Safety and Pharmacokinetics of Intravitreally Repeatedly Injected Panitumumab in Non-Human Primates - A Study Performed Under Good Laboratory Practice

Safety and Pharmacokinetics of Intravitreally Repeatedly Injected Panitumumab in Non-Human Primates - A Study Performed Under Good Laboratory Practice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Safety and Pharmacokinetics of Intravitreally Repeatedly Injected Panitumumab in Non-Human Primates - A Study Performed Under Good Laboratory Practice Ya Xing Wang, Frank G. Holz, Martin Coenen, Xinmin Sun, Wankun Xie, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8501876/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 17 You are reading this latest preprint version Abstract Background Epidermal growth factor (EGF) has been suggested to play a role in myopic axial elongation, and EGF receptor blockade may be of potential therapeutic benefit. Here we examined the ocular and systemic toxicity of panitumumab, a clinically used EGF receptor blocker in oncology, when repeatedly administered intravitreally in non-human primates. Methods The experimental study included six non-human cynomolgus primates (3 males) which underwent five (n = 1 animal) or three (n = 5 animals) 4-weekly intravitreal injections of panitumumab (dose: 0.78 mg (78µL)) or of phosphate buffered solution (PBS) (78µL). Results The study group with panitumumab injections consisted of 7 eyes and the control group with PBS injections of 5 eyes. Two animals of the study group developed on Day 59 (two days after the third injection) signs of a slight intraocular inflammation (cells in anterior chamber and vitreous) and reduction of intraocular pressure, with most of the signs having resolved at study end (Day 86). Panitumumab reached the serum peak concentration at 24h after the first dose (C max 18.3 to 946ng/mL; serum exposure 2120 to 37300 h*ng/mL). Four weeks after the third injection (Day 86), panitumumab concentrations in aqueous humor ranged from 12.8 ng/mL to 65.0 ng/mL, and in the vitreous from 1.74 ng/mL to 531 ng/mL, with a panitumumab accumulation factor between 0.891 and 0.012. TUNEL staining did not reveal pathological results. Conclusions Except for mild and reversible intraocular inflammation in some eyes, repeated intravitreal application of 0.78mg panitumumab did not result in ophthalmological or systemic adverse effects in non-human primates. Intravitreal panitumumab Epidermal growth factor Panitumumab High myopia Myopic macular degeneration Introduction Axial elongation is the major process changing axial hyperopia of the newborn to emmetropia of the young adult, and in the case of an overshooting, to axial myopia of adolescents and adults. The underlying mechanism leading to axial elongation has not fully been uncovered yet. Several molecules have been reported to be associated with axial elongation in experimental studies, conducted mostly in chicken and guinea pigs. 1 – 17 These molecules include dopamine, atropine, TGF-β, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and amphiregulin and other epidermal growth factor (EGF) family members, to name a few. 1 – 17 Recent studies conducted in young guinea pigs with or without lens-induced myopization showed that repeated intravitreal application of antibodies against EGF family members, such as amphiregulin, neuregulin-1, betacellulin, epigen und epiregulin, and EGF itself, were associated with a decrease in axial elongation, while repeated intravitreal injections of EGF family members– namely, amphiregulin, neuregulin-1, and EGF - were associated with an increase in axial elongation over time. 11 , 12 , 14 – 17 As a corollary, the intravitreal application of EGF receptor antibodies was associated with a reduction in axial elongation in young guinea pigs. 14 In young adult monkeys without externally induced myopization, intravitreally applied amphiregulin antibody was associated with a decrease, and intravitreally applied amphiregulin was associated with an increase, in axial elongation. 18 , 19 These observations prompted the hypothesis that EGF and EGF family members may be associated with the process of axial elongation, and that their blockade may potentially reduce further axial elongation, in particular in adult highly myopic patients with ongoing axial elongation and progressing myopic macular degeneration. 20 , 21 Longitudinal studies have shown that axial elongation can continue in highly myopic eyes even in the age of 50 + years, and that ongoing axial elongation is a major, perhaps the only modifiable risk factor for progression of myopic macular degeneration, and potentially of high myopia-associated, non-glaucomatous or glaucomatous/glaucoma-like optic neuropathy. 22 – 24 In view of a potential clinical application of intravitreally applied EGF blockers or EGF receptor (EGFR) blockers to reduce further axial elongation in highly myopic eyes, we herein explored the safety and tolerability of intravitreally repeatedly injected panitumumab as an EGF receptor blocker in non-human primates. Panitumumab as other EGF receptor blockers has been in clinical therapeutic systemic use for about 20 years for refractory EGFR-expressing metastatic colorectal cancer in patients with non-mutated (wild-type) KRAS. 25 Methods The experimental study included 6 non-human cynomolgus primates (3 males) with an age of 3.5 to 5.0 years and a body weight of 2.74 to 3.44 kg. They were supplied by the Hua Zhen Laboratory Animal Breeding Centre (Conghua, Guangzhou, China) (License No.: SCXK (Yue) 2020-0028). The animals were housed at a temperature of 18°C to 26°C and a relative humidity of 40% to 70% throughout the study. An approximately 12-hour light and dark cycle was provided. Fruit and certified non-human primate diet were provided daily. Tap water meeting the State Standard for drinking water was provided ad libitum during study period. Animal care was compliant with the Guide for the Care and Use of Laboratory Animals (8th Edition, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, D.C., 2011) and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99–198). The study was conducted at JOINN Laboratories (Suzhou, China) in compliance with good laboratory practice (GLP) regulations. The study was reviewed and approved by the Institutional Animal Care and Use Committees at JOINN Laboratories to ensure that the study was carried out in an ethical manner. In a first part of the study, one non-human primate received 5 intravitreal injections of panitumumab (dose: 0.78 mg; volume: 78 µL) into the right eye and 5 intravitreal injections of PBS into the left eye on Days 1, 29, 50, 78 and 106 respectively. The animal was sacrificed on Day 135. In the second part of the study, five animals received three intravitreal injections of panitumumab (Vectibix®) in the dose of 0.78 mg and volume of 78 µL. The injections were carried out in intervals of 4 weeks on Days 1, 29, and 57, and the animals were sacrifice on Day 86. Prior to the injections, the animals received mydriatic eye drops, and were then anesthetized by an intramuscular injection of Zolazepamum (Zoletil® 50; 4–12 mg/kg, 50 mg/mL) (Virbac Co., Carros, France). The most recently measured body weight data were used for calculation of the dose of the anesthetics. The animals were kept warm using appropriate means (for example, cover blanket) during anesthesia until regaining consciousness. Panitumumab was taken from the Vectibix® bottle using a filter needle. The needle was exchanged to the injection needle, with care being taken to consider the dead volume of the injection needle. The animals received levofloxacin eye drops and/or ofloxacin eye ointment 2–3 times daily for three consecutive days after the intravitreal injections. The animals were observed at least twice daily during the study period, and any abnormal findings were recorded. Once per week, they were taken out of their cages and clinical signs of toxicity were searched, including changes on the body surface and visible mucosa, neurological and behavioral activities, respiratory parameters, and gastrointestinal reactions. Body weight was measured once per week. In addition, the animals were systemically and ophthalmologically examined at baseline, in regular intervals during the study period, and at four weeks after the last injection at study end (Day 86), when the animals were sacrificed. The ophthalmological examinations were carried out under general anesthesia induced by an intramuscular injection of Zoletil® 50 (4–12 mg/kg, 50 mg/mL). The ophthalmological