Targeting the tumor-associated folate receptor with an 111In- DTPA conjugate of pteroic acid

Article abstract of DOI:10.1021/ja043006n

The cell membrane folate receptor is a potential molecular target for tumor-selective drug delivery. To probe structural requirements for folate receptor targeting with low molecular weight radiometal chelates, specifically the role of the amino acid frag

Full text of DOI:10.1021/ja043006n

Published on Web 04/27/2005
Targeting the Tumor-Associated Folate Receptor with an
111In-DTPA Conjugate of Pteroic Acid
Chun-Yen Ke, Carla J. Mathias, and Mark A. Green*
Contribution from the Department of Industrial and Physical Pharmacy, Purdue UniVersity,
West Lafayette, Indiana 47907
Received November 19, 2004; E-mail: magreen@purdue.edu
Abstract: The cell membrane folate receptor is a potential molecular target for tumor-selective drug delivery.
To probe structural requirements for folate receptor targeting with low molecular weight radiometal chelates,
specifically the role of the amino acid fragment of folic acid (pteroylglutamic acid) in mediating targeting
selectivity, the amide-linked conjugate pteroyl-NHCH2CH2OCH2CH2OCH2CH2NH-DTPA was prepared by
a three-step procedure from pteroic acid, 2,2′-(ethylenedioxy)-bis(ethylamine), and t-Bu-protected DTPA.
This conjugate, 1-{2-[2-[(2-(biscarboxymethyl-amino)ethyl)-carboxymethyl-amino]ethyl]-carboxymethyl-
amino}-acetylamino-3,6-dioxa-8-pteroylamino-octane (1), was employed for synthesis of the corresponding
111In(III) radiopharmaceutical. Following intravenous administration to athymic mice, the 111In complex of 1
was found to selectively localize in folate receptor-positive human KB tumor xenografts and to afford
prolonged tumor retention of the 111In radiolabel (5.4 ( 0.8, 5.6 ( 1.1, and 3.6 ( 0.6% of the injected dose
per gram of tumor at 1, 4, and 24 h, respectively). The observed tumor localization was effectively blocked
by co-administration of folic acid with the 111In-1 complex, consistent with a folate receptor-mediated
targeting process. In control studies, tumor targeting with this pteroic acid conjugate appears as effective
as that seen using 111In-DTPA-folate, a radiopharmaceutical that has progressed to clinical trials for
detection of folate receptor-expressing gynecological tumors.
Introduction
endocytosis of, a range of folate conjugates that are not
substrates for the ubiquitous reduced folate carrier.35-39 Thus,
The cell membrane folate receptor (or folate-binding protein,
FBP)1,2 is a potential molecular target for tumor-selective drug
delivery,3-7 since the folate receptor is found in only a limited
range of normal tissues but commonly overexpressed by
malignant cells.8-34 The folate receptor binds, and allows
(18) Weitman, S. D.; Weinberg, A. G.; Coney, L. R.; Zurawski, V. R.; Jennings,
D. S.; Kamen, B. A. Cancer Res. 1992, 52, 6708-6711.
(19) Weitman, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.; Frasca, V.;
Zurawsky, V. R.; Kamen, B. A. Cancer Res. 1992, 52, 3396-3401.
(20) Holm, J.; Hansen, S. I.; Hoier-Madsen, M.; Birn, H.; Helkjaer, P.-E. Arch.
Biochem. Biophys. 1999, 366, 183-191.
(21) Ross, J. F.; Chaudhuri, P. K.; Ratnam, M. Cancer 1994, 73, 2432-2443.
(22) Wu, M.; Gunning, W.; Ratnam, M. Cancer Epidemiol. Biomarkers PreV.
1999, 8, 775-782.
(23) Mantovani, L. T.; Miotti, S.; Menard, S.; Canevari, S.; Raspagliesi, F.;
Bottini, C.; Bottero, F.; Colnaghi, M. I. Eur. J. Cancer 1994, 30A, 363-
369.
(1) Elnakat, H.; Ratnam, M. AdV. Drug DeliVery ReV. 2004, 56, 1067-1084.
(2) Sabharanjak, S.; Mayor, S. AdV. Drug DeliVery ReV. 2004, 56, 1099-
1109.
(3) Wang, S.; Lee, R. J.; Mathias, C. J.; Green, M. A.; Low, P. S. Bioconjugate
Chem. 1996, 7, 56-62.
(4) Mathias, C. J.; Lee, R. J.; Wang, S.; Waters, D. J.; Low, P. S.; Green, M.
A. J. Nucl. Med. 1996, 37, 1003-1008.
(24) Reddy, J. A.; Haneline, L. S.; Srour, E. F.; Antony, A. C.; Clapp, D. W.;
Low, P. S. Blood 1999, 93, 3940-3948.
(5) Mathias, C. J.; Wang, S.; Low, P. S.; Waters, D. J.; Green, M. A. Nucl.
Med. Biol. 1999, 26, 23-25.
(25) Nakashima-Matsushita, N.; Homma, T.; Yu, S.; Matsuda, T.; Sunahara,
N.; Nakamura, T.; Tsukano, M.; Ratnam, M.; Matsuyama, T. Arthritis
Rheum. 1999, 42, 1609-1616.
