Inhibition of pre-existing natural periodontitis in non ... in non-human primates by a locally...

Inhibition of pre-existing naturalperiodontitis in non-humanprimates by a locallyadministered peptide inhibitor ofcomplement C3Maekawa T, Briones RA, Resuello RRG, Tuplano JV, Hajishengallis E, KajikawaT, Koutsogiannaki S, Garcia CAG, Ricklin D, Lambris JD, Hajishengallis G.Inhibition of pre-existing natural periodontitis in non-human primates by a locallyadministered peptide inhibitor of complement C3. J Clin Periodontol 2016;doi: 10.1111/jcpe.12507.

AbstractAim: Human periodontitis is associated with overactivation of complement,which is triggered by different mechanisms converging on C3, the central hub ofthe system. We assessed whether the C3 inhibitor Cp40 inhibits naturally occur-ring periodontitis in non-human primates (NHPs).Materials and Methods: Non-human primates with chronic periodontitis wereintra-gingivally injected with Cp40 either once (5 animals) or three times (10 ani-mals) weekly for 6 weeks followed by a 6-week follow-up period. Clinical peri-odontal examinations and collection of gingival crevicular fluid and biopsies ofgingiva and bone were performed at baseline and during the study. A one-wayrepeated-measures ANOVA was used for data analysis.Results: Whether administered once or three times weekly, Cp40 caused a signifi-cant reduction in clinical indices that measure periodontal inflammation (gingivalindex and bleeding on probing), tissue destruction (probing pocket depth andclinical attachment level) or tooth mobility. These clinical changes were associatedwith significantly reduced levels of pro-inflammatory mediators and decreasednumbers of osteoclasts in bone biopsies. The protective effects of Cp40 persisted,albeit at reduced efficacy, for at least 6 weeks following drug discontinuation.Conclusion: Cp40 inhibits pre-existing chronic periodontal inflammation andosteoclastogenesis in NHPs, suggesting a novel adjunctive anti-inflammatory ther-apy for treating human periodontitis.

Tomoki Maekawa1,2,Ruel A. Briones3, Ranillo R.G.

Resuello4, Joel V. Tuplano4,Evlambia Hajishengallis5, Tetsuhiro

Kajikawa1, Sophia Koutsogiannaki6,Cristina A.G. Garcia3, Daniel Ricklin6,

John D. Lambris6,† and GeorgeHajishengallis1,†

1Department of Microbiology, School of

Dental Medicine, University of Pennsylvania,

Philadelphia, PA, USA; 2Research Center for

Advanced Oral Science, Graduate School of

Medical and Dental Sciences, Niigata

University, Niigata, Japan; 3College of

Dentistry, Manila Central University,

Caloocan City, Philippines; 4Simian

Conservation Breeding and Research Center

(SICONBREC), Makati City, Philippines;5Division of Pediatric Dentistry, Department

of Preventive and Restorative Sciences,

School of Dental Medicine, University of

Pennsylvania, Philadelphia, PA, USA;6Department of Pathology and Laboratory

Medicine, University of Pennsylvania

Perelman School of Medicine, Philadelphia,

PA, USA

†J.D.L. and G.H. co-supervised this work.

Key words: complement; compstatin;

cytokines; inflammation; non-human

primates; periodontitis

Accepted for publication 27 December 2015

Conflict of interest and source of funding statementG.H. and J.D.L. have a joint patent application that describes the use of complement inhibitors for therapeutic purposes inperiodontitis. J.D.L. is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors for clinical appli-cations. This work was supported by grants from the National Institutes of Health (AI068730 and AI030040 to J.D.L.;DE015254, DE017138, DE021685 and DE024716 to G.H.) and the European Commission (FP7-DIREKT 602699 to J.D.L.).

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1

J Clin Periodontol 2016; doi: 10.1111/jcpe.12507

info:doi/10.1111/jcpe.12507

Periodontitis is a prevalent chronicoral disease that affects nearly halfof adults in the USA and the UKand perhaps worldwide (Demmer &Papapanou 2010, White et al. 2012,Eke et al. 2015). The disease is dri-ven by exaggerated inflammationinduced by dysbiotic microbial com-munities forming on subgingivaltooth sites (Lamont & Hajishengallis2015) and can lead to tooth loss andimpaired mastication and nutritionalstatus (Chapple 2014). Tooth lossmay occur as a result of excessivedestruction of periodontium. In itssevere form that affects almost 10%of adults (Eke et al. 2012, Whiteet al. 2012), chronic periodontitis isnot merely a common cause of toothloss, but is also associated with cer-tain systemic conditions, such asatherosclerosis, diabetes, rheumatoidarthritis, chronic obstructive pul-monary disease and adverse preg-nancy outcomes (Tonetti et al. 2007,Kebschull et al. 2010, Lalla & Papa-panou 2011, Han et al. 2014, Kozielet al. 2014, Hajishengallis 2015). Theserious public health impact of thisoral disease and its economic burden(Beikler & Flemmig 2011, Chapple2014) call for innovative treatmentsadjunctive to existing therapies (suchas mechanical removal of the tooth-associated biofilm and antimicrobialtreatment), which are not always suf-ficient to control periodontitis(Armitage 2002, Colombo et al.2012, Rams et al. 2014). In thisstudy, we have tested a complement-targeted therapeutic approach in ahighly relevant preclinical model ofperiodontitis.

