Towards highly efficient and selective contrast agents for MRI

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Towards Highly Efficient and

Selective Contrast Agents for MRI

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Towards Highly Efficient and Selective

Contrast Agents for MRI

Proefschrift

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus prof. dr. ir. J.T. Fokkema voorzitter van het College voor Promoties,

in het openbaar te verdedigen

op maandag 24 mei 2004 om 13:00 uur

door

Luca FRULLANO

Licentiaat in de scheikunde (Università degli Studi di Torino, Italië) Geboren te Turijn, (Italië)

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Dit proefschrift is goedgekeurd door de promotoren:

Prof. dr. R.A. Sheldon Prof. S. Aime

Toegevoegd promotor: Dr. ir. J.A. Peters

Samenstelling promotiecommissie:

Rector Magnificus, voorzitter

Prof. dr. R.A. Sheldon Technische Universiteit Delft, promotor Prof. S. Aime Universiteit van Turijn, promotor

Dr. ir. J.A. Peters Technische Universiteit Delft, toegevoegd promotor Prof. dr. ir. H. van Bekkum Technische Universiteit Delft

Prof. dr. R.N. Muller Universiteit van Mons-Hainaut, België Dr. C. Platas-Iglesias Universiteit van La Coruña, Spanje Dr. W.A.P. Breeman Erasmus Universiteit Rotterdam Prof. Dr. W.R. Hagen Technische Universiteit Delft, reservelid

ISBN 90-9017880-5

All rights reserved. No part of the material protected by this copyright notice may be reproduced or utilised in any form or by any other means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the author.

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1

1 General Introduction

1.1 MRI and Contrast Agents

Since 1973, when the first MR image was published in Nature1_{, MRI has}

developed unrelentingly, becoming one of the most powerful diagnostic techniques available today. The future perspectives offered to MRI by the use of Contrast Agents (CAs), like an increased image quality, the possibility of targeting a wide variety of diseased tissues and the development of agents responsive to various physical and chemical parameters, have attracted a great deal of attention both from academia and from pharmaceutical industry. The importance that MRI has acquired has been recently acknowledged by the Nobel Prize Committee which awarded the 2003 Nobel Prize in Physiology or Medicine to Paul C Lauterbur and Peter Mansfield for their pioneering contributions leading to the application of MRI in medical diagnosis.

Magnetic Resonance Imaging, as a diagnostic technique, is able to generate extremely accurate 2D and 3D images of organs and tissues by means of NMR

parameters.2-4_{In Table 1.1 the advantages and disadvantages of MRI are}

compared to those of other common imaging techniques.5_{MRI images are}

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Table 1.1 Strength and weakness of various imaging techniques.5 Imaging Modality Spatial Resolution (SR) Time

resolution Tissue Contrast

Sensitivity Contrast Medium Magnetic Resonance Imaging (MRI) − Clinical scanner (0.2 – 0.3 mm) − High-resolution research scanner (10s of µm) 1-2 s − Excellent soft tissue contrast. − Poor contrast for

dense bones − mM Gd − nM Fe Positron emission tomography (PET) − mm s-min − Very high contrast between labeled and unlabeled tissues − pM tracer Single Photon Emission Computed Tomography (SPECT) − Sub-mm min − Very high contrast between labeled and unlabeled tissues − Sub-nM tracer Computed Tomography Contrast-enhanced (CT) − Clinical scanner (0.2 – 0.3 mm) − High-resolution research scanner (10s of microns) 1-2 s − Excellent bone contrast − Moderate soft tissue contrast. − mM Ultrasound (US) − Moderate (0.2 mm) for clinical scanners. − Some high frequency transducers may provide higher SR (µm) ms

− Good soft tissue contrast but less than MRI. − 1 bubble Optical Imaging (OI) − mm (bioluminescence) − µm (fluorescence) min (biolum.) ms (fluoresc.) − Very high contrast between labelled and unlabelled tissues − ~100 cells/voxel (biolum.) − pM (fluoresc.)

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the nuclei of choice because of their high NMR sensitivity, high natural abundance and because the human body is constituted by water for more than 70% in weight. The creation of an MRI image is possible by spatially encoding the resonance of the water protons present in the tissues. Spatial encoding can be obtained using two methods referred to as frequency encoding and phase

encoding.

With frequency encoding (see Fig. 1.1), magnetic field gradients are superimposed on the homogeneous main magnetic field. As a result, protons positioned in different locations resonate at different frequencies. Then, by a mathematical process named ‘back-projection’, it is possible to reconstruct the image. However, this procedure is currently little used in clinical MRI due to the high sensitivity to inhomogeneities in the main magnetic field.

Currently, frequency encoding is applied in combination with phase encoding (see Fig. 1.2). With phase encoding, the spatial discrimination in one direction is achieved applying a magnetic field gradient in that direction prior to the acquisition and repeatedly changing its magnitude. The rate of dephasing is dependent on the location of the individual spin and the strength of the gradient, thus, the phase of the spin contains spatial information.

Fig. 1.1 Frequency encoding, obtained by applying a gradient Gx over the

main magnetic field, allows the spatial discrimination of the NMR resonances arising from molecules present in different positions. More projections would allow a more precise determination of the position.

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The contrast in the MRI image is determined by the difference in signal intensities in the various voxels and it may be influenced by numerous factors, which can be divided in intrinsic and extrinsic ones (Table 1.2). The presence of many factors influencing the contrast can represent a drawback due to the difficulty of controlling all of them, but, consequently, an MRI image encodes a much larger amount of information with respect to other imaging techniques.

Fig. 1.2 (A) Pulse sequence showing the application of a 90° pulse simultaneously to a slice selection gradient (Gs), followed by a phase

selection gradient (Gφ) applied for a short time prior to the acquisition and finally a frequency encoding gradient (Gf) applied during the

signal acquisition. The phase selection gradient is changed stepwise in order to allow the spatial discrimination in the x dimension (B) Effect of the application of the pulse sequence represented in A on the NMR signal arising from nuclei in the grey pixel in 1.

The contrast in an MRI scan can be influenced by the selection of the pulse sequence. One of the most commonly used pulse sequence is the so called

‘spin-echo’ sequence, illustrated in Fig. 1.3 The signal intensity upon applying this

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Table 1.2 Main contrast parameters in magnetic resonance imaging.

Intrinsic Extrinsic

− proton density − T1 relaxation − T2 relaxation − cross relaxation

− dia- and ferro-magnetic perturbations − chemical shift − temperature − diffusion − perfusion − physiological motion − bulk flow − viscosity

− changes of tissue composition

− magnetic field strength − magnetic field

homogeneity − hard and software

parameters − RF sequences − pulse sequence

parameters − contrast agents

Fig. 1.3 Spin echo sequence

(

)

{

1 exp TR-TE / 1

}

{

exp TE /

(

2

)

}

SI= ⋅ ⋅ −K ρ _− T_ ⋅ − T _Eq._1.1

Here K is a parameter dependent on the flow, perfusion and diffusion, ρ

represents the proton density, TR is the repetition time, TE the echo time, and

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By the choice of the appropriate values of TR and TE, one can emphasise the T1

or T2 contribution to the image contrast obtaining T1 or T2 ‘weighed’ images. T1

weighed images are the most common. In these images, tissues with shorter T1

give a brighter response (higher signal intensity) and those with longer T1 give

a darker response.

