Patent Application: US-53053808-A

Abstract:
the invention provides methods and materials for identifying agents for preventing and / or treating anthrax and similar diseases . embodiments provide strains and model systems for studying non - lethal and lethal exposure to anthrax and similar disease vectors . embodiments provide materials and methods for using the strains and model systems for differential profiling , such as proteomic profiling , such as differentiation phosphorylation profiling , to target identification and therapeutics discovery and development . embodiments provide pharmaceutically acceptable compositions , and methods for using them to prevent and / or treat anthrax and similar diseases comprising an agent that decreases the activity of caspase ¼ , such as yvad , and / or an agent that increases the phosphorylation of akt , such as ib - meca or cl - ib - meca , together with , in particular embodiments , an antibiotic , such as ciprofloxacin . kits comprising the same are provided as well , among other things .

Description:
preventing and treating diseases , such as anthrax , that cannot be adequately prevented or treated with currently available therapeutics will require not only new therapeutics that target the infectious agent but also new therapeutics that eliminate or mitigate pathogenic host responses to infection . the present invention provides , in embodiments , methods for the identification of novel therapeutic targets and novel therapies . the invention provides highly effective post - exposure agents and treatment strategies for preventing and / or treating microbial infections and diseases that target one or more host responses , rather than the infectious organism . in embodiments the treatments are mediated through specific pro - survival pathways . in embodiments the microbial infection is anthrax and the microbe is bacillus anthraces . in embodiments the therapeutic agents and methods have no direct anti - microbial effect and / or no direct effect on the action of microbial toxins . in embodiments the agents are one or more of a modulator of host cell inflammatory response and / or a mediator of host cell apoptopic response . in embodiments the agents are one or more of a caspase inhibitor and / or a a3ar agonist . 44 in embodiments the agents and / or therapies have a synergistic effect on post - exposure survival in combination with one or more anti - microbial agents . in embodiments in this regard the antibiotic dose is low . in embodiments the antibiotic is a member of the ciprofloxacin class or the tetracycline class of antibiotics . in embodiments the antibiotic is ciprofloxacin . embodiments of the invention provide systems biology methods for identifying novel therapeutic targets , novel therapeutics , and novel therapies . in embodiments the methods comprise system wide analysis of proteins under non - pathogenic and pathogenic conditions . in embodiments the methods comprise system wide analysis of signaling proteins under pathogenic and non - pathogenic conditions . in embodiments the methods comprise system wide analysis of post - translation modification of proteins under pathogenic and non - pathogenic conditions . in embodiments the methods comprise system wide analysis of phosphorylation of proteins under pathogenic and non - pathogenic conditions . in embodiments the methods comprise system wide analysis of post - translational modification of signaling proteins under pathogenic and non - pathogenic conditions . in embodiments the methods comprise system wide analysis of phosphorylation of signaling proteins under pathogenic and non - pathogenic conditions . in embodiments the methods comprise using arrays for system wide analysis . in embodiments the arrays comprise a plurality of antibodies . in embodiments the antibodies are specific for a corresponding plurality of proteins . in embodiments the antibodies are specific for a plurality of proteins . in embodiments the antibodies are specific for a corresponding plurality of modifications of a corresponding multiplicity of proteins . in embodiments the antibodies are specific for post - translational modifications of signaling proteins . in embodiments the antibodies are specific for specific phosphorylations of specific signaling proteins . in embodiments the methods comprise analysis of host responses in host cells exposed to and / or infected with either one of a matched pair of isogenic strains of a disease vector , wherein one member of the pair is pathogenic and the other member is not pathogenic . in embodiments the host cells are small airway epithelial cells . in embodiments the host cells are human small airway epithelial cells . as an example of an embodiment of the invention for determining potential novel therapeutic targets , the impact of anthrax infection on host signaling pathways was studied using cell culture conditions that mimic a human exposure route . in the case of inhalation anthrax , the outcome of the spore interaction with the epithelial surface of the lungs has long been recognized as one of the factors contributing to bacterial virulence . persistence of the dormant spores in the lungs remains a challenging problem in the current antibiotic - based prophylaxis of anthrax , 8 as exposed persons are required to take antibiotics for at least 60 days ( see inglesby et al ., anthrax as a biological weapon 2002 : updated recommendations for management , jama 287 ( 17 ): 2236 - 52 ( 2002 ), which is herein incorporated by reference in its entirety , particularly in points pertinent to the foregoing . in a novel aspect of the invention , human lung epithelial cells were used as an in vitro model of early inhalation exposure , and cell signaling events were monitored before and during exposure to the germinating anthrax spores and vegetative cells . in addition , a novel protein microarray platform was employed for multiplexed analysis of phosphorylation - driven cell signaling cascades , as recently described for tissue - based cancer studies . 