P-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and 111In Immuno-Single Photon Emission Computed Tomography Imaging

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Article

p-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator

for Radiometalpharmaceuticals and 111In Immuno-SPECT Imaging

Sarah Spreckelmeyer, Caterina F. Ramogida, Julie Rousseau, Karen Arane, Ivica

Bratanovic, Nadine Colpo, Una Jermilova, Gemma Dias, Iulia Dude, Maria de

Guadalupe Jaraquemada Peláez, Francois Benard, Paul Schaffer, and Chris Orvig

Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00311 • Publication Date (Web): 06 Jul 2017

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Bioconjugate Chemistry

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p-NO -Bn-H neunpa and H neunpa-Trastuzumab: Bifunctional Chelator

for Radiometalpharmaceuticals and In Immuno-SPECT Imaging

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Sarah Spreckelmeyer, Caterina F. Ramogida, Julie Rousseau, Karen Arane, Ivica Bratanovic, Nadine Colpo, Una

§

#

†

#

§

Jermilova, Gemma M. Dias, Iulia Dude, Maria de Guadalupe JaraquemadaꢀPeláez, François Bénard, Paul Schaffer,

†

Chris Orvig *

†

Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vanꢀ

couver, British Columbia, V6T 1Z1, Canada

‡

Dept. Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy, University of Groningen, Antonius

Deusinglaan 1, Groningen 9713 AV, The Netherlands

§

Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, V6T 2A3, Canada

#

th

BC Cancer Agency, 675 West 10 Avenue, Vancouver, British Columbia, V5Z 1L3, Canada

*

Eꢀmail: orvig@ubc.chem.ca

Potentially nonadentate (N O ) bifunctional chelator pꢀSCNꢀBnꢀH neunpa and its immunoconjugate H neunpaꢀTrastuzumab for

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In radiolabeling are synthesized. The ability of pꢀSCNꢀBnꢀH neunpa and H neunpaꢀTrastuzumab to radiolabel quantitatively

InCl at ambient temperature within 15 min or 30 min, respectively, is presented. Thermodynamic stability determination with

In , Bi and La resulted in high pM values. In vitro human serum stability assays have demonstrated both In complexes to

have high stability over 5 days. Mouse biodistribution of [ In][In(pꢀNO ꢀBnꢀneunpa)] , compared to that of [ In][In(pꢀNH ꢀBnꢀ

CHXꢀA"ꢀDTPA)] , at 1 h, 4 h and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse

biodistribution study, the immunoconjugates InꢀneunpaꢀTrastuzumab and InꢀCHXꢀA”ꢀDTPAꢀTrastuzumab demonstrate a

similar distribution profile, but with slightly lower tumor uptake of InꢀneunpaꢀTrastuzumab compared to InꢀCHXꢀA”ꢀDTPAꢀ

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+

3+

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ꢀ

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Trastuzumab. These results were also confirmed by ImmunoꢀSPECT imaging in vivo. These initial investigations reveal the acyclic

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bifunctional chelator pꢀSCNꢀBnꢀH neunpa to be a promising chelator for In (and other radiometals) with high in vitro stability,

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and also show H neunpaꢀTrastuzumab to be an excellent In chelator, with promising biodistribution in mice.

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Introduction

vant to

In and

Lu, macrocyclic DOTA (1,4,7,10ꢀ

tetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic acid) is the goldꢀ

standard chelator, while acyclic chelator DTPA (diethylenetriꢀ

amine pentaacetic acid) and chiral analogue CHXꢀA”ꢀDTPA

Early detection and specific therapy are the key factors for the

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successful treatment of cancer. In (t ₁_/₂= 2.8 days) and/or

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Lu (t ₁_/₂= 6.6 days) are important radioisotopes in nuclear

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are ubiquitous in In radiopharmaceutical development (Figꢀ

ure 1). Recent studies developed bifunctional somatostatin

medicine that match either the requirements for single photon

emission tomography (SPECT) and performing dosimetry, or

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analogues of DOTA with increased stability in vivo. As an

for therapeutic purposes, respectively. In being a cyclotron–

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acyclic goldꢀstandard, the commercially available radiopharꢀ

produced radiometal (via the Cd(p,n) In reaction) emits

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maceutical OctreoScan ( InꢀDTPA octeotride) reached apꢀ

gamma rays (245 and 171 keV) and Auger electrons. Lu

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proval in 1994 (Figure 1). Since the success of OctreoScan,

being a reactorꢀproduced radiometal ( Lu(n,gamma) Lu)

emits primarily beta particles (490 keV) that can be used for

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several more bifunctional acyclic In chelators that contain

different biomolecules have been developed, hoping to overꢀ

come the limitations of OctreoScan. These include an inꢀ

creased physiological uptake which restricts the detection of

small lesions, prolonged imaging protocol and relatively high

1

therapy.

A common method to incorporate metallic radioisotopes (i.e.

radiometals) into radiopharmaceuticals is via chelation of the

desired radioisotope using a bifunctional chelator (BFC). As

implied by the name, BFCs possess two properties – they must

chelate the radiometal of interest in a tight and stable metalꢀ

ligand complex, and the BFC must incorporate a point of

attachment for conjugation to a targeting vector (e.g. biomoleꢀ

cule of interest in disease progression such as a peptide or

antibody). Both macrocyclic and acyclic chelators are used in

the clinic, and are also of interest in the field of medicinal

inorganic chemistry research. The pros and cons of cyclic vs

4

radiation dose to the patients, as well as low image quality.

Our group has developed several promising acyclic chelators

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for In and/or Lu, based on picolinic acid binding motifs,

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ꢀ8

which we have since dubbed the “pa”ꢀfamily of chelators.

Of note, octadentate H octapa (N O ) and its bifunctional

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analogue pꢀSCNꢀBnꢀH octapa showed exceptional complexaꢀ

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tion properties (quantitative In or Lu radiolabeling in 10ꢀ

0 minutes at ambient temperature) and favorable in vivo

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stability of resulting complexes. Furthermore, chiral ligands

acyclic chelators are widely known and beyond debate. Releꢀ

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Bioconjugate Chemistry

H CHXdedpa (N O ) and H CHXoctapa (N O ) showed promꢀ

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ising Ga and In radiolabeling properties, respectively, and

subsequently impressive stability in human serum.

Results and Discussion

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Synthesis and characterization of the ligand

Our group continues to design ligands that may incorporate

large metal ions (such as radioactive actinides/lanthanides for

imaging/therapy), which possess ideal properties for radioꢀ

pharmaceutical incorporation, e.g. fast, mild, and quantitative

complexation of radiometals at low ligand concentrations;

formation of resultant thermodynamically stable and kineticalꢀ

ly inert metalꢀcomplexes; and a convenient point of attachꢀ

ment to targeting vectors. Herein, we report the synthesis and

characterization of a novel nonadentate (CN = 9) acyclic cheꢀ

lator H neunpa (N O , referred to herein as either pꢀNO ꢀBnꢀ

The synthesis of the previously reported analogue H decapa

used Nꢀbenzyl protection, Nꢀalkylation with an alkyl halide,

benzyl deprotection via hydrogenation, a second alkyl halide

Nꢀalkylation, and finally deprotection in refluxing HCl (6M).

The Nꢀbenzyl protection was found to be the yieldꢀlimiting

step because the deprotection always resulted in partly elimiꢀ

nating the picolinic acid moieties. The use of Oꢀ

nitrobenzenesulfonyl (nosyl) was found to give better cumulaꢀ

tive yields compared to Nꢀbenzyl protection. Based on that,

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the bifunctional analogue H neunpa, was synthesized with a

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H neunpa or H neunpa) and bifunctional analogue pꢀSCNꢀBnꢀ

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general reaction scheme that follows Nꢀnosylꢀprotection, biꢀ

functionalization on the middle nitrogen atom via Nꢀ

alkylation, Nꢀalkylation with picolinic acid, nosylꢀdeprotection

with thiophenol, a second alkyl halide Nꢀalkylation and esterꢀ

deprotection with LiOH to yield pꢀNO ꢀBnꢀH neunpa 6

H neunpa that was designed as a bifunctional analogue of

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H decapa (N O ), reported by our group in 2012. The carboxꢀ

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ylic acid group on the middle nitrogen atom has been replaced

by pꢀnitrobenzeneꢀethylene to keep its symmetry, and act as

the bifunctional arm to attach the ligand to a biomolecule

through a thiourea bond (Figure 1). We hypothesized that the

extended diethylenetriamine backbone and nine coordinating

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(Scheme 1). The isothiocyanate (NCS) analogue for mAb

conjugation, pꢀSCNꢀBnꢀH neunpa 9, was synthesized from the

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intermediate 5 followed by nitroꢀreduction, esterꢀdeprotection

with LiOH and isothiocyanate formation with thiophosgene

(Scheme 1).

atoms of H neunpa may favorably form complexes with large

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3+

metal ions such as In (92 pm, CN = 8) , Lu (103 pm, CN =

9), or Bi (117 pm, CN = 8). Radiolabeling of In and Lu

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Starting from the diethylenetriamine backbone, the two primaꢀ

ry amines were protected with the 2ꢀnitrobenzenesulfonyl

groups to yield compound 1. Compound 1 is highly polar due

to the two nosyl groups, thus a highly polar solvent like methꢀ

anol is needed to separate it from the column. The second step

is Nꢀalkylation with 4ꢀ(2ꢀbromoethyl)nitrobenzene. In order to

maintain symmetry of the ligand, the ideal spot for bifunctionꢀ

alization is the middle nitrogen. After that, Nꢀalkylation with

to H neunpa was assessed and compared to goldꢀstandards

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DOTA and CHXꢀA”ꢀDTPA, and an in vivo biodistribution

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study of H neunpa and CHXꢀA”ꢀDTPA labeled with In was

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performed. Thermodynamic stability constants of selected

metalꢀneunpa complexes were also determined. Moreover,

coupling of the HER2/neu targeting monoclonal antibody

(mAb) Trastuzumab was performed via the reaction between

the antibody’s primaryꢀamine(s) with the isothiocyanate funcꢀ

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methylꢀ6ꢀbromomethyl picolinate was performed to yield

tional group of pꢀSCNꢀBnꢀH neunpa. The bioconjugate was

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compound 3. The most challenging step was the nosylꢀ

deprotection, constantly resulting in low yields of compound

4. The deprotected product is unfortunately highly polar and

likely adsorbs on the surface of potassium carbonate, as seen

by the red color of the salt. It was not possible to remove the

large fractions of the deprotected product completely from the

salt, which explains the low yield reported in the Experimental

Section. Subsequently, alkyl halide Nꢀalkylation was perꢀ

labeled with In, and in vivo biodistribution and SPECT/CT

imaging studies were conducted and compared directly to a

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InꢀCHXꢀA”ꢀDTPAꢀTrastuzumab conjugate.

formed to yield product 5 with 71 % yield. pꢀNO ꢀBnꢀ

2

H neunpa 6 was synthesized in a final step of ester deprotecꢀ

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tion with LiOH. This compound was further used for radioꢀ

labeling experiments as well as potentiometric stability titraꢀ

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tions. The H NMR spectrum of the final product is shown in

Figure 2.

pꢀSCNꢀBnꢀH neunpa 9 was synthesized starting from the

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intermediate 5 of the previous reaction route. Reduction of the

nitro group with palladium on carbon yielded the amineꢀ

functionalized product 7. The hydrolysis of the two tertꢀbutyl

esters and two methyl esters was performed differently from

previous reports.

peratures, compound 8 was synthesized by adding 10 eq. of

lithium hydroxide to the reaction mixture at room temperature

to yield the product, with a 50 % yield. The final step is the

synthesis of the isothiocyanateꢀfunctionalized product 9. This

was achieved by the reaction of excess thiophosgene with the

aromatic primary amine to yield the final product with a 59 %

Figure 1. Structures of cyclic (DOTA) and acyclic (OctreoScan,

CHXꢀA"ꢀDTPA) commercial chelators, and acyclic “pa”ꢀligands

H CHXdedpa, H CHXoctapa, H octapa, H decapa, and novel

5,12

Instead of acidic hydrolysis at high temꢀ

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nonadentate chelator pꢀSCNꢀBnꢀH neunpa discussed in this work

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yield. Overall, the synthesis of pꢀSCNꢀBnꢀH neunpa from

diethylenetriamine has a cumulative yield of 2.3 %, comparaꢀ

ble to the overall synthesis yield of H decapa (2.5 %).

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Bioconjugate Chemistry

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Scheme 1. Synthetic scheme for p-SCN-Bn-H neunpa and

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p-NO -Bn-H neunpa

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NMR spectrum cannot be used for proper assignments (Figure

S1).

