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Pegaptanib sodium TM (Macugen ) 申屾 殷月恒 郑啸 何琄 LOGO ABC of Macugen Biotechnology drug(FDA, 2004) Ophthalmologic(眼科学) treatment An angiogenesis-inhibiting antiVEGF165 aptamer(适体) first aptamer approved for use in man Bombastic drug in the market Contents 1 AMD 2 Mechanism:Aptamer 3 Drug preparation 4 Pharmacological index 5 Market and the future Part 1 Age Related Macular Degeneration (AMD) 年龄相关性黄斑变性 1.1 Symptoms 1.2 Term definitions 1.3 Pathology of Wet-AMD 1.1 Symptoms of AMD 1.1.1 If you see world like this..…. 1.2 Term definition Age related macular degeneration (AMD): AMD affects the macula(黄斑), the part of the eye that allows you to see fine detail. A disease that blurs the sharp, central vision needed for "straight-ahead" activities such as reading, sewing, and driving. wet and dry forms 1.1.2 Anatomy of the Eye macula supplied with oxygen-rich blood that nourishes the cells. 1.2.1 Age-related Macular Degeneration (AMD) "wet" or neovascular Retina of an Eye with Wet AMD "dry" or atrophic Retina of an Eye with Dry AMD 1.2.2 Neovascular (Wet) AMD accounts for 90% of the severe vision loss caused by macular degeneration macula damage occurs rapidly advanced AMD : loss of central vision can occur quickly. more severe than the dry form. 1.1.3 Progression in eyesight Early stage Later on 1.3.6 Illustration 1.3 Pathology of Wet-AMD LOGO 1.3.1 Neovascular (Wet) AMD The oxygen supply to the macula is disrupted the body responds by growing new, abnormal blood vessels (Angiogenesis). Growth of abnormal blood vessels behind the retina under the macula. very fragile leak blood and fluid raise the macula from its normal place at the back of the eye. 1.3.2 Angiogenesis(新生血管) the growth of new blood vessels. uncontrolled in neovascular AMD 1.3.4 Mediated by: 碱性成纤维细胞生长因子(bFGF) 血小板衍生生长因子( PDGF) α与β转化生长因子( TGF) 表皮生长因子( EGF) 胰岛素样生长因子( IGF) Ischemia缺血 Hypoxia缺氧 Balance 血管内皮生长因子(VEGF) –main promoter 色素上皮衍生因子( PEDF)—main inhibitor 1.3.5 Vascular endothelial growth factor (VEGF) a cytokine(细胞因子) 血管内皮生长因子 1.3.5 VEGF a secreted protein that selectively binds and activates its receptors located primarily on the surface of vascular endothelial cells. 6 isoforms VEGF165 Receptors: flt - 1 (fms- like tyrosine kinase) KDR ( kinase insert domain-containing receptor) Biological Function: induces angiogenesis increases vascular permeability Increases inflammation contribute to the progression of the neovascular (i.e., wet) form of agerelated macular degeneration (AMD) 1.3.5 VEGF---deactivated form 1.3.5 VEGF---activated form (dimer) From target to bullet From disease to remedy ------Part2 Aptamer LOGO 1.3.3 Choroidal NeoVascularisation (CNV ) 脉络膜新生血管 视网膜下新生血管( subretinal neovascularization, SRNV) 或称视网膜下 脉络膜源性新生血管( subretinal choroidal neovascularization , SRCNV) 一般是由于炎症、外伤、病变、变性等原 因, 在脉络膜微循环障碍的情况下, 玻璃膜 (Bruch 膜)发生皲裂, 从而诱发脉络膜毛细 血管向色素上皮下,进而向神经上皮下生长 (是血管内皮细胞的侵润、迁移) , 称为视网 膜下脉络膜源性新生血管, 一般都是形成新 生血管膜。 Macugen– new genesis of macular Age-Related Macular Degeneration Diabetic Macular Edema 1.3.6 新生血管形成的早期关键步骤 蛋白酶被激活 蛋白酶溶解血管基底膜和周细胞 外基质的蛋白, 使之水解 微血管内皮细胞通过血管基底膜 侵润、迁移进入临近细胞外间质 形成新生血管芽 A quick view on retina (视网膜) Part2 Mechanism-Aptamer Macugen (pegaptanib sodium) is the first drug developed by Pfizer and Eyetech for the treatment of the "wet" forms of AMD The VEGF Family 2.1 Macugen is an aptamer What aptamer is------the single-stranded or double stranded oligonucleotide which combine with protein or other small molecules 2.2 Merits of the aptamer Aptamers has the advantages of both antibodies and small molecule compounds : 1 High stability 2 Wide range of targets 3 Strong binding capacity 4 Easy preparation 5 Safe and effective 2.2.1 High stability *be heated up to 80 or 90 degrees. *frozen into dry powder(冻干粉) can be kept at room temperature for several years, *It can survive in rather harsh environments with various solvents. 