examination included measurement of intraocular pressure (IOP), dark-adapted electroretinography (ERG), optical coherence tomography (OCT) of the fundus, and fluorescein angiography as well as color fundus photography. If several examinations were performed on the same day, the sequence of examinations was: animal restrain→ general ocular examination (prior to receive the mydriatic agent)→ dark adapted ERG→light-adapted ERG→ ocular tonometry→ general ocular examination (in medical mydriasis)→ OCT →ocular photography → fluorescein angiography. The general ophthalmological examinations (including inspection of the external eye, examination of the cornea and of the anterior and posterior ocular segment) were conducted at baseline, at Days 1, 29 and 57 before the injections were carried out, at one day after each injection (i.e., Days 2, 30, 58), at three days after each injection (i.e., Days 4, 32, 60), at seven days after the injections (i.e., Days 8, 36, 64), and at the end of the study period. Tonometry was performed at baseline, prior to each injection (Days 1, 29, 57), about 15 min after each injection, two days after each injection (Days 3, 31, 59), and at the end of the study. Dark-adapted and light-adapted ERG and fundus fluorescein angiography were conducted at baseline, at two days after the last injection (Day 59) and at study end. For the purpose of the ERG examination, the animals were placed in a dark room for at least 30 minutes for dark adaptation. After taking the dark-adapted ERG, the animals were placed in a light room for at least 10 minutes to perform the light-adapted ERG. For the fluorescein angiography, the animals received an intravenous fluorescein sodium injection in a dose of 8–10 mg/kg body weight (concentration: 100 mg fluorescein/mL). Blood samples were collected from the subcutaneous hindlimb vein at baseline, and at 1, 2, 6, 24, 48, 72, 120, 168, 240, 336, 672 hours after the intravitreal injections on Days 1 and 57. Aqueous humor and vitreous humor (volume: 0.1–0.2 mL) were sampled from both eyes at study end. The samples were immediately put into an ice-filled container and transferred to a refrigerator with a temperature of -60ºC. The concentrations of panitumumab in the plasma, aqueous humor and vitreous body samples were analyzed using the validated ELISA method. The lower limit of quantification of the method were 1.00 ng/mL. Toxicokinetic parameters were calculated using the non-compartmental analysis (NCA) method in WinNonlin (version number: 8.0.0.3176). At study end, the animals were sacrificed by an intramuscular injection of using Zoletil 50 (12 mg/kg, 50 mg/mL) and femoral artery exsanguinations. After death, the corpses were macroscopically examined and an autopsy was performed. Samples tissues were fixated in a solution of 10% neutral buffered formalin for histopathological evaluation. The eyes were enucleated and fixated in Davidson’s fixative. The tissues were processed by routine histological methods including embedding in paraffin, sectioning, mounting on slides and staining with hematoxylin and eosin (H&E). TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining was carried out in two slides of each eye to compare the density of apoptotic cells between the left eyes and rights eyes. Panitumumab is a fully human monoclonal antibody specific to EGF receptor. 25 The dose of panitumumab used in this study was based on the results of previous studies on guinea pigs and on the design of an eventual future clinical study of intravitreally applied panitumumab to prevent further axial elongation in highly myopic adult patients with progressive myopic macular degeneration. 11 , 12 , 14 – 17 , 26 In the latter study, the maximal dose of intravitreally injected panitumumab is 1.8 mg. 26 Taking into account the volume of 10.306 mL of a highly myopic human eye with an axial length of 27 mm and the volume of 2.953 mL of a non-human primate eye with diameter of 17.8 mm, the panitumumab dose for the non-human primate eye corresponding to the dose of 1.8 mg panitumumab in the highly myopic human eye at the same panitumumab concentration would be (2.953 mL / 10.306 mL) * 1.8 mg or 0.52 mg panitumumab in the non-human primate eye. The dose of 0.78 mg panitumumab used in our study would correspond to a dose of (10.306 mL / 2.953 mL) * 0.78 mg or 2.72 mg panitumumab in the human myopic eye with an axial length of 27 mm. In a concentration of 10 mg panitumumab / mL as taken out of the Vectibix® bottle and diluted as recommended by the manufacturer, the dose of 0.78 mg panitumumab corresponded to a volume of 78 µL panitumumab. A reason not to use a higher dose than 0.78 mg panitumumab in the study was, that an intravitreally injected volume higher than 0.78 µL might have led to an IOP-rise related tissue damage in the retina and optic nerve. Results During the study period, none of the animals died. No test article-related abnormal findings were noted in clinical observations, body weight data, food consumption, hematology, blood coagulation, clinical chemistry or urinalysis in the animals throughout the study. The mean eye diameters as directly measured post mortem in the 5 animals of the second study part were 18.15 ± 0.91 mm, 17.41 ± 0.94 mm and 17.85 ± 0.53 in the horizontal, vertical and sagittal direction, respectively. The mean eye diameter of all directions was 17.80 ± 0.61 mm. Assuming a mostly spherical eye shape, the mean eye volume was 2965 ± 309 mm 3 or 2.965 ± 0.309 mL. The intraocular concentration of panitumumab was 0.78 mg / 2.965 mL or 0.26 mg / mL of intraocular volume. Upon slit lamp-based biomicroscopy, minor retrocorneal precipitates were noted in animal #2370454 and animal #2370455 at Day 32, with complete resolution at Day 85. Gray-white dot-like particles of equal size in the anterior chamber and on the lens surface with a minor to moderate severity were noted after the second and third injection in animal #2370454; the particles had disappeared on Day 85. In a similar manner, gray-white dot-like particles of equal size were detected in the anterior vitreous at Day 36 in animal #2370454. The particles could still be seen until Day 85. The severity was minimal to slight. On Day 59 (two days after the third intravitreal injection), the fundus images of two animals of the test article group were slightly blurred, showing dilated retinal veins, slightly unsharp optic disc borders in fluorescein angiography, dot-like hyperreflective signals in the posterior vitreous upon OCT examination, and some unevenness of the retinal surface on OCT images. These eyes showed some retrocorneal precipitates, particles in the anterior chamber and anterior vitreous, and precipitates on the surface of the anterior lens capsule. Most of these changes had resolved at the end of the study, except for the hyperreflective bodies in the posterior vitreous (as assessed by OCT) and except for the precipitates on the anterior lens surface. In particular, fundus photography and fluorescein angiography and OCT imaging of the retina were unremarkable at study end. The IOP in animals receiving the test article was significantly lower than that of the animals of the negative control at Day 59. In the animal of the first study part, with panitumumab administrations on Days 1, 29, 50, 78 and 106, respectively, the drug accumulation factors on Day 50 and Day 106 were 0.871 and 0.905, respectively, indicating no significant drug accumulation in the serum after repeated administration. At Day 135, 28 days of the last injection, the drug concentration in vitreous humor and aqueous humor of the right eye of the animal were below the lower limit of quantification of the method, and the drug concentrations in the vitreous humor and aqueous humor of the left eye were 8.64 ng/mL and 1.59 ng/mL, respectively. In the animals of the second part of the study, the drug after its first intravitreal application reached the peak concentration at 24 h in the serum, with the C max ranging from 18.3 to 946 ng/mL, and the serum exposure ranged from 2120 to 37300 h*ng/mL. At the time of the third injection (D57), the drug concentration in the serum of one animal (#2370454) was lower than the lower limit of quantification (1.00 ng/mL) at each time point, and the drug reached the peak at 24 h and 1 h in the serum of the other two animals, respectively, with the C max being 87.2 ng/mL and 28.2 ng/mL, and the serum exposure being 5570 h*ng/mL and 448 h*ng/mL, respectively. Before necropsy on D86, the drug concentrations of Vectibix® in the aqueous humor ranged