ZA200602053B - Use of peptides derived from the A alpha or B beta chain of human fibrinogen for the treatment of shock - Google Patents

Description

. - 2 0 0 6 / 0 2 0 8 ackeround of the inverntion

The present inventi on is directed to a pharmaceut=ical preparation for the treatment of shack.

Shock is an acute ¢ omplication of many different pathological conditions chzaracterized by the inabilaty of the cardiovascular systerm to maintain an adequate perfusion pre=ssure. Infectious agents can directly or indirectly caus € a failure of the cardiova_scular sys-tem. Bacteria, bacterial toxins, virus and last but not le€ast an inadequate cellular or huroral host response involving inflammation and coagtlation can lead to a loss Of vascular ton. e, loss of vascular barrier function, loss of myocardial contractility and loss of <organ fun__ction, which alone or irr combination leads to shock amd finally to the death of &he patient.

Tre=atment of bacterial infection relies on antibiotic treatment, which kills the bactezna but this does not treat toxinemaa and does not correct for the inadequate cellular or hurmoral respoonse. In Gram-negative bacteria lipopolysaccharide (ZILPS or endotoxin) is respeonsible for the initiation of Gram-negative shock. Grain-positive bacteria can cause multipele organ fail" ure and septic shock without endotoxemia but the cell wall of Gram-positive baactena also con. tains toxins like lipoteichoic acid (LTA) and peptidogz lycan (PepG). LTA and FPepG act in syn ergy to releasc cytokine s such as tumor necrosis factom (TNF) a and interferon &IFN) v, to indmuce INOS and finally cause shock and organ failure.

Endotoxemia, sepsis and septic shock are associat ed with the generation of extensive amounts of nitric oxide (NCD). The excessive vasodilataticon and vascular hyporeactzivity to presssor agents associated with circulatory shock can be re=versed with inhibitors of the indi_1cible isoform of NO synthase (INOS) (Southan and Ss zabo, Biochem Pharmacol. 199 6;51:383-94, Thiemerme ann Gen Pharmacol 1997; 29: 159-66), but iNOS inhibi tors do not redi_1ce the organ injury caused by toxins (Wray et al. Shoe—k 1998,9:329-335).

Treatment of shock caused by viral infections is even a greater challenge, si_nce anti- vira-1drugs are not available for most infections. Treatmerts aiming to eliminate thes infe ctious agent alone are n ot sufficient in patients with stock due to an infectious agent, beczause secondary events initiated by the infectious agent involving an inflammatory reac~ tion and alterations in the coagulation system may hawe become independent ard lead to the leath of the patient irrespective of the question whethe=r the causative infectiouss agent has “been neutralized or not. A specific treatment is not available, thus current procesdures aim to re=lieve symptoms, which includes mechanical ventilaticon, fluid replacement, the use of card. 10 active drugs, strict control of oxygen saturation, hemoglobin, glucose and remal function. The control of the inflammation reaction only, e .g. with high dosage sterosids or the ve . 200 6/020 inh-bition of coagulation with antithrombin, does not produce improvement of survival. The onlzy molecule which so far has been proven to be of no#table cffectiveness in recucing mowmtality is activated protein C’, which interacts with czoagulation/fibrinolysis z=and the nfl ammation processes.

Shock during the course of an infection is mostly associated with overt or non-overt chamges in plasma fibrinogen accompanied by fibrin formation and by a raise o£ fibrin fragments. This activation of clotting as well as fibrinolytic pathways may resul tin overt or non -overt disseminated intravascular coagulation (DIC) resulting in vessel occliision and end -organ damage, and in consumption of coagulation {actors resulting in bleed_ing. Sepsis 1s the commonest cause of DIC. Importantly, fibrinogen, fmbrin and fibrin fragments play not onlyy a role in blood coagulation, but have several bindimg sites for cellular and rmatrix proteins, which allow them to interact with white blood cells, platelets, endothelial cells and matrix structures. This leads to cell activation, cell migr.ation, cytokine release a_nd ultimately an inflammatory reaction. The role of fibrinogen or fi brin in inflammation 1s -amply docmmented (reviewed by Altieri Thromb Haemost 82:77 81-786; Herrick et al. Itt J Biochem

Cell Biol 31:741-46). The D-region of the molecule con tains many binding site for matrix mol ecules, endothelial cells, platelets and inflammatory cells. The E-region of fm brin binds to

CD 1c (Loike et al. Proc Natl Acad Sci USA 88:1044-4.8).