A: ligand

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B: La complex

C: In complex

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.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4

f1 (ppm)

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Figure 2. H NMR spectra of A: pꢀNO ꢀBnꢀH neunpaꢀpꢀBnꢀ

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ꢀ

NO (400 MHz, CDCl , 25 °C); B: [La(pꢀNO ꢀBnꢀneunpa)] (400

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2

ꢀ

MHz, CDCl , 25 °C); C: [In(pꢀNO ꢀBnꢀneunpa)] (400 MHz,

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DMSOꢀd , 25 °C)

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IR

ꢀ

Due to the insolubility of [Bi(pꢀNO ꢀBnꢀneunpa)] , an IR

2

experiment on the solid was performed (Figure 3). Shifts of

various peaks of the ligand itself compared to the Bi complex

Synthesis and characterization of nonꢀradioactive metal comꢀ

plexes

ꢀ1

can be observed. The OH stretch at 2500 cm disappeared

after complexation, suggesting that the carboxylic acids are

ꢀ

1

bound to the metal ion; the carboxyl stretch at 1700 cm disꢀ

appeared as well, supporting this assumption. The two stretchꢀ

NMR

ꢀ

1

ꢀ1

3+

es of the nitro functional group (1500 cm and 1400 cm )

Three complexation experiments were performed with La ,

ꢀ1

3+

1

stayed the same. The stretch at 1200 cm in the ligand spectra

can be assigned as a CꢀN stretch that shifts to lower energies

In and Bi . H NMR spectra of the pꢀNO ꢀBnꢀH neunpa

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ligand precursor, and corresponding La and In complexes can

ꢀ

1

ꢀ

(1000 cm ) when bound to the metal ion.

be found in Figure 2. The [La(pꢀNO ꢀBnꢀneunpa)] complex

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100

shows H NMR upfield shifts of the alkylꢀregion; this effect

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has been previously observed in our group. The aromatic

region is more resolved and shows a splitting of the peaks.

Integration of all peaks gives the same number of protons

compared to the uncomplexed ligand. Furthermore, the HSQC

spectra of this complex (Figure S2) shows the same number of

carbons compared to the bare ligand, suggesting that there is

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p-NO -Bn-H neunpa

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only one isomer in solution. In contrast, the H NMR spectrum

ꢀ

[Bi(pꢀNO

ꢀBnꢀneunpa)]^ꢀ

2

of [In(pꢀNO ꢀBnꢀneunpa)] shows more splitting in the aroꢀ

2

matic and alkyl regions. The aromatic peaks are sharp and

well resolved and integrating the peaks suggests one major

static isomer. In addition, the COSY spectrum of this complex

shows clear coupling of several peaks in the complex alkyl

region (Figure S12), leading to the assumption there are fluxꢀ

ional isomers in solution. Comparing these results to those

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Figure 3. IR spectra of pꢀNO ꢀBnꢀH neunpa and [Bi(pꢀNO ꢀBnꢀ

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2

ꢀ

1

ꢀ

with [In(decapa)] , which gave a complex H NMR spectrum

with multiple isomers presumably due to several unbound

neunpa)]

Thermodynamic Stability

The extended diethylenetriamine backbone, along with the

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carboxylates , we can see an improvement in terms of isomerꢀ

ization by replacing one carboxylate group with the functionꢀ

alization arm on the middle nitrogen atom of the diethylenetriꢀ

nonadentate N O binding motif of H neunpa, were specificalꢀ

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ly designed to accommodate binding of larger metal ions. As

amine backbone. Due to insolubility of the Bi complex, the H

such, the protonation constants of H neunpa as well as the

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Bioconjugate Chemistry

Page 4 of 17

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+

3+

stability constants of the respective La , Bi and In comꢀ

plexes were determined at 25 ºC in 0.16 M NaCl aqueous

solution. The stepwise protonation constants (log K) obtained

are presented in Table 1 together with protonation and

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Table 1. Stepwise Protonation Constants (log K_H_h_L) of H neunpa ( 25 ºC, I = 0.16 M NaCl)

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ꢀ

5ꢀ

neunpa (this decapa

work) work)

(this

4ꢀ 10

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equilibrium reaction

octapa

DTPA

CHXꢀA’’ꢀDTPA

DOTA

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0.92(2)

.29(2)

.79(2)

11.03(3)

9.20(3)

6.86(4)

4.43(4)

3.46(5)

2.84(6)

2.52(4)

ND

8.59(4)

5.59(6)

3.77(2)

2.77(4)

2.79(4)

ND

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9.40

4.85

3.10

2.20

12.30

9.24

5.23

3.32

2.18

12.60(1)

9.70(1)

4.50(1)

4.14(1)

2.32(1)

L + H ⇆ HL

+

HL + H ⇆ H L

2

+

H L + H ⇆ H L

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+

4.02(3)

H L + H ⇆ H L

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+

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.97(2)

.39(5)

H L + H ⇆ H L

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+

H L + H ⇆ H L

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+

ND

H L + H ⇆ H L

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+

H L + H ⇆ H L

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a

Literature data of related systems are presented for comparison. L = Ligand and charges of ligand species and metal complexes were

omitted for simplicity.

stability constants reported for the related ligands H decapa,

The higher protonation constants in the case of H decapa

5

H octapa, DTPA and CHXꢀA”ꢀDTPA. A straightforward

could be attributed to the higher negative charge of the ligand.

The speciation plots for H neunpa and H decapa are shown in

4

comparison of the ability of different ligands to coordinate a

specific metal ion (rather than the thermodynamic stability

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Figure S4 in the Supporting Information.

constants alone), is the conditional stability constant or pM

Potentiometric titrations of H neunpa were carried out in the

4

n+

3+

value. pM is defined as (ꢀlog [M ] ) and is calculated at

free

xꢀ

presence of La , Bi , and In in order to determine the stabilꢀ

ity constants of the corresponding metal complexes. For lanꢀ

thanum, combined potentiometricꢀspectrophotometric titraꢀ

tions demonstrated that the complexation started from pH 2,

based on the distinctive features of the spectra compared to the

electronic spectra of H neunpa (Figures S3 and S5). The therꢀ

modynamic stability of [La(neunpa)] was determined to be

log K_M_L= 19.81(4) and pM = 16. This value is close to the

values obtained for [La(octapa)] log K = 19.92(6) and

La(DTPA)] log K = 19.48 . Similar to the free ligand, the

deprotonation of the [La(H neunpa)] and La(Hneunpa) speꢀ

cies is marked by the appearance of a single isosbestic point at

91 nm between the pH range 2.42ꢀ8.23 and suggests that the

deprotonations occur at the two terminal amines of the diethyꢀ

lenetriamine backbone (Figure S5a). The [La(neunpa)] speꢀ

cies further deprotonates presumably due to the deprotonation

of a coordinated water molecule with pK 9.78 to form the

monohydroxo complexes (Figure S5b). Species distribution

diagrams for the lanthanum(III) complexes of H neunpa are

plotted in Figure S6. The thermodynamic stability constant of

the bismuth(III) complexes of H neunpa could not be deterꢀ

mined by direct potentiometric titrations as this requires the

knowledge of the concentration of the free and bound metal

ion at equilibrium, and even at pH 2 the Bi(III) complex was

already significantly formed. The ligandꢀligand competition

method using Na H EDTA as a known competitor was used to

n+

specific conditions ( [M ] = 1 µM, [L ] = 10 µM, pH 7.4 and

5 ºC), taking into consideration both metalꢀligand association

2

and ligand basicity. The protonation constants of the new

synthesized ligand H neunpa were determined by potentiomꢀ

4

etric titrations at pH 1.8ꢀ11.5 and by combined potentiometricꢀ

4

1

6,17

ꢀ

spectrophotometric titrations

over the pH range 2.5ꢀ11.5.

In Figure S3 are shown the sets of spectra obtained as a funcꢀ

ꢀ4

ꢀ

19

tion of pH, at 7.18 x 10 M ligand concentration. The first and

second protonation processes occur at the two terminal amines

ML

2ꢀ

20

[

ML

+

of the diethylenetriamine backbone (log K = 10.92(2) and log

1

2

K = 9.29(2)), as suggested by the appearance of a single isosꢀ

2

bestic point at 284 nm between pH 8.33 and 11.32 in the UVꢀ

potentiometric titration (Figure S3c). The third protonation

2

ꢀ

process (log K = 6.79(2)) is assigned to the central nitrogen

3

atom in the backbone and is supported by the appearance of an

isosbestic point at 293 nm in the pH region between 5.39 and

8

.33 (Figure S3b). The fourth and fifth protonation processes

13,18

are attributed to the picolinate moieties

(log K = 4.02(3)

4

and log K = 2.97(2)). The UVꢀpotentiometric titration showed

5

also in this case a single isosbestic point at 296 nm for these

protonation processes (Figure S3a). The sixth protonation step

4

is attributable to the carboxylic acid substituent (log K =

6

2

.39(5)) and was calculated from potentiometric titrations. The

value of log K could not be determined, as the value was

7

below the threshold of the electrode (pH < 2). H neunpa, the

4

2

bifunctional analogue of the previously reported H decapa (for

5

yield the stability constants presented in Table 2 and speciaꢀ

tion plots in Figure S7. Particularly high thermodynamic staꢀ

bility of [Bi(neunpa)] was found, log K = 28.76(9) and pBi

which we correct here the protonation constants, Table 1)

presents overall fairly similar protonation constants, although

the fourth and fifth protonation processes attributed to the

picolinate moieties differ by 0.41 and 0.49 units respectively.

ꢀ

ML

=

[

27. The thermodynamic stability constant of the

ꢀ

2

ꢀ

Bi(neunpa)] complex is lower than those of [Bi(DTPA)]

4

ACS Paragon Plus Environment

Page 5 of 17

Bioconjugate Chemistry

2

ꢀ

14

ꢀ

and [Bi(CHXꢀAꢀDTPA)] complexes and lower than that for

[Bi(DOTA)] ; however, it is interesting to note that

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

3+

3+ a

Table 2. Stepwise Stability Constants (log K) of H neunpa complexes with La , Bi and In

4

4ꢀ

5ꢀ 10

4ꢀ

equilibrium reaction

neunpa

decapa

octapa

DTPA

CHXꢀA’’ꢀDTPA DOTA

19

20

21

3+

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

La + L ⇆ LaL

19.81(4)

19.92(6)

19.48

22.0

+

8

3

9

2

1

3

.05(5)

.28(6)

.78(4)

8.76(9)

0.26(5)

.8(1)

LaL + H ⇆ LaHL

+

LaHL + H ⇆ LaH L

2

+

LaLOH + H ⇆ LaL

14

22

3+

35.2(4)

34.9(4)

30.3

Bi + L ⇆ BiL

+

BiL + H ⇆ BiHL

+

BiHL + H ⇆ BiH L

2

+

10.57(7)

8.17(2)

5.07(2)

BiLOH + H ⇆ BiL

10

23,24

24

3

+

2

27.56(5)

5.47(3)

2.73(6)

9.83(7)

26.8(1)

29.0

23.9(1)

In + L ⇆ InL

1

0

+

2.9(2)

InL + H ⇆ InHL

+

3

9

.40(3)

.41(3)

InHL + H ⇆ InH L

2

+

InLOH + H ⇆ InL

3+

pLa

16

19.7

3+

25

pBi

27

3+

10

23.6

23.1

26.5

25.7

18.8

a

Literature data for related systems are presented for comparison. L = Ligand and charges of ligand species and metal complexes were

omitted for simplicity.

3

+

177

111

H neunpa and DOTA have the same pBi value of 27 (Table

The radiolabeling properties of Lu and In with H neunpa

were investigated, and compared directly to results obtained

for the goldꢀstandards DOTA and CHXꢀA”ꢀDTPA.

4

2+

2

). Despite the high formation constant of [In(H neunpa)]

2

log K_M_L_H₂= 36.64(3), the system is well determined by direct

potentiometric titration taking advantage of the indiumꢀ

chloride competing species. The system as in the case of lanꢀ

Initial radiolabeling experiments revealed that pꢀNO ꢀBnꢀ

H neunpa could quantitatively complex In (radiochemical

yield, RCY > 99%) in 10 minutes at room temperature (RT),

pH 4, at ligand concentrations of 10 M. Subsequently, conꢀ

centrationꢀdependent labeling was performed by decreasing

the ligand concentration 10ꢀfold while keeping the In activiꢀ

ty constant. Quantitative radiolabeling was achieved at ligand

concentrations as low as 10 M (Figure 4), at 10 min and

ambient temperature. At decreasing ligand concentrations of

0 , 10 , and 10 M, radiochemical yields gradually deꢀ

creased to 71.1, 10.5, and 1.5%, respectively. These results

demonstrate the ability of pꢀNO ꢀBnꢀH neunpa to rapidly and

efficiently complex In in high specific activities at ambient

2

1

11 3+

4

^t^h^aⁿ^u^m⁽^I^I^I⁾^aⁿ^d^bⁱ^s^m^u^t^h⁽^I^I^I⁾^c^o^m^p^l^e^x^e^s^c^oⁿ^t^aⁱⁿⁱⁿ^g^M^L^H2^,

ꢀ

4

MLH, ML and ML(OH) complex species (Figure S8) presentꢀ

^e^d^a^hⁱ^g^h^l^o^g^KML = 28.17(2) and pM = 23.6, which is signifiꢀ

cantly higher than for DOTA (Table 2), slightly higher than

111

for the previously reported H decapa, 2.1 pM units lower than

5

ꢀ

ꢀ7

for DTPA and 2.9 pM units lower than for [In(octapa)] . To

our knowledge thermodynamic formation constants of the

2

ꢀ

8

ꢀ9

ꢀ10

[In(CHXꢀA”ꢀDTPA)] have not been yet reported. It is noteꢀ

1

10

worthy that, as with other previously reported ligands , the

trend of the stability constants and pM values and the human

serum stability data do not correlate well, and despite the

2

4

111

ꢀ

2ꢀ

higher pM values for [In(octapa)] species or [In(DTPA)] vs

[

temperatures. H octapa showed similar radiolabeling efficienꢀ

4

ꢀ

7

In(neunpa)] , [In(neunpa] showed an exceptional serum staꢀ

cies at 10 M, results at lower ligand concentrations are not

1

0

bility 97.8(1) % after one day, 5.5 units higher than the

reported. In sharp contrast to the two “pa” ligands is the

macrocyclic goldꢀstandard DOTA which is reported to require

heating samples at 100°C for 30 minutes to achieve high radiꢀ

ochemical yields. The acyclic chelator CHXꢀA”ꢀDTPA is a

relatively recent addition to the list of potential In chelators;

ꢀ

[

In(octapa)] complex, 7.9 units higher than the [In(CHXꢀA”ꢀ

2

ꢀ

2

ꢀ

DTPA)] complex and 9.5 units higher than the [In(DTPA)]

complex.