(6) Wang, S.; Luo, J.; Lantrip, D. A.; Waters, D. J.; Mathias, C. J.; Green, M.
A.; Fuchs, P. L.; Low, P. S. Bioconjugate Chem. 1997, 8, 673-679.
(7) Mathias, C. J.; Wang, S.; Waters, D. J.; Turek, J. J.; Low, P. S.; Green, M.
A. J. Nucl. Med. 1998, 39, 1579-1585.
(26) Spinella, M. J.; Brigle, K. E.; Sierra, E. E.; Goldman, I. D. J. Biol. Chem.
1995, 270, 7842-7849.
(27) Holm, J.; Hansen, S. I.; Hoier-Madsen, M.; Helkjaer, P.-E.; Bzorek, M.
Acta Pathol. Microbiol Immunol. Scand. 1995, 103, 862-868.
(28) Baggnoli, M.; Canevari, S.; Figini, M.; Mezzanzanica, D.; Raspagliesi, F.;
Tomassetti, A.; Miotti, S. Gynecol. Oncol. 2003, 88, S140-S144.
(29) Maziarz, K. M.; Monaco, H. L.; Shen, F.; Ratnam, M. J. Biol. Chem. 1999,
274, 11086-11091.
(8) Garin-Chesa, P.; Campbell, I.; Saigo, P. E., Jr.; Lewis, J. L.; Old, L. J.;
Rettig, W. J. Am. J. Pathol. 1993, 142, 557-567.
(9) Stein, R.; Goldenberg, D. M.; Mattes, M. J. Int. J. Cancer 1991, 47, 163-
169.
(10) Li, P. Y.; Del Vecchio, S.; Fonti, R.; Carriero, M. V.; Potena, M. I.; Botti,
G.; Miotti, S.; Lastoria, S.; Menard, S.; Colnaghi, M. I.; Salvatore, M. J.
Nucl. Med. 1996, 37, 665-672.
(30) Shen, F.; Wu, M.; Ross, J. F.; Miller, D.; Ratnam, M. Biochemistry 1995,
34, 5660-5665.
(11) Buist, M. R.; Kenemans, P.; den Hollander, W.; Vermorken, J. B.; Molthoff,
C. J. M.; Burger, C. W.; Helmerhorst, T. J. M.; Baak, J. P. A.; Roos, J. C.
Cancer Res. 1993, 53, 5413-5418.
(31) Matherly, L. H.; Wong, S. C.; Angeles, S. M.; Taub, J. W.; Smith, G. K.
Proc. Am. Assoc. Cancer Res. 1994, 35, 307.
(32) Shen, F.; Zheng, X.; Wang, J.; Ratnam, M. Biochemistry 1997, 36, 6157-
(12) Toffoli, G.; Cernigoi, C.; Russo, A.; Gallo, A.; Bagnoli, M.; Boiocchi, M.
Int. J. Cancer 1997, 74, 193-198.
6163.
(33) Wang, H.; Ross, J. F.; Ratnam, M. Nucleic Acid Res. 1998, 26, 2132-
2142.
(13) Williams, W. M.; Huang, K. C. Am. J. Physiol. 1982, 242, F484-F490.
(14) Selhub, J.; Franklin, W. A. J. Biol. Chem. 1984, 259, 6601-6606.
(15) Selhub, J.; Emmanouel, D.; Stavropoulos, T.; Arnold, R. Am. J. Physiol.
1987, 252, F750-F756.
(34) Holm, J.; Hansen, S. I.; Hoier-Madsen, M.; Bostad, L. Biochem. J. 1991,
280, 267-271.
(35) Leamon, C. P.; Reddy, J. A. AdV. Drug DeliVery ReV. 2004, 56, 1127-
(16) Selhub, J.; Nakamura, S.; Carone, F. A. Am. J. Physiol. 1987, 252, F757-
F760.
1141.
(36) Ke, C.-Y.; Mathias, C. J.; Green, M. A. AdV. Drug DeliVery ReV. 2004,
56, 1143-1160.
(17) Kamen, B. A.; Caston, J. D. J. Biol. Chem. 1975, 250, 2203-2205.
9
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J. AM. CHEM. SOC. 2005, 127, 7421-7426
7421

A R T I C L E S
Ke et al.
Chart 1. Structural Formulas of Folic Acid, Pteroic Acid, DTPA-Folate, and DTPA-NOON-Pte (1)
both in vitro and in vivo, these folate conjugates selectively
target folate receptor-expressing cells. Internalization of the
folate conjugates appears to occur via a receptor-mediated
pathway, since the uptake is specific for the attached folate
molecule, saturable at nanomolar concentrations, inhibited at
low temperatures, competitively blocked by excess free folate
or FBP antibody, and eliminated by removal of FBP from the
cell surface.3,6,40-50
radiometal-labeled (111In, 66-68Ga, 99mTc) folate-chelate con-
jugates.36,55 The present study was undertaken to better probe
the structural requirements for folate receptor targeting with low
molecular weight radiometal chelates, specifically examining
the role of the amino acid fragment of folic acid (pteroylglutamic
acid) in mediating folate-receptor affinity.
Results and Discussion
Characterizations of the folate “binding pocket” and structural
requirements for folate receptor ligands have been inves-
tigated51-53 to improve the binding affinity and selectivity of
ligands to the receptor; however, the detailed structure of the
folate receptor has not been crystallographically established.
