Celastrol efficacy by oral administration in the adjuvant-induced arthritis model

ORIGINAL RESEARCH published: 08 Sep embe 2020 doi: 10.3389/ med.2020.00455 F on ie s in Medicine | www. on ie sin.o g 1Sep embe 2020 | Volume 7 | A icle 455 Edi ed by: Pe e Ko s en, Uni e si y Medical Cen e Gö ingen, Ge many Re iewed by: Ja ie Rod íguez-Ca io, Uni e sidad de O iedo Mie es, Spain Ka a ina Baue o a, Cen e o Expe imen al Medicine, Slo ak Academy o Sciences (SAS), Slo akia *Co espondence: B uno Vidal [email p o ec ed] †These au ho s sha e senio au ho ship Special y sec ion: This a icle was submi ed o Rheuma ology, a sec ion o he jou nal F on ie s in Medicine Recei ed: 11 Decembe 2019 Accep ed: 08 July 2020 Published: 08 Sep embe 2020 Ci a ion: Cascão R, Vidal B, Ca alho T, Lopes IP, Romão VC, Goncal es J, Moi a LF and Fonseca JE (2020) Celas ol E icacy by O al Adminis a ion in he Adju an -Induced A h i is Model. F on . Med. 7:455. doi: 10.3389/ med.2020.00455 Celas ol E icacy by O al Adminis a ion in he Adju an -Induced A h i is Model Ri a Cascão1, B uno Vidal1*, Tânia Ca alho2, Inês Pascoal Lopes1, Vasco C. Romão 1,3, João Goncal es4, Luis Fe ei a Moi a5† and João Eu ico Fonseca 1,3† 1Unidade de In es igação em Reuma ologia, Faculdade de Medicina, Ins i u o de Medicina Molecula -João Lobo An unes, Cen o Académico de Medicina de Lisboa, Uni e sidade de Lisboa, Lisbon, Po ugal, 2Compa a i e Pa hology Uni , Ins i u o de Medicina Molecula -João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon, Po ugal, 3Se iço de Reuma ologia e Doenças Ósseas Me abólicas, Hospi al de San a Ma ia, Cen o Hospi ala Uni e si á io Lisboa No e, Lisbon, Po ugal, 4Faculdade de Fa mácia, iMed – Resea ch Ins i u e o Medicines, Uni e sidade de Lisboa, Lisbon, Po ugal, 5Inna e Immuni y and In lamma ion Labo a o y, Ins i u o Gulbenkian de Ciência, Oei as, Po ugal Backg ound: We p e iously demons a ed ha celas ol has signi ican an i-in lamma o y and bone p o ec i e e ec s when adminis e ed ia he in ape i oneal ou e. Fo u he p eclinical e alua ion, an e ec i e o al adminis a ion o celas ol is c ucial. He e we aimed o s udy he he apeu ic dose ange o i s o al adminis a ion. Me hods: Celas ol (1–25 µg/g/day, N=5/g oup) was adminis a ed o ally o emale adju an -induced a h i is (AIA) a s a e 8 days o disease induc ion o a pe iod o 14 days. A g oup o heal hy (N=8) and a h i ic (N=15) gende - and age-ma ched Wis a a s was used as con ols. Du ing he ea men pe iod, he in lamma o y sco e, ankle pe ime e , and body weigh we e measu ed. A he end o he ea men , he animals we e sac i iced, blood was collec ed o clinical pa hology, nec opsy was pe o med wi h collec ion o in e nal o gans o his opa hological analysis, and paw samples we e used o disease sco ing. Resul s: Doses highe han 2.5 µg/g/day o celas ol educed he in lamma o y sco e and ankle swelling, p ese ed join s uc u e, hal ed bone des uc ion, and diminished he numbe o syno ial CD68+mac ophages. Bone eso p ion and u no e we e also educed a 5 and 7.5 µg/g/day doses. Howe e , he dose o 7.5 µg/g/day was associa ed wi h hymic and li e lesions, and highe doses showed se e e oxici y. Conclusion: O al adminis a ion o celas ol abo e 2.5 µg/g/day amelio a es a h i is. This da a suppo s and gi es ele an in o ma ion o he de elopmen o a p eclinical es o celas ol in he se ing o a ch onic model o a h i is since heuma oid a h i is is a long- e m disease. Keywo ds: heuma oid a h i is, adju an -induced a h i is, celas ol, dose, e icacy Cascão e al. E icacy and Toxici y o Celas ol INTRODUCTION The he apeu ic e ec o celas ol has been demons a ed in se e al in lamma o y diseases. Celas ol is a pen acyclic- i e pene compound ha can be ound in oo ex ac s om T ip e ygium wil o dii (TW) (1), an he b used in Chinese medicine (2–4). In he las 5 yea s, inc easing e idence o he he apeu ic po en ial o celas ol in he ea men o heuma oid a h i is (RA) has eme ged. S udies ha e sugges ed ha he an i- in lamma o y p ope ies o celas ol can be mainly a ibu ed o he egula ion o cy okine p oduc ion (5–8), he modula ion o in lamma o y cells (8–13), he inhibi ion o os eoclas ogenesis, and bone p o ec ion (8,14–16), mos ly due o i s abili y o down egula e he NF-kB pa hway. Speci ically, we ha e demons a ed ha he in ape i oneal adminis a ion o celas ol supp essed in lamma o y signs (7), p ese ed join s uc u e, wi h ab oga ion o he in lamma o y in il a e