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    • 专利摘要:
    The invention further relates to a pharmaceutical composition comprising a first and a second polypeptide for use in the prevention or treatment of a periodontal disease, wherein the first polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%>SI) with SEQ ID 1 and, the second peptide has at least 90%>, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with SEQ ID 2.
    公开号:SE1650188A1
    申请号:SE1650188
    申请日:2016-02-12
    公开日:2017-08-13
    发明作者:Bengtsson Torbjörn；Khalaf Hazem
    申请人:Bengtsson Torbjoern；Khalaf Hazem；
    IPC主号:
    专利说明:

    160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx l USING BACTERIOCINS IN THE PREVENTION ANDTREATMENT OF PERIODONTITIS Field of the InventionThis invention pertains in general to the field of treatment periodontal diseases.More particularly the invention relates to a use of a bacteriocin for the prevention and/or treatment of infectious periodontal diseases, including periodontitis.
    Background of the Invention Periodontitis is a common chronic inflammatory disease caused by anaccumulation of different pathogenic biofilm-forming bacteria in dental pockets, leadingto an exaggerated immune response that destructs periodontal ligament and causesalveolar bone loss. Porphyromonas gíngívalís is considered a keystone pathogen amongthese pathogenic bacteria and is well associated with the development of periodontitis.P. gíngívalís is an anaerobic gram-negative, black-pigmented bacterium that has beenwidely associated as a putative organism in causing aggressive periodontitis andprogression into systemic diseases. The ability of P. gíngívalís to invade host cells,including gingival epithelial cells and heart and aortic endothelial cells demonstrate apossible mechanism for its establishment and subsequent pathogenesis by evading thehost immune system. P. gíngívalís express multiple virulence factors (fimbriae, capsule,LPS, proteinases and toxic end-products) that contribute significantly to theirpathogenicity by altering host immune responses. However, P. gíngívalís virulence isprimarily associated with the elevated proteolytic activity of cysteine proteinases thatare divided into arginine-specific (Rgp) and lysine-specific (Kgp). Besides theimmunomodulatory effects of P. gíngívalís, the bacterium is also able to form biofilmscontaining extracellular polymeric substances that provides antibiotic resistance and aniche for long-terrn survival within the host by evading host immune responses.
    These biofilms are difficult to treat and conventional methods are still beingused to treat patient with periodontitis, including mechanical removal of hard and softsubgingival biofilms, scaling and chemical treatment to control plaque (metallic salts,antibiotics and phenols). These methods are less efficient since within a few hours newbiofilms are formed that are prone to cause infections. Furthermore, periodontitis hasbeen associated with other systemic conditions, such as atherosclerosis, followingidentification of periodontal pathogens, including P. gíngívalís, in atherosclerotic plaques. It has recently been shown that P. gíngívalís induces gene expression of 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 2 angiopoietin 2 in aortic smooth muscle cells, which increases the migration of the cellsthat is associated with the pathogenesis of atherosclerosis.
    Bacteria growing as adherent biofilms almost always lead to a significantdecrease in susceptibility to antibiotic therapy compared with cultures grown insuspension. Several mechanisms have been suggested for the tolerance to antibiotics ofthe biofilm. One such suggested mechanism includes the prevalence of dorrnant cells,with decreased bacterial metabolic activity and increased doubling times, inside thebiofilm, due to a gradient of nutrients and oxygen that will form from the top to thebottom of the biofilm. However, since there is no generally agreed mechanism for theresistance of biofilm bacteria, it has been hard to device strategies for combating biofilmbacteria, and various elements in the process of biofilm formation are currently studiedas targets for novel drugs and delivery technologies.
    It is known that antibiotic therapy is the first altemative to combat bacterialinfections in humans, but the occurrence of bacterial resistance to conventionalantibiotics is a growing public health problem. Consequently, there is today an intensesearch for new antimicrobials with effective activity and with less possibility to induceantimicrobial resistance. Thus there is a need for altemative method to antibiotic therapy in the prevention and treatment of infectious diseases, including periodontitis.
    Summary of the Invention Accordingly, the present invention preferably seeks to mitigate, alleviate oreliminate one or more of the above-identified deficiencies in the art and disadvantagessingly or in any combination and solves at least the above mentioned problems byproviding a pharrnaceutical composition comprising a first and a second polypeptide foruse in the prevention or treatment of a periodontal disease, wherein the first polypeptidehas at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with SEQ ID 1and, the second peptide has at least 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity (%SI) with SEQ ID 2.
    More particularly, the first and second peptides are present in a ratio of 1:20 to1:1, preferably 1:12 to 1:4, most preferably 1:8. The first polypeptide is the ot peptideand the second polypeptide is the ß peptide, respectively, of the bacteriocin NC8 ußfrom L. plantarum NC8.
    According to one aspect of the invention, the composition is for use for promoting and enhancing wound healing and regeneration. 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 3 According to another aspect of the invention, a composition comprising a firstand a second polypeptide is for use in coating a medical device to limit P. gingivaliscolonization on its surface, wherein the first polypeptide has at least 90%, 95%, 96%,97%, 98% or 99% sequence identity (%SI) with SEQ ID l and, the second peptide hasat least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with SEQ ID 2.
