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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

– 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

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

Ideal Drug Delivery Process

4Absorption Distribution MetabolismFormulation

Ideal Mail Delivery Process

5Receiving DeliveringProcessing

Mail Delivery Process


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.

Oral Delivery

� Oral delivery involves

transport of drug via

gastrointestinal tractgastrointestinal tract

� Drugs are readily

metabolized in liver

11

Intravenous Delivery

� Bioavailability

a problem with

intravenous deliveryintravenous delivery

�Instability

�Toxicity

12

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

• 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.



• Examples


Outline



– Permeability





– Conclusion15

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


O OH OH

O

O

OHOO

H3C

H3CO

HONH

Molecule-Based Drug Carrier Example

1 2

17

OCH3

O

O

OH

OH H O

OH

O

OH

OH3C

OH

HN

O

O

-CO2


1 2

3 4

Macromolecule-Based Drug Carrier Example

Gemtuzumab Ozogamicin

antibody

18


5

6

Macromolecule-Based Drug Carrier Example

67

19

Cleaves DNA double helix

Bergman

rearrangement


1,4-benzenediyl diradical

Calicheamicin

6

8



• Examples


Outline



– Permeability





– Conclusion 20

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.

Molecular Umbrellas are PassivePassive

Membrane Transport Carriers

22http://io.uwinnipeg.ca/~simmons/cm1503/Image129.gif

Human Serum Albumin are ActiveActive

Membrane Transport Carriers

23



• Examples


Outline



– Permeability





– Conclusion 24

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.

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

Concept of Molecular Umbrellas

Hydrophobic

Hydrophilic

Drug molecule

27

Molecular Umbrella Conformations

These molecules are composed of two or more facial amphiphiles (“walls”) attached to a

central scaffold.28

Molecular Umbrellas (MU)

29

Hydrophilic

drug

Hydrophilic

Side

Hydrophobic

Side


30

Hydrophilic

drug

Hydrophilic

Side

Hydrophobic

Side


31

Hydrophilic

drug

Hydrophilic

Side

Hydrophobic

Side


32

Hydrophilic

drug

Hydrophilic

Side

Hydrophobic

Side


33

Hydrophilic

drug

Hydrophilic

Side

Hydrophobic

Side


34

Hydrophilic

drug

Hydrophilic

Side

Hydrophobic

Side

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

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

Synthesis of MU

13 14

37


15 16

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


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

Permeability Through Lipid Bilayers

• The larger the molecule, the

less permeable it is

• The more hydrophilic the

molecule, the less

permeable it is

39

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

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

Probing MU Permeability

Molecular umbrellas

Reaction chamber

Renaming molecular

umbrella detected by

UV spectroscopy

Liposome

42


Permeability Results

Di-walled MU

Tetra-walled MU

K = 835

Km = 208

• Permeability increases with increasing umbrella size

43


Octa-walled MU

Km = 1206

Km = 835

Peptide transport by MU vehicle

5-thiol(2-nitrobenzoyl) handle

Glutathione (GSH)

44

USSG = Umbrella- Glutathione

GSH = Gluthathione

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


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


GSH = Gluthathione

GSSL = Gluthathione-linker

GSSG = Gluthathione dimer

Linker = 5-thiol(2-nitrobenzoyl)

Peptide Release Chemistry

47


MU-Assisted Opioid Peptide

o-dithiobenzyl carbamate linker

DADLE = H-Tyr-D-Ala-Gly-Phe-D-Leu-OH

48


Jing, B.; Janout, V.; Regen, S. Bioconjugate Chem. 2003, 14 (6), 1191-1196.

20

Release of Opioid Peptides

o-thiobenzoquinone

Methide (OTBM)

49

Methide (OTBM)



Higher

conc. of

(OTBM)

Release of Opioid Peptides

UV absorbance spectra

o-thiobenzoquinone(OTBM)

50



o-thiobenzoquinone

methide (OTBM)

moiety generated after

release of DADLE

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

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

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

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

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

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



• Examples


Outline



– Permeability





– Conclusion57

Human Serum Albumin

58

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

The Difference Between Normal

and Tumor Tissue

60

Crystal Structure of HSA

61

Crystal Structure of HSA

62

Cys 34

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

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

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.

Drug Binding Site of HSA

66

Cys 34


67

Cys 34


68

Cys 34

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

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.

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


72

Cys 34

Tumor Inhibition Studies

Tumor growth inhibition of MDA-MB-435 cancer cell line under therapy with

doxorubicin and DOXO-EMCH

73



Bio-Distribution Studies

15

20

25

30

DOXO-EMCH

DOXO

Ra

dio

scti

vit

y le

ve

ls

74



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

• 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

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

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

Thank youThank you

78

Thank youThank you

molecular umbrellas and human serum albumin as … umbrellas and human serum albumin as ... the...

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