from 12.8 ng/mL to 65.0 ng/mL, and the drug concentrations in the vitreous humor ranged from 1.74 ng/mL to 531 ng/mL. The accumulation factor (AF, AUC last, D57 /AUC last, D1 ) in the two monkeys with detectable panitumumab in the serum (#2370453, #2370455) were 0.891 and 0.012, respectively. The results showed that no significant drug accumulation was observed in the serum of the animals after repeated administration. No test article-related abnormal findings were noted in organ weights, during the macroscopic observation and in the histologic examination of the animals. No significant apoptosis was histologically detected on the histological slides with TUNEL staining. Signs of phototoxicity on the external eye regions or general body surface, and signs of phototoxicity in the eye, such as a bleaching of the retinal pigment epithelium, retinal edema or retinal thinning in the foveal region, decreased photoreceptor density in the fovea or in other regions of the fundus, or changes in the retinal pigment epithelium layer in the fovea or extrafoveal regions, were not detected, neither on the fundus photographs, the OCT images, or histologically. The ERG did not reveal significant differences between the study eyes and control eyes. Discussion In this experimental study, conducted under good laboratory practice (GLP) conditions, three 4-weekly repeated intravitreal applications of panitumumab (Vectibix®) in 5 animals or five 4-weekly repeated intravitreal applications of panitumumab in one animal, with a recovery period of 4 weeks after the last injection, were not associated with any systemic side effects. Two monkeys showed signs of a mild intraocular inflammation after the third intravitreal injection. At study end, these inflammatory signs had dissolved except for minor retrocorneal and epilental precipitates and hyperreflective bodies in the posterior vitreous cavity. The findings suggest that the intravitreal injection of Vectibix® had been relatively well tolerated by the animals without any tissue damage. These observations are in line with the results of previous experimental investigations in which antibodies to EGF and the EGF receptor, such as cetuximab and panitumumab, were repeatedly intravitreally injected into guinea pigs and rabbits. 11 , 12 , 14 – 17 , 27 , 28 The results also agree with the observations made in a clinical study in which elderly highly myopic patients with myopic macular degeneration repeatedly received intravitreal injections of Vectibix® in doses of 0.6 mg, 1.2 mg and 1.8 mg. 26 In the latter clinical study, 11 patients with a mean age of 66.8 ± 6.3 years received intravitreal injections of panitumumab in doses of 0.6 mg (4 eyes; 1x1 injection, 3x2 injections), 1.2mg (4 eyes; 1x1 injection, 2x2 injections, 1x3 injections) and 1.8 mg (3 eyes; 1x1 injection, 2x2 injections), respectively. Treatment-related systemic adverse events or intraocular inflammatory reactions were not detected in any eye. Correspondingly, best corrected visual acuity (1.62 ± 0.47 logMAR (logarithm of the minimal angle of resolution) versus 1.28 ± 0.59 logMAR; P = 0.08) and intraocular pressure (13.8 ± 2.4 mm Hg versus 14.3 ± 2.6 mm Hg; P = 0.20) remained unchanged during the study period. In a similar manner, those 9 patients with a follow-up of more than 3 months (mean: 6.7 ± 2.7 months) did not show a significant change in axial length (30.73 ± 1.03 mm versus 30.77 ± 1.19 mm; P = 0.56). 26 Axial myopia can be regarded as the result of an overshooting of the physiological process of emmetropization. The process of emmetropization describes the physiological axial elongation of the eye by which the marked axial hyperopia present at birth is transformed into emmetropia in adulthood. According to recent clinical studies, axial elongation usually ceases in the third decade of life in about two-thirds of moderately myopic individuals, while one-third of such individuals can show continuous axial elongation in later life. 29 It may hold true in particular for highly myopic patients with myopic macular degeneration or high myopia-associated optic neuropathies. 22 – 24 , 30 , 31 Continuous axial elongation was a main risk factor for the progression of myopic macular degeneration in clinical longitudinal studies. 22 , 23 , 30 , 31 It may be the only modifiable factor to prevent development and progression of myopic macular degeneration. Experimental studies suggested that axial elongation occurs through a growth and enlargement of Bruch´s membrane (BM) in the fundus periphery. 20 , 21 Arguments in favor of that hypothesis include histomorphometric and clinical findings of an axial elongation-associated thinning of the subfoveal choroid, a decrease in the density of photoreceptors and retinal pigment epithelium (RPE) cells and in retinal thickness in fundus midperiphery (while all three parameters are independent of axial length in the macular region), a presumed shift of BM opening of the optic nerve head canal into the macular direction (explaining the development of parapapillary gamma zone in the temporal parapapillary region), a gamma zone-associated elongation of the disc-fovea distance (while the vertical distance between the temporal vascular arcades is independent of axial elongation), and an axial elongation-related decrease in angle kappa between the temporal vascular arcade and the optic disc as the angle vertex. 20 , 21 In the same manner, BM thickness was independent of, and its volume increased with axial length. 19 , 20 , 32 Supporting the hypothesis were also results of biomechanical investigations in which the elastic modulus of BM was comparable or higher than that of the sclera for an intraocular pressure of approximately 15 mm Hg. 33 The location of the midperiphery as the site of BM enlargement fits with results of experimental studies and clinical observations that the afferent, sensory part of the feedback mechanism governing the process of axial elongation is located in the mid-periphery of the fundus. 34 – 38 EGF belongs to the list of molecules which have been discussed to play a role in the process of axial elongation. These molecules include dopamine, atropine, TGF-β, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and amphiregulin and other epidermal growth factor (EGF) family members, to name a few. 1 – 17 Intravitreal application of antibodies to EGF, EGF family members (such as amphiregulin, neuregulin-1, betacellulin, epigen und epiregulin) and the EGF receptor were associated with a decrease in axial elongation, while intravitreal applications of EGF family members were related with an increase in axial elongation. 11 , 12 , 14 – 18 , 39 These studies were conducted in guinea pigs and non-human primates. The results of the study presented herein fit into the series of the previous investigations in that EGF may play a role in axial elongation, that a blockade of the EGF pathway may perhaps be a potential therapeutic option to reduce further axial elongation of highly myopic eyes in adult patients with myopic macular degeneration, and that repeatedly intravitreally applied panitumumab as an EGF receptor blocker may clinically be well tolerated. Various limitations have to be taken into account. Firstly, the number of animals included into the study was small so that the finding can only be a hint, but definitely not a proof, for safety of intravitreally applied panitumumab. Clinical safety may be assessed in large clinical trials. Secondly, two monkeys showed a temporary, slight intraocular inflammation after the third intravitreal application of panitumumab. At study end, the inflammation had completely subsided. While the reasons for the inflammation have remained unclear, a potential cause might have been the difference between humans and non-human primates, taking into account that panitumumab is a fully humanized antibody. In conclusion, under the conditions of this study, 0.78 mg Vectibix® was repeatedly intravitreally injected into Cynomolgus monkey eyes 3 times (5 animals) or five times (one animal) in 4-weeks intervals, with a following 4-week recovery period. A slight, temporary and reversible ocular inflammation with retrocorneal precipitates, particles in the anterior chamber and vitreous, and precipitates on the lens surface, as visualized by OCT, fundus photography and fluorescein angiography, and a reduction of IOP could be observed in some eyes after the third intravitreal injection, with resolution at study end. No test article-related systemic toxicity was observed. The observations may support the