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Molecular Umbrellas Molecular Umbrellas and Human Serum Albumin as and Human Serum Albumin as Novel Drug Delivery VehiclesNovel Drug Delivery VehiclesNovel Drug Delivery VehiclesNovel Drug Delivery Vehicles
HovigHovig KouyoumdjianKouyoumdjian
Wednesday, Wednesday, Wednesday, Wednesday, Wednesday, Wednesday, Wednesday, Wednesday, February 18, 2009February 18, 2009February 18, 2009February 18, 2009February 18, 2009February 18, 2009February 18, 2009February 18, 2009
Page 2
– Introduction to Drug Delivery
• Delivery methods
• Examples
– Novel concepts of drug carriers
Outline
• Molecular Umbrellas (MU)
– Concept, chemistry
– Permeability
– MU bioconjugation and drug delivery
• Human Serum Albumin (HSA)
– Biochemical and chemical characteristics
– HSA bioconjugation and drug delivery
– Conclusion2
Page 3
Relevance of Drug Delivery
• Drug synthesis is execution of a synthetic scheme
• Drug delivery complements drug synthesis by bridging chemistry of synthesis to the biochemistry of uptakeof uptake
• It involves numerous aspects to formulate a drug, like:– Solubility
– Toxicity
– Efficacy
• It’s a package deal…
3
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Ideal Drug Delivery Process
4Absorption Distribution MetabolismFormulation
Page 5
Ideal Mail Delivery Process
5Receiving DeliveringProcessing
Page 6
Mail Delivery Process
6Receiving DeliveringProcessing
Page 7
Mail Delivery Process
7Receiving DeliveringProcessing
Page 8
Mail Delivery Process
8Receiving DeliveringProcessing
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Mail Delivery Process
9Receiving DeliveringProcessing
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Aspects of Drug Delivery
• The ability to specifically formulate drugs to achieve better absorption, distribution, metabolism and excretion (ADME) properties
• Drugs can be delivered as free molecules or bound to carriers
• Pharmacologically active compounds usually delivered combined to a carrier
10
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; 2nd ed.; Elsevier Academic Press, 2004.
Page 11
Oral Delivery
� Oral delivery involves
transport of drug via
gastrointestinal tractgastrointestinal tract
� Drugs are readily
metabolized in liver
11
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Intravenous Delivery
� Bioavailability
a problem with
intravenous deliveryintravenous delivery
�Instability
�Toxicity
12
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Enhanced Bioavailability
� Bioavailability is obtained
when drug is resistant to
liver metabolism
� Two conditions should be
met for better delivery
�Site specificity
�Prolonged release
13
Page 14
• The concept of prodrug was introduced in late 1950s
• Prodrug concept is converting the pharmacologically active
compound to an inactive one through conjugating it to a
carrier
Prodrug Concept
• Ideal carrier Characteristics
– Protect the drug until it reaches site of action
– Allow the release of the drug chemically or enzymatically
– Nontoxic
– Biodegradable
– Biochemically inert and non-immunogenic
14Albert, A. Nature 1958, 182, 421-3.
Page 15
– Introduction to Drug Delivery
• Delivery methods
• Examples
– Novel concepts of drug carriers
Outline
• Molecular Umbrellas (MU)
– Concept, chemistry
– Permeability
– MU bioconjugation and drug delivery
• Human Serum Albumin (HSA)
– Biochemical and chemical characteristics
– HSA bioconjugation and drug delivery
– Conclusion15
Page 16
Examples of Drug Reactivation
• Drugs can be reactivated via chemical or enzymatic process
• Molecule carriers
O OH
OH
O
OH
OH• Molecule carriers
– ex. Doxorubicin-carrier
• Macromolecule carriers– ex. Gemtuzumab Ozogamicine
16
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; 2nd ed.; Elsevier Academic Press, 2004.
O OH OH
O
O
OHOO
H3C
H3CO
HONH
Page 17
Molecule-Based Drug Carrier Example
1 2
17
OCH3
O
O
OH
OH H O
OH
O
OH
OH3C
OH
HN
O
O
-CO2
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; 2nd ed.; Elsevier Academic Press, 2004.
1 2
3 4
Page 18
Macromolecule-Based Drug Carrier Example
Gemtuzumab Ozogamicin
antibody
18
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; 2nd ed.; Elsevier Academic Press, 2004.
