Patent Application: US-51941703-A

Abstract:
the invention relates to the use of a peptide which binds to lipopolysaccharide or lipoteichoic acid , for manufacturing a pharmaceutical composition for treating sepsis or septic shock , wherein the peptide comprises the amino acid sequence of apolipoprotein ci or a part thereof that comprises at least the amino acids of the c - terminal helix of apoci . the use of human apoci is preferred . the peptide can be administered clinically to patients who have sepsis or threaten to develop sepsis . measurement of the apoci content in blood can be utilized for determining the severity and prognosis of the course of the septic condition or for monitoring an anti - sepsis treatment .

Description:
we have found , as example 1 and fig1 show , that the level ( the concentration ) of apolipoprotein ci ( apoci ) in septic blood is strongly lowered , which does not hold , or holds to a much lesser extent , for the ( numerous ) other apolipoproteins . this lowered apoci content in septic blood is confirmed by the literature ( barlage , 2001 ), although the authors have not discussed a possible role of apoci in sepsis . apoci is the smallest apolipoprotein ( 6 . 6 kda , 57 amino acids ) which is chiefly synthesized by the liver and circulates in the blood in a fairly high concentration (. about . 6 mg / dl ), both in free , unbound form and as a constituent of lipoproteins ( chylomicrons , vldl , hdl ) ( jong , 1999 ). the amino acid sequence of apoci is known ( see table 1 ). to date , no mutations have become known that lead to different variations (‘ polymorphisms ’) of apoci . using transgenic mice that overexpress the human apoci , we have demonstrated that apoci plays a role in the lipid metabolism , since apoci overexpression leads to increased lipid levels in the blood ( jong , 1996 ; jong , 1998 ), but a role , if any , of apoci in sepsis was not yet known . we have developed the hypothesis that the apoci depletion associated with sepsis is a direct consequence of bacterial infection . the low apoci concentration might be the consequence of binding of apoci to bacterial products , specifically lps , which may possibly lead to accelerated clearance of apoci from the blood . via an extensive literature study , it was found that lps - binding proteins have common characteristics , with the presence of a combination of positively charged and hydrophobic amino acids being essential . two of these proteins are the limulus anti - lps factor ( lalf ) ( hoess , 1993 ), a factor in the ‘ lal assay ’ routinely used to measure the lps activity , and cap18 larrick , 1994 ), which is synthesized by polymorphonuclear cells . in fact , apoci contains a large number of positively charged lysines ( one in each six amino acids ), alternated with hydrophobic amino acids . to our surprise , human apoci contains a peptide sequence in its c - terminal portion ( kvkeklk ) seq id no . : 4 which is virtually identical to the lps - binding sequences of lalf ( kwkykgk ) seq id no . : 5 and cap18 ( kikeklk ), seq id no . : 6 where symbols in bold - type and italics respectively represent positively charged and hydrophobic amino acids . we have subsequently demonstrated that apoci , both in pure form and as a constituent of plasma in fact binds strongly to lps ( see example 2 and fig2 , 3 , 4 and 5 ). we have compared the amino acid sequences of apoci , such as occur in man ( swissprot p02654 ), baboon ( swissprot p34929 ), dog ( swissprot p56595 ), rat ( swissprot p19939 ) and mouse ( swissprot p34928 ), as can be found under the numbers indicated ( http :// www . expasy . ch / sprot /). see table 1 . the consensus lps - binding sequence underlined in table 1 as found in human apoci ( kvkeklk ) seq id no : 4 has been found to occur virtually unmodified in the animal variants . human apoci forms two amphipatic helices of class 2 ( amino acid sequences 7 - 29 and 38 - 52 ), which facilitate the binding of apoci with lipoproteins . on the basis of structural homology , it is assumed that animal apoci takes a comparable structure . on the basis of the disappointing results of the earlier discussed , failing experimental antiseptic therapies , we have furthermore developed the hypothesis that therapeutic reduction of the proinflammatory response is not only ineffective but even undesirable , because in this way the causative bacterial proliferation is not inhibited . although the cause of sepsis is very heterogeneous and the time of intervention may be determinative of the success of the treatment , we deem it