We have recently described a novel role for the Bioeta,s.42 sequence of fibrin in inflammation (WO 02/48180). This sequence is also loc ated within the E-regiorm of fibrin and is only active when fibrinopeptide is cleaved. Fibrin fragments containing this sequence at thmeir free N-terminus of the beta chain bind to endothe=lium and cause inflammation, and a peptide matching the amino acids 15-42 of the Bbeta chaain of fibrin blocks bind ing of fibrin fragaments to endothelial surfaces and blocks inflammatison in vitro (WO 02/481280). In vivo, this peptide prevents myocardial inflammation and reducces myocardial infarct sm zes in situations of ischemia / reperfusion (WO 02/48180).

Fibrin fragments occur in any situation of impairecd fibrin formation and irmpaired fibri nolysis. Specifically in situations of shock due to an infectious agent, this al tered fibrin formation and fibrinolysis is a major problem. For many diseases a direct correl=ation between the outcome and the impairment of fibrin formaation/ fibrinolysis has beeen documented. E.g. Dengue (van Gorp et al. J] Med Virol 2002, 67:549-54, Mairulu et al.

Lancet Inf Dis 2003; 3:33-41). Adult respiratory distress syndrome (ARDS) isa form of acutee lung injury that is characterized by florid extravascular fibrin deposition (I dell Am J

Respir Med. 2002; 1:383-91). Thrombosis in the pulmonary vasculature and dissseminated intra_vascular coagulation have also been observed in ass. ociation with ARDS.

The reasons for the persistence/global emergence ©f Dengue fever (DF) armd hem-orragic Dengue fever (DHF) as a major public healttn problem are complex, vector

Cm control measures were not succeessful to eliminate DF/DHF. Currently the main focus of poublic sector funding for dengue research (estimated to be US$ 15 million in 2001) is on zrolecular epidemiology, immune pathophysiology, second gesneration vaccine discovery esearch, and new or uimprove=d approaches to vector control.

Several candidate vacci nes are in clinical trial in USA a.nd Thailand. but still no dru. g to treat infected patients is on . the market, even worse, currently no commercial chemo- t=herapy R&D activities appea-r to be under way. The world health organization has publisBhed

Strategic directions to fight DZF/DHF which - as high priority aims - include the developm- ent oof antivirals directed at protease or other poorly studied enzyrmes; the development of antm- rnediators directed at causes of increased vascular permeabilitty or altered haemostasis.

Summary of the invention “The invention relates to the uzsc of a peptide of general Formuala ©)

Ri } 0) _N—CH o—C—2Zy—2Z>

R2 wwherein

IR, and R,, being equal or diffferent, denote hydrogen, a satura_ted or unsaturated hydrocartoon roiety comprising from 1 to 10, in particular from 1 to 3, car bon atoms, =~, denotes a histidine or prolmne moiety, ~Z, denotes an arginine moietss/, a peptide moiety or a protein moiety comprising an initial arginine moiety, in particular comprising from 2 to 30 amino acids, which peptide has the biologi cal property of matching the inducible VE-cadherin binding

Lnotif on the BB-chain (i.e. Bf{3i5.4;) of human fibrin, for the preparation of a pharmaceutical preparation for the treatment - of shock.

AA peptide of general Formulas Il

O

Ri, I (10)

N—CH;—C—Zy——Arg—2Z3 — Za Zs

Ra ms preferably used, wherein oo ,2u CGA G200 8

Z, denotes a his®idine or proline moiety,

Arg denotes an arginine moiety,

Z3 denotes a pro line or valine moiety,

Z4 denotes a leucine or valine moiety,

Zs denotes a pepa tide moiety or a protein moiety in particular corprising from 2 to 230 amino acids or an alcotmol moiety comprising from 1 to 10, in particular— from 1 to 3, carbo n atoms

Or an organic or inorganic base moiety.

Furthermore, a peptide is preferably used in which Zs is a peptid-e moicty derived from the

Aalpha-chain or the Bbeta-chain of the fibrin.