clinical development of intravitreal application of panitumumab in highly myopic patients with ongoing axial elongation or with progressive myopic macular degeneration. Declarations Funding: Research Development Fund of Beijing Municipal Health Commission (2019-4); National Natural Science Foundation of China (82271086). The funder of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. Conflicts of interest/Competing interests: Songhomitra Panda-Jonas, Jost B. Jonas: European patent EP 3 271 392, JP 2021-119187, and US 12,024,557: „ Agents for use in the therapeutic or prophylactic treatment of myopia or hyperopia; European patent application 23196899.1 „EGFR Antagonists for the treatment of diseases involving unwanted migration, proliferation, and metaplasia of retinal pigment epithelium (RPE) cells. All other authors: None. Availability of data and material: All data are available upon reasonable request from the corresponding author. Authors' contributions: Study design: YXW, SPJ, LD, JBJ; Funding: WYX; Conducting the study: WYX, XS, WX, LD, JBJ; Analysis of data: WYX, FGH, MC, XS, WX, SPJ, LD, JBJ; Statistical analysis: WYX, FGH, MC, XS, WX, SPJ, LD, JBJ; Writing the first manuscrt draft: WYX, XS, WX, SPJ, JBJ; Revision and final approval of the manuscript: WYX, FGH, MC, XS, WX, SPJ, LD, JBJ. Ethics approval: Animal care was compliant with the Guide for the Care and Use of Laboratory Animals (8 th Edition, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, D.C., 2011) and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198). The study was conducted at JOINN Laboratories (Suzhou, China) in compliance with good laboratory practice (GLP) regulations. The study was reviewed and approved by the Institutional Animal Care and Use Committees at JOINN Laboratories to ensure that the study was carried out in an ethical manner. Consent to participate: Not applicable Consent for publication: Not applicable References Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-beta 2 in experimental myopia in chicks. Invest Ophthalmol Vis Sci. 1995;36(6):1183–7. Chua WH, Balakrishnan V, Chan YH, et al. Atropine for the treatment of childhood myopia. Ophthalmology. 2006;113(12):2285–91. Gao Q, Liu Q, Ma P, Zhong X, Wu J, Ge J. Effects of direct intravitreal dopamine injections on the development of lid-suture induced myopia in rabbits. Graefes Arch Clin Exp Ophthalmol. 2006;244(10):1329–35. Barathi VA, Weon SR, Beuerman RW. Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation. Mol Vis. 2009;15:1277–93. Jobling AI, Wan R, Gentle A, Bui BV, McBrien NA. 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Intraocular cetuximab: Safety and effect on axial elongation in young Guinea pigs with lens-induced myopization. Exp Eye Res. 2024;238:109715. Lee SS, Lingham G, Sanfilippo PG, et al. Incidence and progression of myopia in early adulthood. JAMA Ophthalmol. 2022;140(2):162–9. Saka N, Ohno-Matsui K, Shimada N, et al. Long-term changes in axial length in adult eyes with pathologic myopia. Am J Ophthalmol. 2010;150(4):562–8. Saka N, Moriyama M, Shimada N, et al. Changes of axial length measured by IOL master during 2 years in eyes of adults with pathologic myopia. Graefes Arch Clin Exp Ophthalmol. 2013;251(2):495–9. Dong L, Shi XH, Kang YK, et al. Bruch's membrane thickness and retinal pigment epithelium cell density in experimental axial elongation. Sci Rep. 2019;9(1):6621. Wang X, Teoh CKG, Chan ASY, Thangarajoo S, Jonas JB, Girard MJA. Biomechanical properties of Bruch's membrane-choroid complex and their influence on optic nerve head biomechanics. Invest Ophthalmol Vis Sci. 2018;59(7):2808–17. Smith EL 3rd, Kee CS, Ramamirtham R, Qiao-Grider Y, Hung LF. Peripheral vision can influence eye growth and refractive development in infant monkeys. Invest Ophthalmol Vis Sci. 2005;46(11):3965–72. Mutti DO, Hayes JR, Mitchell GL. Refractive error, axial length, and relative peripheral refractive error before and after the onset of myopia. Invest Ophthalmol Vis Sci. 2007;48(6):2510–9. Sankaridurg P, Holden B, Smith E III. Decrease in rate of myopia progression with a contact lens designed to reduce relative peripheral hyperopia: one-year results. Invest Ophthalmol Vis Sci. 2011;52(13):9362–7. Berntsen DA, Barr CD, Mutti DO, Zadnik K. Peripheral defocus and myopia progression in myopic children randomly assigned to wear single vision and progressive addition lenses. Invest Ophthalmol Vis Sci. 2013;54(8):5761–70. Wildsoet CF, Chia A, Cho P, et al. IMI - Interventions Myopia Institute: Interventions for controlling myopia onset and progression report. Invest Ophthalmol Vis Sci. 2019;60(3):M106–31. Zhang R, Dong L, Wu H, et al. mTORC1 signaling and negative lens-induced axial elongation. Invest Ophthalmol Vis Sci. 2023;64(10):24. Additional Declarations Competing interest reported. Songhomitra Panda-Jonas, Jost B. Jonas: European patent EP 3 271 392, JP 2021-119187, and US 12,024,557: „ Agents for use in the therapeutic or prophylactic treatment of myopia or hyperopia; European patent application 23196899.1 „EGFR Antagonists for the treatment of diseases involving unwanted migration, proliferation, and metaplasia of retinal pigment epithelium (RPE) cells. All other authors: None. 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Holz","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"G.","lastName":"Holz","suffix":""},{"id":573903284,"identity":"f3ba2b59-57c6-4605-9b4f-130cbb4f46fb","order_by":2,"name":"Martin Coenen","email":"","orcid":"","institution":"University Hospital Bonn","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Coenen","suffix":""},{"id":573903285,"identity":"303b6421-fa26-4b92-978e-8d0a35f64410","order_by":3,"name":"Xinmin Sun","email":"","orcid":"","institution":"JOINN Laboratories","correspondingAuthor":false,"prefix":"","firstName":"Xinmin","middleName":"","lastName":"Sun","suffix":""},{"id":573903290,"identity":"4bd32811-c387-4867-a8bb-af31bcb8d37b","order_by":4,"name":"Wankun Xie","email":"","orcid":"","institution":"JOINN Laboratories","correspondingAuthor":false,"prefix":"","firstName":"Wankun","middleName":"","lastName":"Xie","suffix":""},{"id":573903293,"identity":"b57401d8-5f83-4290-8661-0c368f938aed","order_by":5,"name":"Songhomitra Panda-Jonas","email":"","orcid":"","institution":"Rothschild Foundation Hospital","correspondingAuthor":false,"prefix":"","firstName":"Songhomitra","middleName":"","lastName":"Panda-Jonas","suffix":""},{"id":573903296,"identity":"9f297e4d-0ef5-4132-a579-6f62f448228b","order_by":6,"name":"Li Dong","email":"","orcid":"","institution":"Beijing Tongren Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Dong","suffix":""},{"id":573903300,"identity":"7d592bd8-a669-4375-8fc6-c64e91e255fb","order_by":7,"name":"Jost B. 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Songhomitra Panda-Jonas, Jost B. Jonas: European patent EP 3 271 392, JP 2021-119187, and US 12,024,557: „ Agents for use in the therapeutic or prophylactic treatment of myopia or hyperopia; European patent application 23196899.1 „EGFR Antagonists for the treatment of diseases involving unwanted migration, proliferation, and metaplasia of retinal pigment epithelium (RPE) cells. All other authors: None.","formattedTitle":"Safety and Pharmacokinetics of Intravitreally Repeatedly Injected Panitumumab in Non-Human Primates - A Study Performed Under Good Laboratory Practice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAxial elongation is the major process changing axial hyperopia of the newborn to emmetropia of the young adult, and in the case of an overshooting, to axial myopia of adolescents and adults. The underlying mechanism leading to axial elongation has not fully been uncovered yet. Several molecules have been reported to be associated with axial elongation in experimental studies, conducted mostly in chicken and guinea pigs.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e These molecules include dopamine, atropine, TGF-β, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and amphiregulin and other epidermal growth factor (EGF) family members, to name a few.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Recent studies conducted in young guinea pigs with or without lens-induced myopization showed that repeated intravitreal application of antibodies against EGF family members, such as amphiregulin, neuregulin-1, betacellulin, epigen und epiregulin, and EGF itself, were associated with a decrease in axial elongation, while repeated intravitreal injections of EGF family members\u0026ndash; namely, amphiregulin, neuregulin-1, and EGF - were associated with an increase in axial elongation over time.