The complement system, a net-work of interacting fluid-phase andcell surface-associated molecules, iscentrally involved in immunity andinflammation through direct effectson immune cells or crosstalk andregulation of other host signallingpathways, such as those activated bytoll-like receptors (TLRs) (Hajishen-gallis & Lambris 2010). The variouscomponents of complement are pro-duced systemically or locally inperipheral tissues and the system canbe triggered via distinct cascademechanisms (classical, lectin or alter-native), all of which converge at thethird component (C3) (Ricklin et al.2010). C3 cleavage and activation byconvertases leads to the generationof effector molecules that mediate

diverse functions, including recruit-ment and activation of inflammatorycells (induced by the anaphylatoxinsC3a and C5a), microbial opsoniza-tion and phagocytosis (via opsoninssuch as C4b and C3b) and directlysis of susceptible microbes (by theC5b-9 membrane attack complex)(Ricklin et al. 2010).

Early clinical and histologicalobservations in human periodontitishave associated periodontal inflam-mation and tissue destruction withincreased complement activity(Schenkein & Genco 1977, Niekrash& Patters 1986, Nikolopoulou-Papa-constantinou et al. 1987, Patterset al. 1989). Indeed, complementcomponents and their activationproducts are readily detected inchronically inflamed gingiva and thegingival crevicular fluid (GCF) ofpatients, whereas they are undetectedor present at lower levels in healthycontrol samples (Attstrom et al.1975, Courts et al. 1977, Schenkein& Genco 1977, Toto et al. 1978,Lally et al. 1982, Nikolopoulou-Papaconstantinou et al. 1987, Raute-maa & Meri 1996). Moreover,induction of experimental gingivitis inhuman volunteers causes progressiveC3 activation in the GCF (Patterset al. 1989). Conversely, a decrease inclinical parameters of inflammationupon successful periodontal therapyleads to reduced C3 activation in theGCF (Niekrash & Patters 1985).

More recently, studies in animalmodels, including mouse strains withcomplement knockout mutations,have established a cause-and-effectrelationship between complementactivation and periodontitis andgleaned insights into the mechanismswhereby complement mediates peri-odontitis (Breivik et al. 2011,Hajishengallis et al. 2011, Lianget al. 2011, Maekawa et al. 2014a,b).In this regard, we have shown thatC3-dependent inflammation in miceis crucial for the long-term suste-nance of the dysbiotic microbiotaand for maximal induction of alveo-lar bone loss (Maekawa et al.2014a). Consistent with these find-ings, local C3 inhibition in a modelof ligature-induced periodontitis inyoung non-human primates (NHPs)prevents the development of gingivalinflammation and alveolar bone loss(Maekawa et al. 2014a). The inhibi-tor we used was Cp40, an improved

analogue of compstatin, which is apeptidic compound that blocks C3activation exclusively in humans andnon-human primates (Qu et al. 2013,Ricklin & Lambris 2013, Mastelloset al. 2015b). Cp40 and other comp-statin analogues bind C3 and inter-fere with its binding to and cleavageby the C3 convertase, thereby block-ing the generation of downstreameffector molecules regardless of theinitiation pathway of complementactivation (Ricklin & Lambris 2013,Mastellos et al. 2015b).

However, whether Cp40 is effec-tive in a therapeutic – rather thanpreventive – setting was notaddressed in our previous publica-tion. The main objective of this studywas therefore to determine whetherlocal C3 inhibition could inhibit pre-existing chronic periodontal disease,which typically affects adults; in thiscontext, ageing is thought to affectthe immuno-inflammatory status ofthe periodontal tissue, thereby con-tributing to increased susceptibilityto periodontitis (Hajishengallis2014a). This notion is consistent withrecent studies in NHPs showing age-dependent differential expression ofimmune and inflammatory genes inthe periodontium (Ebersole et al.2015, Gonzalez et al. 2015). Byscreening a population of adultNHPs (cynomolgus monkeys) in theSimian Conservation Breeding andResearch Center (SICONBREC,Makati, Philippines), we identifiedanimals with naturally occurringchronic periodontitis and treatedthem with Cp40. Our results showthat locally administered Cp40 canreverse pre-existing chronic peri-odontal inflammation in the absenceof additional treatments, such asscaling and root planing, thus identi-fying an anti-inflammatory therapythat can potentially contribute to thetreatment of human periodontitis.

