79162913 the inventor of omniscan steven quay s 1990 pubished admission that it was not safe for...

8/2/2019 79162913 the Inventor of Omniscan Steven Quay s 1990 Pubished Admission That It Was Not Safe for Human Use

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Magnetic Resonance Imaging, Vol. 8, pp. 467-48 1, I990Printed n the USA. All rights eserved. 0730-725x/90$3.00 + .oaCopyright 1990PergamonPressplc

l Original ContributionTHE RELATIONSHIP BETWEEN THERMODYNAMICS ANDTHE TOXICITY OF GADOLINIUM COMPLEXESWILLIAM P. CACHER IS, STE VEN C. QUAY, AND SCOTT M. ROCKLAGE

Salutar, Inc., Sunnyvale, California 94086, USAThe suitability of gadolinium complexes as magnetic resonance imaging contr ast agents depends on a number offactors. A ther modynamic relat ionship t o toxicity exists if one assumes t hat the chemotoxicity of the intact complexis minimal but that the toxicity of the components of the complex (free metal and uncomplexed ligands) is sub-stantial. Release of Gd3+ from the complex is responsible for the toxicity associated with gadoliium complexes;this release appears to be a consequence of Zn2+ , Cu2+, and Ca2+ tra nsmetallatiou in vivo. This hypothesis issupported by acute toxicity experiments, which demonstra te that despite a 50-fold r ange of LDse values for fourGd complexes, all become lethally toxic when they release precisely the same quantity of Gd3+, and by subchronicrodent toxicity exper iments, which demonstra te a set of gross and microscopic findings similar to those knownto be caused by Zn2+ deficiency. Finally, this hypothesis predicts that subtle changes in formulation can furtherenhance the intrinsic safety of these complexes.Ke_~words: Thermodynamics; Toxicity; Gadolinium; Contrast.

INTRODUCTIONThe evaluation of paramagnetic complexes as contrastagents for magnetic resonance imaging has focusedupon the safety and efficacy of these agents. Safetyhas been evaluated by acute toxicity (LD,,), sub-chronic toxicity, local tissue tolerance, cardiovascularpharmacology, mutagenic potential, absorption, dis-tribution, metabolism and excretion studies. Thesecomplexes are composed of relatively toxic compo-nents, i.e., metal ion and ligand, bonded together byionic forces and subject to dissociation. An under-standing of the in vivo affinity, i.e., stability, of theseconstituents for each other is important to the futuredevelopment of magnetopharmaceuticals. One mech-anism to explain the toxicity of these complexes is invivo dissociation and/or metabolism to yield toxicmetal ions and free ligands.

Many workers have used the in vitro thermodynamicstability constant of the complex as a measure of thein vivo affinity of metal ion and ligand. The relation-ship between thermodynamic stability constant and in

vivo toxicity, however, does not hold for GdDTPA-BMA, GdDTPA, GdDTPA-BP and GdEDTA. A morecomplete thermodynamic evaluation is needed to as-certain in vivo stability and to explain the observedtoxicity of these complexes.

MATERIALS AND METH ODSSynthesis of GdDTPA-BA4A

A full description of ligand (DTPA-BMA) andcomplex (GdDTPA-BMA) synthesis is given in Ref. 2.Synthesis of N,N-2-Bis(2-pyridylrnethyl)diethylenetriamine

Diethylenetriamine (76 g, 0.74 mol) and pyridine-2-carboxaldehyde (174 g, 1.62 mol) in 2.5 L of abso-lute ethanol were heated for 2 hr at 50C with stirring.After cooling to room temperature, 25 g of 10% pal-ladium on charcoal was added and the schiff basehydrogenated at slightly greater than 1 atm of hydro-gen, over a 48-hr period. The catalyst was removed byfiltration, the filtrate adjusted to pH 4 with HCl gas

RECEIVED 9/30/89; ACCEPTED 2/17/90. GdDTPA-BP and Dilip Worah, Debra Kesler and DeanAcknowledgments-The authors thank William Dow and Kessler for performing the animal studies.David Love for synthesizing and formulating GdDTPA- Address all correspondence to Scott M. Rocklage, Salu-BMA, Joan Carvalho for synthesizing and formulating tar, Inc., 428 Oakmead Parkway, Sunnyvale, CA 94086.467


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468 Magnetic Resonance Imaging 0 Volume 8, Number 4, 1990

and then lowered to pH 1 using 12 N HCl. The result-ing precipitate was removed by suction filtration,washed with absolute ethanol until the washings werecolorless, and recrystallized from 95% ethanol. ThisHCl salt was then dissolved in 500 mL of water andneutralized with 5 N NaOH, then raised to pH 12.5,and the free base extracted with methylene chloride(4 x 500 mL). The methylene chloride solution wasdried to give 136 g (65%) of a pale yellow oil. NMR(D,O): 6 2.46 (s, 8H), 3.55 (s, 4H), 7.10 (m, 4H), 7.55(t, J= 12.5 Hz, 2H), 8.25 (d, J= 10 Hz, 2H), 7.50 (t,J = 10 Hz, 2H), 8.40 (d, J = 10 Hz, 2H).Synthesis of N,N-2-Bis(2-pyridylmethyl)-N,N,W-Tris(t-Butylcarboxymethyl)Diethylenetriamine