likely that specifically enhancement of the inflammatory response in an early stage of sepsis increases the antibacterial effectiveness and hence raises the chance of survival . while proteins , such as cap18 , lalf , cap37 , prophenin and polyphemusin , which do not form part of lipoproteins , are also capable of binding lps , they do not , in contrast with apoci , occur in a high concentration in the blood . further , these proteins lead to suppression of the lps - related inflammation cascade , which according to our hypothesis is unfavorable for the course of the disease . the bacterial infection will , as a result , aggravate rather than improve . surprisingly , we have now established that the binding of lps to human apoci leads to a considerably longer residence time of lps in the blood ( see example 4 and fig6 ), owing to its binding to plasma lipoproteins , facilitated by the amphiphilic structure of apoci . also , we have established that administration of lps with apoci to mice leads to a strongly increased proinflammatory response compared with mice that were administered lps alone , which appears from a fourfold increased tnfα production ( see example 5 and fig7 ). the finding that apoci enters into a direction interaction with lps is new and points to a direct role of apoci in the endogenous control of bacterial sepsis . because apoci is a constituent of lipoproteins ( chylomicrons , vldl and hdl ) and is consequently cleared slowly from the blood , we therefore suppose that the binding of lps to apoci extends the residence time of lps in the blood , so that the activation of white blood cells is additionally stimulated in a natural way . as a result , the production of proinflammatory cytokines ( tnfα , il - 1β and il - 6 ) will increase and consequently the antibacterial inflammation cascade will work extra strongly . this mechanism can also partly explain why lipoproteins play a protective role in sepsis . accordingly , the essence of the present invention is that through administration of apoci , or an adequate fragment thereof , as defined herein , a timely , extra forceful inflammation cascade is obtained which enables early neutralization of both lps and the lps - producing bacteria , so that the stage of full - blown sepsis or septic shock will not be reached . the small size of apoci enables obtainability of the protein on a large scale through so - called solid - phase peptide synthesis . this holds to a greater extent for smaller peptides ( fragments of apoci ), which bind lps and stimulate lps - induced activation of white blood cells . since apoci is an endogenous plasma protein , it will be tolerated well and not generate any ( additional ) immune reactions . ( for that reason , use of human apoci will be preferred in humans , while in animals , preferably the corresponding animal apoci will be used ). it is noted that according to the invention , apoci , or a suitable fragment thereof , is used as the active substance to control or prevent sepsis or septic shock . according to the invention , the apoci is not used as carrier for another substance . according to the invention , the apoci , or fragment thereof , is preferably used as free peptide , not bound in complexes with lipids . as is demonstrated in example 1 and fig1 , patients having severe sepsis exhibit a strongly lowered apoci level . in the example given ( 17 patients admitted to the clinic with severe sepsis ) the apoci level was on average 1 . 6 mg / dl . the apoci levels in patients who eventually survived the sepsis ( 47 %: 8 / 17 ) exhibited a progressive rise after 3 days and normalized after about 4 weeks . by contrast , the apoci levels in the patients who eventually died from the sepsis within 4 weeks ( 53 %: 9 / 17 ), did not exhibit any rise . these data show that monitoring of the apoci concentration in blood plasma has a prognostic value for the chance of survival of the septic patient ( see also fig1 ), and can be used for monitoring the effect of antiseptic therapies . as set out above , the invention comprises the application of apoci and peptides derived therefrom which bind to lps for the therapeutic treatment of gram - negative bacterial sepsis . however , since apoci and peptides derived therefrom also bind to lipoteichoic acid ( lta ), the toxic equivalent of lps in gram - positive bacteria , the invention comprises the use of apoci in bacterial infections in general , hence also in gram - positive bacterial sepsis . according to a particular preferred embodiment , the peptide consists of the whole amino acid