Lanthanide(III) Lanthanide(III) ChelatesChelates of DTPAof DTPA--Based Based GlycoconjugatesGlycoconjugates: : LectinLectin--Mediated Mediated

Medical Imaging AgentsMedical Imaging Agents

João Paulo André

DepartmentDepartment ofof ChemistryChemistry, , UniversityUniversity ofof Minho, Braga, PortugalMinho, Braga, Portugal

SummarySummary

1.1. Objective: Develop new Lanthanide (III) Objective: Develop new Lanthanide (III) glycoconjugateglycoconjugate chelateschelates of of DPTADPTA capable of targeting the liver capable of targeting the liver asialoglycoproteinasialoglycoprotein receptor receptor

(ASGPR)(ASGPR) - lectinlectin with with physicophysico--chemical and biological properties chemical and biological properties

capable ofcapable of making them potential agents for liver imagingmaking them potential agents for liver imaging2. 2. SynthesisSynthesis and and physicophysico--chemicalchemical characterizationcharacterization ofof dendrimericdendrimeric

DTPA DTPA basedbased glycoconjugateglycoconjugate ligandsligands

3.3. PhysicoPhysico--chemicalchemical characterizationcharacterization ofof theirtheir LnLn(III) (III) chelateschelates: :

-- NMR NMR studystudy ofof thethe Eu(III) and Sm(III) Eu(III) and Sm(III) chelateschelates-- RelaxivityRelaxivity (NMRD) (NMRD) studystudy ofof thethe Gd(III) Gd(III) chelateschelates and and theirtheir lectin lectin bindingbinding; ; determinationdetermination ofof molecular molecular parameterparameterss

Active targeting for medical diagnostic

Cell surface Cell surface receptorreceptor((internalization internalization ––Intracellular deliveryIntracellular delivery))

linker

Vector molecule(peptide, antibody, carbohydrate,...)

Bifunctional chelator

Paramagnetic (Gd(III)) : MRI

Metal ion

Gamma emitter: scintigraphy

Lectins : non-enzymatic proteins or glycoproteins which bind to carbohydrates and act as recognition determinants in many biological processes

TargetingTargeting LectinsLectins

-- ASGPASGP--R recognizes terminal R recognizes terminal ββ--galactosylgalactosyl residues on residues on desidesiaalylatedlylatedglycoproteinsglycoproteins

-- Control of intracellular traffic of Control of intracellular traffic of glycoproteinsglycoproteins and and liverliver clearanceclearance from the from the circulatory system of circulatory system of desialylateddesialylated glycoproteinsglycoproteins and apoptotic cells and apoptotic cells

-- Interactions of tumour cells with the immune system, masked by Interactions of tumour cells with the immune system, masked by sialylsialylterminalsterminals

-- Adhesion of infectious agents to host cellsAdhesion of infectious agents to host cells-- Recruitment of leucocytes to inflammatory sites (Recruitment of leucocytes to inflammatory sites (SelectinsSelectins) )

Molecular fit between pairs of complementary structuresMolecular fit between pairs of complementary structures::lectins lectins carbohydratescarbohydrates ((ligandsligands))

This action can be blocked by appropriate sugars This action can be blocked by appropriate sugars in vivoin vivo and and in vitroin vitro

LLiveriver ASGP ASGP RReceptoreceptorStructuralStructural aspectsaspects

-Composed of two homologous subunits, smaller H1 and bigger H2 in humans (ratio3:1).-Each subunit is a type II transmembrane lectin specific to Gal/N-AcGal.-Each subunit has a short aa cytoplasmatic N terminus, a hydrophobic membrane domain, and an exoplasmatic carboxy terminus exhibiting a carbohydrate recognition domain (CRD) which requires Ca2+ ions.

