Published on Web 02/24/2004
`
`Physicochemical Properties of Mixed Micellar Aggregates
`Containing CCK Peptides and Gd Complexes Designed as
`Tumor Specific Contrast Agents in MRI†
`Antonella Accardo,‡ Diego Tesauro,‡ Paola Roscigno,§ Eliana Gianolio,^
`Luigi Paduano,§ Gerardino D’Errico,§ Carlo Pedone,‡ and Giancarlo Morelli*,‡
`Contribution from the Centro InteruniVersitario per la Ricerca sui Peptidi BioattiVi (CIRPeB) &
`Department of Biological Chemistry,UniVersity of Naples “Federico II” Via Mezzocannone,
`6 Naples, I-80134, Italy, Department of Chemistry, UniVersity of Naples “Federico II”
`Via Cynthia, Naples, I-80126, Italy, and Department of Chemistry IFM,UniVersity of Turin,
`Via Giuria, 7 Turin, I-10125, Italy
`
`Received October 23, 2003; E-mail: morelli@chemistry.unina.it
`
`Abstract:New amphiphilic molecules containing a bioactive peptide or a claw moiety have been prepared
`in order to obtain mixed micelles as target-specific contrast agents in magnetic resonance imaging. The
`first molecule, C18H37CONH(AdOO)2-G-CCK8 (C18CCK8), contains a C18 hydrophobic moiety bound to
`the C-terminal cholecystokinin octapeptide amide (CCK 26-33 or CCK8). The second amphiphilic
`compound, C18H37CONHLys(DTPAGlu)CONH2 (C18DTPAGlu) or its gadolinium complex, (C18DTPAGlu-
`(Gd)), contains the same C18 hydrophobic moiety bound, through a lysine residue, to the DTPAGlu chelating
`agent. The mixed aggregates as well as the pure C18DTPAGlu aggregate, in the presence and absence
`of Gd, have been fully characterized by surface tension measurements, FT-PGSE-NMR, fluorescence
`quenching, and small-angle neutron scattering measurements. The structural characterization of the mixed
`aggregates C18DTPAGlu(Gd)-C18CCK8 indicates a spherical arrangement of the micelles with an external
`shell of (cid:24)21 Å and an inner core of (cid:24)20 Å. Both the DTPAGlu(Gd) complexes and the CCK8 peptides
`point toward the external surface. The measured values for relaxivity in saline medium at 20 MHz proton
`Larmor frequency and 25 (cid:176)C are 18.7 mM -1 s-1. These values show a large enhancement in comparison
`with the isolated DTPAGlu(Gd) complex.
`
`Introduction
`
`The more promising medical diagnostic imaging procedures
`currently in use are based on nuclear medicine ((cid:231)-scintigraphy
`and positron emission tomography, PET) and magnetic reso-
`nance imaging (MRI) techniques. The critical point for these
`techniques is that both require a different amount of reporter
`compounds (contrast agents) to be accumulated in the area of
`interest.1,2 In fact, contrast agents help to discriminate between
`
`† Abbreviations: AdOO, 8-amino-3,6-dioxaoctanoic acid; Boc, tert-
`butoxycarbonyl; tBu, tert-butyl; C12E5, polyoxyethylene-5-lauryl ether;
`CCK, cholecystokinin; CCK8, C-terminal octapeptide of cholecystokinin;
`CCKA-R, CCKB-R, cholecystokinin receptor types A and B; cmc, critical
`micellar concentration; DCM, dichloromethane; DIPEA, N,N-diisopropy-
`lethylamine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
`DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DpyCl,
`dodecylpyridinium chloride; DTPAGlu, N,N-bis[2-[bis(carboxyethyl)amino]-
`ethyl]-L-glutamic acid; EDT, ethanedithiol; Fmoc, 9-fluorenylmethoxycar-
`bonyl; FT-PGSE-NMR, Fourier transform pulsed gradient spin-echo nuclear
`magnetic resonance; GPCR, G-protein coupled receptor; HATU, O-(7-
`azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HOBt, 1-hydroxybenzo-
`triazole; ICP,
`inductively coupled plasma; MRI, magnetic resonance
`imaging; Mtt, 4-methyltrityl; NMRD, nuclear magnetic relaxation dispersion;
`PyBop, benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium; Rt, retention
`time; SANS, small-angle neutron scattering; TFA, trifluoroacetic acid; TIS,
`triisopropylsilane; TMS, tetramethylsilane.
`‡ Department of Biological Chemistry, University of Naples.
`§ Department of Chemistry, University of Naples.
`^ Department of Chemistry IFM, University of Turin.