    Brief Description of the Drawings These and other aspects, features and advantages of which the invention iscapable of will be apparent and elucidated from the following description ofembodiments of the present invention, reference being made to the accompanyingdrawings, in which; Figures lA and B, wherein lA shows a graph of inhibition of P. gingivalisgrowth by Lactobacillus . Cultures were acquired after coculture of P. gingivalis withLactobacillus (L. brevis, L. plantarum 44048 and L. plantarum NCS) for 4 days, andprevention of P. gingivalis growth by Lactobacillus was shown to be species dependent.In lB, images acquired after coculture of P. gingivalis with sterile filtered supematantsfrom Lactobacillus, for 4 days, at 40>< magnif1cation, are shown. White lines indicatethe inhibition zone; Figure 2, visualizes the antimicrobial activity of NCS otß from L. plantarumNC8 on wild type (WT) P. gingivalis ATCC 33277 and W50, respectively, usingSytox® Green fluorescent dye. Representative images of three independentexperiments; Figure 3 is a bar graph of Zeta potential and size of liposomes and W50microvesicles. The zeta potential and size of liposomes with different lipid compositionwas measured to identify the best match with microvesicles from P. gingivalis W50. Allmeasurements are done in triplicates; Figures 4A to C, shows dose- and time dependent release of CF in response tobacteriocin NC8 uß, where 4A shows the release of 5(6)-carboxyfluorescein (CF) fromliposomes (5:95 POPS:POPC) after 30 min with addition of NC8 u (square), ß(diamond) and otß l:l (ball). The interaction kinetics was recorded every minute forNCS ot in 4B, and NC8 ß in 4C, with the liposomes and is displayed in increasingbacteriocin concentration (0.005-2 uM) indicated by the arrow. CF release without addition of bacteriocins were < l % and all data point are average of n = 3; 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 4 Figures 5A and B, shows CD spectra illustrating the secondary structurechange of NC8 u and ß. 5A shows the CD spectra of 100 uM NC8 u and 5B NC8 ß insolution (dashed lines) and with 1 mM (total lipid concentration) 5:95 POPSiPOPCliposomes (solid lines) in 10 mM PB pH 7, 20°C; Figure 6 shows bar graphs representing NC8 uß binding to P. gíngívalís in adose-dependent manner. Binding of NC8 uß to P. gíngívalís was analyzed by SPR. BothP. gíngívalís ATCC 33277, shown in 6Aand W50, in 6B, were found to bind toimmobilized NC8 uß (280 nM). The binding was verified by pre-incubating the bacteriawith different concentrations of soluble NC8 uß prior to analysis, which resulted in asignificantly reduced binding to the immobilized bacteriocins. Results are presentedfrom three independent experiments.; Figures 7A and B shows bar graphs representing NC8 uß reduction of thebinding of P. gíngívalís to immobilized anti-P. gíngívalís antibodies. Binding of P.gíngívalís to the immobilized anti-P. gíngívalís antibodies was analyzed by SPR. Pre-incubation of P. gíngívalís ATCC 33277 (figure 7A) and W50 (figure 7B) withincreasing concentrations of soluble NC8 uß prior to analysis reduced the bacterialbinding to its antibodies in a dose-dependent manner. Results are presented from threeindependent experiments. One-way ANOVA with Tukey's multiple comparison test(*p<0.05; **p<0.01; ***p<0.001); Figure 8 shows bacteriocin NC8 otß damaging the membrane of P. gíngívalís.In 8A P. gíngívalís ATCC 33277 were either left untreated or treated with 280 nM ofNC8 otß in a ratio of 1:1 for 2 min and 10 min. Bacterial ultrastructure was examinedusing a Hitachi HT 7700 transmission electron microscope and showed typicalcoccobacillus shapes of untreated P. gíngívalís, where the outer membrane (OM) andcellular membrane (CM) could be clearly distinguished. Treatment with NC8 otßefficiently damaged P. gíngívalís cells by causing rupture of the bacterial membraneand leakage of intracellular content (full arrow), eVentually resulting in completelydetached outer membrane (arrow heads). Scale bar = 200 nm; Figure 9, Hydropathy scores of NC8 u and ß. The amino acid sequence ofNC8 a) ot and b) ß and their corresponding hydropathy score (Kyte J and Doolittle RF.Joumal of molecular biology. 1982;157(1): 105-32).
    Figure 10 shows how the two-peptide bacteriocin NC8 uß introduces epithelialcells to proliferate (top), and summarized in bar graph (bottom left). Initial characterization of the molecular mechanisms indicate that the intra cellular signaling 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 5 protein Akt and the growth factor TGF-Bl are involved in this process (bar graphbottom right); Figure 11 shows how NC8 otß prevents gingival epithelial cell death caused bythe pathogen P. gíngívalís, where cell viability in trials with and without NC8 otß issummarized in the bottom graph; and Figure 12 visualizes how the antimicrobial activity of NC8 otß from L.plantarum NC8 on wild type (WT) P. gíngívalís is enhanced when the ratio ouß isshifted towards a higher proportion of ß, i.e. from 1:2 to 1:8, using Sytox® Greenfluorescent dye.
    Description of embodiments The following description focuses on an embodiment of the present inventionapplicable to using bacteriocins in the prevention or treatment of a periodontal disease,such as periodontitis. However, it will be appreciated that the invention is not limited tothis application but may be applied as an altemative method to antibiotic therapy in theprevention and treatment of a wide range of infectious diseases, related toPorphyromonas gíngívalís (P. gíngívalís). Furthermore, the bacteriocins can be used forcoating of surfaces, such as for coating of medical instruments to hamper P. gíngívalíscolonization.
    Attractive substances from which novel antibiotics may be developed are thebacteriocins, a group of bacterially produced peptides used to fight other bacteria.Bacteriocins have a net positive charge and express amphipathic structures that interactwith negatively charged microbial membranes and kill microbes usually through pore-forrning mechanisms. These mechanisms are more difficult to evade by developingresistance, compared to metabolic enzymes which usually are targets for conventionalantibiotics. Bacteriocins are divided into three classes depending on their characteristics.Class I (lantibiotics) are small peptides (< 5l antimicrobial peptides secreted by bacteria as part of their defence mechanism. 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 6 Lactobacillus species are part of the normal flora of humans, and have beenreported to be important contributors to the balance of oral microflora by preventingpathogen colonization and thus maintaining a healthy state. However, the mechanismsby which Lactobacillus interacts with periodontal pathogens and promotes healthbenefits are sparsely investigated. The oral cavity harbors a wide variety of bacteriafrom the genus Lactobacillus that play an important role in the maintenance of a healthybalance of the oral ecosystem and are suggested to have protective effects in thepathogenesis of periodontal disease. However, many Lactobacillus species areoutcompeted by gram-negative pathogens, including P. gingivalis, during diseaseprogression. However, it has been reported that certain Lactobacillus species, includingL. rhamnosus and L. salivarius, possess antimicrobial activity against periodontalpathogens, such as P. gingivalis. Further, it has been reported that bacteriocins from L.paracasei were able to kill P. gingivalis at a minimal concentration of 0. 14 mM(Pangsomboon K. et.al. Archives of oral biology. 2006;51(9):784-93). In comparison,results for a pharrnaceutical composition from the present invention (SPR analysis,figure 6A,B and figure 7 A,B ) show that concentrations within the nanomolar rangeeffectively alters P. gingivalis.
    It was envisaged by the present inventors that specific Lactobacillus speciesindeed may be able to suppress periodontal pathogens primarily through expression andsecretion of certain bacteriocins. Further it was envisaged that prevention ofperiodontitis can be accomplished through preventing colonization of the pathogen P.gingivalis by use of bacteriocins. Thus, it was hypothized that deterrnining the anti-bacterial activity of Lactobacillus species and their bacteriocins against P. gingivalismay lead to alternative methods to antibiotic therapy for the treatment and/or preventionof periodontitis.
    The oral cavity harbors a wide variety of Lactobacillus species, including L.gasseri, L. salivarius, L. brevis, L. plantarum and L. rhamnosus. Although the majorityof these species are frequently found in the mouth, we show that they possess differentspecificity towards inhibition of P. gingivalis.