To a solution of N, N-2-bis(2_pyridyl)diethylenetri-amine (23.6 g, 82.6 mmol) and diisopropylethylamine(53.4 g, 0.4 mol) in 1.2 L of methylene chloride atroom temperature was added dropwise t-butylbromo-acetate (50 g, 0.2 mol) in 300 mL of methylene chloride.After stirring for 24 hr, the solution was evaporatedto dryness and placed under vacuum for 2 hr to re-move excess diisopropylethylamine. The crude solidwas taken up in 1.5 L of methylene chloride, washedwith 0.2 N NaOH, water (2 x 250 mL), brine (200mL) and dried (MgS04). The methylene chloride wasremoved, 200 mL of ethyl acetate added and this so-lution passed through 300 g of silica gel in a buchnerfunnel using EtOAc to elute the product. The purefractions (TLC: MeOH/CH2C12: 3/7) were combinedto give 40.6 g (79.5%) of the ester, NMR (CDC&) 61.26 (s, 9H), x 1.31 (s, 18H), 2.62 (s, 8H), 3.12 (s,2H), 3.17 (s, 4H), 3.78 (s, 4H), 7.02 (t, J = 10 Hz,2H), 7.35 (d, J = 10 Hz, 2H).Synthesis of N,N-2-Bis(2-Pyridylmethyl)Diethylenetriamine-N,W,N-Triacetic Acid

The tris(t-butylcarboxymethyl)ester (24.8, 0.1 mol)was dissolved in a solution of 600 mL of methylenechloride containing 380 mL of trifluoroacetic acid.The solution was stirred for 48 hr, evaporated underreduced pressure and diluted with 50 mL of water.This solution was applied to 200 mL of AG50-X8, H+form, 100-200 mesh and after washing with water un-til neutral, the product was eluted with 1 N NH40H.After removal of NH40H solution, the product wastaken up in 24 mL of water, adjusted to pH 10 andthe solution applied to AGl-X8, acetate, 100-200mesh. The column was washed with three bed volumesof water and the product eluted with 2 N HOAc togive 12.0 g (69%) of product after several lyophiliza-tions. NMR (D,O) 6 3.02 (t, J= 6 Hz, 4H), 3.08 (t,J = 6 Hz, 4H), 3.14 (s, 4H), 3.41 (s, 2H), 4.08 (s, 4H),

7.52 (m, 4H), 8.05 (t, J= 10 Hz, 4H), 8.40 (d, J=10 Hz, 2H).Potentiometric Measurements

Potentiometric titrations, to determine the acid pro-tonation constants as well as the metal ion stabilityconstants of DTPA-BMA and DTPA-BP, were car-ried out with an automatic titrator system.3 The sys-tem was controlled by a BASIC computer programwhich displays the data in tabular form concurrentwith a high resolution plot. Components of the au-totitrating system included a Fisher digital pH meter,Corning glass and AgCl reference combination elec-trode, and a Metrohm digital autoburette. In each ex-periment, temperature was maintained at 25.OC + 0.1and ionic strength was kept constant at 0.10 M withNaCl. The concentration of metal ions and ligand wasmaintained between 3 x lop3 M and 5 x 10m3M, and0.1000 M HCl was used as the titrant to minimize ionicstrength changes during the course of a titration. Ad-dition of a small amount of NaOH prior to the titra-tions was used to raise the solutions to pH 11.

Stability constants for Gd3+, Zn2+, Cu2+, andCa2+ complexes of DTPA-BP and the Zn2+, Cu2+and Ca2+ complexes of DTPA-BMA were determinedby direct titration. For these systems the complexeswere found to be greater than 25% dissociated at pH2, while the data for the GdDTPA-BMA system re-vealed the complex to be dissociated less than 1% atpH 2. Thus GdDTPA-BMA was studied using a li-gand-ligand competition titration. In this experimenta 1: 1: 1 molar ratio of Gd3+, DTPA-BMA, andEDTA was titrated. EDTA forms a complex withGd3+ whose stability constant is accurately known.At high pH, Gd3+ is primarily bound to EDTA. TheGd3+ ion was readily transferred to DTPA-BMA asthe pH was lowered to 2. The rate of metal transferwas sufficiently fast that equilibrium pH measure-ments could be made in a reasonable time period af-ter each addition of acid.Computations

Proton association constants for DTPA-BMA werecalculated using a BASIC computer program writtenfor polyprotic weak acid equilibrium calculations. Thestability constants of the metal ion complexes werecalculated using a BASIC program designed for metalion, ligand and proton systems containing a variety ofspecies. Both programs employed a modified Newton-Raphson algorithm which solved the simultaneousnonlinear mass balance equations, yielding -log[H+]values. The evaluated equilibrium constants were variedby a combined Simplex/Marquardt nonlinear regres-


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Thermodynamics and toxicity 0 W.P. CACHERIS ? AL. 469

sion algorithm. The average difference between ob-served and calculated -log [H+] was co.04 throughoutall titrations.