Materials and Methods

Non-human primates

All animal procedures were per-formed according to protocolsreviewed and approved by the Insti-tutional Animal Care and Use Com-mittees of the University ofPennsylvania and of the SICON-BREC, an Association for Assess-ment and Accreditation of

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

2 Maekawa et al.

Laboratory Animal Care Interna-tional-accredited facility where theNHP work was performed. Fifteenadult male cynomolgus monkeys(Macaca fascicularis) (7–15 yearsold; 5.0–7.6 kg body weight) wereselected for the study after screeningthe SICONBREC breeding coloniesfor animals with chronic periodonti-tis. The inclusion criteria were thepresence of at least 30% of sites withprobing pocket depth and clinicalattachment level ≥4 mm, associatedwith bleeding on probing, and radio-graphic evidence of bone loss (usinga digital X-ray dental system; Vat-ech, Fort Lee, NJ, USA). The ani-mals were socially housed instainless steel cages and were used inthe study after they were acclima-tized to the protocol procedures for4 weeks. Environmental enrichmentwas provided through daily handlingby animal care technicians, environ-mental enrichment items and visualcontact with other study animals.Each animal was offered a measuredamount of an approved feed mix-ture. Fresh, potable drinking waterwas available to the animals ad libi-tum. Clinical periodontal examina-tions, dental X-rays, collection ofGCF and periodontal tissue biopsieswere performed in a manner similarto a human clinical study, exceptthat the animals were anaesthetizedduring the procedures.

C3 inhibitor

The compstatin analogue Cp40 (y-I[CV(1MeW)QDW-Sar-AHRC](NMeI)-NH2; with y = D-tyrosine; Sar = sar-cosine/N-methyl glycine) was pro-duced as a disulphide-bridged, cyclicpeptide by solid-phase peptide syn-thesis methodology as previouslydescribed (Qu et al. 2013).

Experimental design

The study involved a 6-week treat-ment period with Cp40 and a 6-weekfollow-up period without Cp40 treat-ment. Cp40 was administered eitheronce a week (5 animals; “19-treat-ment”) or three times per week (10animals; “39-treatment”). Specifi-cally, using a 30-g short needle, Cp40was injected locally into the gingiva(100 lg/site in 50-ll volume) of ante-rior and posterior teeth on both sidesof the maxilla (15 sites total; 13 sites

corresponding to palatal inter-dentalpapillae and two sites correspondingto the distal gingiva of the secondmolars). Clinical readings madebefore Cp40 administration served asbaseline controls for each animal.The mandible was not treated withCp40 but was monitored by clinicalperiodontal examination and sam-pling of biological specimens (seebelow) throughout the study, for com-parative purposes. Clinical examina-tions, sample collection and standardlaboratory techniques (immunofluo-rescence histochemistry, histologicalTRAP staining and assessment ofhost immune responses) are describedin Appendix S1.

Statistical analysis

For the comparison of mean valueswithin the groups during the time-course studies, one-way repeated-measures ANOVA with Greenhouse-Geisser correction was performedusing the GraphPad Prism program,version 6.0h (La Jolla, CA, USA).In case of significant differences,Bonferroni’s or Tukey’s multiplecomparisons test was performed.

Results

Locally administered Cp40 inhibits clinical

periodontal inflammation in NHPs

All animals enrolled in the studywere systemically healthy and main-tained good systemic health duringthe observation period. No adverseeffects were noted during the courseof the study. To determine whethercomplement is causally linked toinflammation associated with natu-rally occurring chronic periodontitis,we targeted the central complementcomponent C3. Specifically, welocally injected the C3-specific inhibi-tor Cp40 into the maxillary gingivaof NHPs. Initially, the drug wasadministered for 6 weeks at a fre-quency of three times weekly (“39-treatment”) and its clinical effects weremonitored for 12 weeks. Cp40caused a significant reduction in sev-eral clinical indices that measureperiodontal inflammation (gingivalindex and bleeding on probing), tis-sue destruction (probing pocketdepth and clinical attachment level)or tooth mobility often associatedwith bone loss (Fig. 1a–e). These

data indicate that, at least by clinicalcriteria, Cp40 blocks inflammatoryprocesses that drive periodontal tis-sue destruction. Interestingly, theseprotective effects were evident asearly as 1 week after treatment initi-ation and progressively improveduntil week 6, when the drug was dis-continued. Remarkably, the protec-tive effects of Cp40 persisted for atleast six additional weeks, since atthe termination of the study (week12) all indices remained at signifi-cantly lower levels relative to theircorresponding baselines (Fig. 1a–e).Importantly, without any exception,all ten cynomolgus monkeys responded favourably to Cp40 with 57–87% reduction in gingival inflamma-tion and 31–58% decrease in thedepths of the periodontal pocketsafter 6 weeks of treatment (FigsS1–S10). The plaque index, a clinicalmeasure of biofilm accumulation ontooth surfaces, was modestly but sig-nificantly reduced by Cp40 at a fewtime points, immediately before andafter week 6 (Fig. 1f). The aforemen-tioned clinical indices were alsomonitored in the untreated jaw(mandible) during the same 12-weekinterval. In contrast to the improvedclinical condition of the Cp40-trea-ted maxilla, the clinical indices in themandible did not show significantdifferences in the course of the studyas compared to their baseline values(Fig. 1a–f).