9 using this technology , which takes advantage of the large and growing number of antibodies that recognize proteins only when they are phosphorylated , it is possible to quantitatively and sensitively measure over 100 kinase substrates from a few thousand cells . to more specifically identify cell signaling pathways that are causally important / related to the infectious process directly , the signal pathway profiling was performed using a toxigenic anthrax strain sterne ( pxo1 + , pxo2 − ) and compared to the impact of bacterial exposure on lung epithelial host cell signaling with the isogenic , non - pathogenic anthrax strain ( delta - sterne ( pxo1 − , pxo2 − ) profiled in the same manner . the pathogenic and non - pathogenic strains provided herein are a means to identify the pathogenic host responses due to the expression of anthrax virulence factors encoded by the pxo1 plasmid , and represent an important advance over previous studies that failed to utilize isogenic matched strains and typically report results using only virulent strains or toxins . 10 , 11 , 12 , 13 , 14 the matched isogenic approach also provides the opportunity to study the late bacteremic stages of infection , when anthrax - encoded secreted toxins along with other pathogenic factors are thought to be involved in the damage to the host vital organs with high epithelial content , such as lung , liver , spleen , and kidney . therefore data collected and results generated by the exposure of primary lung epithelial cells to a pathogenic and non - pathogenic strain provide general guidance regarding therapeutic mitigation of anthrax , and validate the biological significance of findings in animal models systems ( e . g ., spore challenged mice ), as described herein . the cultured lung epithelial cells challenged with anthrax spores serve as sensors of infection , and are sensitive to pathogenic factors encoded by the toxigenic plasmid pxo1 . the ability to broadly measure the activation and phosphorylation of cell signaling pathways using a novel proteomic assay provides critical information about which specific signaling networks , out of the myriad of potential candidates , are altered . the use of isogenic matched non - pathogenic and pathogenic strains for host challenge studies also is highly valuable for the facile determination of specifically affected networks . ultimately , the information gleaned from the in vitro cell line experiments provides a rationale for pharmacological approaches to use in animal models . the mouse studies described herein , for example , reveal that the pathway effects observed in cell culture are not simply correlative findings , but underpin the most important direct biological outcome of anthrax exposure : mortality . among these pathways , interference with the cell survival program through modulation of mapk and akt activation may represent an important part of anthrax pathogenic strategy . this is in agreement with the recent report on the inhibition of t lymphocyte akt phosphorylation in vivo by letx , 15 and allows rational explanation of some poorly understood pathophysiological features of anthrax , as discussed below . liver damage and cardiovascular collapse are considered to be the major causes of death of anthrax toxin - challenged animals see , for instance , cui et al . am . j . physiol . regul . integr . comp . physiol . 286 : r699 - 709 ( 2004 ) and moayeri et al ., curr . opin . microbial . 7 : 19 - 24 ( 2004 ), each of which is herein incorporated in its entirety in this regard . lethality during cardiovascular anthrax lethal toxin infusion is associated with circulatory shock but not with inflammatory cytokine or nitric oxide release in rats . the results herein described demonstrate that enhanced gsk - 3β phosphorylation at low moi is followed by its down - regulation at higher number of bacteria . the results show further that edtx through generation of camp is able to modulate akt , and this suggests , based on the fact that gsk - 3β is a specific substrate of akt kinase activity , that the downstream activity of gsk - 3β regulating glycogenolysis by liver and skeletal muscles may be responsible for the glucose level perturbation in anthrax . 16 letx has also been shown to modify transcription of the gsk - 3β - mediated genes in macrophages by an unknown mechanism . 