`
`10.1021/ja039195b CCC: $27.50 © 2004 American Chemical Society
`
`normal and pathological regions. The quantity of reporter
`compound to be accumulated in the area of interest strongly
`varies between these imaging techniques. While the very
`sensitive nuclear medicine techniques require very low tissue
`concentration (10-10 M) of radionuclide to give diagnostically
`significant, low resolved images, MRI gives very resolved
`images but, due to its very low sensitivity, needs higher
`concentration (10-4 M) of contrast agents such as paramagnetic
`Gd(III) complexes. To reach the required local concentration
`of the contrast agent, many carriers have been developed such
`as liposomes3 and other microparticulates,4 micelles,5 dendrim-
`ers,6 linear polymers,7,8 proteins,9 or peptides,10 all of these
`derivatized with the metal complex of interest. Among these
`
`(1) Weissleder, R.; Mahmood, U. Mol. Imaging Radiol. 2001, 219, 316.
`(2) Aime, S.; Cabella, C.; Colombatto, S.; Geninatti Crich, S.; Gianolio, E.;
`Maggioni, F. J. Magn. Reson. Imaging 2002, 16, 394.
`(3) Glogard, C.; Stensrud, G.; Hovland, R.; Fossheim, S. L.; Klaveness, J. Int.
`J. Pharm. 2002, 233, 131.
`(4) Morel, S.; Terreno, E.; Ugazio, E.; Aime, S.; Gasco, M. R. Eur. J. Pharm.
`Biopharm. 1998, 45, 157.
`(5) Anelli, P. L.; Lattuada, L.; Lorusso, V.; Schneider, M.; Tournier, H.; Uggeri,
`F.; Magn. Reson. Mater. Phys., Biol. Med. 2001, 12, 114.
`(6) Wiener, E. C.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow, O.
`A.; Tomalia, D. A.; Lauterbur, P. C. Magn. Reson. Med. 1994, 3, 1.
`(7) Aime, S.; Botta, M.; Garino, E.; Geninatti Crich, S.; Giovenzana, G.;
`Pagliarin, R.; Palmisano, G.; Sisti, M. Chem. Eur. J. 2000, 6, 2609.
`(8) Bligh, S. W.; Harding, C. T.; Sadler, P. J.; Bulman, R. A.; Bydder, G. M.;
`Pennock, J. M.; Kelly, J. D.; Latham, I. A.; Marriott, J. A. Magn. Reson.
`Med. 1991, 17, 516.
`J. AM. CHEM. SOC. 2004, 126, 3097-3107 9 3097
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`MPI EXHIBIT 1094 PAGE 1
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`A R T I C L E S
`
`Accardo et al.
`
`Figure1. Schematic representation of mixed micellar aggregates.
`
`carriers, micellar aggregates, formed by amphiphilic molecules
`and structurally constituted by a hydrophobic core and a
`hydrophilic shell, have recently drawn much attention owing
`to their easily controlled properties and good pharmacological
`characteristics.11 For example,
`the self-assembling micellar
`organization of Gd(III)(DOTA) complex derivatized with a
`liphophilic tail allows obtaining a high relaxivity MRI contrast
`agent.12
`There is an increasing interest in developing new contrast
`agents with enhanced properties. First, the new contrast agents
`should have high contrast activity of the metal complex. This
`is critical for MRI applications: high relaxivity of the para-
`magnetic Gd complexes allows reducing the very high concen-
`tration of the reporter compound to be realized in the area of
`interest. Second, the new contrast agents should be selective
`for a specific target: this property allows addressing the reporter
`compound only to the targets, such as membrane receptors
`overexpressed by cancer cells. Therefore, the total amount of
`the contrast agent to be injected for diagnostic analysis could
`this result is very important in (cid:231)-scintigraphy in
`be reduced:
`which radioactive isotopes are used.
`This paper is concerned with the development of new contrast
`agents based on mixed micelles that fulfill both these properties.
