5 ng / ml . all wells except control wells received concanavalin a ( cona ) at 10 pg ( 0 . 01 ml ). the final volume for all wells was 0 . 21 ml . after incubation , the cells were pulsed in 5 % co 2 at 37 ° c . with 1 μci of ( 3 h )- thymidine ( i . e ., the cells were incubated for a controlled period of time with thymidine , one of the building blocks of dna , which contained tritium , a radioactive isotope of hydrogen ). the cells were harvested , and washed to remove any tritiated thymidine that had not been incorporated into new dna . the radioactivity levels of the treated cells were then measured , using a scintillation counter . the results , in table 2 , are reported as counts / min . as shown in table 2 , lymphocytes which had been activated by cona treatment synthesized high levels of new dna . however , treatment by either fdp or cyclosporine was able to block cona activation of the lymphocytes . table 2______________________________________inhibition of rat t - lymphocyte proliferationafter stimulation with con - a , by varying concentrations of fdptreatment activation index______________________________________controls 527 ± 139 ( no con - a or fdp ) 10 μg con - a ; no fdp 30718 ± 4316con - a plus 1 : 10 fdp 101 ± 16con - a plus 1 : 100 fdp 190 ± 42con - a plus 1 : 250 fdp 383 ± 170con - a plus 1 : 500 fdp 3592 ± 974con - a plus 1 : 1000 fdp 12288 ± 1880con - a plus 1 : 10 , 000 fdp 19046 ± 3237______________________________________ as shown in table 2 , fdp inhibited the cona - induced activation and proliferation of t - cells in a dose dependent manner , as evidenced by the much lower levels of labelled thymidine which were incorporated into the newly synthesized dna of the cells treated by both fdp and cona , compared to cells treated by cona alone . linear regression analysis of the data demonstrated a strong correlation between increasing fdp concentrations , and increased suppression of lymphocyte activation ( r = 0 . 886 , p & lt ; 0 . 02 ). as expected , cyclosporin a also inhibited t - cell activation and proliferation in these tests . however , as noted above , cyclosporin a is a powerful and dangerous immunosuppressant drug , and does not offer a safe and practical treatment for asthma sufferers . accordingly , these data indicate that fdp can help suppress the inflammatory process in asthma sufferers , by helping control and prevent activation of t - lymphocytes in bronchial mucosa . the applicant also investigated the expression of cytokines by t - lymphocytes . isolated lymphocytes were obtained from rats , and incubated with stimulation by cona treatment , as described above ; treated cells were pre - treated with fdp , as described above , while control cells were not treated with fdp . after 5 days of incubation , rna was extracted from the lymphocytes , using rna - stat60 . the integrity of rna samples was evaluated by use of agarose - formaldehyde gel electrophoresis , and its quantity was determined spectrophotometrically . the rna was transcribed into complementary dna ( cdna ), using a reverse transcriptase enzyme . 10 μl of solution containing 1 μg of total cellular rna was heated at 65 ° c . for 10 min , and then chilled on ice . to this solution was added 10 μl of a mixture containing 2 × reverse transcriptase buffer ( 100 mm tris - hcl , ph 8 . 3 , 150 mm kc1 , 6 mm mgcl 2 ), 1 μg oligo ( dt ) 12 - 18 , 100 μg / ml of acetylated bovine serum albumin , 0 . 5 mm mixed deoxynucleotides ( dntp ), 10 units of rnaasin , and 100 units of mmlv reverse transcriptase enzyme . the mixture was incubated for 45 min at 35 ° c ., then chilled on ice . the cdna sequences for three specific interleukins ( il - 1 , il - 2 , and il - 6 ) were then amplified , using polymerase chain reaction ( pcr ) techniques , using previously published sense and anti - sense primer sequences for rat il - 1 , rat il - 2 , and rat il - 6 . 10 μl of cdna was amplified by pcr in a total volume of 100 μl . the pcr mixture contained a final volume of 1 × pcr buffer ( 10 mm tris - hcl , ph 8 . 3 , 50 mm kcl , 1 . 5 mm mgcl 2 ), 50 mm dntp , 0 . 1 μm each of the 5 &# 39 ; and 3 &# 39 ; primers , and 2 . 5 units of thermophilus aquaticus dna polymerase ( purchased from promega ). the reaction mixture was amplified for 30 cycles in an automatic dna thermal cycler . each cycle had 3 steps : the double - helical strands were separated ( denatured ) at 94 ° c . for 45 seconds , the primers were allowed to anneal to the strands for 60 ° c . for 45 seconds , and any annealed primers were extended by the dna polymerase at 72 ° c . for 2 minutes . after 30 cycles , an additional primer extension step was performed at 72 ° c . for 7 minutes . 20 μl of each pcr reaction product was electrophoresed on a 1 . 8 % agarose gel in 0 . 