sequence of apoci ( or comprises this sequence ), in particular that of human apoci , which consists of 57 amino acids according to the amino acid sequence tpdvssaldklkefgntledkarelisrikqselsakmrewfsetfqkvkeklkids , seq id no . : 2 according to another preferred embodiment , the peptide is , or comprises , the amino acid sequences of homologues of human apoci such as they occur in the animal kingdom ( in particular in mammals , for instance horse , cow , dog , rabbit , rat , mouse ). according to another preferred embodiment , the peptide is , or comprises , at least the c - terminal helix of apoci . in human apoci , the c - terminal helix is formed by the amino acids 38 - 52 , with the amino acid sequence mrewfsetfqkvkek , seq id no . : 11 . preferably , a useful fragment of human apoci comprises at least the amino acids mrewfsetfqkvkeklk , seq id no . : 1 . examples of suitable peptides are , for instance , the peptides mrewfsetfqkvkeklk , seq id no . : 1 and sakmrewfsetfqkvkeklkids , seq id no . : 3 . these peptides comprise the c - terminal helix of apoci and the whole putative lps binding sequence kvkeklk , seq id no . : 4 . instead of a fragment of human apoci , a corresponding fragment of an animal apoci can be used . a property important according to the invention of apoci and the peptides defined herein , is , besides their binding to lps , their binding to lipoproteins . the complete human apoci protein ( amino acids 1 - 57 ) is commercially available ( biodesign international , maine , usa and intracel corporation , maryland , usa ). also , procedures have been described to isolate human apoci as pure protein from blood ( tournier , 1984 ; jackson , 1986 ). finally , there is the possibility of constructing an adenoviral expression vector that codes for human or animal apoci , so that apoci can be produced on a large scale by astrocytoma cells in culture ( kypreos , 2001 ). because apoci is a very small protein , there is also the possibility of obtaining it from the individual amino acids by means of solid phase peptide synthesis ( clark - lewis , 1986 ). this technique also enables systemization of all human apoci analogs and other apoci - derived peptides as described herein . human apoci is water - soluble and so can be administered intravenously in an aqueous solution ( for instance in a physiological saline solution ). single - time ( bolus ) injection is a possibility , as is continuous infusion . also , apoci has lipid - binding properties , which further permits intravenous administration as a constituent of commercially available triglyceride - rich lipid emulsions ( e . g . intralipid ®, intrafat ® and lipofundin ®) which are clinically routinely used as efficient parenteral energy source . in this case , too , bolus injection and continuous infusion both are possibilities . oral administration of peptides according to the invention and of apoci in particular is in principle also possible ( buclin , 2002 ). pharmaceutically acceptable carriers that are suitable for these different administration routes are naturally known to those skilled in the art . the plasma concentration of apoci in healthy individuals is on average 6 mg / dl ( curry , 1981 ), while we have demonstrated that apoci in septic patients is strongly lowered . it is expected that a plasma concentration of 0 . 05 - 500 mg / dl ( preferably 0 . 5 - 50 mg / dl ) can be therapeutically effective . based on an average plasma volume of 2 . 5 l given a body weight of 70 kg , it can be calculated that a dose of 0 . 02 mg / kg - 200 mg / kg ( preferably 0 . 2 mg / kg - 20 mg / kg ) can be effective in the case of a single - time ( bolus ) injection . when administered as infusion , the dose will be strongly dependent on the rate of clearance of the apoci from the blood , so that the dosing can vary within wide limits , for instance from 0 . 01 to 10 mg / kg / minute . it is expected that apoci will bind freely circulating lps and lta and will keep them present in the blood longer , so that lps and lta will be able to induce an increased proinflammatory antibacterial reaction , so that bacteria and lps are neutralized faster and more effectively . both preventive application of apoci in individuals at increased risk of developing sepsis ( for instance in the case of major surgery , or with immune deficiency ) and application of apoci in sepsis or septic shock will lead to the prevention of death resulting from sepsis . the apoci level in blood plasma can be established in any suitable manner . preferably , the apoci level is determined with a sandwich enzyme - linked immunosorbent assay ( sandwich elisa ). to this end , for instance 96 - wells plates ( medium - binding plates ; costar ) are coated with a ( 10 , 000 - fold dilution of a ) polyclonal antibody to human apoci generated in goat ( goat - anti human apoci ; academy bio - medical company , houston , usa ; cat . 