Moreover, a pep tide 1s preferably used in which

Zs is a peptide moiety comprising the amino a cid sequence

Asp Lys Lys Argz Glu Glu Ala Pro Ser Leu Ar=g Pro Ala Pro Pro llc Ser Gly Gly Gly~ Tyr Arg

Z, is a histidine amoiety,

Arg Is an arginirae moiety,

Z3 1s a proline n—oiety, and 741s a leucine m_oiety.

Furthermore, a pe eptide is preferably used in w hich

Zs is a peptide ra olety comprising the amino a cid sequence

Glu Arg His Gln. Ser Ala Cys Lys Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp T-mp Asn

Tyr Lys

Z) is a proline moiety,

Arg is an arginine moiety,

Z3 is a valine moiety, and

Za is a valine mo-1ety.

Furthermore, thes invention relates to the use o fa peptide which e=xhibits the N-term inal sequence

Gly-His- #Arg-Pro-Leu-Asp-Lys-Lys-Ar-g-Glu-Glu-Ala-Proo-Ser-Leu-Arg-Pro— Ala- -Pro-Pro— Pro-lle-Ser-Gly-Gly-Gly-Tyr—Arg

LU Uuiulund and which has the bic logical property of matching the insducible VE-cadherin binding motif on the BB-chain (i.e. “Bf )s.42) of human fibrin for the pre—paration of a ph .ammaceutical preparation for the treatment of shock.

A further preferred ermbodiment of the use according to tkhe invention is characterized in that the peptide 1s

Gly-His-Arg-"Pro-Leu-Asp-Lys-Lys-Arg-Glu-Glum-Ala-Pro-Ser-L- eu-Arg-Pro-Ala- -Pro-Pro-Pro- lle-Ser-Gly-Gly-Gly-Tyr-Arg.

It has been shown that in particular shock conditions car be treated with the above- mentioned peptides, “wherein shock is associated with ore or more from . the group comprising bacterial toxins, disseminated intravascular coagulopathy, n- ecrotizing fasciitis, haemorrhagic shock following viral infection, in particu lar caused by fillovirus, arenaviridae, bunyaviridae, flavivi rus, dengue, acute hemorrhagic respoiratory failure caused by infectious agents or autoimmurne diseases, organ failure after orgarm injury, in parti- cular myocardial infarction, vascular szurgery, clamping of organs, haemorrhagic shock, lung infarction, liver infarction, gut infarction, surgical procedures and stroke=, and organ dys. function of grafted organs.

Detailed Description of the Invention

Peptides and Protein_s

Peptides were produced by solid-phase peptide synthesis and purim fied with reversed- phase HPLC using naucleosil 100-10C18 columns (PiChnem, Graz, Aust- ria). It should be noted that the beta 1 5-42 region is 100% similar amongs species when as llowing for conservative amino acid substitutions. The N-terminal clisulfide knot otf fibrinogen (NDSK) composed of amino acids Aal-51, BB1-118 and y1-78 was prepared ass previously described (WO 02/48180). Thee N-terminal disulfide knot of fibrirn (NDSK-II, wh _1ch lacks fibrinopeptides A ard B) composed of amino acids Ao. 17-51, BB15-11 8 and y1-78 was prepared by treating NDSK with thrombin (20 U/ Img of NDSK) for =3 h at 37 °C. Residual thrombin was neutraalized with 10 mM disopropyl fluor-ophosphate (Fli aka, Milwaukee, WI) for 2 h at 37°C. All products were then dialyzed into ptosphate buffere=d saline (PBS).

ELISA

Peptide BB) s.4; bincds to VE-cadherin

The interac tion of the Bbeta chain (E3beta;s_42) of +200 6/02053 morphologic charges (Bunce et al. J Clin Invest 89:842-50; Bach set al. Exp Cell Res 238:324-34; Challupowicz et al. J Cell Bio 1130:207-15; Hamaguchi et al. Blood 81:234& -56;

Francis et al. Bloaod cells 19:291-306), proliferation (Sporn et al. F3lood 86:1802-10), the release of von Wm llebrand factor (Ribes et al. J Clin Invest 79:117—23, Ribes et al. J Chin