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e As a corollary, the intravitreal application of EGF receptor antibodies was associated with a reduction in axial elongation in young guinea pigs.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e In young adult monkeys without externally induced myopization, intravitreally applied amphiregulin antibody was associated with a decrease, and intravitreally applied amphiregulin was associated with an increase, in axial elongation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThese observations prompted the hypothesis that EGF and EGF family members may be associated with the process of axial elongation, and that their blockade may potentially reduce further axial elongation, in particular in adult highly myopic patients with ongoing axial elongation and progressing myopic macular degeneration.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Longitudinal studies have shown that axial elongation can continue in highly myopic eyes even in the age of 50\u0026thinsp;+\u0026thinsp;years, and that ongoing axial elongation is a major, perhaps the only modifiable risk factor for progression of myopic macular degeneration, and potentially of high myopia-associated, non-glaucomatous or glaucomatous/glaucoma-like optic neuropathy.\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e In view of a potential clinical application of intravitreally applied EGF blockers or EGF receptor (EGFR) blockers to reduce further axial elongation in highly myopic eyes, we herein explored the safety and tolerability of intravitreally repeatedly injected panitumumab as an EGF receptor blocker in non-human primates. Panitumumab as other EGF receptor blockers has been in clinical therapeutic systemic use for about 20 years for refractory EGFR-expressing metastatic colorectal cancer in patients with non-mutated (wild-type) KRAS.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe experimental study included 6 non-human cynomolgus primates (3 males) with an age of 3.5 to 5.0 years and a body weight of 2.74 to 3.44 kg. They were supplied by the Hua Zhen Laboratory Animal Breeding Centre (Conghua, Guangzhou, China) (License No.: SCXK (Yue) 2020-0028). The animals were housed at a temperature of 18\u0026deg;C to 26\u0026deg;C and a relative humidity of 40% to 70% throughout the study. An approximately 12-hour light and dark cycle was provided. Fruit and certified non-human primate diet were provided daily. Tap water meeting the State Standard for drinking water was provided \u003cem\u003ead libitum\u003c/em\u003e during study period. Animal care was compliant with the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (8th Edition, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, D.C., 2011) and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99\u0026ndash;198). The study was conducted at JOINN Laboratories (Suzhou, China) in compliance with good laboratory practice (GLP) regulations. The study was reviewed and approved by the Institutional Animal Care and Use Committees at JOINN Laboratories to ensure that the study was carried out in an ethical manner.\u003c/p\u003e \u003cp\u003eIn a first part of the study, one non-human primate received 5 intravitreal injections of panitumumab (dose: 0.78 mg; volume: 78 \u0026micro;L) into the right eye and 5 intravitreal injections of PBS into the left eye on Days 1, 29, 50, 78 and 106 respectively. The animal was sacrificed on Day 135. In the second part of the study, five animals received three intravitreal injections of panitumumab (Vectibix\u0026reg;) in the dose of 0.78 mg and volume of 78 \u0026micro;L. The injections were carried out in intervals of 4 weeks on Days 1, 29, and 57, and the animals were sacrifice on Day 86.\u003c/p\u003e \u003cp\u003ePrior to the injections, the animals received mydriatic eye drops, and were then anesthetized by an intramuscular injection of Zolazepamum (Zoletil\u0026reg; 50; 4\u0026ndash;12 mg/kg, 50 mg/mL) (Virbac Co., Carros, France). The most recently measured body weight data were used for calculation of the dose of the anesthetics. The animals were kept warm using appropriate means (for example, cover blanket) during anesthesia until regaining consciousness. Panitumumab was taken from the Vectibix\u0026reg; bottle using a filter needle. The needle was exchanged to the injection needle, with care being taken to consider the dead volume of the injection needle. The animals received levofloxacin eye drops and/or ofloxacin eye ointment 2\u0026ndash;3 times daily for three consecutive days after the intravitreal injections.\u003c/p\u003e \u003cp\u003eThe animals were observed at least twice daily during the study period, and any abnormal findings were recorded. Once per week, they were taken out of their cages and clinical signs of toxicity were searched, including changes on the body surface and visible mucosa, neurological and behavioral activities, respiratory parameters, and gastrointestinal reactions. Body weight was measured once per week. In addition, the animals were systemically and ophthalmologically examined at baseline, in regular intervals during the study period, and at four weeks after the last injection at study end (Day 86), when the animals were sacrificed. The ophthalmological examinations were carried out under general anesthesia induced by an intramuscular injection of Zoletil\u0026reg; 50 (4\u0026ndash;12 mg/kg, 50 mg/mL). The ophthalmological examination included measurement of intraocular pressure (IOP), dark-adapted electroretinography (ERG), optical coherence tomography (OCT) of the fundus, and fluorescein angiography as well as color fundus photography. If several examinations were performed on the same day, the sequence of examinations was: animal restrain\u0026rarr; general ocular examination (prior to receive the mydriatic agent)\u0026rarr; dark adapted ERG\u0026rarr;light-adapted ERG\u0026rarr; ocular tonometry\u0026rarr; general ocular examination (in medical mydriasis)\u0026rarr; OCT \u0026rarr;ocular photography \u0026rarr; fluorescein angiography. The general ophthalmological examinations (including inspection of the external eye, examination of the cornea and of the anterior and posterior ocular segment) were conducted at baseline, at Days 1, 29 and 57 before the injections were carried out, at one day after each injection (i.e., Days 2, 30, 58), at three days after each injection (i.e., Days 4, 32, 60), at seven days after the injections (i.e., Days 8, 36, 64), and at the end of the study period. Tonometry was performed at baseline, prior to each injection (Days 1, 29, 57), about 15 min after each injection, two days after each injection (Days 3, 31, 59), and at the end of the study. Dark-adapted and light-adapted ERG and fundus fluorescein angiography were conducted at baseline, at two days after the last injection (Day 59) and at study end. For the purpose of the ERG examination, the animals were placed in a dark room for at least 30 minutes for dark adaptation. After taking the dark-adapted ERG, the animals were placed in a light room for at least 10 minutes to perform the light-adapted ERG. For the fluorescein angiography, the animals received an intravenous fluorescein sodium injection in a dose of 8\u0026ndash;10 mg/kg body weight (concentration: 100 mg fluorescein/mL).\u003c/p\u003e \u003cp\u003eBlood samples were collected from the subcutaneous hindlimb vein at baseline, and at 1, 2, 6, 24, 48, 72, 120, 168, 240, 336, 672 hours after the intravitreal injections on Days 1 and 57. Aqueous humor and vitreous humor (volume: 0.1\u0026ndash;0.2 mL) were sampled from both eyes at study end. The samples were immediately put into an ice-filled container and transferred to a refrigerator with a temperature of -60\u0026ordm;C. The concentrations of panitumumab in the plasma, aqueous humor and vitreous body samples were analyzed using the validated ELISA method. The lower limit of quantification of the method were 1.00 ng/mL. Toxicokinetic parameters were calculated using the non-compartmental analysis (NCA) method in WinNonlin (version number: 8.0.0.3176).