Notably, pteroic acid, a fragment of folic acid lacking the distal
glutamyl residue (Chart 1), does not appreciably bind to the
high-affinity folate receptor.54
Pteroic acid was conjugated with diethylenetriaminepentaace-
tic acid (DTPA) via an amide-linked hydrophilic spacer, 2,2′-
(ethylenedioxy)-bis(ethylamine), yielding the bifunctional che-
late: pteroyl-NHCH2CH2OCH2CH2OCH2CH2NH-DTPA (1).
The three-step synthesis is outlined in Scheme 1. Briefly, pteroic
acid and 2,2′-(ethylenedioxy)-bis(ethylamine) were coupled
using
benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphonium
hexafluorophosphate (PyBOP) and N-hydroxybenzotriazole
(HOBt) in the presence of N-methylmorpholine (NMM) as a
base. Due to the poor solubility of pteroic acid and its
derivatives, dimethyl sulfoxide (DMSO) was used as a solvent
for all the synthetic steps. After removal of DMSO by trituration
with methanol and diethyl ether and vacuum evaporation of
volatile reagents, pteroyl-ethylenedioxy-bis(ethyldiamine) (in-
termediate 2, yellow solid) was produced in 57% yield.
DTPA dianhydride is a commercially available and commonly
used starting material for synthesis of DTPA bioconjugates.
However, to avoid the undesired cross-linking side reaction that
is possible with DTPA dianhydride,56-59 tri-tert-butyl-protected
DTPA was instead prepared and utilized.60 The yellow solid 2
was mixed with DTPA tri-tert-butyl ester using the same
coupling conditions as the previous step; however, a strong base,
We, and others, have shown that the folate receptor can be
targeted for medical diagnostic imaging with a variety of
(37) Lu, Y.; Sega, E.; Leamon, C. P.; Low, P. S. AdV. Drug DeliVery ReV. 2004,
56, 1161-1176.
(38) Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. AdV. Drug DeliVery
ReV. 2004, 56, 1177-1192.
(39) Zhao, X. B.; Lee, R. J. AdV. Drug DeliVery ReV. 2004, 56, 1193-1204.
(40) Leamon, C. P.; Low, P. S. J. Biol. Chem. 1992, 267, 24966-24971.
(41) Leamon, C. P.; Low, P. S. Proc. Natl. Acad. U.S.A. 1991, 88, 5572-5576.
(42) Antony, A. C.; Kane, M. A.; Portillo, R. M.; Elwood, P. C.; Kolhouse, J.
F. J. Biol. Chem. 1985, 260, 14911-14917.
(43) Henderson, G. B.; Tsuji, J. M.; Kumar, H. P. J. Membr. Biol. 1988, 101,
247-258.
(44) Pizzorno, G.; Cashmore, A. R.; Moroson, B. A.; Cross, A. D.; Smith, A.
K.; Marling-Cason, M.; Kamen, B. A.; Beardsley, G. P. J. Biol. Chem.
1993, 268, 1017-1023.
(45) Sirotnak, F. M. Cancer Res. 1985, 45, 3992-4000.
(46) Leamon, C. P.; Low, P. S. Biochem. J. 1993, 291, 855-860.
(47) Turek, J. J.; Leamon, C. P.; Low, P. S. J. Cell Sci. 1993, 106, 423-430.
(48) Leamon, C. P.; Pastan, I.; Low, P. S. J. Biol. Chem. 1993, 268, 24847-
24854.
(49) (a) Lee, R. J.; Low, P. S. J. Biol. Chem. 1993, 269, 3198-3204. (b) Lee,
R. J.; Low, P. S. Biochim. Biophys. Acta 1995, 1233, 134-144.
(50) Leamon, C. P.; Low, P. S. J. Drug Targeting 1994, 2, 101-112.
(51) McAlinden, T. P.; Hynes, J. B.; Patil, S. A.; Westerhof, G. R.; Jansen, G.;
Schornagel, J. H.; Kerwar, S. S.; Freisheim, J. H. Biochemistry 1991, 30,
5674-5681.
(55) Ke, C.-Y.; Mathias, C. J.; Green, M. A. Nucl. Med. Biol. 2003, 30, 811-
817 and references therein.
(56) Deal, K. A.; Motekaitis, R. J.; Martell, A. E.; Welch, M. J. J. Med. Chem.
1996, 39, 3096-3106.
(57) Arano Y.; Uezono, T.; Akizawa, H.; Ono, M.; Wakisaka, K.; Nakayama,
M.; Sakahara, H.; Konish, J.; Yokoyama, A. J. Med. Chem. 1996, 39,
3451-3460 and references therein.
(52) Leamon, C. P.; DePrince, R. B.; Hendren, R. W. J. Drug Targeting 1999,
7, 157-169.
(58) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Supuran, C. T. J. Med. Chem.
2002, 45, 1466-1476.
(53) Linder, K. E.; Wedeking, P.; Ramalingam, K.; Nunn, A. D.; Tweedle, M.
F. J. Nucl. Med. 2000, 41 (Suppl.), 470.
(59) Lee, R. T.; Lee, Y. C. Bioconjugate Chem. 2001, 12, 845-849.