31a - glb ). next , human blood plasma or serum is added ( 100 , 000 - 300 , 000 - fold dilution ), whereby the apoci present therein is quantitatively bound by this primary antibody . the apoci is recognized by a secondary antibody ( 15 , 000 - fold dilution ) which is coupled to horseradish peroxidase ( hrp ) ( hrp - goat - anti human apoci ; academy bio - medical company , houston , usa ; cat . 31h - g1b ). the hrp is subsequently quantified by means of a stain reaction with 3 , 3 ′, 5 , 5 ′- tetramethylbenzidine ( tmb ) as substrate , and the stain intensity is compared with a calibration curve which is constructed with pure human apoci ( labconsult , brussels , belgium ; cat . a50366h ). those skilled in the art , of course , know many variants of this apoci determination . thus , other assay techniques than elisa can be used , other detection systems ( enzymes and substrates ), etc . the finding that apoci ( and peptides derived therefrom ) play a protective role in gram - negative ( and gram - positive ) sepsis is supported by a number of experiments . to that purpose , use is made of : a . blood of septic patients ; b . mice that do not synthesize apoci ( apocl . sup .−/− mice ); c . transgenic mice that overexpress human apoci ( apoc1 mice ); d . apoci which has been purified from human blood . predictive value of the apoci level for the chance of survival in sepsis we have measured the apoci levels in the blood plasma of 17 septic patients for - four weeks after admission to the clinic . for that purpose , we used the human - apoci - specific sandwich elisa as described above under f . of the 17 septic patients who were admitted to the clinic with severe sepsis , blood samples were taken over a period of 28 days . in the blood plasma , the contents of apoci , triglycerides ( tg ) and total cholesterol ( tc ) were measured . in fig1 a the apoci content is plotted against time . in fig1 b the apoci content has been corrected for the triglyceride content as a measure for the amount of triglyceride - rich lipoproteins , and in fig1 c the apoci content has been corrected for both triglycerides and cholesterol as a measure for the total amount of circulating lipoproteins . a distinction was made between the patients who survived the sepsis ( closed symbols ; n = 9 .+−. sem ) and the patients who died from the sepsis within 30 days ( open symbols ; n = 8 + sem ). the dotted lines indicate the values as found in healthy individuals . all patients who were included in the investigation exhibited a strongly lowered apoci level in the blood ( ca . 1 . 6 mg / dl ). the patients who died from the consequences of sepsis within four weeks , exhibited a lower apoci level ( ca . 1 . 3 mg / dl ) at the time of admission than did the patients who survived the sepsis ( ca . 1 . 9 mg / dl ). while the apoci level virtually normalized within four weeks in patients who survived the sepsis ( ca . 5 mg / dl ), the apocii level did not exhibit any rise in the patients who eventually died from sepsis . these data show that the plasma level of apoci has a predictive value for the chance of survival in sepsis . also , they underline our hypothesis that apoci falls short in patients who die from sepsis , and which can therefore have a therapeutic use . in this example , it is demonstrated that purified human apoci binds strongly to lps and that this binding is resistant to electrophoretic forces ( agarose gel electrophoresis ). it was found this involved the loss of the naturally occurring micellary structure of lps . the results are shown in fig2 , 3 and 4 . lps re595 ( salmonella minnesota ) was radioactively labeled with 125 i as described ( rensen , 1997 ) and incubated ( 30 min at 37 ° c .) with increasing amounts of human apoci which has been isolated from human blood ( tournier , 1984 ). next , the incubation volumes were transferred to an agarose gel ( position r f 0 ), after which , for 2 hours , an electrophoretic field was applied ( so - called agarose gel electrophoresis ). after electrophoresis , the 125 i - lps was visualized by means of autoradiography . through its negative charge , lps tends to migrate to the positive pole ( anode ); which , however , is prevented in that the lps forms large micellary structures that cannot pass the relatively small pores formed by the agarose matrix . however , addition of a small amount of apoci ( only 0 . 