Invest 84: 435-42* ; Erban and Wagner, J B iol Chem 267, 2451-58) and possibly IL-8 (Q1 et al. Blood 90:3593-3602) and membrane expression of CD54 (Harley et al. Art Thromb asc

Biol 20:652-658) . VE-cadherin has been identified as a binding ligand of the sequence

Bbeta)s..; and EL _ISAs have been developed to demonstrate this in_teraction of endothelia 1 cells and/or VE-cadherin with fibrin or fib rin fragments. Martinez et al have used anti-pean cadherin antibodies to capture cadherins from endothelial cells followed by incubation w ith fibrin (Martinez e=tal. Ann NY Acad Sci 9 36:386-405), HUVEC monolayers (which express

VE-cadherin) hav=e been overlaid with rad x o-1abeled fibrin fragmerts or peptide Bbetays., 2 (Bach et al. J Bioll Chem 273:30719-28; Harley et al. Art Thromb “Vasc Biol 20:652-658) , and recombinant “VE-cadherin was used bys Gorlatov and Medved (Biochemistry 41:4107 - 16). Others have Lased ELISA for detectiora of fibrin fragments witknin the blood, mostly by using antibodies to distinct sequences withrin the fibrinogen molecule including antibodies against the Bbetay sa; motif (reviewed in F areced et al. Clin Chem 8 :1845-53).

We have de veloped a modified ELIS A working with the sanme principles described by others, but the purx-pose of the herein descri bed ELISA is not to quaxntify fibrin degradatior products, but to search for proteins, peptid €s or compounds which interfere with the bindang of the Bbeta;s4> sequence and the VE-cadlerin. The principle is that the VE-cadherin, eit her as a truncated protein, as a full protein or coupled with other protei ns which do not interfere with the Bbeta)s.q— binding site, is allowed to interact with the Bbewta; sa; sequence of fibrin.

Into this system omne can introduce any other additional substance a_nd measure if this substance inhibits VE-cadherin / Bbeta,s.4= binding.

In detail, 96 well protein immobilizer plates (Exiqon, Vedbaesk, DK) were coated w ith recombinant humaan VE-cadherin FC fusion protein (8 nM/ml; R&D Systems, Minneapol is) in PBS and were 1 eft overnight at 4 °C. Plates were then washed arnd incubated with pepti de

Bp )s.42 (CHRPLIOKKREEAPSLRPAPPPI SGGGYR) tagged with a FLAG-sequence (DYKDDDDK) ast the C-terminus of the peptide or with a FLAG-taagged random peptide (DRGAPAHRPPFRGPISGRSTPEKEKLLIPG) at a concentration ofZ 0-30 pMol. After washing, bound FLAG-tagged peptide was detected by incubation —with a peroxidase-labe 1led anti-FLAG antiboady (Sigma, St. Louis, US A) and chromogenic sutostrate. Optical density was determined bx an ELISA plate reader set at a wavelength of 45 0 nm. Data represent tte mean of three inde=pendent experiments, ea ch performed in triplicatzes. The table below

©. 2006/0 2053 slhh.ows that the peptide Bf 5.4; bound to VE-cadhe=rin in a concentration-de=pendent manner.

In contrast, the random peptide demonstrated onl—y insignificant binding.

Dense dependent binding of peptide Bbeta;s.4r to WW E-cadherin _ vi | lo bash pals Je poss bs po

Te bb panos unk bs hob

FIL _AG |mean 0.010.0210.0810.33 }0.92]1.3 1.5 1.93 2.1 M

Ep

TL | al bbb bake

FL_AG [mean 0.01 0.01]0.03 ]0.12/0.2 0.35 {0.5 bob bob bolo joosr] fo lo

Pe=ptide BB s.42 and fibrin fragments compete for binding to VE-cadherin.

In a next step, we analyzed whether this EL. -ISA can be used to scree for other pe ptides/compounds to compete with the binding of the Bf)s.42 sequence t=o0 VE-cadherin. As ex pected, peptide BB s.4> completely inhibited birding of the flag-tagged oeptide Bf5.4; and wzas used as the positive control and random peptides or solvent had no effect and were used as negative controls. Shorter peptides partially inlmibited the binding of BB 15.42 to VE- ca-dherin. NDSK-II inhibited Bf315.42 binding in a concentration-dependent fashion. An eq uilibrium between Bf s.42 and NDSK-II (50% 12nhibition) was reached a” t a molar ratio of 24-:1. NDSK had little or no effect.

