Patent Application: US-201515514044-A

Abstract:
ecotin variants and their use in treating viral hemorrhagic fever are described . described herein are methods for treating systemic inflammatory response syndrome or viral hemorrahagic fever by administering an ecotin polypeptide . described herein is a polypeptide comprising the amino acid sequence of any of seq id nos : 2 - 9 and 11 - 18 . also described : is a polypeptide comprising the amino acid sequence of any of seq id no : 11 - 18 preceded by a methionine ; a polypeptide comprising the amino acid sequence of any of seq id no : 11 - 18 with up to 5 single amino acid changes or deletions provided that the polypeptide does not comprise the amino acid sequence of seq id no : 10 .

Description:
described below are studies on wild type ecotin ( nb101 ; seq id no : 1 ) and an ecotin variant ( nb109 ; seq id no : 2 ). nb109 differs from ecotin in one amino acid residue , m84r , at the p1 position of the so - called reactive center loop (“ rcl ”; amino acids 82 - 88 ; amino acid number of mutations refers to the mature ectotin sequence , i . e ., seq id no : 1 lacking the first 20 amino acids ( mktilpavlfaafattsawa ; seq id no : 19 ) as shown in seq id no : 10 ). nb101 is a broad - spectrum protease inhibitor targeting serine elasase ( also called neutrophil elastase ( ne ) or granulocyte elastase ( ge )) coagulation factors ( xa , xiia , viia ), and kallikrein ( table 1 ). in addition to its potential anti - inflammatory function via ne inhibition , nb101 directly targets two components of the “ sirs triangle ”; coagulation and kallikrein . however , nb101 does not inhibit fibrinolysis . thus , all potential point mutations at the p1 position of the rcl were screened resulting in nb109 . distinct from nb101 , nb109 inhibits plasmin and thrombin . as a result , it directly targets all three components of the “ sirs triangle ”. nb109 shares the chemical and physical properties with ecotin . nb109 has an equivalent number of negatively charged residues ( asp + glu ) and positively charged residues ( arg + lys ), and the calculated pi is 6 . 85 [ 61 ]. one unit of compound activity is defined as the amount of compound required to inhibit 50 % trypsin under the standard assay conditions . based on this definition , nb109 has a specific activity of 1 × 10 5 unit / mg , which is equivalent to nb101 . nb101 and nb109 were tested to determine their ability to inhibit blood coagulation , in particular the intrinsic pathway of blood coagulation via inhibition of inflammation and kallikrein - kinin system . the agents were test on human blood coagulation in vitro by performing pt ( prothrombin time ; extrinsic coagulation pathway ) and aptt ( activated partial thromboplastin time ; intrinsic coagulation pathway ) assays . both molecules exhibited a potent dose - dependant anti - coagulation effect , and nb109 was approximately 2 times more potent than nb101 ( fig1 ), probably due to its activity against thrombin . in addition , both nb109 and nb101 exhibited stronger inhibition ( roughly two fold ) towards the intrinsic coagulation pathway ( as measured by aptt ) than the extrinsic pathway ( measured by pt ) ( fig1 ). it is important to note that pt and aptt elevations are expected pharmacological effects of the candidates . pt or aptt elevation per se does not signify spontaneous bleeding as an adverse effect . spontaneous bleeding tendency is associated with uninhibited fibrinolysis and increased vascular permeability [ 62 ]. nb101 and nb109 may have a reduced risk of spontaneous bleeding because they inhibit either vascular hyper - permeability or both vascular hyper - permeability and fibrinolysis . nb101 and nb109 were tested in the murine endotoxemia model , which is a lethal shock model induced by two consecutive systemic exposures of endotoxin ( lps ) administered 24 hr apart . pathophysiologically , this model is characterized by inflammation , hemorrhage , tissue necrosis , and dic [ 63 ]. the vehicle - treated mice all suffered a rapid death within one day of lps challenge , but treatment with nb101 and nb109 had significant survival benefit ( fig2 ). in this study , nb101 and nb109 all increased animal survival in a similar manner , and they both compared very favorably against the current standard anti - dic treatment , low molecular weight heparin ( lmwh ). lmwh given twice before the lps elicitation only improved 30 - hr survival of the treated mice by 25 % ( 50 % survival in the treated group and 25 % survival in the control group ) [ 64 ]. cecal ligation and puncture ( clp ) is another commonly used animal model of sirs . in the clp model , sirs is produced by peritonitis following intestinal injury and infection by multiple bacteria that normally reside in the intestines . it is considered to better mimic the natural cause of sepsis [ 65 ]. in a preliminary study , nb109 achieved significant ( p & lt ; 0 . 