Models of subunit organization of rat ASGP-R (RHL)

Organization of membrane bound C-type lectins

Mannose macrophagereceptor

Chicken hepaticlectin

Kupffer cell receptor L-selectin

CrystCrystal al StructureStructure ofof CRD CRD ofof H1 H1 SubunitSubunit ofof ASGPASGP--RRCa(2) binding site (8 O atoms)

Gal binds at Ca(2) binding sitereplaces axial water molecules 11 and 13 by (3)OH and (4)OH groups of the carbohydrate

Overlay of sugar binding site of H1-CRD of ASGPR and CDR of MBP mutant containing N-AcGal

Structure of H1- CRD (aas 147-290)

Burkhard,…J Mol Biol, 2000

LLiveriver ASGP ASGP RReceptoreceptorReceptor Receptor mediatedmediated eendocytosisndocytosis via chlathrinvia chlathrin--coated pit pathwaycoated pit pathway

Ligands are cleared from the circulation by receptor-mediated endocytosis and degraded in lysosomes, while the receptor is recycled to the cell surface.

1. Ligand (L) binding2. Receptor (R)-ligand (L) internalization3. Uncoating4. Segregation of R and L5. L delivery to lysossomes6. L degradation7. Reactivation of R8. Recycling of R to cell surface9. Formation of new coated pits

TTargetargetinging LLiveriver ASGP ASGP RReceptorseceptorsPrevious studiesPrevious studies

Target ASGPTarget ASGP--R: attach R: attach galactosylgalactosyl target residues to a target residues to a carrier containing efficient reporter groupscarrier containing efficient reporter groups

reporter reporter -- carrier carrier –– vector vector -- technique technique -- referencereference

-- 111111In In –– DOTA DOTA -- gal Gamma Imaging gal Gamma Imaging Meade, et.al., 2003Meade, et.al., 2003-- 99m99mTc Tc -- DTPADTPA-- GSA GSA

((galactosylatedgalactosylated HSA) SPECT HSA) SPECT Vera et. al., 2001Vera et. al., 2001-- MION MION -- ASF (ASF (asialofetuinasialofetuin) MRI Brady, ) MRI Brady, WeisslederWeissleder, et al., 1993, et al., 1993-- USPIO USPIO –– AG (AG (arabinogalactanarabinogalactan) MRI Brady, ) MRI Brady, WeisslederWeissleder, et. al., 1991, et. al., 1991-- SL (spin label)SL (spin label)nn –– AG MRI AG MRI GallezGallez, et. al., 1994, et. al., 1994-- (GdDTPA)(GdDTPA)858858galgal22842284-- PL PL

((polylysinepolylysine –– 2136 amino groups) 2136 amino groups) MRI Vera, et. al., 1995MRI Vera, et. al., 1995

-- SPECT quantification of SPECT quantification of asialoglasialoglyycoproteincoprotein receptorreceptor (ASGP(ASGP--R)R)correlated with hepatic function in normal vs. pathologycorrelated with hepatic function in normal vs. pathology

-- Tumours (Tumours (egeg. . hepatomahepatoma) have reduced ASGP) have reduced ASGP--R, uptake of R, uptake of targeted drugs much decreased vs. normal targeted drugs much decreased vs. normal

-- ASGPASGP--R present in hepatic carcinoma metastasesR present in hepatic carcinoma metastases-- Some of these findings also detected by MRISome of these findings also detected by MRI

Our approach:Our approach:-- GalactosylGalactosyl residues were employed as a targeting device as residues were employed as a targeting device as

part of part of dendrimericendrimeric glycoconjugateglycoconjugates where they were s where they were attached to reporter groups containing the attached to reporter groups containing the LnLn(III)(III)--bindingbindingmoiety, because moiety, because galactosegalactose binding binding ASGPASGP--RR are exposed on are exposed on the surface of liver the surface of liver parenchimalparenchimal cells (~500,000/cell)cells (~500,000/cell)

TTargetargetinging LLiveriver ASGP ASGP RReceptorseceptorsPreviousPrevious resultsresults and and oourur approachapproach