`The basic idea behind the new contrast agents is that the two
`different monomers synthesized, one of which contains a
`bioactive molecule able to address the aggregates on the specific
`biological target and the other containing a chelating moiety
`able to form stable complexes with the metal of interest, and
`both presenting also a lipophilic tail, could self-assemble with
`each other in a mixed micelle. The hydrophobic chains allow
`
`the monomers to form mixed micelles, leaving the hydrophilic
`heads on the surface of the aggregates available for the
`appropriate task. In Figure 1 is reported a schematic representa-
`tion of the mixed micellar structure formed in aqueous solution
`by the two monomers. The first monomer C18H37CONH(AdOO)2-
`G-CCK8 (C18CCK8) contains a C18 hydrophobic moiety bound
`to the C-terminal cholecystokinin octapeptide amide (CCK 26-
`33 or CCK8).13 Moreover, to ensure that the bioactive peptide
`remains on the external surface of the micelle when the
`aggregate is formed, two oxoethylene linkers and a glycine
`residue were introduced between the lipophilic tail and the
`CCK8 peptide. The choice of the CCK8 peptide is based on
`the knowledge that this peptide displays high affinity for both
`cholecystokinin receptors, CCKA-R and CCKB-R.14 These
`receptors belong to the G-protein coupled receptors (GPCRs)
`superfamily and are localized in the cell membrane. Both
`CCKA-R and CCKB-R are very promising targets for specific
`contrast agents due to their overexpression in many tumors:
`CCKA-R is overexpressed in pancreatic cancer and CCKB-R is
`found in small cell lung cancer, colon, and gastric cancers,
`medullary thyroid carcinomas, astrocitomas, and stromal ovarian
`tumors.15
`The second monomer, C18H37CONHLys(DTPAGlu)CONH2
`(C18DTPAGlu), or its gadolinium complex (C18DTPAGlu(Gd)),
`contains the same C18 hydrophobic moiety bound, through a
`lysine residue, to the DTPAGlu as chelating agent.16 This
`monomer has been designed to keep the chelating agent on the
`external surface of the mixed micelle. The chelating moiety
`could complex radioactive metal isotopes such as 111In(III) for
`application of the mixed micelles as reporter compounds in
`
`(9) Schmiedl, U.; Ogan, M.; Paajanen, H.; Marotti, M.; Crooks, L.; Brito, E.;
`Brasch, R. C. Radiology 1987, 162, 205.
`(10) Liu, S.; Edward, D. S. Chem. ReV. 1999, 99, 2235.
`(11) Torchilin, V. P. AdV. Drug DeliVery 2002, 54, 235.
`(12) Andre, J. P.; Toth, E.; Fischer, H.; Seelig, A.; Macke, H. R.; Merbach, A.
`E. Chem. Eur. J. 1999, 5, 2977.
`
`(13) Silvente-Poirot, S.; Dufresne, M.; Vaysse, N.; Fourmy, D. Eur. J. Biochem.
`1993, 215, 513.
`(14) Wank, S. A. Am. J. Physiol. 1995, 269, G628.
`(15) Reubi, J. C.; Schaer, J. C.; Waser, B. Cancer Res. 1997, 57, 1377.
`(16) Anelli, P. L.; Fedeli, F.; Gazzotti, O.; Lattuada, L.; Lux, G.; Rebasti, F.
`Bioconjugate Chem. 1999, 10, 137.
`
`3098 J. AM. CHEM. SOC. 9 VOL. 126, NO. 10, 2004
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`Mixed Micellar Aggregates as Contrast Agents
`
`A R T I C L E S
`
`diagnostic (cid:231)-scintigraphy, or paramagnetic ions such as Gd-
`(III) for application as contrast agent in MRI. A DTPAGlu-
`CCK8 conjugate has been recently used to give in vitro and in
`vivo very stable complexes with the radioactive isotope 111In-
`(III),17 while Gd(III) complexes of DTPAGlu and of DTPAGlu-
`CCK8 conjugate have been completely characterized for both
`their stability and relaxometric properties.18 The supramolecular
`aggregation of the C18DTPAGlu(Gd) complexes in the mixed
`micelle gives rise to a slow rotation and, consequently, increases
`the proton relaxivity with respect to that shown by monomeric
`Gd(III)-chelate complexes.12 Finally,
`the presence in the
`micelles of two separate monomers gives the unique opportunity
`to tune the ratio between the two active components in order to
`find the right compromise between the number of bioactive
`peptides on the micellar surface to address the micelle on the
`target receptors and the number of the metal-chelate complexes
`able to give high contrast activity of the supramolecular adduct.
`In this paper we present a complete physicochemical char-
`acterization of mixed micelles formed in aqueous solution by
`the two monomers C18CCK8 and C18DTPAGlu in the presence
`or in the absence of a nonionic surfactant (polyoxyethylene-5-
`lauryl ether, C12E5) used to stabilize the micelle formation.
`Moreover, the mixed micelles containing Gd(III) ions complexed
`by the chelating DTPAGlu agent have been studied for their
`relaxometric behavior in view of their use as CCK receptor
`specific contrast agents for MRI applications.