In the absence of a contrast agent, the contrast in the image is mainly determined by differences in proton concentrations and by endogenous variations in NMR relaxation times (T1 and T2). The differences in proton

concentrations among different tissues are usually insufficient to give rise to an acceptable contrast, whereas endogenous differences in relaxation times may result often in a reasonable contrast. However, the presence of paramagnetic contrast agents (CAs), may cause a dramatic enhancement of the proton relaxation rates, and therefore, improve the contrast. Furthermore, CAs may help to obtain physiological information from the MRI image.

N O O -O N N O O -O -O -O O -O Gd3+ N N N N O O -O O -O O -C H3 OH Gd3+ [Gd(DTPA)] 2-Magnevist® [Gd(HP-DO3A)] ProHance® N N N N O O -O O -O O -O O -Gd3+ N O O -NH N N O O -O O NH O -O C H3 CH3 Gd3+ [Gd(DOTA)] -Dotarem® [Gd(DTPA-BMA)] 2-Omniscan®

Fig. 1.4 Structure of some clinically applied CAs.

The most important class of contrast agents used in medical diagnosis consists of Gd3+_{complexes with polyaminopolycarboxylate ligands}6-9_{(Fig. 1.4). The first}

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CA to be approved for in vivo applications was GdDTPA (Magnevist®_{) that was}

introduced on the market in 1988. Since then other Gd3+_{complexes have been}

approved for use in clinical practice and several new CAs are currently in clinical trials. Other families of contrast agents have been studied and applied for specific types of imaging like gastrointestinal or lung imaging. Their description is out of the scope of this thesis, comprehensive reviews can be found in the literature.10, 11

The efficiency of the MRI CAs is measured in terms of a parameter named relaxivity (r) that indicates the ability of the CA to shorten the relaxation times of the water protons. Relaxivity is defined in Eq. 1.2, where Ti,obs is the observed

longitudinal (i=1) or transversal (i=2) NMR relaxation time, Ti,p and Ti,d are the

paramagnetic and the diamagnetic contribution to the observed relaxation time, respectively, [M] is the millimolar concentration of the paramagnetic species and ri is the longitudinal or transversal relaxivity.

[ ]

, , , , 1 1 1 1 1, 2 i i obs i p i d i d r M i T =T + T = + T = Eq. 1.2

Apart from the normal requirements for pharmaceuticals, CAs must show other specific properties. In the first place, CAs must be efficient relaxation agents for the water protons, that is CAs must be able to markedly change the relaxation times of the water protons in the tissues at concentrations that can easily be tolerated (about 10-4_{M). It is important to note that, in the case of CAs for MRI,}

the response is not only dependent on the local concentration but is also related to the relaxivity of the CA in each specific physiological compartment.

Gd3+_{ions give rapid hydrolysis at physiological pH, producing insoluble}

Gd(OH)3 that accumulates in bones and liver from where it is slowly released.

The toxicity of these ions arises from their ability to compete for binding sites usually occupied12_{by endogenous ions such as Ca}2+_{, Cu}2+_{, and Zn}2+_.13-15_{For this}

reason, the complexes used as MRI CAs must be characterised by a high thermodynamic and kinetic stability to avoid the release of the toxic Gd3+_ions

via trans-metallation with the various competing ions and to avoid problems arising from the toxicity of the free ligand. The stabilities of several complexes of clinical relevance are listed in Table 1.3 along with the stability of the corresponding complexes of some competing ions.6, 16

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Table 1.3 Stability constant of various Gd3+_{complexes (K}_GdL_{) with ligands of}

clinical relevance compared with the stability of the related complexes with some competing cations. The conditional stability constant at pH 7.4 in water at 25 °C for the Gd3+_{complexes is also}

indicated (KcGdL)6

Ligand log KGdL log KcGdL log KCaL log KCuL log KZnL

EDTA 17.7 17 17.37 18 14.70 6 14.8 17 10.61 18_18.7818_16.56 DTPA 22.46 18_17.706_10.7518_21.3818_18.2918 DTPA-BMA 16.85 19_14.906_7.1719_13.0319_12.0419 DOTA 25.3 17 24.6 20 24.0 21 22.1 22 18.33 23 18.6 17 17.23 24_22.6324_21.0524 DO3A 21.0 17_14.9723 14.5 17 11.74 23_22.8723_19.2623 HP-DO3A 23.8 17_17.2123 17.1 17 14.83 23_22.8423_19.3623

Currently, the contrast agents approved for clinical use are mostly non-specific and, once injected, they rapidly equilibrate between the intravascular and the interstitial compartments. Only two of the agents commercialised at present, Multihance®_{and Eovist}®_{, tend to accumulate in a specific compartment, in this}

case the liver. However, it may be expected that this situation will change in the near future because of the ever-growing interest for contrast agents with a range of innovative properties. Some of the new CAs that are being developed are, (i) agents able to target specific biologically important chemical functionalities (targeting contrast agents), (ii) agents able to respond to specific physiological parameters like temperature, pH, enzyme activity, etc. , (responsive or smart contrast agents), and (iii) agents that are able to remain compartmentalised in the circulatory system (Blood pool agents).

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1.2 Paramagnetic Relaxation

As early as in the 1940s, Bloch noted that the long proton relaxation time of water constitutes a drawback for the observation of aqueous systems.25_He

suggested the use of small quantities of paramagnetic agents to overcome the problem. This was demonstrated by the longitudinal relaxation time of the water protons in a concentrated solution of ferric nitrate, which was 10-4_-10-5_s,

about five orders of magnitude lower than the value observed for pure water. Thus, since the early years of NMR, paramagnetic ions have been exploited to shorten the relaxation times of other nuclei, opening the way to their future use as MRI contrast agents.

Fig. 1.5 Representation of the motions that cause fluctuating magnetic fields

on the nuclei surrounding the paramagnetic centre. (A) electron spin relaxation, (B) molecular rotation, (C) chemical exchange. (s= electronic spin; I=nuclear spin).

The relaxation enhancement arises from fluctuations of the local magnetic field experienced by the nuclei in proximity to the paramagnetic centre. This fluctuating magnetic field originates from various phenomena (see Fig. 1.5).26

A

B

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(i) changes in the electron magnetic moment due to the electron relaxation generate fluctuating magnetic fields (Fig. 1.5 A). (ii) rotation of the molecule gives rise to a variation of the magnetic field that is experienced by the nuclei around the paramagnetic centre. This can originate from the magnetic moment of the various unpaired electrons or from the net magnetisation arising from differences in population of the Zeeman levels (Curie spin relaxation) (Fig. 1.5 B). (iii) ligands that exchange between the complex and the bulk cross the magnetic field force lines and thus experience a modulation of the magnetic field (Fig. 1.5 C).

