synthesis and characterization of new low-molecular-weight lysine-conjugated gd-dtpa contrast agents

Full Paper

Received: 1 July 2010, Revised: 4 September 2010, Accepted: 7 July 2010, Published online in Wiley Online Library: 29 December 2010

(wileyonlinelibrary.com) DOI:10.1002/cmmi.422

Synthesis and characterization of newlow-molecular-weight lysine-conjugatedGd-DTPA contrast agentsSophie Laurenta, Carmen Burteaa, Luce Vander Elsta

and Robert N. Mullera*

Various blood pool contrast agents (CAs), characteriz

Contrast M

ed by intravascular distribution, have been developed to assistcontrast enhanced magnetic resonance angiography (MRA). Among these CAs, the DTPA derivatives conjugated tosynthetic polypeptides, such as polylysine, represent attractive candidates for blood pool imaging. However, due tothe presence of charged residues located on their backbone, these agents are retained in the kidneys and thiscompromises their long blood half-life. In order to overcome this major drawback of the polylysine compounds, twonew low-molecular-weight CAs were synthesized in the present work by conjugating four or six1-p-isothiocyanatobenzyl-DTPA moieties to tri- or penta-Lys peptides [(Gd-DTPA)4Lys3 and (Gd-DTPA)6Lys5], respect-ively. All the –NH2 groups of Lys were thus blocked by covalent conjugation to DTPA. The stability and relaxometricproperties of these compounds, as well as their pharmacokinetic and biodistribution characteristics, were thenevaluated. The half-life in blood of these new polylysine derivatives, as determined in rats, is twofold longer than thatof Gd-DTPA. The compounds could thus be optimal blood pool markers for MRA, which typically uses fast acquisitiontimes. The absence of positive molecular charge did not limit their retention in kidneys 2h after administration. Onthe other hand, (Gd-DTPA)4Lys3 is retained in kidneys to a lesser extent than (Gd-DTPA)6Lys5. Their moderateretention in blood and their higher stability and relaxivity in comparison with Gd-DTPA highlight these polylysinederivatives as optimal compared with previously developed polylysine compounds. Copyright # 2010 John Wiley &Sons, Ltd.

Keywords: Gd-DTPA-conjugated polylysine compounds; MRI contrast agents; blood pool contrast agents; MR angiography

* Correspondence to: R. N. Muller, Department of General, Organic and Bio-medical Chemistry, NMR and Molecular Imaging Laboratory, University ofMons, Avenue Maistriau 19, Mendeleev Building, B-7000 Mons, Belgium.E-mail: [email protected]

a S. Laurent, C. Burtea, L. Vander Elst, R. N. Muller

Department of General, Organic and Biomedical Chemistry, NMR and

Molecular Imaging Laboratory, University of Mons, Avenue Maistriau 19,

Mendeleev Building, B-7000 Mons, Belgium 2

1. Introduction

The diagnosis of a diversity of vascular diseases (e.g. traumaticinjuries, ulcers, infectious diseases, tumors, embolism andatherosclerosis) requires the visualization of the vascular system,which is possible by magnetic resonance angiography (MRA)among other clinical imaging techniques. MRA can be carried outin the absence of any contrast agent (CA), but the accuratediagnosis of vascular diseases could be hindered by artifactstriggered by the turbulent blood flow (1). To solve thisshortcoming, various blood pool CAs, characterized by intravas-cular distribution, have thus been developed to assist theimaging technique currently known as contrast enhanced MRA(CE-MRA) (2). These CAs are basically characterized by enhancedr1 and low r2 relaxivities, a prolonged vascular residence timeand a limited extravasation to allow repeated image acquisitionsafter a single administration (3). Among the strategies thathave been approached to attain this aim (1,4), one may citereversible binding to serum albumin (5–7), mimicking plasmaproteins (macromolecules and colloids) (8,9), and mimickingcirculating blood cells (liposomes and micelles) (10,11).In order to develop macromolecular blood pool CAs, synthetic

