Monitoring new long-lasting intravitreal formulation for glaucoma with vitreous images using optical coherence tomography

pha maceu ics A icle Moni o ing New Long-Las ing In a i eal Fo mula ion o Glaucoma wi h Vi eous Images Using Op ical Cohe ence Tomog aphy Ma ia Jesus Rod igo 1,2,3,* , Amaya Pé ez del Paloma 4,5 , Albe o Mon olío4,5 , Sil ia Mendez-Ma inez 1,2 , Manuel Subias 1,2 , Ma ia Jose Ca diel 6, Te esa Ma inez-Rincon 1,2, JoséCegoñino 4,5, JoséMa ia F aile 7, Eugenio Vispe 8, JoséAn onio Mayo al 7, Vicen e Polo 1,2 and Elena Ga cia-Ma in 1,2,3   Ci a ion: Rod igo, M.J.; Paloma , A.P.d.; Mon olío, A.; Mendez-Ma inez, S.; Subias, M.; Ca diel, M.J.; Ma inez-Rincon, T.; Cegoñino, J.; F aile, J.M.; Vispe, E.; e al. Moni o ing New Long-Las ing In a i eal Fo mula ion o Glaucoma wi h Vi eous Images Using Op ical Cohe ence Tomog aphy. Pha maceu ics 2021,13, 217. h ps://doi.o g/10.3390/ pha maceu ics13020217 Academic Edi o : Anuj Chauhan Recei ed: 30 No embe 2020 Accep ed: 2 Feb ua y 2021 Published: 5 Feb ua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Depa men o Oph halmology, Miguel Se e Uni e si y Hospi al, 50009 Za agoza, Spain; [email p o ec ed] (S.M.-M.); [email p o ec ed] (M.S.); [email p o ec ed] (T.M.-R.); polo@uniza .es (V.P.); [email p o ec ed] (E.G.-M.) 2Miguel Se e Oph halmology Resea ch G oup (GIMSO), A agon Heal h Resea ch Ins i u e (IIS A agon), Uni e si y o Za agoza, 50009 Za agoza, Spain 3 RETICS: Thema ic Ne wo ks o Co-Ope a i e Resea ch in Heal h o Ocula Diseases, 28040 Mad id, Spain 4Bioma e ials G oup, A agon Ins i u e o Enginee ing Resea ch (I3A), Uni e si y o Za agoza, 50018 Za agoza, Spain; amaya@uniza .es (A.P.d.P.); amon olio@uniza .es (A.M.); jcegoni@uniza .es (J.C.) 5Depa men o Mechanical Enginee ing, Uni e si y o Za agoza, 50018 Za agoza, Spain 6Depa men o Pa hology, Lozano Blesa Uni e si y Hospi al, 50009 Za agoza, Spain; mjca dielga [email p o ec ed] 7Ins i u e o Chemical Syn hesis and Homogeneous Ca alysis (ISQCH), Facul y o Sciences, Uni e si y o Za agoza-CSIC, C/Ped o Ce buna 12, 50009 Za agoza, Spain; jm aile@uniza .es (J.M.F.); mayo al@uniza .es (J.A.M.) 8Ch oma og aphy and Spec oscopy Labo a o y, Ins i u e o Chemical Syn hesis and Homogeneous Ca alysis (ISQCH), Facul y o Sciences, Uni e si y o Za agoza-CSIC, Ped o Ce buna 12, 50009 Za agoza, Spain; e p@uniza .es *Co espondence: [email p o ec ed]; Tel.: +34-619788942 o +34-976765558; Fax: +34-976566234 Abs ac : In a i eal injec ion is he gold s anda d he apeu ic op ion o pos e io segmen pa holo- gies, and long-las ing elease is necessa y o a oid einjec ions. The e is no e ec i e in a i eal ea men o glaucoma o o he op ic neu opa hies in daily p ac ice, no is he e a non-in asi e me hod o moni o d ug le els in he i eous. He e we show ha a glaucoma ea men combining a hypo ensi e and neu op o ec i e in a i eal o mula ion (IF) o b imonidine–Laponi e (BRI/LAP) can be moni o ed non-in asi ely using i eo e inal in e ace imaging cap u ed wi h op ical cohe - ence omog aphy (OCT) o e 24 weeks o ollow-up. Quali a i e and quan i a i e cha ac e isa ion was achie ed by analysing he changes in i eous (VIT) signal in ensi y, exp essed as a a io o e inal pigmen epi helium (RPE) in ensi y. Vi eous hype e lec i e agg ega es mixed in he i eous and ended o se le on he e inal su ace. Rela i e in ensi y and agg ega e size p og essi ely dec eased o e 24 weeks in ea ed a eyes as he BRI/LAP IF deg aded. VIT/RPE ela i e in ensi y and o al agg ega e a ea co ela ed wi h b imonidine le els measu ed in he eye. The OCT-de i ed VIT/RPE ela i e in ensi y may be a use ul and objec i e ma ke o non-in asi e moni o ing o BRI/LAP IF. Keywo ds: b imonidine; Laponi e; d ug deli e y; glaucoma; nanomedicine; moni o ing; op ical cohe ence omog aphy; i eous; in a i eal 1. In oduc ion B imonidine is an ocula hypo ension d ug widely used in oph halmological clinical ca e. Fo adequa e he apeu ic con ol, opical adminis a ion is ecommended wice a day because o i s sho hal -li e (app oxima ely 12 h) [ 1 ]. Recen ly, b imonidine also exhibi ed a neu op o ec i e e ec in e inal ganglion cells (RGCs), pho o ecep o s and o he e inal Pha maceu ics 2021,13, 217. h ps://doi.o g/10.3390/pha maceu ics13020217 h ps://www.mdpi.com/jou nal/pha maceu ics Pha maceu ics 2021,13, 217 2 o 16 cells [ 2 ] in animal and human s udies [ 3 ] and wi h bo h opical and in a i eal adminis a- ion [ 4 ]. The sho hal -li e o b imonidine means ha pe iodic in a i eal adminis a ion is needed o achie e he apeu ic e icacy. Howe e , epea ed injec ion could lead o com- plica ions [ 5 ]. The e o e, de elopmen o sus ained d ug deli e y sys ems is necessa y. Laponi e®Na+0.7[(Si8Mg5.5Li0.3)O20(OH)4]−0.7 is a biocompa ible and biodeg adable clay used in biomedicine [ 6 ]. I s sa e y in in a i eal injec ion has been demons a ed in animal s udies [ 7 – 9 ]. Laponi e ® o ms a anspa en gel when dispe sed in an aqueous medium. I is able o in e ac wi h o he molecules, ac ing as a ca ie and eleasing he d ug in a con olled manne [ 10 ]. In his ega d, ou esea ch g oup ecen ly demons a ed ha an in a i eal o mula ion (IF) con aining b imonidine–Laponi e (BRI/LAP) p oduced a hypo ensi e and neu op o ec i e e ec in a ch onic glaucoma animal model o e 6 mon hs o ollow-up [9]. The i eous humou is a 3-D s uc u e mainly composed o wa e , collagen ib es and hyalu onan ha occupies 80% o eye olume. I allows ligh ansmission and in e - enes in eye me abolism [ 11 ]. A i eous sample biopsy is a use ul means o diagnosing in lamma o y, in ec ious o oncological ocula diseases as well as o ca ying ou oxi- cological pos -mo em analysis [ 12 , 13 ]. Fu he mo e, he i eous humou is becoming inc easingly impo an in clinical p ac ice as i ac s as a he apeu ic a ge o pos e io pole pa hologies in ol ing macula oedema, such as diabe ic e inopa hy, ascula occlusions o aged- ela ed macula degene a ion. Howe e , as an in a i eal ea men o glaucoma is no ye a ailable in clinical p ac ice, many esea che s a e wo king o esol e his limi a ion wi hin a medium- e m ho izon. The po en ial in e es in using in a i eal adminis a ion is o ci cum en ocula ba ie s while a oiding sys emic ad e se e en s and, by ac ing as a ese oi , main aining he apeu ic d ug le els nea he si e o ac ion [14]. Op ical cohe ence omog aphy (OCT) is an easy- o-handle, cos -e icien , and objec i e echnology ha p o ides high- esolu ion c oss-sec ional images. I has long been widely used in clinical p ac ice and esea ch o s udy neu o e inal s uc u e. Imp o ed OCT de- ices wi h swep -sou ce o enhanced i eous imaging ha e made i possible o s udy he i eous in no mal and in lamed s a es and o s udy changes o i a e ea men [15–17] . Howe e , i has ne e been used o moni o d ug le els in IFs. Cu en ly, loss in IF he a- peu ic e icacy is e alua ed on he basis o changes in neu o e inal s uc u e o dec eases in subjec i e isual acui y measu emen s, which indica e an inc ease in disease ac i i y. I would be bene icial, howe e , o ind an objec i e i eous moni o ing ma ke wi h which o quan i y he deg ada ion o he injec ed molecule and which could e en an icipa e he loss o he apeu ic e ec be o e s uc u al e inal changes a e de ec ed by OCT, especially as compa ed wi h o he mo e expensi e o in asi e echniques used in esea ch, such as posi on emission omog aphy o magne ic esonance imaging [18,19]. In p e ious OCT-based s udies by his g oup, BRI/LAP IF was isualised in he i eous ca i y as hype e lec i e agg ega es [ 9 ]. Expanding on his inding, his s udy uses OCT o analyse changes in he hype e lec i i y signal om he BRI/LAP IF in he i eous humou in a eyes o e a 24-week pe iod. This pape desc ibes a moni o ing ma ke o he IF, co ela es he OCT i eous signal o d ug le els and discusses he he apeu ic e ec o BRI/LAP IF [ 9 ]. I p oposes se ial OCT as a mo e a o dable and simple me hod o moni o ing BRI/LAP IF in i o in animal esea ch. 2. Ma e ials and Me hods 2.1. Da a Collec ion OCT images o he i eo e inal in e ace and d ug le el da a we e ob ained om he expe imen s ca ied ou in a p e ious in e en ional s udy conduc ed by he au ho s [ 9 ] (CC BY 4.0 license). In ha s udy, ch onic glaucoma was bila e ally induced by biweekly injec ions o hype onic solu ion in o he episcle al eins, acco ding o he well-es ablished Mo ison model [ 20 ], which led o episcle al ein scle osis and he e o e ocula hype en- sion (OHT). The ea e , a single 3- µ L BRI/LAP IF injec ion (10 mg BRI/LAP/mL) in o he i eous ca i y o igh eye (RE) o Long–E ans a s was pe o med a baseline. Le Pha maceu ics 2021,13, 217 3 o 16 eyes (LEs) se ed as non- ea ed hype ensi e con ols. In aocula p essu e was measu ed wi h a ebound onome e (Tonolab ® iCa e, Helsinki, Finland) o oden esea ch. The ma e ial and me hods used o he s udy we e deeply de ailed in [ 9 ]. In his s udy, OCT examina ions o he 43 ea ed a s a baseline and a weeks 1, 2, 4, 6, 8, 12, 24 a e in a i - eal injec ion we e analysed o quan i y BRI/LAP IF e olu ion o e 6 mon hs. Ano he 23 non- ea ed heal hy a s we e examined o compa ison as non-hype ensi e con ols. The expe imen was app o ed be o ehand by he E hics Commi ee o Animal Resea ch o Za agoza Uni e si y (PI34/17, 27 h June 2017) and was ca ied ou in s ic acco dance wi h he Associa ion o Resea ch in Vision and Oph halmology’s S a emen o he Use o Animals. 2.2. Op ical Cohe ence Tomog aphy Images we e acqui ed using a high- esolu ion OCT de ice (HR-OCT Spec alis, Heidelbe g ® Enginee ing, Heidelbe g, Ge many) wi h a plane powe polyme hylme hac y- la e (PMMA) con ac lens o 270 µ m hickness and 5.2 mm diame e (Can o +Nissel ® , No hamp onshi e, UK) adap ed o he a s’ co nea o ob ain highe quali y images. The oden e sion o his sys em acqui es c oss-sec ional images by means o 61 b-scans measu ing a ound 3 mm in leng h and cen ed on he op ic ne e. I has a esolu ion o 3 mic ons pe pixel gene a ed. A o al o 1536 × 496 pixels pe image we e analysed. The e inal pos e io pole p o ocol wi h au oma ic segmen a ion, eye- acking so wa e and ollow-up applica ion we e used o ensu e ha he same poin s we e e-scanned h oughou he s udy. The “enhance dep h imaging” mode was disabled in all cases. 