    The ability of different Lactobacillus strains to inhibit P. gingivalis growth wasassessed on fastidious anaerobe agar plates, by exposing different P. gingivalis strainsto Lactobacillus strains. It Was found that L. plantarum NC8 and 44048 Were able tosignificantly suppress P. gingivalis growth. This is illustrated in figure 1A, where L.brevis 30670, L. plantarum NC8 and 44048 are compared, showing that L. brevis 30670is ineffective in inhibiting P. gingivalis growth while L. plantarum NC8 and 44048 both 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utaraft_EBR.2016021201383320043.aocx 7 significantly suppressed P. gingivalis growth. L. plantarum NC8 has been isolated fromferrnented grass, is plasmid-free and expresses the characterized bacteriocin NC8 aß,whereas L. plantarum 44048 is isolated from human vaginal tract and is catalasepositive, however, no bacteriocin has so far been isolated from this strain. Although L.plantarum 44048 was originally isolated from the vaginal tract, these two strains of L.plantarum are frequently found in the same environment such as ferrnented dairyproducts.
    Lactobacillus are able to lower the pH in their environment, why it had to beconfirmed that the antimicrobial activity of lactic acid bacteria was not due to acidic pH.The growth media from all three Lactobacillus strains of figure lA were sterile filteredand the pH was found to be acidic (3.4-4. l). However, antimicrobial activity against P.gingivalis was only found in media derived from L. plantarum 44048 and L. plantarumNCS, as shown in Figure lb. This suggests that the inhibition is most likely due tosecretion of specific active antimicrobial peptides, since acidic pH was not acontributing factor for the observed antimicrobial activity.
    Lactobacillus plantarum is a highly versatile lactic acid bacterium found insaliva and gastrointestinal tract as well as ferrnented vegetables, meat and dairyproducts. L. plantarum NC8 has been used as a model strain in many laboratoriesworldwide, and is a naturally plasmid-free L. plantarum strain. L. plantarum NCS haspreviously been shown to produce a two-peptide bacteriocin, NC8 aß, classified as aclass IIb bacteriocin. Class IIb bacteriocins (two-peptide bacteriocins) require twodifferent peptides, norrnally in an equal amount, for optimal antibacterial activity. NCSaß is composed of the peptides NC8 a and ß, here referred to as SEQ ID l and SEQ ID2, respectively. NC8 aß is heat-stable and reported to possess antimicrobial activitytowards gram-positive bacteria. The corresponding genes are designated plNC8A andplNC8B and produce bacteriocin precursors of 47 and 55 amino acids, respectively. Themature NC8 a (cf SEQ ID l) and NC8 ß (cf. SEQ ID 2) peptides contain 29 and 34amino acids, respectively. The mature NC8 a and NC8 ßpeptides share little homologywith known proteins, however class II bacteriocins commonly share a N-terrninalconsensus sequence and most often a C-terrninal sequence is responsible for species-specific activity, causing cell-leakage by perrneabilizing the target cell wall.
    The antimicrobial activity of NC8 aß on P. gingivalis was probed using afluorescent dye (Sytox® Green), which can only cross damaged membranes andfluoresce upon binding to nucleic acids. Thus P. gingivalis were incubated in the presence or absence of NC8 aß, as seen in figure 2. The uptake of Sytox Green by wild 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 8 type P. gíngívalís ATCC 33277 and W50 was markedly enhanced With increasingconcentrations of NC8 otß), which indicates a disordered integrity of P. gíngívalísmembranes. Strikingly, the effect of NC8 otß bacteriocins was observed to be instant, asfluorescence was detected immediately after addition of NC8 otß reaching maximumintensity already after l min.
    Having seen the instant effect of NC8 otß on P. gíngívalís membranes, acarboxyfluorescein (CF) release assay was used in order to investigate the effect of thebacteriocins on the integrity of model lipid membranes. Liposomes were prepared byextrusion through a polycarbonate membrane with a l00 nm pore size resulting in largeunilamellar vesicles (LUVs) with a hydrodynamic diameter of about l23 nm and a zetapotential of -24 mV. The lipid composition (5:95 POPS:POPC) was chosen to mimicthe zeta potential of microvesicles present in the supematant retrieved cultures of P.gingivalis W50 (-24.8 mV) (Fig 3). The CF release was monitored over time usingconcentrations of bacteriocins ranging from 0.005 to 2 uM. The relative release after 30min incubation with NC8 ot, NC8 ß and NC8 otß (molar ratio l:l) is shown in Fig 4a.
    A 30 min incubation with 2 ttM NC8 ot caused about 70 % release of CF whilethe same concentration of NC8 ß induced almost complete release of the liposomalcontent (97.5 %). It is noteworthy that even for low concentrations of NC8 ß the releaseof CF was high. As little as 0.05 ttM (~ 0.002 peptide/lipid) NC8 ß caused about 80 %CF release in 30 min. For NC8 otß, the release kinetics and extent of release was similarto NCS ß alone for concentrations < 0.2 uM, whereas higher concentrations resulted inslightly higher release for NC8 otß.
    The CF release assay thus indicates that both peptides bind to lipid membranesresulting in a significant reduction in membrane integrity. NCS ot alone did howevercause significant leakage of CF albeit requiring a higher concentration than NCS otß.The release kinetics for NC8 ot and ß (Fig 4b and c respectively) show that membranedisruption was almost immediate after exposure to the bacteriocins. Within the firstcouple of minutes the CF release had already saturated. The maximum release increasedwith increasing concentration of bacteriocin. At lower concentrations a large fractionsof liposomes were unaffected.
    This prompted a secondary structure investigation of the two peptides of NC8otß using CD spectroscopy (Fig 5). In the absence of lipid membranes, NC8 ot showedno signs of ordered secondary structure (i.e. random coil). NC8 ß, on the other hand,showed CD spectra indicative of ot-helical secondary structure. In the presence ofPOPS:POPC (5:95) LUVs, both peptides underwent a structural transition, which was 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 9 quite prominent for NC8 ß that adopted a distinct ß-sheet conforrnation. NC8 otdemonstrated a shift in the CD spectrum indicating a certain amount of ot-helicalstructure.