The dynamic behaviour of these phenomena can be described by defining several correlation functions, which describe the time evolution of the various phenomena. A correlation function can be approximated by an exponential

depending on a parameter named correlation time (τc) in the absence of

constraints. Fourier transformation from the time to the frequency domain results in a Lorentzian function of the form:

2 2 ( ) 1 c c J ω τ ω τ = + Eq. 1.3

Fig. 1.6 Spectral density functions J(ω) as a function of frequency obtained for

τc=1×10-9 s (a) and τc =5×10-10 s (b)

This function, the spectral density function, carries the information regarding the various frequencies available in the lattice and their associated relative intensities. The relaxation rate of a nucleus depends on the intensity of a frequency, corresponding to the Larmor frequency ω, present in the lattice. In

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general, the longer the correlation time the higher the percentage of small frequencies.

The various phenomena that are able to induce fluctuating magnetic fields in the nuclei of interest are characterised by their respective correlation times, which combine to the correlation times τc and τe (Eq. 1.4 and Eq. 1.5).

, ,

1 1 1 1

dip

c i s i M r

τ =τ +τ +τ i=1,2 dipolar or “through space” interaction Eq. 1.4

, ,

1 1 1

sc

e i s i M

τ =τ +τ i=1,2 scalar or “through bonds” interaction Eq. 1.5

The time scale of the various correlation times are presented in Fig. 1.7.

Fig. 1.7 Range of typical values of the correlation times

The addition of a paramagnetic solute to a solution produces an increment of the relaxation rate, which adds up to its diamagnetic value (Eq. 1.2).

The paramagnetic effect on the water molecules arises from two different contributions (Eq. 1.6). The ‘inner sphere’ contribution describes the paramagnetic relaxation induced in water molecules that are directly coordinated to the metal centre, while the ‘outer sphere’ contribution describes the contribution in water molecules freely diffusing around the paramagnetic centre. A third contribution often referred to as ‘second sphere contribution’ has been proposed by some groups. It concerns paramagnetic relaxation induced in water molecules that are not directly coordinated to the metal centre, but that are not freely diffusing around the complex due to hydrogen bonds or other weak interactions. This contribution, although not always accepted, is often

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described using the same equations as those used for the inner sphere contribution, but now with a longer distance between the paramagnetic ion and the water protons. The different types of water molecules are sketched in Fig. 1.8.

1 1 1

is os

p p p

r =r +r _Eq._1.6

Fig. 1.8 Schematic representation of the various types of water molecules

surrounding the metal complex together with the parameters influencing the relaxivity. Inner , second , and outer sphere water molecules.

The next paragraphs will describe these contributions more in detail.

1.2.1 Inner Sphere Relaxation

The theory of the relaxation of nuclei in the inner coordination sphere of a

paramagnetic centre has initially been developed by Solomon27_{and has}

subsequently been extended by Bloembergen and Morgan.28, 29

The following equations describe analytically the contribution of the water molecules directly coordinated to the metal centre to the overall relaxation rate.

[ ]

(

)

1, 1, 1 1 55.56 is is M M q M R T T τ ⋅ = = ⋅ + Eq. 1.7 k=1/τM τr τs

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[ ]

(

)

(

)

(

)

(

)

2 2 2 2, 2 2 2, ₂ 1 1 1 1 55.56 ₁ ₁ M M M Mf is is M _M _M _Mf T T q M R T _T τ ω τ _τ _ω  ₊ _{+ ∆}  ⋅ _ _ = = ⋅  ₊ _{+ ∆}    Eq. 1.8

Here, [M] is the molar concentration of the paramagnetic agent, q is the number of water molecules in the first coordination sphere of the metal ion, T1M and T2M

are their longitudinal and transversal proton relaxation time, respectively, τM is

the mean residence lifetime of these molecules in the inner coordination sphere, and ∆ωMf is the chemical shift difference between the free and the bound water

molecules.

When τM>>T1M, the inner sphere contribution is not transferred to the bulk

water and this condition is referred to as ‘slow exchange region’. When

T1M>>τM, the coordinated water molecules are labile and the paramagnetic

contribution can be transferred to the bulk water.

The relaxation time T1M of the metal ion coordinated water molecules is

described in terms of three distinct contributions from the scalar or contact (Ti,c),

the dipolar or pseudocontact (Ti,p) and the Curie (Ti,χ) mechanisms.

, , , , 1 1 1 1 1, 2 i M i sc i dip i i T T T Tχ = + + = _Eq._1.9 Dipolar Contribution

The dipolar contribution arises from the through-space transmitted interaction of the nuclear dipole with the unpaired electron dipoles. The increment of the nuclear relaxation rate due to the interaction with unpaired electron spins is proportional to the square of the interaction energy and to the appropriate spectral density function f(ω,τc/e) (Eq. 1.10).

(

)

2 1 , 3 f , / µ µ _{ω τ} − _∝ ⋅  _⋅     N s i M c e T r Eq. 1.10

Here µN and µs are the nuclear and electronic magnetic moments and r

represents the distance between them. From this general equation and considering the transition probability between the various levels, it is possible to derive the Solomon equations.27

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(

)

(

)

(

)

(

)

1 1 2 2 2 2 0 6 2 1 2 2 2 2 2 2 2 1 2 2 2 2 2 2 0 1 2 2 2 6 2 2 1 2 1 1 2 15 4 3 6 1 1 1 1 3 7 2 15 4 1 1 I M M I dip dip c c c I c I s c I s c c c I c s c g S S R T r g S S r γ β µ π τ τ τ ω τ ω ω τ ω ω τ γ β µ τ τ π ω τ ω τ +   = = _ _ ×     × + +  + + − + +     +     ≅ _ _ _ + _ + +   _ _ Eq. 1.11

(

)

(

)

(

)

(

)

2 2 2 2 2 2 0 6 2 1 2 2 1 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 0 1 2 1 2 2 6 2 2 1 2 1 1 1 15 4 3 6 6 4 1 1 1 1 1 3 13 1 ₄ 15 4 1 1 I M M I dip dip c c c c c I c S c I s c I s c c c c I c s c g S S R T r g S S r γ β µ π τ τ τ τ τ ω τ ω τ ω ω τ ω ω τ γ β µ _τ τ τ π ω τ ω τ +   = = _ _ ×     × + + + + ≅ + + + − + +     + _ _   ≅ _ _ _ + + _ + +  _{ } _ Eq. 1.12

Here γI is the gyromagnetic ratio of the relaxing nucleus, g is the electron g

factor, S is the electronic spin of the paramagnetic ion (for lanthanides it is necessary to use the total angular moment J), β is the Bohr magneton, r is the distance between the relaxing nucleus and the electron dipole, while ωI and ωs

are the nuclear and electronic resonance frequency, respectively. Since |ωs|/|ωI| is 658, the terms |ωs ± ωI| can be approximated by ωs.