10 the physiological effects of camp on liver and other organs mimic stimulation of the vascular adrenergic receptors ( ars ). the epinephrine - like activity of culture filtrates from b . anthracis , and other observations such as the “ sudden death ” phenomenon commonly observed in anthrax patients , implicate central nervous system ( cns ) involvement in the action of anthrax toxins , and indicate the presence of a low - molecular - weight , endogenously produced ar agonist , which has not yet been identified . 37 , 17 the results set forth herein indicate that the agonist is camp , produced under cns control as a mediator of physiological stress and / or as a result of edtx enzymatic activity . mediation of anthrax pathogenesis by the action of camp on the akt signaling pathway explains , at least partly , the ability of the β - ar agonist isoproterenol to prevent the life threatening drop in blood pressure caused by intensive vasodilation in monkeys administered lethal doses of crude anthrax toxin . 18 elevation of camp quickly improves endothelial barrier function . however , prolonged generation of camp by edtx produces the opposite effect , resulting in low blood oxygenation and , ultimately , to septic shock . as a result , therapies attempting to utilize camp to protect the vasculature barrier function have often failed . 19 in addition , chronic exposure of cardiomyocytes to camp leads to hypercontracture and toxicity , an effect that mimics the effect of catecholamines , such as norepinephrine , acting through β1 - ar . 36 finally , letx inhibition of p38 signaling should block the anti - apoptotic effect of β2 - ar stimulation acting through pi3 kinase and akt . 20 the invention is further described by way of the following examples . the examples are by way of illustration only and are not limitative . a full understanding of the invention requires reading the entirety of the application disclosure , including all of the foregoing and the following text ( including the description , abstract , and claims ) and the figures . such an understanding furthermore requires ( and assumes ) that the same will be read , understood , and interpreted with the knowledge , understanding , and insight of a person skilled in the art ( s ) pertinent to the invention and necessary to its understanding and application . antibodies against total and phosphorylated forms of the following proteins used for reverse phase protein microarray and western blot analyses were obtained from cell signaling technology ( beverly , mass .). antibodies ( identified by their specificities ) were used at the following dilutions : confluent hsaecs ( seeded at 10 6 / well in 12 - well plates ) were starved in the same media as above but containing 1 % fcs for 16 hours and then challenged with spores . as shown in the figures , as described below , cells were cultured for up to 12 hours after challenge . supernatants were removed , and cells were lysed and immediately boiled for 10 min in 100 μl of a 1 : 1 mixture of t - per reagent ( pierce , rockford , ill .) and 2 × tris - glycine sds sample buffer ( novex / invitrogen ) in presence of 2 . 5 % β - mercaptoethanol and protease inhibitors . lysed samples were stored at − 80 ° c . prior to use . nine nl of each sample were arrayed by a direct contact pin arrayer ( aushon biosystems , burlington , mass .) onto nitrocellulose slides ( whatman , mass .). samples were printed in duplicate and in five - point 1 : 2 dilution curves to ensure a linear detection range for the antibody concentrations used . slides were kept at − 20 ° c . before analysis with antibodies . to estimate total protein content , selected slides were stained with sypro ruby protein blot stain ( molecular probes , eugene , oreg .) and visualized on a fluorchemk imaging system ( alpha innotech , san leandro , calif .). antibodies were pre - validated for specificity by western blotting and peptide competition . slides were stained with pre - validated specific antibodies on an automated slide stainer ( dako , carpinteria , calif .) using a biotin - linked peroxidase catalyzed signal amplification . the arrayed slides were placed into 1 × re - blot solution ( chemicon , temecula , calif .) for 15 min , washed two times for 5 min each in pbs , placed into i - block solution ( applied biosystems , foster city , calif .) in pbs / 0 . 1 % tween - 20 for at least 2 hours and then immunostained using the automatic slide stainer ( autostainer , dako cytomation , carpinteria , calif .) using manufacturer - supplied reagents . briefly , the slides were incubated for 5 min with hydrogen peroxide , rinsed with high - salt tris - buffered saline ( csa buffer , dako ) supplemented with 0 . 