    It is known that the antimicrobial activity of bacteriocins is dependent onbinding to common epitopes on bacterial surfaces. Although binding of bacteriocinsalone does not attribute for damage of bacterial membranes, we show that theantimicrobial effect of NC8 uß on P. gíngívalís is evident (Fig 2). It is thereforeimportant to determine if these effects are specific through binding of NC8 otß to P.gíngívalís. Both P. gíngívalís ATCC 33277 (Fig 6a) and W50 (Fig 6b) were shown tobind to immobilized bacteriocin NC8 otß (280 nM). Furthermore, pre-incubation of P.gíngívalís with increasing concentrations of NC8 otß caused a dose-dependent decreasein binding of the bacteria to the immobilized NC8 otß peptides. Interestingly, pre-incubation of the bacteria with low concentrations of NC8 otß (2.8 nM) were able tosignificantly decrease the binding, compared to untreated bacteria, indicating highspecificity and binding affinity. Similar results were obtained with channels in theBiacore immobilized with 2.8, 28 and 2800 nM of NC8 otß and with similar pre-treatments (data not shown).
    The binding of NC8 otß to P. gíngívalís was Verified using channelsimmobilized with anti-P. gíngívalís antibodies. Both ATCC 33277 (Fig 7a) and W50(Fig 7b) bound to the immobilized antibodies. This binding was significantly reduced ina dose-dependent manner following pre-incubation of the bacteria with increasingconcentrations of NC8 otß. The results suggest that the damage caused by NC8 otßbacteriocins alters the integrity of P. gíngívalís membranes, resulting in modifiedepitopes with reduced binding to the antibodies, compared to untreated bacteria.
    Having established that the binding and antimicrobial effect of NC8 otß on P.gíngívalís is evident, the nature of the damage caused by these peptides was Visualized.P. gíngívalís ATCC 33277 were either left untreated or treated with 280 nM of NC8 otßfor 2 and 10 min, followed by morphological studies using TEM. Untreated P.gíngívalís cells showed typical coccobacillus morphology in which the outer and innermembrane are apparent and can be distinguished (Fig 8). Treatment of the bacteria withNC8 otß for 2 min resulted in leakage of intracellular content, which may be due to poreformation. Most striking was that the bacteriocin treatment caused detachment of theouter membranes of P. gíngívalís. After 10 min of treatment, a large number of distortedand completely separated membranes were observed. These findings confirm that the effects of NC8 otß on P. gíngívalís are specific and instant. 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx Although bacteriocins are generally considered to target bacteria that areclosely related to the bacteriocin-producing species, our results shows that bacteriocinNC8 uß may be used to suppress P. gíngívalís growth and prevent its establishment.The reported narrow-spectrum activity of most bacteriocins could suggest that theirmechanism of action is primarily mediated through binding to specific receptors. Diepand colleagues (Diep DB et. al. Proc Natl Acad Sci U S A. 2007;104(7):2384-9) showedthat the class II bacteriocin lactococcín A from Lactococcus lactís, binds to themembrane-located components IIC and IID of the mannose phosphotransferase systemon target bacteria. Although P. gíngívalís contains functional glucose/galactosetransporters, these processes have mainly been associated with the LPS synthesispathway. Genetic analysis of P. gíngívalís suggest that amino acids are the primarysource for growth, while the uptake/metabolism of carbohydrates is limited.Furthermore, plantaricin EF (two-peptide bacteriocin, subclass IIb) from L. plantarumC11 did not exploit the mannose phosphotransferase system to kill susceptible bacteria,suggesting that the two-peptide bacteriocins have a different mechanism of action.
    The calculated net charge for the individual u and ß peptides of bacterocinNC8 uß at pH 7 are 4.1 and 5.2, respectively. These positively charged peptides may beattracted to the negatively charged components lipid A and phosphate groups of the LPSmolecule, which initiates the initial interaction prior to pore formation. By incorporatingLPS or lipid A into liposomes, to resemble the outer membrane of gram-negativebacteria, Matsuzaki and collegues (Matsuzaki K. et.al. FEBS Lett. 1999;449(2-3):221-4) were able to show that the antimicrobial peptide magainin was attracted to thenegatively charged lipid A and formed a helix upon binding. This appears to be acommon mechanism applied by a range of different naturally occurring and syntheticcationic antimicrobial peptides, and could thus be used to reduce LPS-inducedinflammation and sepsis. Scott and colleagues (Scott MG. J Immunol. 2000;164(2):549-53) showed that neutralization of the inflammatory effects of LPS by human ot-defensin-1, human ß-defensin-2 and other synthetic peptides, was due to prevention of LPSinteraction with the acute-phase LPS-binding protein.
    Studies with model membranes, summarized in figure 9, suggest that thebacteriocins may interact with lipid membranes without a specific target epitope. Theinteraction is most likely due to an initial electrostatic attraction between the cationicbacteriocins and the anionic lipid membrane, which is commonly seen in themechanisms of many antimicrobial peptides. It has previously been shown that P. gíngívalís cells are highly negatively charged at pH>3. An increased concentration of 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx ll bacteriocins on the liposome surface will in turn cause an increase in perrneability of theliposomes. This increased perrneability is related to the structural changes that wereobserved when the bacteriocins interact with the liposome surface, likely due topartition-folding coupling due to their amphipathic properties. The studies on the modelmembrane showed that both single chains of bacteriocin NC8 otß individually causedisruption of the membrane integrity, although the effect was more pronounced for NC8ß.
    Efforts have been made to screen for and synthesize antimicrobial peptidesagainst P. gíngívalís, considering its role as a keystone pathogen in periodontitis. In astudy where 236 lactic acid bacterial isolates from food were included, none of thetested strains was able to affect the growth of gram-negative periodontal pathogens,including P. gíngívalís (Zoumpopoulou G. et.al., Intemational joumal of molecularsciences. 2013;l4(3):4640-54.). Antimicrobial agents shown to suppress P. gíngívalísgrowth includes L. paracaseí HL32 antimicrobial agents (Pangsomboon K. et.al.,Joumal of applied microbiology. 2009;106(6):1928-40) and a short biosyntheticallyproduced peptide, Pep-7 (Suwandecha T.20l5;l97(7):899-909).
    In this study, it is shown that L. plantarum NC8 and 44048 are able to suppress et.al. Archives of microbiology.
    P. gíngívalís growth. Furthermore, bacteriocin NC8 uß from L. plantarum NC8 are ableto bind, at the nanomolar range, to P. gíngívalís and cause cellular distortion throughdetachment of the outer membrane and bacterial lysis. It is known that the gram-negative anaerobic bacterium P. gíngívalís is a major causative agent of chronicperiodontitis. P. gíngívalís has also been widely associated as a putative organism incausing aggressive periodontitis and progression into systemic diseases. The ability ofP. gíngívalís to invade host cells, including gingival epithelial cells and heart and aorticendothelial cells, demonstrate a possible mechanism for its establishment andsubsequent pathogenesis by evading the host immune system. P. gíngívalís expressmultiple virulence factors (fimbriae, capsule, LPS, proteinases and toxic end-products)that contribute significantly to their pathogenicity by altering host immune responses.Soluble or immobilized NC8 uß bacteriocins may thus be used to prevent P. gíngívalíscolonization and pathogenicity, and thus supplement the host immune system againstinvading pathogens associated with periodontitis and other systemic inflammatorydiseases, including atherosclerosis.