10

111

3+

in contrast to DOTA it can efficiently complex In isotopes at

ambient temperatures yet exhibits comparable in vivo stability

Radiolabeling Experiments with Unmodified Chelators

5

ACS Paragon Plus Environment

Bioconjugate Chemistry

Page 6 of 17

2

,26

to DOTA conjugates , making it a more appealing chelator

for radiolabeling of heatꢀsensitive biomolecules such as afꢀ

90.1% intact. The initial drop in stability after 1 h may be due

to the presence of two isomers in the labeling reaction of pꢀ

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

2

7ꢀ31

111

fibodies or antibodies.

ies with pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA at ligand concentrations

Our initial In radiolabeling studꢀ

NH ꢀBnꢀCHXꢀA”ꢀDTPA (vide supra, major isomer 88.5%

2

88

and minor isomer 11.5%). Studies with YꢀCHXꢀDTPA have

demonstrated that thermodynamic stability of the resultant

metal complex can be significantly affected by the absolute

configuration, possibly due to unfavourable steric hindrance of

2

ꢀ

4

of 10 M corroborate the efficient and mild labeling of this

ligand which yielded RCYs >99%; however, two evident

peaks in the HPLC radioꢀchromatogram are observed – one

major product at 8.6 min and a minor product at 8.0 min (Figꢀ

ure S10), with the ratio between the major and minor product

being 7.7. The appearance of two distinct peaks in the radioꢀ

chromatogram may indicate the formation of distinct Inꢀ

chelate isomers. Contrary to H neunpa, at pꢀNH ꢀBnꢀCHXꢀ

A”ꢀDTPA concentrations of 10 and 10 M, In labeling

yield decreased to 75.0 and 3.4%, respectively. The ratio of

major to minor product in the HPLC radioꢀchromatogram also

changed drastically at lower ligand concentrations, with the

32

certain stereoisomers; therefore, it is feasible that the minor

isomer is kinetically labile with respect to transchelation to

1

11

ꢀ

serum proteins. Indeed, [ In(pꢀNO ꢀBnꢀneunpa)] displayed

2

1

11

111

marginally higher stability than [ In(pꢀNH ꢀBnꢀCHXꢀA”ꢀ

DTPA)] , [ In(DOTA)] , and [ In(octapa)] after 1 d (97.8

± 0.1%, 89.9 ± 0.6, 88.3 ± 2.2%, 92.3 ± 0.04%, respectively).

2

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

2ꢀ

111

ꢀ

111

ꢀ

4

2

ꢀ7

ꢀ8

111

Table 3. Human serum stability challenge data performed

at 37°C (n = 3), with stability shown as percentage of in-

ꢀ

7

ratio being close to unity (0.95) for 10 M labeling.

111

tact In-complex

1

50

00

Complex

1 h (%)

1 d (%)

5 d (%)

1

11

[

In(pꢀNO ꢀBnꢀ

97.9 ± 0.3 97.8 ± 0.1

97.8 ± 0.7

2

ꢀ

1

neunpa)]

111

[

In(pꢀNH ꢀBnꢀ

91.8 ± 1.8 89.9 ± 0.6

90.1 ± 0.9

2

ꢀ

5

0

CHXꢀA”ꢀDTPA)]

1

11

ꢀ a

b

[

In(octapa)]

93.8 ± 3.6 92.3 ± 0.04

89.6 ± 2.1 88.3 ± 2.2

ND

0

111

ꢀ a

b

In(DOTA)]

ND

0.00010.001 0.01 0.1

1

10 100 1000

log [ꢁM]

111

c

InCl (control)

4.0

7.2

3.4

3

1

11

a

Figure 4. Radiolabeling results of

(10min, RT, pH 4).

InꢀpꢀNO ꢀBnꢀneunpa

Mouse serum stability data performed at ambient temperature;

2

10

b

c

data included from ref for comparison. ND = not determined. n

1 only.

=

111

Unlike the facile labeling kinetics of [ In(pꢀNO ꢀBnꢀ

neunpa)] , initial radiolabeling studies with Lu were unsucꢀ

2

ꢀ

177

Initial Biodistribution Studies

Mouse biodistribution studies over the course of 24

hours (n = 4 each time point) were performed with [ In(pꢀ

177

cessful. Attempted Lu labeling at ligand concentrations of

ꢀ4

111

1

0

M in 10 minutes at room temperature, pH 4 or 5.5, disꢀ

ꢀ

111

2ꢀ

played a radiochemical yield of 12.4%; heating the sample to

0 °C for 1 hour did not improve RCY. Conversely, goldꢀ

standard DOTA was quantitatively radiolabeled (RCY > 99%)

with Lu when heated to 40 °C for 1 hour at the same ligand

concentration (10 M). The inability of pꢀNO ꢀBnꢀH neunpa

to complex Lu isotopes at mild temperatures (< 40 °C)

precluded further study with this isotope, since it was immediꢀ

ately obvious from the initial results that H neunpa was a poor

match for Lu and presented no potential advantage comꢀ

pared to the goldꢀstandard DOTA.

NO ꢀBnꢀneunpa)] and

[

In(pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA)]

2

4

and the data are summarized in Table 4. Both Inꢀcomplexes

were rapidly excreted through the kidneys and activity cleared

quickly from all other organs. Notably, uptake of [ In(pꢀ

1

77

111

ꢀ

4

ꢀ

2

4

NO ꢀBnꢀneunpa)] in the intestines was significantly higher

2

1

77

111

2ꢀ

than for [ In(pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA)] after 15 min

(17.9 ± 5.5% ID/g vs 3.6 ± 1.6% ID/g) and 1 h (39.8 ± 2.9%

ID/g vs 10.7 ± 1.4% ID/g). One explanation for the difference

in intestine uptake is that the monoꢀanionic Inꢀneunpa comꢀ

plex is more lipophilic than the diꢀanionic InꢀpꢀNH ꢀBnꢀ

2

4

1

77

111

1

11

2

CHXꢀA”ꢀDTPA complex, as evinced by shifts in the radioꢀ

HPLC retention times (t = 12.9 min and 8.6 min, respectiveꢀ

R

Stability Studies with the Unmodified Chelators

ly) and the absolute logP values of each complex (ꢀ1.65 ±

0.04, and ꢀ3.85 ± 0.17, respectively), thus shifting the excreꢀ

tion of the radiotracer from renal to intestinal elimination

because highly charged polar substances are generally elimiꢀ

nated via the kidneys while less hydrophilic compounds tend

to be eliminated via the intestinal tract. Nonetheless, the reꢀ

maining Inꢀcomplex in the intestines at 1 h was rapidly

excreted by 4 h for both complexes, and the uptake in intesꢀ

tines of [ In(pꢀNO ꢀBnꢀneunpa)] and [ In(pꢀNH ꢀBnꢀCHXꢀ

111

In order to probe the kinetic inertness of the [ In(pꢀNO ꢀBnꢀ

neunpa)] complex, a 5 d in vitro competition experiment was

performed in the presence of human blood serum. Serum

contains many endogenous ligands that can compete for In(III)

binding in vivo, such as apoꢀtransferrin and albumin, and any

2

ꢀ

1

11

1

11

chelateꢀbound In must therefore be sufficiently stable to

withstand transchelation to such proteins. The in vitro stability

1

11

ꢀ

of [ In(pꢀNO ꢀBnꢀneunpa)] at 1 h, 1 and 5 d time points was

111

ꢀ

111

2

1

11

tested alongside goldꢀstandard

DTPA)] for comparison (Table 3). The [ In(pꢀNO ꢀBnꢀ

neunpa)] complex exhibited exceptional stability, remaining

[

In(pꢀNH ꢀBnꢀCHXꢀA”ꢀ

2ꢀ

2

A”ꢀDTPA)] were no longer statistically different (p > 0.05)

at later time points (0.265 ± 0.206% ID/g vs 0.160 ± 0.047%

ID/g, for 4 h; 0.216 ± 0.114% ID/g vs 0.129 ± 0.06% ID/g, for

2ꢀ

111

2

ꢀ

1

11

9

7.8% intact over 5 days, while the [ In(pꢀNH ꢀBnꢀCHXꢀA”ꢀ

2

2ꢀ

2

4 h, respectively). It has been suggested that administration

DTPA)] complex showed an initial ~8% drop in stability

after 1 h and subsequently stabilized for 5 days to remain

111

of an unstable Inꢀcomplex would result in demetalation of

the complex in vivo and subsequent accumulation of transcheꢀ

6

ACS Paragon Plus Environment

Page 7 of 17

Bioconjugate Chemistry

1

11 3+

111

lated or “free” In activity in the liver, spleen, and bone

organs suggests both Inꢀcomplexes are exceptionally robust

33

111

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

over time; therefore, the rapid excretion of [ In(pꢀNO ꢀBnꢀ

and stable in vivo (0.035 ± 0.008% ID/g vs.

2

ꢀ

111

2

neunpa)] and [ In(pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA)] from these

2

1

11

scid

Table 4.

Decay corrected % ID/g values from biodistribution of

In-complexes in healthy NOD.Cg-Prkdc

tm1Wjl

scid

tm1Wjl

Il2rg

/SzJ female NOD.Cg-Prkdc Il2rg

/SzJ mice (4 months old), n = 4; statistical analysis Student's T-test: bold

=

p < 0.05.

organ

15 min

1 h

4 h

24 h

111

-

[

In][In(p-NO -Bn-neunpa)]

2

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

Blood

1.979 (0.425)

0.174 (0.113)

1.644 (0.321)

0.983 (0.362)

17.941 (5.475)

0.792 (0.379)

2.684 (0.190)

0.287 (0.196)

1.251 (0.364)

0.585 (0.089)

5.681 (1.343)

2.695 (0.392)

0.419 (0.032)

0.394 (0.101)

0.743 (0.351)

0.059 (0.033)

4.129 (2.183)

0.077 (0.007)

0.009 (0.001)

0.101 (0.011)

0.056 (0.034)

0.022 (0.004)

0.0020 (0.0009)

0.059 (0.072)

0.012 (0.011)

0.0064 (0.0011)

0.0009 (0.0007)

0.014 (0.005)

Fat

Uterus

Ovaries

Intestine

Spleen

Liver

0.0080 (0.0067)

0.216 (0.114)

39.760 (2.865)

0.073 (0.024)

0.312 (0.090)

0.026 (0.006)

0.054 (0.019)

0.037 (0.029)

0.484 (0.322)

0.388 (0.526)

0.075 (0.089)

0.016 (0.004)

0.072 (0.029)

0.012 (0.002)

0.143 (0.095)

0.265 (0.206)

0.032 (0.027)

0.071 (0.016)

0.010 (0.006)

0.012 (0.002)

0.012 (0.010)

0.158 (0.105)

0.072 (0.101)

0.011 (0.007)

0.0030 (0.0018)

0.0099 (0.0069)

0.0013 (0.0006)

0.029 (0.023)

0.029 (0.010)

0.035 (0.008)

Pancreas

Stomach

Adrenal glands

Kidney

Lungs

0.0047 (0.0023)

0.062 (0.019)

0.0009 (0.0018)

0.077 (0.058)

0.056 (0.096)

Heart

0.0061 (0.0103)

0.0020 (0.0016)

0.0102 (0.0060)

0.0009 (0.0016)

0.0078 (0.0060)

Muscle

Bone

Brain

Tail

111

2-

[

In][In(p-NH -Bn-CHX-A"-DTPA)]

2

Blood

2.370 (0.221)

0.323 (0.070)

1.643 (0.121)

1.279 (0.177)