Biospeciation calculations were performed with aBASIC program which employed the modifiedNewton-Raphson algorithm. The non-linear equa-tions resulted from the mass balance equations for aneight-component system consisting of Gd3+, Zn+,Cu2+, Ca2+, ligand, monoaminomonocarboxylateamino acids (which were combined since their stabil-ity constants are very similar), citrate, and human se-rum albumin. The program required equilibriumconstants which were taken from the Iiterature,4-6 at37C and p = 0.15 M. Values that were not availableat p = 0.15 M were replaced by values at p = 0.10 M.Equilibrium constants that were not known at 37Cwere calculated from the enthalpy and entropy offormation7 (measured at 25C). The enthalpy and en-tropy of formation are relatively temperature indepen-dent over small temperature ranges7 and were reliedupon for accurate equilibrium constants at 37C. Inthe case of GdEDTA it was necessary to take into ac-count ternary complexes of GdEDTA with aminoacids and citrate.8 It can be shown from the relativelysmall value for the equilibrium ML + M = M2L (logK = 4.48 for ZqDTPA) that the binuclear complexesof DTPA are not formed in significant concentrationsin vivo, and these were excluded from the biospecia-tion calculations. A distribution volume of 180 mL/kgwas used to convert dosage to in vivo concentration.The in vivo concentrations of the components usedwere: Ca2+, 2.5 mM: Zn2+, 50 PM: Cu2+, 1 PM:amino acids, 2.76 mM: citrate, 0.11 mM: and albu-min, 0.4 mM.9 The root of the set of equationsyielded the free concentrations of all components.These concentrations were used along with the stabil-ity constants to determine full speciation for each dos-age of the complexes.Transmetallation Kinetics

A measurement of the rate of Gd3+ release fromGdDTPA-BMA, GdDTPA and GdDTPA-BP wasattempted by a method used by Margerum forGdEDTA and GdCDTA.O A solution of 1.288 x10e5 M GdDTPA-BMA (or GdDTPA) was preparedat pH 5.0 with an acetate buffer (0.01 M buffer, p =0.1 M). A lo-fold excess of Cu2+ was added and theformation of the Cu2+ complex was followed by UVabsorption at 268 nm.Animal Studies

Acute toxicity (LD,,) studies were conductedin Swiss-Webster mice. Five groups of mice, each

containing four males and four females, were in-jected intravenously with either GdDTPA-BMA,Na2[GdDTPA] , GdDTPA-BP , Na [CaDTPA-BMAI ,Na[CaDTPA-BP] or formulations containing eitherGdDTPA-BMA + 5 mole % Na[CaDTPA-BMAI ,Na2 [GdDTPA] ] + 5 mole % Na3 [CaDTPAl ,GdDTPA-BP + 5 mole % Na[CaDTPA-BP] orGdDTPA-BMA + 5 mole % Na[CaDTPA-BMA] +123 mole % NaCl. For each LD,, determination,each group of eight mice was given a different dosageof compound. Dose volumes larger than about 40mL/kg body weight were administered as divideddoses, at one-half hour apart. Animals were observed,once daily, for mortality or morbidity for 14 days andthe LDso value was calculated. The calculations weremade using a weighted probit method. Osmolarityof the various formulations were measured on a Wes-car model 550 vapor pressure osmometer.

A subchronic toxicity study was conducted inSprague-Dawley rats. Three groups of rats, each con-sisting of 10 male and 10 female rats, receivedGdDTPA-BMA intravenously three times a week forthree consecutive weeks at dosage levels of 0.1, 2.0,and 5.0 mmol/kg. A fourth group received normal sa-line and served as the negative control. All animalswere observed twice daily for morbidity and mortal-ity. Clinical observations for obvious toxicologic ef-fects were also performed at least once daily. Grossand microscopic evaluations were conducted at studytermination.

RESULTSPotentiometry

The potentiometric titration curves for DTPA-BMA and ZnDTPA-BMA are shown in Fig. 1. TheDTPA-BMA curve shows a very sharp decrease be-tween pH 9 and 5.5. This is due to the large separationof the pK, values of the two most basic groups of theligand, 9.37 and 4.38 (shown by NMR to be theamines). The third amine of the ligand has a pK, of3.31 and the carboxylates all have pK, values below2. The ZnDTPA-BMA curve drops rapidly from theinitial pH of 10 to pH 5, since the only protonation ofthe complex takes place with a pK, of 3.99. At pH 2,the protonated ZnHDTPA-BMA and CuHDTPA-BMA complexes are ca. 35% and 25% dissociated, re-spectively. The CaDTPA-BMA complex undergoes asimilar protonation with a pK, of 4.45, but becomescompletely dissociated at pH 2.7.