2.2.2 Wide range of targets metal ions ,organic molecules, nucleic acid, peptide(缩氨酸), protein, cells, cell aggregates, subcellular, macromolecular polymers SELEX technology through reverse, appropriate body also can be screened even if we do not know the characters of the targets. 2.2.3 Strong Binding Capacity When the target goal exists, single-stranded DNA or RNA folding adapt to form hairpin(发 夹) ,pseudoknot(假结), bulge(凸环), G2quartet(G2四分体) special three-dimensional spatial structures, closely integrated with the target molecules through hydrogen bonding, hydrophobic accumulation, van der Waals force. 2.2.4 Easy preparation Making Macugen, for example, is a straightforward 15-step synthetic process, unlike an antibody, where have to build a gigantic fermentation plant. 2.2.5 Safe and effective * Macugen, for instance, binds to only one of six isoforms (isoform 165) of vascular endothelial growth factor (VEGF). * Lack of immunogenicity 2.3 Drawbacks • High manufacturing cost • Low concentration in cells • Short serum half-life Do not get into cell very well is another drawback to aptamers. The solution is TLR9, target Tolllike receptor 9 on dendritic(树突) cell. This receptor can evolved specifically to bring certain nucleic acids ( like aptamers) into intracellular compartments Short serum half-life aptamers are quickly eliminated by the kidneys. ARC-183 (an anticoagulant), deliberately designed for a short duration of action, has a half-life of about 2 minutes. Solution: the half-life can be increased by attaching polyethylene glycol (PEG) molecules to the oligonucloeotides Part3--- The development of Macugen C294H342F13N107Na28O188P28[C2H4O]n (where n is approximately 900) 3.1Structural Formula Molecular Weight : approximately 50 kilodaltons. Active Conformation VEGF165 Aptamer(secondary structure) 3.2 SELEX Systemic Evolution of Ligand by Exponential Enrichment(指数富集配基的系 统进化), Larry Gold,the University of Colorado,1990s A new combinational chemistry methodology for in vitro selection of specific aptamers 3.2 SELEX Procedure DNA or RNA library from solid-phase synthesis One round of selection and amplification Repeated in vitro selection and amplification Specific aptamer is obtained Cloning,sequencing,evaluation,modification ,etc illustration Affinity selection Library Modification SELEX Repetition Aptamer Amplification 3.3 Discovery Step1 Preliminar y selection with the SELEX method Step2 Affinity Selection of Aptamer Fragments— minimal aptamers 2’-OMe Substitution at purines Step 3 Binding Rate Constants Specificity of Aptamers VEGF Receptor Binding Inhibition Vascular Permeability Assay 2’ -F-pyrimidine RNA libraries containing 30 or 40 random nucleotides 3.3.1 Preliminary selection 12 rounds of SELEX 46 of these sequences were grouped into three major families based on conserved primary structure motifs Family 1 3 a highly conserved share a conserved sequence primary structure 5’-GAAN(3–4)UUGG-3’ ; motif ; no predicted secondary also share a structure common to all is predicted secondary evident 2 structure share a strongly conserved sequence 5’-GAAN(3– 4)UUGG-3’ ;share the ability to form a short base paired stem 3.3.2 Aptamer fragments identification Use both a biochemical approach (family 1)and predictions based on conserved secondary structure motifs(family2&3) to derive a high affinity truncated(截短) aptamer from one member of each sequence family Truncation Comparison 3.3.3 2’-OMe substitution 3.3.3.1 From experience Substitution at the 2’-OH positions of RNA oligonucleotides by 2’-OMe improves their stability against nucleases allows for more efficient chemical synthesis observed that high affinity RNA ligands generally accept a high percentage of 2’OMe purine substitutions with little or no loss of affinity for the target protein 3.3.3.2 find out the right positions? In such an affinity selected pool, positions that do not tolerate substitution are biased for 2’-OH and thus show higher sensitivity to hydrolysis. Step 1 5’radiolabeled libraries were prepared in which five or six 2’-OHpurine positions