3.644 (1.632)

0.627 (0.069)

3.388 (0.293)

0.539 (0.148)

1.037 (0.115)

0.592 (0.174)

7.643 (1.741)

1.677 (0.227)

0.697 (0.089)

0.500 (0.122)

0.717 (0.187)

0.063 (0.018)

3.562 (1.334)

0.091 (0.035)

0.016 (0.007)

0.116 (0.045)

0.077 (0.033)

10.713 (1.428)

0.074 (0.031)

0.271 (0.093)

0.036 (0.017)

0.058 (0.025)

0.064 (0.048)

1.152 (0.276)

0.120 (0.045)

0.041 (0.013)

0.022 (0.008)

0.057 (0.011)

0.017 (0.004)

0.349 (0.063)

0.013 (0.014)

0.0037 (0.0014)

0.082 (0.092)

0.024 (0.016)

0.160 (0.047)

0.036 (0.008)

0.053 (0.005)

0.014 (0.009)

0.018 (0.003)

0.022 (0.003)

0.632 (0.076)

0.023 (0.003)

0.011 (0.001)

0.0038 (0.0003)

0.0112 (0.0014)

0.0068 (0.0008)

0.410 (0.498)

0.0011 (0.0003)

0.0024 (0.0017)

0.035 (0.007)

Fat

Uterus

Ovaries

Intestine

Spleen

Liver

0.0188 (0.0047)

0.129 (0.060)

0.032 (0.008)

0.023 (0.006)

Pancreas

Stomach

Adrenal glands

Kidney

Lungs

0.0053 (0.0020)

0.042 (0.030)

0.0156 (0.0043)

0.301 (0.043)

0.012 (0.002)

Heart

0.0069 (0.0011)

0.0016 (0.0007)

0.0066 (0.0015)

0.0018 (0.0006)

0.0505 (0.0324)

Muscle

Bone

Brain

Tail

7

ACS Paragon Plus Environment

Bioconjugate Chemistry

Page 8 of 17

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

1

11

Table 5. Chemical and in vitro characterization data of In-neunpa -/- CHX-A''-DTPA-Trastuzumab radioimmunoconju-

gates

Immunoconjug

ate

Radiolabeling conditions Chelate/mA Specific activity Immunoreactive

Serum stability over

5 days (%)

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

and yield

b

(mCi/mg)

fraction (%)

1

11

Inꢀneunpaꢀ

Trastuzumab

pH 6, r.t., 15 or 30 min, 5.5 ± 1.1

92.6 %

28.0

>99

94.7 %

1

11

InꢀCHXꢀA”ꢀ

pH 6, r.t., 30 min, 91.6%

4.6 ± 0.7

20.8

>99

ND

DTPAꢀ

Trastuzumab

0

.023 ± 0.006% ID/g for liver; 0.029 ± 0.01% ID/g vs 0.032 ±

.008% ID/g for spleen; 0.010 ± 0.006% ID/g vs 0.007 ±

.002% ID/g for bone, at 24 h, respectively). Furthermore,

In(pꢀNO ꢀBnꢀneunpa)] had improved kidney clearance

compared to [ In(pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA)] at 24 h

assessed at 15 min. Calculated RCYs after 15 min were 15.0,

84.4, 92.6% at pH 5.0, 5.5, or 6.0, respectively (Figure S11).

RCY was also assessed after 90 min for pH 5.0 and 6.0 reacꢀ

tions; yields increased to 38% and remained constant at 92%

for pH 5.0 and 6.0, respectively. These initial radiolabeling

111

ꢀ

[

2

111

2ꢀ

2

(

<

0.077 ± 0.058% ID/g vs 0.301 ± 0.043% ID/g, respectively, p

0.05). Although these initial biodistribution data appear

tests suggest an optimal radiolabeling pH of 6.0 for H neunpaꢀ

Trastuzumab, in order to generate Inꢀconjugates of high

4

111

promising it may be that the predicted ꢀ1 and ꢀ2 charge of the

Inꢀneunpa/ꢀCHXꢀA”ꢀDTPA complexes, respectively, at physiꢀ

ological pH, could be mediating the rapid elimination of the

metalꢀcomplexes from the body; therefore, the Inꢀcomplexes

may not have ample opportunity to dissociate in vivo giving

the appearance of a stable complex. In order to further scrutiꢀ

nize the in vivo stability of Inꢀneunpa and InꢀCHXꢀA”ꢀ

DTPA an immunoꢀconjugate should be prepared (vide infra)

and accordingly, biodistribution of each complex can be moniꢀ

tored over the course of several days instead of hours.

radiochemical yield (>90%) and purity in only 15 min at RT.

This is in agreement with a solution equilibrium study, which

ꢀ

reflects the maximum of the [In(neunpa)] species formed at

pH 6 (see distribution diagram in Figure S7). The kinetic

111

inertness of InꢀneunpaꢀTrastuzumab was assessed in an in

vitro human serum challenge assay at 37°C. Much like the

unconjugated precursor, InꢀneunpaꢀTrastuzumab was excepꢀ

1

11

111

tionally inert to transchelation when incubated with human

serum, with 95.0 ± 1.1, 96.0 ± 2.5, 94.7 ± 0.6, and 94.8 ± 1.6%

111

of the Inꢀbioconjugate remaining intact after 1, 2, 5, and 7

days, respectively.

1

11

Inꢀlabeled Trastuzumab conjugates were then prepared for

in vivo studies. Both immunoconjugates were radiolabeled

Preparation of Bioconjugates and In Vitro Characterization

The promising radiolabeling efficiencies and in vitro kinetic

inertness of [ In(pꢀNO ꢀBnꢀneunpa)] provided motivation to

prepare and test the radiolabeling properties, and in vivo beꢀ

haviour of a H neunpaꢀbioconjugate. The HER2/neuꢀtargeting

antibody Trastuzumab was chosen as the biovector because it

is well established to target HER2ꢀexpressing tumors such as

the SKOVꢀ3 ovarian cancer cell line. To provide a basis for

comparison, the goldꢀstandard CHXꢀA”ꢀDTPA was also conꢀ

jugated to Trastuzumab and tested in parallel in the radiolabelꢀ

ing and in vivo experiments.

111

ꢀ

with In in NH

(

4

OAc buffer (0.15 M, pH 6) for 30 min at RT

Table 5), resulting in exceptionally high radiochemical yields

>90%) and radiochemically pure products (>99% after spin

2

4

111

purification) for both

InꢀneunpaꢀTrastuzumab and

Inꢀ

CHXꢀA”ꢀDTPAꢀTrastuzumab. Final specific activities were

determined to be 28.0 and 20.8 mCi/mg (1036 and 770

111

MBq/mg) for InꢀneunpaꢀTrastuzumab and InꢀCHXꢀA”ꢀ

DTPAꢀTrastuzumab, respectively. In vitro cellular binding

111

assays with SKOVꢀ3 cancer cells showed both

Inꢀ

immunoconjugates absolutely reactive towards the tested cell

111

The novel bifunctional chelator pꢀSCNꢀBnꢀH neunpa 9 and

4

line (>99% immunoreactivity). Both Inꢀimmunoconjugates

have thus the ability to still bind to HER2.

goldꢀstandard pꢀSCNꢀBnꢀCHXꢀA”ꢀDTPA were conjugated to

Trastuzumab, by incubation at room temperature at 5:1 molar

ratio of ligand to antibody under slightly basic conditions (pH

.0). Final immunoconjugates were purified by spin filtration

and stored at ꢀ20°C until use. A radiometric isotopic dilution

assay was employed to determine the number of accessible

chelates per antibody; an average of 5.5 ± 1.1 H neunpa cheꢀ

lates per antibody and 4.6 ± 0.7 CHXꢀA”ꢀDTPA chelates per

antibody were conjugated to Trastuzumab.

34

Biodistribution and SPECT/CT Imaging Studies

9

1

11

In order to compare directly the pharmacokinetics of Inꢀ

111

neunpaꢀTrastuzumab to InꢀCHXꢀA”ꢀDTPAꢀTrastuzumab in

vivo, biodistribution and single photon emission computed

tomography (SPECT) in conjunction with helical Xꢀray CT

imaging experiments were performed on female mice bearing

subcutaneous SKOVꢀ3 ovarian cancer xenografts on the left

shoulder. Either tracer was injected via the tail vein (~37

MBq, ~35 – 50 µg, in 200 µL saline), and after 1, 3, and 5

4

111

Preliminary

In radiolabeling efficiency of H neunpaꢀ

4

Trastuzumab was tested at pH 5.0, 5.5, and 6.0 in NH OAc

buffer (0.15 M) at RT, and the radiochemical yield (RCY) was

4

8

ACS Paragon Plus Environment

Page 9 of 17

Bioconjugate Chemistry

days (n = 4 per time point) the mice were imaged (n = 2, Figꢀ

ure 4) and sacrificed to collect organs and tumors to be countꢀ

ed on a calibrated γꢀcounter.

tumor). This distribution of antibodyꢀlinked tracer is well

known and is due to the metabolism and circulation of antiꢀ

bodies (or antibodyꢀchelate conjugates).

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

35

1

11

SPECT/CT overlays of

InꢀCHXꢀA”ꢀDTPAꢀTrastuzumab

111

and InꢀneunpaꢀTrastuzumab immunoconjugates are shown

in Figure 5 at 1, 3 and 5 days post injection. These images

were corrected for decay to allow qualitative comparison for

80

1

3

d CHX-DTPA-trastuzumab

5d CHX-DTPA-trastuzumab

1

11

the two radiolabeled immunoconjugates. For InꢀCHXꢀA”ꢀ

1d neunpa-trastuzumab

3d neunpa-trastuzumab

1

11

60

40

DTPAꢀTrastuzumab and

InꢀneunpaꢀTrastuzumab, day 1

5d neunpa-trastuzumab

images show significant activity in the blood, the heart, the

spleen and the tumor. The activity in the blood, the heart and

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

11

the spleen decreases over time. The

InꢀCHXꢀA”ꢀDTPAꢀ

*

Trastuzumab shows a higher activity in the tumor at all three

time points, giving highly localized activity to the tumor site.

**

1

11

On the other hand, InꢀneunpaꢀTrastuzumab shows a lower

uptake of activity into the tumor at day one post injection.

Over time, the activity in the tumor decreased to being barely

visible after 5 days post injection. Activity in the tumors for

**

20

111

the InꢀneunpaꢀTrastuzumab is still present at day 3 and 5

0

Blood

Spleen

Liver

Kidney

Bone

Tumor

postꢀinjection but in order to be able to compare the two tracꢀ

ers, an appropriate scale bar was required to prevent

Organ

111

Figure 6. Biodistribution of InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab

111

compared to InꢀneunpaꢀTrastuzumab in specific organs. Data

are expressed as mean ± SD (n=4). For statistical analysis * (p ≤

0

.05) and ** (p ≤ 0.01), twoꢀway ANOVA.

The blood, liver, kidney and bone show the lowest %ID/g

1

11

regarding all the different organs. The blood from

Inꢀ

neunpaꢀTrastuzumab treated mice is cleared faster than the

111

goldꢀstandard InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab between 1

111

d and 3 d. Additionally, InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab

shows an increase in accumulation in the tumor over time,

111

whereas InꢀneunpaꢀTrastuzumab shows a decrease of uptake

into the tumor over time, which is consistent with the

SPECT/CT overlay observations. Regarding the tumor:organ

111

ratios (Figure 7), InꢀneunpaꢀTrastuzumab and InꢀCHXꢀ

A’’ꢀDTPAꢀTrastuzumab show interestingly only significant

different values 5 d after injection for each ratio, tumor:blood,

tumor:heart and tumor:muscle. Furthermore, the addition of

the several chelating ligands onto Trastuzumab (5.5 ± 1.1

111

Figure 5.

SPECT/CT overlays of

InꢀCHXꢀA”ꢀDTPAꢀ

111

Trastuzumab (left) and InꢀneunpaꢀTrastuzumab immunoconjuꢀ

gates. Fused ꢁSPECT/CT images in female mice with subcutaneꢀ

ous SKOVꢀ3 xenografts on left shoulder, imaged at 1, 3 and 5

days post injection. Tumors are highlighted with arrows.

H neunpa chelates per antibody and 4.6 ± 0.7 CHXꢀA”ꢀDTPA

4

chelates per antibody) can modify the overall charge of the

ꢀ

antibody. Specifically, one negative charge per [In(neunpa)]

complex and two negative charges per [In(CHXꢀA”ꢀDTPA)]

2

ꢀ

1

11

oversaturation of the high uptake of the InꢀCHXꢀA”ꢀDTPAꢀ

Trastuzumab within tumors. Reducing the max value of the

scale bar by a factor of 2.8 shows the remaining activity within

complex labeled to Trastuzumab is generated; this induces a

twoꢀfold increase of negative charge on the CHXꢀA”ꢀDTPAꢀ

Trastuzumab conjugates compared to neunpaꢀTrastuzumab

conjugates, assuming an equal number of accessible chelates

111

the tumors for the InꢀneunpaꢀTrastuzumab (data not shown).