The results of the potentiometric measurements forDTPA-BMA, DTPA, DTPA-BP and EDTA and theGd3+, Zn2+, Cu2+ and Ca 2+ thermodynamic stability


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470 Magnetic Resonance Imaging 0 Volume 8, Number 4, 19901211109a76543210

0 1 2 3 4 5Moles of acid/moles of base

Fig. 1. Potentiometric titration curves of DTPA-BMA inthe presence and absence of equimolar Znzf. Concentra-tion of metal ion and ligand = 5.00 x 10e3 M (~1= 0.10 MNaCl, 25C).

constants are given in Table 1. The value obtained forthe thermodynamic stability constant of the GdDTPA-BMA complex was obtained by a competitive titrationtechnique with EDTA. The most basic ligand, DTPA(CPK, = 30), has the highest thermodynamic stability

constant with Gd3+ while DTPA-BMA (CpK, = 17) isless basic than EDTA (CpK, = 22) and has the secondlowest Gd3+ thermodynamic stability constant.

ThermodynamicsThe ligands DTPA-BMA, DTPA, DTPA-BP andEDTA are polyprotic. A comparison of thermody-namic stability constants with the stability at pH 7.4is an important first step in understanding in vivo sta-bility. Each ligand has a different response to protoncompetition for Gd 3+ binding which is based on itsintrinsic basicity. Specifically, the thermodynamic sta-bility constant (Ktherm) is defined as

M-+LPML

K [ML1therm = IMl ILl -The conditional stability constant (Kcond)specifies thedegree of metal chelation at a given pH, and is givenby

Kcod= + ([L] + [HLI + [z&L] + . . . )

where Kcondaccounts for free ligand species in vari-ous protonated forms. This expression correctly de-

Table 1. Protonation constants and metal chelate stability constants (25C, p = 0.10 M (NaCl)).Uncertainty (u) in Log K values are given in parenthesesLog K

EquilibriumWWLIWID-bW WI WIW,WP-bU WIV-WlMW WIW,WW,LI[WP-L&lM-WIWlWW WI Ll[GdHL]/[GdL][H]G4Wal Ll[CaHL]/[CaL][H][ZnL]/[ZnL][L][ZnHL]/[ZnL](H][ZnH2L]/[ZnHL][H][cw~K~l Kl[CuHL]/[CuL][H][CuH,L]/[CuHL][H]

aThis work.bReferences 4 and 6.Not measured.

DTPA-BMA=9.37 (0.01)4.38 (0.01)3.31 (0.04)1.43 (0.12)

16.85 (0.05)c7.17 (0.04)4.45 (0.02)

12.04 (0.03)4.04 (0.04)13.03 (0.03)3.36 (0.02)

DTPA-BPa9.53 (0.11)6.46 (0.07)4.76 (0.08)3.41 (0.08)2.28 (0.10)

16.83 (0.11)c7.97 (0.05)5.30 (0.03)

14.02 (0.14)5.36 (0.07)3.67 (0.04)

17.50 (0.17)3.74 (0.03)2.92 (0.03)

DTPAb10.498.604.282.642.0

1.622.462.3910.756.11

18.705.6021.384.81

EDTAb10.176.112.681.951.5617.271.5310.61c16.863.018.863.0


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Thermodynamics and toxicity 0 W.P. CACHERIS ET AL. 4 7 1

scribes the affinity of the metal ion for the ligand butis not equivalent to the thermodynamic expression.The conditional stability constant, at a given pH, canbe calculated from the thermodynamic value if theprotonation constants of the ligand are known

K cond - Kthermewhere LT is the total concentration of the uncom-plexed ligand, i.e. ([L] + [HLI + [H2L] + . . . ).Thus,

K cond - &xxm (1 + &I [H+l + KHIKw[~+I~+ . . . )-

= & m m ~ Hwhere KH values are stepwise proton association con-stants for the ligand and the sum of these terms is(~6. Figure .2 describes the variation of the condi-tional stability constants for GdDTPA-BMA andGdDTPA with pH. At a pH high enough to ensurecomplete deprotonation of the ligands (pH > 1 ), theconditional stability constants of the complexes areequal to their thermodynamic values and GdDTPA isfound to be more stable than GdDTPA-BMA by afactor of 105.2. However, at pH 7.4, GdDTPA is

?of ;i

CdDTPA-BMA

O...,...,...,,,2 3 4 5 6 7 0 9 10 11 12

PHFig. 2. pH dependence of conditional stability constant forGdDTPA-BMA and GdDTPA. GdDTPA has a higher con-ditional stability constant than GdDTPA-BMA at pH 11 bya factor of 105.6. At pH 7.4 this factor is reduced to 103.4and at pH 4 these complexes have the same conditional sta-bility constant values.

more stable than GdDTPA-BMA by only a factor of102-9. The complexes are equally stable at pH 4.Solubility