5
6
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Macromolecule-Based Drug Carrier Example
67
19
Cleaves DNA double helix
Bergman
rearrangement
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action; 2nd ed.; Elsevier Academic Press, 2004.
1,4-benzenediyl diradical
Calicheamicin
6
8
Page 20
– Introduction to Drug Delivery
• Delivery methods
• Examples
– Novel concepts of drug carriers
Outline
• Molecular Umbrellas (MU)
– Concept, chemistry
– Permeability
– MU bioconjugation and drug delivery
• Human Serum Albumin (HSA)
– Biochemical and chemical characteristics
– HSA bioconjugation and drug delivery
– Conclusion 20
Page 21
Recent Vehicles in Drug Delivery
– Facially amphiphiles
• Molecular Umbrellas
– Membrane Passive Transport
– Albumin-based drug carriers
• Human Serum Albumin
– Membrane Active transport
21
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
Page 22
Molecular Umbrellas are PassivePassive
Membrane Transport Carriers
22http://io.uwinnipeg.ca/~simmons/cm1503/Image129.gif
Page 23
Human Serum Albumin are ActiveActive
Membrane Transport Carriers
23
Page 24
– Introduction to Drug Delivery
• Delivery methods
• Examples
– Novel concepts of drug carriers
Outline
• Molecular Umbrellas (MU)
– Concept, chemistry
– Permeability
– MU bioconjugation and drug delivery
• Human Serum Albumin (HSA)
– Biochemical and chemical characteristics
– HSA bioconjugation and drug delivery
– Conclusion 24
Page 25
Molecular Umbrellas
• One of the major goals in modern medicinal
chemistry is to find ways of transporting polar
drugs across hydrophobic barriers such as the drugs across hydrophobic barriers such as the
plasma membrane of cells
25
Takeuchi, T.; Kosuge, M.; Tadokoro, A.; Sugiura, Y.; Nishi, M.; Kawata, M.; Sakai, N.; Matile, S.; Futaki, S. ACS Chem.
Biol. 2006, 1, 299-303.
Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A. J. Am. Chem. Soc. 2004, 126, 9506-9507.
Page 26
Facially Amphiphiles
• Facially amphiphils are
molecules with both
hydrophilic and
hydrophobic sides
26
hydrophobic sides
• They mimic the α-helices’
facial amphiphilic
character in penetrating
the cell membrane
Page 27
Concept of Molecular Umbrellas
Hydrophobic
Hydrophilic
Drug molecule
27
Page 28
Molecular Umbrella Conformations
These molecules are composed of two or more facial amphiphiles (“walls”) attached to a
central scaffold.28
Page 29
Molecular Umbrellas (MU)
29
Hydrophilic
drug
Hydrophilic
Side
Hydrophobic
Side
Page 30
Molecular Umbrellas (MU)
30
Hydrophilic
drug
Hydrophilic
Side
Hydrophobic
Side
Page 31
Molecular Umbrellas (MU)
31
Hydrophilic
drug
Hydrophilic
Side
Hydrophobic
Side
Page 32
Molecular Umbrellas (MU)
32
Hydrophilic
drug
Hydrophilic
Side
Hydrophobic
Side
Page 33
Molecular Umbrellas (MU)
33
Hydrophilic
drug
Hydrophilic
Side
Hydrophobic
Side
Page 34
Molecular Umbrellas (MU)
34
Hydrophilic
drug
Hydrophilic
Side
Hydrophobic
Side
Page 35
Sizes of Molecular Umbrellas
• MU can be synthesized as:
– Di-walled, tetra-walled and octa-walled MU
OHOH OH
OHOH
OH
35
NH
NH
OH
OH OHNH
O
O
O
NHN
NH
O
SO3 SO3
SO3O
NHO
O
NH
OH
OH
OHNH
OH
OH
OH
NH
O
O
O
NH
NH
OHOH
OHNH
O
O
O
O
NH
OH
OH
OHNH
OH
OH
OH
NH
O
O
O
Octa-walled MU
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Synthesis of MU
11 12
36
Mehiri, M.; Chen, W.-H.; Janout, V.; Regen, S. L. J. Am. Chem. Soc. 2009, 131, 1338-1339.
13
Page 37
Synthesis of MU
13 14
37
Mehiri, M.; Chen, W.-H.; Janout, V.; Regen, S. L. J. Am. Chem. Soc. 2009, 131, 1338-1339.