1

11

3+

Comparing the biodistribution pattern of

Trastuzumab with

Inꢀneunpaꢀ

are occupied by In in each immunoconjugate. Consequently,

this variance in overall charge of the Trastuzumab conjugate

1

11

InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab, both

1

11

tracer bioconjugates show the same general uptake profile, i.e.

significant uptake in blood, spleen, liver, kidney, bone and

tumor at day 1 (Figure 6 and Table S1). Three days and 5 days

after immunoconjugate injection, the spleen and tumor still

have the highest uptake of radiotracer compared to all other

organs, but with significant difference (p < 0.01) between

might affect the biodistribution of the resultant Inꢀtracer.

The immunoreactivity results are comparable for H neunpaꢀ

4

and CHXꢀA”ꢀDTPAꢀTrastuzumab conjugates, showing that

the reactivity between Trastuzumab and its receptor is not

altered due to the structural modification post chelateꢀ

conjugation. We wonder if the stability of the Trastuzumabꢀ

receptorꢀcomplex might not be as stable because of the charge

difference discussed before. This could lead to a decreased

uptake into the cancer cells. To conclude from these observaꢀ

1

11

111

InꢀCHXꢀA”ꢀDTPAꢀTrastuzumab

and

Inꢀneunpaꢀ

1

11

Trastuzumab (49.65 ± 6.79 %ID/g for InꢀCHXꢀA’’ꢀDTPAꢀ

Trastuzumab and 21.47 ± 6.61 %ID/g for

1

11

Inꢀneunpaꢀ

111

Trastuzumab after 5 days in the spleen and 59.14 ± 7.70

tions, different pharmacokinetic mechanisms for Inꢀneunpaꢀ

1

11

111

%

2

ID/g for

InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab and 16.01 ±

111

Trastuzumab and

InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab might

.24 %ID/g for InꢀneunpaꢀTrastuzumab after 5 days in the

9

ACS Paragon Plus Environment

Bioconjugate Chemistry

Page 10 of 17

take place after 5 days. These differences will be investigated great success, >99 % and 92.6 %, respectively. Human serum

111

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

further in order to fully understand the mechanism of tumor

uptake.

stability experiments revealed that the [ In(pꢀNO ꢀBnꢀ

2

ꢀ 111

neunpa)] complex and

conjugate were 97.8 and 94.7 % intact after 5 days, respectiveꢀ

ly. H neunpaꢀTrastuzumab was highly immunoreactive (>99

InꢀneunpaꢀTrastuzumab immunoꢀ

The slightly inferior uptake for this radiometalꢀneunpa antiꢀ

body conjugate is disappointing but the complete chemistry

4

%

) as indicated by a cellular binding assay. Biodistribution

and biology results suggest strongly that H neunpa is an attracꢀ

4

111

ꢀ

study of [ In(pꢀNO ꢀBnꢀneunpa)] in mice showed higher

tive chelating ligand with a built In conjugatable moiety and

2

3

+

3+

uptake into the intestine within the first hours compared to

should be investigated further with Bi and in other In ꢀ

biovector conjugates.

111

2ꢀ

[

In(CHXꢀA”ꢀDTPA)] due to its higher lipophilicity. Small

animal SPECT/CT imaging and biodistribution studies of

1

11

InꢀneunpaꢀTrastuzumab were performed using female

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

scid

tm1Wjl

25

NOD.CgꢀPrkdc Il2rg

grafts, and it was found that InꢀneunpaꢀTrastuzumab sucꢀ

cessfully identified the tumor from surrounding tissues and

/SzJ mice bearing SKOVꢀ3 xenoꢀ

T:Blood(¹In-CHX-A''-DTPA-Trastuzumab)

11

111

T:Blood( In-neunpa-Trastuzumab)^*^*

1

11

20

15

111

other organs. Compared to the goldꢀstandard InꢀCHXꢀA’’ꢀ

DTPAꢀTrastuzumab, our immunoconjugate showed slightly

lower tumor uptake which decreased over time and a lower

tumor:blood ratio after 5 days post injection, although high

quality SPECT/CT images were obtained. A different pharmaꢀ

cokinetic behavior of both immunoconjugates can be the result

of different charges on the immunoconjugates. Thermodynamꢀ

1

0

5

0

1d

3d

5d

ⁱ^c^s^t^a^bⁱ^lⁱ^t^y^e^x^p^e^rⁱ^m^eⁿ^t^s^s^u^p^p^o^r^t^t^h^e^s^e^fⁱⁿ^dⁱⁿ^g^s^,^sⁱⁿ^c^e^p^ꢀ^N^O2^ꢀ

time

BnꢀH neunpa was found to bind strongly to large, highly

charged metal ions like In , La and Bi . Indeed, these reꢀ

4

3+

8

0

T: Heart (¹In-CHX-A''-DTPA-Trastuzumab)

T: Heart ( In-neunpa-Trastuzumab)

11

111

sults suggest H neunpa as a strong Bi(III) chelator and, conꢀ

4

**

sidering the higher 3.6 units pM value respect to its In(III)

60

2

12

complex, it could be of interest for Bi(III) isotopes ( Bi and

213

Bi) in targeted alpha therapy (TAT). These encouraging

4

2

0

results suggest H neunpa and its immunoconjugate have promꢀ

ise for studies with other radiometals and targeting vectors.

These experiments are currently underway.

4

Experimental

1

d

3d

5d

time

Materials and Methods

250

All solvents and reagents were from commercial sources

(Sigma Aldrich, TCI) and were used as received unless otherꢀ

T: Muscle (¹¹¹In-CHX-A''-DTPA-Trastuzumab)

T: Muscle (¹¹¹In-neunpa-Trastuzumab)

**

2

00

50

wise noted. pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA and pꢀSCNꢀBnꢀCHXꢀ

2

A”ꢀDTPA were purchased from Macrocyclics (Dallas, TX)

and used as received. Human serum was purchased frozen

1

13

from Sigma Aldrich. H and C NMR spectra were recorded

at room temperature on a Bruker AV400 instrument; the NMR

spectra are expressed on the δ (ppm) scale and are referenced

to the residual solvent signal of the deuterated solvent. All

spectra were recorded with sweep widths of 0ꢀ14 ppm or ꢀ20ꢀ

100

50

0

1d

3d

5d

1

13

2

20 ppm for H and C NMR, respectively, and deviations in

time

the presented spectra are magnifications for visualization

purpose only. Assignments of the peaks in the NMR spectra

are approximate. Mass spectrometry was performed on a Waꢀ

ters ZQ spectrometer equipped with an electrospray source.

The HPLC system used for purification of ligands and precurꢀ

sors consisted of a Waters 600 controller equipped with a

Waters 2487 dual λ absorbance detector connected to a Pheꢀ

nomenex synergi hydroꢀRP 80Å 250mm x 21.1 mm semiꢀ

Figure 7. Tumor:Organ ratios of CHXꢀA’’ꢀDTPA and neunpa.

Data is expressed as mean ± SD (n=4). For statistical analysis **

(p ≤ 0.01), twoꢀway ANOVA.

Summary

111

177

preparative column. Analysis of In and Lu radiolabeled

chelate complexes was carried out using a Phenomenex Synꢀ

ergi 4 µ HydroꢀRP 80 Å analytical column (250 mm x 4.60

mm 4 µm) using an Agilent HPLC system equipped with a

model 1200 quaternary pump, a model 1200 UV absorbance

detector (set at 250 nm), and a Raytest Gabi Star NaI(Tl) deꢀ

tector. The radiochemical purity and specific activity of the

The acyclic chelator pꢀNO ꢀBnꢀH neunpa and the bioconjuꢀ

2

4

gated analogue H neunpaꢀTrastuzumab (5.5 ± 1.1 chelates per

4

antibody) have been synthesized, characterized (HRꢀESIꢀMS,

1

13

H NMR, C NMR, 2DꢀHSQC and cold metal complexation

1

11

177

studies) and evaluated via radiolabeling with In and Lu.

Unfortunately, low radiochemical yields of pꢀNO ꢀBnꢀ

2

177

H neunpa with

Lu were obtained (pH 4ꢀ5.5, ambient –

111

4

final In radioimmunoconjugates was determined by using a

sizeꢀexclusion chromatography (SEC) column (Phenomenex,

40°C, max. RCY 12.4 %). The radiolabeling yields of pꢀNO ꢀ

2

1

11

BnꢀH neunpa and H neunpaꢀTrastuzumab with In were a

4

1

0

ACS Paragon Plus Environment

Page 11 of 17

Bioconjugate Chemistry

13

BioSepꢀSECꢀsꢀ3000) on an Agilent HPLC system equipped

6.62 Hz, 4H). C NMR (101 MHz, CDCl , 25°C): 158.0,

3

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

with a model 1200 quaternary pump, a model 1200 UV abꢀ

sorbance detector (set at 280 nm), and a Bioscan (Washington,

DC) NaI scintillation detector (the radiodetector was connectꢀ

ed to a Bioscan BꢀFCꢀ1000 flowꢀcount system, and the output

from the Bioscan flowꢀcount system was fed into an Agilent

157.6, 147.5, 146.8, 129.6, 129.5, 124.0, 117.3, 114.4, 55.0,

52.5,

37.6,

and

33.5.

HRꢀESIꢀMS

calcd.₊for

+

[C H N O S +H] : 623.1230; found 623.1237 [M+H] .

24

26

6

10 2

Dimethylꢀ6,6ꢀ(((((4ꢀnitrophenethyl)azanediyl)bis(ethaneꢀ2,1ꢀ

diyl))bis(((2ꢀnitrophenyl)sulfonyl)azanediyl))bis(methylene))ꢀ

dipicolinate, 3

3

5900E interface, which converted the analog signal to a digiꢀ

tal signal). Instant thin layer chromatography paper strips

impregnated with silica gel (iTLCꢀSG, Varian) were used to

111

To a solution of 2 (13.59 g, 21.8 mmol) in dry DMF (80 mL)

was added methylꢀ6ꢀbromomethyl picolinate (11.55 g, 50.2

mmol, 2.3 eq) and sodium carbonate (5.32 g, 50.2 mmol, 2.3

eq). The bright orange reaction mixture was stirred at 60°C

overnight, filtered to remove excess sodium carbonate, and

concentrated in vacuo. The crude product was purified by

silica chromatography (CombiFlash Rf automated column

system; 2 x 80 g silica; solid (pause) preparation; A: hexane,

B: ethyl acetate, 100% A to 100% B gradient) to yield product

analyze crude Inꢀimmunoconjugate labeling reactions and

complex stability and counted on either a BioScan System 200

imaging scanner equipped with a BioScan Autochanger 1000

or on a Raytest miniGita with Beta GMC detector radioꢀTLC

plate reader using TLC control Mini Ginastar software. PDꢀ10

desalting columns (Sephadex Gꢀ25 M, 50 kDa, GE

Healthcare) and centrifugal filter units with a 50 kDa molecuꢀ

lar weight cutoff (Ultracelꢀ50: regenerated cellulose, Amicon

Ultra 4 Centrifugal Filtration Units, Millipore Corp.) were

used for purification and concentration of antibody conjugates.

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

3 as an orange/brown oil (70 %, 14 g). H NMR (400 MHz,

1

11

CDCl

7.78 (t, J = 7.8 Hz, 2H), 7.67ꢀ7.60 (m, 6H), 7.54 (d, J = 7.8 Hz,

H), 7.18 (d, J = 8.4 Hz, 2H), 4.67 (s, 4H), 3.89 (s, 6H), 3.29

3

, 25°C): 8.02ꢀ8.00 (m, 4H), 7.96 (d, J = 7.7 Hz, 2H),

InCl was cyclotron produced and provided by Nordion as a

3

1

17

~

0.05 M HCl solution. LuCl was purchased from Perkinꢀ

3

2

Elmer and provided as a solution in dilute HCl.

13

(t, J = 6.8 Hz, 4H), 2.58ꢀ2.51 (m, 8H). C NMR (101 MHz,

CDCl , 25°C): 165.3, 157.0, 148.1, 147.6, 146.4, 138.1, 132.1,

3

1

5

29.7, 126.0, 125.9, 124.4, 124.4, 123.5, 55.3, 53.9, 52.9,

2.7,

46.9,

and

33.4.

HRꢀESIꢀMS

^c^a^l^c^d^.+

for

N,Nꢀ(2ꢀNitrobenzensulfonamide)ꢀ1,2ꢀtriaminodiethane, 1

+

[

C H N O S +H] : 921.2184; found 921.2184 [M+H] .