An important consideration in understanding thestability of such complexes in vivo is their solubilitywith respect to precipitating anions such as PO:-,OH-, and CO:-. Ligands that have too weak an af-finity for gadolinium to prevent precipitation in vivowould be expected to be toxic. For example, simplegadolinium salts have been demonstrated to precipi-tate in vivo.12 Martell has defined a solubility con-stant for chelating agents that predicts the solubility ofmetal ions in the presence of chelating agents and pre-cipitating ion13. This solubility constant was initiallydesigned to predict the ability of a ligand to diminishmetal ion overload when the metal may be present insome insoluble form. The solubility constant is de-fined as

KSO,= mLT

which describes the amount of metal, as the complex,in the presence of the ligand. Alternatively, KsOl maybe expressed as

&,I = &erm [Ml [LlLT .

where LT is the total concentration of the ligandnot bound to the metal ion M, i.e., (1L] + [HL] +[l&L] + . . . + [CaL] .+ [ZnL] + [CuL] + . . . ) . Inbiological systems the ligand can exist in different pro-tonated forms and also be bound to other metal ions:

LT = [Ll + KH I [H+l [Ll + KHIK HZ[H+I~[LI

+ . . . + KCaL [Ca2+] [Ll + KZnL [Zn2+] [L]+ KcuL[Cu2+] [L] + . . .

thusKsO, = Ktherm [Ml ((.y~l + a& + (~2: + a& + . . . )-Iwhere

a; = 1 + KH, [H+] + K,,K,,[H+] + . . .a& = KCaL [Ca2+]cr.& = KZnL [Zn+]CX&= Kc-,/ [Cu2+] .


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412 Magnetic Resonance Imaging 0 Volume 8, Number 4, l!XKlKSol s often reported as a logarithm

log&l1 = 1ogKtlerm + h%[Ml

A negative value of log KS,,, predicts that the ligandwill not be able to solubilize GdP04 while a positivevalue indicates the ligand has the ability to dissolve theprecipitate.

The amount of free metal derived from the solubil-ity of GdP04, in the presence of a ligand, can be cal-culated by14

log[M] = -logaM- logan (PO:-) + log&, - log [PO:-]

where

and an(POj-) is the proton competition factor forphosphate at pH 7.4. Log[M] values were calculatedfrom the above expressions for GdP04 in the pres-ence of DTPA-BMA, DTPA, DTPA-BP, and EDTA.These calculations were performed, at a [PO:-] of1.13 mM, by determining the amount of [L] Zn2+],[Cuzf] , and [Cat+] in vivo via a biospeciation model(vide infra). Table 2 shows the results of these calcu-lations for a 0.1 mmol/kg dosage of these complexes(distribution volume = 180 mL/kg). Values of free li-gand and endogenous metal ions are shown along withfree metal (log[M]) . The value of log[M] and free en-dogenous metal ions were then used to calculate KSolfor these complexes. Log KSolvalues of 6.7, 10.2, 5.7,and 2.0 were calculated for GdDTPA-BMA,GdDTPA, GdDTPA-BP and GdEDTA, respectively.These values for KSol predict stability towards phos-phate precipitation in vivo, and this has been con-firmed by biodistribution data obtained forGdDTPA-BMA, GdDTPA, and GdEDTA.

Figure 3 illustrates the variation in the value of

-10

-15 IIncreasing Thermodynamic Stability Constant +

Fig. 3. Values of log Ksol for several Gd3+ complexes. Thecomplexes are arranged in order of increasing thermody-namic stability constant. Complexes with positive log Ksolvalues are expected to be soluble in vivo with respect toGdP04 precipitation while those with negative Ksol valuesare not. The right hand legend estimates the percent of gad-olinium as the indicated complex (the remainder is insolubleGdP04).

KSolwith complexing strength of the ligand. GdC& isknown to precipitate in vivo,r2 consistent with a verylarge negative value of KSO,.Gd(OAch and GdNTAwould also be expected to be unstable towards PO:-precipitation in vivo. Figure 4 shows the results ofsimiliar calculations for GdDTPA-BMA, GdDTPA,GdDTPA-BP and GdEDTA collected in the urine. Allfour complexes show stability towards PO:- precipi-tation from pH 5 to 8 when collected and concen-trated in the urine. The concentration of thecomplexes in the urine was estimated to be 30 mMbased on a 7 mmol dose (0.10 mmol/kg x 70 kg), as-suming that 50% of the complex is excreted in 1.58hrr6 and that urinary output is 75 mL/hr. GdCl,,Gd(OAc)3 and GdNTA were not included since these

Table 2. Constants obtained for solubility prediction of gadolinium complexes in the presence of phosphate.K SP = 1O-22.26, [PO:-] = 1.13 mM, dosage = 0.1 mmol/kg, volume of distribution = 180 ml/kg