and natural polymers (e.g. serum albumin, dextran, polylysine)were often conjugated to diethylenetriamine-pentaacetic acid(DTPA). Since they contain a large number of available chelatingresidues, the DTPA derivatives conjugated to synthetic polypep-tides such as polylysine represent attractive candidates for blood

edia Mol. Imaging 2011, 6 229–235 Copyrigh

pool imaging (3), and they have been extensively assessed inpulmonary (12), renal (13), cardiac (14,15) and gastrointestinalhemorrhage (16) imaging. However, the relatively long circulationtime of these compounds (t1/2 in rabbit plasma of 1.4 h) iscurrently compromised by their kidney retention (93% elimi-nated after 7 days) as reported by Schuhmann-Giampieri et al.(17) due to the presence of highly charged residues located ontheir backbone (3). Other compounds have been subsequentlydeveloped, having in mind a reduction of the immunogenicityand of the tissue distribution in conjunction with a significantprolongation of the blood half-life. Bogdanov et al. (18) reportedthe synthesis of a monomethoxy ether of poly(ethylene glycol)covalently attached to poly(L-lysine), this last one serving asthe carrier of the Gd-DTPA which has substituted 25–33% of theamino groups. The blood half-life of this agent was of 14 h,as determined in rats, but its elimination from the body was

t # 2010 John Wiley & Sons, Ltd.

29

Figure 1. Structure of complexes (Gd-DTPA)4Lys3 (n¼ 1) and(Gd-DTPA)6Lys5 (n¼ 2).

S. LAURENT ET AL.

230

extremely slow, i.e. 85% of the injected dose was eliminated in12 days via the kidneys.Aiming to overcome this major drawback of the polylysine

compounds, which often contain more than 500 lysine residuesper molecule, two new low-molecular-weight CAs were synthes-ized in the present work by conjugating four or six1-p-isothiocyanatobenzyl-DTPA (p-SCN-Bz-DTPA) moieties totri- or penta-Lys peptides [(Gd-DTPA)4Lys3 and (Gd-DTPA)6Lys5],

Figure 2. NMRD profiles of (Gd-DTPA)4Lys3 and (Gd-DTPA)6Lys5 as compare

wileyonlinelibrary.com/journal/cmmi Copyright # 2010 Jo

respectively. All the –NH2 groups of Lys were thus blocked bycovalent conjugation to DTPA. The stability and relaxometricproperties of these compounds, as well as their pharmacokineticand biodistribution characteristics, were then evaluated.

2. Experimental Procedures

2.1. Instrumentation

2.1.1. Spectroscopy

The products were identified by 1H NMR (in D2O) on aBruker-AMX-300 instrument (Bruker, Karlsruhe, Germany). Theabbreviations used were: ‘s’ for singlet, ‘d’ for doublet, ‘t’ fortriplet, ‘q’ for quartet, ‘m’ for massif, and ‘br’ for broad.

2.1.2. Mass spectrometry

Electrospray mass spectra were obtained on a Q-TOF 2 massspectrometer (Micromass, Manchester, UK). Samples weredissolved in a MeOH–H2O mixture (50:50).

2.1.3. HPLC

High-performance liquid chromatography (HPLC) was used tocontrol the purity of the ligands and complexes. HPLC wasperformed on a Waters 600 multisolvent delivery system equippedwith a Rheodyne injection valve (20mL loop) and controlled by theMillenium software (Waters, Milford, USA). Novapak C18 column(4.56� 150mm) was used. Elution was performed with a gradientof water–acetonitrile (time 0, 98:2; time 20min, 0:100; time 25min,0:100; and time 27min, 98:2) at a flow rate of 1mlmin�1. A UV/diode array detector was used to monitor the elution of the ligandor of the complex (254 or 270nm).

d with Gd-DTPA.

hn Wiley & Sons, Ltd. Contrast Media Mol. Imaging 2011, 6 229–235

Table 1. Parameters obtained by the theoretical fitting of the relaxometric data

Complexes r1 at 0.47 T (s�1 mM�1) r1 at 1.4 T (s�1 mM

�1) tR (ps) tSO (ps) tV (ps) r (nm)

