Medicinal Application of Dendrimers

Literature Seminar
Shogo HASHIZUME (M2)‘11. 12. 6. (Tue.)1

Prologue - What is the Dendrimer??
Dendrimer = a family of nanosized, 3D polymer
a class of macromolecules
having highly branched architecture

Fig. A: Schematic 2D presentation of dendrimer
Fig. B: 3D presentation of dendrimer
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Prologue - What is the Dendrimer??
- Dendrimers consist of three main components…
Surface: functional peripheral group
Interior: affects host-guest properties
Core: affects 3D shape of dendrimer- Count branching points as the “generation”

This is a “G2 dendrimer.”

Higher generation dendrimers form more dense, dimensional shape.
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Prologue - Polymer vs Dendrimer
What makes dendrimers great over simple linear/branched polymer ??

It is “monodispersity”
PolymerDendrimer

Simple chain growth procedure

Stepwise growth

Mixture of different products
will be obtained…

Nearly monodisperce productwill be obtained !!
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Prologue - Backholding of Dendrimers
But…

Dendrimers always branch as simply extending outward…??
Doesn’t occur ???
Backholding actually occurs.

Baker, J. R. Jr. et al.
Macromolecules 2002, 35, 4510.

For example…

Conformational simulation of PAMAM at…

high pHneutral pHlow pH

5Prologue - Early Development of Dendrimers- First synthesis dendritic arms

- Synthesis of dendrimers at higher
generations with well-defined structures

Vogtle, F. et al.
Synthesis 1978, 2, 155.

But, synthetical difficulties limited
to only low generation compound.
Tomalia, D. A. et al.
Polymer J. 1985, 17, 117.

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Prologue - Examples of Dendrimers
Since then, over 100 dendrimer structure have been realized.

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Today’s Contents
0. Prologue
1. How to Synthesize Dendrimers
2. Dendrimers as Drugs
3. Dendrimers as Drug Carriers
4. Perspectives
Other applications: Tissue engineering
Transfection
Magnetic resonance imaging (MRI)etc.
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Today’s Contents
0. Prologue
1. How to Synthesize Dendrimers
2. Dendrimers as Drugs
3. Dendrimers as Drug Carriers
4. Perspectives

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Divergent or Convergent ??
Roughly, two synthetic strategies of dendrimer…
- Divergent method- Convergent method

10Divergent Method
Tomalia, D. A. et al.
Polymer J. 1985, 17, 117.

Tomalia’s PAMAM synthesis

However, many side reactions…
- Incomplete Michael additionO
MeO2C

MeO2COMeNNHNON

CO2Me

CO2Me

- Retro-Michael reaction @ high temp.
OMeO2C

HOMe

MeO2C
NHN

- Hydrolysis of methyl ester
O
MeO2C

OOMe

N

MeO2C

OHN

N o more reacti ve
f or amid ati on11

Divergent Method
Divergent synthesis needs many steps
Improving yields & eliminating purif. steps are the keys.

Need optimization of conditions & smart ligation system
Examples:- Using anhydride coupling

Frechet, J. M. J. et al. JACS 2001, 123, 5908.

Up to generation 6 dendrimer obtained
without chromatographic purification !!

- Using click chemistry

will be showed later12

Convergent Method
Constructing from periphery toward the core

Only limited active sites per a reaction(= more defect-free product)First convergent synthesis:

Frechet, J. M. J. et al. JACS 1990, 112, 7638.

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Convergent Method
However, high generation dendrimers are difficult by convergent method.
In the case of Frechet’s synthesis (JACS,1990)…
MeHO[G-4]-BrOH+84% yieldOHMeHO[G-6]-Br

OH+51% yield

OH

It’s due to steric crowding at dendrons focal point.

stericallycongested

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Convergent Method
One solution is “double-staged” approach.

Frechet, J. M. J. et al.
JACS 1991, 113, 4252.