Fig. 1.9 Plot of the spectral density functions f1 and 0.5·f2 (corresponding to

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The terms between square brackets in Eq. 1.11 and Eq. 1.12 represent the probabilities of the various possible transitions and contain the spectral density function f(ω,τc). In the fast motion limit, with small τc, R1 and R2 are almost

equal, while outside this region, for long τc, R1 decreases with τc, whereas R2 is

constantly increasing due to the presence of the frequency independent term 4τc

in Eq. 1.12 (Fig. 1.9).

The dipolar contribution is the only one playing a significant role in the longitudinal relaxation rate enhancement of water protons by means of Gd3+

based MRI contrast agents, while other contributions can be important for the transversal relaxation rate enhancement.

Contact Contribution

The contact contribution, arising from the unpaired spin density delocalised on the resonating nuclei, represents an effect transmitted through bonds. Due to its nature the correlation time modulating this contribution is not influenced by the reorientational correlation time.

The contact contribution to the paramagnetic relaxation is given by the Bloembergen equations (Eq. 1.13 and Eq. 1.14).28

(

)

1 2 2 1 2 2 2 1 2 1 3 1 M sc e M sc s e A R S S T τ ω τ     = = + _{ } _ _ +   =  Eq. 1.13

(

)

2 2 2 2 1 2 2 2 1 1 1 3 1 M sc e M sc e s e A R S S T τ τ ω τ     = = + _{ } _ + _ +   =  Eq. 1.14

The contact contribution to R1, as a function of τe, is characterised by a maximum while R2 constantly increases upon increasing the correlation time τe

(Fig. 1.10).

Since ωs2τe2 is extremely large, for Gd3+ complexes, the contact contribution to R1

is usually negligible (except for directly coordinated nuclei in the case of Ln3+

ions other than Gd3+_{), while it is often the dominating contribution to R}₂_for

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Fig. 1.10 Plot of the spectral density functions f1 and 0.5·f2 (corresponding to

the expressions in parentheses in Eq. 1.13 and Eq. 1.14) against τe for

various proton Larmor frequencies (in MHz).

Curie Mechanism

The Curie or susceptibility mechanism describes the dipolar interaction that takes place between the nuclear spins and the magnetic moment generated by the thermally averaged excess of electron population in the electronic spin levels. In the presence of an external magnetic field the electronic spin levels are populated according to the Boltzmann law and a slight excess of population is present in the lower level generating a static magnetic moment aligned with the external field.

The Curie mechanism is modulated only by the reorientational correlation time (or possibly by τM) and is not dependent on the electronic relaxation time since

the electronic magnetic moment is already an average over the electron states. The Curie contribution plays a role only when τr is at least four orders of

magnitude larger than τs. This process is described by the following

equations:30-32

(

)

2 ₂ ₂ ₂ 2 ₂ ₄ ₄ ₂ ₂ 2 0 0 1 6 2 2 2 6 2 2 2 3 2 ( 1) 3 5 4 1 5 4 3 1 I r I r M z I r I r g g S S R S r kT r µ γ β τ µ ω β τ π ω τ π ω τ +     = _ _ = _ _ ⋅ + +     Eq. 1.15

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(

)

2 ₂ ₂ ₂ 2 0 2 6 2 2 2 ₂ ₄ ₄ ₂ ₂ 0 2 6 2 2 1 3 4 5 4 1 1 ( 1) 3 4 5 4 ₃ 1 I r M z r I r I r r I r g R S r g S S kT r µ γ β _τ τ π ω τ µ ω β _τ τ π ω τ     = _ _ _ + _= +       +   = _ _ _ + _ +   _ _ Eq. 1.16

where k is the Boltzmann constant, T is the absolute temperature, r is the distance between the nuclear spin and the paramagnetic ion, and <Sz> is the

expectation value for the z projection of the nuclear spin. In the case of lanthanides the total angular momentum <Jz> should be used instead of <Sz>.

This mechanism contributes significantly to the longitudinal relaxation only for large molecules characterised by long τr and at high magnetic field.

Electronic Relaxation

As pointed out before, the electronic relaxation time can play a role in the modulation of the dipolar and the contact contribution to the inner sphere relaxation.

Several mechanisms can contribute to the electronic relaxation,26, 33-36_{some of}

them correspond with mechanisms observable also in the solid state and are based on the coupling between the electronic spin transition and the lattice vibrational levels (phonons). These mechanisms include direct coupling, Raman processes, and Orbach processes.

Apart from these, three other mechanisms are characteristic of solutions. In the first, electronic relaxation is induced by anisotropy of g (electronic g factor) and

A (scalar coupling constant) and is proportional to τr. In the second, the

relaxation results from the fact that upon sudden change in rotational motion of a molecule the electronic cloud may be slightly distorted and thus generates an instantaneous electric dipole moment which can couple with the spin angular moment and cause relaxation. The third mechanism arises from the modulation of the Zero Field Splitting (ZFS) in solution. Collisions between the solvent molecules and the paramagnetic species cause a transient ZFS of the spin levels and this allows the coupling of rotation with spin transitions. This mechanism defined by Bloembergen and Morgan29_{and Rubinstein et al.}37_{can be described}

by the following equations.

(

)

2 1 2 2 2 2 1 1 2 4 1 3 50 1 1 4 τ τ ∆ ω τ ω τ   = = _ + − _{ }_{ +} + _ +   v v e e s v s v R S S T Eq. 1.17

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(

)

2 2 2 2 2 2 2 5 2 1 4 1 3 3 50 1 1 4 τ τ ∆ _τ ω τ ω τ   = = _ + − _{ }_ + + _ + +   v v e v e s v s v R S S T Eq. 1.18

Here, ∆2_{is the mean squared fluctuation of the ZFS and}τ_v_{is the correlation time}

for the distortion of the metal coordination cage.

For the various lanthanide ions, apart from Gd3+_{, the electronic relaxation is}

believed to be related with Orbach type processes or in some cases with Raman processes. In the case of Gd3+_{the dominant mechanism is the modulation of the}

ZFS.

For Gd3+_{complexes, the value of the low field limit of the transversal electronic}

relaxation time (τs0) is given by:

s0

(

12 v

)

1

2

τ = ∆ τ − Eq. 1.19

The relaxivity of Gd3+_{complexes at low field is mainly dependent on}τ_s0_{, which}

in turn is highly influenced by the symmetry of the Gd3+ _{complex and by the}

nature of the coordinating groups. For example, comparing the relaxivity at low field (< 1 MHz) of GdDOTA and GdDTPA, the more symmetrical GdDOTA shows a higher relaxivity (ca. 12 s-1_mM-1_{) with a}τ_s0_{of 700 ps, while the}

relaxivity of GdDTPA is much lower in the same region (ca. 7.4 s-1_mM-1_{) due to}

a shorter electronic relaxation time τs0 (ca. 80 ps).