DTPA- derived dendrimeric glycoconjugatesSynthetic Approach

Convergent synthesis

M n

+

+

+

carboxylic acid groupcarboxylic acid group

sugar moietysugar moiety

amine groupamine group

Pro-chelator: DTPA bisanhydrideSugar block: carboxylate functionalized

glycodendromer (G0, G1, G2)

DTPADTPA--bisamidebisamide glycoconjugatesglycoconjugates

N NN

O

R1

HOOH

HO

R2

NH

S

O

NH

OH

O

O

O

HO

O

HOO

HN

ON

HN

HNS

O

O

O

OHHO

OH

HO

S

OHO

HO

OH

OH

NH

2-DTPA(Gal)4

O

R1

OH

OH

HO

R2

NH

SO

HN

N NN

OH

O

O

O

HO

O

HOO

HN

HN O

N

HN

NH

S

O

O OOH

OH

OH

HO

S

O

OHHO

OH

HO

1a- R1= OH and R2= H - DTPA(Gal)21b- R1= H and R2= OH - DTPA(Glc)21c- R1=H and R2= β−D-galactopyranosyl - DTPA(Lac)2

n = 2

n = 4

DDendrimericendrimeric LnLn((DTPADTPA--bisamidebisamide) ) glycoconjugatesglycoconjugates

Ln(DTPALn(DTPA))-- (Sugar)(Sugar)nn

Sugar = Sugar = GalGal, , GlcGlc, , LacLac

n = 2, 4 n = 2, 4

OR1

AcO

OAc

OAc

R2 HNS

ONHR

OR1

AcO

OAc

OAc

R2 S

O

OH

H2NNHR

3

4 5- R=Boc

6- R= H

+

a- R1= OH and R2= H b- R1= H and R2= OH c- R1=H and R2= β−D-galactopyranosyl

a

b

HNO

N

HNNH

S

OO

O

OAcOAc

OAcAcO

SO

OAc

AcOOAc

AcO

NHR

O

N

HNNH

S

OO

O

OAcOAc

OAcAcO

S

O

OAc

AcOOAc

AcO

RO

H2N NHR

+

4 9- R = Boc

10 - R = H

7- R = tertBu

8 - R = Ha

a

b

SynthesisSynthesis ofof DTPADTPA--bisamidebisamide glycoconjugatesglycoconjugates

BocBoc protectedprotectedHMDHMD--functionalizedfunctionalized

monovalentmonovalent thioglycosidesthioglycosides

BocBoc protectedprotectedHMDHMD--

functionalizedfunctionalizeddivalentdivalent

thioglycosidesthioglycosidesand and deprotectiondeprotection

TFA-DCM

TFA-DCM

TFA-DCM

i) DIPEA/DCM

ii) DCC/HBT, DCM

DCC/HBT, DCM

and and deprotectiondeprotection

N NN

HOO

O

O

O

O

O

O 11

+ 6

11 + 10

a- R1= OH and R2= H - DTPA(Gal)2b- R1= H and R2= OH - DTPA(Glc)2c- R1=H and R2= β−D-galactopyranosyl - DTPA(Lac)2

2 - DTPA(Gal)4a, b

a,b 1

SynthesisSynthesis ofof DTPADTPA--bisamidebisamide glycoconjugatesglycoconjugates

DTPADTPA--bisamidebisamide glycoconjugatesglycoconjugates: mono and : mono and divalentdivalent dendromersdendromers

a) i) DIPEA/DCM; ii) DMF

b) i) KOMe/EtOH; ii) Amberlist 15, NH3(aq)

iii) RPC8 silica, elution with H2O/MeOH

NMR cNMR characteriharacterizzationation ofof Ln-DTPA-bisamideschelates

L = DTPAGalL = DTPAGal22LaL 25 ºC

N NN

O

R1

HOOH

HO

R2

NH

S

O

NH

OH

O

O

O

HO

O

HOO

HN

ON

HN

HNS

O

O

O

OHHO

OH

HO

S

OHO

HO

OH

OH

NH

2-DTPA(Gal)4

O

R1

OH

HO

NH

SO

HN

N NN

OH

O

O

O

HO

O

HOO

HN

HN O

N

HN

NH

S

O

O OOH

OH

OH

HO

S

O

OHHO

OH

HO

1a- R1= OH and R2= H - DTPA(Gal)21b- R1=H and R2= β−D-galactopyranosyl - DTPA(Lac)2