15 16
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Synthesis of MU
14 + 16
NH
XX X
NH
X
XX
NH
O
O
O
NHN
NH
O
NH
O
O
NH
NH
XXX
NH
XXX
NH
O
O
O
O
NH
38
Mehiri, M.; Chen, W.-H.; Janout, V.; Regen, S. L. J. Am. Chem. Soc. 2009, 131, 1338-1339.
14 + 16
SO3 SO3
SO3O
O
NH
X
X
XNH
X
X
X
O
O
O
NH
X
X
XNH
X
X
X
O
O
O
18
17
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Permeability Through Lipid Bilayers
• The larger the molecule, the
less permeable it is
• The more hydrophilic the
molecule, the less
permeable it is
39
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Permeability Formula
Pm permeability
Dm membrane diffusion coefficient of the solute
L
KDP
mm
m
.
=
Dm membrane diffusion coefficient of the solute
Km partition coefficient of water-membrane
L membrane thickness
The larger the molecule the smaller the D value
The more hydrophilic the molecule the smaller the K value
Pidgeon, C.; Ong, S.; Liu, H.; Qiu, X.; Pidgeon, M.; Dantzig, A. H.; Munroe, J.; Hornback, W. J.; Kasher, J. S.; et al.
J. Med. Chem. 1995, 38, 590-4. 40
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Liposome
• Liposomes are the
spherical analogs of lipid
bilayers
41http://images.google.com/imgres?imgurl=http://upload/commons/thumb/c/c6/Phos
pholipids_aqueous_solution_structures.svg/
• They are used as
mimetic representation
of the cell membrane
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Probing MU Permeability
Molecular umbrellas
Reaction chamber
Renaming molecular
umbrella detected by
UV spectroscopy
Liposome
42
Mehiri, M.; Chen, W.-H.; Janout, V.; Regen, S. L. J. Am. Chem. Soc. 2009, 131, 1338-1339.
Page 43
Permeability Results
Di-walled MU
Tetra-walled MU
K = 835
Km = 208
• Permeability increases with increasing umbrella size
43
Mehiri, M.; Chen, W.-H.; Janout, V.; Regen, S. L. J. Am. Chem. Soc. 2009, 131, 1338-1339.
Octa-walled MU
Km = 1206
Km = 835
Page 44
Peptide transport by MU vehicle
5-thiol(2-nitrobenzoyl) handle
Glutathione (GSH)
44
USSG = Umbrella- Glutathione
GSH = Gluthathione
Page 45
Proof of MU-Mediated Transport
The transport confirmed by quantifying the
amount of cystine (GSSG) that had been produced
and entrapped in the liposomes
45
USSG = Umbrella- Glutathione
GSH = Gluthathione
GSSG = Gluthathione dimer
USH = Umbrella-SH
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
Page 46
Proof of MU-Mediated Transport
GSSL
GSH
GSH
GSH
GSH
GSH
GSH
X
Glutathione-linker control conjugate did not
produce any detectable (GSSG)
Glutathione-linker control
(GSSL)
46
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
GSH = Gluthathione
GSSL = Gluthathione-linker
GSSG = Gluthathione dimer
Linker = 5-thiol(2-nitrobenzoyl)
Page 47
Peptide Release Chemistry
47
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
Page 48
MU-Assisted Opioid Peptide
o-dithiobenzyl carbamate linker
DADLE = H-Tyr-D-Ala-Gly-Phe-D-Leu-OH
48
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
Jing, B.; Janout, V.; Regen, S. Bioconjugate Chem. 2003, 14 (6), 1191-1196.
20
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Release of Opioid Peptides
o-thiobenzoquinone
Methide (OTBM)
49
Methide (OTBM)
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
Jing, B.; Janout, V.; Regen, S. Bioconjugate Chem. 2003, 14 (6), 1191-1196.
Page 50
Higher
conc. of
(OTBM)
Release of Opioid Peptides
UV absorbance spectra
o-thiobenzoquinone(OTBM)
50
Janout, V.; Zhang, L.-h.; Staina, I. V.; Di Giorgio, C.; Regen, S. L. J. Am. Chem. Soc. 2001, 123, 5401-5406.