In a second, independent experi-ment, we investigated whether Cp40could retain its efficacy if adminis-tered only once per week (“19-treat-ment”). Similar clinical analysesrevealed that a single weekly admin-istration of Cp40 could significantlyreduce indices of clinical inflamma-tion and tissue destruction (Fig. 2),with almost comparable efficacy andsimilar time course pattern to that ofthe 39-treatment (see superimposi-tion of the data in Fig. S11). More-over, similarly to the 39-treatment,all five cynomolgus monkeys used inthe 19-treatment study respondedfavourably to the drug with noexception (Figs S12–S16).

Decreased levels of pro-inflammatory

mediators following local treatment with

Cp40

GCF was collected for monitoringchanges in the cytokine and immune


C3 inhibition in periodontitis 3

mediator levels during the 6-weekcourse of Cp40 treatments, as wellas during the follow-up period toweek 12. Multi-cytokine analysis ofthe GCF revealed that the 39-treat-ment with Cp40 resulted in signifi-cantly lower levels of pro-inflammatory and osteoclastogeniccytokines, as compared to their base-line values (Fig. 3). The pro-inflam-matory cytokines measured includedIL-1b, IL-6, IL-8 and IL-17(Fig. 3a–d), all which have beenassociated with periodontal inflam-mation in humans (Graves 2008,Moutsopoulos et al. 2012, Zenobia

& Hajishengallis 2015), and receptoractivator of NF-jB ligand(RANKL) (Fig. 3e), a key osteoclas-togenic cytokine involved in boneloss disorders including periodontitis(Bostanci et al. 2007, Miossec &Kolls 2012). In contrast, the GCFlevels of osteoprotegerin (OPG), anatural antagonist of RANKL (Bos-tanci et al. 2007, Miossec & Kolls2012), were increased upon Cp40treatment relative to baseline(Fig. 3f). Cp40 also caused a signifi-cant decrease in the GCF levels ofC3a and C5/C5a as seen as early as1 week after treatment (Fig. 3g,h

respectively). These favourablechanges in the host response profile(inhibition of pro-inflammatorymediators and upregulation of OPG)were most pronounced at 6 weeks,although significant changes per-sisted for the entire or most of thestudy duration (12 weeks), despitedrug withdrawal at week 6 (Fig. 3).The same mediators were monitoredin GCF samples collected from theuntreated jaw (mandible) during thesame 12-week interval but did notshow significant differences relativeto baseline values (Fig. 3). Impor-tantly, Cp40 retained its capacity tosignificantly suppress the GCF levelsof pro-inflammatory mediators andupregulate OPG even when adminis-tered only once per week (Fig. 4).

The anti-inflammatory action ofCp40 was also evident in the peri-odontal tissue (Fig. 5), thus confirm-ing the GCF findings. Specifically,immunofluorescence histochemistryof biopsy specimens taken before(0 week) and after (6 weeks) treat-ment showed that Cp40 causeddecreased expression of IL-17,RANKL and elevated expression ofOPG in the connective tissue adja-cent to the alveolar bone at 6 weeksrelative to baseline (Fig. 5). More-over, Cp40 treatment caused adecrease in the complement cleavagefragments C3d and C5a (Fig. 5), fur-ther confirming its ability to inhibitcomplement activation.

The inhibitory action of Cp40 onthe various pro-inflammatory and/orpro-osteoclastogenic cytokines wasmediated, at least in part, at thetranscriptional level since the expres-sion of gingival IL-1b, IL-6, IL-8,IL-17 and RANKL mRNA was sig-nificantly inhibited in animals with39 or 19 weekly treatments(Fig. 6a,b respectively). Conversely,and in accord with the protein data,OPG mRNA expression wasincreased (Fig. 6). The characteristicelevation of IL-1b in human peri-odontitis (compared to periodontalhealth) has been correlated withincreased NLRP3 (NALP3) inflam-masome mRNA expression levels(Bostanci et al. 2009). In this regard,Cp40 inhibited the gingival NLRP3mRNA expression in NHPs (Fig. 6),suggesting its potential to interferewith NLRP3 inflammasome-depen-dent processing of IL-1b; this notionis consistent with the reduced levels

Fig. 1. Cp40 decreases inflammatory clinical parameters of naturally occurring chronicperiodontitis in NHPs after local administration three times weekly. Cp40 was injected– three times weekly for 6 weeks – into the inter-dental papillae and the distal gingivaof the second molars of the maxilla (“Cp40”), whereas the mandible was not treated(“Untreated”). Each animal was clinically examined at the indicated time points andthe following clinical parameters were recorded: (a) gingival index; (b) bleeding onprobing; (c) probing pocket depth; (d) clinical attachment level; (e) mobility index and(f) plaque index. The data were expressed relative to the baseline values (at week 0),set as 100 (Raw data are shown for each animal in Figs S1–S10). Results aremeans � SD (n = 10 monkeys). *p < 0.05 and **p < 0.01 compared to baseline (one-way repeated-measures ANOVA and Bonferroni’s multiple comparisons test). NHPs,non-human primates.