    Thus, in a first embodiment, a pharrnaceutical composition comprising a first and a second polypeptide is used in the prevention or treatment of a periodontal disease, 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 12 wherein the first polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99%sequence identity (%SI) With SEQ ID 1 and, the second peptide has at least 90%, 95%,96%, 97%, 98% or 99% sequence identity (%SI) with SEQ ID 2. In one embodiment,the first polypeptide of the composition is a peptide according to SEQ ID 1 and thesecond polypeptide of the composition is the a peptide according to SEQ ID 2.
    Bacteriocin NC8 otß is composed of the peptides NC8 ot (SEQ ID 1) and ß(SEQ ID 2) and is classified as a class IIb bacteriocin. Class IIb bacteriocins (two-peptide bacteriocins) require two different peptides, norrnally in an equal amount, foroptimal antibacterial activity. Therefore, it was surprisingly found that thatantimicrobial activity of a pharrnaceutical composition comprising bacteriocin NC8 otßwas highest when the second peptide (i.e. ßpeptide) were present is in an molar amountequal to or larger than the amount of the first peptide (i.e. ot peptide). Antimicrobialactivity experiments indicated that the Molar ratio between the first and second peptidesof the composition should be from 1:1 to 1:20, preferably 1:4 to 1:12, most preferably1:8, as can be seen in figure 12. Thus, in one embodiment, the first and second peptidesof the composition are present in a molar ratio from 1:1 to 1:20, preferably 1:4 to 1:12,most preferably 1:8.
    In one embodiment, the first polypeptide of the composition is the ot peptideand the second polypeptide of the composition is the ß peptide, respectively, of thebacteriocin NC8 otß.
    In one embodiment, the periodontal disease is selected from the groupconsisting of periodontitis or peri-implantitis.
    Thus in one embodiment, bacteriocin NC8 otß is used to supplement the hostimmune system against invading pathogens associated with periodontitis and othersystemic inflammatory diseases, including atherosclerosis.
    Gingivitis, which is inflammation of the gum tissue, may often precedeperiodontitis and is usually a response to bacterial biofilms adherent to tooth surfaces.
    In absence of treatment, gingivitis can progress to periodontitis. Thus, bacteriocin NC8otß may also be used for preventing gingivitis, by suppressing P. gíngívalís growth andprevent its establishment of biofilms.
    Commonly, antibiotics treatment for periodontitis is administered orally. Suchtreatment may lead to unwanted side effects, such as knocking out the protective floraor developing antibiotics resistance. Such treatment may also lead to changes in theintestinal bacterial composition, which may result in superinfection by fungi and other infective organisms. NC8 otß bacteriocins may beneficially be administered locally, in 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 13 the form of a solution, cream, a gel or in immobilized form (as described further undercoating below). The forrnulations would further include solvent and/or a variety ofexcipients, for instance to stabilize the peptides and suppress aggregation, such assolubilizers, surfactants, bulking agents (such as carbohydrates), thickeners (such aspolymers) to increase solution viscosity, preservatives, vehicles, salts or sugars tostabilize proteins and to obtain physiological tonicity and osmolality and/or bufferingagents to control pH.
    Thus in one embodiment, NC8 otß bacteriocins are administered locally in theforrn of a solution, cream, a gel or in immobilized forrn. In one further embodiment, thecomposition comprising the first and second peptides further comprises solvent and/orexcipients such as solubilizers, surfactants, bulking agents, thickeners, preservatives,vehicles, salts, sugars, and/or buffering agents.
    In one embodiment, the composition is forrnulated as a solution, cream, a gelor immobilized as a coating on a device. In one further embodiment, the composition isadministered locally on the site of infection, such as topically.
    The composition is forrnulated for administration as a single dose or multipledoses, such as two, three, four, five doses. Further, an effective dose of the first andsecond polypeptide may range between 0.01 to 100 ug, such as 0.01-10 ug or 1-50 ugof each polypeptide. The effective dose was further confirmed by the SPR analysis(figures 6A,B and figures 7 A,B ) where it is shown that concentrations within thenanomolar range effectively alters P. gíngívalís.
    Thus in one embodiment, the composition is forrnulated for administration as asingle dose or multiple doses, such as two, three, four, five doses. In a furtherembodiment, an effective dose of the first and second polypeptide may range between0.01 to 100 ug, such as 0.01-10 ug or 1-50 ug of each polypeptide.
    Bacterial infection and inflammation in the periodontium is sometimes linkedto dental implants (i.e. peri-implantitis), caused by the bacterial adherence andcolonization in the peri-implant area. Peri-implantitis is characterized by progressiveperi-implant bone loss associated with soft-tissue inflammatory lesion caused bybacterial infection. Treatment may include removing dead tissue, antibiotics, andimproved dental hygiene. Preventive measures include polish the implant surfaces, tominimize bacterial adherence, which is a time consuming and costly procedure. Thus,implant coating or treatment with antibacterial material would minimize the incidenceof peri-implantitis and avoid the high-cost of producing a highly polished surface onimplant. Thus, a coating comprising the first and second peptide of the invention (i.e. 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 14 bacteriocin NC8 otß) may be used to impart bacterial resistance to a coating for a dentalimplant by preventing P. gíngívalís colonization on the coated surface. Similarly, such acoating could be used for any medical device, or part of a medical device, Where P.gíngívalís colonization on the surface should be prevented. The medical device mayalso be a band-aid comprising the first and second peptide of the invention (i.e.bacteriocin NC8). This Would help facilitate local administration on a Wound orinfection site. If the coating material is thin gel-like material Where bacteriocin NC8abis either tethered to a polymeric scaffold via a flexible linker or physically entrapped ina biopolymeric matrix, its bactericidal property Will be retained, or even improvedbecause of its high local concentration.
    Non-natural or modified amino acids can be introduced that enable convenientcoupling chemistries, including click-chemistry approaches. Bacteriocins can also bemodified With either N- or C-terrninal azide groups to enable copper-free click reactionWith e.g. cyclooctyne conjugated polymers. Using biodegradable polymers such ashyaluronic acid (HA), the release rate Will be dependent on the hydrolysis rate of thebiopolymer backbone and can be tuned to a certain extent by using different polymers.
    Thus, in one embodiment a use of a composition comprises a first and a secondpolypeptide for coating a medical device, Wherein the first polypeptide has at least 90%,95%, 96%, 97%, 98% or 99% sequence identity (%SI) With SEQ ID l and, the secondpeptide has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) WithSEQ ID 2. By coating a medical device to be used periodontally, such as dental implant,colonization of P. gíngívalís on the device surface may be limited. In one embodiment,the medical device is for use in the oral cavity. In a further embodiment, the medicaldevice is a prosthesis or dental implant. The device may be fully or partially coated.