\u003c/p\u003e \u003cp\u003eAt study end, the animals were sacrificed by an intramuscular injection of using Zoletil 50 (12 mg/kg, 50 mg/mL) and femoral artery exsanguinations. After death, the corpses were macroscopically examined and an autopsy was performed. Samples tissues were fixated in a solution of 10% neutral buffered formalin for histopathological evaluation. The eyes were enucleated and fixated in Davidson\u0026rsquo;s fixative. The tissues were processed by routine histological methods including embedding in paraffin, sectioning, mounting on slides and staining with hematoxylin and eosin (H\u0026amp;E). TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining was carried out in two slides of each eye to compare the density of apoptotic cells between the left eyes and rights eyes.\u003c/p\u003e \u003cp\u003ePanitumumab is a fully human monoclonal antibody specific to EGF receptor.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The dose of panitumumab used in this study was based on the results of previous studies on guinea pigs and on the design of an eventual future clinical study of intravitreally applied panitumumab to prevent further axial elongation in highly myopic adult patients with progressive myopic macular degeneration.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In the latter study, the maximal dose of intravitreally injected panitumumab is 1.8 mg.\u003csup\u003e26\u003c/sup\u003e Taking into account the volume of 10.306 mL of a highly myopic human eye with an axial length of 27 mm and the volume of 2.953 mL of a non-human primate eye with diameter of 17.8 mm, the panitumumab dose for the non-human primate eye corresponding to the dose of 1.8 mg panitumumab in the highly myopic human eye at the same panitumumab concentration would be (2.953 mL / 10.306 mL) * 1.8 mg or 0.52 mg panitumumab in the non-human primate eye. The dose of 0.78 mg panitumumab used in our study would correspond to a dose of (10.306 mL / 2.953 mL) * 0.78 mg or 2.72 mg panitumumab in the human myopic eye with an axial length of 27 mm. In a concentration of 10 mg panitumumab / mL as taken out of the Vectibix\u0026reg; bottle and diluted as recommended by the manufacturer, the dose of 0.78 mg panitumumab corresponded to a volume of 78 \u0026micro;L panitumumab. A reason not to use a higher dose than 0.78 mg panitumumab in the study was, that an intravitreally injected volume higher than 0.78 \u0026micro;L might have led to an IOP-rise related tissue damage in the retina and optic nerve.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDuring the study period, none of the animals died. No test article-related abnormal findings were noted in clinical observations, body weight data, food consumption, hematology, blood coagulation, clinical chemistry or urinalysis in the animals throughout the study.\u003c/p\u003e \u003cp\u003eThe mean eye diameters as directly measured post mortem in the 5 animals of the second study part were 18.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 mm, 17.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 mm and 17.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 in the horizontal, vertical and sagittal direction, respectively. The mean eye diameter of all directions was 17.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 mm. Assuming a mostly spherical eye shape, the mean eye volume was 2965\u0026thinsp;\u0026plusmn;\u0026thinsp;309 mm\u003csup\u003e3\u003c/sup\u003e or 2.965\u0026thinsp;\u0026plusmn;\u0026thinsp;0.309 mL. The intraocular concentration of panitumumab was 0.78 mg / 2.965 mL or 0.26 mg / mL of intraocular volume.\u003c/p\u003e \u003cp\u003eUpon slit lamp-based biomicroscopy, minor retrocorneal precipitates were noted in animal #2370454 and animal #2370455 at Day 32, with complete resolution at Day 85. Gray-white dot-like particles of equal size in the anterior chamber and on the lens surface with a minor to moderate severity were noted after the second and third injection in animal #2370454; the particles had disappeared on Day 85. In a similar manner, gray-white dot-like particles of equal size were detected in the anterior vitreous at Day 36 in animal #2370454. The particles could still be seen until Day 85. The severity was minimal to slight.\u003c/p\u003e \u003cp\u003eOn Day 59 (two days after the third intravitreal injection), the fundus images of two animals of the test article group were slightly blurred, showing dilated retinal veins, slightly unsharp optic disc borders in fluorescein angiography, dot-like hyperreflective signals in the posterior vitreous upon OCT examination, and some unevenness of the retinal surface on OCT images. These eyes showed some retrocorneal precipitates, particles in the anterior chamber and anterior vitreous, and precipitates on the surface of the anterior lens capsule. Most of these changes had resolved at the end of the study, except for the hyperreflective bodies in the posterior vitreous (as assessed by OCT) and except for the precipitates on the anterior lens surface. In particular, fundus photography and fluorescein angiography and OCT imaging of the retina were unremarkable at study end.\u003c/p\u003e \u003cp\u003eThe IOP in animals receiving the test article was significantly lower than that of the animals of the negative control at Day 59.\u003c/p\u003e \u003cp\u003eIn the animal of the first study part, with panitumumab administrations on Days 1, 29, 50, 78 and 106, respectively, the drug accumulation factors on Day 50 and Day 106 were 0.871 and 0.905, respectively, indicating no significant drug accumulation in the serum after repeated administration. At Day 135, 28 days of the last injection, the drug concentration in vitreous humor and aqueous humor of the right eye of the animal were below the lower limit of quantification of the method, and the drug concentrations in the vitreous humor and aqueous humor of the left eye were 8.64 ng/mL and 1.59 ng/mL, respectively.\u003c/p\u003e \u003cp\u003eIn the animals of the second part of the study, the drug after its first intravitreal application reached the peak concentration at 24 h in the serum, with the C\u003csub\u003emax\u003c/sub\u003e ranging from 18.3 to 946 ng/mL, and the serum exposure ranged from 2120 to 37300 h*ng/mL. At the time of the third injection (D57), the drug concentration in the serum of one animal (#2370454) was lower than the lower limit of quantification (1.00 ng/mL) at each time point, and the drug reached the peak at 24 h and 1 h in the serum of the other two animals, respectively, with the C\u003csub\u003emax\u003c/sub\u003e being 87.2 ng/mL and 28.2 ng/mL, and the serum exposure being 5570 h*ng/mL and 448 h*ng/mL, respectively.\u003c/p\u003e \u003cp\u003eBefore necropsy on D86, the drug concentrations of Vectibix\u0026reg; in the aqueous humor ranged from 12.8 ng/mL to 65.0 ng/mL, and the drug concentrations in the vitreous humor ranged from 1.74 ng/mL to 531 ng/mL. The accumulation factor (AF, AUC\u003csub\u003elast, D57\u003c/sub\u003e/AUC\u003csub\u003elast, D1\u003c/sub\u003e) in the two monkeys with detectable panitumumab in the serum (#2370453, #2370455) were 0.891 and 0.012, respectively. The results showed that no significant drug accumulation was observed in the serum of the animals after repeated administration.\u003c/p\u003e \u003cp\u003eNo test article-related abnormal findings were noted in organ weights, during the macroscopic observation and in the histologic examination of the animals. No significant apoptosis was histologically detected on the histological slides with TUNEL staining. Signs of phototoxicity on the external eye regions or general body surface, and signs of phototoxicity in the eye, such as a bleaching of the retinal pigment epithelium, retinal edema or retinal thinning in the foveal region, decreased photoreceptor density in the fovea or in other regions of the fundus, or changes in the retinal pigment epithelium layer in the fovea or extrafoveal regions, were not detected, neither on the fundus photographs, the OCT images, or histologically. The ERG did not reveal significant differences between the study eyes and control eyes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this experimental study, conducted under good laboratory practice (GLP) conditions, three 4-weekly repeated intravitreal applications of panitumumab (Vectibix\u0026reg;) in 5 animals or five 4-weekly repeated intravitreal applications of panitumumab in one animal, with a recovery period of 4 weeks after the last injection, were not associated with any systemic side effects. Two monkeys showed signs of a mild intraocular inflammation after the third intravitreal injection. At study end, these inflammatory signs had dissolved except for minor retrocorneal and epilental precipitates and hyperreflective bodies in the posterior vitreous cavity. The findings suggest that the intravitreal injection of Vectibix\u0026reg; had been relatively well tolerated by the animals without any tissue damage.