and cellula p oli e a ion (7,8), and hal ed ocal bone damage in he adju an -induced a h i is (AIA) a model (8,17). This inhibi o y e ec o cellula in il a ion and p oli e a ion may p o e o be o in e es o ea he de elopmen o he syno ial umo -like pannus issue cha ac e is ic o RA, one o he main con ibu o s o bone damage. Impo an ly, we ha e epo ed ha his compound is able o signi ican ly dec ease he numbe o sublining CD68+syno ial mac ophages (8), a bioma ke o ea men e icacy in RA (18–20). So a , we obse ed ha he in ape i oneal adminis a ion o celas ol o AIA a s was no associa ed wi h o e signs o oxici y (8). Despi e he he apeu ic po en ial o celas ol, u he clinical applica ion seems o be limi ed by poo wa e solubili y (21), low o al bioa ailabili y (22), possible side e ec s (23–26), and a iabili y in dose egimens (9,27–30). The e o e, in o de o ad ance he p eclinical de elopmen o celas ol as a candida e he apeu ic compound o RA ea men , we analyzed he he apeu ic dose ange o o al adminis a ion o pu e celas ol using he AIA a model. MATERIALS AND METHODS Animals The AIA model has been he mos ex ensi ely used a h i ic a model o s udy an i-a h i ic agen s because i has an excellen ack eco d o p edic ing bo h ac i i y and oxici y. AIA a s sha e key ea u es ela ed o RA, making hem a c i ical ool in d ug de elopmen , and exhibi he g ea es magni ude o disease when compa ed wi h o he models o a h i is (31). Eigh -week-old emale Wis a AIA a s weighing 230–250 g we e pu chased om Cha les Ri e Labo a o ies In e na ional (Massachuse s, USA). Cha les Ri e Labo a o ies pe o med he induc ion o adju an disease using F eud’s comple e adju an , supplemen ed wi h mycobac e ium, and injec ed in he igh oo pad. The AIA a s we e main ained unde speci ic-pa hogen- ee condi ions, andomly housed pe g oup unde s anda d labo a o y condi ions (a 22◦C unde 10-h ligh /14-h da k condi ions), and gi en ee access o ood (RM3, SDS Die s, UK) and wa e (ul apu e). In addi ion, o minimize animal discom o , pape sha ings we e used as bedding ma e ial in Double Decke GR1800 cages (Techniplas , UK) wi h i e animals each. The c i e ia o a humane sac i ice we e de e mined as p e iously published (8), and he animals we e sac i iced when p esen ing he maximum in lamma o y sco e in mo e han wo limbs o when weigh loss exceeded 20%. In acco dance wi h Di ec i e 2010/63/EU, all animal p ocedu es we e app o ed by he ins i u ional animal wel a e body (ORBEA- iMM) and licensed by he Po uguese compe en au ho i y (DGAV—Di ecção Ge al de Alimen ação e Ve e iná ia, license numbe : 0421/000/000/2016). Celas ol P epa a ion and Adminis a ion Celas ol (Sigma, Missou i, USA) s ock solu ion o 10 mg/ml was p epa ed using e hanol 100% as sol en ( ehicle). A ecen s udy has es ed he solubili y o celas ol in di e en ehicles, and we ha e p e iously demons a ed ha e hanol is one o he mos e icien sol en s o his compound (21). This celas ol s ock solu ion was u he dissol ed in PEG400 (Sigma, S . Louis, USA) (1, 2.5, 5, 7.5, 12.5, and 25 µg/g in 1 ml) and adminis a ed by o al ga age o AIA a s o 14 consecu i e days (N=5 a s/g oup). The sample size in each g oup was calcula ed using ee sample size calcula ing G∗Powe e sion 3.1.9.2 so wa e [ ype o powe analysis: a p io i;αe o p obabili y: 0.05; powe (1-βe o p obabili y): 0.95; e ec size d: 2.59; ac ual powe : 0.976]. This calcula ion was based on ou own p e ious da a (7,8,17). Ou s udy was app o ed by he ins i u ional animal wel a e body, licensed by he Po uguese compe en au ho i y, and complied wi h good e hical, scien i ic, legal, and economic easons o using labo a o y animals, including he 3R p inciple ( eplace, educe, and e ine). Focused on he “ educe” ule, we used he minimum numbe o animals, calcula ed by he ee sample size calcula ing G∗Powe so wa e, in o de o pe o m his s udy. The need o daily adminis a ions is suppo ed by he s udy o Zhang e al., which showed ha he hal -li e o pu e celas ol is ∼10 h (22). Based on his publica ion, we ha e also calcula ed he o al dose o 2.5 µg/g/day as he equi alen o he in ape i oneal dose o 1 µg/g/day ha we had p e iously ound o be e ec i e in he ea men o a h i is in he same a model (7,8). The calcula ion was based on he ac ion o he celas ol dose abso bed in he po al blood a e o al adminis a ion, which was 17.6%. In ape i oneal dose calcula ion ook in o conside a ion ha he in apo al dose will ha e highe bioa