4 Maekawa et al.

of IL-1b protein in the GCF ofCp40-treated animals (Figs 3a and4a).

Cp40 causes a reduction in the numbers

of periodontal osteoclasts in NHPs

The RANKL/OPG ratio in the GCFis a potential indicator of periodonti-tis (Bostanci et al. 2007, Belibasakis& Bostanci 2012). We thereforedetermined whether the ability ofCp40 to decrease the GCF levels ofRANKL and enhance the levels ofOPG (Figs 3 and 4), hence to lowerthe RANKL/OPG ratio, could have

an impact on osteoclastogenesis. Tothis end, we counted the numbers ofosteoclasts (TRAP-positive multinu-cleated cells; Fig. S17) in bone biop-sies taken at baseline and at 6 and12 weeks following Cp40 treatment.We found that in the maxillae of theanimals, Cp40 caused a significantdecrease in the numbers of osteo-clasts after 6 weeks of 39 or 19weekly treatments (Fig. 7a,b, leftpanels). This favourable effect per-sisted through to week 12, eventhough the drug was not adminis-tered in the last 6 weeks (Fig. 7a,b,left panels). In contrast, the numbers

of osteoclasts in the mandibles,which were not treated, did not dis-play significant differences in thecourse of the study (Fig. 7a,b, rightpanels).

Discussion

To the best of our knowledge, thisstudy marks the first time that apharmacological intervention –whether host-modulation or antimi-crobial – is shown to inhibit inflam-mation in the context of naturallyoccurring periodontitis in NHPs.Previously conducted studies, includ-ing our own, have used induciblemodels of the disease involvingplacement of ligatures with or with-out exogenous inoculation of peri-odontal pathogens (Offenbacheret al. 1987, Pierce & Lindskog 1987,Nisengard et al. 1989, Persson et al.1994, Li et al. 1996, Assuma et al.1998, Moritz et al. 1998, Cappelliet al. 2000, Roberts et al. 2004, Pageet al. 2007, Maekawa et al. 2014a,Shin et al. 2015). The immune sys-tem and periodontal anatomy of thecynomolgus monkey is similar tothat of humans, and periodontitis inthese animals exhibits clinical, micro-biological and immuno-histologicalfeatures that are highly similar tothose observed in human periodonti-tis (Kornman et al. 1981, Page &Schroeder 1982, Brecx et al. 1985,Ebersole et al. 2002, 2014). There-fore, the cynomolgus model, espe-cially in the setting of naturallyoccurring periodontitis, is consider-ably more predictive of drug efficacyin humans compared to widely usedmodels, such as those in rodents,rabbits or dogs. The successful Cp40inhibition of natural periodontalinflammation and osteoclastogenesisin NHPs additionally showsunequivocally that C3 is critical forthe pathogenesis of this oral disease,as suggested by earlier correlativehuman clinical studies (Attstromet al. 1975, Schenkein & Genco1977, Toto et al. 1978, Niekrash &Patters 1985, Nikolopoulou-Papa-constantinou et al. 1987, Patterset al. 1989, Hajishengallis 2010). Inconjunction with the earlier humanstudies, the present work places peri-odontitis to a growing list of diseaseswith substantial complementinvolvement, including paroxysmalnocturnal haemoglobinuria (PNH),

Fig. 2. Single weekly administration of Cp40 decreases inflammatory clinical parame-ters of naturally occurring chronic periodontitis in NHPs. Cp40 was injected – onceweekly for 6 weeks – into the inter-dental papillae and the distal gingiva of the secondmolars of the maxilla (“Cp40”), whereas the mandible was not treated (“Untreated”).Each animal was clinically examined at the indicated time points and the followingclinical parameters were recorded: (a) gingival index; (b) bleeding on probing; (c)probing pocket depth; (d) clinical attachment level; (e) mobility index and (f) plaqueindex. The data were expressed relative to the baseline values (at week 0), set as 100(Raw data are shown for each animal in Figs S12–S16). Results are means � SD(n = 5 monkeys). *p < 0.05 and **p < 0.01 compared to baseline (one-way repeated-measures ANOVA and Bonferroni’s multiple comparisons test). NHPs, non-human pri-mates.



age-related macular degeneration,atypical haemolytic uraemic syn-drome and rheumatoid arthritis(Ricklin & Lambris 2013). Consis-tent with the role of C3 as a poten-tial therapeutic target inperiodontitis, a recent report hasidentified C3 among the top 21 mostpromising candidate genes involvedin periodontitis, by using an integra-tive gene prioritization method anddatabases from genome-wide associ-ation studies and microarray experi-ments (Zhan et al. 2014).