1.2.2 Outer Sphere Relaxation

The outer sphere contribution to the relaxation times arises from the electron nuclear dipolar interaction between the paramagnetic centre and the water molecules diffusing around the metal centre.

For small-sized monoaquo Gd3+_{complexes, the outer sphere contribution can}

account for 40-50% of the observed relaxivity, but for macromolecular systems this contribution is of less importance.

The most commonly accepted model to describe the outer sphere contribution, for τd, the diffusion correlation time (Eq. 1.22), and τs having comparable values,

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(

)

(

)

2 2 2 2 0 1, 1, 1 2 1 32 ( 1) 405 4 1000 3 , , 7 , , A os I s os H d s H d s N M r S S T d D J J µ π _{γ γ} π ω τ τ ω τ τ ⋅     = =_ _{ }⋅ _ + × ⋅ ⋅     ×_ + _ = Eq. 1.20

(

)

1 2 3 1 2 2 1 1 ₄ , , Re 4 1 1 ₉ ₉ d d sj d sj d d d d d d sj sj sj i J i i i τ ωτ τ ω τ τ τ τ τ ωτ ωτ ωτ τ τ τ  _ _   ₊ _ ₊ _     _ _  =           + + + + + +  _ _ _ _ _ _            Eq. 1.21

Here NA is the Avogadro’s number, M is the molar concentration of the

complex, J(ω,τd, τsj) is the spectral density function incorporating the

dependence on the correlation time τd and the electronic relaxation time, d is

distance of the closest approach of the solvent protons and the paramagnetic centre, D=DI+DS is the sum of the diffusion coefficients of the complex and of

the water molecules and τd is the diffusion correlation time, which is given by

the following equation.

2 τ = + d I S d D D Eq. 1.22

The outer sphere relaxation is thus described by equations that take into account the modulation of the magnetic field on the relaxing nuclei due to electronic relaxation and diffusion.

Often the outer sphere relaxation contribution is estimated from the relaxivity of a complex of similar size and shape as the complex under study, but that does not coordinate water molecules.

1.3 The Search for High Relaxivities

In the previous paragraphs the dynamic and structural determinants of the relaxivity of paramagnetic complexes have been introduced. The search for more efficient contrast agents involves the optimisation of the various parameters governing the relaxivity.

(28)

These parameters include the various correlation times (reorientational, exchange lifetime and electronic relaxation time), the number of water molecules directly coordinated to the metal and the distance r between the water protons and the paramagnetic centre.

The search for high relaxivities involves the optimisation of all these parameters, but the fine tuning of the distance between the paramagnetic metal and the water protons is difficult to achieve, and a careful control of the electronic relaxation time is not easy as well. For this reason the most attention has been focused on the optimisation of the other parameters.

1.3.1 Number of Coordinated Water Molecules (q)

The number of water molecules directly coordinated to the metal centre (q) cannot be increased over a certain limit because this would affect the overall stability of the complex. Thus, only systems containing up to three water molecules in the inner coordination sphere have been investigated, but currently all the commercial contrast agents have only one molecule of water directly coordinated to the Gd3+_ion.

An alternative approach used to maximise the paramagnetic relaxation is to increase the number of water molecules in the second coordination sphere through the introduction of groups on the ligand that are able to give hydrogen bonding. In this way the number of inner sphere water molecules is not altered and the stability of the complex is not influenced but at the same time the paramagnetic relaxation is enhanced.

1.3.2 Exchange Lifetime (τM)

Lanthanide aqua ions are among the most labile complexes with an exchange rate in the order of nanoseconds. The complexes currently used as MRI CA are characterised by exchange lifetimes more than two orders of magnitude higher than that of the aqua ion Gd(H2O)83+.40 For small Ln3+ complexes, characterised

by a short reorientational correlation time, the exchange lifetime is rarely a limiting factor, but for immobilised complexes, i.e. complexes interacting with slowly tumbling macromolecules, τM can be long enough to quench the

(29)

Fig. 1.11 3-D representation of the dependence of the longitudinal relaxivity

upon τM and T1e for a slowly tumbling Gd3+ complex (τr=30 ns) at

0.47 T (q=1, r=3Å).

The ideal water exchange lifetime is found in the following range:

1 1 1 1 1 , τ τ τ < < M M r s T

The optimal value of τM can be calculated to be 30-40 ns. The water exchange

lifetime must be rapid enough to allow an efficient transfer of the inner sphere relaxivity to the bulk water but at the same time not that rapid that it shortens the correlation times for the dipole-dipole interactions. Simulations predict, for complexes with an optimal value of the exchange lifetime, relaxivities 20-25 times higher than observed for the CAs currently on the market.

The exchange lifetime of a complex might be influenced by the binding with a macromolecule, for example a protein, due to the possibility of establishing hydrogen bonding interactions or due to possible steric blocking of the water exchange pathway.

The exchange of protons from the coordinated water molecules to the bulk water may occur following two different mechanisms: i) exchange of whole water molecules with the bulk, ii) exchange of the water protons independently

-12 -10 -8 -6 -4 20 40 60 80 100 120 -12 -10 -8 -6 -4 r 1 (s -1 m M -1 ) log

Τ

1e (s ) log

τ

M (s)

(30)

from the exchange of the entire molecule (prototropic exchange).41_{The second}

mechanism is catalysed under basic or acidic conditions and thus can become important especially under these conditions.

The water exchange rate in nine coordinated Gd3+_{complexes with}

polyaminopolycarboxylate ligands depends upon several factors. The exchange rate is higher for negatively charged compounds. Also the structural properties of the complexes play an important role. In the [Gd(H2O)8]3+ aqua ion, the

difference in energy between the eight and nine coordinated state is very small and so little energy is required to reach the transition state (coordination number 9) in an associatively activated process. Instead, all the nine

coordinated Gd3+_{polyaminopolycarboxylates are characterised by a}

dissociatively activated exchange. This can be explained in terms of the higher steric hindrance caused by the eight fold coordination of a ligand molecule that prevents a second water molecule to enter the coordination site before departure of the bound water molecule. Moreover, for these complexes the eight coordinated state is energetically unstable, and thus a high energy is necessary to reach the transition state and this is reflected in the lower exchange rate compared to the aqua ion. In general, the dissociative mechanism is favoured in complexes showing high steric crowding around the water binding site.42

1.3.3 Determination of τM

Accurate estimations of the exchange lifetime can be obtained by analysing the temperature dependence of the transversal relaxation rate of the water 17_O

resonance with the technique developed by Swift and Connick.43, 44

According to this theory, the paramagnetic contribution (R2Op) to the transverse relaxation rate of the 17_{O is given by the following equation (analogue to Eq. 1.8}

describing the proton relaxation).45

(

)

2 2 2 2 2 2 O O O O O M ex M p M ex _O _O M ex R k R R P k R k Ο 2 Μ Ο 2 Μ ∆ω ∆ω + + = + + Eq. 1.23

Here PM represents the molar fraction of waters bound to the metal complex, kex

their exchange rate, 2

O M

R their 17_{O transverse relaxation rate and} Ο

Μ

(31)

NMR chemical shift difference between the coordinated water molecules and the bulk water.

