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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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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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