The complement inhibitioncapacity of Cp40 was confirmed byfindings of decreased GCF levels ofC3a and C5/C5a. The detecting anti-body utilized in the latter assay doesnot distinguish between C5 and theactivation product C5a and, there-fore, C5 levels could be reduced indi-rectly by Cp40 through inhibition ofinflammation (Zou et al. 2013).However, the ability of Cp40 toreduce C3a definitely confirmed itspotential to inhibit complement acti-vation in our study. In this regard, itis uncertain if intra-gingivally admin-istered Cp40 inhibited complementactivation in the gingival tissueresulting in lower levels of availableC3a to transudate to the periodontalpocket or whether Cp40 also dif-fused to the periodontal pocketwhere it could additionally blockcomplement activation in the GCF.Although both possibilities arelikely, the ability of Cp40 to blockcomplement in the gingiva was con-firmed by immunohistochemistrythat showed decreased levels of thecomplement cleavage products (C3dand C5a) in the connective tissue inthe vicinity of the bone.

Cp40 is the first compstatin ana-logue with subnanomolar targetaffinity (KD = 0.5 nM) and featuresa plasma half-life that exceeds expec-tations for most peptidic drugs (Quet al. 2013, Mastellos et al. 2015b).The relatively long half-life of theCp40 peptide was attributed to a“target-driven” model, according towhich an initial rapid clearance ofexcess free peptide (i.e. not bound toC3) is followed by slow clearance ofC3-bound peptide. As the measuredhalf-life values of different comp-statin analogues correlate positivelywith their binding affinities for C3(Qu et al. 2013), it can be inferredthat the tight binding of Cp40 to C3

Fig. 3. Decreased GCF levels of pro-inflammatory and pro-osteoclastogenic mediatorsin NHPs with natural periodontitis after local treatment with Cp40 three times weekly.At the indicated time points, GCF was collected from monkeys with natural periodon-titis which were treated three times weekly for 6 weeks with Cp40 in the maxilla(“Cp40”) but not in the mandible (“Untreated”). Total cytokine or mediator content(a, IL-1b; b, IL-6; c, IL-8; d, IL-17; e, RANKL; f, osteoprotegerin (OPG); g, C3a; h,C5/C5a) in the eluted GCF samples was measured using a Bio-Plex system or ELISA(C3a only). Data are means � SD (n = 10 monkeys). *p < 0.05; **p < 0.01 comparedto baseline (one-way repeated-measures ANOVA and Bonferroni’s multiple comparisonstest). GCF, gingival crevicular fluid; NHPs, non-human primates; OPG, osteoprote-gerin; RANKL, receptor activator of NF-jB ligand.


6 Maekawa et al.

delays its clearance. Similarly, theexpected tight binding of Cp40 toabundant C3 in the inflamed peri-odontium could contribute todelayed elimination of the drug fromthe tissues, in turn accounting – atleast in part– for the sustained pro-tective effect of Cp40 observed in thisstudy. Alternatively, or additionally,it is likely that the observed suppres-sion of inflammation by Cp40 tipsthe balance towards tissue homeosta-sis, which might then resist patholog-ical processes for some time even inthe absence of the drug. In thisregard, the clinical periodontalindices, the GCF levels of pro-inflam-matory mediators and the numbersof periodontal osteoclasts persisted atlower levels than their correspondingbaseline values for at least 6 weeksfollowing drug withdrawal.

Given that complement is not alinear cascade of events but essen-tially a hub-like network that istightly connected to other compo-nents of the immune system (Ricklinet al. 2010), it is plausible that C3inhibition has a far-reaching impactabove and beyond complement itself.For instance C3a- and C5a-inducedsignalling pathways cross-talk withand amplify TLR-mediated inflam-matory responses in various tissuesincluding the periodontium (Zhanget al. 2007, Hajishengallis & Lambris2010, Abe et al. 2012). Complementinhibition, therefore, is likely to miti-gate inflammation initiated throughTLR activation, either by microbialligands (e.g. bacterial lipopolysaccha-ride or lipoproteins) or by endoge-nous molecules acting as dangersignals upon their release due toinflammatory tissue destruction (e.g.biglycan, hyaluronan fragments andheparan sulphate fragments) (Miyake2007, Schaefer 2010). These consider-ations may in part explain why theinhibition of a single molecule, C3,has a strong influence on the courseof natural periodontal inflammation.

It was also recently shown that,in human monocytes, C3a regulatesthe release of intracellular ATP intothe extracellular space, thereby con-trolling NLRP3 inflammasome acti-vation and subsequent secretion ofIL-1b, which in turn promoteshuman CD4+ T cell production ofIL-17 (Asgari et al. 2013). In thisregard, a clinical study has shownenhanced expression of NLRP3

Fig. 4. Single weekly administration of Cp40 decreases the levels of pro-inflammatoryand pro-osteoclastogenic mediators in the GCF of NHPs with natural periodontitis.At the indicated time points, GCF was collected from monkeys with natural periodon-titis which were treated once weekly for 6 weeks with Cp40 in the maxilla (“Cp40”)but not in the mandible (“Untreated”). Total cytokine or mediator content (a, IL-1b;b, IL-6; c, IL-8; d, IL-17; e, RANKL; f, OPG; g, C3a; h, C5/C5a) in the eluted GCFsamples was measured using a Bio-Plex system or ELISA (C3a only). Data aremeans � SD (n = 5 monkeys). *p < 0.05; **p < 0.01 compared to baseline (one-wayrepeated-measures ANOVA and Bonferroni’s multiple comparisons test). GCF, gingivalcrevicular fluid; NHPs, non-human primates; OPG, osteoprotegerin; RANKL, recep-tor activator of NF-jB ligand.