ailabili y. The e o e, he ela ionship be ween he a ea unde he cu e and he doses adminis e ed in he o al and he in apo al dose was used o de e mining he in ape i oneal dose. T ea men was ini ia ed a e 8 days o disease induc ion, a he acu e clinical s age o a h i is p og ession ( he apeu ic in e en ion) (32). Heal hy non-a h i ic (N=8) and a h i ic un ea ed (N=15) emale age-ma ched Wis a a s we e used as con ols. The a h i ic un ea ed g oup ecei ed an equal olume o ehicle h ough o al ga age. The ehicle p opo ion o e hanol and PEG400 used was in he same p opo ion as he one used in he celas ol- ea ed g oups. The a s we e sac i iced a e 22 days o disease p og ession by CO2na cosis, and blood, in e nal F on ie s in Medicine | www. on ie sin.o g 2Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol o gans, as well as paw samples we e collec ed. S udies using he AIA model a e gene ally comple ed a his ime poin due o a pla eau e ec o in lamma o y mani es a ions (7,31). A h i is Se e i y E alua ion Disease ac i i y was clinically e alua ed du ing he pe iod o ea men by wo independen in es iga o s using an in lamma o y sco e and by measu ing he ankle pe ime e as eadou o a icula swelling. The in lamma o y sco e was measu ed by coun ing he sco e o each limb join in a scale o 0–3 (0—absence, 1—e y hema, 2—e y hema and swelling, and 3—de o mi ies and unc ional impai men ). The o al sco e o each animal was de ined as he sum o he pa ial sco es o each a ec ed join (7,33). Body weigh was also egis e ed, e e y 2 days, h oughou he expe imen al p ocedu e. Clinical Pa hology and His ological Analysis Blood was collec ed om he hea and used o se um biochemis y measu emen o c ea ine kinase (CK), u ea, lac a e dehyd ogenase (LDH), alanine ansaminase (ALT) (BioAssay Sys ems, Cali o nia, USA), and p o- ANP (Biomedica, Viena, Aus ia) by enzyme-linked immunoso ben assay (ELISA) echnique, acco ding o he manu ac u e ’s ins uc ions. The ELISA measu emen was pe o med using he pla e eade In ini e M200 (Tecan, Mannedo , Swi ze land). Nec opsy was pe o med, and he le hind paw was collec ed o ibio a sal join his opa hological analysis and disease sco ing, and he li e , spleen, kidney, lung, hymus, hea , gas oin es inal ac , and long bone (hume us) we e collec ed o ou ine his opa hological analysis o assess signs o celas ol-induced oxici y. B ie ly, all o gans and issues we e immedia ely ixed in 10% neu al bu e ed o malin, he bones we e u he decalci ied in 10% o mic acid, and all samples we e p ocessed o pa a in embedding. Fo his ological disease ac i i y sco ing, se ial 5-µm sec ions o he ibio a sal join s we e s ained wi h hema oxylin and eosin (H&E) and immunohis ochemis y was pe o med using he ollowing an ibodies: mouse monoclonal an i-CD68 (Abcam, Camb idge, UK), mouse monoclonal an i-os eocalcin (os eoblas ma ke ; indica o o os eoblas ic ac i i y; Abcam, Camb idge, UK), abbi polyclonal an i-ca hepsin K (os eoclas ma ke ; ma u e os eoclas enzyme; Bio by , Camb idge, UK), and abbi polyclonal an i-Ki67 (Abcam, Camb idge, UK) an ibodies. The issue sec ions we e incuba ed wi h he p ima y an ibody and wi h EnVision+(Dako, Glos up, Denma k). Colo was de eloped in a solu ion con aining diaminobenzadine- e ahyd ochlo ide (Sigma, Missou i, USA) and 0.5% H2O2in phospha e-bu e ed saline bu e (pH 7.6). The slides we e coun e s ained wi h hema oxylin and moun ed. His ological disease ac i i y sco ing in he ibio a sal join s was pe o med by an independen esea che blinded o he expe imen al g oups using ou semi-quan i a i e sco es, as p e iously published: sublining laye in il a ion sco e (0—none o di use in il a ion, 1—lymphoid cell agg ega e, 2—lymphoid ollicles, and 3—lymphoid ollicles wi h ge minal cen e o ma ion), lining laye cell numbe sco e (0— ewe han h ee laye s, 1— h ee o ou laye s, 2— i e o six laye s, and 3—mo e han six laye s), bone e osion sco e (0—no e osions, 1—minimal, 2—mild, 3— mode a e, and 4—ma ked), and global disease se e i y sco e (0—no signs o in lamma ion, 1—mild, 2—mode a e, and 3—se e e) (7,34,35). The p oli e a ion o syno iocy es was also analyzed using a semi-quan i a i e sco e (0–4) o Ki67 immunos aining (0—no s ained cells, 1−0–25% s aining, 2−25–50% s aining, 3−50–75% s aining, and 4—mo e han 75% s ained cells) (7). Images we e acqui ed in a Leica DM2500 (Leica Mic osys ems, We zla , Ge many) coupled o a Leica MC170 HD mic oscope came a. Fo