2.3. B imonidine–Laponi e (BRI/LAP) Fo mula ion and Analysis BRI/LAP was p epa ed [ 9 ] by addi ion o Laponi e (100 mg) o a solu ion o B i- monidine (10 mg) in e hanol (10 mL) wi h s i ing and hen sol en e apo a ion unde a acuum, o ob ain he BRI/LAP o mula ion in powde o m, which was gamma- ay s e ilised. BRI/LAP was injec ed in he o m o a yellow colloidal dispe sion in balanced saline solu ion (BSS) (10 mg/mL). The b imonidine con en in he a eyes was analysed [ 8 , 9 ] using an ul a-high-p essu e liquid ch oma og aphy mass spec ome e (UHPLC-MS, Wa e s, Mil o d, MA, USA). The eyes we e i s cu in o pieces, sonica ed wi h a solu ion o o mic acid in ace oni ile, hen wi h ammonium o ma e in phospho ic acid and in e nal s anda d (2-b omoquinoxaline), cen i uged and he supe na an was cleaned up by solid phase ex ac ion. 2.4. In Vi o Release o BRI om BRI/LAP Fo mula ion Release was s udied in a model o i eous humou made up o 0.5% sodium hyalu ona e in saline solu ion (pH 7.1–7.4). The elease es s we e pe o med by dispe sing BRI/LAP (5 mg, weigh a io 1/10, b imonidine amoun 454.5 µ g) in he ex ac ion medium (0.5 g o he i eous model) unde s i ing a 120 pm a 37 ◦ C. A e 24 h he dispe sion was cen i uged a 14,000 pm o 20 min. The liquid phase was sepa a ed and he solid was e-dispe sed in esh ex ac ion medium o a new cycle. The liquid phase was dilu ed wi h ace oni ile (0.5 mL) con aining he in e nal s anda d, cen i uged again and analysed by HPLC as desc ibed abo e. 2.5. Analysis o he In a i eal Fo mula ion Using OCT The BRI/LAP IF agg ega es p esen in he OCT scans o he i eo e inal in e ace we e s udied. This echnique ocuses on he analysis o opaci ies in he i eo e inal in e ace by OCT, which does no equi e a co ec ion ac o o i s his ological co ela ion [ 21 ] and ensu es a cha ac e isa ion o he ac ual agg ega e. These agg ega es we e de ined as being do s dispe sed in he i eous humou o i eo e inal in e ace whose la ge size, i egula i y o g ea e signal hype e lec i i y di e en ia ed hem om backg ound speckle noise. Pha maceu ics 2021,13, 217 4 o 16 OCT image con as was no adjus ed a any ime. OCT aw images we e expo ed as Audio Video In e lea e (AVI) ideos. These ideos we e analysed using a cus om p og am implemen ed in Ma lab ( e sion R218a, Ma hwo ks Inc., Na ick, MA, USA). This code allows us o ind he inne limi ing memb ane (ILM) and he inne and ou e laye s o he e inal pigmen epi helium (RPE) by g ayscale con e sion. This makes i possible o delimi i eous space and RPE space in each b-scan (Figu e 1). The i eous was de ined as he space be ween he uppe mos ex en o he b-scan and he ILM, whe eas he RPE was de ined as he space be ween he inne and ou e laye o he RPE [ 16 , 17 ]. The mean in ensi y alue o hese wo spaces was calcula ed as he a e age o he in ensi y o all he pixels wi hin each egion, ob aining he VIT/RPE ela i e in ensi y in each b-scan. Thus, he VIT/RPE ela i e in ensi y o each eye is he a e age o he 61 b-scans. Fu he mo e, as he BRI/LAP agg ega es end o be deposi ed on he ILM– e inal ne e ib e laye (RNFL), he inne and ou e limi s o he ILM–RNFL we e de e mined o ob ain he e olu ion o ILM–RNFL hickness h oughou he 24 weeks o ollow-up. Pha maceu ics 2021, 13, x FOR PEER REVIEW 5 o 17 Figu e 1. Quan i a i e assessmen o VIT/RPE ela i e in ensi y in he same igh eye o a a a (A) wo weeks and (B) six weeks pos -injec ion wi h b imonidine–Laponi e in a i eal o mula ion. Abb e ia ions: IF: in a i eal o mula ion; VIT: i eous; RPE: e inal pigmen epi helium; ILM: inne limi ing memb ane. 2.6. S a is ical Analysis All da a we e eco ded in an Excel da abase, and s a is ical analysis was pe o med using SPSS so wa e e sion 20.0 (SPSS Inc., Chicago, IL, USA). To assess sample dis ibu ion, he Kolmogo o –Smi no es was used. Howe e , gi en he non-pa ame ic dis ibu ion o mos o he da a, he Mann–Whi ney U es was employed o e alua e he di e ences be ween bo h coho s, and a pai ed Wilcoxon es was used o compa e he changes eco ded in each eye o e he s udy pe iod. p alues < 0.05 we e conside ed o indica e s a is ical signi icance. 3. Resul s A o al o 186 OCT ideos om 43 ea ed a s (43 hype ensi e REs ea ed wi h BRI/LAP IF and 29 non- ea ed hype ensi e LEs) and 23 non- ea ed heal hy a s (23 REs/23 LEs) we e analysed. The REs injec ed wi h he BRI/LAP IF showed hype e lec i e Figu e 1. Quan i a i e assessmen o VIT/RPE ela i e in ensi y in he same igh eye o a a a ( A ) wo weeks and ( B ) six weeks pos -injec ion wi h b imonidine–Laponi e in a i eal o mula ion. Abb e ia ions: IF: in a i eal o mula ion; VIT: i eous; RPE: e inal pigmen epi helium; ILM: inne limi ing memb ane. Pha maceu ics 2021,13, 217 5 o 16 The size o he agg ega es in each b-scan was also de e mined by calcula ing he numbe o pixels ha each agg ega e con ains in he image. In he analysed b-scans, he e a e a o al o 761,856 pixels and he image a ea is 2906 mm 2 . The e o e, he a io