(60) Achilefu, S.; Wilhelm, R. R.; Jimenez, H. N.; Schmidt, M. A.; Srinivasan,
A. J. Org. Chem. 2000, 65, 1562-1565.
(54) Kamen, B. A.; Capdevila, A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 5983-
5987.
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7422 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

A Folate Receptor-Targeted Pteroic Acid Conjugate
A R T I C L E S
Scheme 1. Synthesis of Compound 1a
a Reagents: (a) 2,2′-(ethylenedioxy)bis(ethylamine), PyBOP, HOBt, NMM, DMSO; (b) DTPA tri-tert-butyl ester, PyBOP, HOBt, NMM, DMSO, MTBD;
(c) 70% TFA/CH2Cl2.
N-methyl-1,5,9-triazabicyclo[4.4.0]decene (MTBD), was added
to improve the solubility of pteroyl-ethylenedioxy-bis(ethyl-
diamine).61 The pteroyl-ethylenedioxy-bis(ethyldiamine)-DTPA
tert-butyl ester, 3, was double-purified using semipreparative
reverse-phase HPLC. The three t-butyl protecting groups were
then removed with 70% trifluoroacetic acid in methylene
chloride, and the desired product, 1, was isolated as a pure
yellow solid by trituration with diethyl ether. Both 1 and 3
exhibit the expected molecular ion peaks in their positive ion
and negative ion electrospray mass spectra.
The pteroic acid-DTPA conjugate 1 (“DTPA-NOON-Pte”)
was used to prepare the corresponding indium(III) chelate
radiolabeled with 111In (67.3 h half-life; abundant γ photons at
171 keV (90.2%) and 245 keV (94.0%)). The radiopharmaceu-
tical was conveniently obtained from 111In chloride via ligand
exchange in acetate buffer. The radiochemical purity of the
resulting crude 111In-DTPA-NOON-Pte was evaluated by radio
HPLC (Figure 1a); the major radioactive HPLC peak, eluting
with a retention time of 14.2 min, was collected. To verify this
peak as the desired 111InIII-1 complex, stable indium(III)[113/115In]
chloride was added to the crude radiopharmaceutical solution
Figure 1. (a) HPLC of the crude no carrier-added 111In-DTPA-NOON-
Pte product. The 111In-DTPA-NOON-Pte product, eluting at 14.2 min,
corresponds to 82% of the total radioactivity. The identities of the
radiochemical impurities, eluting as 2.8 and 10.6 min, are unknown. The
UV signals arise from the excess of 1 in the formulation, which may be
present as a mixture of the free ligand and its complexes with trace metal
contaminants. (b) HPLC of the carrier-added 111In-DTPA-NOON-Pte
product, obtained by addition of carrier indium(III) chloride to an aliquot
of the crude reaction mixture that produced (a), showing the appearance of
carrier [In-DTPA-NOON-Pte]1- in the output from the UV detector. In
both cases, the eluate passes through the UV-vis detector ∼15 s before
the radioactivity detector.
(containing compound 1 in excess) generating carrier-added
111In-DTPA-NOON-Pte at a concentration sufficient for detec-
tion by both the UV and radioactivity detectors connected in a
series at the HPLC column outlet. As expected, the radiochro-
matogram of the carrier-added 111In-DTPA-NOON-Pte prepa-
ration was identical with that of the no-carrier-added 111In-
DTPA-NOON-Pte, but with the appearance of UV intensity for
the carrier-added product (Figure 1b). Electrospray mass
spectrometry confirmed the chemical identity of the nonradioac-
tive carrier product, [113/115In-DTPA-NOON-Pte]1-, eluting at
14 min.
The HPLC-purified 111In-DTPA-NOON-Pte complex was
isolated from the HPLC solvent system by solid-phase extraction
and then repeatedly analyzed by HPLC to assess complex
stability. This HPLC analysis showed the isolated [111In-DTPA-
NOON-Pte]1- radiopharmaceutical to remain stable for at least
7 days at room temperature (Figure 2). Radio TLC of the HPLC-
purified 111In-DTPA-NOON-Pte product (Figure 3) confirms
the absence of 111In chloride and 111In acetate, both of which
irreversibly adsorb to C18 (Rf ) 0).
To assess the ability of the 111In-DTPA-NOON-Pte complex
to target the folate receptor in vivo, the biodistribution and
pharmacokinetics of this radiopharmaceutical were evaluated
in athymic mice bearing subcutaneous folate receptor positive
human KB cell tumor xenografts.6,7 The 111In-DTPA-NOON-
Pte agent rapidly cleared from the blood (Table 1) and was found
to selectively localize in the folate receptor positive tumors (5.4
( 0.8 and 5.6 ( 1.1% of the injected 111In dose per gram of
tumor at 1 h and 4 h postinjection, respectively). Prolonged
tumor retention of the radiolabel was observed, with 3.6 ( 0.6%
of the injected 111In dose per gram of tumor still remaining at
24 h postinjection. Tumor localization of the 111In radiolabel
(61) Luo, J.; Smith, M. D.; Lantrip, D. A.; Wang, S.; Fuchs, P. L. J. Am. Chem.
Soc. 1997, 119, 10004-10013.
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A R T I C L E S
Ke et al.