VE-cadherin was coated to the plastic surfaece at a concentration of 8 nM. Then inclicated peptides were added at concentrations o=f 200 uM, NDSK or ND. SK-II were added at indicated concentrations. Detection of binding eof the FLAG-tagged Bbe ta)s.4; (12 nM) was performed as described above.

Blocking reagent % inhi bition of 15-42FL AG-binding to VE-cadherin mean + SD peptide 15-42 (28 mer) 10010 peptide random (&3mer) 3£3 peptide random (2 8mer) 103 solvent 0+0 peptide 15-18 (diiaer) 200 uM 65+ 12 peptide 15-26 (12-mer) 200 uM 64 + 10 peptide 15-30 (16Gemer) 200 pM 61 +13 peptide 15-34 (20 mer) 200 pM 67 £17 peptide 15-37 (244 mer) 200 pM 17+ 19 peptide 16-42 (277 mer) 200 pM 55 +13 peptide 15-18 (drier) 12 uM 7x2 peptide 15-26 (12mer) 12 uM 6x1 peptide 15-30 (16 mer) 12 pM 6+3 peptide 15-34 (2 mer) 12 pM 7x1 peptide 15-37 (2c mer) 12 uM 7+2 peptide 16-42 (2°~/mer) 12 uM 5+2

NDSK-II 0.06 ph 1+0

NDSK-II1 0.12 ph 39 +18

NDSK-II 0.20 pu=1 42 + 14

NDSK-II 0.60 p= 52 +16

NDSK-II 1.2 pM 63 +13

NDSK-I1 2.4 pME 79 +9

NDSK-II 4.0 pMT 82 +12

NDSK 0.06 uM 0-0

NDSK 0.12 uM 2 +1

NDSK 0.20 uM 1 +1

NDSK 0.60 pM 7+6

NDSK 1.2 pM 15+13

NDSK 2.4 1M 16 +9

NDSK 4.0 pM 20+ 10 anti-VE-cadherien Ab (TEA1/31, 1 mg/ml) 2+1

. 20u5/020 53

Effectivesness of peptide bbeta;s 4; for the treatment of der gue virus infected mice.

Material s and Methods.

Vizarus. Dengue virus type 2 (DEN-2), strain P23085 , was obtained from the Staate

Collection of Viruses, Moscow, Russia in the form of infe cted ICR mouse brain lyophilized suspensieon. The obtained Dengue virus has undergone passsages in the brain of the suckling

ICR micee as described earlier (Atrasheuskaya et al. FEMS Immunology and Medical

Microbiology. 35, 33-423). Ten % brain suspension servecd as a virus stock and was sstored at -40°C. T he virus titer was determined by the serial dilutioras of brain suspension. Bra_in suspensicon was inoculated i.p. in groups of 10 mice (4-weeek old BALB/c) each and t-he mortality~ was recorded. The virus titer was calculated, and_ it was at 7.4 1g LDS0/ml.

All work: with the infectious virus was performed in the maximum containment bioszafety level-3 (E3SL.-3) laboratory of the SRC VB «Vector» (Russ. 1a).

Animals.

Fovar-week old inbred male BALB/c mice (haplotypes H-2d) were received fromm the vivarium of the State Research Center of Virology and Bico technology «Vector». Anir=mals were placed in individual cages with food and water availaz ble ad libitum.

Assays.

Blood was taken from mice under methoxyflurane aresthesia from the orbital s inus before infection and after the challenge with DEN-2. Threes mice for each time point =were used for Faarvesting blood.

Cir culating platelets (PLT), red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB) and hematocrit (HCT) were determined by using Cell --Dyn 900

Hematology analyzer (Sequoia-Turner corporation, USA, CCA).

Part of th e harvested blood was centrifuged for obtaining s: erum, which was stored at 80°C till the en.d of experiment. Serum levels of cytokines were mmeasured by using enzyme= immunoa_ssay kits produced by R&D Systems (Minneapoli s,USA) according to the manufact-ure’s instructions. Detection limits were as follow—s: TNF-a, less 5.1 pg/ml; interleukin (IL)-18, 3.0 pg/ml; IL-6, 3.1 pg/ml; IFNy- less 220 pg/ml.