a b

c d

ef

g h

i j

kl

m1

2

3 R2

OH

45

6

SmL 60 ºC EuL 7 ºCGal and bridges

Gal and bridges

DTPA backbone

DTPA backbone

4 diastereoisomeric pairs of enantiomers

1 23

4

5

6,6’

f,k g,jh,i

a

lmb

c

d e

Coordination polyhedrons of theeight enantiomers ofLn-DTPA-bisamides chelates

Conformational interconversion inthe diethylenetriamine backbone ofLn-DTPA-bisamides chelates

Geraldes et al, Inorg. Chem., 1993

SBM SBM TheoryTheory ofof InnerInner--SphereSphere ProtonProton RelaxivityRelaxivity

r r r1 1is 1os= + InnerInner-- and and outerouter--spheresphere contributionscontributions

rq

T1is1mH

m

= × ×+

11000 5555

1. τ InnerInner--spheresphere termterm exchangeexchange –– parametersparameters: : qq, , ττMM

InnerInner--spheresphere TT1M1M parametersparameters::

( )2 2 2 2

S I d1H d2H6 2 2 2 2

GdH I d1H S d2H1m

1 2 3 7115 4 1 1

oH S S

rTµ γ γ τ τπ ω τ ω τ

⎡ ⎤⎛ ⎞= + +⎜ ⎟ ⎢ ⎥+ +⎝ ⎠ ⎣ ⎦

h rrGdHGdH, , ττMM,, ττRR

eRmd

1111

iiH T++=

τττ

∆∆22, , ττvvTT1e, 1e, TT2e2e

{ }1 125

4 1 3 11

41 41

2T

S Se

ZFS

vS2

v2

S2

v2

⎛⎝⎜

⎞⎠⎟ = + −

++

+

⎛

⎝⎜⎜

⎞

⎠⎟⎟∆ τ

ω τ ω τ( ) 1 526

1 0 372718

1 1242

2T e

ZFS

vS2

v2 S v

⎛⎝⎜

⎞⎠⎟ =

++

+

⎡

⎣⎢⎢

⎤

⎦⎥⎥

∆ τω τ ω τ

..

..

FreedFreed TheoryTheory ofof OuterOuter--SphereSphere ProtonProton RelaxivityRelaxivity

OuterOuter--spheresphere termterm –– parametersparameters:: aaGdHGdH,, DDGdHGdH, , ∆∆22, , ττvv

( )[ ]2 2 2 2

A 0 S I1os os I 1e os S 2e

GdH GdH

32 1 3 ( , ) 7 ( , ) 405 4Nr S S J T J T

a Dπ µ γ γ ω ω

π⎛ ⎞= + +⎜ ⎟⎝ ⎠

h

( )

1 2

GdHGdH

os 1 2 3 2

GdH GdH GdHGdH GdH GdH

e e e

11 4, Re

4 11 9 9

jeje

j j j

iT

J T

i i iT T T

τωτω

τ τ τωτ ωτ ωτ

⎡ ⎤⎛ ⎞⎢ ⎥+ +⎜ ⎟⎜ ⎟⎢ ⎥⎝ ⎠= ⎢ ⎥

⎛ ⎞ ⎛ ⎞ ⎛ ⎞⎢ ⎥+ + + + + +⎜ ⎟ ⎜ ⎟ ⎜ ⎟⎢ ⎥⎜ ⎟ ⎜ ⎟ ⎜ ⎟⎝ ⎠ ⎝ ⎠ ⎝ ⎠⎣ ⎦

FreeFree diffusiondiffusion modelmodel

NMRD of Gd-DTPA - glycoconjugate

25 ºC

60 ºC

Gd(III)DTPALac2

Parameters from the analysis of the NMRD profilesParameters from the analysis of the NMRD profilesGd(III)-DTPA-glycoconjugates

