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by

Venu Gopal. S

II/II M. Pharmacy (Pharm.Chemistr

ANTISENSEANTISENSE

THERAPEUTICSTHERAPEUTICS

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Outline

Introduction

o History

Medicinal Chemistry

Mechanism of Action

Pharmacokinetics

Uses

Advantages

Limitations

References

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INTRODUCTION

Antisense drugs/ Antisense Oligonucleotides (ASO) are the nucleic

acid sequences of DNA/ RNA or chemically modified derivatives ofthese, which are 12-25 nucleotides in length.

These are chemically modified to bind to specific complementary

areas of disease producing m-RNA molecule in a sequence specific

manner via by Watson crick base pairing.

They are chemically engineered to have good drug properties.

They act by formation of the ASOmRNA heteroduplex that leads

to mRNA degradation or induces splicing or blocking translation ofmRNA by ribosomes.

They can be used to treat number of diseases.

Many of the ASOs are under Phase-I, II, III clinical trials. 3

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4

Sense

strand

of DNA

Antisens

e strand

of DNA

Sense & Antisense strands

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In 1966,H. G. Khorana and his group synthesized

oligoribonucleotides that were used to confirm the

Genetic code.In 1970, Khorana first synthesized gene, the 77bp

yeast tRNAAla gene.

In 1978, the beginning of antisense technology i.e. tracing of

sequence specific binding ofmodified DNA to complementaryDNA.

Later introducing these molecules into cells & preventing

them from nuclease activity was developed and testing on

animal models was done.

HISTORY In early 1950, Alexander Todds group pioneered H-

phosphonate and phosphate triester methods of

oligonucleotide synthesis.

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Relation of Antisense Technology to

other segments of biopharmaceutical

industry

ANTISENSE

TECHNOLOGIES

DRUG DELIVERY SYSTEMS GENE THERAPY

PHARMACEUTICALS

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DRUGS Fomivirsen

First ASO to reach marketIt is 21mer Phosphorothioate

oligonucleotide.

Used against Viral retinitis caused

by Human Cytomegalovirus.

Oblimersen

Inhibits the production of a

protein Bcl-2.

Used in Melanoma, Lymphocytic

Leukemia,Hodgkinslymphoma.

Alicaforsen

Used in ulcerative colitis,

psoriasis, rheumatoid arthritis

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Designing the ASOs

We need to consider at least four parameters in ASO design in

order to increase the Hit rate: (i)prediction of the secondary structure of RNA; Secondary

structure of the target mRNA with minimal overall freeenergy

as a potential ASO target site.

(ii)identification of preferable RNA secondary local structures;

Local structures accessible to ASOs are located at the terminal

end, internal loops, joint sequences, hairpins and bulges of 10 or

more consecutive nucleotides.

(iii)motifs searching and GC(Guanine & Cytosine) content

calculation; GC content is strongly correlated to thermodynamic

stability of the ASOmRNA duplexes and RNase H activity.

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(iv)binding energy (G037) prediction: To design a potent ASO, the

binding energy between the ASO and mRNA should beG0

37 -8kcal/mol.

(http://128.151.176.70/) a domain has been developed to

calculate binding energy of ASO/mRNA.

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Screening of ASO

After designing, some screening strategies areemployed to obtain

potent ASOs. They are:a. mRNA walking (RNA-RNA interactions)

b. Oligonucleotide array

c. RNase H mapping

d. Computational algorithms in ASO design is most economical

and very often generates potent ASO.

Some computational databases, algorithms are freely available infollowing sites

(http://sfold.wardsworth.org/cgi-bin/index.pl),

(http://www.bioit.org.cn/en/database%20and%20software.htm

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labour intensive and

requireexpensive

equipment

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SYNTHESISOligonucleotide synthesis is the

chemical synthesis of relatively short

fragments of nucleic acids with a

defined sequence.

Synthesis is carried out in the

opposite, 3' to 5' direction.

The process is implemented as solid-phase synthesis using

Phosphoramiditemethod and

building blocks derived from

protected deoxynucleosides (dA, dC,

dG, and dT), ribonucleosides (A, C, G,and U) or chemically modified

nucleosides e.g. LNA

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MATERIALS

Nucleic Acid Synthesizer

Acetic anhydride and N-methyl

imidazole

Conc. NH4OH

All phosphorus linkages must be

blocked with a Cyanoethyl group.

Four DNA phosphoramidite

monomers (bases) with all the 5-

hydroxyl groups blocked with DMT

group (Dimethoxytrityl, [bis-(4-

methoxyphenyl) phenyl methyl]).

The solid support is loaded into the reaction column of nucleic acid

synthesizer. In each step, the solutions will be pumped through the

column by computer control. 13

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Mainly four steps are involved:

A. Deblocking

B. Base CondensationC. Capping

D. Oxidation/ Stabilization

a) Deblocking

The first base, which is attached to the solid support, is at first inactivebecause all the active sites have been blocked or protected.