Dergue virus in the animals’ blood was identified by- RT-PCR as described earl? ier (Harris et. al. J. Clin. Microbiol. 36, 2634-2639). Total RNA from the blood was isola. ted using a kizt from Quiagen (Germany). Primers were as follos wing: upper

S’AATA"TGCTGAAACGCGAGAGAAACCG (position 1 36-161), lower 5’AAGG_AACGCCACCAAGGCCATG (position 237-2583), amplifying a 119 bp pro duct.

To quanti_fy the virus load, DEN-2 was titrated onto Vero E=6 cell cultures as describeadd earlier (Harris et al. J. Clin. Microbiol. 36, 2634-2639). On. day 0 and 22 after the cha llenge

° L 2008/02023 blood of the surviving mice was analyzed for an ti-DEN-2 antibodies (IgG) by ELISA as described earlier &Ignatyev et al. J. Biotechnolo=y. 44, 111-118).

Design of experirment.

Inbred four—week old male BALB/c mice =were divided into 6 main groups. Each group contained 50 mices. All animals were infected intraperitoneally (i.p.) with the mouse-adapted

DEN-2 strain P22 085 (as described above) with a dose of 1 LDsgand exammined daily for signs of morbidit=y. Mice from the first subgroups of all main groups (Al--F1) were used for the mortality cont=rol. Each subgroup contained 220 mice. Animals of the s econd subgroups (A2 - F2) were ussed for obtaining serum sample=s. Each subgroup containeed 30 mice.

Group descriptior. n= 50 in cach group

Control group received only virus.

Treatment vith peptide Bf 5.42 was performed twice per day, 4800 pg/kg cach by intraperitoneal ing ection from day 3-post infecticon to day 8-post infection

Blood and serum samples were obtained at the selected time points: day 1. ,3,5, 7,11, 22 after the challengee.

Statistical analysis was conducted using Student’s t or Chi-square test. P-values <0.05 were considered signifmcant.

Table 1. Mortality and IgG titer. p<<0.05 betwee=n groups

ET ee (%) (%) ; unrated {40 60 |6soosn24s [1160

Bp 0 100 |Almicesurvived fj120

Table 2

Jews [BBueteaed fas [sl laws 33

IL-6 pam] 100 viremia lg PFU/m! [controls |Bbie gay | 0 | 0

6 62:04 [0:04 2s | oo | o

Gram-negative shock

Male Wistar rats voveighing 230-280 g were housed in the Tierversu.chsanlage (University of Diisseldo rf) and fed on a standard diet with water ad libiti=¢m. All procedures were carried out in acco-rdance with the AAALAC guidelines and Guide _ for the Care and

Use of Laboratory Anin—als (Department of Health and Human Services, National Institutes of Health, Publication Mo. 86-23). In addition, all experiments have beer approved by an ethical and research boaard of the University of Diisseldorf and the county. As previously described (Zacharowsk) . et al. Crit Care Med 2000, Zacharowski et al. (zit Care Med 2001; 29:1599-1608), rats weme anaesthetized with sodium thiopentone (120 m g/kg i.p.) and anesthesia was maintaired with supplementary doses of sodium thiopent_onc as required.

The trachea was cannulated to facilitate respiration and rectal temperature was maintained at 37 °C witch a homoeothermic blanket. The right carotid art ery was catheterized and connected to a pres sure transducer for the measurement of phasic and mean arterial blood pressure (MAP) &and heart rate (HR) which were displayed on a damta acquisition system (MacLab 8e, AIDI Instruments, Germany) installed on an IBM cosmputer. The right jugular vein was-cannu-lated for the administration of drugs. The bladder— was also cannulated to facilitate urine flow &and to prevent the possibility of the development of post-renal failure.

All animals received a *total fluid replacement of 1.0 ml/kg/h (0.9% sodi um chloride, saline, as an i.v. infusion nto &he jugular vein) throughout the experiment. Upo n completion of the surgical procedure, carcliovascular parameters were allowed to stabilize for 15 min and constantly recorded over 6 h. In this model of LPS-induced multiple orgs an failure, a period of 6 h is essential to achieve a significant rise in the serum levels of AST and ALT, while a significant rise in the sesrum levels of urea and creatinine can already be observed after 2 h.