Gd-(DTPA)4Lys3 110 79 25 0.30mol Gd3þ 5.7 5.0mol CA 22.8 20

Gd-(DTPA)6Lys5 128 83 33 0.30mol Gd3þ 5.9 5.7mol CA 35.4 34.2

Gd-DTPA 3.8 3.6 59 82 23 0.31

The following parameters were fixed: D¼ 3.3� 10�9 m2 s�1, d¼ 0.36 nm, q¼ 1. tM of Gd-(DTPA)4Lys3 and Gd-(DTPA)4Lys3 was set to100ms, a value that does not limit the relaxivity at 310 K. The longitudinal relaxivity at 0.47 and at 1.4 T is expressed per mol ofgadolinium or per mol of CA.

LOW-MOLECULAR-WEIGHT LYSINE-CONJUGATED GD-DTPA DERIVATIVES

2.1.4. Relaxometry

Proton nuclear magnetic relaxation dispersion (NMRD) profileswere measured on a Stelar Spinmaster FFC, fast field cycling NMRrelaxometer [Stelar, Mede (PV), Italy] over a magnetic field strengthrange from 0.24mT to 0.24 T. Measurements were done on samplesof 0.6ml. Additional relaxation rates at 20, 60 and 200MHz wererespectively obtained on a Minispec MQ-20, a Minispec MQ-60(Bruker, Karlsruhe, Germany) and a high-resolution spectrometer(Bruker MSL 200, Bruker, Karlsruhe, Germany). Fitting of the 1HNMRD was performed with a data-processing software that usesdifferent theoretical models describing observed nuclear relaxationphenomena (Minuit, CERN Library) (19,20).

2.1.5. Transmetallation kinetics

The evolution of the water proton paramagnetic longitudinalrelaxation rate (Rp1) of a buffered solution (phosphate buffer, pH¼7) containing 2.5mM of gadolinium complex and 2.5mM ofZnCl2 (21) was measured on a spin analyzer Minispec MQ-20

Figure 3. Transmetallation of (Gd-DTPA)4Lys3 and (Gd-DTPA)6Lys5 as compa

Contrast Media Mol. Imaging 2011, 6 229–235 Copyright # 201

(Bruker, Karlsruhe, Germany) at 20MHz and 378C. The tempera-ture was kept constant through a perchlorinated liquid flow. Thesamples (0.3ml) were kept in 7mm o.d. pyrex tubes at 378C in adry block between measurements (up to 4320min) (22).

2.2. Synthesis of (Gd-DTPA)4Lys3 and of (Gd-DTPA)6Lys5

All chemicals were purchased from Sigma-Aldrich (Bornem,Belgium) and were used without purification. The p-SCN-Bz-DTPAligand was purchased from Macrocyclics (Dallas, TX, USA).

2.2.1. Conjugation of Lys3 and Lys5 to p-SCN-Bz-DTPA

The peptide in aqueous solution was added to a water solution ofp-SCN-Bz-DTPA (8 and 12 equivalents). The pH of the solution wasset between 9 and 10 and the mixture was stirred during 48 h atroom temperature. The pH was then adjusted to 7. The productwas purified by dialysis on a Spectra/Por Biotech Cellulose Ester(CE) membrane with a cut-off of 1000 Da (VWR, Leuven, Belgium)and by column chromatography on silica gel 60 RP18 (40–63 mm;

red with Gd-DTPA.

0 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/cmmi

231

Figure 4. Plasma pharmacokinetic profiles of (Gd-DTPA)4Lys3 and

(Gd-DTPA)6Lys5 in Wistar rats. The data are represented as gadolinium

(A) or contrast agent (B) plasma concentrations and are compared toGd-DTPA.

S. LAURENT ET AL.

232

Merck, Darmstadt, Germany) with methanol–water 40:60 (v/v) aselution solvent.