5 × tris - borate - edta buffer . dna bands , visualized by ethidium bromide staining and ultraviolet transillumination , were photographed with positive / negative 665 - type film , and the photographic plates ( negatives ) were scanned by a densitometer . the results of these tests on rat lymphocytes indicated that fdp - treated cells showed no detectable levels of rna encoding the il - 2 protein , despite cona stimulation . by contrast , control cells which did not receive fdp treatment showed substantial levels of rna which encoded the il - 2 protein . in addition , the amounts of il - 2 released by human t - cells were also evaluated , using cona stimulation in the presence or absence of fdp . these tests used a commercially available human immunoassay kit ( inter test - 2x il - 2 elisa kit , sold by genzyme corp ., cambridge , mass .) which uses monoclonal antibodies that bind specifically to human il - 2 . the results , in table 3 , indicate that fdp , at all tested concentrations , abolished il - 2 production by the stimulated human t - lymphocytes . il - 2 concentrations are expressed in picograms per milliliter of media . even despite activation by cona , there was no detectable il - 2 in any of the fdp - treated human cell populations . table 3______________________________________fdp suppression of interleukin - 2 productionby human t - cells stimulated by con - acell treatment after 24 hours after 72 hours______________________________________media only nd ndmedia + cona ( 10 μg ) 916 ± 112 958 ± 62 . 9media + cona + fdp ( 1 : 100 ) nd ndmedia + cona + fdp ( 1 : 500 ) nd ndmedia + cona + fdp ( 1 : 1000 ) nd nd______________________________________ ( nd = not detectable by elisa assay ) example 5 : effects of fdp on acute experimental asthma induced in dogs by methacholine several different approaches have been used to study pulmonary gas exchanges during asthma - like attacks in lab animals . one such animal model involves bronchospasms which can be experimentally induced , in dogs , by inhalation of a drug called methacholine . to carry out these tests , healthy mongrel dogs weighing 19 to 22 kg were anesthetized with sodium pentobarbital , 30 mg / kg iv , and intubated with endotracheal tubes . initially , the ventilation was maintained with a harvard ventilator , which served also to deliver methacholine , with or without aerosolized fdp . subsequently , the animals were continuously ventilated prior to and following drug administration . arterial venous and pulmonary artery ( swan - ganz ) were inserted , and used to obtain blood samples , monitor vascular pressures and measure cardiac output using thermodilution method . arterial end pulmonary pressure , electrocardiogram , were constantly monitored on a dr - 8 multichannel recorder . arterial blood , hemoglobin , ph , po 2 , pco 2 , and potassium were measured with radiometer abl - 4 ( copenhagen ). upon completion of the surgical preparation , the dogs were allowed 30 min to stabilize hemodynamically . several arterial blood samples were obtained and ventilation was adjusted so that arterial blood gases and ph were within physiologic range . the bronchial challenge procedure entailed a 90 sec administration of aerosolized 0 . 5 % methacholine hydrochloride ( sigma ). in treatment animals , fdp ( 10 % w / v ) was administered for 3 minutes , in the same manner . aerosols were delivered via harvard respirator and microembulizer . blood gas measurements were made at 5 , 15 , and 30 minutes after drug inhalation was completed . bronchoprovocation was either with methacholine alone ( n = 7 ), or following pretreatment with fdp ( n = 7 ). all seven dogs treated with methacholine but not fdp suffered bronchospasm attacks . these attacks were accompanied by a significant decrease in blood pressure during methacholine administration , and there was also significant decrease in pao 2 ( mean and sem pao 2 values after 5 minutes were 47 ± 2 . 85 mm hg ). these reductions in pao 2 levels lasted for more than 30 minutes . hemodynamic responses were more highly varied , but all dogs treated with methacholine but not fdp suffered from various levels of hemodynamic disruption . by contrast , pretreatment with fdp resulted in smaller declines and disruptions in pao 2 values . mean pao 2 values after 5 minutes were 70 ± 5 . 62 mm hg , and by 30 minutes after methacholine administration , pao 2 values had returned to their normal baseline levels . these data are shown in table 4 . differences between fdp - treated and control dogs were compared by the two - tailed student &# 39 ; s unpaired ` t ` test , and probability levels lower than 5 % were considered to be significant . table 4______________________________________arterial blood oxygen partial pressure in dogs , before and after methacholine inhalationbaseline 5 min 15 min 30 min______________________________________controls 99 ± 5 . 74 47 ± 2 . 85 53 ± 3 . 86 62 ± 6 . 2 ( no fdp ) pre - treated 89 ± 1 . 74 69 ± 6 . 49 81 ± 3 . 36 89 ± 3 . 16with fdp ( p = 0 . 007 ) ( p & lt ; 0 . 001 ) ( p = 0 . 002 ) ______________________________________ in addition , five dogs were treated with fdp after ( but not before ) the administration of methylcholine . the pao 2 levels in these dogs dropped to 64 ± 4 . 26 mm hg , 72 ± 1 . 84 mm hg , and 80 ± 4 . 04 mm hg , when measured at 5 , 15 and 30 minutes ( respectively ) after fdp treatment . these pao 2 levels were substantially higher and better than the pao 2 levels of dogs that received only methylcholine ( p & lt ; 0 . 006 , p & lt ; 0 . 02 and p & lt ; 0 . 01 , respectively ). there were no consistent alterations in pco 2 . one animal was excluded from the data , since it developed a pulmonary embolism due to heart worms . example 6 : effects of fdp on lung function in human volunteers in five human volunteers ( 3 males , 2 females ) who were generally healthy and who did not suffer from asthma , a 10 % ( w / v ) solution of fdp in sterile water was inhaled for 5 consecutive breaths , using a standard multifit nebulizer ( misty ox , sold by the medical molding corp . of america , costa mesa , calif .) attached to a medical grade oxygen delivery system . when adjusted to deliver 56 - 68 l / min with a 5 - 6 l / min oxygen flow , the final oxygen content was 28 %. the output tube from the nebulizer was connected to a standard face mask , which was placed on the face of the subject . the bottle of the nebulizer contained 100 ml of the fdp solution . the fdp dose delivered in the mist , during the 5 consecutive breaths , was calculated to be about 12 mg . prior to the fdp inhalation , in each volunteer , 3 consecutive peak expiratory flow rate ( pefr ) measurements were made with the aid of an astech peak flow meter ( center laboratories , port washington , n . y . 