Three groups were stucied:

Rats were subjected to sham operation: (sham).

12 F. 2006/020%3

Rats were subjected to Gram-negative shock. Lipopolysacchar-ide from E. coli, serotype 0.12 7:B8 (6 mg/kg i.v.) was given over 5 min i.v., 1 h later, armimals received saline (2.4 ml/kzg): (LPS + saline).

Rats were subjected to Gram-negative shock. Lipopolysacchar—ide from E. coli, serotype 0.12 7:B8 (6 mg/kg i.v.) was given over 5 min 1.v., animals rec eived BB5.42 (2.4 mg/kg): (LPS + BBs.a2).

Survival n=20 in each group, p<0.05 25%

Six hours after the induction of Gram-negative shock, bl ood was collected from th e catheter placed in the right carotid artery. The blood sample w as centrifuged (1610 x g fcor 3 min at room temperature) to separate plasma. The following narker enzymes were meas-ured in th_e plasma as biochemical indicators of multiple organ injumy/dysfunction:

Liver injury was assessed by measuring the rise in plasma levels of alanine amirotransferase (ALT, a specific marker for hepatic parenchs/mal injury) and aspartate amuotransferase (AST, a no n-specific marker for hepatic injury).

Renal dysfunction was estimated by measuring the rises in plasma levels of urea (2an indicator of impaired excretory function of the kidney and/or inereased catabolism) and creatinine (an indicator of reduced glomerular filtration rate, and hence, renal dysfunctio n).

Plasmma levels of glucose and amylase were measured as indire=ct markers of pancreatic func tion and injury.

In ac3dition, arterial pO; was measured as indirect marker of lung function/injury. Laboratory values ie jo [Ben

ALT

52

Co tar 529.0 30.8

Creamunine [05 Jos Jos ] I

SEAT me _ : "2008/0205 1 =

A

1315 [753 [45 8 1713.3 [1837.4 19 45.1 131.5 122.7 1776.8 p®2 |900 lev.0 leg

SEEM mean arterial pressure sham LPS LPS +

BBis5.42 0 |1209 55 [1189 54 128.9 52 1115 106 [90.7 55 837 46 1132 73 [1002 5.1 1023 4 116.4 54 |90 7.4 1032 4 1087 89 [811 7.9 1012 3 1041 96 (60.1 10.8 |971 54 6 |1041 96 [341 77 107.7 96 he . art rate sham LPS LPS

CC] BBis.42 mean SEM os |4822 17 457 18.1 4369 6.4 461.7 121 [511.2 27.4 (4848 18.6 488 1 13.6 |523.3 273 [4841 10.3 506.6 26.6 | 518.5 249 [5098 12 488.9 174 | 516.7 321 5167 13.4 sham LPS LFS

Bf 315.0 mean SEM

I EE

6 |4437 0.7 530.1 277 |52416 244

Organ (lung, liver, heart and k idney) biopsies of all groupos studied were taken at the end of tthe experiment. The biopsies were fixed in buffered formaldehyde solution (4% in phosphate buffered saline) at room t emperature and sent to Vienna. Standard H&E—stained sections revealed no differences. However, in sections stained for fibrin deposits using aci dic fuchsin -— orange G, we found significantly elevated numbers oe f fibrin thrombi in controls receiving LPS alone as compared wath animals treated with LP®S plus Bf5.42 (p<0.05). In sham trezated animals no fibrin thrormbi were present. mean number of fibrin thrombi withain vessels

EEE TE hear 47+ 41 78 +37 lung 1 +0

Claims (1)

1. CLAIMS:

   1. The vase of a peptide of general Formula Oo Ry, | 0) N—CHy—C—Z2y—2; R2 wherein R; and R,, I>eing equal or different, denote hydmrogen, a saturated or unsaturated hydrocarbon moiety com prising from 1 to 10 carbon atoms, Z, denotes a histidine or proline moiety, Z, denotes aan arginine moiety, a peptide moietsy or a protein moiety comprising an initial arginine momiety, which pepti de has the biological property of matching the inducible VE-cadheri n binding motif on the BB-chain (i.e. BP;s4;) of human fa brin, for the preparation of a pha.rmaceutical preparation for the treatment of shock.