1 % tween - 20 , blocked with avidin block solution for 10 min , rinsed with csa buffer , and then incubated with biotin block solution for 10 min . after another csa buffer rinse , a 5 min incubation with protein block solution was followed by air - drying . the slides then were incubated with either a specific primary antibody diluted in dako antibody diluent or , as a control , with only dako antibody diluent for 30 min . the slides were then washed with csa buffer and incubated with a secondary biotinylated goat anti - rabbit igg h + l antibody ( 1 : 5000 ) ( vector labs , burlingame , calif .) for 15 min . for amplification purposes , the slides were washed with csa buffer and incubated with streptavidin - horseradish peroxidase ( hrp ) for 15 min , followed by a csa buffer rinse . slides were then incubated in diaminobenzidine ( dab ) chromogen diluted in dako dab diluent for 5 min , washed in deionized water and imaged using a umax powerlook iii scanner ( umax , dallas , tex .) at 600 dpi . the images were analyzed with software alphaease fc ( alpha innotech , san leandro , calif .). for each antibody , average pixel intensity value for the negative control ( staining with only second antibody ) was subtracted from the average pixel intensity and the resulting quantity then was divided by the corresponding value of the sypro - stained total protein slides assays described above . two separate independent experiments were performed , and the averages of the measurements were analyzed . positive and negative controls , consisting of a431 cells , respectively , treated and not treated with egf , were printed on every slide array and served as reference standards for antibody performance . western blots were used to independently confirm the reverse phase protein microarray data . 20 μl of cell lysates were used for western blots , which were stained with 1 : 1000 - diluted primary antibody and 1 : 7500 - diluted secondary antibody . primary and secondary antibodies were the same as used for the reverse phase protein microarray . reverse - phase protein microarray and western blot data are presented as the average of two independent experiments . for reverse - phase protein microarray assays each sample was printed in duplicate . dba / 2 male mice ( jackson labs ), 6 to 8 weeks old , received food and water ad libitum , and were challenged with anthrax spores ( 1 × 10 7 spores , i . p .) on day 0 . ciprofloxacin ( sigma , st louis , mo .) treatment ( 50 mg / kg , once daily , i . p .) was initiated at day + 1 , simultaneously with administration of inhibitors , and continued for 10 days . two doses ( 2 . 5 mg / kg and 12 . 5 mg / kg ) of yvad ( acetyl - tyrosyl - valyl - alanyl - aspartyl - chloromethylketone from bachem bioscience , pa .) and 3 doses ( 0 . 05 , 0 . 15 and 0 . 3 mg / kg ) of cl - ib - meca ( 1 -[ 2 - chloro - 6 -[[( 3 - iodophenyl ) methyl ] amino ]- 9h - purin - 9 - yl ]- 1 - deoxy - n - methyl - β - d - ribofuranuronamide from sigma , st louis , mo .) were administered . animals received yvad on days + 1 to + 4 , and cl - ib - meca on days + 1 to + 10 once daily , s . c . to more specifically identify cell signaling pathways that are causally important or related to the infectious process directly , signal pathway profiling was carried out using matched isogenic strains of b . anthracis : pathogenic strain sterne ( pxo1 + , pxo2 − ) and non - pathogenic anthrax strain ( delta - sterne ( pxo1 − , pxo2 − ). pathogenic effects of b . anthracis were determined by exposing lung epithelial cells to each of the strains , separately , and monitoring subsequent changes in cell physiology , as described , for instance , in other examples herein . the use of these matched , isogenic pathogenic and non - pathogenic strains provides a means to identify pathogenic host responses to anthrax virulence factors encoded by the pxo1 plasmid . these strains and their use is an important advance over previous studies of b . anthracis infections that utilized only virulent strains or toxins without matched isogenic controls , such as those provided by the strains discussed above . 21 , 22 , 23 , 24 , 25 the matched strains also provide the ability to study the late bacteremic stages of infection , when anthrax - encoded secreted toxins along with other pathogenic factors are thought to be involved in the damage to vital organs with high epithelial content , such as lung , liver , spleen , and kidney . results from in vitro studies of exposure of primary lung epithelial cells to matched pathogenic and non - pathogenic strains , accordingly , should provide valuable guidance for animal studies and for prophylactic and therapeutic intervention to prevent , mitigate , or cure anthrax , as provided further in other examples herein . the dynamics of cell signaling phosphorylation in the human small airway epithelial cells ( hsaecs ) after exposure to anthrax spores was determined using an array of 43 different antibodies , as described in example 1 . the specificity of each antibody ( set forth above , in example 1 ) had been validated in previous studies . see espina et al ., j . immuno . meth . 290 ( 1 - 2 ): 121 - 133 ( 2004 ) and in references therein , all of which are herein incorporated by reference in their entireties , particularly in parts pertinent to the foregoing antibodies and their validation and the like . the panel was selected based on the ability of the antibodies to broadly monitor the molecular networks involved in host response pathways most likely to be affected by bacterial exposure : namely survival , apoptosis , inflammation , growth , differentiation , and immune responses . changes in phosphorylation of host cell signaling proteins were determined by comparing phosphorylation of signaling proteins in hsaecs exposed either to pathogenic b . anthracis strain sterne ( pxo1 + , pxo2 − ) or non - pathogenic b . anthracis strain sterne ( pxo1 − , pxo2 − ) (“ delta sterne ”). phosphorylation of the proteins was determined in all cases using antibody panels as described above . two independent experiments were performed and the results were averaged . 