5 molecules of apoci with respect to 1 molecule of lps ) already led to complete migration of the lps to the positive pole , which indicates that apoci binds to lps thereby forming relatively small apoci / lps complexes ( fig2 a ); also , the effect of increasing amounts of lps on the migration of a fixed amount of apoci was determined . for that purpose , the apoci was visualized in the gel after electrophoresis by means of immunoblotting with a horseradish peroxidase - conjugated antibody directed against human apoci ( academy bio - medical company , houston , usa ; cat . 31h - g1b ). the binding of lps to apoci led to accelerated migration of apoci to the positive pole , which indicates that apoci in effect enters into a physical interaction with lps ( fig2 b ) the affinity of the binding of lps to apoci proves to be so high that lps prevents demonstration of apoci during immunoblotting of the agarose gel with the apoci - specific antibody in a dose - dependent manner ( fig2 b ). a particular amount of human apoci ( 0 . 75 ng / ml ) was incubated with lps in increasing molar ratios , after which the apoci concentration was determined with the aid of the human apoci - specific sandwich elisa as described above under f . the lps - induced inhibition of the stain reaction from which the apoci concentration is read , was determined as a percentage of the maximum staining in the absence of lps ( n = 2 .+−. sem ) ( fig3 ). again , the binding of lps to apoci proved to prevent the reaction of apoci with the apoci - specific antibody in a dose - dependent manner . addition of a 200 - fold molar excess of lps to apoci led to more than 40 % inhibition of the staining reaction , that is , a nearly twofold underestimation of the apoci concentration . although apociii has a size ( 79 amino acids ) and structure ( two amphipatic helices ) equal to apoci , lps had no effect on the quantification of concentrations of apociii with the aid of a comparable apociii - specific sandwich elisa ( fig3 ). finally , the effect of apoci on the monomerization rate of lps was studied . fluorescent ( fluorescein isothiocyanate ; fitc )- labeled lps hardly exhibits fluorescence , because the micellary structure of lps leads to the fluorescing fitc molecules being too closely packed together , giving rise to the phenomenon of fluorescence quenching . fitc - lps re595 ( 100 ng ) was incubated with pure human apoci ( in molar ratios 1 : 1 , 1 : 5 , 1 : 10 and 1 : 20 ), after which the occurrence of fluorescence was followed in time with the aid of a fluorimeter . apoci proved to cause the increase in fitc fluorescence to increase strongly in a dose - dependent manner , which indicates that apoci is capable of monomerizing the lps ( i . e . to liberate it from the micellary form ) ( fig4 a ). by contrast , the apolipoprotein ciii ( apociii ), having a comparable size and structure , proved hardly effective ( fig4 b ). we have determined the effect of the apoci level in murine plasma on monomerization of fitc - lps as described in example 2 . for that purpose , fitc - lps was incubated with plasma that had been isolated from wild - type mice , genetically modified mice which are deficient for apoci ( apocl . sup .−/−), or genetically modified mice which express the human apoci hemizygote ( apoc1 . sup .+/ 0 ) or homozygote ( apoc1 . sup .+/+). fitc - lps re595 ( 100 ng ) was incubated with plasma ( 0 . 125 %) isolated from the blood of the various mouse types , after which the occurrence of fluorescence was followed in time with the aid of a fluorimeter . the expression of human apoci in mouse plasma led to an apoci - dose dependent increase of the fitc - lps monomerization , which was 32 % at a maximum . by contrast , the monomerization of fitc - lps proved to be reduced by 60 % owing to the absence of mouse - apoci ( n = 4 .+−. sem ). this indicates not only that mouse - apoci , like human apoci , is capable of monomerizing lps , but especially also that the endogenous presence of apoci contributes for a considerable part to the total lps - disaggregated capacity of mouse plasma . apoci extends the residence time of lps in the blood by preventing the uptake of lps by the liver we have administered lps intravenously to wild - type mice in the absence or presence of purified apoci . addition of apoci proved to extend the residence time of lps in the blood strongly through increased interaction with hdl . wild - type ( c57b1 / 6 ) mice were anesthetized and , via the lower vena cava , injected intravenously with radioactively (. sup . 