Higher generation
dendrimers

Using as “flexible core”
to reduce the steric hinderance
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Combined Divergent-Convergent Method“Double exponential” method

Wilkins, C. L.; Moore, J. S. et al.
JACS 1995, 117, 2159.

Orthogonallyprotected monomer

But this method has combined disadvantages of divergent/convergent method- Higher generation dendrimers cannot be synthesized due tosteric hinderance at the late stage.
- Increasing of protection/activation chemistry with generation
requires highly efficient reaction scheme.
16Synthesis Using “Click Chemistry”
Improving yields of each step is the key to “monodispersity.”
Cu-catalyzed cycloadditionof an alkyne and an azide.

Using reliable reactions

R'+R

NN
N

R'Cu cat.
RNN
N

Both of divergent/convergent approaches are possible.
Hawker, C. J.; Sharpless, K. B.; Fokin, V. V. et al.
ACIE 2004, 43, 3928.
Chem. Commun. 2005, 5775.

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Today’s Contents
0. Prologue
1. How to Synthesize Dendrimers
2. Dendrimers as Drugs
3. Dendrimers as Drug Carriers
4. Perspectives

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As Antimicrobial- Cationic dendrimers with amphiphilic properties
Cooper, S. L. et al. Biomacromolecules, 2000, 1, 473.
PAMAM

Activities depend on generation number and alkyl chain length of ammonium.
higher generation = more surface groups
lower generation = more potent for permeability

However,they have cytotoxicities against eukaryotic cells due to cationic nature.

Can we reduce the positive charge on surface ??
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To Overcome The Drawbacks- Partially PEGylated PAMAM dendrimer

Cai, C. et al.
Biomacromolecules, 2007, 8, 1807.

ca. 43% of amines PEGylated

Effective cytotoxicity against some species ofbacteria without toxicity against eukaryotic cells.
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To Overcome The Drawbacks- Anionic amphiphilic dendrimers

Grinstaff, M. W. JACS 2008, 130, 14444
Cytotoxicity against Gram-positive bacteria

Cytotoxicity against eukaryotic HUVEC cells

Selective cytotoxicity
as antimicrobial21

As Antiviral (Anti-HIV) - Anionic Dendrimers
Polyanions are known as anti-HIV by inhibiting gp120(a glycoprotein on the surface of HIV envelope).

V3 loop in gp120 is a positively charged region.
- VivaGel (sulfonated polylysine dendrimer)- Poly(phosphor-hydrazone) dendrimers
with terminal phosphonic acid & alkyl chain

McCarthy, T. D. et al.
Mol. Phrm. 2005, 2, 312
Blanzat, M.; Turrin, C.-O. et al.
Org. Biomol. Chem. 2009, 7, 3491
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As Antiviral (Anti-HIV) - Mannose-Binding Dendrimer

Inhibiting the interaction
between HIV and dendric cells.
Wang, S.-K.; Liang, P.-H.; Wong, C.-H. et al. PNAS 2008, 105, 3690.
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Today’s Contents
0. Prologue
1. How to Synthesize Dendrimers
2. Dendrimers as Drugs
3. Dendrimers as Drug Carriers
4. Perspectives

24Advantages of Dendrimers as Drug Carriers
Chemotherapeutic drugs have some problems…

- Low solubitlity in water (due to hydrohobicity)- Easily metabolized

etc.

Dendrimers as one of the solutions…

- Solubilizing in water (Dendrimers’ solubilities regulatable)- Less filtered out of bloodstream (unable to exceed the renal threshold)- Tumor selectivity (they uptake larger macromolecules)
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How Dendrimers Carry Drugs ??
Roughly devided…

Physical Encapsulation

Chemical Conjugation

Directly coupled
Encapsulating inhydrophobic interior

Coupled via linkage

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Physical Encapsulation
Capture & release of drugs in dendrimers is effected byhydrophobic forces, hydrogen bonding steric hinderance, electrostatic interactions
Several dendrimers are known to encapsulate drugs as guest molecules.
- DOX & MTX in PEGylated G3-NH2 or G4-NH2 PAMAM dendrimer
Kono, K. et al. Bioconj. Chem. 2000, 11, 910.
OO

OHOHOHOMe OOHOMeONH2 OHdoxorubicin (DOX)
N

H2NN

NN

MeN
H
N

NH2

CO2H

O
methotrexate (MEX)

CO2H

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Release of Encapsulated Drugs
However, too rapid release of drugs from dendrimers is problematic.
Example:Release of DOX fromPEGylated G4-PAMAM dendrimer

DOX was released 10 times fasterin isotonic buffer than in non-isotonic buffer.