40 40 8 14 2

Diethylenetriamine (4.19 mL, 38.8 mmol) was dissolved in

2

^T^H^F⁽²⁴⁰^m^L⁾^aⁿ^d^c^o^o^l^e^d^t^o⁰^°^C^.^S^o^dⁱ^u^m^c^a^r^b^oⁿ^a^t^e^N^a^C^O3

(9.04 g, 2.2 eq.) was added, followed by a slow addition of 2ꢀ

Dimethylꢀ6,6ꢀ(((((4ꢀnitrophenethyl)azanediyl)bis(ethaneꢀ2,1ꢀ

diyl)bis(azanediyl))bis(methylene))dipicolinate, 4

nitrobenzensulfonyl chloride (18.9 g, 85.3 mmol, 2.2 eq.),

causing the reaction mixture to turn pale yellow. The reaction

mixture was stirred overnight at room temperature. The offꢀ

white mixture was filtered to remove sodium carbonate and

the filtrate was rotary evaporated to dryness. The crude prodꢀ

To a solution of 3 (7.48 g, 8.1 mmol) in dry THF (100 mL)

was added thiophenol (1.91 mL, 18.7 mmol, 2.3 eq.) and poꢀ

tassium carbonate (3.71g, 26.8 mmol, 3.3 eq.). The reaction

mixture was stirred at 50°C for 72 hours, changing color to

light orange. The excess salts were removed by centrifugation

^u^c^t^w^a^s^p^u^rⁱ^fⁱ^e^d^b^y^sⁱ^lⁱ^c^a^c^h^r^o^m^a^t^o^g^r^a^p^h^y⁽^C^o^m^bⁱ^F^l^a^s^h^Rf

automated column system 220 g HP silica; solid (pause) prepꢀ

aration; A: hexanes, B: ethyl acetate, C: methanol, 100 % A to

(5 min, 4000 rpm) followed by several washes with DMF. The

filtrate was concentrated in vacuo in a (maximum) 50°C waꢀ

terbath temperature. The resulting crude dark orange oil was

purified by neutral alumina chromatography (CombiFlash Rf

automated column system; 6 x 40 g neutral alumina; liquid

injection A: dichlormethane, B: methanol, 100 % A to 20 % B

1

00 % B gradient followed by 100 % C) to yield the product 1

1

as a yellowꢀorange solid (88 %, 16.15 g). H NMR (400 MHz,

acetoneꢀd , 25°C): 8.13ꢀ8.11 (m, 2H), 7.94ꢀ7.89 (m, 6H), 3.11

(t, J= 7.32 Hz, 4H), 2.67 (t, J= 5.80 Hz, 4H). C NMR (101

MHz, acetoneꢀd , 25°C): 134.0, 132.7, 130.7, 125.0, 47.7, and

6

1

3

6

gradient) to yield product 4 as an orange oil (32.4 %, 1.45 g).

+

1

4

3.1. HRꢀESIꢀMS calcd. for [C H N O S +H] : 474.0753;

16 19 5 8 2

+

H NMR (400 MHz, CDCl , 25°C): 8.01 (d, J = 8.6 Hz, 2H),

3

found 474.0749 [M+H] .

7.90 (d, J = 7.6 Hz, 2H), 7.73 (t, J = 7.8 Hz, 2 H), 7.45 (d, J =

7.7 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 3.99 (s, 4H), 3.91 (s,

13

6H), 2.79ꢀ2.72 (m, 12H). C NMR (101 MHz, CDCl , 25°C):

N,Nꢀ(((4ꢀNitrophenyl)azanediyl)bis(ethaneꢀ2,1ꢀdiyl))bis(2ꢀ

nitrobenzenesulfonamide), 2

3

1

65.6, 158.9, 148.6, 147.4, 164.4, 137.8, 129.7, 126.0, 123.9,

23.7, 55.9, 54.0, 53.0, 52.7, 47.0, and 33.3. HRꢀESIꢀMS

To a solution of 1 (16.15 g, 34.1 mmol) in DMF (60 mL) was

+

calcd. for [C H N O +H] : 551.2618; found 551.2617

28

34

6

added K CO (6.13 g, 44.3 mmol, 1.3 eq.) and 4ꢀ(2ꢀ

+

2

3

[

M+H] .

bromoethyl)nitrobenzene (10.20 g, 44.3 mmol, 1.3 eq.). After

stirring the reaction mixture for 3 days at 40°C, the bright

yellow solution was cooled to room temperature and the exꢀ

cess K CO was removed by centrifugation. After drying the

N,Nꢀ[(tertꢀButoxycarbonyl)methylꢀN,Nꢀ

[6(methoxycarbonyl)pyridineꢀ2ꢀyl]methyl]ꢀNꢀ(4ꢀ

nitrophenethyl)ꢀ1,2ꢀtriaminodiethane, 5

2

3

solution in vacuo, the crude dark red product was purified by

silica chromatography (Combi Flash Rf automated column

system; 80 g HP silica; solid (pause) preparation; A: hexane,

To a solution of 4 (1.45 g, 2.6 mmol) in acetonitrile (60 mL)

was added tertꢀbutylbromoacetate (894 µL, 6.1 mmol, 2.3 eq.)

and sodium carbonate (642 mg, 6.1 mmol, 2.3 eq.). The reacꢀ

tion mixture was stirred at 60°C overnight, filtered to remove

excess sodium carbonate and concentrated in vacuo. The

crude product was purified by silica chromatography (Comꢀ

biFlash Rf automated system; 40g HP silica; A: dichloroꢀ

methane, B: methanol, 100% A to 20% B gradient) to yield

B: ethyl acetate, 100% A to 100% B gradient) to yield product

1

2

as an orange fluffy solid (64.0 %, 13.59 g). H NMR (400

MHz, CDCl , 25°C): 8.11ꢀ8.09 (d, J= 8.58 Hz, 2H), 8.08ꢀ8.06

3

(

m, 2H), 7.84ꢀ7.81 (m, 2H), 7.76ꢀ7.73 (m, 4H), 7.32ꢀ7.30 (d,

d= 8.58 Hz, 2H), 5.68 (s, 2H, NH), 3.07ꢀ3.05 (t, d= 5.63, 4H),

.86ꢀ2.82 (t, J= 6.88, 2H), 2.74ꢀ2.72 (m, 2H), 2.70ꢀ2.67 (t, J=

2

1

ACS Paragon Plus Environment

Bioconjugate Chemistry

Page 12 of 17

1

product 5 as an orange oil (72 %, 1.48 g). H NMR (400 MHz,

8.05ꢀ8.04 (d, J = 6.6 Hz, 2H), 7.96ꢀ7.94 (d, J = 5.8 Hz, 2H),

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

CDCl , 25°C): 8.11 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 7.7 Hz,

7.63ꢀ7.61 (d, J = 6.6 Hz, 2H), 7.42ꢀ7ꢀ40 (d, J = 5.8 Hz, 2H),

7.32 (s, 2H), 4.08 (s, 4H), 3.71 (s, 4H), 3.59 (s, 2H), 3.53 (s,

3

2H), 7.89 (t, J = 7.7 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H), 7.43 (d,

J = 8.5 Hz, 2H), 4.18 (s, 4H), 3.96 (s, 6H), 3.90 (s, 4H), 3.73

(m, 2H), 3.54 (s, 4H), 3.45 (br s, 4H), 3.24 (m, 2H), 1.38 (s,

13

4H), 3.35 (s, 4H), 3.14 (m, 2H). C NMR (400 MHz, MeOD):

173.5, 167.4, 159.0, 148.7, 140.3, 139.1, 131.8, 128.3, 125.6,

1

3

13

1

8

8H). C NMR (101 MHz, CDCl , 25°C): 168.7, 165.1,

124.4, 116.7, 58.7, 56.5, 55.8, 53.1, 50.1, 30.4; CꢀDEPT

3

56.7, 147.3, 147.3, 143.8, 139.1, 130.1, 127.5, 125.0, 124.0,

3.2, 57.5, 56.0, 54.5, 53.3, 50.4, 48.8, 29.8, 28.0. HRꢀESIꢀ

NMR (400 MHz, MeOD): 140.3↑, 131.5↑, 128.1↑, 125.6↑,

124.2↑, 58.4↓, 56.4↓, 55.5↓, 51.5↓, 49.9↓, 30.2↓. HRꢀESIꢀMS

+

MS calcd. for [C H N O H] :779.3980; found 779.3973

calcd. for [C H N O +H] : 609.2673; found 609.2671

[M+H] .

4

0

54

6

10

30 37

6

8

+

[

M+H] .

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

pꢀNO ꢀBnꢀH neunpa · 2.2 HCl · 3.1 H O, 6

pꢀSCNꢀBnꢀH neunpa, 9

2

4

2

4

To compound 5 (0.23 g, 0.3 mmol) in THF/H O (3 mL, 3:1)

Compound 8 (0.04 g, 0.1 mmol) was dissolved in 0.1 M HCl

(1 mL) and dichloromethane (1 mL). Thiophosgene (0.05 mL,

0.6 mmol, 10 eq) was added and the solution was stirred vigꢀ

orously at room temperature overnight in the dark. The solꢀ

vents were concentrated in vacuo and the product purified by

semiꢀpreparative RPꢀHPLC (10mL/ min, gradient A: 0.1%

TFA in deionized water, B: acetonitrile, A: 95% to B: 100%

2

was added lithium hydroxide (0.07 g, 3.0 mmol, 10 eq.) and

the mixture was stirred for 16 h at room temperature. Solvents

were evaporated and the crude product was purified by semiꢀ

preparative reverseꢀphase (RP) HPLC (10mL/ min, gradient

A: 0.1% TFA in deionized water, B: acetonitrile, A: 95% to B:

1

00% for 25 min., t = 14.00 min) and the product 6 was obꢀ

R

1

tained as a yellow oil (61 %, 0.12 g). H NMR (400 MHz,

for 25 min., t = 17.00 min) to yield product 9 as an orange oil

R

1

CDCl , 25°C): 8.11 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 7.7 Hz,

(59 %, 0.02 g). H NMR (400 MHz, MeOD, 25°C): 8.04ꢀ8.02

3

2

H), 7.89 (t, J = 7.7 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H), 7.43 (d,

(d, J = 6.7 Hz, 2H), 7.95ꢀ7.92 (t, J = 8.2 Hz, 2H), 7.61ꢀ7.57 (t,

J = 7.5 Hz, 2H), 7.27ꢀ7ꢀ25 (d, J = 7.5 Hz, 2H), 7.18ꢀ7.16 (d, J

= 7.5 Hz, 2H), 4.05 (s, 4H), 3.66 (s, 4H), 3.55 (s, 2H), 3.50 (s,

J = 8.5 Hz, 2H), 4.18 (s, 4H), 3.90 (s, 4H), 3.73 (m, 2H), 3.54

13

(

s, 4H), 3.45 (br s, 4H), 3.24 (m, 2H). C NMR (101 MHz,

13

CDCl , 25°C): 168.7, 165.1, 156.7, 147.3, 147.3, 143.8, 139.1,

4H), 3.26 (br s, 4H), 3.07 (m, 2H). C NMR (400 MHz,

3

1

30.1, 127.5, 125.0,124.0, 57.5, 56.0, 54.5, 48.8. HRꢀESIꢀMS

MeOD): 173.5, 167.4, 159.0, 148.7, 140.3, 137.5, 131.5,

128.3, 126.9, 125.6, 115.9, 58.7, 56.5, 55.8, 51.9, 50.1, 30.5;

+

calcd. for [C H N O +H] : 639.2415; found 639.2415

30

34

6

10

+

13

[

·

M+H] . Elemental analysis: calcd % for pꢀNO ꢀBnꢀH neunpa

CꢀDEPT NMR (400 MHz, MeOD): 140.3↑, 131.4↑, 128.3↑,

2

4

2.2 HCl ·3.1 H O: C 46.55 N 10.86 H 5.2; found: C 46.72, N

126.9↑, 125.6↑, 58.7↓, 56.35↓, 56.5↓, 51.9↓, 50.1↓, 30.5↓. HRꢀ

2

+

10.64, H 5.37.

ESIꢀMS calcd. for [C H N O +H] : 651.2237; found

651.2239 [M+H] .

31

35

6

8

+

N,Nꢀ[(tertꢀButoxycarbonyl)methylꢀN,Nꢀ

[

6(methoxycarbonyl)pyridineꢀ2ꢀyl]methyl]ꢀNꢀ(4ꢀ

Na[La(pꢀNO ꢀBnꢀneunpa)]

2

aminophenethyl)ꢀ1,2ꢀtriaminodiethane, 7

Compound 6 (10.2 mg, 16.0 mmol) was dissolved in water

and lanthanum perchlorate (7.7 mg, 17.6 mmol, 1.1 eq.) was

added. The pH was adjusted to 4 using 0.1 M NaOH. The

successful Laꢀcomplexation as a white precipitate was conꢀ

firmed by HRꢀESIꢀMS immediately after adding La(ClO)₄.

Compound 5 (0.11 g, 0.1 mmol) was dissolved in glacial aceꢀ

tic acid (3 mL) and Pd/C 10% was added, the vessel sealed

and purged with H gas, charged with a H balloon and left to

2

stir for 2 h at room temperature. The reaction mixture was then

filtered through Celite and concentrated under reduced presꢀ

sure to yield compound 7. The aromatic amine was confirmed

by a purple ninhydrin staining. The solution was filtered and

After centrifugation, the precipitate was washed with water.