(DTPA)4Lys3: yield, 14%; dH (D2O, 258C): 1.3–2.0 (24H, m, 12�CH2 lys); 2.5–3.0 (24H, m, 12� CH2N); 3.2–3.3 (48H, m, 20�CH2COOH, 4� CH2f); 4.4–4.7 (7H, m, 7� CH); 7.8–7.2 (16H, m,f). MS-ESI: C106H150N22O44S4: 2564 [MþH]þ.(DTPA)6Lys5: yield, 11%; dH (D2O, 258C): 1.3–2.0 (36H, m, 18�CH2 lys); 2.5–3.0 (36H, m, 12� CH2N); 3.2–3.3 (72H, m, 30�CH2COOH, 6� CH2f); 4.4–4.7 (11H, m, 11� CH); 7.8–7.2(24H, m, f). MS-ESI: C162H230N34O66S6: 3900 [MþH]þ.

2.2.2. Complexation with gadolinium

The ligand (0.5mmol) was solubilized in water (2ml). An equimolaraqueous solution of GdCl�36H2O was added dropwise and the pHwas kept between 5.5 and 6.5. The solution was stirred and heatedat 608C during 48h. The pH was then brought to 9 with NaOH inorder to precipitate the non-complexed Gd3þ ions as gadoliniumhydroxide, which can be eliminated by filtration. The pH of thefiltrate was adjusted to 7. The filtrate was treated with Chelex resinduring 2h in order to eliminate the residual free Gd3þ ions. Anarsenazo test confirmed the absence of free Gd3þ ions. The finalconcentration of the gadolinium complex was determined byrelaxometry on a 20MHz Minispec MQ-20.

(Gd-DTPA)4Lys3: MS-ESI, C106H138Gd4N22O44S4, 3183 [MþH]þ.HPLC, 10.37min.(Gd-DTPA)6Lys5: MS-ESI, C162H212Gd6N34O66S6, 4830 [MþH]þ.HPLC: 10.81 min

2.3. Biological characterization

The blood pharmacokinetics and biodistribution of the contrastagents were evaluated on male Wistar rats (250� 20 g; n¼ 3/group; Harlan, Horst, The Netherlands). All the animal exper-iments fulfilled the requirements of the Ethical Committee of ourinstitution.

2.3.1. Blood plasma pharmacokinetics

The rats were anesthetized with 50mg kg�1 b.w., i.p, of nembutal(Sanofi, Brussels, Belgium). They were tracheotomized, and theleft carotid artery was catheterized for blood collection. Gdcomplexes were injected as a bolus through the femoral vein atthe expected clinical dose according to their NMR efficacy (i.e.relaxivity) (23,24): 0.05mmol Gd kg�1 b.w (8.33mmol CA kg�1

b.w.) for (Gd-DTPA)6Lys5 and 0.075mmol Gd kg�1 b.w.(18.75mmol CA kg�1 b.w.) for (Gd-DTPA)4Lys3). Gd-DTPA wasused as a control and injected at a dose of 0.1mmol kg�1 b.w.Blood samples (�0.2ml) were collected (with saline replacement)before and at 1, 2.5, 5, 15, 30, 45, 60, 90 and 120min afterinjection. The gadolinium content of the blood samples wasdetermined by relaxometry at 378C and 60MHz on a BrukerMinispec. A two-compartment distribution model was used tocalculate the pharmacokinetic parameters such as the elimin-ation half-life (Te1/2), the steady-state volume of distribution(VDss) and the total clearance (Cltot). The gadolinium concen-trations in blood were converted to plasma concentrations byassuming a hematocrit value of 0.53 (blood volume, 58ml kg�1;plasma volume, 31ml kg�1) (25).


2.4. Biodistribution

The biodistribution of the Gd complexes was evaluated at theend of the pharmacokinetics experiment. The organs (liver,kidneys, heart, spleen, lungs) were weighted, dried overnight at608C, and subsequently digested (up to 0.4 g each sample) inacidic conditions (3ml HNO3, 1ml H2O2) by microwaves(Milestone MSL-1200, Sorisole, Italy). The gadolinium contentwas determined by inductively coupled plasma–atomic emissionspectroscopy (ICP-AES, Jobin Yvon JY70þ , Longjumeau, France).The results were calculated as percentages of the injected doseg�1 (%ID g�1).