were partially 2’OMe substituted. Step2 Incubated with VEGF, collect the substituted oligonucle ot-ides bound by the protein Step 3 Selected pool and the starting unselected library were partially hydrolyzed by alkali and the products displayed on a polyacrylamide( 聚丙烯酰胺)gel Filled circles represent band intensity ratios where the position was partially 2’-OMe substituted in the library; open circles show the average band intensity ratio for all libraries in which the position was unsubstituted. Filled circles that fall well above the range for a particular position are indicative of a bias for 2’OH (against 2’-OMe) in the affinity selected pool. 3.3.4 binding constant determination t22-OMe showed the fastest rate of dissociation; t2-OMe and t44-OMe showed slightly slower rates of dissociation respectively. 3.3.5 specificity All three minimal 2’OMe-substituted aptamers bind to human VEGF165 and its mouse homologue(VEGF164) with comparatively high affinity 3.3.6VEGF Receptor Binding Inhibition Two VEGF receptors, Flt-1 (fms-like tyrosinekinase) and KDR (kinase insert domaincontaining receptor),have been identified on human vascular endothelial cells Assesse the capacity of the minimal 2’OMe-substituted aptamers to inhibit VEGF binding to each of the receptors individually Cell-associated VEGF decreased, in each case,with increasing concentration of the aptamer 3.3.7 Vascular Permeability Assay The Miles assay offers a simple and rapid means of monitoring the ability of various compounds to inhibit the activity of VEGF in vivo. Intradermal injection of VEGF in adult guinea pigs induces a rapid increase in the permeability of dermal microvessels that may be monitored by quantitating the leakage of intravascular Evans Blue dye into the skin. t44-OMe inhibited the response by 58% at 1 mM and 48% at 0.1 mM thus the most effective antagonist of VEGFinduced vascular permeability. Preincubation of VEGF with 1 or 0.1 mM of each 2’-OMe-substituted aptamer showed varying degrees of inhibition of the vascular permeability response The addition of 40-kDa PEG at the 5’-end of t44OMe resulted in a slight apparent reduction (4-fold) in binding affinity to VEGF but a marked enhancement in inhibitory activity in the Miles assay Comparison of candidate anti-VEGF aptamers In summary--step1 step2 step3 Lead compound are obtained by SELEX Lead modification from two structure &2’OMe substitution aspects:simplified Lead optimization in parallel with and in accordance to bioactivity assessment Part4--- Pharmaceutical index, Market and the Future 4.1Administration intravitreous(玻璃体内) injection 0.3 mg once every six weeks (9 injections per year) inspected visually for particulate matter and discoloration adequate anesthesia and a broadspectrum topical microbicide monitoring for intraocular pressure, endophthalmitis(眼内炎) 4.2Pharmacodynamic Properties binds with high specificity and affinity to extracellular VEGF165 ---a modified oligonucleotide that associated with the progression of wet AMD inhibiting its activity 4.3 Pharmacokinetic Properties Absorption: The rate of absorption from the eye is the rate-limiting step in the disposition in animals and is likely to be in humans. Distribution/Metabolism/Excretion: In animals, pegaptanib distributes primarily into plasma volume and is not extensively distributed to peripheral tissues after intravenous administration. Pegaptanib is metabolized by endo- and exonucleases(核酸内/外切酶). In rabbits, pegaptanib is eliminated as parent drug and metabolites primarily in the urine. 