    In one embodiment, a medical device, Which has been at least partly coatedWith a composition comprising a first and a second polypeptide, Wherein the firstpolypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI)With SEQ ID l and, the second peptide has at least 90%, 95%, 96%, 97%, 98% or 99%sequence identity (%SI) With SEQ ID 2. In this Way, colonization of P. gíngívalís onthe device surface may be limited. In one embodiment, the first polypeptide of thecomposition is a peptide according to SEQ ID l and the second polypeptide of thecomposition is the a peptide according to SEQ ID 2.
    Periodontitis is associated With destruction of tooth-supporting tissues, Which iscaused by proteolytic enzymes released by periodontal pathogens, e.g. P. gíngívalís, andhost immune cells. Except the importance to prevent and inhibit the groWth of 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx periodontal pathogens, e.g. by bacteriocin NC8 otß, there is a tremendous important taskto develop novel therapeutic procedures to stimulate wound healing and generation ofnew tooth-supporting tissues.
    To enhance periodontal wound healing and regeneration, various surgicaltechniques are employed, including implantation of bone grafts and substitutes, guidedtissue regeneration and biological factors and combinations thereof Several studiesindicate that regeneration of periodontal tissue occurs if endogenous mesenchymal stemcells and/or progenitor cells within periodontal ligaments are stimulated. eg. bycytokines and growth factors. Growth factors, such as FGF (Fibroblast growth factor),TGF (Transforrning growth factor), PDGF (Platelet-derived growth factor), IGF(Insulin-like growth factor) and EGF (Epiderrnal growth factor), may stimulateproliferation and differentiation of undifferentiated mesenchymal cells into cells thatforrn hard tissues, such as osteoblasts and cementoblasts, thereby acceleratingperiodontal wound healing and regeneration. Members of the transforrning growthfactor ß (TGFß) superfamily have been implicated in the differentiation ofcementoblasts.
    As can be seen from figure ll, it is surprisingly shown that NC8 otß markedlyincreases the proliferation of both gingival epithelial cells and gingival fibroblasts, andthat this effect is associated with an enhanced release of TGFß. Furthermore, it is shownthat bacteriocin NC8 otß antagonizes the harrnful and toxic effects of P. gíngívalís ongingival epithelial cells and fibroblasts (Figure l2). Tissues that line the inside of themouth, the esophagus and part of the rectum are composed of nonkeratinized stratifiedsquamous epithelium, while gingival fibroblasts are the most abundant cell types inperiodontal connective tissues. Furthermore, they have distinct functional activities inthe repair of periodontal tissues as well as in inflammatory periodontal diseases. Thisindicates that bacteriocin NC8 aß stimulates periodontal wound healing andregeneration. The findings also suggests that bacteriocin NC8 otß can be used inpromoting and enhancing wound healing in general, for example in injuries caused byacute and chronic skin and soft tissue infections. Thus, a composition comprising thefirst and the second polypeptides of the invention can be administered locally on awound to promoting and enhancing wound healing, for instance in the forrn of a gel asdescribed above. Furthermore, a band aid comprising the composition, such as a gel orcoating as described above, could be administered locally on a wound to promote and enhance wound healing. 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 16 Thus in one embodiment, the composition comprising the first and secondpeptide of the invention is used for promoting and enhancing Wound healing andregeneration. In one further embodiment, the Wound is a periodontal Wound.
    In one embodiment, the Wound healing is promoted and enhanced throughincreasing the proliferation of both gingival epithelial cells and gingival fibroblasts.
    In one embodiment, the composition is used for antagonizing harrnful and toxiceffects of P. gíngívalís on gingival epithelial cells and fibroblasts.
    In summary, bacteriocin NC8 aß has a great and unique potential indifferent applications in preventing and treating periodontal disease, andassociated disorders, both through its antibacterial and proliferative Woundhealing effects.
    Sequence identity (%SI) as described herein may be assessed by any convenientmethod. Programs that compare and align pairs of sequences, like ALIGN (Myers andMiller, CABIOS, 4:11-17, 1988), FASTA (Pearson, Methods in Enzymology, 183263-98,1990) and gapped BLAST (Altschul et al., Nucleíc Acíds Res., 25:3389-3402, 1997), orBLASTP (Devereux et al., Nucleíc Acíds Res., 12:387, 1984) can be used for thispurpose. If no such resources are at hand, according to one embodiment, sequenceidentity (%SI) can be calculated as (%SI) = 100% * (Nr of identical residues in pairwisealignment) / (Length of the shortest sequence).
    Although the present invention has been described above With reference to (a)specific embodiment(s), it is not intended to be limited to the specific form set forthherein. Rather, the invention is limited only by the accompanying claims and, otherembodiments than the specific above are equally possible Within the scope of theseappended claims, e. g. different than those described above.
    In the claims, the term "comprises/comprising" does not exclude the presenceof other elements or steps. Furthermore, although individually listed, a plurality ofmeans, elements or method steps may be implemented by e. g. a single unit or processor.Additionally, although individual features may be included in different claims, thesemay possibly advantageously be combined, and the inclusion in different claims doesnot imply that a combination of features is not feasible and/or advantageous. Inaddition, singular references do not exclude a plurality. The terms "a", "an", “f1rst”,“second” etc do not preclude a plurality. Reference signs in the claims are providedmerely as a clarifying example and shall not be construed as limiting the scope of theclaims in any Way.
    Experimental 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 17 The following examples are mere examples and should by no mean beinterpreted to limit the scope of the invention. Rather, the invention is limited only by the accompanying claims.
    Materials and Methods Bacterial culture conditionsP. gingivalis Wild type strains ATCC 33277 (ATCC, Manassas, VA) and W50, and the W50-derived Kgp proteinase and Rgp proteinase mutant strains (KlA and ES,respectively) Were a kind gift from Dr. M.A. Curtis (Molecular Pathogenesis Group,Queen Mary, University of London). P. gingivalis strains Were groWn in anaerobicconditions (80% N2, 10% C02, and l0% H2) at 37 °C in an anaerobic chamber(Concept 400 Anaerobic Workstation; Ruskinn Technology Ltd., Leeds, UnitedKingdom). The bacterial concentration Was adjusted to correlate With approximately 109 CFU/ml, Which Was deterrnined by viable count by culturing the bacteria onfastidious anaerobe agar (45.7 g/liter, pH 7.2, Acumedia, Neogen, Lansing, USA),supplemented With 5% defibrinated horse blood for 5 days.