Table 1. Biodistribution of 111In-DTPA-NOON-Pte in KB Tumor-Bearing Athymic Mice at Various Times Following Intravenous
Administration
percentage of injected 111In dose per gram (tissue wet mass)a
1 h
4 h
4 h
−
blockedb
24 h
blood
heart
lungs
liver and gall bladder
spleen
kidney (one)
stomach, intestines, and contents
muscle
tumor
0.18 ( 0.01
3.5 ( 0.4
1.5 ( 0.2
4.3 ( 0.7
0.31 ( 0.09
61 ( 5
1.7 ( 0.2
3.5 ( 0.5
5.4 ( 0.8
0.050 ( 0.009
2.9 ( 0.9
1.4 ( 0.3
1.9 ( 0.6
0.29 ( 0.07
81 ( 7
1.5 ( 0.9
2.8 ( 0.8
5.6 ( 1.1
0.016 ( 0.010
0.015 ( 0.010
0.041 ( 0.027
0.051 ( 0.029
0.028 ( 0.017
0.90 ( 0.59
0.035 ( 0.002
1.2 ( 0.5
0.72 ( 0.21
1.2 ( 0.6
0.27 ( 0.08
105 ( 7
1.7 ( 1.1
1.2 ( 0.2
0.080 ( 0.057
0.12 ( 0.07
1.9 ( 0.6
3.6 ( 0.6
tumor/blood
tumor/kidney
tumor/liver
30 ( 5
0.088 ( 0.013
1.3 ( 0.4
111 ( 11
0.069 ( 0.013
3.0 ( 0.5
7.5 ( 2.4
0.12 ( 0.03
2.2 ( 0.6
1.5 ( 0.7
105 ( 20
0.035 ( 0.008
3.7 ( 1.4
tumor/muscle
1.6 ( 0.4
2.1 ( 0.2
2.0 ( 0.6
tumor mass (g)
animal mass (g)
animal quantity and gender
0.15 ( 0.10
29.5 ( 1.2
3M
0.080( 0.031
28.6 ( 1.6
4M
0.077 ( 0.010
28.2 ( 1.1
4M
0.104 ( 0.097
28.6 ( 0.8
4M
a Values shown represent the mean ( standard deviation. b Folate receptors blocked by co-injection of folic acid dihydrate at a dose of 4.1 ( 0.4 mg/kg.
(Table 1). This is similar to the current (Table 2) and prior6,7
findings for 111In-DTPA-folate and contrasts with the com-
bination of fecal and urinary clearance observed for 67Ga-
deferoxamine-folate.3-5
The substantial retention of 111In in the kidneys (Table 1) is
fully consistent with binding of 111In-DTPA-NOON-Pte to
tissue folate receptors, since folate receptors are abundant in
the renal proximal tubules.7,13-16 This interpretation is supported
by the expected, and observed, marked reduction in renal 111In
levels when 111In-DTPA-NOON-Pte was co-administered with
excess folic acid. Prior studies of 111In-DTPA-folate and
67Ga-deferoxamine-folate in this mouse tumor model have
Figure 2. Repeat HPLC analyses of the HPLC-purified no carrier-added
[
matogram ca. 90 min following radiopharmaceutical isolation. Top:
Chromatogram 7 days later, confirming the radiochemical stability of this
product.
shown that tumor/nontarget contrast can be substantially
manipulated by co-administering modest doses of folic acid with
the radiopharmaceutical;4-5,7 however, for the purposes of the
present study, no effort was made to employ such a strategy to
further optimize 111In-DTPA-NOON-Pte targeting selectivity
in vivo.
111In-DTPA-NOON-Pte]1- (tR ) 14.2 min, Figure 1a). Bottom: Chro-
The behavior of the 111In-DTPA-NOON-Pte radiopharma-
ceutical in this mouse tumor model (Table 1) is virtually
identical to that observed for 111In-DTPA-folate (Table 2),
an agent that has met with success in clinical trials for imaging
folate receptor-expressing gynecologic cancers.62 Thus, although
pteroic acid itself is a poor ligand for the folate receptor, pteroic
acid conjugates appear viable as candidates for folate receptor-
directed drug targeting. The present results indicate that the
glutamate residue of folate (pteroylglutamate) need not be
viewed as an essential component of drug conjugates designed
for tumor targeting via the folate receptor. This finding is
consistent with the reported retention of folate receptor affinity
by conjugates based structurally upon substitution of glycine
for the glutamate of folate.52 The slight decline in the tumor
concentration of 111In 24 h following 111In-DTPA-NOON-Pte
administration is similar to the findings previously reported for
111In-DTPA-folate at 24-48 h postinjection.7
Figure 3. Radio TLC of the HPLC-purified no carrier-added [111In-DTPA-
NOON-Pte]1-
CN).
(
111In-1) (C18 plate eluted with 25% NH4OAc:75% CH3-
clearly appears to be mediated by the cellular folate receptor,
since the tumor uptake of radiotracer drops dramatically (0.12
( 0.07% of the injected 111In dose per gram at 4 h, Table 1)
when 111In-DTPA-NOON-Pte was co-injected with an excess
of folic acid, which will compete for available folate receptor
sites. Urinary excretion appears to be the primary whole body
clearance pathway for the 111In-DTPA-NOON-Pte, since the
radiotracer was never appreciably found in the intestinal contents
The affinity of the In-DTPA-NOON-Pte conjugate for the
folate receptor has not been directly measured; however, the
(62) Siegel, B. A.; Dehdashti, F.; Mutch, D. G.; Podoloff, D. A.; Wendt, R.;
Sutton, G. P.; Burt, R. W.; Ellis, P. R.; Mathias, C. J.; Green, M. A.;
Gershenson, D. M. J. Nucl. Med. 2003, 44, 700-707.