he assessmen o celas ol-induced oxici y, 4-µm sec ions o he li e , spleen, kidney, lung, hymus, hea , gas oin es inal ac , and hume us we e s ained wi h H&E and analyzed by a pa hologis (TC) blinded o he expe imen al g oups. The slides we e scanned and images we e acqui ed by a Hamama su NanoZoome SQ slide scanne . The classi ica ion o lesions ollowed p e iously published c i e ia (36–42). Measu emen o Se um Bone Tu no e and Reso p ion Ma ke s Bone u no e was analyzed by quan i ying he le els o a a e- esis an acid phospha ase 5b (TRACP-5b), p ocollagen ype 1 amino- e minal p opep ide (P1NP), and C- e minal c oss-linked elopep ide o ype I collagen (CTX-I) in se um using ELISA (Immunodiagnos ic Sys em, Boldon, UK). All comme cial assays we e pe o med acco ding o he manu ac u e s’ ins uc ions, and s anda d cu es we e gene a ed using he supplied e e ence concen a ions. Measu emen was pe o med using a pla e eade In ini e M200 (Tecan, Mannedo , Swi ze land). S a is ical Analysis No mali y dis ibu ion was assessed by D’Agos ino and Pea son es . The ea ed g oups (celas ol 1, 2.5, 5, and 7.5 ug/g) we e compa ed agains he un ea ed a h i ic g oup wi h he Mann–Whi ney es wi h Bon e oni co ec ion o accoun o mul iple compa isons, as p e iously epo ed (17). Thus, applying he Bon e oni co ec ion, we di ided he global signi icance le el a 0.05 by he numbe o independen es s (n=4) o ge he Bon e oni c i ical alue o p<0.0125, below which a es would be signi ican . In addi ion, he Mann–Whi ney es was also used o compa e di e ences be ween he o he wo independen g oups: heal hy non- a h i ic and un ea ed a h i ic a s. Fo pai ed samples (e.g., di e en ime poin s), we ha e used he Wilcoxon ma ched-pai s signed- ank es . In hese cases, p<0.05 we e conside ed o be signi ican . All s a is ical analyses we e pe o med using he G aphPad P ism V.5.01 (G aphPad, Cali o nia, USA). Da a we e p esen ed as median wi h in e qua ile ange. F on ie s in Medicine | www. on ie sin.o g 3Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol RESULTS O al Celas ol Imp o ed he Clinical Ou come and Amelio a ed he His opa hological Aspec s o AIA Ra s The onse o a h i is in he con ala e al ankle join o he injec ion si e occu ed app oxima ely a day 8 pos -disease induc ion. As demons a ed in Figu e 1A, all animals showed clinical signs o a h i is by he 4 h day o disease induc ion, and a e 10 days, he un ea ed a h i ic g oup showed accele a ed disease p og ession. In con as , a e 6 and 7 days o ea men , he a s ea ed wi h 2.5 and 5 µg/g/day (p=0.0111 and p=0.006) and 7.5 µg/g/day(p=0.010) o celas ol showed a signi ican lowe in lamma o y sco e compa ed o un ea ed a h i ic a s. O no e, o al celas ol a 1 µg/g/day had no e ec in a h i is p og ession. A e 3 days o ea men , all animals (5 ou o 5) ea ed wi h a dose o 25 µg/g/day and 3 animals (3 ou o 5) ea ed wi h 12.5 µg/g/day we e eu hanized due o p og essi e weigh loss (o e 15% o body weigh ), onse o dia hea and espi a o y dis ess. In he g oup o animals ea ed wi h 7.5 µg/g/day o celas ol, only one animal (1 ou o 5) was eu hanized a e 10 days o ea men wi h he same clinical signs. A e 14 days o ea men , he dosage anging om 2.5 o 7.5 µg/g/day showed a signi ican an i-in lamma o y e ec , as assessed by he e alua ion o he clinical in lamma o y sco e (p=0.0023 wi h 2.5 µg/g/day, p=0.0016 wi h 5 µg/g/day, and p=0.0028 wi h 7.5 µg/g/day s. a h i ic animals, as shown in Figu e 1B) and also by he measu emen o ankle pe ime e (p=0.0053 wi h 2.5 µg/g/day, p=0.0053 wi h 5 µg/g/day and p=0.0118 wi h 7.5 µg/g/day s. un ea ed a h i ic animals, as shown in Figu e 1C). O no e, no di e ences we e obse ed in body weigh a e 14 days o ea men , compa ing each dose o celas ol (up o 7.5 µg/g/day) wi h he ehicle con ol animals (Figu e S1A). Addi ionally, when compa ing, wi hin he same g oup, he i s and las day o ea men , we we e able o obse e an inc ease in body weigh in heal hy a s (p=0.0078, Figu e S1B) and a signi ican weigh loss in he un ea ed a h i ic g oup, as expec ed (p=0.0074, Figu e S1C). Howe e , no weigh loss was obse ed in AIA animals ea ed wi h celas ol using doses up o 7.5 µg/g/day (Figu es S1D–G). As shown in Figu e 2, syno ial hype plasia and ma ked in lamma o y cell in il a ion we e seen in ibio a sal join s o un ea ed a h i ic a s (p=0.0019 and p=0.0001 s. heal hy con ols, espec i ely), also associa ed wi h signi ican bone e osion (p=0.0008 s. heal hy con ols). In con as , in celas ol- ea ed a s, in lamma o y in il a es we e educed in all dose egimens, excep o he 1 µg/g/day g oup (Figu e 2A,p=0.0010 in 2.5 µg/g/day, p=0.0006 in 5 µg/g/day and p=0.0016 in 7.5 µg/g/day s. a h i ic a s). We also obse ed a educ ion in he numbe o cells p esen in he syno ial lining laye wi h he dose o 2.5 µg/g/day (Figu e 2B,p=0.0110 s. a h i ic a s), and a ma ked endency o dec ease in he 5 and 7.5 µg/g/day doses (p=0.0220; p=0.0225, espec i ely, s. a h i ic a s). These concen a ions o celas ol we e also e ec i e in p e en ing bone a icula des uc ion (Figu e 2C,p=0.0053 in 2.5 µg/g/day, p=0.0069 in 5 µg/g/day, and p=0.0118 in 7.5 µg/g/day s. a h i ic a s), wi h animals p esen ing a no mal join s uc u e a he end o he s udy pe iod (Figu e 2D,p=0.0028 in 2.5 µg/g/day, p=0.0048 in 5 µg/g/day, and p=0.0041 in 7.5 µg/g/day s. a h i ic a s). In addi ion, we obse ed ha animals ea ed wi h celas ol in he dose ange be ween 2.5 and 7.5 µg/g/day ha e educed syno ial cell p oli e a ion, as assessed in he ibio a sal join s by Ki67 immunos aining (p=0.0020, p=0.0055, and p=0.0026 s. a h i ic animals, espec i ely) (Figu e 2E). Finally, we obse ed ha un ea ed a h i ic a s had a highe numbe o in il a ing CD68+syno ial mac ophages as compa ed o heal hy con ols (p=0.0001) (Figu e 2F). Celas ol adminis a ion was associa ed wi h a signi ican dec ease in he numbe o CD68+mac ophages in il a ing he a h i ic join (p=0.0014 in 2.5 µg/g/day, p=0.0095 in 5 µg/g/day s. a h i ic a s; o he 7.5 µg/g/day he e is a s ong endency al hough no eaching signi icance, p=0.0213). O al Celas ol Reduced he Numbe o Join Os eoclas s and Os eoblas s The immunolocaliza ion o os eoclas s and os eoblas s in subchond al bone issue a he ibia/ alus egion was pe o med o e alua e a possible celas ol dose-dependen e ec on bone emodeling. Un ea ed a h i ic a s showed inc eased os eoclas numbe s (ca hepsin k+cells) in he ibio a sal bones (p=0.004 s. heal hy con ols, Figu e 3A). Impo an ly, celas ol adminis a ion was associa ed wi h a signi ican dec ease in he numbe o os eoclas s, o le els simila o heal hy con ols (p=0.004 in 5 µg/g/day and p=0.004 in 7.5 µg/g/day s. a h i ic a s). Un ea ed a h i ic a s also showed inc eased numbe s o os eoblas s (os eocalcin-posi i e cells) (p=0.0005 s. heal hy con ols, Figu e 3B), a pheno ype also e e sed by celas ol adminis a ion, wi h a signi ican educ ion in he numbe o os eoblas s as compa ed o AIA a s (p=0.0005 in 5 µg/g/day and p=0.0005 in 7.5 µg/g/day) eaching le els simila o heal hy con ols. In he case o a s ea ed wi h celas ol a he dose o 2.5 µg/g/day he e is a endency owa d a dec ease in he numbe o os eoblas s ha did no each s a is ical signi icance (p=0.02 s. un ea ed AIA a s). O al Celas ol Reduced Bone Tu no e and Reso p ion Ma ke s in AIA Ra s In un ea ed a h i ic a s, he e was no signi ican inc ease in TRACP-5b le els a he end o he s udy pe iod (Figu e 4A). Impo an ly, bo h 5 and 7.5 µg/g/day doses o celas ol educed TRACP-5b le els, when compa ed wi h un ea ed a h i ic a s (p=0.0031 and p=0.0065, espec i ely), sugges ing a dec ease in bone eso p ion. In addi ion, he ele a ed le els o P1NP obse ed in un ea ed a h i ic a s in compa ison wi h heal hy con ols (p=0.0074, Figu e 4B) p esen ed a ma ked endency o dec ease wi h he celas ol dose o 5 µg/g/day (p=0.0145 s. un ea ed a h i ic animals). In acco dance, celas ol adminis a ion was able o signi ican ly educe he CTX-I le els (p=0.0079 o 5 µg/g/day, Figu e 4C) and also induced a s ong endency o dec ease wi h he 7.5 µg/g/day dose, F on ie s in Medicine | www. on ie sin.o g 4Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol FIGURE 1 | O al celas ol signi ican ly imp o ed he clinical ou come in adju an -induced a h i is a s. The in lamma o y sco e measu ed h oughou he ea men pe iod (A), he in lamma o y sco e (B), and he ankle pe ime e (C) e alua ed by day 22 a e disease induc ion showed ha he ange o dosage be ween 2.5 and 7.5 µg/g/day was e ec i e in p e en ing a h i is p og ession. Heal hy N=8, a h i ic N=17, celas ol 1 µg/g/day N=5, celas ol 2.5 µg/g/day N=5, celas ol 5 µg/g/day N=5, celas ol 7.5 µg/g/day N=5, celas ol 12.5 µg/g/day N=5, and celas ol 25 µg/g/day N=5. The da a a e shown as median wi h in e qua ile ange. The di e ences we e conside ed as s a is ically signi ican o p<0.05, acco ding o Mann–Whi ney es s o compa isons be