Chester Clinic

 

Unbekannt

Graefe's Arch Clin Exp Ophthalmol(2004) 242:582–586 Pharmacokinetics and safety Hubert KalbacherNils Alteheld of intravitreally delivered etanercept Kan KoizumiTim U. KrohneAntonia M. Joussen Received: 8 September 2003 Abstract Background: The anti-in- choroid. Conclusions: Intravitreally Revised: 11 November 2003 flammatory drug etanercept may be delivered etanercept is safe and re- Accepted: 16 February 2004 an effective therapeutic agent in dia- sults in high concentrations in the Published online: 17 March 2004 betic retinopathy. In order to further retina and choroid over a long period
Springer-Verlag 2004 evaluate its potential, the pharmaco- S. Fauser ()) · N. Alteheld · K. Koizumi · kinetics and safety of this drug after T. U. Krohne · A. M. Joussen intravitreal delivery were investigat- Abteilung fr Netzhaut- ed. Methods: After intravitreal ad- und Glaskrperchirurgie des Zentrums ministration of etanercept in rabbits, fr Augenheilkunde und Zentrumfr Molekulare Medizin (ZMMK), clinical examination, electroretinog- Universitt zu Kln, raphy (ERG), visually evoked poten- tials (VEP) and histology were eval- 50931 Cologne, Germany uated. The pharmacokinetics and distribution of etanercept were ana- Tel.: +49-221-4787719 lyzed using fluorescence-coupled Fax: +49-221-4787930 protein at 0, 2, 4, and 8 weeks after injection in vitreous, retina, and choroid. Results: No adverse effects and signs of toxicity were found.
Etanercept showed peak concentra- Universitt Tbingen, tions after 4 weeks in the retina and Tbingen, Germany and binds to a2-integrins (including CD18) expressed onleukocytes [3, 11].
Increased leukocyte adhesion to the vascular endothelium Various mediators contribute to the up-regulation of is one of the earliest events of inflammation and neovas- endothelial cell and leukocyte adhesion molecules. Tumor cularization. Besides the well-known inflammatory dis- necrosis factor a (TNFa) is a proinflammatory cytokine eases such as uveitis in various forms, diseases such as that has been implicated in this process. TNFa expression diabetic retinopathy also show signs of inflammation. In is upregulated in the extracellular matrix, endothelium, diabetic retinopathy increased leukocyte adhesion to the and vessel walls of fibrovascular tissue of eyes with vascular endothelium has been demonstrated in multiple proliferative diabetic retinopathy, and TNFa protein settings [3, 5, 13, 16]. The altered binding results in early levels are elevated in the vitreous from eyes with this blood–retina barrier breakdown, capillary nonperfusion, condition [7, 9, 10, 17]. The administration of neutralizing and endothelial cell injury and death. The adhesion is antibodies against ICAM-1 or CD18 in a rat model of mediated in part by intercellular adhesion molecule-1 diabetic retinopathy markedly reduces leukocyte adhesion (ICAM-1) [12], which is expressed on endothelial cells and, as a consequence, blood–retina barrier breakdownand endothelial injury [5]. Administration of a soluble TNF-receptor–Fc fusion protein (etanercept) has a similar added to the biological samples, and they were vortexed and effect, causally linking TNFa to leukocyte adhesion.
centrifuged at 10,000 g. Next, 20 l of each sample was analyzedon a Pharmacia 75HR 5/20 high-performance gel filtration column.
Additionally, TNFa might also have a direct effect on This column operated at a flow rate of 0.4 ml/min using PBS pH retinal cell apoptosis and thus reduce endothelial cell 7.2. The effluent was passed through a Merck ultraviolet detector and a fluorescence spectrophotometer (490/520 nm) set up in Etanercept is a soluble fusion protein consisting of the series. The signals were recorded on a D2500 integrator (Merck/ extracellular ligand-binding portion of human TNF re- ceptor (p75) linked to an Fc portion of human IgG1. Itcontains 934 amino acids and has an apparent molecular Administration of etanercept weight of 150 kDa. Its main therapeutic indication is thetreatment of rheumatoid and psoriatic arthritis. Ongoing Soluble p75 TNF-a receptor–Fc fusion protein (etanercept; Enbrel,Wyeth Pharmaceuticals, Mnster, Germany) was reconstituted with clinical studies are testing the effect of TNFa inhibitors in sterile water according to the manufacturer's instructions. Animals systemic treatment for various diseases such as uveitis, received either etanercept or solvent alone. The drug was admin- Behet's disease, and Wegener's granulomatosis.