\u003c/p\u003e \u003cp\u003eThese observations are in line with the results of previous experimental investigations in which antibodies to EGF and the EGF receptor, such as cetuximab and panitumumab, were repeatedly intravitreally injected into guinea pigs and rabbits.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The results also agree with the observations made in a clinical study in which elderly highly myopic patients with myopic macular degeneration repeatedly received intravitreal injections of Vectibix\u0026reg; in doses of 0.6 mg, 1.2 mg and 1.8 mg.\u003csup\u003e26\u003c/sup\u003e In the latter clinical study, 11 patients with a mean age of 66.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3 years received intravitreal injections of panitumumab in doses of 0.6 mg (4 eyes; 1x1 injection, 3x2 injections), 1.2mg (4 eyes; 1x1 injection, 2x2 injections, 1x3 injections) and 1.8 mg (3 eyes; 1x1 injection, 2x2 injections), respectively. Treatment-related systemic adverse events or intraocular inflammatory reactions were not detected in any eye. Correspondingly, best corrected visual acuity (1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 logMAR (logarithm of the minimal angle of resolution) versus 1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 logMAR; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08) and intraocular pressure (13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mm Hg versus 14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mm Hg; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.20) remained unchanged during the study period. In a similar manner, those 9 patients with a follow-up of more than 3 months (mean: 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 months) did not show a significant change in axial length (30.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 mm versus 30.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 mm; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.56).\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAxial myopia can be regarded as the result of an overshooting of the physiological process of emmetropization. The process of emmetropization describes the physiological axial elongation of the eye by which the marked axial hyperopia present at birth is transformed into emmetropia in adulthood. According to recent clinical studies, axial elongation usually ceases in the third decade of life in about two-thirds of moderately myopic individuals, while one-third of such individuals can show continuous axial elongation in later life.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e It may hold true in particular for highly myopic patients with myopic macular degeneration or high myopia-associated optic neuropathies.\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Continuous axial elongation was a main risk factor for the progression of myopic macular degeneration in clinical longitudinal studies.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e It may be the only modifiable factor to prevent development and progression of myopic macular degeneration. Experimental studies suggested that axial elongation occurs through a growth and enlargement of Bruch\u0026acute;s membrane (BM) in the fundus periphery.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Arguments in favor of that hypothesis include histomorphometric and clinical findings of an axial elongation-associated thinning of the subfoveal choroid, a decrease in the density of photoreceptors and retinal pigment epithelium (RPE) cells and in retinal thickness in fundus midperiphery (while all three parameters are independent of axial length in the macular region), a presumed shift of BM opening of the optic nerve head canal into the macular direction (explaining the development of parapapillary gamma zone in the temporal parapapillary region), a gamma zone-associated elongation of the disc-fovea distance (while the vertical distance between the temporal vascular arcades is independent of axial elongation), and an axial elongation-related decrease in angle kappa between the temporal vascular arcade and the optic disc as the angle vertex.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e In the same manner, BM thickness was independent of, and its volume increased with axial length.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Supporting the hypothesis were also results of biomechanical investigations in which the elastic modulus of BM was comparable or higher than that of the sclera for an intraocular pressure of approximately 15 mm Hg.\u003csup\u003e33\u003c/sup\u003e The location of the midperiphery as the site of BM enlargement fits with results of experimental studies and clinical observations that the afferent, sensory part of the feedback mechanism governing the process of axial elongation is located in the mid-periphery of the fundus.\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEGF belongs to the list of molecules which have been discussed to play a role in the process of axial elongation. These molecules include dopamine, atropine, TGF-β, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and amphiregulin and other epidermal growth factor (EGF) family members, to name a few.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Intravitreal application of antibodies to EGF, EGF family members (such as amphiregulin, neuregulin-1, betacellulin, epigen und epiregulin) and the EGF receptor were associated with a decrease in axial elongation, while intravitreal applications of EGF family members were related with an increase in axial elongation.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e These studies were conducted in guinea pigs and non-human primates. The results of the study presented herein fit into the series of the previous investigations in that EGF may play a role in axial elongation, that a blockade of the EGF pathway may perhaps be a potential therapeutic option to reduce further axial elongation of highly myopic eyes in adult patients with myopic macular degeneration, and that repeatedly intravitreally applied panitumumab as an EGF receptor blocker may clinically be well tolerated.\u003c/p\u003e \u003cp\u003eVarious limitations have to be taken into account. Firstly, the number of animals included into the study was small so that the finding can only be a hint, but definitely not a proof, for safety of intravitreally applied panitumumab. Clinical safety may be assessed in large clinical trials. Secondly, two monkeys showed a temporary, slight intraocular inflammation after the third intravitreal application of panitumumab. At study end, the inflammation had completely subsided. While the reasons for the inflammation have remained unclear, a potential cause might have been the difference between humans and non-human primates, taking into account that panitumumab is a fully humanized antibody.\u003c/p\u003e \u003cp\u003eIn conclusion, under the conditions of this study, 0.78 mg Vectibix\u0026reg; was repeatedly intravitreally injected into Cynomolgus monkey eyes 3 times (5 animals) or five times (one animal) in 4-weeks intervals, with a following 4-week recovery period. A slight, temporary and reversible ocular inflammation with retrocorneal precipitates, particles in the anterior chamber and vitreous, and precipitates on the lens surface, as visualized by OCT, fundus photography and fluorescein angiography, and a reduction of IOP could be observed in some eyes after the third intravitreal injection, with resolution at study end. No test article-related systemic toxicity was observed. The observations may support the clinical development of intravitreal application of panitumumab in highly myopic patients with ongoing axial elongation or with progressive myopic macular degeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding: Research Development Fund of Beijing Municipal Health Commission (2019-4); National Natural Science Foundation of China (82271086). The funder of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript.\u003c/p\u003e\n\u003cp\u003eConflicts of interest/Competing interests: Songhomitra Panda-Jonas, Jost B. Jonas: European patent EP 3 271 392, JP 2021-119187, and US 12,024,557: \u0026bdquo; Agents for use in the therapeutic or prophylactic treatment of myopia or hyperopia; European patent application 23196899.1 \u0026bdquo;EGFR Antagonists for the treatment of diseases involving unwanted migration, proliferation, and metaplasia of retinal pigment epithelium (RPE) cells. All other authors: None.