3. Results and discussion

3.1. Synthesis of polylysine-conjugated Gd-DTPAderivatives

Mass spectrometry confirmed the structures of the complexes(Fig. 1) resulting from reacting the peptides [(Lys)3 or (Lys)5] with



an excess of p-SCN-Bz-DTPA ligand, dialyzing the reactionmixture, and then complexing the ligand with GdCl�36H2O.

3.2. Relaxometric characterization

The relaxivities (310 K, 20MHz) were found to be equal to 5.7 and5.9 s�1mM

�1 for (Gd-DTPA)4Lys3 and (Gd-DTPA)6Lys5, respectively(Fig. 2). The continuous increase of the relaxivity at 20MHz whentemperature decreases (from 45 to 58C) demonstrates that thewater exchange is not a limiting factor (data not shown). Theproton NMRD profiles of the complexes were recorded at 310 Kand compared with the NMRD profiles of the parent compoundGd–DTPA (Fig. 2). The observed relaxivity of the new complexesis larger than that of the parent compound Gd-DTPA. The protonNMRD profiles were fitted by the classical description, whichtakes into account two contributions: the innersphere modeldescribed by Solomon (26) and Bloembergen (27), referring tothe short distance interactions, and the outersphere contributiondescribed by Freed (28), accounting for the larger distanceinteractions. Parameters obtained by the theoretical adjustmentof NMRD profiles are summarized in Table 1 [fixed parameters:number of coordinated water molecules, q¼ 1; distance ofclosest approach, d¼ 0.36 nm; relative diffusion coefficient,D¼ 3.3� 10�9 m2 s�1 (29); distance between the Gd3þ ionand the protons of the innersphere water molecule, r¼ 0.30 nm(or 0.31 nm for Gd-DTPA)]. The higher relaxivities at 310 K ascompared with Gd-DTPA are related to a slower rotationalcorrelation time associated with a larger molecular weight.In the presence of HSA, complexes show a weak increase of

their relaxation rates that demonstrates negligible affinity for theprotein. For example, at 20MHz, the r1 values are 7.3 s

�1mM�1 for

(Gd-DTPA)4Lys3 and 7.0 s�1mM�1 for (Gd-DTPA)6Lys5 in 4% HSA

solution. In aqueous solution, the r1 values are 5.7 s�1mM�1 for

(Gd-DTPA)4Lys3 and 5.9 s�1mM�1 for (Gd-DTPA)6Lys5. Transme-

tallation of the two complexes by Zn2þ ions showed a betterstability than that of the commercially used Gd-DTPA derivative(Fig. 3). The stability of the new complexes was tested bymeasuring the exchange between the gadolinium ion and thezinc ion, the latter one being known as the blood ion most likelyto be exchanged with the gadolinium ion in plasma becauseof its similar radius. This experiment was performed in thepresence of phosphate ions, which form an insoluble complexwith the gadolinium ions. During the transmetallation process,

Table 2. Pharmacokinetic parameters of (Gd-DTPA)4Lys3 and (Gd-D

Pharmacokineticparameters (Gd-DTPA)4Lys3

Te1/2 (min) 26.4� 3.9*

Cltot (ml kg�1 min�1)mol Gd3þ 7.32� 1.19mol CA 9.02� 1.32

VDss (l kg�1) 0.30� 0.03*

The results are represented as means� SEM. The statistical signific*p< 0.05;**p< 0.01.


the released gadolinium ions precipitate with the phosphate ionsand they no longer contribute to the proton paramagneticrelaxation rate of the solution. As a result, the paramagneticrelaxation rate of the solution decreases (21). For Gd-(DTPA)4Lys3and Gd-(DTPA)6Lys5, a decrease of 29 and 30% of the protonparamagnetic relaxation rate was observed after 4 days, attestingto a better stability of the complexes as compared with thecommercially used Gd-DTPA derivative (Fig. 3).