Jing, B.; Janout, V.; Regen, S. Bioconjugate Chem. 2003, 14 (6), 1191-1196.
o-thiobenzoquinone
methide (OTBM)
moiety generated after
release of DADLE
Page 51
Nucleotide Transport
5-thiol(2-nitrobenzoyl) handle
51
Janout, V.; Jing, B.; Regen, S. L. Bioconjugate Chem . 2002, 13, 351-356.
Antisense nucleotides therapeutic applications
lung cancer, Crohn’s disease, malignant melanoma, chronic lymphocytic leukemia,
multiple myeloma, and cytomegalovirus retinitis
21
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MU-Nucleotide Chemistry
O3SOHN
OSO3
OSO3
OSO3
O
NHN
O
O3SO
OSO3
NO2
S
O
SN
NN
N
NH2
O
OHOHHH
OP
O
O
PO
O
OP
O
O
O
GS21
52Janout, V.; Jing, B.; Regen, S. L. Bioconjugate Chem. 2002, 13, 351-356.
O3SOHN
OSO3
OSO3
OSO3
O
NHN
O
O3SO
OSO3
NO2
S
O
SG
N
NN
N
NH2
O
OHOHHH
OP
O
O
PO
O
OP
O
O
O
S
+22
Page 53
Chlorambucil Drug Mechanism
Chlorambucil 23 24
53
Vijayaraghavan, S.; Jing, B.; Vrablik, T.; Chou, T.-C.; Regen, S. L. Bioconjugate Chem. 2003, 14, 667-671.
2526
27
Page 54
MU-Chlorambucil Synthesis
+
OH
OH
OH
+
54
Vijayaraghavan, S.; Jing, B.; Vrablik, T.; Chou, T.-C.; Regen, S. L. Bioconjugate Chem. 2003, 14, 667-671.
28
Page 55
in vitro-Transport Evidence
29
55
Mehiri,M,; Jing, B.; Ringhoff, D.; Janout,V.; Cassimeris, L.; Regen, L. Bioconjugate Chem. 2008, 19 (8), 1510-1513
Coumarin 343 fluorescently-labeled molecular umbrella transport
evidence through HeLa cells using confocal Microscopy
29
Page 56
Summary for Molecular Umbrellas
• Molecular umbrellas proved to be a promising
technique in drug delivery
• MU have been found capable of transporting • MU have been found capable of transporting
certain hydrophilic peptides and nucleotides
across liposomal membranes by passive
diffusion.
56
Page 57
– Introduction to Drug Delivery
• Delivery methods
• Examples
– Novel concepts of drug carriers
Outline
• Molecular Umbrellas (MU)
– Concept, chemistry
– Permeability
– MU bioconjugation and drug delivery
• Human Serum Albumin (HSA)
– Biochemical and chemical characteristics
– HSA bioconjugation and drug delivery
– Conclusion57
Page 58
Human Serum Albumin
58
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Advantages of HSA
HSA is an ideal candidate for drug delivery due to its:
preferential uptake in tumor and inflamed tissue
ready availability
biodegradability
lack of toxicity and immunogenicity
pH stability
soluble properties
heat tolerance 59
Page 60
The Difference Between Normal
and Tumor Tissue
60
Page 61
Crystal Structure of HSA
61
Page 62
Crystal Structure of HSA
62
Cys 34
Page 63
Methotrexate (MTX) plays an important role in the treatment of a variety of
malignancies and is well understood with respect to its mechanism of action at
the molecular level.
HSA-MTX Conjugate
limited by its:
- toxic dose-related side effects
- lack of selectivity, which is closely related to its pharmacokinetic
properties
- bioavailability complications
Methotrexate
63
30
Page 64
Synthesis of MTX Conjugates
31
64
Warnecke, A.; Fichtner, I.; Sass, G.; Kratz, F. Arch. Pharm. Chem. Life Sci. 2007, 340, 389 – 395.
32
33
Page 65
Maleimide-Sulfhydryl Conjugation
• Specific reaction when pH 6.5-7.5
• SH is 1000 times more reactive than NH2 at pH = 7.0
65
Partis, M.; Griffiths, D.; Roberts, G.; Beechey, R. J. Protein Chem. 1983, 2, 263-277.
Page 66
Drug Binding Site of HSA
66
Cys 34
Page 67
Drug Binding Site of HSA
67
Cys 34
Page 68
Drug Binding Site of HSA
68
Cys 34
Page 69
MTX-HSA
shows better
accumulation
MTX-HSA Accumulation Studies
DU-145 cancer cell line treated with MTX-HSA or MTX.