The 17_{O transverse relaxation rate of the bound water molecules,}

2

O M

R , is the result of four different contributions (Eq. 1.24), the scalar (sc), dipolar (dip) quadrupolar (quad) and Curie (Cur) contributions.

2 2 2 2 2

O O sc O dip O quad O Cur

M M M M M

R =R +R +R +R Eq. 1.24

For small sized Gd3+_{complexes the scalar mechanism is the only one}

influencing the overall transverse relaxation rate of the bound water molecules. For a complex characterised by a fast reorientational correlation time, in the order of 100 ps, the contribution deriving from all the other mechanism is always lower than 1% in the temperature range 273-363K.

The scalar contribution to the transverse relaxation rate, which depends on the Gd3+_{unpaired electron density localised on the}17_{O nucleus, can be calculated}

with the following equation (analogous to Eq. 1.14 describing the scalar contribution to the proton transversal relaxation).

(

)

2 2 2 1 2 2 2 1 1 3 1 τ τ ω τ     = _{ } + _ + _ +  = _ _ O sc e M e s e A R S S Eq. 1.25

Here S is the electronic spin quantum number, A/ħ is the Gd-17_O_water_scalar

coupling constant and τei (i=1,2) are the correlation times that modulate the

scalar interaction and these can be expressed by the following equation.

1 O 1 1

ei M si

τ− ₌τ − ₊τ−

Eq. 1.26

Here the symbols maintain the meaning given before. For a Gd3+_complex

bound to a macromolecule both the dipolar and the quadrupolar mechanism can become important and have to be considered (Eq. 1.27-29).45_{This introduces}

a dependence of the transverse relaxation rate on the reorientational correlation time τr.45

(

)

2 6 2 2 1 1 τ τ ω τ − +   = _ + _ +   dip O dip d M d Gd O s d K S S R r Eq. 1.27

(32)

1 O 1 1 1 di M s r τ− ₌τ − ₊τ− ₊τ− _Eq._1.28

(

)

2 2 2 3 2 1 O quad quad M r I R K I I τ + = − Eq. 1.29

Here rGd-O is the distance between the Gd3+ ion and the water oxygen atom, Kdip

is a constant that includes the nuclear gyromagnetic ratios and Kquad_{is a}

constant that includes the quadrupolar coupling constant and the asymmetry parameter, and finally, I represents the nuclear spin quantum number (5/2 for

17_O).

The temperature dependence of 2

O M

R follows the temperature dependence of

τM, τr, τv and ∆ω as described by the following equations: ΟΜ

( )

₁ 298.15 1 _exp 1 1 298.15 298.15 j j j T H R T τ ∆ τ − − ₌   ₋     _ _   Eq. 1.30

(

1

)

3 _B g S S B A k T Ο Μ β ∆ω = + = Eq. 1.31

Here τj indicates the various correlation times, ∆Hj represents the activation

enthalpy for the related dynamic process, T is the absolute temperature, B is the magnetic field strength, kB is the Boltzmann constant, g is the g factor for the

free electron, µB is the Bohr magneton, and the other parameters have already

been defined above.

1.3.4 Reorientational Correlation Time (τr).

At the magnetic fields strengths currently employed in MRI (0.5-1.5 T or 20-60 MHz), the relaxivity of Gd3+_{complexes (characterised by long}τ_s_{) is mainly}

determined by the value of the reorientational correlation time that dominates the overall correlation time τc. The lengthening of τr for these systems represents

the major source of relaxivity enhancement and different strategies have been adopted to reach this goal.

(33)

dendrimers. However, the first approach may present some complications. In the first place, during the catabolic pathway, problems of toxicity can arise due to the fact that, for example, proteolytic degradation occurs in lysosomes where the pH is low (about 4-5) and this can induce metal dissociation from the

complex.9_{Other problems that can be encountered are possible allergic}

reactions to these foreign molecules, or possible alteration of the hydrophobic– hydrophilic domains of the biological macromolecule. The non-covalent interaction between the complex and a macromolecule seems thus a more attractive alternative. With this approach the bound complex, characterised by higher relaxivity, is in equilibrium with the free complex that can be excreted via the normal excretory mechanism for small molecules. In this way the equilibrium is ultimately shifted to the free species that will be safely excreted. Simulations based on the Solomon-Bloembergen theory show that relaxivities up to 120 s-1_mM-1_{can be achieved for immobilised monoaquo Gd}3+_complexes

(Fig. 1.11). However, these values have not been reached yet, either because of the bad tuning of other important parameters or because the immobilised complexes retain a certain degree of internal mobility and thus are not only characterised by the global motion of the macromolecule. Another limitation that has been recently investigated arises from the intrinsic mobility of the coordinated water molecules.46, 47

1.3.5 Determination of τr

The reorientational correlation time is often determined by fitting the magnetic field dependence of the water proton relaxation rate (NMRD) according to the Solomon-Bloembergen-Morgan and the Freed theories. However the numerous parameters that influence proton relaxivity prevent an accurate determination of τr. For this reason it is advisable to determine this parameter through

independent measurements.

For a small spherical molecule τr can be estimated using the Debye-Stokes

equation (Eq. 1.32): 3 4 3 eff r B r k T πη τ = _Eq._1.32

(34)

where η is the microviscosity of the solution and reff is the effective radius of the

molecule, both these parameters are difficult to estimate.

An alternative approach to estimate the reorientational correlation time exploits the τr dependence of the longitudinal relaxation time of the water 17O nuclei.

The relaxation of these nuclei in solution of paramagnetic complexes is governed by quadrupolar (quad) and dipolar (dip) mechanisms which are described by the equations Eq. 1.33 and Eq. 1.11 respectively:48, 49

(

)

(

)

(

)

(

)

2 2 2 2 2 2 2 2 1 2 2 2 2 1 3 2 3 1 3 0.2 0.8 10 2 1 1 1 4 3 2 3 1 3 10 2 1 r r q I r I r r I T I I I I I π _χ _η τ τ ω τ ω τ π _χ _η _τ   + = + _ + _≅ − _ + + _ + ≅ + − Eq. 1.33

Here I is the nuclear spin, χ is the quadrupolar coupling constant, η is an

asymmetry parameter, ω is the nuclear Larmor frequency, I is the nuclear spin number and rGdO is the Gd–Owater distance. However, the quadrupolar coupling

constant and the Gd–Owater distance can only be estimated leading to a certain

degree of uncertainty in the τr determination.