3.3. Pharmacokinetic parameters

The pharmacokinetic profiles of the two compounds comparedwith that of Gd-DTPA are presented in Fig. 4 either as gadolinium(Fig. 4A) or as CA (Fig. 4B) plasma concentration.The pharmacokinetic parameters were calculated using abi-exponential fit of the plasma concentration vs time curves(Table 2). The pharmacokinetic parameters show a significantlyprolonged blood residence time for (Gd-DTPA)4Lys3 (Te1/2¼ 26.4min, p< 0.05) and for (Gd-DTPA)6Lys5 (Te1/2¼ 28.5min,p< 0.01), which is almost double that of Gd-DTPA (Te1/2¼ 14.9min). The Cltot is not significantly different from thatof Gd-DTPA (8.66ml kg�1min�1), although it is slightly inferiorfor (Gd-DTPA)6Lys5 (6.94ml kg�1min�1 when expressed in molesof gadolinium; 5.91ml kg�1 min�1 when expressed in moles ofCA). The VDss value (0.3 l kg�1 for (Gd-DTPA)4Lys3 and 0.25 l kg�1

for (Gd-DTPA)6Lys5) is moderately larger than that of Gd-DTPA(0.2 l kg�1) and indicates some extravasation towards theinterstitial space.

3.4. Biodistribution

The biodistribution two hours after injection of the twoGd-DTPA-conjugated polylysine compounds is presented inFig. 5, where they are calculated either as gadolinium (Fig. 5A)or as CA (Fig. 5B) concentration. When calculated as gadoliniumconcentration, the results show that both compounds areaccumulated in the kidneys in significantly (p< 0.01) higherconcentrations as compared with Gd-DTPA. This characteristicis directly related to the length of the Lys chain, i.e.(Gd-DTPA)6Lys5 was found in higher concentration (14.6% ofID g�1) than (Gd-DTPA)4Lys3 (3.8% of ID g�1). In addition,(Gd-DTPA)6Lys5 was also found in significantly (p< 0.01) higherconcentrations in the spleen and lungs. However, when theresults are calculated as CA concentration, the accumulation of

TPA)6Lys5 as compared with Gd-DTPA, determined in Wistar rats

Contrastagent

(Gd-DTPA)6Lys5 Gd-DTPA

28.9� 2.4** 14.9� 1.2

6.94� 1.18 8.66� 1.185.91� 1.450.25� 0.012* 0.20� 0.013

ance was calculated vs Gd-DTPA with Student’s t-test:


233

Figure 5. Biodistributions of (Gd-DTPA)4Lys3 and (Gd-DTPA)6Lys5 as compared with Gd-DTPA. The results are calculated as either gadolinium (A) or CA

(B) concentration and are represented as means� SEM. The Student t-test was calculated versus Gd-DTPA: *p< 0.05; **p< 0.01.

S. LAURENT ET AL.

234

both compounds in the heart, spleen and lungs is inferior to thatof Gd-DTPA. The concentration of (Gd-DTPA)6Lys5 is still higher inkidneys (p< 0.01), but this could be related to the Te1/2, which isthe longest among the tested compounds.

4. Conclusions

Even though the volume of distribution of the two compoundsindicates a leakage into the interstitial space, their half-life inblood is twofold larger than that of Gd-DTPA. The prolongedcirculating times highlight these compounds as potential bloodpool markers for MRA, which typically uses fast acquisition times,e.g. less than 5min. The CAs preferred for MRA have longerelimination half-lives (2,3,30) that allow repeated acquisition ofimages after a single administration, limiting thus the toxic effectsassociated with higher doses. The elimination half-lives of ourpolylysine derivatives is comparable to that of P792 (23) andMS-325 (5) as determined in rats, the volume of distribution beingclose to that of MS-325 in the same species. When calculated permole of CA, the r1 values are in the range of macromolecular P792


(23) and the HSA-bound form of MS-325 ones (8). The length ofthe Lys chain does not seem to significantly influence theduration of the retention in blood. The absence of positivemolecular charge did not limit the retention of the twocompounds in kidneys. On the other hand, (Gd-DTPA)4Lys3is retained in kidneys to a lesser extent than (Gd-DTPA)6Lys5,which could be an advantage from the pharmacological point ofview. However, the presence of the two compounds in thekidneys 2 h after their injection could be simply related to theirdelayed excretion consequent to the prolonged half-life in blood.Their moderate retention in blood and the higher stability andrelaxivity in comparison with Gd-DTPA suggest that these newpolylysine derivatives could be optimal vs previously developedpolylysine blood pool agents (17,18), which are characterized byexcessive blood retention, i.e. 1.4–14 h, and belated excretion.