`
`Materials and Methods
`
`Materials. Protected NR-Fmoc-amino acid derivatives, coupling
`reagents, and Rink amide MBHA resin were purchased from Calbio-
`chem-Novabiochem (Laufelfingen, Switzerland). The Fmoc-8-amino-
`3,6-dioxaoctanoic acid (Fmoc-AdOO-OH) was purchased from Neo-
`system (Strasbourg, France). The DTPAGlu pentaester, N,N-bis[2-
`[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid
`1-(1,1-dimethylethyl) ester,16 was provided by Bracco Imaging SpA
`(Milan, Italy) and was used without further purification. For its synthesis
`refer to published methods.16 C12E5 (stated purity >99%) was
`purchased from Sigma-Aldrich (Milwaukee, WI), as reagent grade, and
`it was used without further purification. All other chemicals were
`commercially available by Sigma-Aldrich, Fluka (Bucks, Switzerland),
`or LabScan (Stillorgan, Dublin, Ireland) and were used as received
`unless otherwise stated. The molar mass of the C12E5 surfactant was
`406.60 g mol-1. All solutions were prepared by weight using doubly
`distilled water. Samples to be measured by FT-PGSE-NMR and SANS
`techniques were prepared using heavy water (Sigma-Aldrich, purity
`>99.8%). The pH of all solutions was kept constant at 7.4.
`Chemical Synthesis. Solid phase peptide synthesis was performed
`on a Shimadzu (Kyoto, Japan) Model SPPS-8 fully automated peptide
`synthesizer. Analytical RP-HPLCs were carried out on a Shimadzu 10A-
`LC using a Phenomenex C18 column (Torrance, CA), 4.6 (cid:2) 250 mm,
`eluted with an H2O/0.1% TFA (A) and CH3CN/0.1% TFA (B). Two
`gradients from 60% to 80% B over 10 min at 1 mL/min flow rate and
`from 80% to 95% B over 15 min at 1 mL/min flow rate were used.
`Preparative RP-HPLCs were carried out on a Waters (Milford, MA)
`Model Delta Prep 4000 equipped with a UV lambda-Max Model 481
`detector using a Vydac C18 column (Columbia, MD), 22 (cid:2) 250 mm,
`eluted with an H2O/0.1% TFA (A) and CH3CN/0.1% TFA (B) linear
`gradient at 20 mL/min flow rate. UV-vis spectra were carried out by
`
`(17) Aloj, L.; Carraro, C.; Zannetti, A.; Del Vecchio, S.; Tesauro, D.; DeLuca,
`S.; Panico, M.; Arra, A.; Pedone, C.; Morelli, G.; Salvatore, M. J. Nucl.
`Med. 2004, 45(3), in press.
`(18) Aime, S.; Gianolio, E.; Morelli, G.; Tesauro, D.; De Luca, S.; Anelli, P. L.
`Manuscript in preparation.
`
`using an UV-vis Jasco (Easton, MD) Model 440 spectrophotometer
`with a path length of 1 cm. Mass spectra were carried out on a Maldi-
`tof Voyager-DE Perseptive Biosystem (Framingham, MA) apparatus
`using R-cyano-4-hydroxycinnamic acid as matrix and bovine insulin
`as internal reference. The monodimensional 1H NMR in DMSO-d6
`spectrum was performed on a Varian (Palo Alto, CA) 400 MHz
`spectrometer.
`C18H37CONH(AdOO)2-G-CCK8 (C18CCK8). Peptide synthesis
`was carried out in the solid phase under standard conditions using Fmoc
`strategy. Rink amide MBHA resin (0.78 mmol/g, 0.1 mmol scale, 0.128
`g) was used. The peptide chain was elongated by sequential coupling
`and Fmoc deprotection of the following Fmoc-amino acid derivatives:
`Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-
`OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp-
`(OtBu)-OH, Fmoc-Gly-OH, and two residues of Fmoc-AdOO-OH. All
`couplings were performed twice for 1 h, by using an excess of 4
`equivalents (equiv) for the single amino acid derivative. The R-amino
`acids were activated in situ by the standard HOBt/PyBop/DIPEA
`procedure.19 DMF was used as a solvent. Fmoc deprotections were
`obtained by 20% solution of piperidine in DMF. Only the two residues
`of Fmoc-AdOO-OH were added in a single coupling by using an excess
`of 2 equiv. When the peptide synthesis was complete, the resin was
`washed and the terminal Fmoc protection removed. To obtain the
`lipophilic monomer, 0.119 g (0.4 mmol) of nonaoctanoic acid were
`coupled on the linker peptide N-terminus by using 0.208 g (0.4 mmol)
`of PyBop, 0.0612 g (0.4 mmol) of HOBt, and 134 (cid:237)L (0.8 mmol) of
`DIPEA in 2 mL of a mixture DMF/DCM ) 1/1. Coupling time was 1
`h under stirring at room temperature.
`The yield for aliphatic acid coupling, monitored by the Kaiser test,
`was in the range 95-98%. For deprotection and cleavage, the fully
`protected resin was treated with TFA containing triisopropylsilane
`(2.0%), ethanedithiol (2.5%), and water (2.5%), and the free product
`precipitated at 0 (cid:176)C by adding ethyl ether dropwise. Purification of the
`crude mixture was carried out by RP-HPLC, Rt ) 21.9 min. Maldi-
`tof-MS confirms the product identity: C18H37CONH(AdOO)2-G-CCK8
`(MW ) 1687), m/z ) 1688.