125i ) labeled lps re595 ( 10 μg / kg ) without , and with , respectively , apoci ( molar ratio lps : apoci = 1 : 0 . 2 , 1 : 0 . 5 , 1 : 1 and 1 : 16 ) and bovine serum albumin ( molar ratio lps : albumin = 1 : 16 ). blood samples and bits of liver tissue were taken at t = 2 , 5 , 10 , 20 and 30 minutes after injection . after this , the mice were sacrificed . next , the amounts of lps in the blood sera and liver samples were quantified on the basis of the amount of radioactive radiation . also , a serum sample was taken 30 minutes after injection of lps : apoci ( 1 : 1 ) and subjected to agarose gel electrophoresis to study the distribution of the 125 i - lps over the various lipoproteins . lipoprotein lipids were visualized with the aid of sudan black , and 125 i - lps by means of autoradiography . in fig6 a and 6b the amounts of 125 i - lps in the liver and the blood serum , respectively , are plotted as a percentage of the injected dose against time ( n = 3 .+−. sem ). fig6 c shows the distribution of 125 i - lps over the lipoproteins . the presence of apoci was found to inhibit the decrease of lps from the blood strongly and dose - dependently . already at a molar ratio of 1 : 1 , the uptake of lps by the liver was reduced 8 - fold . the strong binding of apoci to lps ( see example 2 ) therefore proves to be relevant in the blood too . this led to an extended residence time of lps in the blood through a strongly increased apoci - mediated binding to hdl ( fig6 c ), thereby improving the interaction with inter alia white blood cells and eventually leading to a strongly stimulated inflammation reaction ( see example 5 ). we have administered lps intravenously to wild - type mice in the absence or presence of purified apoci . addition of apoci proved to increase the lps - induced inflammatory response strongly . wild - type ( c57b1 / 6 ) mice were injected , by way of the tail vein , with lps re595 ( 25 μg / kg ) without , and with , apoci ( molar ratio lps : apoci = 1 : 5 ). blood samples were taken at t = 0 , 30 , 60 , 90 , 120 and 180 minutes after injection . in the samples , subsequently , the amount of tnfα was quantified with the aid of a mouse - tnfα specific elisa according to the accompanying instructions ( immunosource , halle , belgium , cat . no . bms607mst ), and the amounts of tnfα in the blood plasma were subsequently plotted against time . addition of apoci to lps proved to lead to a fourfold increase of the lps - induced tnf level ( 10 . 8 .+− 0 . 4 . 5 versus 2 . 8 .+− 0 . 1 . 7 ng / ml ), while an equal amount of apoci without lps has no tnfα inducing effect ( n = 6 .+-. sem ). we have measured the correlation between plasma apoci levels in patients who did or did not develop endotoxemia during a heart operation with cardiopulmonary bypass , and the tnfα level during the operation . the apoci level proved to be strongly positively correlated to the tnfα response in case of a developed endotoxemia . during a cardiopulmonary bypass , temporarily a low oxygen pressure arises in the blood which is accompanied by an endotoxemia through leakage of lps from the intestines to the blood . we investigated the correlation between the preoperative plasma apoci level and the plasma tnfα level in 160 patients who had a heart operation involving a cardiopulmonary bypass . plasma samples were taken before the operation for the purpose of measurements of lipid parameters ( apoci , apociii , apoe , triglycerides and total cholesterol ). also , plasma samples were taken after inducing anesthesia ( time 1 ), after removal of the aorta clamp ( time 2 ) and half an hour after termination of the extracorporal perfusion ( time 3 ). at times 1 , 2 and 3 the tnfα level was determined , with a distinction being made between patients who did ( lps & gt ; 5 pg / ml ) or did not ( lps & lt ; 5 pg / ml ) develop endotoxemia at both time 2 and 3 . r = spearman &# 39 ; s rank correlation coefficient . *= statistically significant ( p & lt ; 0 . 05 ). a strongly significant relation was found to exist between the apoci level and the tnfα level in patients who during a heart operation with cardiopulmonary bypass developed endotoxemia , but not in patients who did not develop endotoxemia . such a correlation is not demonstrated for other apolipoproteins ( apociii , apoe ) or neutral lipids ( triglycerides as a measure for the amount of triglyceride - 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