GdDTPA-BMA GdDTPA-BP GdDTPA GdEDTAIL1LogtMl[Ca2+][Zn2+][Cu2]Log &or

1.35 x 1O-9 M 3 42-84 x LO- M 2.84 x lo-l3 M 1 40-714 x 10-r M-9.3 -8.92.33 x 1O-3 M 2.34 x 1O-3 M 2.33 x lo- M 2.40 x 1O-3 M2.46 x 10-s M 2.20 x 1O-8 M 2.37 x lo- M 1.34 x lo-* M1 786:7

x 10-l M 1 78517

x 10-l M 1 7810:2

x 10-l M 1.84 x lo- M2.0


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Thermodynamics and toxicity 0 W.P. CACHERIST AL. 473

GdDTPA-BYA GdDTPA CdDTPA-BP CdEDTA

pH 50 pH 65 pH 80Fig. 4. Values of log Kso, for GdDTPA-BMA, GdDTPA,and GdEDTA in urine at pH 5.0, 6.5, and 8.0. The complexconcentration was assumed to be 30 mM based on a 7-mmoldose (0.1 mmol/kg x 70 kg) with 50% of the complex be-ing collected in 1.58 hours with urinary output of 75 mL/hr.All three complexes are expected to be stable towardGdP04 precipitation under these conditions.

are insoluble in plasma and would not be expected tobe present in the urine.Biospeciation

In order to better understand the toxicity of gado-linium complexes we have employed a broad thermo-dynamic approach. The Gd3+ stability constant is notthe only stability constant relevant to a considerationof the toxicity of Gd3+ complexes. Based on the as-sumption that most of the toxicity of such complexesarises from release of the highly toxic Gd3+ ion invivo, it is important to know the stability of the ligandwith endogenous metal ions such as Zn+, Cu+, andCa2+ since these metal may displace the Gd3+ fromthe complex. In addition, the stability constants ofGd3+ with biological ligands such as amino acids, cit-rate and albumin must be known. Figure 5 shows thevariation of Gd3+ released from GdDTPA-BMA withconcentration of the complex, as a function of theconditions of the experiment. The in vitro calculationsshow that very little Gd3+ is released if the equilib-rium M + L Ft ML is considered in isolation. Protoncompetition at pH 7.4 increases the quantity of gad-olinium ion released from about 0.1 mM to 1 nM. Ad-ditional equilibria with endogenous metal ions andligands are very important, increasing the amount of

Gd3+ released from the complex by a factor of 103-104.Recognition of these additional equilibria is importantin predicting the amount of dissociation of a complexin vivo and in explaining the subsequent differences intoxicity amongst complexes.The difference in stability constant between Gd3+and other endogenous metal ions for the same ligandis termed selectivity. In a thermodynamic context,the different stability constants for these endogenousmetal ions provide an explanation of the toxicityof GdDTPA-BMA, GdDTPA, GdDTPA-BP, andGdEDTA. EDTA shows very little selectivity betweenmetal ions. The Gd3+ stability constant for EDTA isgreater than that for Zn2+ only by a factor of 10.4.DTPA-BP has a greater selectivity for Gd3+ overZn2+ by a factor of 102.8. In DTPA, this factor in-creases to 103.8, however, with DTPA-BMA theGd3+/Zn2+ selectivity is even greater than for DTPA,with Gd3+ being preferred by 104.*. The selectivityof these ligands with Gd3+, Zn2+, Cu+, and Ca2+ isillustrated in Fig. 6.

Among the endogenous metal ions, Zn+, Cu2+and Ca2+, Zn2+ was expected to have the greatest sig-nificance in effecting Gd3+ release from the com-plexes in vivo. Cu 2+ has a favorable stability constantwith respect to all three ligands, but is present in suchsmall concentrations in the blood (l-10 PM) that itcannot displace much Gd3+. Ca2+ is not expected todisplace Gd3+ from these complexes, despite its high

11 t I

10 I\- vitro, thermodynamic 1

5 i,,,,...,,,,,,,.....,.,,,...,...,.....,.........J0 1 2 3 4 5[GdDTPA-BMA] mM

Fig. 5. Comparison of in vitro and in vivo -log[Gd3+]versus concentration of GdDTPA-BML. The in vitro valueswere obtained by consideration of thermodynamic and con-ditional (pH 7.4) equilibria. The in vivo values were deter-mined by biospeciation calculations.


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474 Magnetic Resonance Imaging 0 Volume 8, Number 4, 1990

5 I I I IEDTA DTPA-BP DTPA DTPA-BMA

UgandFig. 6. Three-dimensional plot of the thermodynamic stability constants of DTPA-BMA, DTPA, DTPA-BP and EDTA withGd3+, Zn2+, Cu2+ and Ca+.

concentration in plasma (2.5-4 mM), since DTPA-BMA, DTPA, DTPA-BP, and EDTA have relativelylow stability constants with this metal ion. Zn+,however, has moderately high stability constants withDTPA-BMA, DTPA, DTPA-BP and EDTA and it ispresent in concentrations (lo-50 PM) that can displacea significant amount of Gd3+. However, as indicatedabove, DTPA-BMA, DTPA, DTPA-BP, and EDTAare very different in their selectivity for Gd3+ overZn2+. This can be seen in Fig. 7, which shows the ra-tio of stability constant for these ligands with Gd3+with Zn2+. This illustrates that of all the ligands con-sidered herein GdDTPA-BMA has the largest selec-tivity for Gd3+ over Zn2+.

