LOGO

申屾 殷月恒 郑啸 何琄

Pegaptanib sodium (MacugenTM )Pegaptanib sodium (MacugenTM )

ABC of MacugenBiotechnology drug （ FDA, 2004 ）Ophthalmologic( 眼科学 ) treatmentAn angiogenesis-inhibiting anti-

VEGF165 aptamer( 适体 )first aptamer approved for use in manBombastic drug in the market

Contents

AMD1

Mechanism ： Aptamer2

Drug preparation3

Pharmacological index4

Market and the future5

Part 1 Age Related Macular Degeneration (AMD)

年龄相关性黄斑变性1.1 Symptoms1.2 Term definitions1.3 Pathology of Wet-AMD

1.1 Symptoms of AMD1.1.1 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

maculasupplied 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 rapidlyadvanced 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

LOGO

1.3 Pathology of Wet-AMD1.3 Pathology of Wet-AMD

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)

血管内皮生长因子 (VEGF) –main promoter

色素上皮衍生因子 ( PEDF)—main inhibitor

Balance

Ischemia 缺血Hypoxia 缺氧

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 VEGFa secreted protein that selectively

binds and activates its receptors located primarily on the surface of vascular endothelial cells.

6 isoformsVEGF165Receptors:

flt - 1 (fms- like tyrosine kinase) KDR ( kinase insert domain-containing

receptor)

Biological Function:

induces angiogenesis increases vascular permeability Increases inflammationcontribute to the progression of the

neovascular (i.e., wet) form of age-related macular degeneration (AMD)

1.3.5 VEGF---deactivated form

1.3.5 VEGF---activated form (dimer)

LOGO

From target to bulletFrom disease to remedyFrom target to bulletFrom disease to remedy

------Part2 Aptamer

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 Toll-like 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

3.1Structural Formula

Molecular Weight : approximately 50 kilodaltons.

C294H342F13N107Na28O188P28[C2H4O]n (where n is approximately 900)

VEGF165 Aptamer(secondary structure)

Active Conformation

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

3.2 SELEX Procedure

DNA or RNA library from solid-phase synthesisOne round of selection and amplificationRepeated in vitro selection and amplificationSpecific aptamer is obtainedCloning,sequencing,evaluation,modification ,etc

illustration

Affinity selection Amplification

Aptamer

SELEX

Library

Modification

Repetition

3.3 Discovery

Step1Step1 Step2Step2 Step 3Step 3

Preliminary selection with the SELEX method

Affinity Selection of Aptamer Fragments—minimal aptamers2’-OMe Substitution at purines

Binding Rate ConstantsSpecificity of AptamersVEGF Receptor Binding InhibitionVascular Permeability Assay

3.3.1Preliminary selection

12 rounds of SELEX

2’ -F-pyrimidineRNA libraries

containing 30 or 40 random

nucleotides

46 of these sequences were grouped into three major families based on conserved primary structure motifs

Family1

share a conserved sequence5’-GAAN(3–4)UUGG-3’ ;no predicted secondary structure common to all is evident

2

share a strongly conserved sequence 5’-GAAN(3–4)UUGG-3’ ;share the ability to form a short base paired stem

3

a highly conserved primary structure motif ; also share a predicted secondary structure

3.3.2 Aptamer fragments identificationUse 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

Substitution at the 2’-OH positions of RNA oligonucleotides by 2’-OMe

improves their stability against nucleasesallows 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

3.3.3.1 From experience

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 1Step 1 Step2Step2 Step 3Step 3

5’-radiolabeled

libraries were

prepared in which five or

six 2’-OH-purine

positions were

partially 2’-OMe

substituted.

Incubated with VEGF, collect

the substitut

ed oligonucleot-ides bound by

the protein

Selected pool and the starting

unselected library were

partially hydrolyzed by alkali and the

products displayed on a polyacrylamid

e( 聚丙烯酰胺 )gel

Filled circles representband 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 areindicative 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 InhibitionTwo VEGF receptors, Flt-1 (fms-like

tyrosinekinase) and KDR (kinase insert domain-containing 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 AssayThe 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 VEGF-induced 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 t44-OMe 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---

step1step1 Lead compound are

obtained by SELEX

step2step2Lead modification from two

aspects:simplified structure &2’-OMe substitution

step3step3Lead optimization in parallel

with and in accordance to bioactivity assessment

Part4---

Pharmaceutical index, Market and the Future

4.1Administration

intravitreous( 玻璃体内 ) injection0.3 mg once every six weeks (9 injections per year) inspected visually for particulate matter and discolorationadequate anesthesia and a broad-spectrum topical microbicidemonitoring for intraocular pressure, endophthalmitis( 眼内炎 )

4.2Pharmacodynamic Properties

---a modified oligonucleotide that binds with high specificity and affinity to extracellular VEGF165 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 VisudyneRanibizumab(Lucentis)Bevacizumab(Avastin)Triple TherapyAnecortave acetate

Photodynamic Therapy(PDT) With VisudyneRanibizumab(Lucentis)Bevacizumab(Avastin)Triple TherapyAnecortave 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

Inexpensive price

Intravitreal injections,

no thromboembolic events

Triple Therapy of Triamcinolone, PDT and Pegaptanib Sodium

Limit contiguous macular damage and preserve vision

Price limits

Anecortave acetate (Retaane)

slowed progression of neovascular wet AMD,

posterior juxtascleral placement

Adverse reactions

4.8 Conclusions of these drugsBeyond 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 studyⅡ 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.

“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.

From Lab to LifeFrom Premise to Patient

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, 649-6519. 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. 613. 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. Cost-effectiveness 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

Covalently conjugate of an oligonucleotide (28 nucleotide totally)

2’-methoxy

2’-fluoro

R-group

Two polyethylene glycol(PEG) units are covalently attached

Lysine residue

conformational stability & enhanced

pharmacokinetics

Incubation with the target molecules1

Filtration of the complex 2

Amplification (eg.RNA to cDNAs; PCR)3

A secondary library obtained4