is 3815 µ m 2 /pixel. To calcula e ha co ec ly, he backg ound speckle noise o he image was dele ed using a denoising il e , which made i possible o dis inguish be ween agg ega es and backg ound noise. This il e was implemen ed in ou cus om code ollowing he de ini ion o agg ega es as la ge do s whose in ensi y is g ea e han he backg ound in ensi y. In o de o ensu e ha we only quan i y he agg ega es p oduced by he BRI/LAP IF, a minimum limi was es ablished, so ha he possible noise [ 22 ] due o he physiological componen s o he eye we e no aken in o accoun . This minimum limi was se a 500 µ m 2 pe agg ega e. Once we had calcula ed he size o each agg ega e, we could compu e he a e age a ea and o al a ea o he BRI/LAP IF in each eye a di e en s ages du ing he ollow-up. The imaging da a we e analysed o clinical and d ug le el in o ma ion by a masked eade . OCT segmen a ion was pe o med by wo di e en esea che s, likewise masked, o e i y ep oducibili y. 2.6. S a is ical Analysis All da a we e eco ded in an Excel da abase, and s a is ical analysis was pe o med using SPSS so wa e e sion 20.0 (SPSS Inc., Chicago, IL, USA). To assess sample dis- ibu ion, he Kolmogo o –Smi no es was used. Howe e , gi en he non-pa ame ic dis ibu ion o mos o he da a, he Mann–Whi ney U es was employed o e alua e he di e ences be ween bo h coho s, and a pai ed Wilcoxon es was used o compa e he changes eco ded in each eye o e he s udy pe iod. p alues < 0.05 we e conside ed o indica e s a is ical signi icance. 3. Resul s A o al o 186 OCT ideos om 43 ea ed a s (43 hype ensi e REs ea ed wi h BRI/LAP IF and 29 non- ea ed hype ensi e LEs) and 23 non- ea ed heal hy a s (23 REs/23 LEs) we e analysed. The REs injec ed wi h he BRI/LAP IF showed hype e lec- i e do s/agg ega es mixing uni o mly in he i eous gel and dispe sed as loa e s, wi h a endency o mo e owa d he i eo e inal in e ace du ing he 24-week ollow-up [ 9 ]. The e was also OCT-guided e idence o he hype e lec i e do s c ossing he i eo e inal in e ace and embedding deeply in he e inal issue. Pa icula quali a i e cha ac e is ics o he beha iou o he BRI/LAP IF obse ed in se e al animals a e shown in Figu e 2. OCT also de ec ed a p og essi e dec ease o e ime in he numbe and size o BRI/LAP agg ega es (Figu e 3). Figu e 4shows he empo al change in agg ega e size. Figu e 4A shows ha o al agg ega e a ea dec eased wi h ime o e he 24 weeks o ollow-up. This d op was e y ma ked du ing he i s 4–6 weeks, a e which he change was mo e g adual. Mo eo e , o al agg ega e a ea inc eased wo weeks a e injec ion. Al hough a simila end was obse ed (agg ega e size inc eased a 2 weeks), his seems o be a he expense o an inc ease in mean agg ega e size. A conside able dec ease was hen de ec ed a ound 8 weeks, and om 12 weeks onwa ds mean agg ega e size emained p ac ically cons an (Figu e 4B). Pha maceu ics 2021,13, 217 6 o 16 Pha maceu ics 2021, 13, x FOR PEER REVIEW 6 o 17 do s/agg ega es mixing uni o mly in he i eous gel and dispe sed as loa e s, wi h a endency o mo e owa d he i eo e inal in e ace du ing he 24-week ollow-up [9]. The e was also OCT-guided e idence o he hype e lec i e do s c ossing he i eo e inal in e ace and embedding deeply in he e inal issue. Pa icula quali a i e cha ac e is ics o he beha iou o he BRI/LAP IF obse ed in se e al animals a e shown in Figu e 2. OCT also de ec ed a p og essi e dec ease o e ime in he numbe and size o BRI/LAP agg ega es (Figu e 3). Figu e 4 shows he empo al change in agg ega e size. Figu e 4A shows ha o al agg ega e a ea dec eased wi h ime o e he 24 weeks o ollow-up. This d op was e y ma ked du ing he i s 4–6 weeks, a e which he change was mo e g adual. Mo eo e , o al agg ega e a ea inc eased wo weeks a e injec ion. Al hough a simila end was obse ed (agg ega e size inc eased a 2 weeks), his seems o be a he expense o an inc ease in mean agg ega e size. A conside able dec ease was hen de ec ed a ound 8 weeks, and om 12 weeks onwa ds mean agg ega e size emained p ac ically cons an (Figu e 4B). Figu e 2. Miles ones in he e olu ion o he b imonidine–Laponi e in a i eal o mula ion (BRI/LAP IF) analysed using op ical cohe ence omog aphy. (A): C oss-sec ional image o he op ic ne e showing hype - e lec i e IF agg ega es a e c ossing he pos e io i eous co ex (PVC). The g een a ow poin s o he pos e io i eous co ex. Th ee hype - e lec i e do s a e ound in he space be ween he PVC and he inne limi ing memb ane (ILM). (B): Hype - e lec i e agg ega es (g een iangle) a he momen o c ossing he pos e io i eous co ex. (C): Hype - e lec i e agg ega es (g een ci cle) a anged one-by-one in a ow. (D): La ge BRI/LAP IF agg ega e (g een hombus) in he i eous humou . A ligh op ical shadow can be obse ed (indica ing po en ial pe cep ion o loa e s) simila o he shadow ha e inal essels p oduce. (E,F): Hype - e lec i e agg ega es pene a ing he e inal laye s. Deposi s in he inne nuclea laye o pe i ascula (g een squa e). Deposi s in he ou e nuclea laye (g een s a ). Red lines indica e he ILM and e inal pigmen epi helium (RPE) bounda ies. (B) Shows an example wi hou bounda ies ( ed lines) so as o pe mi measu emen . (A,B) Show images ob ained a 6 weeks o ollow-up. (C,D) Show images ob ained a 8 weeks o ollow-up. (E,F) Show images ob ained a 12 weeks o ollow-up. Rep esen a i e images a e ex ac ed om di e en animals. Figu e 2. Miles ones in he e olu ion o he b imonidine–Laponi e in a i eal o mula ion (BRI/LAP IF) analysed using op ical cohe ence omog aphy. ( A ): C oss-sec ional image o he op ic ne e showing hype - e lec i e IF agg ega es a e c ossing he pos e io i eous co ex (PVC). The g een a ow poin s o he pos e io i eous co ex. Th ee hype - e lec i e do s a e ound in he space be ween he PVC and he inne limi ing memb ane (ILM). ( B ): Hype - e lec i e agg ega es (g een iangle) a he momen o c ossing he pos e io i eous co ex. ( C ): Hype - e lec i e agg ega es (g een ci cle) a anged one-by-one in a ow. ( D ): La ge BRI/LAP IF agg ega e (g een hombus) in he i eous humou . A ligh op ical shadow can be obse ed (indica ing po en ial pe cep ion o loa e s) simila o he shadow ha e inal essels p oduce. ( E , F ): Hype - e lec i e agg ega es pene a ing he e inal laye s. Deposi s in he inne nuclea laye o pe i ascula (g een squa e). Deposi s in he ou e nuclea laye (g een s a ). Red lines indica e he ILM and e inal pigmen epi helium (RPE) bounda ies. ( B ) Shows an example wi hou bounda ies ( ed lines) so as o pe mi measu emen . ( A , B ) Show images ob ained a 6 weeks o ollow-up. ( C , D ) Show images ob ained a 8 weeks o ollow-up. ( E , F ) Show images ob ained a 12 weeks o ollow-up. Rep esen a i e images a e ex ac ed om di e en animals. Pha maceu ics 2021, 13, x FOR PEER REVIEW 7 o 17 Figu e 3. P og essi e dec ease in hype - e lec i e agg ega es ( ed a ows) o he b imonidine– Laponi e in a i eal o mula ion (BRI/LAP IF) de ec ed in he i eous– e inal in e ace using op ical cohe ence omog aphy o e 24 weeks o ollow-up. Figu e 4. Tempo al change in agg ega e a ea. (A) To al a ea o he agg ega es loca ed in he i eous; (B) mean a ea o he agg ega es. OCT analysis e ealed he dec easing in ensi y o he hype e lec i e IF agg ega es in he i eous o e 24 weeks o ollow-up (Figu e 5A). The igu e shows ha he in ensi y peaked a he end o he second week and hen dec eased un il i s abilised a ound week 12. I should be no ed ha he in ensi y index ose sligh ly om weeks 4 o 6 and ha his coincided wi h a dec ease in o al agg ega e a ea (Figu e 4A) and a sligh inc ease in mean agg ega e a ea (Figu e 4B). Compa ison o he in ensi y indices o REs ( ea ed wi h BRI/LAP IF) and LEs (non- ea ed) (Figu e 5A) shows ha he in ensi y is e y much lowe in he LEs (0.30 s., 0.25; p < 0.001). As can be seen, VIT/RPE ela i e in ensi y in eyes wi h glaucoma ea ed wi h BRI/LAP IF is highe han in eyes wi h Figu e 3. P og essi e dec ease in hype - e lec i e agg ega es ( ed a ows) o he b imonidine– Laponi e in a i eal o mula ion (BRI/LAP IF) de ec ed in he i eous– e inal in e ace using op ical cohe ence omog aphy o e 24 weeks o ollow-up. Pha maceu ics 2021,13, 217 7 o 16 Pha maceu ics 2021, 13, x FOR PEER REVIEW 7 o 17 Figu e 3. P og essi e dec ease in hype - e lec i e agg ega es ( ed a ows) o he b imonidine– Laponi e in a i eal o mula ion (BRI/LAP IF) de ec ed in he i eous– e inal in e ace using op ical cohe ence omog aphy o e 24 weeks o ollow-up. Figu e 4. Tempo al change in agg ega e a ea. (A) To al a ea o he agg ega es loca ed in he i eous; (B) mean a ea o he agg ega es. OCT analysis e ealed he dec easing in ensi y o he hype e lec i e IF agg ega es in he i eous o e 24 weeks o ollow-up (Figu e 5A). The igu e shows ha he in ensi y peaked a he end o he second week and hen dec eased un il i s abilised a ound week 12. I should be no ed ha he in ensi y index ose sligh ly om weeks 4 o 6 and ha his coincided wi h a dec ease in o al agg ega e a ea (Figu e 4A) and a sligh inc ease in mean agg ega e a ea (Figu e 4B). Compa ison o he in ensi y indices o REs ( ea ed wi h BRI/LAP IF) and LEs (non- ea ed) (Figu e 5A) shows ha he in ensi y is e y much lowe in he LEs (0.30 s., 0.25; p < 0.001). As can be seen, VIT/RPE ela i e in ensi y in eyes wi h glaucoma ea ed wi h BRI/LAP IF is highe han in eyes wi h Figu e 4. Tempo al change in agg ega e a ea. ( A ) To al a ea o he agg ega es loca ed in he i eous; ( B ) mean a ea o he agg ega es. OCT analysis e ealed he dec easing in ensi y o he hype e lec i e IF agg ega es in he i eous o e 24 weeks o ollow-up (Figu e 5A). The igu e shows ha he in ensi y peaked a he end o he second week and hen dec eased un il i s abilised a ound week 12. I should be no ed ha he in ensi y index ose sligh ly om weeks 4 o 6 and ha his coincided wi h a dec ease in o al agg ega e a ea (Figu e 4A) and a sligh inc ease in mean agg ega e a ea (Figu e 4B). Compa ison o he in ensi y indices o REs ( ea ed wi h BRI/LAP IF) and LEs (non- ea ed) (Figu e 5A) shows ha he in ensi y is e y much lowe in he LEs (0.30 s., 0.25; p< 0.001). As can be seen, VIT/RPE ela i e in ensi y in eyes wi h glaucoma ea ed wi h