The consequences of Gd3+ selectivity over otherendogenous metal ions for a specific ligand can be un-derstood through in vivo speciation calculations. Acomputer calculation of the speciation of Gd3+ com-plexes was performed incorporating the metal ionsZn2+, Cu2+, and Ca2+. Fe3+ was not considered sinceit is very tightly bound by the storage proteins ferritinand hemosiderin, and is essentially unavailable for in-teraction with Gd3+ complexes. The in vivo ligandsused in the biospeciation calculations included aminoacids, citrate and human serum albumin.

Figure 8 shows the result of such calculations. Theamount of Gd3+ released from the complex as afunction of dosage is shown for GdDTPA-BMA,GdDTPA, GdDTPA-BP, and GdEDTA. GdEDTAreleases a relatively large amount of Gd3+ at low dos-

ages. GdDTPA-BMA releases about one-half as muchGd3+ as does GdDTPA at any given dosage. The re-sults of the biospeciation model are consistent with theobserved LDso values (IV, mice) of GdDTPA-BMA(14.8 mmol/kg), Na2 [GdDTPA] (5.6 mmol/kg),GdDTPA-BP (2.8 mmol/kg), and NMG[GdEDTA](0.3 mmol/kg). The LD5,-,dosage for each of these

EDTA DTPA-BP DTPA DTPA-BMALlgand

Fig. 7. Selectivity of DTPA-BMA, DTPA, DTPA-BP andEDTA for Gd3+ over Zn+. . . . .Selectrvity is given as KG&K =r. GdDTPA-BMA has the largest selectivity of the threeligands.


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Thermodynamics and toxicity l W.P. CACHERIS TAL . 475

complexes is indicated in Fig. 8 and relates adminis-tered dosage to the quantity of Gd3+ released.Despite a 50-fold range in LDso values based on ad-ministered dosage, all four complexes become lethallytoxic to half the population of mice when they releaseca. 13-15 PM Gd 3+. Since Gd3+ has been shown toinhibit Ca2+ binding to mammalian cardiac sarco-plasmic reticulum at about 20 PM Gd3+, the actualmechanism of toxicity could involve hemodynamicdisruption.r7 The conditional stability of such com-plexes, in the form considered for in vivo speciation ofthe complexes, thus provides an excellent correlationwith observed toxicity. Figure 9 shows that the Gd3+rel .~sed from GdDTPA-BMA is found primarily asthe &rate complex.

The correlation between Gd3+ selectivity of a li-gand and toxicity can also be understood by definitionof a Gd3+ selectivity constant. The Gd3+ selectivityconstant accounts fur Gd3+ selectivity of the ligandby modifying the thermodynamic stability constant ofa Gd3+ complex to incorporate ligand equilibria withH+, Zn2+, Ca2+ and Cu2+. More explicitly,

30 , I

0 ' I0.1 1 10Dosage (mmole/kg)

Fig. 8. Results of the biospeciation calculations forGdDTPA-BMA, GdDTPA, GdDTPA-BP and GdEDTA.The amount of Gd3+ released from the complex as a func-tion of complex dosage is shown (distribution volume = 180mL/kg). GdEDTA is expected to release a relatively largeamount of Gd3+ at small dosages. At each dosage,GdDTPA-BMA is expected to release about one-half of theGd3+ as compared to GdDTPA. All four complexes be-come lethally toxic to half the population (mice when ca.13-15 PM Gd3 is released.

25

0-0.1 1 1 0Dosage (mmole/kg)

Fig. 9. Speciation of Gd3+ released from GdDTPA-BMAas a function of dosage. The Gd3+ released is primarilyfound as the citrate complex.

whereCYH 1 + KH, [H+] + Km Krr2 [H+] + . . .

a~:=KCaL Ca2+]cc&= KZnL Zn2+]a& = KCuL Cu2+] .

Table 3 indicates the calculated Gd3+ selectivityconstants at pH 7.4 for DTPA-BMA, DTPA, DTPA-BP and EDTA as well as the LDso values for theGd3+ complexes. The concentrations of Ca2+, Zn2+,and Cu2+ used were 2.5 mM, 50 PM and 1 PM, respec-

Table 3. Thermodynamic and selectivity stabilityconstants for gadolinium complexes andLDso values (mice, iv). Uncertainty (a) inLD,, values are given in parentheses.Complex Log Krherm Log &et LD50aGdDTPA-BMA 16.85 9.04 14.8 (0.7)Na*[GdDTPA] 22.46 7.04 5.6 (0.2)GdDTPA-BP 16.83 5.32 2.8 (0.1)NMG[GdEDTA] 17.27 423 0.3 (0.1)mmol/kg.


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476 Magnetic Resonance Im aging 0 Volume 8, Number 4, 1990tively. These represent the total in vivo concentrationsof these metal ions, since equilibria with endogenousligands were not considered. Figure 10 shows that un-like the thermodynamic Gd3+ stability constants, acorrelation can be drawn between the Gd3+ selectivityconstant and LDSO or each of these four complexes.

