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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
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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