Acknowledgements

This work was financially supported by FNRS and ARC (researchcontract no. 05/10-335) program of Research of the French



Community of Belgium, and the COST (Cooperation in Scienceand Technology) Action D18 and D38 of the European Com-munity RTD Framework Program.

References1. Burtea C, Laurent S, Vander Elst L, Muller RN. Contrast agents –

magnetic resonance. In Handbook of Experimental Pharma-cology, Semmler W, Schwaiger M (eds). Springer: Berlin, 2008;135–165.

2. Bremerich J, Bilecen D, Reimer P. MR angiography with bloodpool contrast agents. Eur Radiol 2007; 17: 3017–3024.

3. Bogdanov AA, Lewin M, Weissleder R. Approaches and agents forimaging the vascular system. Adv Drug Deliv Rev 1999; 37:279–293.

4. Geraldes CF, Laurent S. Classification and basic properties ofcontrast agents for magnetic resonance imaging. Contrast MediaMol Imag 2009; 4: 1–23.

5. Parmelee DJ, Walovitch RC, Ouellet HS, Lauffer RB. Preclinicalevaluation of the pharmacokinetics, biodistribution, and elimin-ation of MS-325, a blood pool agent for magnetic resonanceimaging. Invest Radiol 1997; 32: 741–747.

6. Muller RN, Raduchel B, Laurent S, Platzek J, Pierart C, Mareski P,Vander Elst L. Physicochemical characterization of MS-325, a newgadolinium complex, by multinuclear relaxometry. Eur J InorgChem 1999; 1949–1955.

7. Parac-Vogt TN, Kimpe K, Laurent S, Vander Elst L, Burtea C, Chen F,Muller RN, Ni Y, Verbruggen A, Binnemans K. Synthesis, charac-terization and pharmacokinetic evaluation of a potential MRIcontrast agent containing two paramagnetic centers with albu-min binding affinity. Chem Eur J 2005; 11: 3077–3086.

8. Corot C, Violas X, Robert P, Gagneur G, Port M. Comparison ofdifferent types of blood pool agents (P792, MS325, USPIO) in a rabbitMR angiography-like protocol. Invest Radiol 2003; 38: 311–319.

9. Corot C, Robert P, Idee JM, Port M. Recent advances in iron oxidenanocrystal technology for medical imaging. Adv Drug Deliv Rev2006; 58: 1471–1504.

10. Parac-Vogt TN, Kimpe K, Laurent S, Pierart C, Vander Elst L, MullerRN, Binnemans K. Paramagnetic liposomes containing amphi-philic bisamide derivatives of Gd-DTPA with aromatic side chaingroups as possible contrast agents for magnetic resonanceimaging. Eur Biophys J 2006; 35: 136–144.

11. Laurent S, Vander Elst L, Thirifays C, Muller RN. Relaxivities ofparamagnetic liposomes: on the importance of the chain typeand the length of the amphiphilic complex. Eur Biophys J 2008;37: 1007–1014.

12. Berthezene Y, Vexler V, Clement O, Muhler A, Moseley ME, BraschRC. Contrast-enhanced MR imaging of the lung: assessments ofventilation and perfusion. Radiology 1992; 183: 667–672.

13. Vexler V, Berthezene Y, Clement O, Muhler A, Rosenau W, MoseleyME, Brasch RC. Detection of zonal renal ischemia with contras-t-enhanced MR imaging with a macromolecular blood poolcontrast agent. J Magn Reson Imag 1992; 2: 311–319.

14. Tombach B, Reimer P, Prumer B, Allkemper T, Bremer C, Muhler A,Heindel W. Does a higher concentration of gadolinium chelates


improve first-pass cardiac signal changes? J Magn Reson Imag1999; 10: 806–812.