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7424 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

A Folate Receptor-Targeted Pteroic Acid Conjugate
A R T I C L E S
Table 2. Biodistribution of 111In-DTPA-Folate (111In-DTPA-en-γGlu-Pte) in Athymic Mice with Subcutaneous KB Cell Tumor Xenografts
percentage of injected 111In dose per gram (tissue wet mass)a
1 h postinjection
4 h postinjection
4 h postinjection blockedb
blood
heart
lungs
liver and gall bladder
spleen
kidney
muscle
0.14 ( 0.03
2.3 ( 0.4
1.3 ( 0.1
4.0 ( 1.5
0.36 ( 0.03
90 ( 9
3.5 ( 0.8
1.0 ( 0.2
5.3 ( 0.4
0.064 ( 0.007
2.0 ( 0.3
1.1 ( 0.3
2.2 ( 0.4
0.35 ( 0.11
85 ( 12
2.9 ( 0.7
1.0 ( 0.2
6.8 ( 1.2
0.029 ( 0.011
0.022 ( 0.010
0.065 ( 0.021
0.12 ( 0.03
0.060 ( 0.021
5.0 ( 5.5
0.023 ( 0.013
0.49 ( 0.20
0.16 ( 0.07
stomach, intestines, and contents
tumor
tumor/blood
tumor/kidney
tumor/liver
38 ( 7
0.060 ( 0.011
1.5 ( 0.5
106 ( 15
0.080 ( 0.012
3.3 ( 1.1
5.5 ( 0.8
0.050 ( 0.023
1.4 ( 0.4
tumor/muscle
1.6 ( 0.3
2.5 ( 0.9
8.4 ( 3.8
tumor mass (g)
animal mass (g)
0.138 ( 0.051
24 ( 2
0.202 ( 0.083
25 ( 1
0.193 ( 0.078
24 ( 1
a Values shown represent the mean ( standard deviation; n ) 4. Each mouse received ∼3 µCi In-111 in a formulation containing 0.2 µg of DTPA-
folate. b Folate receptors blocked by co-injection of folic acid dihydrate at a dose of 0.50 ( 0.08 mg/kg.
obtained by reaction of 342 µCi 111In with 15 µg of DTPA-folate,
literature has shown that folate conjugates, such as DTPA-
had a radiochemical purity >98% by C18 TLC.6,64 High-field NMR
folate, can exhibit folate receptor affinities very similar to that
spectra were recorded in DMSO-d6 on a Bruker ARX 300 (1H at 300
of folic acid itself.63 In screening candidate folate receptor-
MHz) spectrometer. 1H chemical shifts are reported in δ ppm relative
to residual DMSO (2.49 ppm) as internal standard. Multiplicities in
targeted radiopharmaceuticals, we have instead focused on
assessment of targeting efficiency and selectivity in vivo, where
1H are reported as (s) singlet, (d) doublet, (t) triplet, (q) quartet, and
success implicitly depends not only on receptor affinity, but
(m) multiplet. The HPLC methods employed a Rainin Rabbit-HP system
also on factors such as the effectiveness of drug delivery to
receptor-rich tissue, levels of nonspecific binding, the rates and
routes of agent metabolism, and the rates and routes of
radionuclide clearance and excretion. The reported data indicate
that, in vivo, 111In-DTPA-NOON-Pte targets the folate receptor
as efficiently, and selectively, as the clinically studied 111In-
DTPA-folate radiopharmaceutical.62
with a Knauer UV-vis detector and a NaI(Tl) radioactivity detector
(Bioscan). The HPLC flow rate was 0.5 mL/min for 4.6 × 250 mm
column, and 2.35 mL/min for 10 × 250 mm column. Program 1: 5%
solvent B at 0 min, ramped to 25% B at 25 min, then to 60% B at 27
min, and reached 100% B at 30 min (solvent A is 56 mM NH4OAC
(99.99+%) in water; solvent B is CH3CN). Program 2: 5% solvent B
at 0 min, to 70% B at 30 min, then ramped to 100% B at 32 min, and
remained 100% B to 40 min (solvent A is 5% CH3CN in 0.1% aqueous
TFA; solvent B is 10% water in CH3CN with 0.1% TFA). Program 3:
(using the same solvents as program 2) 5% solvent B at 0 min, ramped
to 40% B at 30 min, then to 100% B at 32 min, and remained 100%
B to 40 min.
Conclusion
Tumor-selective drug targeting via the folate receptor is
feasible with pteroic acid conjugates lacking amino acid
fragments, such as the glutamic acid moiety of folic acid. This
finding is expected to somewhat simplify folate receptor-based
drug targeting strategies, obviating the need to selectively control
conjugation via the R- or γ-carboxylates of folic acid, while
also avoiding amino acid chirality and/or racemization issues
that can exist in preparation of receptor-targeted conjugates
based on folic acid itself.