24 of the 43 signaling endpoints were statistically changed upon exposure to either strain . 6 of those exhibiting the most significant changes were further tested and verified by western blot analysis . among these the most prominent difference was that phosphorylation of proteins of the pro - survival signaling pathway was considerably greater in cells exposed to the pathogenic strain than it was in cells exposed to the non - pathogenic strain . this was particularly true for phosphorylation of mitogen - activated protein kinases ( mapkks ) erk1 / 2 ( p44 / 42 mapk ), their downstream target p90 rsk , other members of the mapk family , such as the stress - activated kinases p38 and jnk , and the global regulators of survival pathways — the serine / threonine kinases akt1 / 2 . representative results in this regard are depicted graphically in fig1 . increased phosphorylation of erk and akt kinases is generally accepted as serving a protective role , directed to the elimination of non - pathogenic bacteria . 26 , 27 , 28 akt is a pluripotent mediator of a number of cellular processes . it provides a crucial link between p13 kinase and anti - apoptotic mediators , and is one of the most important mediators of cell survival . 29 down - regulation of erk and akt phosphorylation in the epithelium upon exposure to the pathogenic strain compared to the non - pathogenic strain pointes to a pathogenic mechanism leading to suppression of epithelial survival and abrogation of the protective functions of epithelial cells . mapkks are known to be specific targets of lethal toxin ( letx ) proteolytic activity 30 , 31 and they are implicated in the induction of apoptosis by letx in macrophages and epithelial cells . 32 , 33 the effect of anthrax exposure on akt phosphorylation in target host cells , however , is a novel observation . and , since akt regulates glycogen synthesis by phosphorylation of glycogen synthase kinase 3 ( gsk3 ), akt inactivation may play an important role in the abnormal glucose levels observed both in animals exposed experimentally to anthrax and in human patients suffering from anthrax infection . also notable is the link between the akt activity and the adenylate cyclase activity of edema toxin ( edtx ). cyclic amp ( camp ) and its effector camp - dependent protein kinase ( pica ) together are an integral component of many intracellular signaling pathways . in many cell types an increase in the level of cellular camp inhibits cell growth and inhibits akt signaling , by blocking the coupling of akt with its upstream regulators . 34 , 35 as a result , edtx adenylate cyclase activity may mediate the decreased akt phosphorylation engendered in cells exposed to toxigenic b . anthracia . in view of the foregoing results , and the importance of akt to survival pathways , the effect of edtx on akt phosphorylation was studied in greater detail . representative results depicted in . fig2 show that that akt phosphorylation in hsaecs exposed to different concentrations of edtx first increases , then decreases , over a period of at least 8 hours and , finally , returns almost to normal within 24 hours . letx , at the same time , does not affect akt phosphorylation in hsaecs ( data not shown ). this pattern of akt phosphorylation matches the camp - mediated physiological response of experimental animals to prolonged intravenous infusion of epinephrine which leads to the same type of vascular collapse that has long been recognized as a major aspect of the terminal phase of anthrax infection . 36 , 37 while no in vitro system can model with complete fidelity the entire complexity of host - pathogen infectious processes that occur in intact organisms , results obtained using hsaecs , among others , are nonetheless reasonably predictive of the results to be expected in vivo and remain particularly valuable for testing therapeutic approaches that target the host cell response . taken together , the results indicate that pharmacologically correcting the altered host cell intracellular signaling , could affect the lethal outcome in anthrax - challenged animals . protective effect of yvad in an animal model , alone and when used in combination with another agent according to smith et al ., antibiotics alone cannot treat anthrax after a certain time of “ no return ” when toxemia becomes a predominant factor . in certain embodiments of the invention several agents are used in combination , with one another and / or with an antibiotic . in the case of anthrax infection , for instance , each of the illustrative agents individually can correct one or more signaling abnormalities caused by either or both letx and edtx , and they can be used alone or in combination with one another and / or in combination with an antibiotic , such as ciprofloxacin , which targets the bacterial proliferation . thus , by way of example , it has been shown that apoptosis is induced by letx in cultured macrophages and in the livers of anthrax - challenged mice . it also has been shown that the general caspase inhibitor , z - val - ala - asp ( ome )- fluoromethylketone (“ z - vad ”) and the specific caspase - ¼ inhibitor , acetyl - tyrosyl - valyl - alanyl - aspartyl - chloromethylketone (“ yvad ”), each has a protective anti - apoptopic effect in both of these models . 