4.4.Adverse reactions: Psychiatric(精神病的)Disorders: Depression and nightmare. Nervous System Disorders: Headache. Eye Disorders Cardiac Disorders Vascular Disorders: Aortic aneurysm and hypertension Respiratory, Thoracic and Mediastinal Disorders Gastrointestinal Disorders: Dyspepsia and vomiting. Skin and Subcutaneous Tissue Disorders Injury, Poisoning and Procedural Complications 4.5 Possible Risks Intraocular injections Multiple intravitreal(玻璃体内) injections (9 injections per year) Adverse events High expenses($3300/mg) 4.7 Treatment OptionsⅠ ——Medicinal Therapy Photodynamic therapy (光动力疗法,PDT) with Visudyne (维速达尔) Pharmacological therapy with anecortave acetate (乙酸阿奈可他) Intravitreal(玻璃体内) pegaptanib sodium Intravitreal ranibizumab(兰尼单抗) Intravitreal bevacizumab(贝伐单抗) Treatment OptionsⅡ ——Surgical Therapy Thermal laser photocoagulation(热激光凝 固) Surgical excision of the neovascular tissue (新生血管组织切除) Subfoveal(视中央凹下) surgery with cell transplantation(细胞移植视中央凹下手术) 360-degree macular translocation(360度 黄斑易位) Transpupillary thermaltherapy (经瞳孔温热疗 法 ,TTT) Radiation treatment Major Therapeutic Approaches Pegaptanib (Macugen®) Photodynamic Therapy(PDT) With Visudyne Ranibizumab(Lucentis) Photodynamic Therapy(PDT) Bevacizumab(Avastin) With Visudyne Triple Therapy Ranibizumab(Lucentis) AnecortaveBevacizumab(Avastin) acetate Triple Therapy Anecortave acetate Therapeutic Drugs Proposed Benefits Possible Risks Photodynamic Therapy(PDT) With Visudyne Decreased rate of vision deterioration rather than improvement Multiple treatments, high expenses, requirements of light Ranibizumab(Lucentis) slowed progression of neovascular “wet” AMD Intravitreal injections, arterial thromboembolic(动脉血 栓栓子)events, adverse reactions,price limits Bevacizumab(Avastin) As above Intravitreal injections, no thromboembolic events Inexpensive price Triple Therapy of Limit contiguous macular Triamcinolone, PDT and damage and preserve vision Pegaptanib Sodium Price limits Anecortave acetate (Retaane) Adverse reactions slowed progression of neovascular wet AMD, posterior juxtascleral placement 4.8 Conclusions of these drugs Beyond symptomatic relief ,various approaches have disease-modifying effects, as the mechanism of action may delay the progression of disease at the cellular or organic level. However, none of them can provide an essential cure but lead to a slower rate of vision decline. Although having exhibited curative effects in various level, futher clinical trials are required for evaluating of efficacy, tolerability and safety,etc. Pegaptanib is the first approved therapy in a new class of ophthalmic preparations that targets VEGF165,thereby providing a prospective treatment for wet AMD. 4.9 Marketing development of Macugen May 3, 2004 Eyetech announces that phase Ⅱ study of Macugen showed positive visual and anatomical outcomes for diabetic macular edema. June 17, 2004 Eyetech/Pfizer file New Drug Application with FDA for Macugen. Aug. 17, 2004 New Drug Application for Macugen accepted by FDA. Dec. 17, 2004 Eyetech/Pfizer announce FDA approval of Macugen as treatment for neovascular (wet) age-related macular degeneration. From Lab to Life From Premise to Patient “It was a very important urgent medical need, a very big market and a very exciting science. It’s a terrific example of going from the laboratory bench to the bedside–from theory to therapy. It’s always important if you have a great idea to stay with and try to figure out what is the best way to test your hypothesis.” ---David R. Guyer, MD, CEO of Eyetech,Inc. Part5.Outlook and Prospect “ More and more, retina specialists rely on Macugen as the first and only VEGF inhibitor approved for the treatment of this devastating disease. Physician acceptance of Macugen continues to be widespread, rapid and strong." --- David R. Guyer References: 1. Pegaptanib for the treatment of age-related macular degeneration, Bo Zhou, Bin Wang, Experimental Eye Research 83 (2006) 615e619 2. Tezel, T.H., Bora, N.S., Kaplan, H.J., 2004. Pathogenesis of age-related macular degeneration. Trends Mol. Med. 10, 417e420. 3. Witmer, A.N., Vrensen, G.F., Van Noorden, C.J., Schlingemann, R.O., 2003. Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 22, 1e29. 4. Robinson, C.J., Stringer, S.E., 2001. The slice variants of VEGF and their receptors. J. Cell Sci. 114, 853e865. 5. Ruckman, J., Green, L.S., Beeson, J., Waugh, S., Gillette, W.L., Henninger, D.D., Claesson-Welsh, L., Janjic, N., 1998. 