1

H NMR (400 MHz, DMSOꢀd , 25°C): 8.15 (d, 2H), 8.01 (m,

6

2H), 7.92 (m, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 8.5

Hz, 2H), 4.01 (s, 4H), 3.69 (m, 5H), 3.51 (d, 4H), 3.25 (s, 7H).

1

the filtrate was concentrated in vacuo. H NMR (400 MHz,

MeOD, 25°C): 7.99 (m, 2H), 7.92 (t, J = 7.9 Hz, 2H), 7.62 (d,

J = 7.7 Hz, 2H), 6.87 (d, J = 7.9 Hz, 2H), 4.01 (s, 2H), 3.46

HSQC (400 MHz, DMSOꢀd , 25°C) in Supporting Inforꢀ

6

+

mation. HRꢀESIꢀMS calcd. for [C H N O La] : 775.1243;

30

32

6

10

13

+

(br.4, 2H), 3.13 (m, 4H), 2.79 (m, 4H), 1.41 (s, 18H).

C

found 775.1236 [M+2H] .

NMR (400 MHz, MeOD): 172.3, 166.7, 160.8, 148.3, 139.7,

1

5

39.5, 130.4, 128.3, 125.4, 125.2, 116.8, 82.7, 59.4, 56.9,

3.4, 52.3, 50.6, 29.9, 28.4. HRꢀESIꢀMS calcd. for

Na[Bi(pꢀNO ꢀBnꢀneunpa)]

2

+

Compound 6 (20.3 mg, 31.8 mmol) was dissolved in water

and bismuth trichloride (11.0 mg, 35.0 mmol, 1.1 eq.) was

added. The pH was adjusted to 4 using 0.1 M NaOH. The

successful Biꢀcomplexation as a white precipitate was conꢀ

[

C H56N O +H] : 749.4238; found 749.4236 [M+H] .

40 6 8

pꢀNH ꢀBnꢀH neunpa, 8

2

4

Compound 6 (0.09 g, 0.13 mmol) was dissolved in THF/H O

firmed by HRꢀESIꢀMS immediately after adding BiCl . After

2

3

(3 mL, 3:1) and lithium hydroxide (0.03 g, 1.26 mmol, 10 eq.)

centrifugation, the precipitate was washed with water. The Biꢀ

was added. The reaction mixture was left at room temperature

for 24 hours. After product formation was confirmed by ESIꢀ

MS analysis, the solution was neutralized with 1 M HCl and

solvents were concentrated in vacuo. For purification, semiꢀ

preperative RPꢀHPLC (10mL/ min, gradient A: 0.1% TFA in

deionized water, B: acetonitrile, A: 95% to B: 100% for 25

complex is not soluble in any solvent; DMSOꢀd was chosen

6

1

13

for NMR analysis. H NMR and C NMR not measurable due

to solubility problems. HRꢀESIꢀMS calcd. for

[C H N O Bi] : 843.1827; found 843.1835 [M+2H] .

+

30

6

10

Na[In(pꢀNO ꢀBnꢀneunpa)]

2

min., t = 11.50 min) was used and product 8 was obtained as a

R

1

yellow oil (50 %, 0.04 g). H NMR (400 MHz, MeOD, 25°C):

1

2

ACS Paragon Plus Environment

Page 13 of 17

Bioconjugate Chemistry

and incubated for 15 min. Each reaction mixture was spotted

In a 20 mL screw cap vial, compound 6 (12 mg, 0.019 mmol)

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

was dissolved in H O:MeOH (2:1, 1.5 mL). In a separate

onto iTLCꢀSG plates, and developed using EDTA (50 mM,

pH 5) as mobile phase. Radioactivity on the plate was measꢀ

ured using a radioꢀTLC plate reader, and number of chelates

attached per antibody was calculated using equation (1).

2

screw cap vial, [In(ClO ) ]·8H O (32 mg) was dissolved in

4

3

2

dist. water (0.5 mL) to make a stock solution (64 mg/mL). An

aliquot (217 ꢁL, 13.8 mg, 0.0249 mmol) of this In(III) stock

solution was added to the chelate solution. The pH of the

#ꢄꢉꢊꢋꢌꢍꢎꢄꢏꢍꢄꢐꢏꢎꢑꢒꢓꢌꢑꢄ(ꢔ ꢖꢗ.ꢘ)

ꢕ

_×_ꢀ_ꢁ_ꢂ_ꢃ_ꢄ_ꢚ_ꢀꢛꢜ

ꢀꢁꢂꢃꢄꢅℎꢆꢃꢇꢈꢆ =ꢄ

solution was adjusted from pH 1 to pH 5 using 1 N NaOH and

ꢍꢊꢍꢏꢒꢄ#ꢄꢙꢊꢋꢌꢍꢎ

o

(1)

0

.1 M HCl. A stir bar was added, the reaction heated to 60 C

in a sand bath and stirred for 3 hours with the lid loosely on.

The mixture was removed from the heat and allowed to cool to

room temperature. A white precipitate had formed, and the

solution was then centrifuged and washed with dist. water (5 x

111

InꢀChelate Radiolabeling Studies.

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

The ligand pꢀNO ꢀBnꢀH neunpa, or gold standard pꢀNH ꢀBnꢀ

2

4

2

CHXꢀA”ꢀDTPA, was made up as a stock solution (1 mg/mL,

1

mL). After drying under high vacuum, the product as a white

ꢀ

3

~10 M) in deionized water. From this stock solution, serial

solid was collected (4 mg, 0.0053 mmol) with an overall yield

ꢀ

4

1

dilutions were prepared to final ligand concentrations of 10

of 28%. H, and COSY NMR (400 MHz, DMSOꢀd ) potential

6

ꢀ

9

ꢀ

3

ꢀ

M – 10 M. A 100 µL aliquot of each ligand stock (10 to 10

M) or water (blank control) was added to screwꢀcap mass

multiple isomers in solutions, see Figure S12 Supporting Inꢀ

9

formation.

HRꢀESIꢀMS

calculated

for

115

+

spectrometry vials and diluted with sodium acetate buffer (pH

[

InC H N O +H+Na] : 773.1038; found: 773.1039

30

6

10

111

+

4, 10 mM, 880 µL). An aliquot of diluted In stock (20 µL,

(M+H+Na) .

~

200 µCi) was added to each vial and allowed to radiolabeled

at ambient temperature for 10 min, then it was analyzed by

RPꢀHPLC to confirm radiolabeling and calculate yields. For

111

human serum stability studies, undiluted InCl stock (~20

3

ꢀ4

µL, 5 mCi) was added to the reaction vial containing 10

M

Bioconjugation of pꢀSCNꢀBnꢀH neunpa and pꢀSCNꢀBnꢀCHXꢀ

4

ligand in sodium acetate buffer. Areas under the peaks obꢀ

served in the HPLC radioꢀtrace were integrated to determine

radiolabeling yields. Elution conditions used for RPꢀHPLC

analysis were gradient: A: 0.1% trifluoroacetic acid (TFA) in

water, B: acetonitrile; 0 to 100% B linear gradient 20 min, 1

A”ꢀDTPA to Trastuzumab.

Trastuzumab (Herceptin, Genentech, San Francisco, CA,

USA) was purified using size exclusion columns (PDꢀ10 deꢀ

salting columns) and centrifugal filter units with a 50 kDa

molecular weight cutoff and phosphate buffered saline (PBS,

pH 7.4) to remove αꢀαꢀtrehalose dehydrate, Lꢀhistidine, and

polysorbate 20 additives. The purified antibody was brought

up in PBS at pH 7.4. For each chelateꢀantibody conjugation,

PBS (905 µL, pH adjusted to 9.0 using 0.1 M Na CO ) and

111

ꢀ

111

mL/min. [ In(pꢀNO ꢀBnꢀneunpa)] (t = 12.9 min), [ In(pꢀ

2

R

2ꢀ

NH ꢀBnꢀCHXꢀA”ꢀDTPA)] (t = 8.0 min (minor product); 8.6

min (major product)) “ In ” (t = 5.3 min).

2

R

111 3+

R

2

3

Partition Coefficients.

Trastuzumab (Genentech, San Francisco, CA, USA) (4 mg, 75

µL in PBS pH 7.4) was added to a low protein binding Eppenꢀ

dorf tube. To the antibody mixture, 5 equivalents of pꢀSCNꢀ

111

Inꢀlabeled complex (30 µL, 20 µCi) was diluted with phosꢀ

phate buffered saline (pH 7.4, 470 µL), and added to 1ꢀoctanol

(500 µL) in a 1.5 mL Eppendorf tube. Samples were vortexed

for 60 seconds and subsequently centrifuged to separate phasꢀ

es (3000 rpm, 5 min). Aliquots (490 µL) of the aqueous and

organic phases were diluted in a standard volume (20 mL) of

water or acetonitrile, respectively, for measurement in an Nꢀ

type Coꢀaxial HPGe gamma spectrometer from Canberra fitted

with a 0.5 mm beryllium window and calibrated (energy and

BnꢀH neunpa or pꢀSCNꢀBnꢀCHXꢀA”ꢀDTPA was added, reꢀ

4

spectively, in small portions (5 x 5 µL in DMSO). The reacꢀ

tion mixture was stirred at ambient temperature overnight, and

subsequently purified by centrifugal filtration. The final bioꢀ

conjugates were stored in 0.25 M sodium acetate at ꢀ20°C.

Final protein concentration was determined by the Bradford

assay.

152

133

efficiency) with a 20 mL Eu and Ba source. The samples

were counted for a minimum of 5 minutes, with a dead time

Chelate number – radiometric isotopic dilution assay.

111

less than 5 %. The amount of Inꢀcomplex (Bq) in each

fraction was quantified using the 171 and 245 keV gamma

The number of accessible chelating ligands conjugated per

antibody was determined using previously described methꢀ

111

lines of In.

36,37

111

ods.

(

Briefly, a 1 µCi/uL [ In]InCl working solution

3

+

111

nonꢀradioactive In spiked with In) was prepared with a

1

11

3+

Inꢀneunpa/CHXꢀA”ꢀDTPAꢀTrastuzumab Radiolabeling for

final In concentration of 500 µM in ammonium acetate buffꢀ

er (0.15 M, pH 6). In duplicate, for each chelateꢀantibody

conjugate, 50 µg of bioconjugate (30 µL) was prepared into

separate 1.5 mL Eppendorf tubes. Aliquots of 20, 25, and 30

In Vivo Studies.

Aliquots of H neunpa/CHXꢀA”ꢀDTPAꢀTrastuzumab (650 µg)

4

were diluted with ammonium acetate buffer (0.15 M, pH 6)

such that the final volume of the reaction was 1 mL, and then

111

µL of the [ In]InCl working solution were added to the two

3

1

11

chelateꢀantibody samples. Positive controls containing 50 µg

InCl (~20 mCi) was added. The mixtures were allowed to

3

1

11

of bioconjugate and 25 µL of buffered InCl only (no nonꢀ

react at ambient temperature for 40 min, and then analysed via

3

3+

111

radioactive In added) were prepared in duplicate. Negative

iTLCꢀSG using 50 mM EDTA (pH 5) as eluent; Inꢀlabelled

1

11

111 3+

controls containing 30 µL of PBS, and 25 µL of [ In]InCl₃

working solution were prepared in duplicate. Samples were

allowed to incubate at room temperature overnight, after

which time EDTA (50 mM, pH 5) was added at 1/9 of the

antibody remained at the baseline, while In ions comꢀ

111

plexed as InꢀEDTA and eluted with the solvent front. Radioꢀ

labeled immunoconjugates were then purified by PDꢀ10 SEC

columns and centrifugal filtration (50k cutꢀoff). The radioꢀ

chemical purity of the final radiolabeled bioconjugates was

3+

reaction volume to scavenge any unspecifically bound In ,

1

3

ACS Paragon Plus Environment

Bioconjugate Chemistry

Page 14 of 17

6

determined using SECꢀHPLC (using an isocratic gradient of

0.1 M sodium phosphate monobasic dehydrate, 0.1 M sodium

phosphate dibasic dodecahydrate, 0.1 M sodium azide and

0.15 M sodium chloride (pH 6.2ꢀ7.0)); the specific activity

was calculated by injecting a known activity, and integrating

areas under the peaks of the UVꢀchromatogram measured

against a standard curve.

pended at 0.23 to 2.3 x 10 cells/mL in PBS (pH 7.4). For each

tested antibody, 50 ꢁL (from a solution of 0.45 µg of each

radioimmunoconjugate diluted in 5 mL of 1% PBSꢀBSA) was

added to each cell concentration tube in duplicate. The radioꢀ

labeled immunoconjugates were incubated for 1 h at 37 °C and

under gentle agitation. Cells were then pelleted and washed

twice with PBS. Each cellꢀbound activity for the different cell

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

111

conditions was determined by measuring the In amount of

activity within the cell pellets using the Wallac WIZARD2

gamma counter with background and decay correction. The

bound fraction was determined as a percentage of total added

activity according to control samples. Immunoreactive fracꢀ

tions were estimated for conditions representing infinite antiꢀ

gen excess by linear regression analysis of a plot of toꢀ

tal/bound activity against 1/[cell concentration]. Results >80%

were considered suitable for in vivo imaging.