To add the next base, the DMT group protecting the 5-OH group must

be removed. This is done with a solution of an acid, such as 2% TCA or

DCA, in an inert solvent (DCM or toluene). The orange-coloured DMT

cation formed is washed out.b) Base Condensation

The next basemonomer cannot be added until it has been activated.

This is achieved by adding tetrazole to the base. Tetrazole cleaves off

one of the groups protecting the phosphorus linkage.14

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Base Condensation

2nd base Tetrazole

1st base

1,2joined15

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c) Capping

When the activated base is added to the reaction column some does

not bind to the active 5-OH site of the previous base.

If this group is left unreacted in one step, it is possible to react in later

additions of different bases leading to an error.

Hence capping is done with a protective group that prohibits the

strand from growing again. Acetic anhydride and N-methylimidazole

are added to the reaction column. These compounds only react withthe 5-hydroxyl group.

Acetic anhydride,

N-methylimidazole

16S li s rt

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Synthetic cycle for preparation of oligonucleotides by

Phosphoramidite method.

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DELIVERY OF ASO Unmodified ASO has net ve charge and cannot pass through plasma

membrane.

PNA & PMO are non charged and do not interact with cell surfaceproteins

Cationic lipid carriers, Dendrimers, Cellular adhesion molecules, Cell

penetrating molecules are used.

All of these cationic delivery systems internalize ASOs via anendocytosis mechanism. They also protect fromendosomes &

lysosomes

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ADME

Absorption:

Delivered directly (IV) or wellabsorbed from injection sites

(SC)

Antisense drugs are rapidly

and effectively absorbed.

In the blood, antisense drugs

bind loosely to proteins.

This binding facilitates their

distribution to tissues and

prevents immediate loss inurine.

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Distribution:

ASOs are highly plasma protein bound (90%)

Binding does not displace small molecules.

short plasma half-life, with most of the disappearance from

plasma accountable by distribution to tissues.

o Binding to plasma proteins prevents renal clearance &

promotes uptake in tissues.

degree of accumulation dependent on antisense drug half-life &

frequency of administration.

Themajor distribution tissues include kidney, liver, spleen, and

bonemarrow.

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Metabolism:

Metabolism process is

different from small

molecules, therebyavoiding drug-drug

interactions.

In tissues, the drugs are

cut by enzymes called

endonucleases. After being

cut by endonucleases, the

drugs may be further

degraded by exonucleases.

Tissue T= 14 to 30days; chemistry &

sequence dependent

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Elimination:

by renal route

Unbound parent drug in

Plasma & nuclease

degraded product of

tissues areeliminated

through urine.

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Mechanism of

Action1. Induce degradation of

RNA by RNAase-H

activation. For RNAase-H

activity duplex b/n RNA

& ASO is required.

2. Inhibition of RNA splicing(premRNA to mRNA)in

nucleus.

3. Inhibiting translation of

mRNA by steric

hindrance.4. degradation of RNA by

other mechanisms

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1.

3.

2.

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SAR

Core of any rational drug discovery

program is Medicinal chemistry.

Modifications in Base, Sugar, Phosphate

moieties of Oligonucleotides results in

compounds with altered ADME

properties.

PYRIMIDINES

large no. ofmodified pyrimidines

have been synthesized and

incorporated

modifications can be done at C2 C4C5

C6 Modification at C2 ,C5,C6 increased

stability of duplex

C4 has significant effect on

hybridization.26

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PURINES

Purine analogs when

incorporated into

oligonucleotides they

result in destabilization

of duplexes.

Modifications are done

at C2 C6 C7 N2 N6

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SUGAR MODIFICATION

Pentafuranose ring is

modified to enhance

hybridization, increase

nuclease resistance, cellular

uptake

Usually done at C2 position

of sugar ring2-O-Methyl (2-OMe) and 2-

O-amino propyl(2-O-AP) has

more stability and enhanced

oral bioavailability.

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BACKBONE MODIFICATION

Replacing phosphate or P-S(Phosphate-

Sugar) unit is to remove ve charge.

Results in increased stability, hybridization,

pharmacokinetics

PNA (Peptide Nucleic Acid) in place of P-S

unit, N-(2 amino ethyl)-glycine units linked

by peptide bonds-PNA has more binding effect to DNA, RNA

-It has better water solubility and target

binding affinity and stability.

LNA (Locked Nucleic Acid)

-Higher affinity than PNA

-resistant to nuclease degradation

PNA (Peptide Nucleic Acid)

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ASO Generations

Chemical modifications are done to enhance ADME properties like

nuclease resistance, tissue half-life, increase affinity, potency and reduce

non-sequence specific toxicity.