`C18H37CONHLys(DTPAGlu)CONH2 (C18DTPAGlu). A sample
`of 624.79 mg (1.00 mmol) of Fmoc-Lys(Mtt)-OH activated by 1 equiv
`of PyBop and HOBt and 2 equiv of DIPEA in DMF was coupled to
`Rink amide MBHA resin (0.78 mmol/g, 0.250 mmol scale, 0.320 g),
`with stirring of the slurry suspension for 1 h. The solution was filtered
`and the resin washed with three portions of DMF and three portions of
`DCM. The Mtt protecting group was removed by treatment with 2.0
`mL of DCM/TIS/TFA (94:5:1) mixture.19 The treatment was repeated
`several times until the solution became colorless. The resin was washed
`by DMF and then the DTPAGlu pentaester chelating agent was linked,
`through its free carboxyl function, to the R-NH2 of the lysine residue.
`This coupling step was performed using 2.0 equiv of DTPAGlu
`pentaester and HATU, and 4 equiv of DIPEA in DMF as solvent. The
`coupling time, compared with the classical solid phase peptide synthesis
`protocol, was increased up to 2 h, and the reaction was tested for
`completion by the Kaiser test. After removal of the Fmoc group by
`20% piperidine in DMF,
`the coupling of nonaoctanoic acid was
`performed in a mixture DCM/DMF (1:1) in the previously described
`condition. For deprotection and cleavage, the fully protected fragment
`was treated with TFA containing TIS (2.0%) and water (2.5%). The
`crude product was precipitated at 0 (cid:176) C, washed several times with small
`portions of water, and recrystallized from methanol and water. The
`product was characterized by 1H NMR spectroscopy and Maldi-tof mass
`spectrometry.
`1H NMR (chemical shifts in (cid:228), TMS as internal standard) ) 4.18
`(m, 1H, CH Lys R), 3.80 (overlapped, 1H, CHGlu R), 3.40 (overlapped,
`8H, CH2COOH) 3.02 (t, 2H, CH2 Lys (cid:15)) 2.81 (m, 8H, N-CH2), 2.22
`
`(19) Chang, W. C.; White, P. D. Fmoc solid phase peptide synthesis; Oxford
`University Press: New York, 2000.
`
`J. AM. CHEM. SOC. 9 VOL. 126, NO. 10, 2004 3099
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`A R T I C L E S
`
`Accardo et al.
`
`(t, 2H, CH2CO), 2.14 (m, 2H, RCH2CH2CO), 1.87 (m, 1H, CH2 Lys
`(cid:226)), 1.76 (m, 1H, CH2 Lys (cid:226)), 1.65 (m, 1H, CH2 Lys (cid:228)), 1.51 (m, 3H,
`CH2 Lys (cid:231),(cid:228)), 1.42 (t, 2H, RCH2CH2CO), 1.25 (m, 30H, CH2 aliphatic),
`0.90 (t, 3H, CH3). Maldi-tof-MS confirms the product identity: C18H37-
`CONHLys(DTPAGlu)CONH2 (MW ) 870), m/z 871.
`Preparation of Gadolinium Complex, C18DTPAGlu(Gd). The
`complexation has been carried out by adding light excess of the GdCl3
`to the aqueous solution of the C18DTPAGlu ligand at neutral pH and
`room temperature. The formation of Gd complex, C18DTPAGlu(Gd),
`was followed by measuring the solvent proton relaxation rate (1/T1).
`The excess of uncomplexed Gd(III) ions, which yields a variation of
`the observed relaxation rate, was removed by centrifugation of the
`solution brought to pH 10; further relaxation rate measurements were
`made to check the complete Gd(III) ions removal.
`Preparation of Solutions. All solutions were prepared by weighting,
`buffering the samples at pH 7.4 with 0.10 M phosphate and 34 mM
`NaCl. pH measurements were made by using the pH meter MeterLab
`PHM 220. The pH meter was calibrated with standards at pH 7 and
`pH 10. In most cases the samples to be measured were prepared from
`stock solutions. All solutions were stirred at room temperature until
`complete dissolution and then used without further treatment.
`In C18DTPAGlu-C18CCK8-water and C18DTPAGlu-C18CCK8-
`C12E5-water solutions the imposed ratio between the solutes was as
`such to have an average of three peptide derivatives per micelle. This
`has been verified through the SANS measurements, as discussed below.
`In C18DTPAGlu-C12E5-water and C18CCK8-C12E5-water
`ternary systems the imposed ratio between stoichiometric solute
`concentrations was 1:3, and 1:9, respectively.
`Surface tension, fluorescence quenching, and SANS measurements
`were also performed on mixtures where C18DTPAGlu was replaced
`with the Gd complex C18DTPAGlu(Gd).