Other techniques that can be employed to determine τr make use of 2H and 13C

NMR of diamagnetic analogues of the investigated Gd3+_{complex (Y}3+_{, La}3+ _or

Lu3+_{). Using}2_{H NMR, a chemical modification of the ligand molecule is}

required. Deuterium atoms must be introduced into the ligand molecule and from the measured 2_{H longitudinal}48, 50_{or transversal}51, 52_{relaxation rates,}

which are dominated by quadrupolar interactions, τr can be determined (Eq.

1.33-34). In this case the asymmetry parameter η is negligible, and the

quadrupolar coupling constant, χ , is usually assumed to be 170 kHz.

( )

₍

₎

2 2 2 2 2 2 2 2 2 1 3 2 3 5 2 1 3 400 2 1 3 1 1 4 r r r I r I r I H T I I η _χ _τ τ τ ω τ ω τ     + = ⋅ ⋅ +_ _ _ + + _ − _ _ _ + + _ Eq. 1.34

Here the symbols maintain the meaning given above.

Alternatively, τr can be determined by measuring the dipole-dipole relaxation

(35)

2 2 2 2 6 1, 1 4 o C H r DD CH N T r µ _{γ γ τ} π   = _ _ = Eq. 1.35

Here ħ is the Dirac constant, N is the number of hydrogen atoms bound to the

13_{C nucleus, r}_CH_{is the distance between the carbon nucleus and a directly}

bound hydrogen atom. The dipole-dipole contribution to the overall relaxation rate, 1/T1,DD, can be obtained using a measurement of the nuclear Overhauser

enhancement of the concerning resonance.48

The latter two methods have the advantage that the measured relaxation rates are directly proportional to the rotational correlation time, but disadvantages of these techniques include the necessity to use a diamagnetic analogue. All methods known to determine τr independently have the disadvantage that they

do not measure the rotation of the metal – coordinated water proton vector, which is the parameter really governing the relaxivity.

1.3.6 NMRD.

The solvent proton relaxation rates in solutions of paramagnetic species, according to the models outlined before, show a magnetic field dependence both in the inner-sphere and in the outer sphere contributions. The parameters affecting the relaxivity can then be obtained through a magnetic field dependent study, that is measuring the solvent proton relaxivity over a wide range of magnetic fields and fitting it to the models described above. The plot of the relaxivity against the magnetic field magnitude (expressed as Proton Larmor Frequency) on a logarithmic scale is known as Nuclear Magnetic Relaxation Dispersion (NMRD) profile.39

However, due to the high number of parameters influencing the relaxivity and to the often featureless appearance of the NMRD curves, the values of the fitted parameters are affected by a relatively high uncertainty. It is thus better to evaluate as many parameters as possible using independent techniques and some of them have been introduced in the previous paragraphs. In brief, EPR and 17_{O NMR have proved to be valuable tools to assess the value of some of}

the parameters involved in relaxation. EPR measurements give the opportunity to evaluate the transverse electronic relaxation rates. 17_{O NMR relaxation rates}

and chemical shifts give access to the number of inner sphere water molecules, the rotational correlation time, the longitudinal electronic relaxation rate, and

(36)

most importantly, this technique allows accurate determination of the water

exchange rate. Other techniques like 2_{H NMR or}13_{C NMR can be used to}

estimate the rotational correlation time.

The set of parameters obtained by these techniques characterises the compounds under study allowing the design of improved models.

Despite the apparently featureless aspect of the NMRD curves some conclusions can be drawn at the first glance. The inner sphere relaxation in the magnetic field region between 10 and 100 MHz is mainly determined by the reorientational correlation time τr giving rise to an evident hump for complexes

characterised by a long τr. Instead, the NMRD region at low field is mainly

influenced by the zero-field electronic relaxation time, τs0. This parameter is in

turn dependent upon the symmetry of the coordination cage and on the nature of the donor atoms. As an example, Fig. 1.12 compares the NMRD profiles of two common CAs, Gd(DOTA) and Gd(DTPA).

Fig. 1.12 r1 NMRD profiles of Gd(DOTA) and Gd(DTPA) at 25°C. The lower

lines represent the outer sphere contribution.

Since the two complexes are monohydrated and of similar size, they are characterised by almost identical τr, and therefore, the two profiles are almost

superimposable ( 6

R GdH

r∝ ⋅q τ r ) in the region at high field. In the low field region, GdDOTA shows a higher relaxivity reflecting its longer zero-field electronic relaxation time (τs0=500 ps) as a result of the higher symmetry of this

(37)

1.4 New Classes of Contrast Agents

The last decade has witnessed an exponential expansion of MRI as a diagnostic technique. The reason for this success has to be found in the unparalleled ability of MRI to provide images of soft tissues. However, in many cases, due to poor contrast the use of a CA becomes necessary. A number of substances have been developed and are already widely used in the hospitals worldwide, but these agents are far from optimal. Firstly, their relaxivity, the main parameter affecting the contrast in the image, is far from the high theoretically reachable values (Fig. 1.11). Secondly, almost all these agents are non-specific, in fact once administered, these agents rapidly equilibrate non-specifically between the intravascular and the interstitial compartments.

For this reason, in recent years, a great deal of attention has been devoted to the

development of new and more efficient contrast agents.53_{The strategies}

adopted to obtain CAs characterised by higher relaxivity has been outlined in the previous paragraphs and they rely upon the optimisation of the various parameters that influence the paramagnetic relaxation. Another approach toward the development of better CAs is the development of new agents that are able either to respond to specific physiological chemical or physical parameters or to localise in specific physiological compartments. The development of such agents would give the radiologists a new powerful diagnostic tool. In particular, researchers around the globe have focused their attention on the development of three different new classes of CAs: i) blood pool agents,53-55_{ii) targeting contrast agents,}53, 56_{iii) responsive (or smart)}

contrast agents.53

1.4.1 Blood Pool Agents

The denomination ‘blood pool agents’ refers to those new agents that show a preferential distribution in the cardiovascular system. The structures that have been proposed as CAs for Magnetic Resonance Angiography (MRA) can be divided in three classes, (i) systems based on liposomes (ii) systems based on polymers or particulates, (iii) small complexes interacting with plasma proteins. Targeting of red blood cells may be an obvious way to approach this problem, but the relatively high amount of paramagnetic agent needed to have an acceptable contrast could alter the red blood cells function. To mimic the

(38)

circulatory characteristics of red blood cells, liposomes57_{have been developed}

and different paramagnetic agents have been encapsulated inside these

vesicles.58-63_{Alternatively, monomeric or polymeric complexes can be}

substituted with hydrophobic groups that are able to take part in the liposome formation.64-67_{By now, none of these agents has found a clinical application}

probably due to the relatively high cost of production and because of safety related problems.55

Fig. 1.13 Structure of potential contrast agents for Magnetic Resonance

Angiography.