15 1 DTPA-BMA

3 10da I I

8 DTPA I2 5 -. DTPA-BP

EDTA0 L...,....,r...,....,....,...,l....,....~..,...,15 16 17 16 19 20 21 22 23 24 25Log KWm+u.,c with Gd

15 .DTPA-BYA 1

10 -

5-

0 EDTA

4 5 6 7 6 9 10Log KYnlb,,, with Gd+

Fig. 10. Correlation between Gd3+ selectivity constant andLD50 values for Gd3+ complexes. The thermodynamic sta-bility constants of GdDTPA-BMA, GdDTPA, GdDTPA-BP and GdEDTA show no correlation with LDso (leftgraph). However, LDso appears to be correlated to the gad-olinium selectivity constant (right graph) with GdDTPA-BMA having the largest Gd3+ selectivity constant and thehighest observed LDso. Data for all complexes are given inTable 3.

KineticsA potential criticism of such a biospeciation model

is the need to assume that thermodynamic equilibriumof the complex is reached in vivo before the com-pound can be excreted from the body. Brucher hasshown that the kinetics of Gd3+ displacement fromGdEDTA by Cu*+ is indeed very fast and occurswithin the stopped-flow time frame (~1 sec).18 Simi-lar experiments have been performed for GdDTPA-BMA, GdDTPA and GdDTPA-BP at pH 5, and theformation of CuDTPA-BMA, CuDTPA andCuDTPA-BP is complete within a similar time frame.

The rate of transmetallation, however, seems to beimportant for sterically rigid complexes, likeGdCDTA and GdDOTA. Both of these complexes donot fit into the thermodynamic correlation discussedhere. CDTA has selectivity comparable to EDTA forGd3+ over Zn*+ Ca*+ and Cu*+. l9 Yet its LDSo value,ca. 2 mmol/kgl: is significantly more favorable thanthat of GdEDTA. GdCDTA fits the thermodynamiccorrelation only if one assumes that the amount ofGd3+ released before clearance from the body isaround 35% of the amount expected if the transmetal-lation reaction were to reach equilibrium. Margerumhas shown that the reaction of Cu*+ with GdCDTAoccurs with a fl,* of 40-100 min at pH 4-5 (35C).This reaction rate would certainly place the amount ofGd3+ released from GdCDTA into the predictedrange if this complex is cleared from the body at thenormal rate for an extracellular agent ( tl,* = 95 min).16GdDOTA is expected to be very toxic based on the bi-ospeciation model, due primarily to the high stabilityconstants for the Ca*+, Zn*+, and Cu*+ complexes ofDOTA* (reflecting its poor selectivity). As withGdCDTA, GdDOTA conforms to the thermodynamiccorrelation if the amount of Gd3+ released within itsbiological lifetime is less than 5% of the amount ex-pected at transmetallation equilibrium. This is not un-reasonable, by analogy with the observed slowdissociation kinetics of CdDOTA.*lToxicity and Formulations

Improvements in safety can be predicted fromslight modifications in formulation if the primarymode of toxicity is Gd3+ release. A small excess of li-gand added to formulations of the complex should becapable of minimizing transmetallation reactions invivo. Figure 11 shows the effect of the amount ofGd3+ released (due to Zn*+ transmetallation) fromthe addition of small quantities of DTPA-BMA saltsto GdDTPA-BMA. Even a 1 mole % addition ofDTPA-BMA salts causes a marked decrease in theamount of Gd3+ released.

Figure 12 indicates that the addition of small


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Thermodynamics and toxicity 0 W.P. CACHERI~T AL. 411

Dosage = 0.1 mmoie/kg

0 1 2 3 4 5 6 7 8 9 10Mole % DTPA-BMA Added to Formulation

Fig. 11. The effect of adding excess DTPA-BMA to formu-lations of GdDTPA-BMA. Even a 1Vo addition of DTPA-BMA significantly decreases amount of Gd3+ expected tobe released in vivo.

amounts of free ligand to the formulation initiallyyields an improvement in the LDse value of morethan a factor of two. Addition of more than 1% ex-cess of free ligand, however, decreases the LDSo valueand a moderately toxic formulation exists when 5mole % free ligand is added. The free ligand (at phys-iological pH), Na2HDTPA-BMA (LDso = 0.16mmol/kg, iv, mice), is as toxic as the Gd3+ ion. Thistoxicity presumably predominates in the toxicity of theformulation when more than 1 mole Vo excess free li-gand is added.

Na[CaDTPA-BMA] has a more favorable LD,evalue of 16.6 mmol/kg (iv, mice). Since in vivo trans-metallation is expected to occur with Zn2+ and notwith Ca2+, the addition of small amounts of

30 -E x c e s s I l g a n d i s

C a D T P A - B MA

,10 -/

79162913 the inventor of omniscan steven quay s 1990 pubished admission that it was not safe for...

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