    The Lactobacillus strains L. plantarum NC8, L. plantarum 44048 and L. brevis 30670(Culture Collection, University of Gothenburg, Sweden) Were groWn on deMan RogosaSharp (MRS, BD Science) supplemented With agar (Difco) at 37 °C for 24 h in a jarcontaining an oxygen-free environment (Anaerobic pouch system EZ, BD Biosciences,CA, USA). Fresh cultures Were used to inoculate MRS broth (Difco, BD Biosciences,CA, USA), and groWn statically and anaerobically for 24 h at 37 °C. Lactobacillus WeregroWn from a 0.5 % inoculum for 24 h at 37 °C under anaerobic and static growthconditions, and Were then used for further experiments. The two-peptide bacteriocinfrom L. plantarum NC8, NC8 a and ß, Were purchased from GL Biochem (Shanghai)Ltd, China. Amino acid sequences for the peptides are: NCSOL- DLTTKLWSSWGYYLGKKARWNLKHPYVQF (SEQ ID l) and Ncsß- svPTSVYTLGIIGLWSAYKHRKTIEKSFNKGFYH (SEQ 1D 2) (MaldonadoA. et al., Appl Environ Microbiol. 2003;69(l):383-9).
    Liposome preparationLiposomes Were prepared by dry film formation, hydration and finally extrusion through a polycarbonate membrane to form monodisperse large unilamellar 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 18 Vesicles. The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3 -phosphatidylcholine (POPC) (Avanti Polar Lipids,Alabaster, USA) Were mixed at molar ratios 1:99, 5:95 and 10:90 While dissolved inchloroforrn. A dry lipid film Was formed by evaporation of the chloroforrn by nitrogenfloW and ovemight lyophilization. The film Was hydrated With either 10 mM phosphatebuffer (PB) pH 7 or 10 mM phosphate buffer saline (PBS) pH 7, and the solution WasVortexed for 1 min and put on a shaker for 1 h before extruded 21 times through a 100nm pore-sized polycarbonate membrane. For fluorescence leakage assay the lipid filmWas hydrated With buffer (PBS) containing self-quenching concentration (50 mM) of5(6)-carboxyfluorescein (CF) (Sigma Aldrich) and liposomes Were prepared asdescribed above. Removal of unencapsulated CF Was done by gel filtration using a PD-25 column (GE Healthcare, Uppsala, Sweden) and liposomes With encapsulated CFWere eluted With PBS.
    Dynamic light scattering and Zeta potential The hydrodynamic radius and zeta potential Was measured on liposomessuspended in 10 mM PB pH 7 and suspensions containing microvesicles from P.gingivalis W50, using a Malvem ZetaSizer Nano S (Malvem Instruments Ltd, UK) anda disposable cuvette. Results are seen in figure 3, identifying the best match With microvesicles from P. gingivalis W50.
    F laorescence leakage assay Leakage of the liposome encapsulated fluorophore CF due to additions of thebacteriocins Was recorded using a fluorescence plate reader (Infinite 200, Tecan,Austria) Where kex = 485 nm and 716m = 520 nm. CF Was encapsulated at self-quenchingconcentration, and CF release results in an increased fluorescence signal. LiposomesWere diluted to 25 uM (total lipid concentration) in PBS, followed by additions of 0,0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 uM of the peptides NC8 a and ß,separately and combined. In order to estimate the maximum release from each sample afinal addition of 0.5 % Triton X-100 Was made at the end of all measurements and thetotal amount of CF (100% release) Was estimated after 15 min incubation. The CFrelease is presented as percentage release for each time interval (measurements takenevery minute). The percentage CF release is calculated as 100 >< (F - F0)/(FT - FO) WhereFO is the initial fluorescence intensity of CF before peptide addition, F is the 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 19 fluorescence intensity of CF at time point t and FT is the maximum fluorescence afterthe addition of Triton X-100. Results are shown in figure 4.
    Circular Dichroism (CD) spectroscopy CD spectra were recorded using a Chirascan spectropolarimeter (AppliedPhotophysics, UK) and a 1 mm quartz cuvette at 20 °C with a sampling interval of 0.5nm. All measurements were done in triplicates and averaged before converted to meanresidue ellipticity (MRE) and curves smoothened using Savitzky-Golay algorithm. CD spectra are shown in figures 5A and 5B.
    Antimicrobial activity of Lactobacillus The ability of different Lactobacillus strains to inhibit P. gingivalis growth wasassessed on fastidious anaerobe agar plates. Briefly, different P. gingivalis strains (108CFU in 100 ul) were spread onto fastidious anaerobe agar plates and allowed to dry.Lactobacillus were diluted in MRS broth and 10 ul drops (106 CFU) were placed ontothe P. gingivalis layer. The plates were incubated for 4 days, after which images wereacquired with Olympus SZX9 at 10>< magnification and the zone of inhibition wasmeasured using the software ImageJ. Results are summarized in figure 1A.
    Antimicrobial activity was also assessed using Lactobacillus culture media(MRS broth), in which different Lactobacillus strains were cultured for 24 h. Thebacteria were removed by centrifugation at 7000 > Antimicrobial activity of bacteriocins NCS aß The antimicrobial activity of NC8 otß on P. gingivalis was visualized using thefluorescent dye Sytox® Green, which can only cross damaged membranes and fluoresceupon binding to nucleic acids. P. gingivalis were incubated in the presence or absenceof NCS otß in 96-well microtiter plates and images were captured with Olympus BX41at 40x magnification, as can be seen in figure 2. The optimal ratio between ot peptideand ß peptide for NC8 otß antimicrobial activity on P. gingivalis was confirmed in asimilar manner. P. gingivalis were incubated with different ratios of ot peptide and ß peptide in 96-well microtiter plates and images were captured with Olympus BX41 at 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 40x magnification. In figure 12, the antimicrobial activity of NC8 otß on P. gíngívalísare shown for u peptide and ß peptide ratios 1:2 and 1:8.
    Transmission electron microscopy (TEM) was used to Visualize the damage ofP. gíngívalís, caused by NC8 uß. Briefly, P. gíngívalís ATCC 33277 were pelleted andwashed with Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KC1, 1.2mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, and 10 mM glucose, pH 7.3). Thebacteria were then treated with 280 nM of NC8 otß in a ratio of 1:1 for 2 min and 10min, followed by fixation in 25% glutaraldehyde in 0.1M phosphate buffer, pH 7.3.Specimens were washed in 0.1M phosphate buffer, postfixed in 2% osmium tetroxide in0.1M phosphate buffer for 2 hours and embedded into LX-112 (Ladd, Burlington,Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leicaultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted withuranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo,Japan). Digital images were taken by using a Veleta camera (Olympus Soft ImagingSolutions, GmbH, Münster, Germany). Representative images of three independent experiments can be seen in figure 8.