Other systems employed to develop cardiovascular agents are represented by particulate or polymeric compounds. Examples of agents of this class include ultrasmall particles of iron oxides (USPIOs),68, 69_Gd3+_{loaded zeolites}70_{or low}

molecular weight Gd3+_{complexes conjugated to different polymers}71, 72

(polyaminoacids like polylysine, polysaccharides such as dextrans and inulin, dendrimers). O N N N O O O O O O O O P O O O O O Gd3+ _ _ N N N N COO OOC OOC COO R R''' R' R'' Gd3+ O O O O -- -R= R=R'= R=R'=R''= R=R'=R''=R'''= R'=R''=R'''= H R''=R'''= H R'''= H Gd3+ O N O O O N N O O O O O O O _ _ Gd(DOTA(BOM)x) (x=1-4) Gd(EOB-DTPA) MS-325

(39)

Finally, a more promising approach may be the non-covalent binding of low molecular weight complexes to plasma proteins. Several complexes have been synthesised for this purpose by attaching a hydrophobic moiety to a chelating agent (Fig. 1.13). The most promising one at the moment is MS-325, which is characterised by a very high interaction constant with HSA (Human Serum Albumin). MS-325 is currently undergoing phase III clinical trials.73, 74

1.4.2 Targeting Contrast Agents

Accumulation of a CA at a specific site, for example at a tumour site, would facilitate the diagnosis. For this reason, much attention has been devoted to the development of systems that are able to recognise specific important biochemical determinants (e.g. receptors on tumour cells).

A major problem encountered during the design of targeting contrast agents is the very low concentration of receptors (10–9_–10–13_{mol/g of tissue) that}

combined with the relative insensitivity of this technique makes it difficult to obtain an image with an acceptable contrast. Furthermore, the occupation of all available receptors would disrupt the metabolic equilibrium leading to disastrous consequences. From these considerations the need for CAs showing the highest possible relaxivity is clearly evident. In Table 1.4, the minimum detectable concentration of some CAs is reported.53, 75_{Notably some of them}

would give rise to a detectable contrast even at the very low receptor concentrations. The challenge of developing targeting contrast agents has been tackled using different approaches.

The finding that some tumours, such as brain tumours, lung carcinoma and some metastases, are characterised by a larger negative charge on the cell surface than normal cells prompted Aime et al. to propose a two step approach to cell surface targeting.76_{First, positively charged oligomers of amino acids}

such as polyornithine or polyarginine are left to interact with the cell surface and then a negatively charged Gd3+_{complex is added to obtain a non-covalent}

Coulomb interaction. Such an approach has the advantage that the injected paramagnetic species can be a small chelate that can be easily eliminated by the kidneys, thus reducing the risks of toxic metal ion release.

The targeting of receptors on cell surfaces is pursued either by the use of labelled antibodies or by low molecular weight targeting complexes. With the first approach, care has to be taken to maintain the affinity of the antibody for

(40)

its antigen, which in turn limits the number of groups that can be attached. Furthermore, the overall charge must be controlled because it could change the biodistribution of the antibody.

Table 1.4 Minimal detectable concentration calculated for various Contrast

Agents.77 Contrast Agent Lowest observable CA concentration (calculated) Proton Larmor Frequency

GdHPDO3A r1=3.7 s-1mM-1 5·10-7 mol/g or 100 µM ca. 90MHz

GdHPDO3A r1=4.1 s-1mM-1a 50 µM 500MHz

GdDTPA r1=4.3 s-1mM-1 100 µM 20 MHz

CA immobilised with r1=200 s-1mM-1 8.5·10-10 mol/g

6th_{generation dendrimer-GdDTPA,}

r1=5800 s-1_mM-1_{(per dendrimer)}

1.9·10-10_mol/g

Superparamagnetic iron oxide,

r1=72000 s-1_mM-1_{(per particle)}

1.6·10-11_mol/g

a_{T=15 °C}

The possibility of extensive substitution of some antibodies with minimal loss of immunoreactivity has been demonstrated along with the possibility to label antibodies with dendrimeric and polymeric chelates, liposomes and nanoparticles again without important loss of affinity for the receptor.53

Low molecular weight targeting CAs have some advantages compared to labelled antibodies. They are, for example characterised by a higher diffusion coefficient, which results in a much faster accumulation at the target site than is possible for labelled antibodies (for the latter it may take days or even weeks). These small complexes can also be eliminated rapidly by glomerular filtration through the kidney and, furthermore, low molecular weight agents increase their relaxivity upon binding, which further improves the contrast of the images.

Besides several examples of low molecular weight systems that target receptors directly, a pre-targeting approach has been proposed. With this approach a biotinylated monoclonal antibody is administered. After localisation at the target site, avidin or streptavidin is added which binds to the antibody. The

(41)

excess of avidin or streptavidin is then removed by addition of biotinylated albumin, and finally a biotynilated contrast agent is injected that will bind the avidin-antibody complex.78

Another example in the field of low molecular weight targeting CAs is based on the abnormal glycation pattern of tumour cell surfaces. Many tumours have been shown to overexpress sialic acid on the cell surface and this has been related to the proliferation and metastatic state of certain types of cancers. The difference between the density of sialic acid residues on cancer cell surfaces (up to 109_{per cell) and on normal cells (only 20·10}6_{for a normal human erythrocyte)}

is remarkably high and has been used to develop targeting contrast agents. Bertozzi et al. used peracetylated N-levulinoylmannosamine as a substrate exploiting the enzymes in the biosynthetic pathway of sialosides to introduce ketone-substituted sialic acid on the tumour cell surface (Fig. 1.14).79_This

ketone group is then used as an anchor for covalent binding of a low molecular weight Ln3+_{complex. Using fluorescent Eu}3+_{complexes it has been shown that}

the concentration of complexes on the cell surface is dependent on the sialic acid expression and that it is high enough (10-20 µM) to be visualised by MRI.

Fig. 1.14 Tumour cell targeting via highly expressed sialic acid residues

(adapted from Lemieux et al.79₎

Cell

O N H OAc AcO AcO CH3 O O AcO

Cell

+ O O

Cell

CO2 O H NH OH O H OH O O O NH2 O O CO2 O H NH OH O H OH O N O O NH2 Eu3+ = -N N N O- _O O -O O -O O -O O -O NH NH O S NH2

Towards highly efficient and selective contrast agents for MRI

Towards Highly Efficient and

Selective Contrast Agents for MRI

Towards Highly Efficient and Selective

Contrast Agents for MRI

Contents

1

1

General Introduction

1.1 MRI and Contrast Agents

Intrinsic Extrinsic

(

)

{

}

{

(

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[ ]

1.2 Paramagnetic Relaxation

[ ]

(

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(

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(

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(

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(

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1.3 The Search for High Relaxivities

Τ

τ

(

)

(

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(

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₍

₎