ween heal hy and a h i ic g oups and p<0.0125 acco ding o Mann–Whi ney es s wi h Bon e oni co ec ion o compa isons be ween a h i ic and celas ol- ea ed g oups. *p<0.05 and p<0.0125. F on ie s in Medicine | www. on ie sin.o g 5Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol FIGURE 2 | O al celas ol ea men amelio a ed he his opa hological aspec s o adju an -induced a h i is (AIA) a s and educed syno ial CD68+mac ophages. Celas ol signi ican ly impai ed in lamma o y cell in il a ion (A), was associa ed wi h a numbe o syno ial lining laye s simila o no mal alues (B), and educed bone e osions (C), hus p ese ing he no mal join s uc u e (D). The celas ol- ea ed AIA a s showed a signi ican educ ion in syno ial cell p oli e a ion as assessed by he Ki67 ma ke (E) and in he numbe o syno ial CD68-posi i e cells (F). Heal hy N=8, a h i ic N=17, celas ol 1 µg/g/day N=5, celas ol 2.5 µg/g/day N=5, celas ol 5 µg/g/day N=5, and celas ol 7.5 µg/g/day N=4. The da a a e exp essed as median wi h in e qua ile ange. The di e ences we e conside ed as s a is ically signi ican o p<0.05, acco ding o Mann–Whi ney es s o compa isons be ween heal hy and a h i ic g oups and p<0.0125 acco ding o Mann–Whi ney es s wi h Bon e oni co ec ion o compa isons be ween a h i ic and celas ol- ea ed g oups. *p<0.05 and p<0.0125. F on ie s in Medicine | www. on ie sin.o g 6Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol FIGURE 3 | O al celas ol educed he numbe o os eoclas s and os eoblas s in a h i ic join s. Ca hepsin k-posi i e cells (A) and os eocalcin-posi i e cells (B) we e iden i ied in a h i ic join s by immunohis ochemis y. Celas ol ea men signi ican ly educes bo h ypes o cells in he doses o 5 and 7.5 µg/g/day. The paw samples we e collec ed a he ime o sac i ice. Heal hy N=8, a h i ic N=17, celas ol 1 µg/g/day N=5, celas ol 2.5 µg/g/day N=5, celas ol 5 µg/g/day N=5, and celas ol 7.5 µg/g/day N=4. The da a a e exp essed as median sco e wi h in e qua ile ange. The di e ences we e conside ed as s a is ically signi ican o p<0.05, acco ding o he Wilcoxon signed- ank es s o compa isons be ween heal hy and a h i ic g oups and p<0.0125 acco ding o Mann–Whi ney and Wilcoxon signed- ank es s wi h Bon e oni co ec ion o compa isons be ween a h i ic and celas ol- ea ed g oups. *p<0.05 and p<0.0125. FIGURE 4 | O al celas ol was associa ed wi h he educ ion o bone u no e and eso p ion ma ke s. TRACP-5b (A), P1NP (B), and CTX-I (C) le els we e quan i ied in a se um samples collec ed a he ime o sac i ice. Celas ol seems o educe he le els o TRACP-5b, P1NP, and CTX-I in he ea ed animals in compa ison wi h he a h i ic a s. Heal hy N=8, a h i ic N=17, celas ol 1 µg/g/day N=5, celas ol 2.5 µg/g/day N=5, celas ol 5 µg/g/day N=5, and celas ol 7.5 µg/g/day N=4. The da a a e exp essed as median wi h in e qua ile ange. The di e ences we e conside ed as s a is ically signi ican o p<0.05, acco ding o Mann–Whi ney es s o compa isons be ween heal hy and a h i ic g oups and p<0.0125 acco ding o Mann–Whi ney es s wi h Bon e oni co ec ion o compa isons be ween a h i ic and celas ol- ea ed g oups. *p<0.05 and p<0.0125. (p=0.0159) when compa ed wi h un ea ed a h i ic a s, which showed an accele a ed bone u no e wi h high le els o CTX-I (p=0.0016 s. heal hy con ols). Also, he CTX-I se um le els had a endency owa d a dec ease a 2.5 µg/g/day, bu i did no each s a is ical signi icance. Highe Dose o Celas ol Was Associa ed Wi h Signs o Toxici y A his opa hological analysis o selec ed o gans showed his ological changes associa ed wi h he disease model and his ological changes associa ed wi h he es compound. F on ie s in Medicine | www. on ie sin.o g 7Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol Conce ning he disease- ela ed his opa hological aspec s, gian -cell g anulomas we e seen in li e , lung, and spleen in all g oups, wi h a ied se e i y un ela ed wi h ea men o dose le els (Figu e S2). G ossly, mul iple g ay o g ay- b own oci we e obse ed o e he pleu al su ace o he TABLE 1 | His opa hological indings in Wis a a s upon celas ol ea men a di e en dose le els. Ta ge o gan E ec Se e i y g adea Celas ol (µg/g/day) 0 1 2.5 5 7.5 Thymus Nec osis, lymphocy e (+2) 0/16 0/5 0/5 0/6 1/6 (+3) 0/16 0/5 0/5 0/6 1/6 Li e In lamma o y cell in il a ion, pe ibilia y (in ahepa ic) (+1) 0/16 0/5 2/5 5/6 2/6 (+2) 0/16 0/5 0/5 0/6 2/6 (+3) 0/16 0/5 0/5 0/6 2/6 Bile duc hype plasia (+1) 0/16 