istered intravitreally in a volume of 0.1 ml in sterile BSS. Injections Due to the immunosuppressive action and reported were performed at 1.5 mm from the limbus, between ora serrata andciliary body. Doses were calculated according to the previous systemic side effects of etanercept, local delivery appears experience with systemic treatment in a rat model and based on the favorable. Concerns have been expressed regarding the recommendation of the manufacturer for treatment of rheumatoid risk of lymphomas and congestive heart failure [2]. For arthritis in humans (Amgen and Wyeth Pharmaceuticals).
the treatment of diabetic macular edema, substances suchas triamcinolone or VEGF antagonists are being investi- gated in clinical trials. However, there is evidencesuggesting that etanercept may be also a potent and even Electroretinograms (ERG) and visually evoked potentials (VEP) more specific therapeutic agent for patients with diabetic were elicited in rabbits under general anesthesia using single bright retinopathy. In the current study, we therefore investigat- xenon flash light stimulation. The ERG was recorded in both eyeswith corneal electrodes (jet electrodes, Universo Plastique, Switzer- ed the pharmacokinetics and safety of local administra- land). The reference electrode (Ag–AgCl, skin) was placed on the tion of the TNFa fusion protein etanercept.
forehead. The VEP was recorded with an occipital skin electrodeand a reference electrode on the forehead (both Ag–AgCl). ERGand VEP were amplified using Grass P122 amplifiers. Averagewaveforms were recorded with a digital storage oscilloscope. The Material and methods recordings of the untreated eye served as control.
Male pigmented rabbits (Charles River, France) weighing approx-imately 2,000 g were used in all experiments. All protocols abided Eyes were enucleated and fixed in 4% formaldehyde for 24 h before by the ARVO (Association for Research in Vision and Ophthal- paraffin embedding and sectioning. Serial sections were stained mology) statement on the "Use of Animals in Ophthalmology and with hematoxylin and eosin and evaluated under a light micro- Vision Research" and were approved by the Animal Care and Use scope. For comparison, defined areas of each retina were captured Committee of the Regierungsprsidium Kln. The animals were fed using a CD-330 charge-coupled device (CCD) camera (Dage-MIT, standard laboratory food and allowed free access to water in an air- Improvision, Heidelberg, Germany) attached to a Zeiss microscope conditioned room with a 12-h light–dark cycle. The animals were (Zeiss, Oberkochem, Germany). The images were captured on an anesthetized with ketamine (40 mg/kg; Ketalar, Parke-Davis, Apple G4 Computer (Apple, Cupertino, CA) and analyzed using Morris Plains, NJ) and xylazine (4 mg/kg; Rompun, Bayer, Openlab software (Improvision). The number of cell layers was Leverkusen, Germany) prior to all experimental manipulations.
determined in a masked fashion.
Fluorescence labeling of etanercept A quantity of 0.5 mg of etanercept in water was ultrafiltrated with a20-kDa membrane by using phosphate-buffered saline (PBS) pH Clinical evaluation 7.2, then 0.1 M borate buffer pH 9.5 to a final volume of 0.5 ml.
The protein concentration was 0.48 mg/ml. Then 0.15 mg fluores-cein isothiocyanate (FITC) on Celite was added to the solution After intravitreal injection of 100 g etanercept, no under stirring in the dark for 1 h. After centrifugation, the solution clinical signs of toxicity were observed in all animals was applied to a Sephadex PD10 column and eluted with PBS. The throughout the follow-up time for up to 2 months. Cornea yellow high-molecular fractions eluting between 3.5 and 5 ml were and lens remained clear. Using indirect ophthalmoscopy, combined and used for the in vivo experiments.
the retina appeared clinically normal. There was novitreous haze or opacities within the vitreous cavity.