kex298/ 106 s-1 3.3±0.2 0.45±0.1 0.40

∆H‡ / kJ mol-1 51.6±1.4 47.6±1.4 40.0

τrH298 / ps 58±11 66±11 332±10

ERH / kJ mol-1 17.3±0.8 21.9±0.5 36.3±0.2

τv298 / ps 25±1 25±1 10±2

Ev / kJ mol-1[g] 1.6±1.8 3.9±1.4 1

∆2 / 1020 s-2 0.46±0.02 0.41±0.02 0.63±0.02

DGdH298/ 10-10 m2 s-1 20±3 23±2 24.0

DTPA DTPA-BMA DTPALac2

EDGdH /kJ mol-1 19.4±1.8 12.9±2.1 20

green parameters are fixed in the fit; Gd-DTPA-BENGALAA: τrH298 = 265 ± 22 ps, Lammers, et al, 1997)

GammaGamma ScintigraphyScintigraphy ofof WistarWistar ratsrats153153Sm(III) Sm(III) labeledlabeled glycoconjugatesglycoconjugates

DOTAGalDOTAGal22 30 min30 min DOTAGlcDOTAGlc22 30 min30 min

- Gal conjugates target liverGal < Gal2 ~ Gal4

- Lac conjugates intermediate- Glc conjugates do not

Uptakes as % ID are small

Biodistribution in Wistar rats153Sm(III)-glycoconjugates

- Gal conjugates target liverGal < Gal2 < Gal4

Cluster effectbloo

d

kidn

ey

liver

sple

en

hear

t

lung

s. in

test

stom

ach

l. in

test

brai

n

gal 2gal 4

0

0.05

0.1

0.15

0.2

0.25

% ID

/g

at 1h

at 24h

-- A series of new DTPA glycosidaseA series of new DTPA glycosidase--resistant resistant dendrimericdendrimeric

glycoconjugatesglycoconjugates was synthesized and chemically characterizedwas synthesized and chemically characterized

-- The solution structure of some of their The solution structure of some of their LnLn complexes was studied by NMRcomplexes was studied by NMR

-- The water The water relaxivityrelaxivity of their of their Gd(IIIGd(III) ) chelateschelates was investigated by NMRDwas investigated by NMRD

-- Preliminary studies of the pharmacokinetics and Preliminary studies of the pharmacokinetics and biodistributionbiodistribution of theof the

153153SmSm--labeled labeled glycoconjugatesglycoconjugates in in WistarWistar rats shows selective liver rats shows selective liver uptake ofuptake of

the the glycoconjugatesglycoconjugates with with galactosegalactose terminal groups but not of the othersterminal groups but not of the others

-- These new These new glycoconjugateglycoconjugate ligandsligands may be selective to the liver ASGPRmay be selective to the liver ASGPR

and are potentially useful for liver targeting and imaging (and are potentially useful for liver targeting and imaging ( 153153SmSm3+3+, , 111111InIn3+3+ forfor

gamma gamma scintigraphyscintigraphy and and Gd(IIIGd(III) for MRI)) for MRI)

ConclusionsConclusions

Collaborations

-J. A. Martins, Paula Baía (Univ. Minho, Braga, PT)

- C. F. G. Geraldes (Dep. Biochemistry, Univ. Coimbra, PT)

- J.J P. Lima, I. Prata, A.C. Santos (Fac. Medicine, Univ. Coimbra, PT)

- M. Anjos Neves (ITN , Sacavém, PT)

- A. E. Merbach, E. Tóth, (EPFL, Lausanne, CH)

Support

F.C.T. (Portugal): grant POCTI/QUI/47005/2002

COST Chemistry D18 Program of the E.U “Chemistry of Metals in Medicine”