(NALP3) inflammasome in peri-odontitis correlating with increasedIL-1b expression levels (Bostanciet al. 2009). Moreover, the forma-tion of sublytic C5b-9 complex onhuman epithelial cells induces intra-cellular Ca2+ fluxes leading toNLRP3 inflammasome activationand IL-1b release (Triantafilou et al.2013). Both of these complement-dependent inflammatory mechanismscan be blocked by Cp40, potentiallyaccounting – at least in part – forthe reduced IL-1b and IL-17 levelsafter Cp40 treatment. In fact, theability of Cp40 to reduce the levelsof IL-1b protein in the GCF might,in part, be related to its capacity toinhibit the expression of NLRP3which is involved in caspase-1–mediated processing and release ofIL-1b protein (Lamkanfi & Dixit2014).

Cp40-mediated inhibition ofIL-17 could in turn contribute to theobserved suppression of RANKL,since IL-17 upregulates RANKL byacting on lymphocytes and stromalcells (e.g. fibroblasts and osteoblasts)(Miossec & Kolls 2012). Besidesinhibiting RANKL expression, Cp40also caused a concomitant increase inthe levels of OPG, both in the gingi-val tissue and the GCF. Although theunderlying mechanisms are uncer-tain, RANKL and OPG were shownto be conversely regulated by severalstimuli (Lee & Lorenzo 1999, Deviet al. 2013, Boespflug et al. 2014).For instance IL-17 was shown toinduce RANKL and suppress OPGexpression in human periodontal

Fig. 5. Expression of inflammatory and osteoclastogenesis-related molecules in periodontal biopsy specimens from Cp40-treatedNHPs. Periodontal biopsy specimens from the maxillae of NHPs treated with Cp40 were processed for fluorescent microscopy. Thespecimens were taken before (week 0) and after (week 6) treatment with Cp40 locally administered three times weekly. Shown arerepresentative overlays of DIC and fluorescent images stained for the indicated molecules. B, bone; CT, connective tissue; DIC,differential interference contrast; NHPs, non-human primates. Scale bar, 100 lm.

Fig. 6. Cp40 inhibits mRNA expression of gingival inflammatory mediators in NHPs.Gingival tissue biopsies were taken from the maxilla of NHPs treated three times (a)or once (b) weekly for 6 weeks and extracted RNA was subjected to real-time PCRprocessed to determine mRNA expression of the indicated molecules at the indicatedtimes. Data were normalized to GADPH mRNA and are presented as fold change inthe transcript levels relative to baseline levels (prior to treatment; week 0), set as 1.Data are means � SD (a, n = 10 monkeys; b, n = 5 monkeys). *p < 0.05; **p < 0.01compared to baseline (one-way repeated-measures ANOVA and Bonferroni’s multiplecomparisons test). NHPs, non-human primates; NS, not significant.


8 Maekawa et al.

ligament cells (Devi et al. 2013). Con-sistently, the ability of Cp40 todecrease the RANKL/OPG ratio wasassociated with decreased osteoclas-togenesis in bone biopsy samples.Therefore, Cp40 has the potential toinhibit naturally occurring bone loss,in accord with its capacity to blockligature-induced bone loss in NHPs(Maekawa et al. 2014a). Althoughinduction of measurable bone losscan be observed within a few weeksin the relatively acute ligature-induced model, bone loss is a rela-tively slow process in naturally occur-ring chronic periodontitis (hencechanges in bone loss could not bedetected radiographically in this studydue to its relatively short duration).

By inhibiting complement at thelevel of C3, Cp40 (and earlier comp-statin analogues) does not interferewith C4b-mediated opsonization of

bacteria via the classical and lectinpathways (Kirjavainen et al. 2008).Nevertheless, for increased safety,Cp40 treatments in disease settingsrequiring long-term systemic inter-vention (e.g. in PNH (Risitano et al.2014)) would necessitate vaccinationagainst encapsulated bacteria (e.g.meningococci) to minimize the riskof potential infections (Mastelloset al. 2015a). Importantly, thesepotential safety considerations areunlikely to apply to the treatment ofperiodontitis through local Cp40administration. In this regard,C3-deficient mice display reducedperiodontal bacterial load comparedto C3-sufficient controls in thecourse of experimental periodontitis(Maekawa et al. 2014a), suggestingthat impaired complement activationdoes not predispose to defectiveimmune surveillance in the periodon-

tium. These findings are in accordwith the notion that inflammationgenerates tissue breakdown products(e.g. degraded collagen peptides orhaeme-containing compounds) thatserve the nutritional needs of peri-odontitis-associated bacteria (Marsh2003, Hajishengallis 2014b). Indeed,studies in mouse and rabbit modelsof periodontitis indicate that thecontrol of inflammation also reducesthe bacterial load (Hasturk et al.2007, Eskan et al. 2012, Abe et al.2014, Moutsopoulos et al. 2014).Conversely, and consistently, thebacterial biomass of human peri-odontitis-associated biofilmsincreases with escalating periodontalinflammation (Abusleme et al. 2013).