005 ) survival advantage in the clp model ( fig3 ). nb101 and nb109 were evaluated in guinea pigs infected with zaire strain of ebov . the vehicle - treated animals invariably died by day 9 . nb101 and nb109 treatment was initiated at 24 hr post infection , and was given by intraperitoneal injections once a day for 7 days . while nb101 did not affect animal survival or body weight loss , nb109 achieved 50 % survival and rescued the surviving animal from fatal body weight loss ( fig4 ). this result provides proof - of - concept . together , the in vitro and in vivo findings indicate that nb109 and nb101 are potentially potent candidates as anti - sirs and anti - vhf compounds and pharmaceutical formulations . nb109 was incubated with a collection of human primary cells , including primary human renal proximal tubule cells , renal cortical epithelial cells , lung vascular endothelial cells , or hepatocytes , as well as cells lines , a549 and beas - 2b , at up to 250 μm . over 4 - day incubation , cytotoxicity was evaluated using the mts assay . nb109 did not cause cytotoxicity and had no effect on viability of the cells . nb109 was examined for indirect hemolysis via activation of complements , or direct hemolysis . as a positive control for the complement - mediated hemolysis , species specific antibodies against red blood cells ( rbc ) were used to activate the classical complement pathway and initiate the signaling cascade leading to the lysis of the rbc . for evaluating direct hemolytic activity of nb109 , the rbc were washed to remove any complement proteins , and then resuspended with heat - inactivated plasma or serum containing nb109 . in the human blood , nb109 did not elicit hemolytic reactions , neither direct nor complement mediated , at concentrations up to 1 mg / ml . systemic safety of nb109 treatment in mice safety and tolerability of nb109 systemic treatment in mice was evaluated in 5 groups of 16 balb / c mice . each of the four groups received one intraperitoneal injection of nb109 at 5 , 15 , 45 , and 90 mg / kg , respectively ; the fifth received pbs . mice were sacrificed at 4 hr and 24 hr post dosing and subjected to necropsy , coagulation analysis , and clinical chemistry . upon necropsy , all animals appeared to be normal without signs of hemorrhage . as expected , coagulation parameters were affected in a dose - dependent manner ; the effects peaked at 4 hr post treatment and returned to the baseline by 24 hr post treatment ( fig5 ), which indicates that nb109 was cleared from the blood within 24 hours . consistent with what was observed in the human blood , aptt was more sensitive to nb109 and the effect was observed at 5 mg / kg whereas pt was not affected until 15 mg / kg . pt returned to the baseline level before aptt did . it should be noted that elevations in pt and aptt are pharmacological effects and are not considered adverse effects . nb109 was given to hartley guinea pigs by intraperitoneal administration at doses of 0 . 1 , 0 . 5 , 1 . 5 , and 5 mg / kg / day for 7 days . safety parameters included clinical signs , serum chemistry , coagulation times , and necropsy . all animals survived nb109 treatment , and all clinical observations for nb109 - treated animals were normal throughout the course of the study . there was no significant difference in body weight change between the groups , and all groups showed significant weight gain ( 19 - 23 % by the end of the study ). necropsy of all nb109 - treated animals was unremarkable . there was a trend of mild and transient elevation of creatine phosphokinase ( cpk ) at ≧ 1 . 5 mg / kg on day 2 , but the values returned to the normal range by day 7 . a mild elevation of ast was seen on day 14 at ≧ 1 . 5 mg / kg , but other liver enzymes and bilirubin were normal . all other clinical laboratory parameters were within the normal range . no changes in coagulation parameters were observed at doses 1 . 5 mg / kg and below ( note that the guinea pig has reduced fvii levels , thus a longer pt than other species ). at 5 . 0 mg / kg , elevated pt and aptt values were observed starting at eight hours after the first dose and continuing on through eight hours after the last dose on day 7 . all pt and aptt values returned to normal by day 14 . a preliminary pharmacokinetic ( pk ) study was conducted in mice in which nb109 was administered by different routes . the data are illustrated in fig6 . initial plasma concentrations were much higher with iv administration relative to ip or sc injection . intraperitoneal injection resulted in considerably higher concentrations than did the same dose by sc injection , meaning that the bioavailability of nb109 by the sc route would be less than ideal . given the variability of the plasma concentration data with iv administration , it was not possible to provide any estimates of pk exposure . however , the plasma concentration for the ip route was amenable to pharmacokinetic modeling ( winnonlin software , cary , n . c .). the half - life of elimination ( t½ ) of nb109 by the ip route was 7 . 