In Vitro Human Serum Stability Data.

The procedures of the serum competition studies followed

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

8,10

111

closely those previously published. The compound [ In(pꢀ

ꢀ

111

2-

NO ꢀBnꢀneunpa)] ,

[ In(pꢀNH ꢀBnꢀCHXꢀA”ꢀDTPA)] , or

2

1

11 3+

blank control “ In ” was prepared using the radiolabeling

1

11

protocol as described above. In triplicate for the Inꢀcomplex,

1

11

solutions were prepared in vials containing 330 µL of Inꢀ

complex (~1.6 mCi), 1000 µL of room temperature human

serum, and 670 µL of phosphate buffered saline (PBS, pH 7.4)

and incubated at 37°C. At time points of 1 h, 1 and 5 days,

SKOVꢀ3 Xenograft Mouse Models.

4

00, 400, and 800 µL aliquots of the human serum competiꢀ

All experiments were conducted in accordance with the guideꢀ

lines established by the Canadian Council on Animal Care and

approved by the Animal Ethics Committee of the University

of British Columbia (protocol # A16ꢀ0104). Female NOD.Cgꢀ

tion mixture were removed from each vial, respectively, dilutꢀ

ed to a total volume of 2.5 mL with PBS, and counted in a

Capintec CRCꢀ55tR dose calibrator; this value is recorded as

scid

tm1Wjl

“

full activity” to be loaded onto the PDꢀ10 column. The 2.5

mL of reaction mixture was loaded onto a preꢀconditioned PDꢀ

0 column, and the empty vial was counted again in the dose

Prkdc Il2rg

/SzJ mice (4 months old) obtained from an

inꢀhouse breeding colony were subcutaneously injected with 8

6

1

x 10 SKOVꢀ3 cells in matrigel (BD Bioscience) on the left

calibrator – this value was recorded as “residual activity” left

in the vial. The loaded effluent was collected in a waste conꢀ

tainer, and then the PDꢀ10 column was eluted with 3.5 mL of

PBS, and collected into a separate vial. The eluent that conꢀ

flank.

111

ꢀ

111

[

In(pꢀNO ꢀBnꢀneunpa)] and

[

In(pꢀNH ꢀBnꢀCHXꢀA”ꢀ

2

2ꢀ

111

DTPA)] in vivo Biodistribution.

tained In bound/associated with serum proteins (size excluꢀ

sion for MW < 5000 Da) was counted in the dose calibrator

and then compared to the total activity that was loaded onto

111

ꢀ

[

In(pꢀNO ꢀBnꢀneunpa)] and

In(pꢀNH ꢀBnꢀCHXꢀA”ꢀ

2

2ꢀ

DTPA)] were prepared according to the radiolabeling protoꢀ

ꢀ

4

1

11

col above using 10 M ligand and ~ 148 MBq (~4 mCi) of

the PDꢀ10 column to obtain the percentage of In that was

bound to serum proteins and therefore no longer chelateꢀbound

by the relationship: 1 – (eluted activity/(full activity – residual

activity) x 100. For serum stability of the radioimmunoconjuꢀ

1

11

InCl in sodium acetate buffer (10 mM, pH 4). Radiolabelꢀ

3

ing yields >99% were confirmed by RPꢀHPLC. Each radioꢀ

labeled tracer was diluted with PBS (pH 7.4) to a concentraꢀ

tion of 10 MBq/mL (370 µCi/mL). Each mouse was intraveꢀ

nously injected through the tail vein with ~ 1 MBq (100 µL) of

111

gates,

InꢀneunpaꢀTrastuzumab was first prepared as deꢀ

scribed above by incubating H neunpaꢀTrastuzumab (200 µg)

4

111

the In complex and then sacrificed by inhalation of isofluꢀ

and InCl (~1 mCi) in NH OAc (0.15 M, pH 6) for 20

3

4

rane followed by CO at 15 min, 1 h, 4 h, or 24 h after injecꢀ

minutes at ambient temperature. After confirming a radiolabelꢀ

ing yield >95% via iTLCꢀSG in 50 mM EDTA (pH 5), in

2

tion (n = 4 at each time point). Blood was withdrawn by cardiꢀ

ac puncture and tissues of interest including fat, uterus, ovaꢀ

ries, intestine, spleen, liver, pancreas, stomach, adrenal glands,

kidney, lungs, heart, muscle, bone (tibia), brain, and tail were

harvested, washed in PBS, dried and weighed. Activity of each

sample was measured by a calibrated gamma counter (Perkin

Elmer, Wizard 2 2480) with decay correction. The activity

uptake was expressed as a percentage of the injected dose per

gram of tissue (%ID/g).

111

triplicate, InꢀneunpaꢀTrastuzumab (330 µL, ~320 µCi) was

incubated with human serum (330 µL) and the mixture was

left at 37°C for 168 h (7 d). At time points of 0, 1, 24, 48, 120,

and 168 h, aliquots (3 – 5 µL) of the competition mixture was

spotted on iTLCꢀSG plates and developed in 50 mM EDTA

(pH 5) as described above.

Cell Culture.

The human ovarian adenocarcinoma HER2ꢀpositive SKOVꢀ3

cells were cultured at 37°C with 5% CO in Dulbecco's modiꢀ

fied Eagle's medium (DMEM, LifeTechnologies, Rockford,

IL, USA) containing 2 mM glutamine and supplemented with

111

Inꢀneunpa/CHXꢀA”ꢀDTPAꢀTrastuzumab SPECT/CT Imagꢀ

2

ing and Biodistribution Studies.

Mice with SKOVꢀ3 ovarian cancer xenografts were adminisꢀ

1

11

tered with ~37 MBq (~1 mCi) of

(1.03 MBq/µg [28.0 µCi/µg]) or

Inꢀneunpaꢀtrastuzmab

InꢀCHXꢀA”ꢀDTPAꢀ

1

0% fetal bovine serum (SigmaꢀAldrich, Oakville, ON, USA)

1

11

and 100 U/mL of penicillinꢀstreptomycin (LifeTechnologies).

Trastuzumab (0.77 MBq/µg [20.8 µCi/µg]) in ~30 µL of PBS

(pH 7.4) via tail vein injection. For each radioimmunoconjuꢀ

gate, mice were imaged (n=2) at 1, 3, or 5 day after injection.

Image acquisition and reconstruction was performed using the

UꢀSPECTꢀII/CT (MILabs, Utrecht, The Netherlands). Approxꢀ

imately 5 min prior to SPECT/CT image acquisition, mice

were anesthetized via inhalation of 2% isoflurane/oxygen gas

In Vitro Immunoreactivity Assay.

1

11

The immunoreactive fractions of

InꢀneunpaꢀTrastuzumab

1

11

and

InꢀCHXꢀA”ꢀDTPAꢀTrastuzumab were determined

3

8

according to the Lindmo cellꢀbinding method using SKOVꢀ3

cells(37, 39) as previously described. Briefly, cells were susꢀ

1

4

ACS Paragon Plus Environment

Page 15 of 17

Bioconjugate Chemistry

and the indiumꢀchloride stability constants included in the

mixture and placed on the scanner bed. Anesthesia was mainꢀ

42

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

tained during imaging as well as body temperature via a heatꢀ

ing pad. A 5 min baseline CT scan was obtained for localizaꢀ

tion with voltage setting at 60 kV and current at 615 µA folꢀ

lowed by a static emission scan using an ultraꢀhighꢀresolution

multiꢀpinhole ratꢀmouse (1 mm pinhole size) collimator. Data

were acquired in list mode, reconstructed using the UꢀSPECT

II software and coꢀregistered for alignment. SPECT images

were reconstructed using maximumꢀlikelihood expectation

maximization (3 iterations), pixelꢀbased ordered subset expecꢀ

tation maximization (16 subsets) and a postꢀprocessing filter

calculations were taken from Baes and Mesmer. The overall

equilibrium (formation) constants log β are referred to the

overall equilibria: pM + qH + rL = MpHpLr (the charges are

omitted), where p might also be 0 in the case of protonation

equilibria and q may be negative. Stepwise equilibrium conꢀ

stants log K correspond to the difference in log units between

the overall constants of sequentially protonated (or hydroxide)

species. The pM values for metal complexes were calculated

43

by using the Hyss software , from the set of stability conꢀ

ꢀ

5

stants for each system at pH 7.4 with [L] = 1.0x10 M and

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

ꢀ6

(Gaussian blurring) of 0.5 mm centered at photopeaks 171

[M] = 1.0x10 M.

keV and 245 keV with a 20% window width. Imaging data

sets were decayed corrected to injection time and converted to

DICOM data for visualization in the Inveon Research Workꢀ

place (Siemens Medical Solutions USA, Inc.). As no calibraꢀ

tion factor was used or attenuation and scatter correction were

performed on the images; they were used only for qualitative

comparison between the two tracers and are presented using a

minꢀmax minꢀmax scale bar of counts corrected for decay. For

biodistribution studies, mice were sacrificed by inhalation of

Corresponding Author

*Email: orvig@chem.ubc.ca

ORCID Chris Orvig 0000-0002-2830-5493

Notes

isoflurane followed by CO (n = 4 at each time point), blood

2

The authors declare no competing financial interest.

was withdrawn by cardiac puncture and tissues were collected

and processed as described above. Tissues collected include

all those listed above in addition to tumor tissue.

Acknowledgments

Funding for this work was provided by the Canadian Institutes

for Health Research (CIHR) and the Natural Sciences and

Engineering Research Council (NSERC) of Canada as a Colꢀ

laborative Health Research Project (CHRP). We acknowledge

Solution Thermodynamics.

Protonation constants and metal stability constants were calcuꢀ

lated from potentiometric titrations using a Metrohm Titrando

809 equipped with a Ross combined electrode and a Metrohm

Dosino 800. The titration apparatus consisted of a 20 mL and

25 ºC thermostated glass cell and an inletꢀoutlet tube for nitroꢀ

gen gas (purified through a 10% NaOH solution) to exclude

111

Nordion (Canada) for their inꢀkind contribution of InCl₃.

TRIUMF receives funding via a contribution agreement with

the National Research Council of Canada. We would also like

to thank Victoria Braun for helping conducting logP measꢀ

urements.

any CO prior and during the course of the titration. The elecꢀ

2

trode was daily calibrated in hydrogen ion concentrations

1

3

using a standard HCl as described before in order to obtain

Supporting Information

the calibration parameters E and pKw. Solutions were titrated

0

The Supporting Information is available free of charge on the

ACS Publications website.

with carbonateꢀfree NaOH (0.157 M) that was standardized

against freshly recrystallized potassium hydrogen phthalate.

111

3D videos of

InꢀCHXꢀA’’ꢀDTPAꢀTrastuzumab and

Inꢀ

Protonation equilibria of the ligand were studied by titrations

neunpaꢀTrastuzumab can be found online under the following

link:

https://www.youtube.com/playlist?list=PLs31b0Z6H1z2CqUpljQ

rQh6vqtNAWBMYb

ꢀ4

of a solution containing H neunpa 7.18 x 10 M at 25 ºC and

4

0

.16 M NaCl ionic strength using a combined potentiometricꢀ

16,17

spectrophotometric procedure.

Spectra were recorded in the

2

00–450 nm spectral range with a 0.2 cm path length fiber

optic on a Varian Cary 60 UV/Vis Spectrophotometer. In the

study of complex formation equilibria, the ligandꢀmetal soluꢀ

tions were prepared by adding the atomic absorption (AA)

Abbreviations

SPECT, single photon emission computed tomography; BFC,

bifunctional chelator; DOTA, 1,4,7,10ꢀtetraazacyclododecaneꢀ

standard metal ion solutions to a H neunpa solution of known

4

1

,4,7,10ꢀtetraacetic

acid;

DTPA,

2ꢀ[Bis[2ꢀ

concentration in the 1:1 metal to ligand molar ratio. The exact

amount of acid present in the lanthanum, bismuth and indium

[

bis(carboxymethyl)amino]ethyl]amino]acetic acid; CHX,

cyclohexyl; CN, coordination number, HER2/neu, human

epidermal growth factor receptor 2; mAb, monoclonal antiꢀ

body; CT, computed tomography.

4

0

standards was determined by Gran’s method titrating

equimolar solutions of either La(III), Bi(III) or In(III) and

Na H ꢀEDTA. Ligand and metal concentrations were in the

2

range of 0.7ꢀ1.0 mM. Each titration consisted of 100ꢀ150

equilibrium points in the pH range 1.8ꢀ11.5, equilibration

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Article Doi

P-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and 111In Immuno-Single Photon Emission Computed Tomography Imaging

DOI: 10.1021/acs.bioconjchem.7b00311

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Article abstract of DOI:10.1021/acs.bioconjchem.7b00311

Full text of DOI:10.1021/acs.bioconjchem.7b00311

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