Wosikowski, K.; Biedermann, E.; Rattel, B.; Breiter, N.; Jank, P.; Loeser, R.; Jansen, G.; Peters, G. J. Clin. Cancer
Res. 2003, 9, 1917-1926.
accumulation
than MTX
69
Page 70
Exogenous and Endogenous HSA
� Commercially available albumin (exogenous) has 20 –60%
free sulfhydryl groups per molecule albumin
� Cysteine-34 position is blocked by sulfhydryl compounds
such as cysteine, homocysteine or glutathione such as cysteine, homocysteine or glutathione
� These drawbacks affect the bioconjugation of HSA to drugs
�Therefore the novel idea of in-situ conjugation of drugs
to HSA came to enhance the capabilities of this technique
70
Kratz, F.; Mueller-Driver, R.; Hofmann, I.; Drevs, J.; Unger, C. J. Med. Chem. 2000, 43, 1253-1256.
Page 71
Doxorubicin Derivative
O O
O OH
OH O
OH
N
OH
HN
O
N
O
O
(6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH)
71
Kratz, F.; Warnecke, A.; Scheuermann, K.; Stockmar, C.; Schwab, J.; Lazar, P.; Drueckes, P.; Esser, N.; Drevs, J.;
Rognan, D.; Bissantz, C.; Hinderling, C.; Folkers, G.; Fichtner, I.; Unger, C. J. Med. Chem. 2002, 45, 5523-5533.
O O OH O
O
NH2
OH
HCl
34
Page 72
Drug Binding Site of HSA
72
Cys 34
Page 73
Tumor Inhibition Studies
Tumor growth inhibition of MDA-MB-435 cancer cell line under therapy with
doxorubicin and DOXO-EMCH
73
Kratz, F.; Warnecke, A.; Scheuermann, K.; Stockmar, C.; Schwab, J.; Lazar, P.; Drueckes, P.; Esser, N.; Drevs, J.;
Rognan, D.; Bissantz, C.; Hinderling, C.; Folkers, G.; Fichtner, I.; Unger, C. J. Med. Chem. 2002, 45, 5523-5533.
Page 74
Bio-Distribution Studies
15
20
25
30
DOXO-EMCH
DOXO
Ra
dio
scti
vit
y le
ve
ls
74
Kratz, F.; Warnecke, A.; Scheuermann, K.; Stockmar, C.; Schwab, J.; Lazar, P.; Drueckes, P.; Esser, N.; Drevs, J.;
Rognan, D.; Bissantz, C.; Hinderling, C.; Folkers, G.; Fichtner, I.; Unger, C. J. Med. Chem. 2002, 45, 5523-5533.
0
5
10
2h 6h 24h 48h
Ra
dio
scti
vit
y le
ve
ls
Duration
Levels of DOXO-EMCH and DOXO in human serum
Page 75
• HSA is proving to be one of the most promising drug
delivery vehicles
• The indigenous nature of HSA makes it more efficient
Summary for HSA
• The indigenous nature of HSA makes it more efficient
and less toxic to the human body
• Methotrexate and Doxorubicine-HSA conjugates
show excellent anti-cancer activities
75
Page 76
Conclusions and Critical Insights• Molecular umbrellas and human serum albumin demonstrated novel
chemical capabilities in transporting some pharmaceutical molecules
• Many biological molecules and anticancer drugs were successfully
delivered by MU and HSA vehicles
• Further research needs to be conducted, especially in delivering
76
• Further research needs to be conducted, especially in delivering
targeted conjugates, such as antisense oligonucleotides and receptor
targeted macromolecules
• Extensive in vivo experiments still required in order to prove the
biocompatibility of drug conjugates
• Computational modeling for HSA delivery are yet to be developed
• Crystal structures of these vehicles are yet to be determined
Page 77
Acknowledgment
• Prof. Xuefei Huang
• Prof. Babak Borhan
• Prof. James Jackson
• Prof. William Wulff
• Dr. Chrysoula Vasileiou
• Jason, Bin, Medha, Gopi, Vivian, Dino, Gilbert, Bo, Steve,
Phil
• Allison, Aman, Monica, Dima
77
Page 78
Thank youThank you
78
Thank youThank you