6 hr . a study of nb109 was conducted in guinea pigs to evaluate the relationship between plasma concentrations of drug and blood coagulation parameters following single and repeated dose administration . nb109 was administered iv to hartley guinea pigs ( n = 3 ) at a dose of 5 mg / kg . there was an excellent correlation between plasma levels of nb109 and prolonged aptt following a single iv dose ( fig7 ). while the aptt closely mirrored the plasma levels of drug which had almost returned to background levels by 8 hr post - injection , the pt remained prolonged at that time - point . repeated dosing was conducted again with a dose of 5 mg / kg daily for 5 days . the plasma drug levels appeared to increase slightly following the third dose , however the variability in data make any conclusions on drug accumulation difficult to determine ( fig8 ). there was a very good correlation between blood coagulation parameters with plasma nb109 . by day 7 ( 96 hr post - dosing ) all parameters had returned to baseline . the constructs shown in tables 2 and 2a were developed and tested and described further below . the amino acid sequence for the constructs are shown in table 3 as seq id nos . 1 - 9 . murine endotoxemia model was used as the first - line screening model due to its simplicity . all of the potentially optimized lead candidates protected animals in this model ; nb142 , nb137 , nb147 , and nb178 appeared to be the most effective ones . interestingly , nb142 is significantly superior to nb101 or nb109 in this model ( error ! reference source not found . 9 ). in addition to having the highest rate of animal survival , nb142 also was most effective at inhibiting inflammatory cytokines il - 6 and tnf - α ( fig9 ) several of the peptides shown in table 3 in mice challenged with injections of poly ( i : c ), an inosine polymer resembling foreign rna molecules . since vhf viruses are all rna viruses , this model is designed to replicate host responses to viral rna molecules . similar to vhf viruses , poly ( i : c ) injection triggers signs of sirs , including release of inflammatory cytokines , elevated d - dimers ( a product of fibrinolysis indicative of dic ), and abundant micro - thrombi in the liver , lung , and kidneys . untreated animals invariably died in five days after the first poly ( i : c ) injection . when treatment was initiated prior to poly ( i : c ) injection , nb109 , nb142 , nb137 , and nb147 all significantly prevented animal death . when nb109 treatment was initiated after poly ( i : c ) injection , it was effective when it was first given at one day after challenge ( fig1 ). nb104 prolonged animal survival even when initiated at 48 hrs after poly ( i : c ) challenge with only two treatments ( fig1 ). this result suggests that both nb142 and nb109 can prevent sirs at the time of induction , but nb142 may be more effective at treating established sirs associated with vhf . in the same model , when given prior to poly ( i : c ) challenge , nb101 , nb109 , and nb142 all significantly inhibited inflammatory cytokines and d - dimer triggered by poly ( i : c ). however , among the three candidates , nb142 was the most effective at inhibiting inflammatory cytokines il - 6 and tnf - α ( error ! reference source not found .). next , nb109 and nb142 were compared in a study of guinea pigs infected with zaire strain of ebov . while vehicle - treated animals invariably died by day 9 , nb142 at 1 mg / kg / day and nb109 at 5 mg / kg / day achieved significant , 67 % survival . again , nb142 showed superior efficacy , with better survival at a lower dose and remarkable body weight gains ( fig1 ). the strength of this study result also lies in the fact that nb109 and nb142 treatment was with an unoptimized treatment dose and regime initiated at 24 hr post infection . preliminary pharmacodynamics of nb142 , nb101 and nb109 nb142 has distinct pharmacodynamics ( pd ) from nb101 and nb109 in vivo . while nb101 and nb109 both cause pt elevations , nb142 does not affect pt ( fig1 ). all three candidates cause elevation in aptt with various potencies . the pd result indicates that nb101 and nb109 inhibit both extrinsic and intrinsic coagulation pathways , whereas nb142 appears to specifically affect the intrinsic coagulation pathway . hematological monitoring of ebov infected rhesus monkeys reveals that consumptive coagulopathy in ebov hf is due to activation of the intrinsic coagulative pathway , rather than extrinsic coagulative pathway [ 66 ]. intrinsic coagulative pathway is directly activated by inflammatory cytokines and kallikrein , and is potentiated by plasmin activation . nb142 has anti - inflammatory effects . it also potently inhibits kallikrein and plasmin while sparing thrombin . thus it may inhibit the upstream events that trigger intrinsic coagulation without exacerbating consumptive coagulopathy . therefore , nb142 may have a preferred pd profile for vhf treatment . peptide can be produced using a high - density , fed - batch e . coli fermentation process followed by periplasmic extraction , an ion - exchange chromatography , and a filtration step to remove endotoxin . two microbial expression systems can be evaluated for lead compound production : e . coli and yeast . nb109 is produced using a time dependent fed - batch e . coli fermentation process using glucose as the carbon source that yields ˜ 0 . 