BRI/LAP IF is highe han in eyes wi h non- ea ed glaucoma due o he p esence o BRI/LAP IF. In e es ingly, he index simila ly inc eased o e he i s wo weeks. This e ec could be p oduced by he induc ion o glaucoma. Fu he mo e, he in ensi y index o non- ea ed eyes ( igh and le con ol eyes; he g ey lines in Figu e 5A ) was also compu ed. In his case, he in ensi y alue emained cons an (0.17). He e, he di e ence obse ed in he in ensi y index be ween eyes wi h glaucoma and heal hy con ols is p oduced by glaucoma induc ion. Finally, we obse ed ha he agg ega es we e ini ially dis ibu ed h oughou he i eous bu as ime wen by (6 o 8 weeks) hey se led on op o he ILM–RNFL. I was no possible o dis inguish be ween he agg ega es and he ILM–RNFL because he in ensi y alues a e e y simila . The e o e, his co-laye was segmen ed in o de o measu e he deposi ed agg ega es. Figu e 5B shows how RE ILM–RNFL hickness inc eased un il i pla eaued a week 12. This inc ease in hickness is di ec ly ela ed o he agg ega es’ dis ibu ion on op o he e ina. In he same plo , LE (un ea ed) ILM–RNFL hickness emained unal e ed o dec eased sligh ly du ing he ollow-up i no agg ega es we e p esen . Fu he mo e, we p e iously no only uled ou ha he hickness inc ease in he ea ed eye was a consequence o neu odegene a ion o cys oid oedema, bu also obse ed unc ional neu op o ec ion and a highe RGC coun [ 9 ]. Pha maceu ics 2021, 13, x FOR PEER REVIEW 8 o 17 non- ea ed glaucoma due o he p esence o BRI/LAP IF. In e es ingly, he index simila ly inc eased o e he i s wo weeks. This e ec could be p oduced by he induc ion o glaucoma. Fu he mo e, he in ensi y index o non- ea ed eyes ( igh and le con ol eyes; he g ey lines in Figu e 5A) was also compu ed. In his case, he in ensi y alue emained cons an (0.17). He e, he di e ence obse ed in he in ensi y index be ween eyes wi h glaucoma and heal hy con ols is p oduced by glaucoma induc ion. Finally, we obse ed ha he agg ega es we e ini ially dis ibu ed h oughou he i eous bu as ime wen by (6 o 8 weeks) hey se led on op o he ILM–RNFL. I was no possible o dis inguish be ween he agg ega es and he ILM–RNFL because he in ensi y alues a e e y simila . The e o e, his co-laye was segmen ed in o de o measu e he deposi ed agg ega es. Figu e 5B shows how RE ILM–RNFL hickness inc eased un il i pla eaued a week 12. This inc ease in hickness is di ec ly ela ed o he agg ega es’ dis ibu ion on op o he e ina. In he same plo , LE (un ea ed) ILM–RNFL hickness emained unal e ed o dec eased sligh ly du ing he ollow-up i no agg ega es we e p esen . Fu he mo e, we p e iously no only uled ou ha he hickness inc ease in he ea ed eye was a consequence o neu odegene a ion o cys oid oedema, bu also obse ed unc ional neu op o ec ion and a highe RGC coun [9]. Figu e 5. (A) Mean VIT/RPE ela i e in ensi y in a s wi h induced bila e al glaucoma (RE ea ed wi h b imonidine– Laponi e) and heal hy con ols o e 24 weeks o ollow-up. (B) Segmen a ion o he ILM–RNFL in a s wi h induced bila e al glaucoma (RE ea ed wi h b imonidine–Laponi e) o e 24 weeks o ollow-up. Abb e ia ions: RE: igh eye; LE: le eye; OH: ocula hype ension; T: ea ed; BRI/LAP IF: B imonidine–Laponi e in a i eal o mula ion; non-T: non- ea ed; hC: heal hy con ol; ILM: inne limi ing memb ane; RNFL: e inal ne e ib e laye . Finally, a 3-D econs uc ion o he 61 b-scans om a speci ic a was pe o med in o de o assess he quali a i e dec ease in agg ega es wi h ime. Figu e 6 shows he same eye a 2 weeks o ollow-up and hen 6 weeks la e (8 weeks o ollow-up). I clea ly shows ha he agg ega es a e widely dispe sed 2 weeks pos -injec ion, and ha 6 weeks la e he agg ega es a e ewe and smalle and ha e p ac ically disappea ed om he i eous humou . Figu e 5. ( A ) Mean VIT/RPE ela i e in ensi y in a s wi h induced bila e al glaucoma (RE ea ed wi h b imonidine– Laponi e) and heal hy con ols o e 24 weeks o ollow-up. ( B ) Segmen a ion o he ILM–RNFL in a s wi h induced bila e al glaucoma (RE ea ed wi h b imonidine–Laponi e) o e 24 weeks o ollow-up. Abb e ia ions: RE: igh eye; LE: le eye; OH: ocula hype ension; T: ea ed; BRI/LAP IF: B imonidine–Laponi e in a i eal o mula ion; non-T: non- ea ed; hC: heal hy con ol; ILM: inne limi ing memb ane; RNFL: e inal ne e ib e laye . Pha maceu ics 2021,13, 217 8 o 16 Finally, a 3-D econs uc ion o he 61 b-scans om a speci ic a was pe o med in o de o assess he quali a i e dec ease in agg ega es wi h ime. Figu e 6shows he same eye a 2 weeks o ollow-up and hen 6 weeks la e (8 weeks o ollow-up). I clea ly shows ha he agg ega es a e widely dispe sed 2 weeks pos -injec ion, and ha 6 weeks la e he agg ega es a e ewe and smalle and ha e p ac ically disappea ed om he i eous humou . Pha maceu ics 2021, 13, x FOR PEER REVIEW 9 o 17 Figu e 6. 