11050 ). briefly , these tests involved having each volunteer take as deep a breath as possible , and then blow as hard as possible into the meter . this was done over a period of a couple of minutes , giving the volunteer some time to catch his breath before and after each effort . the mean of these three measurements was considered as the baseline pefr value for that person . baseline heart rates , systolic and diastolic blood pressures , and body temperatures were also measured . fdp inhalations ( 5 breaths in each set ) were then made , approximately 15 , 30 , 45 , 75 , 105 and 165 minutes after the baseline measurements had been made . on several occasions , fdp inhalations were also made , 2 and 5 minutes after the baseline measurements were taken . after each such inhalation , three different expirations were measured , all within a couple of minutes after the fdp inhalation had been completed . the various measurements made after fdp inhalation were compared with the baseline values , before fdp administration . the t - test was used to compare paired observations , and differences were considered significant only when the two - tailed p value was & lt ; 0 . 05 . the results showed that inhalation of 10 % solution of fdp in aerosolized form caused significant increases in pefr values , in all test subjects . the baseline pefr value ( before inhalation of fdp ) was 471 ± 29 . 6 l / min . following fdp administration , the mean pefr increased to 529 ± 35 . 6 l / min ( p & lt ; 0 . 005 ). in addition , heartbeat rates declined significantly ; baseline rates of 83 . 8 ± 3 . 76 beats / min dropped after fdp treatment to 79 . 6 ± 3 . 26 beats / min ( p & lt ; 0 . 025 ). other measurements also indicated that fdp inhalation did not elicit any changes in blood pressure or body temperature . there were fluctuations in the various measured pefr values , but such fluctuations appeared to be what one would expect in a random sample drawn from a general population of normal subjects . pefr values in the various different volunteers ranged from 353 to 565 l / min in baseline measurements , and from 378 to 645 l / min in post - treatment measurements . one of the volunteers , at the end of the initial study , inhaled a 20 % solution of fdp for 10 min on demand , using the same settings in the nebulizing delivery system . the calculated amount of fdp inhaled was 700 mg . after this inhalation session , his pefr increased from a baseline value of 616 l / min , to 667 l / min . in a subsequent test on a different day , using a 10 % fdp solution at a higher flow rate than for the earlier test , the same subject inhaled 750 mg of fdp over 10 minutes . a similar increase in pefr was observed . no side effects were detected during either test . on two occasions , the effects of albuterol sulfate inhalation solution ( 0 . 005 % w / v in 0 . 9 % nacl ) on pefr was evaluated , using identical procedures . the albuterol sulfate did not induce any significant change in pefr . these tests were all done under the supervision of a trained physician , with informed consent from all participants . in all of these tests , no side effects were noticed by any of the volunteers , other than the above - noted decreases in heart rates and increases in pulmonary ventilation rates . in the tests on human volunteers described in example 6 , one test subject had a chronic cough , which appeared to be secondary to allergic bronchitis . this subject participated in several studies over 10 days . prior to fdp inhalation , her baseline pefr was 390 l / min . this value increased with each and every fdp treatment . at 10 days after the first inhalation , the pefr of this subject had increased to 520 l / min ( p & lt ; 0 . 01 ). upon interrogation , she reported that her breathing was substantially improved , and her coughing had nearly disappeared . this observation indicates that fdp , when administered in this manner , can exert a cumulative improvement in pulmonary ventilation , and in at least some patients , offers a useful treatment for bronchitis and chronic coughing , it is also anticipated that fdp is likely to provide some relief to people who suffer from emphysema , and possibly from other chronic pulmonary problems as well . thus , there has been shown and described a new and useful method for asthma . although this invention has been exemplified for purposes of illustration and description by reference to certain specific embodiments , it will be apparent to those skilled in the art that various modifications , alterations , and equivalents of the illustrated examples are possible . any such changes which derive directly from the teachings herein , and which do not depart from the spirit and scope of the invention , are deemed to be covered by this invention . alexander , a . j ., et al , &# 34 ; cyclosporin a and corticoid - 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