   2. The use according to claim 1, wherein the saturated or unsaturated hydrocarbon moiety of RX | and/or R; comprises from 1 to 3 carbon atoms.

   3. The use according to claim 1, wherein =Z; comprises from 2 to 30 amino acids. 4, The= use according to claim 1, character—ized in that the peptide exhibits the general Formula Il 0 Ri, Il (In N—CH;—C—Zg—Arg —L3—Ld4—L5 R2 wherein 7, denotes a histidine or proline moiety, Amended s heet 15/06/2007

   Aarg denotes an arginine moiety, Zs denotes a proline or valine moiety, Z_, denotes a leucine or valine moiety, Zs denotes a peptide moiety or a protein moiety, om an alcohol moiety comprising from 1 to 1 Ocarbon atoms, or an organic or inorganic base nmoiety.

   Se. The use according to claim 4, characterize in that Zs is a peptide noiety or protein moiety comprising from 2 to 30 amino acids.

   5. The use according to claim 4, characterized in that Zs 1s an alcohol moiety comprising from 1 to 3 carbon acids.

   7. The use according to any one of claims 4 t- 0 6, characterized in thatz Zs is a peptide r-moiety derived from the Aalpha-chain of the fibrim.

   =. The use according to any one of claims 4 to 0, characterized in thas Zs is a peptide moiety derived from the Bbeta-chain of the fibrin . *9Q, The use according to claim 4, characterize=d in that Zs is a peptide moiety comprising the amino acid sequence Asp Lys Lys Arg Glu Glu Ala Pro Ser Leu Arg P=ro Ala Pro Pro Ile Ser G" 1y Gly Gly Tyr Arg Z, is a histidine moiety, Arg is an arginine moiety, Z3 is a proline moiety, and Zs 1s a leucine moiety.

   10. The use according to claim 4, characterized in that Zs is a peptide moiety comprising the amino acic sequence Amemnded sheet 15/06/2007

   Glu Arg His Gln Ser Ala Cys Lys Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp Trp Asn Tyr Lys Z, is a proline moiety, Arg is an arginine moiety, Z3 is a valine moiety, and Z4is a valine moiety.

   11. The use of a peptide which exhibits the N-terminal sequenc=e Gly-His-Arg-Pro-Leu-Assp-Lys-Lys-Arg-Glu-Glu-Ala-Prco-Ser-Leu-Arg-Pro-Ala- -Pro-Pro-Pro-Ile-Ser-Gly~-Gly-Gly-Tyr-Arg which peptide has the biological property of matching the inducible VE-cadherin binding motif on the BB-chain (i.e. BBs x2) of human fibrin, for the preparation of a pharmaceutical preparation for the treatment of shock.

   12. The use according to clairm 11, characterized in that the pepotide is Gly-His-Arg-Pro-Leu-A__sp-Lys-Lys-Arg-Glu-Glu-Ala-Pr -o-Ser-Leu-Arg-Pro-Ala- -Pro-Pro-Pro-lle-Ser-Glsy-Gly-Gly-Tyr-Arg.

   13. The use according to any of claims 1 to 12, wherein shock is associated with one or more from the group comprisin_g bacterial toxins, disseminated intravascular coagulopathy, necrotizing fasciitis, haemorrha_gic shock following viral infecticon, acute hemorrhagic respiratory failure caused by infectious agents or autoimmune di_seases, organ failure after organ injury, vascular surgery, clamping of organs, haemorrhagic shock, lung infarction, liver infarction, gut infarction, surgical procedures and stroke, armd organ dysfunction of grafted organs.

   14. The use according to claimm 13, wherein the viral infection is caused by filovirus, arenaviridae, bunyaviridae, flaxsivirus or dengue. Amended sheet 15/06/2007

   15. The use according to claim 13, wherein the organ failure after organ injury is myocardial irmfarction.

   16. The u se of a peptide according to claim 1, substant: ally as herein described wasith reference to any one of the illustrative examples. Amended sheet 15/06/2007