Although Cp40 was successfullyapplied as a stand-alone treatment inthe current NHP study, it can beenvisioned as an adjunctive therapyto the management of human chronicperiodontitis. Future clinical trialscould investigate the potential ofCp40 to inhibit periodontal inflam-mation and bone loss compared toscaling and root planing, whereas invery severe cases of the disease, Cp40could be combined with scaling androot planing and compared to peri-odontal surgery, in an effort to obvi-ate the need for a surgical approach.It should be noted that future host-modulation interventions, such asCp40, would not necessarily beimplemented in a therapeutic settingbut could also be provided on a pre-ventive basis to high-risk individuals,such as cigarette smokers and dia-betic patients (Heitz-Mayfield 2005,Genco & Genco 2014), before theonset of periodontitis. A clinicallydeveloped Cp40-based drug (AMY-101; Amyndas Pharmaceuticals, Gly-fada, Attica, Greece.) is currentlyunder evaluation as a potential treat-ment of complications of ABO-incompatible kidney transplantationand PNH (Mastellos et al. 2015b).Whether Cp40/AMY-101 can findapplication for the treatment ofhuman periodontitis is a possibilitythat – based on the results of thisNHP study– merits investigation infuture clinical trials.

Acknowledgements

We thank Dr. Niki M. Mout-sopoulos (NIDCR/NIH) for com-ments and suggestions.

Fig. 7. Decreased numbers of osteoclasts after treatment of natural NHP periodontitiswith Cp40. At the indicated time points, alveolar bone biopsies were obtained frommonkeys with natural periodontitis. The animals were treated three times (a) or once(b) weekly for 6 weeks with Cp40 in the maxilla but not in the mandible(“Untreated”). TRAP-positive multinucleated cells (osteoclasts) were enumerated in 10randomly selected sections for each bone biopsy specimen from the maxilla or man-dible of each of the animals. The numbers of osteoclasts were averaged for eachbiopsy specimen and are represented by dots. Scatter dot plots with mean � SD areshown for each group of monkeys (a, n = 10; b, n = 5 animals). Indicated p-valuesdetermined by one-way repeated-measures ANOVA and Tukey’s multiple comparisonstest. NHP, non-human primate.



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Supporting Information

Additional Supporting Informationmay be found in the online versionof this article:

Appendix S1. Supplementary meth-ods.Figure S1. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #1.Figure S2. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #2.Figure S3. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #3.Figure S4. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #4.

Figure S5. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #5.Figure S6. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #6.Figure S7. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #7.Figure S8. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #8.Figure S9. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #9.Figure S10. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (39 treat-ment): Raw data for monkey #10.Figure S11. Cp40 decreases inflam-matory clinical parameters of natu-rally occurring chronic periodontitisin NHPs.Figure S12. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (19 treat-ment): Raw data for monkey #1.Figure S13. Effects of Cp40 oninflammatory clinical parameters of

NHP chronic periodontitis (19 treat-ment): Raw data for monkey #2.Figure S14. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (19 treat-ment): Raw data for monkey #3.Figure S15. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (19 treat-ment): Raw data for monkey #4.Figure S16. Effects of Cp40 oninflammatory clinical parameters ofNHP chronic periodontitis (19 treat-ment): Raw data for monkey #5.Figure S17. Detection of osteoclastsin non-human primate periodontitis.

Address:John D. LambrisPerelman School of MedicineUniversity of Pennsylvania422 Curie BoulevardPhiladelphia, PA 19104-6100USAE-mail: [email protected] HajishengallisSchool of Dental MedicineUniversity of Pennsylvania240 South 40th StreetPhiladelphia, PA 19104-6030USAE-mail: [email protected]

Clinical Relevance

Scientific rationale for the study:Most interventional studies in ani-mal models of periodontitis areperformed in a preventive (ratherthan therapeutic) setting andinvolve inducible models of the dis-ease. To increase the predictivevalue of preclinical intervention for

drug efficacy in humans, we usednon-human primates with naturallyoccurring periodontitis to test thetherapeutic potential of a comple-ment inhibitor (Cp40).Principal findings: Cp40 inhibitedpre-existing periodontal inflamma-tion (determined by both clinicaland laboratory assessment) and

osteoclastogenesis in non-humanprimates, a clinically relevantmodel of human periodontitis.Practical implications: These find-ings pave the way for testing Cp40in future clinical trials for the treat-ment of human periodontitis.


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