Synthesis of 1-{2-[2-[(2-(Biscarboxymethyl-amino)ethyl)-car-
boxymethyl-amino]ethyl]-carboxymethyl-amino}-acetylamino-3,6-
dioxa-8-pteroylamino-octane (1). To a suspension of pteroic acid
(0.025 g, 0.080 mmol), PyBOP (0.125 g, 0.240 mmol), and HOBt
(0.037 g, 0.240 mmol) in DMSO (0.8 mL), NMM (0.049 g, 0.48 mmol)
was added under N2. After being stirred at room temperature for 10
min, 2,2′-(ethylenedioxy)bis(ethylamine) (0.237 g, 1.600 mmol) was
added. The suspension gradually turned clear after the addition of this
amine, with the reagents completely dissolved by 1 h. The reaction
was stirred at room temperature for 22.5 h, then concentrated under
vacuum overnight. The resulting brown oil was triturated with MeOH,
and the resultant suspension was centrifuged to produce a light yellow
solid. The solid was washed with MeOH and Et2O and dried in a
vacuum desiccator to produce 20 mg of product 2. Electrospray MS
calcd for C20H26N8O4: m/z 442, positive mode (M + H+) found 443,
negative mode (M - H+) found 441. 1H NMR (DMSO-d6, 300 MHz):
δ ppm 2.73 (2H, m), 3.20-3.70 (10H, m), 4.46 (2H, s, broad), 6.61
(2H, d, J ) 8.3 Hz), 6.88 (1H, m), 7.02 (2H, m), 7.59 (2H, d, J ) 8.3
Hz), 8.05 (1H, m), 8.61 (1H, s). For identification purposes, analytical
HPLC was performed with a 4.6 × 250 mm C18 reverse-phase column
with flow rate 0.5 mL/min using program 3. The desired product has
a retention time of 17.3 min detected with UV detector @ 254 nm.
Experimental Section
Materials and General Methods. Unless otherwise specified, all
reagents were purchased and used without further purification. Distilled,
deionized water (18 MΩ) was used throughout. DMSO was distilled
from CaH2 under reduced pressure and stored over activated 4 Å
molecular sieves. 111In chloride was obtained from Mallinckrodt
Medical, Inc., St. Louis, MO. The tri-tert-butyl-protected DTPA was
synthesized as described by S. Achilefu et al.60 C18 Sep-Pak Light
solid-phase extraction cartridges were purchased from Millipore, Inc.
A Capintec CRC-127R radioisotope calibrator was used for assays of
111In radioactivity in the microcurie to millicurie range, whereas low-
level (<0.01 µCi) samples of 111In were counted in a Packard A5530
automatic γ scintillation counter with a 3-in. large-bore NaI(Tl) crystal.
111In-DTPA-folate was prepared as described previously for use in
control biodistribution studies.7,64 The resulting 111In-DTPA-folate,
To a solution of tri-tert-butyl DTPA (0.075 g, 0.134 mmol), PyBOP
(0.084 g, 0.163 mmol), and HOBt (0.025 g, 0.163 mmol) in DMSO
(0.4 mL), NMM (0.0247 g, 0.244 mmol) was added under N2 at room
temperature. This premixed solution was then added under N2 to the
(63) Leamon, C. P.; Parker, M. A.; Vlahov, I. R.; Xu, L. C.; Reddy, J. A.;
Vetzel, M.; Douglas, N. Bioconjugate Chem. 2002, 13, 1200-1210.
(64) Mathias, C. J.; Green, M. A. Nucl. Med. Biol. 1998, 25, 585-587.
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A R T I C L E S
Ke et al.
suspension of the yellow solid 2 (0.0120 g, 0.0271 mmol) in DMSO
(0.2 mL). MTBD (0.0532 g, 0.347 mmol) was added after the reaction
mixture was stirred at room temperature overnight. The reaction mixture
was stirred at room temperature for another day and then concentrated
under vacuum. The resulting brown residue was triturated first with
Et2O and then with MeOH to produce crude product as a yellow solid.
This yellow solid was purified using semipreparative HPLC twice on
a C18 column (10 × 250 mm) with program 2. The peak with retention
time 30.5 min, detected by a UV detector at 254 nm, was collected
and concentrated to produce the desired pure product 3. Electrospray
MS calcd for C46H71N11O13: m/z 985, positive mode (M + H+) found
temperature to produce the desired 111In-1 complex for further study.
The complex was reconstituted in water for the reported stability study
(Figure 2) or in saline for evaluation of radiopharmaceutical biodis-
tribution following intravenous administration to tumor-bearing athymic
mice.
The stable In(III) complex of 1, [113/115In-DTPA-NOON-Pte]1-, was
prepared in a manner analogous to the radiolabeled chelate, but at a
1:1 In:1 stoichiometry. Electrospray MS calcd for [115In-DTPA-
NOON-Pte]1- (InC34H43N11O13): m/z 928.21; negative mode (M-)
found 928.46; positive mode found (M- + 2H+) 930.14, (M- + H+
+
Na+) 952.18, and (M- + 2Na+) 974.16. High-resolution positive ion
electrospray mass spectrum for [InC34H43N11O131-+ 2H+]: calcd
930.2237, found 930.2244.