3-D econs uc ion o he e olu ion o he agg ega es a 2 weeks and 8 weeks o ollow-up. The econs uc ion is shown om wo di e en pe spec i es a each poin in ime. Abb e ia ions: N: nasal; I: in e io ; S: supe io ; IF: in a i eal o mula ion. In o de o in es iga e i he i eous OCT da a could se e as an objec i e ma ke o non-in asi e moni o ing o he IF, he cu e o he b imonidine le els ex ac ed om ou p e ious s udy [9] (CC BY 4.0 license) was co ela ed wi h he VIT/RPE ela i e in ensi ies and wi h he cu e o he o al agg ega e a ea (as an exp ession o he o al amoun o IF injec ed) ob ained using OCT a weeks 1, 4, 8, and 24 a e in a i eal injec ion. Bo h he b imonidine le els and he VIT/RPE ela i e in ensi y cu es showed a nega i e linea endency wi h a di ec co ela ion (y = −0.0003x + 0.1016 R 2 = 0.5616 s. y = −0.0002x + 0.2543 R 2 = 0.4301, espec i ely). Mo eo e , he loga i hmic cu es o he b imonidine le els and he o al agg ega e a ea we e e y simila (Figu e 7). Figu e 7. (A) Dec easing b imonidine le el cu es in a eyes exp essed in ng/mL (mean ± s anda d de ia ion; n = 3 eyes in each s udy ime) (da a om [9] (CC BY 4.0 license)) (in blue) and o al agg ega e a ea in he a eye i eous exp essed in pixels (mean ± s anda d de ia ion; n = 9 eyes a week 1, n = 21 eyes a week 4, n = 8 eyes a week 8 and n = 5 eyes a week 24), ob ained using op ical cohe ence omog aphy (in ed) o e 24 weeks o ollow-up. Loga i hmic cu es in dashes. (B) Figu e 6. 3-D econs uc ion o he e olu ion o he agg ega es a 2 weeks and 8 weeks o ollow-up. The econs uc ion is shown om wo di e en pe spec i es a each poin in ime. Abb e ia ions: N: nasal; I: in e io ; S: supe io ; IF: in a i eal o mula ion. In o de o in es iga e i he i eous OCT da a could se e as an objec i e ma ke o non-in asi e moni o ing o he IF, he cu e o he b imonidine le els ex ac ed om ou p e ious s udy [ 9 ] (CC BY 4.0 license) was co ela ed wi h he VIT/RPE ela i e in ensi ies and wi h he cu e o he o al agg ega e a ea (as an exp ession o he o al amoun o IF injec ed) ob ained using OCT a weeks 1, 4, 8, and 24 a e in a i eal injec ion. Bo h he b imonidine le els and he VIT/RPE ela i e in ensi y cu es showed a nega i e linea endency wi h a di ec co ela ion (y = − 0.0003x + 0.1016 R 2 = 0.5616 s. y = − 0.0002x + 0.2543 R 2 = 0.4301, espec i ely). Mo eo e , he loga i hmic cu es o he b imonidine le els and he o al agg ega e a ea we e e y simila (Figu e 7). Pha maceu ics 2021,13, 217 9 o 16 Pha maceu ics 2021, 13, x FOR PEER REVIEW 9 o 17 Figu e 6. 3-D econs uc ion o he e olu ion o he agg ega es a 2 weeks and 8 weeks o ollow-up. The econs uc ion is shown om wo di e en pe spec i es a each poin in ime. Abb e ia ions: N: nasal; I: in e io ; S: supe io ; IF: in a i eal o mula ion. In o de o in es iga e i he i eous OCT da a could se e as an objec i e ma ke o non-in asi e moni o ing o he IF, he cu e o he b imonidine le els ex ac ed om ou p e ious s udy [9] (CC BY 4.0 license) was co ela ed wi h he VIT/RPE ela i e in ensi ies and wi h he cu e o he o al agg ega e a ea (as an exp ession o he o al amoun o IF injec ed) ob ained using OCT a weeks 1, 4, 8, and 24 a e in a i eal injec ion. Bo h he b imonidine le els and he VIT/RPE ela i e in ensi y cu es showed a nega i e linea endency wi h a di ec co ela ion (y = −0.0003x + 0.1016 R 2 = 0.5616 s. y = −0.0002x + 0.2543 R 2 = 0.4301, espec i ely). Mo eo e , he loga i hmic cu es o he b imonidine le els and he o al agg ega e a ea we e e y simila (Figu e 7). Figu e 7. (A) Dec easing b imonidine le el cu es in a eyes exp essed in ng/mL (mean ± s anda d de ia ion; n = 3 eyes in each s udy ime) (da a om [9] (CC BY 4.0 license)) (in blue) and o al agg ega e a ea in he a eye i eous exp essed in pixels (mean ± s anda d de ia ion; n = 9 eyes a week 1, n = 21 eyes a week 4, n = 8 eyes a week 8 and n = 5 eyes a week 24), ob ained using op ical cohe ence omog aphy (in ed) o e 24 weeks o ollow-up. Loga i hmic cu es in dashes. (B) Figu e 7. ( A ) Dec easing b imonidine le el cu es in a eyes exp essed in ng/mL (mean ± s anda d de ia ion; n= 3 eyes in each s udy ime) (da a om [ 9 ] (CC BY 4.0 license)) (in blue) and o al agg ega e a ea in he a eye i eous exp essed in pixels (mean ± s anda d de ia ion; n= 9 eyes a week 1, n= 21 eyes a week 4, n= 8 eyes a week 8 and n= 5 eyes a week 24), ob ained using op ical cohe ence omog aphy (in ed) o e 24 weeks o ollow-up. Loga i hmic cu es in dashes. ( B ) Posi i e linea co ela ion be ween d ug le els and o al agg ega e a ea. Da a a e exp essed as means ± s anda d de ia ion; op ical cohe ence omog aphy (OCT) da a in ed; b imonidine da a in blue;  : 1 week;  : 4 weeks; • : 8 weeks; N : 24 weeks. A sho in i o s udy was pe o med o compa e wi h he esul s ob ained in he in i o s udy. A model o i eous humou (VHM) o med by sodium hialu ona e in saline solu ion was chosen as medium o elease, and he p ocedu e was analogous o ou p eceden s udy wi h he DEX/LAP sys em [ 23 ], wi h equilib a ion o he BRI/LAP o mula ion in he VHM o 24 h, cen i uga ion o sepa a e he liquid phase wi h he eleased BRI o analysis and e-suspension o he solid in a new ba ch o VHM. As can be seen in Figu e 8, he eleased amoun was highe in he i s ex ac ions, indica ing he p esence o a ac ion o BRI loosely bound o LAP, whe eas he eleased amoun in he successi e ex ac ions is