38 as depicted in fig3 , only four doses of yvad in combination with ciprofloxacin , administered on days + 1 through + 4 post infection protect up to 70 % of animals over the course of the experiment from days + 1 through + 10 . under the same conditions , with the same delay in beginning treatment until day + 1 post - exposure ( which better models likely clinical circumstances ), administration of ciprofloxacin by itself protected only 30 % of animals . protective effect of cl - ib - meca in an animal model alone and when used in combination with other agents host akt pathway responses to anthrax exposure and infection provide an example of a host cell response that may be targeted for protection and therapy , as shown in this example . since host cell akt phosphorylation is decreased as a result of exposure to pathogenic b . anthracis , beneficial effects thus may be obtained by counteracting this effect , and restoring akt phosphorylation to its normal levels . the results in this example show that pharmacologically altering camp - mediated host cell akt signaling is protective against the pathological effects of anthrax exposure and infection . akt activity is a function of its phosphorylation . phosphorylation of akt in part , depends on camp levels ; although , the effect is indirect . in many cells , down regulation of cellular camp decreases phosphorylation akt , and that of other regulatory signaling kinases such as erk1 / 2 and gsk3β ( s9 ). 39 , 40 ( in other cells , however , the effect is just the opposite .) cellular camp levels are influenced and often , in part , regulated directly , by a3ars . accordingly , phosphorylation of akt can be increased , and its activity restored in cells exposed to anthrax by stimulating a3ars to decrease cellular camp levels . increased akt activity , for the reasons set forth above , protects cells against the deleterious results of anthrax infection . in addition , the stimulation of a3ars , by itself , may provide prophylactic and / or therapeutic effects against anthrax . in particular , modulation of a3ar activities is known to be cardioprotective during hypoxia , 41 to inhibit apoptosis , to protect against endotoxemia 42 and colitis , 43 and to decrease renal and hepatic injury , and mortality in sepsis . 44 the results in this example show that pharmacological stimulation of adenosine a3 receptors ( a3ars ), which leads to akt phosphorylation and activation protects animals from developing anthrax after exposure to b . anthracis . the results show , furthermore , that the protective and therapeutic effect of the treatment is increased when the agents for stimulating the a3ars are used in combination with other therapeutic agents , such as antibiotics . by way of illustration in this regard , the results in this example show that two a3ar agonists ib - meca ( n 6 -( 3 - iodobenzyl ) adenosine - 5 ′- n - methyluronamide ) and cl - ib - meca , its cl - substituted derivative , protect mice against post - exposure anthrax . as shown in fig3 c treatment with cl - ib - meca , at the optimal dose of 0 . 15 mg / kg , resulted in 40 % protection of exposed animals , even without co - administration of the antibiotic , ciprofloxacin . this was a remarkable result . moreover , administration of cl - ib - meca in combination with ciprofloxacin was more effective than administration of ciprofloxacin , at a variety of doses , and the difference was statistically significant for the results obtained by administration of 0 . 3 mg / kg of cl - ib - meca ( p & lt ; 0 . 05 ). as shown in fig3 e and 3f , even better results were obtained using triple combinations of yvad , cl - ib - meca , and ciprofloxacin , which target generation of il - 1β , a3ar activity , and bacterial growth , respectively . as seen in the figures , the triple combination was synergistic and protected up to 90 % of exposed animals , combinations of yvad and cl - ib - meca without ciprofloxacin were only marginally more protective when used together than when either one or the other was used by itself . these results indicate that the down - regulation of the erk and akt survival axis as observed in host lung epithelial cells is not a simple correlative finding , but is casually important in disease pathogenesis and overall anthrax - induced mortality . these findings also indicate that an optimal combination post - exposure anthrax therapy based entirely on the modulation of the host response to infection could be highly effective , and highly synergistic with the current state - of - care antibiotic , ciprofloxacin . the following references are each individually incorporated herein by reference in their entirety , particularly in parts pertinent to the invention herein disclosed , particularly as to the subject matter of their specific citation described and indicated where cited herein above . 1 holty j e , bravata d m , liu h , olshen r a , mcdonald k m , owens d k . systematic review : a century of inhalational anthrax cases from 1900 to 2005 . ann intern med . 2006 feb . 21 ; 144 ( 4 ): 270 - 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