2 gm purified nb109 per liter of fermentation . the lead compounds can also be produced with a dissolved oxygen - dependent feed control system that uses glycerol as a carbon source . this fermentation process has resulted & gt ; 9 grams per liter expression of a different protein drug candidate . this latter process can be easily scaled up . it uses a semi - defined medium composed of usp - grade reagents that are certified animal - free . as an alternative to the bacterial expression system , yeast strains such as p . pastoris and h . polymorpha can also be evaluated as a system for production lead compounds . these have the advantages of higher eukaryotic expression systems such as better protein processing , folding and secretion when compared to microbial systems , and still have rapid growth and tightly regulated promoters . peptides can be expressed by secretion into yeast media to greatly simplify the purification process . as part of the present invention , strains of p . pastoris have been generated to secrete lead compounds into yeast media . these strains are methanol - inducible and amenable to fermentation . further optimization of the p . pastoris system is possible by investigating multiple secretion leader sequences such as a - mating factor , a - amylase , glucoamylase , inulinase , and invertase yeast signal sequences , and transforming multiple wild type and protease deficient yeast strains . screening of colonies can be performed from supernatants of small scale cultures grown in 96 - and 24 - well formats . selected clones can be grown in shaker flask culture before transfer to fermentation . the fermentation process can be established using available bioflo 3000 and bioflo iv fermenters with volumes of 4 to 20 liters . methanol feed for induction of expression can be quantified by an available ysi 2700 select biochemistry analyzer with methanol probe . fermentation optimization can vary media and feed composition , ph , temperature , feed time course , and time of induction to achieve desired levels of protein expression . the purification process from e . coli fermentation involves a periplasmic extraction followed by an ion - exchange chromatography step for purification and an ion - exchange filtration step for endotoxin reduction . this purification has worked for peptides described herein . the details of this process are presented in fig1 . additional downstream steps can include , but are not limited to , affinity chromatography , hydrophobic interaction chromatography , size - exclusion chromatography , and additional ion - exchange steps . initial screening can be performed in 96 - well filter plates for high throughput without using robotics . binding conditions to be evaluated can include chromatography resins , salts , ionic strength , and ph . micro - eluates can be analyzed for overall concentration by uv absorbance using an available 96 - well uv spectrophotometer and purity by 48 - sample sds - page ( invitrogen , carlsbad , calif .) with coomassie staining . select conditions can be scaled up to chromatography using standard 1 - 10 ml columns on available fplcs . yield and purity of the process intermediates can be monitored using a subset of the release tests described below , including sds - page , hplc and activity . development can also focus on adapting the purification process to the yeast expression system and adding additional purification steps to enhance purity . additional steps may include , but are not limited to , hydrophobic interaction chromatography , reversed - phase chromatography , and additional ion - exchange steps . the lead compounds can be developed into a sterile , non - preserved , unit - dose parenteral product . current data indicate that the lead compounds can be very robust and stable over a broad range of ph and temperature . based on the 1 mg / kg / day effective dose of nb142 in the guinea pig ebov model , the human treatment dose could be approximately 0 . 2 mg / kg / day . for a maximum of 7 - day course , the estimated total drug consumption would be 84 mg ( for 60 kg individual ) to 280 mg ( for 200 kg individual ). a number of embodiments of the invention have been described . nevertheless , it will be understood that various modifications may be made without departing from the spirit and scope of the invention . 1 . borio l , inglesby t , peters c j , schmaljohn a l , hughes j m , jahrling p b , ksiazek t , johnson k m , meyerhoff a , o &# 39 ; toole t , ascher m s , bartlett j , breman j g , eitzen e m , jr ., hamburg m , hauer j , henderson d a , johnson r t , kwik g , layton m , lillibridge s , nabel g j , osterholm m t , perl t m , russell p , tonat k ( 2002 ) hemorrhagic fever viruses as biological weapons : medical and public health management . jama 287 : 2391 - 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