IPHASE/汇智和源 核酸适配体药物体外代谢研究解决方案

Basic composition

Nucleotide (four members: A, G, T/U and C)

Amino acid (20 members)

Materials

Nucleic acid (single-stranded DNA or RNA)

• Protein (polymer peptide)

•Antibodies consist of two light chains and two   heavy chains

Molecular weight and/or size

• 6–30kDa (20–100nt)

• ~2nm

• 150–180kDa

• ~15nm

Secondary structure

•Hairpin, stem, loop, bulge,   G-quadruplex or kissing complex

• α-Helix and β-fold

Binding pattern and/or mechanism of action

• Surface recognition

• Three-dimensional interactions via van der Waals   forces, hydrogen bonding and electrostatic interactions similar to the way   antibodies bind to antigen

• Reversal of activity via complementary antidote   oligonuclotides

• Binding pocket (key and lock model)

• Three-dimensional interaction; antibodies recognize   epitopes located on the target antigen

Affinity

•High

• Multivalent aptamers can confer increasing affinity   and additional functions

•High

•Affinity between antibody and antigen depends on   the number of identical epitopes on the target antigen

Specificity

•High

• The aptamer is able to identify single point mutations and   conformational isomers

•High

•Antigens may have multiple epitopes, which allow different antibodies to   bind to the same antigen

Potential targets

•Wide range: ions, organic and inorganic molecules, nucleic acids,   peptides, proteins, toxins, viral particles, whole cells, entire organs and   live animals

• Limited to immunogenic molecules

•No toxins or other molecules that do not cause strong immune responses

Generation and discovery

• In vitro SELEX (2–15 selection rounds)

• ~2–8 weeks

•Aptamer can be selected in hours or days via high-throughput automated   SELEX

• In vivo biological system

• ~6 months or longer

Manufacturing and costs

•Chemical solid-phase synthesis

•Controllable and completely in vitro procedure

• 2 days for milligrams; 2 weeks for grams

•No or low risk of contamination

• Facile regulatory affairs and cGMP

• Low cost for DNA; high cost for long RNA (>60nt) with special   modifications

•Costs lowered with the development of new technology

• In vivo (animal-based production)

• Potential contamination due to cells or animal-based production

• 3 months for 5–20 grams

• From mammalian cells: high cost

• From transgenic plants or animals: low cost

Batch-to-batch variation

•None or low

• Significant

Physical and

thermal stability

•Very stable and long shelf-life

• Resistant to high temperature (even up to 95°C) and cycles of   denaturation and renaturation

•Aptamers can be lyophilized for long-term storage and transport at room   temperature

•Unstable and limited shelf-life

• Susceptible to temperature (even at room temperature or 37°C) and   irreversible denaturation

• Requires refrigeration for storage and transport

Chemical modification and

conjugation

•Convenient and controllable

•Various types available, including sugar, backbone, base and other   modifications

•Aptamers can be rationally modified without loss of binding affinity

• Restricted and uncontrollable

• Limited types and chemical reactions

• Stochastic modifications very likely to cause negative consequences,   such as loss of activity

Tissue uptake and penetration

• Faster

• Slower

Immunogenicity

•None or low immunogenicity

•High immunogenicity

• Increased immune reaction with repeated dosing

Nuclease degradation

•Vulnerable

• Limited half-life in vivo (~10min for unmodified version)

• Resistant and not affected by nucleases in vivo

Kidney filtration

• Faster

• Short circulation time in vivo (~30min for unconjugated version)

• Slower

• Long circulation time (up to 1 month)

Patents and distribution

• Exclusive patents in SELEX technology

• Limited initial distribution

• Expired protection or no early patents

• More widespread distribution

Development and market

• The development pathway is less explored

• Insufficient education and investment (R&D support)

•Commercialization has focused on diagnostic-based aptamer products

•Well-developed infrastructure

•Abundant support from finance and education

• Rapid and sustained increase in drug market share

Pegaptanib

RNA

VEGF

AMD

上市（现已退市）

2004

Eyetech/Pfizer公司

Zimura

RNA

C5

AMD

Ⅰ期

2016

Ophthotech公司

Fovista

DNA

PDGF

AMD

Ⅲ期

2016

Ophthotech公司

AS1411

DNA

核仁蛋白

肾癌

Ⅱ期

2008

Antisoma研究中心

NOX-A12

RNA

CXCL12

CLL

Ⅰ期

2012

NOXXON制药公司

NOX-E36

RNA

CCL2

DN

Ⅰ期

2015

NOXXON制药公司

ARC1779

DNA

VWF

TTP

Ⅱ期

2008

Archemix公司

ARC19499

RNA

TFPI

血友病

Ⅰ期

2010

维也纳医科大学

NU172

DNA

凝血酶

心脏病

Ⅱ期

2013

ARCA生物制药公司

REG1

RNA

FIXa

冠状动脉疾病

Ⅱ期

2007

Regado生物科学公司

NOX-H94

RNA

铁调素

ACI

Ⅱ期

2013

NOXXON制药公司

血清/血浆

代谢稳定性，血浆蛋白结合试验

金属螯合剂抗凝的血浆不适合代谢稳定性研究。

肝S9

代谢稳定性，代谢产物鉴定

肝S9一定程度上可以代替肝组织匀浆使用。

肝微粒体

代谢稳定性

肝微粒体的核酸酶活性较低，可以根据实际情况进行选择。

肝组织匀浆

代谢稳定性，代谢产物鉴定

酶体系比较全面，推荐用于Aptamer药物的体外筛选评价。

肝细胞

代谢稳定性，代谢产物鉴定

肝细胞酶体系最为完善，适用于肝靶向的Aptamer药物研究。

溶酶体

代谢稳定性

溶酶体具有丰富的酶体系，包括核酸酶和各种水解酶等，是研究Aptamer药物代谢稳定性的高效实验体系。

亚细胞组分

肝溶酶体

酸化肝匀浆液

肝/肠/肾/肺S9

肝/肠/肾/肺微粒体

肝/肠/肾/肺胞质液

原代肝细胞

悬浮肝细胞

贴壁肝细胞

专属血浆

血浆稳定性

血浆蛋白结合