Kono, K. et al. Bioconj. Chem. 2000, 11, 910.

These premature drug release will cause non-selective toxicity in vivo.
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Chemical Conjugation via Direct Coupling
Several conjugates between dendrimer and drug molecule have been reported…

- G3.5 PAMAM-cisplatin conjugate- Carrying 20-25 wt% Pt- 10 times aqueous solubility- But almost no toxicity toward
cancer cells (<1% Pt release)

Duncan, R et al.
Anticancer Drugs 1999, 10, 767.

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Chemical Conjugation via Direct Coupling- Polyester dendrimer-DOX conjugate

Showed decreased activity
compared to free DOX
Frechet, J. M. J.; Szoka, F. C. Jr. et al. Biocoj. Chem. 2002, 13, 453.

Both in vivo and in vitro cancer activity are decreased due tolimited release of the loaded drug.
30Conformation of Drugs on Dendrimer’s Surface
Conformation of drug molecules on dendrimer’s surface is critical to activity.

- G2.5 or G3 PAMAM-MTX conjugate
ON

H2N

H
N

G2.5 PAMAM

NN

N
N

Me

N

N

N

N

N

H
N

NH2H
N

NH2Me

CO2H

CO2H

OO

G3 PAMAM
OCO2H

G2.5 PAMAM-MTX conjugate
3-fold more activeNH

G3 PAMAM-MTX conjugate
10-fold less active

Kannan, R. M. et al. Bioconjugate Chem. 2006, 17, 275.

- G5 PAMAM-NH2 or -OH -MTX conjugate
N

N

H2NN

N

N

H2N

MeN

N
H
N

NH2CO2H

NN

MeN
H
N

NH2O

CO2H

O

PAMAM
O

O

10-fold lower IC50

PAMAM
O

N
H

Baker, J. R. Jr. et al. Pharm. Res. 2002, 19, 1310.

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Chemical Conjugation via Linkages
Releasing drugs near the target is ideal…

pH-Sensitive hydrazone-linkage

at pH 7.4 : <10% releaseat pH 5.0 : 100% release
This hydrazone group is hydrolyzed
under acidic condition.

Hydrazone-linked DOX-dendrimer conjugate also shows
improving activity in in vivo experiment.
Rihova, B. et al. Controlled Release 2001, 74, 225.

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Next Challenge ??

Dendrimers as drug carriers work depending on pH environment.

Can we apply to more specific environment ??
such as cancer-specific intracellular enzymes…

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Today’s Contents
0. Prologue
1. How to Synthesize Dendrimers
2. Dendrimers as Drugs
3. Dendrimers as Drug Carriers
4. Perspectives

34Perspectives
Dendrimers have potential to be prominent materials
for medical application.
But, still not reach the success of linear polymers(I think) due to synthetic difficulties.

More fundamental, detailed studies are necessary…

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Perspectives
Further studies about…

synthesis, physical properties, structural activity relationship

Correlative fundamental and application studies are necessary
in this new, undeveloped area.
Application
Fundamentalstudies

Feedback

Practical(not only medicinal)
application

36References (Reviews)- El-Sayed, M. E. H. et al. Chem. Rev. 2009, 109, 3141.
- Turrin, C.-O. et al. New J. Chem. 2009, 33, 1809.
- Svenson, S. Eur. J. Pharm. Biopharm. 2009, 71, 445.
- Grinstaff, M. W. et al. Chem. Soc. Rev. 2011, 40, 173.

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