`Surface Tension Measurements. The surface tension, (cid:231), was
`measured with an accuracy of 0.1 dyn cm-1, by the Du Nouy ring
`method, using a KSV Sigma 70 digital tensiometer, equipped with an
`automatic device to select the rising speed of the platinum ring and to
`set the time lag between two measurements. The temperature was kept
`constant at 25.00 ( 0.01 (cid:176) C. The tensiometer was calibrated with water
`and acetone.20
`Self-Diffusion Measurements. The self-diffusion coefficients were
`measured by the FT-PGSE-NMR technique at 25 (cid:176) C.21 A spectrometer
`operating in the 1H mode at 80 MHz and equipped with a pulsed
`magnetic field gradient unit, made by Stelar (Mede, Pavia, Italy), was
`employed. The temperature was controlled within 0.1 (cid:176)C through a
`variable-temperature controller, Model VTC-87. The expression for
`individual spin-echo peak amplitude for a given line is
`
`A ) A0 exp[-(cid:231)mg
`
`2g2D(cid:228)2(¢ - (cid:228)/3)]
`
`(1)
`
`where A0 is a constant for a specific set of experimental conditions,
`(cid:231)mg is the gyromagnetic ratio of the proton, D is the self-diffusion
`coefficient of the species responsible for the NMR signal, g is the
`magnitude of the applied gradient, and ¢ and (cid:228) are spacing and duration
`of the gradient pulses, respectively. Echo delays were kept constant so
`that the relaxation effect must not be accounted for. Equation 1 was
`fitted by nonlinear least-squares routine to the attenuation of the echo
`amplitude sampled as a function of g.
`Fluorescence Quenching. To evaluate the mean aggregation number
`of surfactants into the micelles, fluorescence quenching measurements
`were performed by a Jasco FP-750 (Easton, MD) spectrofluorimeter
`at 25 (cid:176) C. The fluorescence probe pyrene and the quencher dodecylpy-
`ridinium chloride (DpyCl) have been used in all systems. The excitation
`wavelength was set at 335 nm, and fluorescence intensity was detected
`at 383 nm. This latter wavelength corresponds to the third out of five
`peaks of vibronic fluorescence that the pyrene spectrum exhibits. The
`
`(20) Arai, H.; Murata, M. J. J. Colloid Interface Sci. 1973, 44, 475.
`(21) Stilbs, P. NMR Spectrosc. 1987, 19, 1.
`
`3100 J. AM. CHEM. SOC. 9 VOL. 126, NO. 10, 2004
`
`the
`aggregation numbers were measured in the assumption that
`intramicellar “static” quenching between the probe and the quencher,
`both following Poisson distribution, is dominant. The pyrene concentra-
`tion was 2 (cid:2) 10-6 mol dm-3, and the fluorescence intensity at 383 nm
`was monitored at decreasing amount of the DpyCl quencher starting
`from a molar concentration ratio R ) [quencher]/[micelle] ) [q]/[M]
`(cid:24) 1.22-24
`The fluorescence intensity in the presence of the quencher is given
`
`by
`
`I ) Io exp(-[q]/[M])
`
`(2)
`
`where Io is the fluorescence intensity in the absence of inhibitor. In the
`approximation of the phase separation model the following equation
`holds:
`
`[M] ) (C - cmc)/Nagg
`
`(3)
`
`where C is the concentration of the aggregating species. Hence, the
`aggregation number, Nagg, is obtained from the slope of ln(I/Io) vs [q]/(C
`- cmc).25,26 Due to the very low concentration of the systems studied,
`in eq 3 molarity has been replaced by molality.
`Water Proton Relaxation Measurements. The longitudinal water
`proton relaxation rates were measured on a Stelar Spinmaster (Mede,
`Pavia, Italy) spectrometer operating at 20 MHz, by means of the
`standard inversion-recovery technique (16 experiments, 2 scans). A
`typical 90(cid:176) pulse width was 4 (cid:237)s and the reproducibility of the T1 data
`was (0.5%. The temperature was kept at 25 (cid:176)C with a Stelar VTC-91
`air-flow heater equipped with a copper-constantan thermocouple
`(uncertainty (0.1 (cid:176) C). The proton 1/T1 NMRD profiles were measured
`over a continuum of magnetic field strength from 0.000 24 to 0.28 T
`(corresponding to 0.01-12 MHz proton Larmor frequency) on a Stelar
`Fast Field-Cycling relaxometer. This relaxometer works under complete
`computer control with an absolute uncertainty in 1/T1 of (1%. Data
`points at 20 and 90 MHz were added to the experimental NMRD
`profiles and were recorded on the Stelar Spinmaster (20 MHz) and on
`a JEOL EX-90 (90 MHz) (Tokyo, Japan) spectrometer, respectively.