much lowe , co esponding o he BRI ac ion mo e igh ly bound o LAP. In any case, he o al amoun eleased a e eigh ex ac ions is lowe han he 22% o he o al BRI p esen in BRI/LAP, con i ming in his way he abili y o his o mula ion o a sus ained elease o a long ime pe iod. Pha maceu ics 2021, 13, x FOR PEER REVIEW 10 o 17 Posi i e linea co ela ion be ween d ug le els and o al agg ega e a ea. Da a a e exp essed as means ± s anda d de ia ion; op ical cohe ence omog aphy (OCT) da a in ed; b imonidine da a in blue; ■: 1 week; ◆: 4 weeks; ●: 8 weeks; ▲: 24 weeks. A sho in i o s udy was pe o med o compa e wi h he esul s ob ained in he in i o s udy. A model o i eous humou (VHM) o med by sodium hialu ona e in saline solu ion was chosen as medium o elease, and he p ocedu e was analogous o ou p eceden s udy wi h he DEX/LAP sys em [23], wi h equilib a ion o he BRI/LAP o mula ion in he VHM o 24 h, cen i uga ion o sepa a e he liquid phase wi h he eleased BRI o analysis and e-suspension o he solid in a new ba ch o VHM. As can be seen in Figu e 8, he eleased amoun was highe in he i s ex ac ions, indica ing he p esence o a ac ion o BRI loosely bound o LAP, whe eas he eleased amoun in he successi e ex ac ions is much lowe , co esponding o he BRI ac ion mo e igh ly bound o LAP. In any case, he o al amoun eleased a e eigh ex ac ions is lowe han he 22% o he o al BRI p esen in BRI/LAP, con i ming in his way he abili y o his o mula ion o a sus ained elease o a long ime pe iod. Figu e 8. Cumula i e elease o BRI om BRI/LAP o he model o i eous humou . 4. Discussion This pape desc ibes use o OCT o pe o m non-in asi e moni o ing o an in a i eal o mula ion (BRI/LAP) used o ea glaucoma. Quali a i e s udy made i possible o obse e he beha iou o BRI/LAP IF in he i eous and e ina o e a 24-week pe iod [9]. In he ea ly s ages o he s udy he BRI/LAP IF was mixed in he i eous, emaining in suspension in small mic oagg ega es ha la e showed a endency o app oach and a ach o he e ina, possibly due o b imonidine opism owa ds he alpha-ad ene gic ecep o s p esen in he ganglion cell laye , inne nuclea laye and ou e nuclea laye [24], melanin, he RPE and he cho oid. In animal s udies, b imonidine has been shown o ha e bo h a unc ional and a s uc u al neu op o ec i e e ec . In a i eal adminis a ion o b imonidine-loaded nanopa icles has shown a neu op o ec i e e ec o e 14 days o moni o ing [4], a hypo ensi e and neu op o ec i e e ec las ing 4 weeks in an acu e glaucoma model [25] and, ecen ly, ou g oup [9] demons a ed a unc ional and s uc u al hypo ensi e and neu op o ec i e e ec las ing 6 mon hs wi h BRI/LAP IF in a ch onic glaucoma model. This was p o ed no only by analysing he neu o e inal hickness wi h OCT and he unc ionally wi h elec o e inog aphy, bu also by analysing images o he i eo e inal in e ace ob ained wi h OCT, which is a no el measu emen me hod ha could p o ide a non-in asi e, objec i e and eliable means o moni o ing he pha macodynamics o he IF. In addi ion, Figu e 8. Cumula i e elease o BRI om BRI/LAP o he model o i eous humou . Pha maceu ics 2021,13, 217 16 o 16 34. Lee, D.; Kim, K.-Y.; Noh, Y.H.; Chai, S.; Lindsey, J.D.; Ellisman, M.H.; Wein eb, R.N.; Ju, W.-K. B imonidine Blocks Glu ama e Exci o oxici y-Induced Oxida i e S ess and P ese es Mi ochond ial T ansc ip ion Fac o A in Ischemic Re inal Inju y. PLoS ONE 2012,7, e47098. [C ossRe ] 35. Tawa i, S.L.; Koch, D.L.; Cohen, C. Elec ical Double-Laye E ec s on he B ownian Di usi i y and Agg ega ion Ra e o Laponi e Clay Pa icles. J. Colloid In e ace Sci. 2001,240, 54–66. [C ossRe ] [PubMed] 36. Fujimo o, J.G. Op ical cohe ence omog aphy o ul ahigh esolu ion in i o imaging. Na . Bio echnol. 2003 ,21, 1361–1367. [C ossRe ] [PubMed] 37. Ha man, R.R.; Kompella, U.B. In a i eal, Sub e inal, and Sup acho oidal Injec ions: E olu ion o Mic oneedles o D ug Deli e y. J. Ocul. Pha macol. The . 2018,34, 141–153. [C ossRe ] [PubMed] 38. Mo oi, S.E.; Reed, D.M.; Sande s, D.S.; AlMaz oa, A.; Kagemann, L.; Shah, N.; Shekhawa , N.; Richa ds, J.E. P ecision medicine o p e en glaucoma- ela ed blindness. Cu . Opin. Oph halmol. 2019,30, 187–198. [C ossRe ] [PubMed] 39. Ko o , E.; Come , G.M.; S e ens, T.; An one i, D.A. Algo i hm o he Measu e o Vi eous Hype e lec i e Foci in Op ical Cohe ence Tomog aphic Scans o Pa ien s wi h Diabe ic Macula Edema. JAMA Oph halmol. 2016,134, 15–20. [C ossRe ] 40. Sakamo o, T.; Ishibashi, T. Hyalocy es: Essen ial cells o he i eous ca i y in i eo e inal pa hophysiology? Re ina 2011 , 31, 222–228. [C ossRe ] 41. Vagaja, N.N.; Chinne y, H.R.; Binz, N.; Kezic, J.M.; Rakoczy, E.P.; McMenamin, P.G. Changes in Mu ine Hyalocy es A e Valuable Ea ly Indica o s o Ocula Disease. In es ig. Oph halmol. Vis. Sci. 2012,53, 1445–1451. [C ossRe ] [PubMed] 42. Liu, X.; Hui, B.T.; Way, C.; Beese, S.; Ad iano, A.; Keane, P.; Moo e, D.J.; Dennis on, A.K. Nonin asi e Ins umen -based Tes s o De ec ing and Measu ing Vi eous In lamma ion in U ei is: A Sys ema ic Re iew. Ocul. Immunol. In lamm. 2020 , 1–12. [C ossRe ] [PubMed]