    SPR analysis Surface plasmon resonance analysis was performed by immobilizing NC8 otßpeptides in 1:1 ratio onto a carboxymethylated dextran (CM-5 sensor chip, GE-Healthcare GmbH, Uppsala, Sweden) using biacore 2000 instrument equipped with fourflow cells (GE-Healthcare GmbH, Uppsala, Sweden). Each channel of the chip wasimmobilized with 3 different concentrations of NC8 uß (2.8, 28, 280 nM) respectively,and the fourth channel was used as a blank for negative reference subtraction betweenthe channels. HBS-EP (0.01 M HEPES, pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005 %surfactant P20) (GE-Healthcare GmbH) was used as running buffer and the flow celltemperature was set to 25 °C in all experiments. Immobilization was a 3-step processperformed using amine coupling kit (GE-Healthcare GmbH, Uppsala, Sweden) wherethe chip surface was activated using 200 mM N-ethyl-N0-(3 diethylaminopropyl)carbodimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) mixture. NC8 ußpeptides diluted in acetate 4.5 buffer (GE-Healthcare GmbH, Uppsala, Sweden) wereimmobilized to the actiVated surface. Ethanolamine-HCl (pH 8.5) was used todeactiVate the surface to enable an efficient binding of samples to the immobilized ligand. The contact time was 7 min, which resulted in immobilization levels between 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 21 1350 and 1750 response units (RU). One thousand RU corresponds to a surface peptideconcentration of about 1 ng/mm2.
    P. gingivalis were washed and prepared as described above after which thebacteria were pre-incubated with 2.8, 28 or 280 nM of NC8 aß for 5 min at roomtemperature before analysis. P. gingivalis without NC8 aß was used as positive control.The binding affinity of the bacteria to the peptides immobilized on each channel of thechip was measured in response units (RU). lRU=lpg/mm2 using Bia-evaluationsoftware. Results are seen in figure 6.
    Immobilization of surfaces with P. gingivalis-specific antibodies produced andtested as described in our previous studies (Nakka SS et. al, Clinical and experimentaldental research (in press). 2015.), were used to study the binding affinity of P.gingivalis with and without NC8 aß to the immobilized antibodies. Samples wereprepared as described above and the responses obtained from Bia-evaluation softwarewere plotted. The immobilization response for anti-P. gingivalis antibodies was 6400 RU. Results are seen in figure 7.
    Statistical analysis All data were analyzed using GraphPad Prism 5.0 (GraphPad Software, LaJolla, CA, USA). One-way ANOVA with Tukey°s multiple comparison test was usedfor the comparisons between the different treatments. P-values are referred to as*,#p<0.05; **,##p<0.01; ***,###p<0.00l.
    权利要求:
    Claims (17)
    [1] 1. A pharrnaceutical coniposition coniprising a f1rst and a second polypeptidefor use in the prevention or treatment of a periodontal disease, Wherein the f1rst polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99%sequence identity (%SI) With SEQ ID 1 and, the second peptide has at least 90%, 95%,96%, 97%, 98% or 99% sequence identity (%SI) With SEQ ID 2.
    [2] 2. The coniposition for use according to claini 1, Wherein the first and secondpeptides are present in a in a Molar ratio of from between 1:1 to 1:20, preferably 1:4 to 1:12, n1ost preferably 1:8.
    [3] 3. The coniposition for use according to claini 1 or 2, Wherein the firstpolypeptide is the u peptide and the second polypeptide is the ß peptide, respectively, ofthe bacteriocin NC8 otß.
    [4] 4. The coniposition for use according to any one of clainis 1 to 3, Wherein theperiodontal disease is selected fron1 the group consisting of periodontitis or peri- in1plantitis.
    [5] 5. The coniposition for use according to any one of clainis 1 to 4, Wherein the periodontal disease is related to/caused by P. gíngívalís.
    [6] 6. The coniposition for use according to any one of clainis 1 to 5, Wherein theconiposition further coniprises solvent and/or excipients, such as solubilizers,surfactants, bulking agents, thickeners, preservatives, vehicles, salts, sugars, and/or buffering agents.
    [7] 7. The coniposition for use according to any one of clainis 1 to 6, Wherein theconiposition is forrnulated as a solution, a cream, a gel, or in ininiobilized forrn as a coating on a device.
    [8] 8. The coniposition for use according to any one of clainis 1 to 7, Wherein the coniposition is adn1inistered locally on the site of infection, such as topically. 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 23
    [9] 9. The composition for use according to claim 7 or 8, Wherein the polypeptideis forrnulated for administration as a single dose or multiple doses, such as two, three, four, five doses.
    [10] 10. The composition for use according to any of claims 7 to 9, Wherein aneffective dose of the first and second polypeptide may range between 0.01 to 100 ug,such as 0.01-10 ug or 1-50 ug of each polypeptide.
    [11] 11. The composition for use according to any one of claims 1 to 10, forpromoting and enhancing Wound healing and regeneration, Wherein the Wound is a periodontal Wound.
    [12] 12. The composition for use according to claim 11, Wherein the Wound healingis promoted and enhanced trough increasing the proliferation of both gingival epithelial cells and gingival fibroblasts.
    [13] 13. The composition for use according to claim 11 or 12, for antagonizing harrnful and toxic effects of P. gíngívalís on gingival epithelial cells and fibroblasts.
    [14] 14. A composition comprising a first and a second polypeptide, for use incoating a medical device to limit P. gíngívalís colonization on its surface, Wherein the first polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99%sequence identity (%SI) With SEQ ID 1 and, the second peptide has at least 90%, 95%,96%, 97%, 98% or 99% sequence identity (%SI) With SEQ ID 2.
    [15] 15. A composition for use according to claim 14, Wherein the medical device is a prosthesis, dental implant or bandage.
    [16] 16. A composition for use according to claim 14 or 15, Wherein the medical device is for use in the oral cavity.
    [17] 17. A medical device, Which has been at least partly coated With a compositioncomprising a first and a second polypeptide, Wherein the first polypeptide has at least90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) With SEQ ID 1 and, the 160211 1:PatrawinTEMPMDS~P133300001_160211_s1utdraft_EBR.2016021201383320043.docx 24 second peptide has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI)With SEQ ID 2.
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    SE543223C2|2020-10-27|
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    SE1650188A|SE543223C2|2016-02-12|2016-02-12|Using bacteriocins in the prevention and treatment of periodontitis|SE1650188A| SE543223C2|2016-02-12|2016-02-12|Using bacteriocins in the prevention and treatment of periodontitis|
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