Detection of etanercept fragments in biological samples Eyes were enucleated and vitreous, retina, and choroid weredissected under a microscope. A quantity of 50 l of PBS was

Fig. 1 Section of a rabbit retina 8 weeks after injection of 100 getanercept into the vitreous. No signs of toxicity are found. Theretina shows normal tissue structures Fig. 2 The b-wave amplitudes in ERG recordings. Both treated anduntreated eyes were measured 4 and 8 weeks after injection of100 g etanercept The histology of both treated and control eyes afterintravitreal administration of 100 g etanercept were notdistinguishable and showed normal anatomy (Fig. 1). Nosigns of toxicity were found. Quantification of retinallayers demonstrated no difference in either group.
A decrease in ERG b-wave amplitude was observed in Fig. 3a–c Etanercept in various tissues at 0, 2, 4, and 8 weeks after both treated and control eyes 4 and 8 weeks after injection injection (amount of protein in relative fluorescence units): a invitreous; b in retina; c in choroid of 100 g etanercept. Comparison of treated and control The vitreous showed no opacities or bands formation.
eyes showed no reduction of b-wave amplitude as an Such signs of toxicity can be found, for example, with effect of treatment (Fig. 2). VEP could be elicited before intravitreal amphotericin B injections. With increasing and after treatment.
doses (10–50 g), retinal ganglion cell loss and focalnecrosis of the nerve fiber layer became also apparent [4].
Daunomycin used for inhibiting proliferative vitreo- retinopathy has been reported to be toxic at doses of5 g in the vitreous [6].
Etanercept was measured in vitreous, retina, and choroid Fluorescence-labeled etanercept was injected in two at 0, 2, 4, and 8 weeks after injection of 5 g and 50 g doses (5 g and 50 g) intravitreally and the amount of fluorescein-coupled etanercept into the vitreous. In the the protein was determined in vitreous, retina, and choroid vitreous, a peak value was measured directly after at 0, 2, 4, and 8 weeks after injection. By using gel injection which gradually declined to the 8th week after filtration, only the amount of intact etanercept was injection. In both the retina and the choroid, the highest determined. Therefore, a decrease in biological activity values were found 4 weeks after injection. But even 8 by decay of etanercept can be ruled out. In the vitreous, weeks after injection, etanercept was still found in retina the amount of etanercept gradually declined up to the 8th and choroid (Fig. 3).
week after injection. In both the retina and the choroid, aslow accumulation was found with a peak at 4 weeks.
After 8 weeks, etanercept was still clearly detectable. The pharmacokinetics of etanercept after intravitreal deliveryensure high concentrations of the drug in the target tissues Experiments with etanercept in a rat model of diabetic over several weeks and offer the chance of a successful retinopathy demonstrated that the drug reduces leukocyte therapy. The relatively high molecular size of etanercept adhesion, blood–retina barrier breakdown and endothelial results in slower clearance than, for example, with injury [8]. Therefore, diabetic retinopathy may be a new triamcinolone [14, 15]. After injection of 400 g triam- therapeutic indication for this agent apart from its main cinolone acetonide, the half-life as determined by HPLC indication in rheumatoid arthritis. As the intravitreal was 1.6 days [15]. Dexamethasone, another glucocorti- administration would minimize systemic side effects and coid, has a half-life of only 2.5 h in physiologic saline [1, deliver the drug locally, the pharmacokinetics and safety of this route of administration were investigated.
In summary, the results show that intravitreally deliv- After intravitreal injection of high doses of etanercept ered etanercept is safe and leads to high concentrations in (100 g per eye), no signs of toxicity were found on the retina and choroid over several weeks.
evaluation of data from clinical examination, histology,ERG, and VEP. Etanercept did not induce any patholog- Acknowledgements Supported by the DFG (Jo 324 /4-1, Jo 324/6- ical changes after an observation time of up to 8 weeks.
1), the ZMMK Kln (TV76), the Ernst und Berta Grimmke Stiftungand the Gertrud und Werner Mller Stiftung.
Function and structure were the same as in control eyes.