Based on the modifications in the chemistry during the course of time,

ASOs are classified t o 3 generations.

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FIRST GENERATION ASO:

Entirely DNA like nucleotides. Known as Phosphorothioates Phosphate-Sugar (PS) backbone is modified in which non-

bridging oxygen atoms in phosphodiester bond is replaced

by a sulphur atom.

This modification confers higher resistance to ASO against

nuclease degradation, leading to higher bioavailability. Thesemodified ASOs promote RNaseH-mediated cleavage of

target mRNA.

Fomivirsen, a 21 bp first generation PS-modified ASO, is

currently the only ASO drug approved for clinical use.

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SECOND GENERATION ASO:

DNA, RNA like nucleotides;more potent

2nd gen chemistry slows degradation of drugs from nucleases.

Since RNA hybridizes more tightly to RNA than DNA, these drugs have

greater affinity to RNA targets.

2-O-Methyl(2-OMe) and 2-O-methoxyethyl(2-MOE)modifications of

PS-modified ASOs are the two most widely studied 2nd gen ASOs.

2-OMe and 2-MOE substitutions do not support RNaseH-mediatedcleavage of target mRNA, which dampens theefficacy of the ASO

To circumvent this shortcoming, a chimeric ASO was developed.

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THIRD GENERATION ASO:

To further enhance pharmacodynamic and pharmacokinetic properties, a

3rd gen of ASOs were developed by chemical modifications of the

furanose ring of the nucleotide.

Peptide nucleic acid (PNA), locked nucleic acid (LNA) and

phosphoroamidatemorpholino oligomer (PMO) are the threemost

studied 3rd gen ASOs.

PNA is a synthetic, non-charged DNA mimic in which phosphodiester

backbone is replaced with a pseudopeptide polymer (N-(2-

aminoethyl)glycine) and bases are attached to the backbone via

methylene carbonyl linkage.

It causes steric hindrance of translational machinery.

PNA is not degraded by nucleases or peptidases. Oral administration will be feasible

Still research going on

contd.32

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ADVANTAGES

It is possible to discover an antisense drug and develop the

data package required for filing with the FDA in less than 15months where as small molecule drug discovery program

generally requires 26 years.

Used to inhibit theexpression ofmolecular targets that arenot easily approached with small molecule-based approaches.

Antisense drugs also has the possibility of being less costly in

production, as the same facility may be used to manufacture

multiple drugs.

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TOXICITY

toxic effects are dependent on ASO backbone chemistry and are

dose dependent. toxicities aremostly due to the non-specific binding of ASOs to

somemRNA that was not the initial target because of sequence

homology.

inhibition of the clotting cascade.

immune stimulation manifested as spleenomegaly, lymphoid

hyperplasia

Thrombocytopenia

Hyperglycaemia

Enhanced liver enzyme levels

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USES

Lung cancer, Prostate Cancer

HIV/AIDS

Cardiovascular disorders(hypertension, dyslipidaemia)

-thalassemia and cystic fibrosis

Duchenne muscular dystrophy

Spinal muscular atrophy

Diabetes mellitus and Obesity

Asthma, Rheumatoid Arthritis,Multiple Sclerosis

Ocular infections, inflammations, Diabetic retinopathy

Targeting Neurological Disorders likeHuntingtons

Disease, ALS Dementias, Neuropathy

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LIMITATIONS

Compared with small molecule drugs, ASO have some limitations.

Cost is one variable in which small molecule drugs, in general,

have an advantage.

Current products targeting systemic diseases must be givenparenterally.

Pharmacokinetic property like tissue and cellular distribution

may not be adequate for treatment of certain diseases, likeneurologic diseases as ASO do not cross the BBB (Blood-Brain

barrier).

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CONCLUSION

ASO has emerged as a valid approach to selectively modulate

geneexpression.

ASO also permit to access new targets and new potential

therapeutic compounds.

However the optimal use of ASO in treatment of diseases

requires solving of problems relating to effective design,

efficient target delivery,enhanced pharmacological activity.

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ReferencesBurger's Medicinal Chemistry and Drug Discovery -- Vol 2.

http://www.idtdna.com/pages/docs/technical-reports/chemical-synthesis-of-oligonucleotides.pdf

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2

003/Holmberg/oligonucleotide_synthesis.htm

Foye's Principles of Medicinal Chemistry.

Journal ofMedical Genetics and Genomics Vol. 3(5), pp. 77 - 83,May

2011.

Antisense & Nucleic Acid Drug Development 12:215224 (2002).

Mol Cancer Ther 2002;1:347-355.

Antisense Drug Technology; Principles, Strategies, and Applications,

Second Edition by Stanley T. Crooke

http://en.wikipedia.org/wiki/Oligonucleotide_synthesis

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