`1H Water Relaxation Rate. The relaxivity of a Gd(III) complex
`results from contributions arising mainly from water molecules in the
`inner and outer coordination spheres:
`
`) r1
`
`His + r1
`
`r1
`
`Hos
`
`(4)
`
`His refers to the contribution from the exchange of the water protons
`r1
`in the first coordination sphere of the paramagnetic metal ion:
`
`His )
`
`r1
`
`nw[C]
`+ (cid:244)M)
`55.6(T1M
`
`(5)
`
`where nw is the hydration number, [C] is the molar concentration of
`the paramagnetic chelate, T1M is the longitudinal relaxation time of the
`inner sphere water protons, and (cid:244)M is their residence lifetime.
`Hos, describes the contribution from water
`The outer sphere term, r1
`molecules which diffuse around the paramagnetic complex (bulk
`solvent).
`The complex theory that governs the relaxation process in a
`paramagnetic chelate is well described by the models developed by
`Solomon-Bloembergen-Morgan for what concerns the inner sphere
`contribution and by Hwang and Freed for the outer sphere one.27
`Small-Angle Neutron Scattering. SANS measurements were
`performed at 25 (cid:176)C on the KWS2 located at the Forschungszentrum
`
`(22) Infelta, P. P.; Gratzel, M.; Thomas, J. K. J. Phys. Chem. 1975, 78, 190.
`(23) Tachiya, M. Chem. Phys. Lett. 1975, 33, 289.
`(24) Gehlen, M. H.; De Schryver, F. C. Chem. ReV. 1993, 93, 199.
`(25) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951.
`(26) McNeil, R.; Thomas, J. K. J. Colloid Interface Sci. 1981, 83, 57.
`(27) Banci, L.; Bertini, I.; Luchinat, C. Nuclear and Electronic Relaxation;
`VCH: Weinheim, 1991; p 91.
`
`MPI EXHIBIT 1094 PAGE 4
`
`MPI EXHIBIT 1094 PAGE 4
`
`

`

`Mixed Micellar Aggregates as Contrast Agents
`
`A R T I C L E S
`
`Julich, Germany. Neutrons with an average wavelength of 7 Å and
`wavelength spread ¢(cid:236)/(cid:236) < 10% were used. A two-dimensional array
`detector at two different sample-to-detector distances, 2 and 8 m,
`detected neutrons scattered from the sample. These configurations
`allowed collecting the scattered intensity in a range of moment transfer
`Q between 0.003 and 0.12 Å-1. Samples were contained in 1 mm path
`length quartz cells, and measurement times ranged between 20 min
`and 2 h. The data were then corrected for background, empty cell, and
`solvent contribution, and then reduced to scattering cross section (in
`absolute units per centimeter), following the standard procedure.28
`Method of Data Analysis. The general scattering intensity, contain-
`ing information about shape, size, and interactions of monodisperse
`scattering bodies, is given by29
`
`I(Q) ) Nb ((cid:229)
`
`i
`
`- Vb
`
`F
`
`s)2P(Q) S(Q) + Iinc
`
`bi
`
`(6)
`
`The CCK8 peptide was synthesized as previously described.32
`At the Asp N-terminal residue of the CCK8 peptide a glycine
`residue and two units of 8-amino-3,6-dioxaoctanoic acid were
`added directly on the solid phase. As a final step, the nonaoc-
`tanoic acid was bound to the N-terminal position of the peptide
`derivative. The cleavage of resin and the deprotection of the
`side chain functions were performed in a TFA/TIS/EDT/water
`mixture. The peptide derivative was isolated in good yields and
`purified by RP-HPLC. The analitycal data (Maldi-tof mass
`spectrum and RP-HPLC) confirm compound identity and purity.
`The two units of the hydrophilic linker increase the affinity for
`the polar solvent of the peptide moiety.
`The amphiphilic chelating moiety C18DTPAGlu was also
`synthesized in the solid phase using a resin support. Fmoc-Lys-
`(Mtt)-OH was anchored to the resin and the side chain was
`selectively deprotected. Then, DTPAGlu pentaester, the chelat-
`ing agent fully protected by tert-butyl (tBu) groups on its
`carboxyl functions with the exception of the carboxyl group on
`the side chain of L-glutamic acid, was linked, by a single
`coupling step, to the (cid:15)-NH2 amino function of the lysine residue.
`Successively, the Fmoc protecting group was removed by the
`R-NH2 amino function of the lysine residue and the nonaoc-
`tanoic acid was bound. After cleavage from the resin the product
`was characterized by 1H NMR spectroscopy and Maldi-tof mass
`spectrometry.