1. Agabeyoglu IT, Wagner JG, Kay DR 4. Cannon JP, Fiscella R, Pattharachayakul 7. Limb GA, Chignell AH, Green W, (1980) A sensitive high-pressure liquid S, Garey KW, De Alba F, Piscitelli S, LeRoy F, Dumonde DC (1996) Distri- chromatographic method for the deter- Edward DP, Danziger LH (2003) Com- bution of TNF alpha and its reactive mination of prednisone, prednisolone parative toxicity and concentrations of vascular adhesion molecules in fibro- and hydrocortisone in plasma. Res intravitreal amphotericin B formulations vascular membranes of proliferative Commun Chem Pathol Pharmacol in a rabbit model. Invest Ophthalmol diabetic retinopathy. Br J Ophthalmol Vis Sci 44:2112–2117 2. Antoni C, Braun J (2002) Side effects of 5. Joussen AM, Murata T, Tsujikawa A, 8. Koizumi K, Poulaki V, Doehmen S, anti-TNF therapy: current knowledge.
Kirchhof B, Bursell SE, Adamis AP Welsandt G, Radetzky S, Lappas A, Clin Exp Rheumatol 20:152–157 (2001) Leukocyte-mediated endothelial Kociok N, Kirchhof B, Joussen AM.
3. Barouch FC, Miyamoto K, Allport JR, cell injury and death in the diabetic (2003) Contribution of TNF-alpha to Fujita K, Bursell SE, Aiello LP, retina. Am J Pathol 158:147–152 leukocyte adhesion, vascular leakage, Luscinskas FW, Adamis AP (2000) 6. Hui YN, Liang HC, Cai YS, Kirchhof and apoptotic cell death in endotoxin- Integrin-mediated neutrophil adhesion B, Heimann K (1993) Corticosteroids induced uveitis in vivo. Invest Oph- and retinal leukostasis in diabetes.
and daunomycin in the prevention of thalmol Vis Sci 44:2184–2191 Invest Ophthalmol Vis Sci 41:1153– experimental proliferative vitreo- retinopathy induced by macrophages.
Graefes Arch Clin Exp Ophthalmol231:109–114 9. Limb GA, Soomro H, Janikoun S, 12. McLeod DS, Lefer DJ, Merges C, Lutty 16. Schrder S, Palinski W, Schmid- Hollifield RD, Shilling J (1999) Evi- GA (1995) Enhanced expression of Schnbein GW (1991) Activated dence for control of tumour necrosis intracellular adhesion molecule-1 and monocytes and granulocytes, capillary factor-alpha (TNF-alpha) activity by P-selectin in the diabetic human retina nonperfusion, and neovascularization in TNF receptors in patients with prolif- and choroid. Am J Pathol 147:642–653 diabetic retinopathy. Am J Pathol erative diabetic retinopathy. Clin Exp 13. Miyamoto K, Khosrof S, Bursell SE, Immunol 115:409–414 Rohan R, Murata T, Clermont A, Aiello 17. Spranger J, Meyer-Schwickerath R, 10. Limb GA, Webster L, Soomro H, LP, Ogura Y, Adamis AP (1999) Pre- Klein M, Schatz H, Pfeiffer A (1995) Janikoun S, Shilling J (1999) Platelet vention of leukostasis and vascular [TNF-alpha level in the vitreous body.
expression of tumour necrosis factor- leakage in streptozotocin-induced dia- Increase in neovascular eye diseases alpha (TNF-alpha), TNF receptors and betic retinopathy via intercellular ad- and proliferative diabetic retinopathy].
intercellular adhesion molecule-1 hesion molecule-1 inhibition. Proc Natl Med Klin 90:134–137 (ICAM-1) in patients with proliferative Acad Sci USA 96:10836–10841 18. Tano Y, Sugita G, Abrams G, diabetic retinopathy. Clin Exp Immunol 14. Schindler RH, Chandler D, Thresher R, Machemer R (1980) Inhibition of in- Machemer R (1982) The clearance of traocular proliferations with intravitreal 11. Lu M, Perez VL, Ma N, Miyamoto K, intravitreal triamcinolone acetonide.
corticosteroids. Am J Ophthalmol Peng HB, Liao JK, Adamis AP (1999) Am J Ophthalmol 93:415–417 VEGF increases retinal vascular ICAM- 15. Scholes GN, O'Brien WJ, Abrams GW, 1 expression in vivo. Invest Ophthalmol Kubicek MF (1985) Clearance of tri- Vis Sci 40:1808–1812 amcinolone from vitreous. Arch Oph-thalmol 103:1567–1569

Source: http://www.kalbacher.uni-tuebingen.de/pdf/2004/hk20044.pdf