15. Roberts HC, Saeed M, Roberts TPL, Muhler A, Brasch RC. MRI ofacute myocardial ischemia: comparing a new contrast agent,Gd-DTPA-24-cascade-polymer, with Gd-DTPA. J Magn ResonImag 1999; 9: 204–208.

16. Gupta H, Weissleder R, Bogdanov AA Jr, Brady TJ. Experimentalgastrointestinal hemorrhage: detection with contrast-enhancedMR imaging and scintigraphy. Radiology 1995; 196: 239–244.

17. Schuhmann-Giampieri G, Schmitt-Willich H, Frenzel T, PressWR, Weinmann HJ. In vivo and in vitro evaluation ofGd-DTPA-polylysine as a macromolecular contrast agent formagnetic resonance imaging. Invest Radiol 1991; 26: 969–974.

18. Bogdanov AA Jr, Weissleder R, Frank HW, Bogdanova AV, Nossif N,Schaffer BK, Tsai E, Papisov MI, Brady TJ. A newmacromolecule asa contrast agent for MR angiography: preparation, properties,and animal studies. Radiology. 1993; 187: 701–706.

19. Muller RN, Declercq D, Vallet P, Giberto F, Daminet B, Fischer HW,Maton F, Van Haverbeke Y. Proc. ESMRMB, 7th Annual Congress,Strasbourg, 1990; p. 394.

20. Vallet P. Relaxivity of nitroxide stable free radicals. evaluations byfield cycling method and optimisation. Ph.D. Thesis. University ofMons-Hainaut, Belgium, 1992.

21. Laurent S, Vander Elst L, Muller RN. Comparative study of thephysicochemical properties of six clinical low molecular weightgadolinium contrast agents. Contrast Media Mol Imag 2006; 1:128–137.

22. Laurent S, Vander Elst L, Copoix F, Muller RN. Stability of MRIparamagnetic contrast media. A proton relaxometric protocol fortransmetallation assessment. Invest Radiol 2001; 36: 115–122.

23. Port M, Corot C, Rousseaux O, Raynal I, Devoldere L, Idee J-M,Dencausse A, Le Greneur S, Simonot C, Meyer D. P792: a rapidclearance blood pool agent for magnetic resonance imaging:preliminary results. MAGMA 2001; 12: 121–127.

24. Port M, Corot C, Raynal I, Idee J-M, Dencausse A, Lancelot E, MeyerD, Bonnemain B, Lautrou J. Physicochemical and biologicalevaluation of P792, a rapid-clearance blood-pool agent formagnetic resonance imaging. Invest Radiol 2001; 36: 445–454.

25. Burtea C, Laurent S, Colet J-M, Vander Elst L, Muller RN. Devel-opment of new glucosylated derivatives of Gd-DTPA for mag-netic resonance angiography. Invest Radiol 2003; 38: 320–333.

26. Solomon I. Relaxation processes in a system of two spins. PhysRev 1955; 99: 559–565.

27. Bloembergen N. Proton relaxation times in paramagneticsolutions. J Chem Phys 1957; 27: 572–573.

28. Freed JH. Dynamic effects of pair correlation functions on spinrelaxation by translational diffusion in liquids. II. Finite jumps andindependent T1 processes. J Chem Phys 1978; 68: 4034–4037.

29. Vander Elst L, Sessoye A, Laurent S, Muller RN. Can the theoreticalfitting of the proton nuclear magnetic relaxation dispersion(proton NMRD) curves of paramagnetic complexes be improvedby independent measurement of their self-diffusion coefficients?Helv Chim Acta 2005; 88: 574–587.

30. Rohrer M. MRI contrast media – introduction and basic propertiesof the blood pool agent Gadofosveset (Vasovist1). In ClinicalBlood Pool MR Imaging, Leiner T, Goyen M, Rohrer M, SchonbergSO (eds). Springer Medizin: Heidelberg, 2008; 4–15.


235

synthesis and characterization of new low-molecular-weight lysine-conjugated gd-dtpa contrast agents

Documents