20-fluoropyrimidine RNA-based aptamers to the 165-amino acid form of VEGF. Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J. Biol. Chem. 273, 20556e20567. 6. Ferrara, N., 2004. VEGF: Basic science and clinical progress. Endocr. Rev. 25, 581e611. 7. Tuerk, C. & Gold, L.Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510 (1990) 8. Karl Thiel: Oligo oligarchy-the surprisingly small world of aptamers. Nature Biotechnology Volume 22, Number 6 June 2004, 649-651 9. Anthony P. Adamis et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nature Reviews Drug Discovery v. 5, no. 2, pp. 123-132 (February, 2006) 10. 沈丽君,惠延年. Macugen 的临床应用. 国际眼科纵览2006年6月第30卷第3期. 11. Lewis Gryziewicz.Regulatory aspects of drug approval for macular degeneration. Advanced Drug Delivery Reviews 57 (2005) 2092– 2098. 12. Peter E. Liggett, MD, Juner Colina, MD,Nauman A. Chaudhry, MD, David Tom, MD,and Gregory Haffner, MD. Triple therapy of intravitreal triamcinolone, photodynamic therapy, and pegaptanib sodium for choroidal neovascularization. America journal of ophthalmology December2006 VOL.142, NO. 6 13. 王雨生,严密.黄斑变性患者的希望. 中华眼底病杂志2007年1月第23卷第1期. 14. 陈有信.脉络膜新生血管治疗研究展望.中华眼底病杂志2007年1月第23卷第1期. 15. 张励,唐由之.年龄相关性黄斑变性的研究进展. 中国中医眼科杂志2005年8月第15 卷 第 3 期 . 16. Miller J W, Schmidt - Erfurth U , Sickenberg M, et al. Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration [J ] 1Arch Ophthalmol , 1999 , 117 (9) :1161 - 11731 17. Regillo C D1. Update on photodynamic [ J ] 1Curr Opin Ophthalmol , 2000 , 11 (3) : 166 – 1701. 18. Newsom R S , McA lister J C , Saeed M, et al1Transpupillary thermotherapy of subfoveal occult choroidal neovascularization [ J ] 1CurrOpin Ophthalmol , Arch Ophthalmol , 2001 , 12 (3) : 212 – 2151. 19. Coscas G1.Perifoveal laser treatment for subfoveal new vessels in age - re lated macular degeneration [J ] 1Arch Ophthalmol , 1991 , 109 (6) :1258 – 12611. 20. Kourlas H , Schiller DS. Pegaptanib sodium for the treatment of neovascular age-related macular degeneration : a review. Clin Ther ,2006 ,28 :362441. 21. Heier J S , Antoszyk AN , Pavan PR , et al. Ranibizumab for treatment of neovascular age-related macular degeneration : A phase IPII multicenter , controlled , multidose study. Ophthalmology , 2006 ,113 :6421. 22. Bressler HM . Treatment of Age-related Macular Degeneration with Photodynamic ( TAP) Study Group. Photodynamic therapy of subfoveal neovascularization in age-related macular degeneration with verteporfin: two-year results of 2 randomized clinical trials-TAP report 2. Arch Ophthalmol , 2001 ,119 :19822071. 23. Javitt JC , Zlateva P , Earnshaw SR , et al. Costeffectiveness model for age-related macular degeneration : comparing Macugen to Visudyne. World Ophthalmology Congress (Brazil) , 2006 ,Abstract . 24. Michels S , Rosenfeld PJ , Puliafilo LA , et al. Systemic bevacizumab(Avastin) therapy for neovascular age-related macular degeneration twelve-week results of an uncontrolled open-label clinical study. Ophthalmology ,2005 ,112 :1035210471 25. American medical association.Treatment for age-related macular degeneration. Medical policy. 2007.09.03. 26. Schmidt-Erfurth U, Michels S, Michels R et al. Anecortave acetate for the treatment of subfoveal choroidal neovascularization secondary to age-related macular degeneration. Eur J Ophthalmol. 2005; 15(4):482-5. 27. Augustin AJ, D'Amico DJ, Mieler WF et al. Safety of posterior juxtascleral depot administration of the angiostatic cortisene anecortave acetate for treatment of subfoveal choroidal neovascularization in patients with age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol. 2005; 243(1):9-12. LOGO 2’methoxy 2’fluoro Covalently conjugate of an oligonucleotide (28 nucleotide totally) R-group Lysine residue Two polyethylene glycol(PEG) units are covalently attached conformational stability & enhanced pharmacokinetics Incubation with the target molecules 1 2 Filtration of the complex Amplification (eg.RNA to cDNAs; PCR) 3 4 A secondary library obtained