0/5 3/5 4/6 4/6 (+2) 0/16 0/5 0/5 1/6 2/6 aSe e i y g ade: (+1), mild; (+2), mode a e; (+3), ma ked. lung and he su ace o he li e and spleen. Mic oscopically, hese changes co esponded o he a iably sized mul i ocal o coalescing g anulomas, cha ac e ized by dense agg ega es o mos ly mac ophages admixed wi h equen mul inuclea ed gian cells and ewe lymphoid cells (Figu e S3). In he li e , hese g anulomas we e p esen in he pa enchyma; in he lung, hey we e mos ly a anged a ound b onchioles, a ound o adjacen o la ge essels; and in he lung in e s i ium and in he spleen hey we e mos ly seen in he ed pulp. Rega ding possible celas ol-induced his opa hological aspec s, lesions we e seen exclusi ely in he hymus and he li e o medium- o high-dose g oups, and he se e i y g ade was ela ed wi h dose (Figu e S4). The hymus showed lymphocy e nec osis, mul i ocal, and mild o mode a e deple ion o lymphocy es in he co ical zone. Se e i y anged om mode a e o ma ked and was seen exclusi ely in wo o six animals o he high-dose g oup (7.5 µg/g/day) (Table 1). In he li e , mild lymphocy e- ich pe ibilia y in lamma o y cell in il a ion, and bile duc hype plasia we e only seen in wo and h ee ou o i e animals, espec i ely, in he 2.5 µg/g/day dose-le el g oup (Table 1). Highe oxici y incidence was obse ed in he 5 and 7.5 µg/g/day dose g oups, wi h a oxici y se e i y g ade anging om mild o mode a e in he medium dose and om mild o ma ked in he high dose. Due o he design o he expe imen and o he use o an acu e a model o a h i is, which apidly p og esses and esol es, i was no possible o assess i changes associa ed wi h celas ol ea men we e e e sible as no analysis FIGURE 5 | Se um biochemis y o li e , enal, and ca diac ma ke s upon o al adminis a ion o celas ol. The se um le els o ALT (A), CK (B), LDH (C), u ea (D), and p o-ANP (E) we e measu ed by ELISA o e alua e li e , kidney, and ca diac oxici y. Only he le els o LDH we e inc eased in animals ea ed wi h 7.5 µg/g/day o celas ol. Heal hy N=8, a h i ic N=10, celas ol 1 µg/g/day N=5, celas ol 2.5 µg/g/day N=5, celas ol 5 µg/g/day N=5, and celas ol 7.5 µg/g/day N=4. The da a a e exp essed as median wi h in e qua ile ange. The di e ences we e conside ed as s a is ically signi ican o p<0.05, acco ding o Mann–Whi ney es s o compa isons be ween heal hy and a h i ic g oups and p<0.0125 acco ding o Mann–Whi ney es s wi h Bon e oni co ec ion o compa isons be ween a h i ic and celas ol- ea ed g oups. *p<0.05 and p<0.0125. F on ie s in Medicine | www. on ie sin.o g 8Sep embe 2020 | Volume 7 | A icle 455 Cascão e al. E icacy and Toxici y o Celas ol was done a e suspension o he compound. No signi ican his ological changes we e seen in any o he o gans. Mo eo e , he se um biochemis y o ALT, u ea, p o-ANP, and CK showed le els wi hin he e e ence ange o he labo a o y o his s ain/species (Figu es 5A,B,D,E). LDH was inc eased a he dosage le el o 7.5 µg/g/day (as shown in Figu e 5C,p=0.0062 s. un ea ed a h i ic a s, espec i ely). The elec oca diog am emained unal e ed in celas ol- ea ed animals (da a no shown). DISCUSSION In his s udy, we aimed o de e mine he he apeu ic o al dose o pu e celas ol and he associa ed oxici y signs in he AIA a model. The doses o celas ol we e ex apola ed om he in ape i oneal dose used in ou p e ious s udies (7,8,17), also aking in o conside a ion he bioa ailabili y and he pha macokine ics o pu e celas ol upon o al adminis a ion in a s (22). We obse ed ha , a e 3 days o ea men , all animals ea ed wi h 25 µg/g/day o celas ol and mo e han 50% o hose ea ed wi h 12.5 µg/g/day showed a signi ican decline in heal h s a us, wi h p og essi e weigh loss, dia hea, and espi a o y dis ess, all o hese un ela ed wi h he disease model, and we e eu hanized. These da a sugges ha he dose o 25 µg/g/day is he le hal dose (LD) and he dose o 12.5 µg/g/day is he LD50 o he o al adminis a ion o pu e celas ol, and hese dose g oups we e he e o e excluded om his s udy. A e 10 days unde celas ol ea men , one ou o i e a s ea ed wi h 7.5 µg/g/day also p esen ed simila clinical signs and was eu hanized. We ha e obse ed ha he o al dose o 1 µg/g/day o celas ol was no e ec i e in he ea men o a h i is. In con as , he o al doses o 2.5, 5, and 7.5 µg/g/day showed signi ican an i- in lamma o y p ope ies as assessed by he e alua ion o he in lamma o y sco e and ankle swelling. Mos o he in lamma o y and bone- ela ed pa ame e s showed a