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAvailability of data and material: All data are available upon reasonable request from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: Study design: YXW, SPJ, LD, JBJ; Funding: WYX; Conducting the study: WYX, XS, WX, LD, JBJ; Analysis of data: WYX, FGH, MC, XS, WX, SPJ, LD, JBJ; Statistical analysis: WYX, FGH, MC, XS, WX, SPJ, LD, JBJ; Writing the first manuscrt draft: WYX, XS, WX, SPJ, JBJ; Revision and final approval of the manuscript: WYX, FGH, MC, XS, WX, SPJ, LD, JBJ.\u003c/p\u003e\n\u003cp\u003eEthics approval: Animal care was compliant with the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u0026nbsp;\u003c/em\u003e(8\u003csup\u003eth\u003c/sup\u003e Edition, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, D.C., 2011) and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198). The study was conducted at JOINN Laboratories (Suzhou, China) in compliance with good laboratory practice (GLP) regulations. \u0026nbsp;The study was reviewed and approved by the Institutional Animal Care and Use Committees at JOINN Laboratories to ensure that the study was carried out in an ethical manner.\u003c/p\u003e\n\u003cp\u003eConsent to participate: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSeko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-beta 2 in experimental myopia in chicks. Invest Ophthalmol Vis Sci. 1995;36(6):1183\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChua WH, Balakrishnan V, Chan YH, et al. Atropine for the treatment of childhood myopia. Ophthalmology. 2006;113(12):2285\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Q, Liu Q, Ma P, Zhong X, Wu J, Ge J. 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Prog Retin Eye Res. 2023;96:101156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang Y, Yokoi T, Nagaoka N, et al. Progression of myopic maculopathy during 18-year follow-up. Ophthalmology. 2018;125(6):863\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee MW, Lee SE, Lim HB, Kim JY. Longitudinal changes in axial length in high myopia: a 4-year prospective study. Br J Ophthalmol. 2020;104(5):600\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu R, Xie S, Igarashi-Yokoi T, et al. Continued increase of axial length and its risk factors in adults with high myopia. JAMA Ophthalmol. 2021;139(10):1096\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDouillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013;369(11):1023\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBikbov MM, Kazakbaeva GM, Holz FG, et al. Intravitreal panitumumab and myopic macular degeneration. Br J Ophthalmol. 2024;108(6):859\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBikbov MM, Khalimov TA, Cerrada-Gimenez M, Ragauskas S, Kalesnykas G, Jonas JB. Compatibility of intravitreally applied epidermal growth factor and amphiregulin. Int Ophthalmol. 2021;41(6):2053\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi HY, Dong L, Shi XH, et al. Intraocular cetuximab: Safety and effect on axial elongation in young Guinea pigs with lens-induced myopization. Exp Eye Res. 2024;238:109715.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SS, Lingham G, Sanfilippo PG, et al. Incidence and progression of myopia in early adulthood. 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Invest Ophthalmol Vis Sci. 2011;52(13):9362\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerntsen DA, Barr CD, Mutti DO, Zadnik K. Peripheral defocus and myopia progression in myopic children randomly assigned to wear single vision and progressive addition lenses. Invest Ophthalmol Vis Sci. 2013;54(8):5761\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWildsoet CF, Chia A, Cho P, et al. IMI - Interventions Myopia Institute: Interventions for controlling myopia onset and progression report. Invest Ophthalmol Vis Sci. 2019;60(3):M106\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang R, Dong L, Wu H, et al. mTORC1 signaling and negative lens-induced axial elongation. Invest Ophthalmol Vis Sci. 2023;64(10):24.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intravitreal panitumumab, Epidermal growth factor, Panitumumab, High myopia, Myopic macular degeneration","lastPublishedDoi":"10.21203/rs.3.rs-8501876/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8501876/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEpidermal growth factor (EGF) has been suggested to play a role in myopic axial elongation, and EGF receptor blockade may be of potential therapeutic benefit. Here we examined the ocular and systemic toxicity of panitumumab, a clinically used EGF receptor blocker in oncology, when repeatedly administered intravitreally in non-human primates.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe experimental study included six non-human cynomolgus primates (3 males) which underwent five (n\u0026thinsp;=\u0026thinsp;1 animal) or three (n\u0026thinsp;=\u0026thinsp;5 animals) 4-weekly intravitreal injections of panitumumab (dose: 0.78 mg (78\u0026micro;L)) or of phosphate buffered solution (PBS) (78\u0026micro;L).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe study group with panitumumab injections consisted of 7 eyes and the control group with PBS injections of 5 eyes. Two animals of the study group developed on Day 59 (two days after the third injection) signs of a slight intraocular inflammation (cells in anterior chamber and vitreous) and reduction of intraocular pressure, with most of the signs having resolved at study end (Day 86). Panitumumab reached the serum peak concentration at 24h after the first dose (C\u003csub\u003emax\u003c/sub\u003e 18.3 to 946ng/mL; serum exposure 2120 to 37300 h*ng/mL). Four weeks after the third injection (Day 86), panitumumab concentrations in aqueous humor ranged from 12.8 ng/mL to 65.0 ng/mL, and in the vitreous from 1.74 ng/mL to 531 ng/mL, with a panitumumab accumulation factor between 0.891 and 0.012. TUNEL staining did not reveal pathological results.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eExcept for mild and reversible intraocular inflammation in some eyes, repeated intravitreal application of 0.78mg panitumumab did not result in ophthalmological or systemic adverse effects in non-human primates.\u003c/p\u003e","manuscriptTitle":"Safety and Pharmacokinetics of Intravitreally Repeatedly Injected Panitumumab in Non-Human Primates - A Study Performed Under Good Laboratory Practice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 14:33:26","doi":"10.21203/rs.3.rs-8501876/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-06T12:51:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-05T09:34:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40921351876216591623072779937551299024","date":"2026-04-04T05:16:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38729545742319180066996137726926975524","date":"2026-04-03T19:49:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271325832305611817908115149646926913740","date":"2026-04-01T13:44:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141571724179155781893017398113441915732","date":"2026-04-01T11:05:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201194449167769854053001223590560907746","date":"2026-04-01T06:25:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36451869499738941297033671581823985773","date":"2026-04-01T03:38:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53734187157781274501353393868662699829","date":"2026-03-31T12:07:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T12:28:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12747892207232678774278180673615915614","date":"2026-01-28T15:49:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194997135831436457369453163735554212002","date":"2026-01-26T04:57:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-13T05:11:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-09T07:19:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-08T12:49:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-08T12:43:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ophthalmology","date":"2026-01-02T15:11:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c48bd627-fefe-4ae5-a751-022f95ab7f75","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T11:25:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 14:33:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8501876","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8501876","identity":"rs-8501876","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}