`Surface Tension. The surface tension was measured on
`C12E5-water, C18DTPAGlu-water, and C18CCK8-water
`binary systems; on C18DTPAGlu-C12E5-water, C18CCK8-
`C12E5-water, and C18DTPAGlu-C18CCK8-water ternary
`systems; and on C18DTPAGlu-C18CCK8-C12E5-water
`quaternary system. The surfactant concentration was raised well
`above the expected critical micellar concentration, cmc. Surface
`tension measurements were also performed on all systems once
`C18DTPAGlu was replaced by C18DTPAGlu(Gd).
`All the above systems show a concentration dependence of
`the surface tension, (cid:231), typical of systems containing amphiphilic
`molecules that form micelles. Addition of solute to pure water
`((cid:231)o ) 72.0 dyn cm-1) has the effect of decreasing (cid:231) to the cmc
`value, at which the surface tension curve changes its slope and
`approaches a constant value. However, there are significant
`differences among the systems studied, as inspection of Table
`1 suggests. For the C18CCK8-water system the typical change
`in the slope of the (cid:231) vs ln m curve, of micellar systems, has not
`been observed.
`Binary Systems: C18DTPAGlu-Water, C18CCK8-
`Water, and C12E5-Water. In Figure 2 the surface tension
`diagram of the C18DTPAGlu-water and C12E5-water binary
`systems along with C18DTPAGlu(Gd)-water system are
`reported. The cmc values detected for the C18DTPAGlu-water
`and C12E5-water systems are about the same, 5 (cid:2) 10-5 and
`4 (cid:2) 10-5 mol kg-1 respectively, although the limit value of the
`surface tension for the two systems is quite different. This is to
`be assigned to the higher hydrophobic contribution of C12E5
`to the surface composition with respect to the other compounds
`studied in this paper.
`The behavior of C18DTPAGlu(Gd)-water is substantially
`similar to that of the corresponding system in the absence of
`
`(32) Ragone, R.; De Luca, S.; Tesauro, D.; Pedone, C.; Morelli, G. Biopolymers
`2001, 56, 47.
`
`J. AM. CHEM. SOC. 9 VOL. 126, NO. 10, 2004 3101
`
`where Nb is the number density of scattering bodies, Vb is the volume
`of each scattering particle, (cid:229)
`ibi is the sum of the scattering lengths
`over the atoms constituting the body, and Fs is the solvent scattering
`length density. P(Q) and S(Q) are the form factor and structure factor,
`respectively. Iinc is the incoherent scattering cross section.
`The form factor contains information on the shape of the scattering
`objects, whereas the structure factor S(Q) accounts for interparticle
`correlations and is normally important for concentrated or charged
`systems.
`The structure of micelles present in the systems under study has
`been established by analyzing the scattering data through eq 6, imposing
`that the number density of scattering bodies and their volume are Nb
`) (C - cmc)La/Nagg and Vb ) NaggVm, where C is the stoichiometric
`solute concentration, Vm is the volume of the free surfactant, Nagg is
`the aggregation number of the micelles, and La is Avogadro’s constant.29
`The micelles were modeled as a spherical “core + shell”, and hence
`the form factor P(Q) is reduced to a sum of two Bessel first-order
`spherical functions:29
`
`P(Q) )(f
`
`3j1(u1)
`u1
`
`+ (1 - f)
`
`3j1(u2)
`u2
`
`)2
`
`(7)
`
`(8)
`
`(9)
`
`(10)
`
`(11)
`
`with
`
`) Qa
`
`u1
`) Q(a + d)
`
`u2
`
`f )
`
`Vf(F
`
`1
`
`- F
`2)
`
`(bi
`
`- Vm
`
`F
`
`s)
`
`i
`
`j1(x) ) sin x - x cos x
`x2
`
`Vf is the micelle core volume; a and d are the radius of the core and
`the thickness of the hydrophilic shell, respectively. F1 and F2 are the
`densities of scattering length of the core and shell.
`The structure factor, S(Q), can be calculated through the Hayter and
`Penfold theory.30,31
`
`Results
`
`Synthesis of Monomers. The peptide synthesis was per-
`formed by the solid phase approach using Fmoc/tBu chemistry.
`
`(28) Berti, D.; Pini, F.; Baglioni, P.; Teixeira, J. J. Phys. Chem. B 1999, 103,
`1738.
`(29) Hayter, J. B.; Penfold, J. J. Colloid Polym. Sci. 1983, 261, 1072.
`(30) Hayter, J. B.; Penfold, J. Mol. Phys. 1991, 42, 109.
`(31) Hayter, J. B.; Hansen, J. B. Mol. Phys. 1991, 42, 651.
`
`MPI EXHIBIT 1094 PAGE 5
`
`MPI EXHIBIT 1094 PAGE 5
`
`(cid:229)
`

`

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