dose-dependen e ec . Howe e , in some o he analyses, his e ec did no each s a is ical signi icance, which migh be due o he smalle sample size in hese g oups. The celas ol doses o 2.5, 5, and 7.5 µg/g/day we e able o educe syno ial cell in il a ion and p oli e a ion and also dec eased bone e osions in join s. Impo an ly, a he doses o 2.5 and 5 µg/g/day and wi h a s ong endency a he dose o 7.5 µg/g/day, celas ol educed he numbe o sublining CD68+ syno ial mac ophages, a bioma ke o clinical esponse (18–20). These da a sugges ha , a hese o al dosages, celas ol is e ec i e o he ea men o a h i is. Addi ionally, he doses o 5 and 7.5 µg/g/day educed he numbe o os eoclas s and os eoblas s p esen in join issues. This obse a ion is in ag eemen wi h he educ ion o he bone eso p ion ma ke TRACP-5b and also wi h he dec ease o he bone eso p ion ma ke CTX-I in he 5 µg/g/day ea ed g oup and a endency o diminish also a he 7.5 µg/g/day dose, sugges ing he con ol o he accele a ed bone u no e cha ac e is ic o a h i is. Ou esul s a e in line wi h o he s, whe e ci cula ing le els o TRACP-5b a e no a ec ed by a h i is induc ion, con a ily o he ma ked os eoclas ic ac i i y occu ing in his a h i ic model. Howe e , he e a e inc eased le els o TRACP-5b in he p o ein ex ac s ob ained om in lamed join s (43). These indings sugges ha TRACP-5b e lec s bone eso p ion mo e accu a ely when measu ed locally a he han sys emically (43,44). Rega ding TRACP-5b, CTX-I, and P1NP esul s, celas ol migh ha e a di ec e ec on bone me abolism in he se ing o in lamma ion. Impo an ly, hese esul s sugges ha he o al adminis a ion o celas ol is also able o con ibu e o he p e en ion o bone damage, as we ha e p e iously demons a ed using AIA a s unde ea men wi h 1 µg/g/day o celas ol ia in ape i oneal ou e (17). Some in lamma o y dis u bances we e s ill no ed in he ea ed animals. This is a consequence o he in lamma o y p ocess ha occu ed du ing he i s days a e disease induc ion ( om day 0 up o day 8) bu be o e celas ol ea men . This is in acco dance wi h ou p e ious obse a ions showing ha in lamma ion induces changes since he i s days o a h i is de elopmen (45,46). Se e al s udies ha e demons a ed ha celas ol has cellula a ge s in he con ex o RA, such as TAK1/IKK and MAPK/MEK pa hways as well as MMP-9, STAT3, RANKL, and MD2/TLR4, in e e ing wi h he p oduc ion o cy okines, chemokines, and in lamma o y media o s; inhibi ing cell in asion and p oli e a ion; and supp essing bone eso p ion (5,9,14,47). In acco dance, Liu e al. ha e obse ed in a mouse model o dexame hasone-induced seconda y os eopo osis ha celas ol no only imp o es lipid me abolism and educes hype calciu ia bu also mi iga es a icula ca ilage lesions, dec eases NF-kB, MMP-1, and MMP-9 exp ession, and educes se um PTH, TRACP-5b, CTX-I, as well as deoxypy idinoline (48). In 2012, ou g oup showed ha celas ol dec eases he sec e ion o bo h IL-1βand TNF in he THP-1 mac ophage-like cell line associa ed no only wi h NF-kB inhibi ion bu also wi h caspase-1 inac i a ion (7). Despi e i s p omising an i-a h i ic e ec s, celas ol has been epo ed o induce weigh loss in mice models o cance (28,49– 51). A hese dosages o celas ol, no body weigh a ia ions we e obse ed, sugges ing no majo oxicological e ec s induced by celas ol o by he sol en s (e hanol and PEG400) used. These wo agen s a e he mos commonly employed (52) and we e iden i ied as he adequa e ones o dissol ing celas ol (22). Howe e , some s udies ha e sugges ed ha e hanol and PEG400 may be dele e ious o he gas oin es inal ac (53, 54), cons i u ing a isk ac o o oxicological side e ec s such as body weigh loss. Since no a ia ions in body weigh we e obse ed in celas ol- ea ed a s and due o he sho - e m ea men du a ion o his s udy, oge he wi h he ac ha we ha e used he minimum PEG and e hanol concen a ions equi ed in o de o ge celas ol solubili y, we can exclude his dele e ious side e ec in he gas oin es inal ac . No clinical chemis y o his opa hological oxici y o his compound has been p e iously shown. In pa allel wi h he assessmen o he he apeu ic e ec o celas ol a di e en dose le els, we also assessed i s possible oxic e ec s a he same doses (1–7.5 µg/g/day). No changes in ALT and blood u ea we e de ec ed, sugges ing ha i did no induce majo li e o enal damage. Celas ol blocks he ion conduc ion o ca diac F on ie s in Medicine | www. on ie sin.o g 9Sep embe 2020 | Volume 7 | A icle 455