1
986, negative mode (M - H+) found 984. H NMR (DMSO-d6, 500
MHz): δ ppm 1.40 (18H, s), 1.45 (9H, s), 3.02 (4H, m), 3.22 (2H, m),
3.31 (6H, m), 3.39-3.55 (18H, m), 4.46 (2H, s, broad), 6.52 (1H, s,
broad), 6.61 (2H, d, J ) 8.3 Hz), 6.88 (2H, s, broad), 7.59 (2H, d, J )
8.3 Hz), 8.04 (1H, d, J ) 5.5 Hz), 8.09 (1H, s, broad), 8.63 (1H, s).
Removal of the tert-Butyl Ester Protecting Groups. To the HPLC-
purified coupling product 3 (2 mg) in a 5-mL round-bottomed flask
was added 70% TFA/CH2Cl2. (The flask was twice rinsed with ultrapure
concentrated HCl and several times with water, then dried in a 110 °C
oven overnight before use.) The reaction mixture was stirred at 0 °C
for 30 min. After being stirred at room temperature for an additional
5 h, the reaction mixture was repeatedly concentrated after two CH2-
Cl2 additions, using rotary evaporator, producing 1 as a yellow residue.
Electrospray MS calcd for C34H47N11O13: m/z 817, positive mode (M
+ H+) found 818, negative mode (M - H+) found 816. 1H NMR
(DMSO-d6, 500 MHz): δ ppm 3.05 (4H, m), 3.22 (2H, m), 3.28-3.60
(24H, m), 4.48 (2H, s), 6.61 (2H, d, J ) 8.6 Hz), 7.36 (2H, s, very
broad), 7.59 (2H, d, J ) 8.6 Hz), 8.05 (2H, t, J ) 5.4 Hz), 8.66 (1H,
s). This product was used for labeling with 111In chloride without further
purification.
Synthesis of 111In Complex of (1). Briefly, ca. 1.2 mCi no carrier-
added 111In chloride in 0.05 mL of 0.05 N HCl was transferred to a
test tube, and 0.05 mL of 0.1 N ammonium acetate (pH 5.5), followed
by 0.02 mL of 0.5 N ammonium acetate (pH 7.4) were added, producing
a solution with pH 7. A sample of ligand 1 was weighed and diluted
in water adjusted with 1 N NaOH to pH 9-10. A quantity of 10 µL of
this solution, containing 95 µg of 1, was added to the 111In acetate
solution (120 µL) and allowed to stand overnight at room temperature,
protected from light.65 The crude 111In-1 complex was purified by radio
HPLC using a 4.6 × 250 mm Dynamax C18 reverse-phase column
(Varian/Rainin) eluting with aqueous NH4OAC/CH3CN using program
1 and a flow rate of 1 mL/min. The fraction with retention time 14.2
min was collected and diluted with water to reduce the percentage of
CH3CN to less than 5%. A C18 Sep-Pak light solid-phase extraction
cartridge was conditioned by washing with EtOH followed by water.
The conditioned C18 Sep-Pak was loaded with the diluted HPLC
fraction and washed with water (3 mL), and then the 111In-1 complex
was eluted with EtOH. The ethanol fractions containing radioactivity
were combined and evaporated to dryness under a N2 stream at room
Biodistribution Studies with 111In Radiotracers. All animal studies
were conducted in accordance with procedures approved by the Purdue
Animal Care and Use Committee. The general procedure for the animal
biodistribution studies has been described previously.4,5,7 The athymic
mice used in these studies were maintained for 3 weeks on a folate-
free diet (to reduce their serum folate to a level near that of human
serum) and had been implanted subcutaneously in the interscapular
region with human KB tumor cells, as described elsewhere.3,4,7 The
diethyl ether anesthetized mice were injected with radiotracer via the
exposed femoral vein and allowed to recover. Syringes used for
radiotracer injections were weighed on an analytical balance before
and after injection to quantify the dose received by each animal.
Following animal sacrifice and dissection, tissue samples were weighed
and counted to quantify 111In. The uptake of radiotracer in each tissue
sample was calculated as both a percentage of the injected dose (%ID)
per organ (Supporting Information) and as a %ID/g of tissue wet weight
(Tables 1 and 2), using reference counts from a weighed and
appropriately diluted sample of the original injectate. Blood was
assumed to account for 5.5% of total body mass. Muscle was assumed
to account for 42% of the total body mass. To competitively block
tissue folate receptors, a subset of the mice were co-injected with 111In
radiopharmaceutical premixed with folic acid dihydrate at the doses
specified (Tables 1 and 2).
Acknowledgment. This work was supported by a grant from
the National Cancer Institute of the U.S. Public Health Service
(R01-CA70849). The athymic mouse, mass spectrometry, and
NMR facilities used in this project are affiliated with the Purdue
Cancer Center and were partially supported by the National
Cancer Institute through Cancer Center Support Grant CA02316.
Supporting Information Available: Data tables expressing
the biodistribution of 111In-DTPA-NOON-Pte and 111In-
DTPA-folate as a percentage of injected 111In dose per organ
or tissue (athymic mouse/KB tumor xenograft model). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(65) Thomas, A. H.; Suarez, G.; Cabrerizo, F. M.; Martino, R.; Capparelli, A.
J. Photochem. Photobiol., A 2000, 135, 147-154 and references therein.
JA043006N
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